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Platelet membrane surface heterogeneity detected by partition in two-polymer phase systems Webber, Timothy James 1984

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PLATELET MEMBRANE SURFACE HETEROGENEITY DETECTED BY PARTITION IN TWO-POLYMER AQUEOUS PHASE SYSTEMS by TIMOTHY JAMES WEBBER B.S., Loyola U n i v e r s i t y , 1972 B.S., C a l i f o r n i a State U n i v e r s i t y , 1976 M.S., C a l i f o r n i a State U n i v e r s i t y , 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard UNIVERSITY OF BRITISH COLUMBIA May 1984 Timothy James Webber, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Ma l l V a n c o u v e r , C a n a d a V6T 1Y3 D e p a r t m e n t o f fCL i i ABSTRACT P l a t e l e t s are heterogeneous with respect to a v a r i e t y of f u n c t i o n a l and p h y s i c a l p r o p e r t i e s , as a r e s u l t of d i f f e r e n t phenomena. Megakaryocytes confer upon t h e i r progeny d i f f e r e n t p l a t e l e t p r o p e r t i e s which then change during c i r c u l a t i o n . Since p l a t e l e t s u r v i v a l time analyses of p l a t e l e t production and p l a t e l e t ageing have provided d i a g n o s t i c i n f o r m a t i o n , e f f o r t s have been expended to o b t a i n comparable i n f o r m a t i o n regarding c i r c u l a t i n g p l a t e l e t heterogeneity through the use of methods which r e f l e c t p l a t e l e t p r o p e r t i e s , e.g., f u n c t i o n , s i z e , d e n s i t y , and membrane surface c h a r a c t e r i s t i c s , which are b e l i e v e d t o change during the s u r v i v a l of p l a t e l e t s w i t h i n the c i r c u l a t i o n . P l a t e l e t membrane p r o p e r t i e s were stud i e d here because c e l l s u rface charge i s thought to be a l t e r e d during c i r c u l a t i o n , and because countercurrent d i s t r i b u t i o n (CCD) i n two-polymer aqueous phase systems has been shown t o be capable of d e t e c t i n g such membrane changes during e r y t h r o c y t e maturation and ageing. This technique, CCD i n phase systems s e n s i t i v e t o membrane charge, was th e r e f o r e assessed f o r a p p l i c a t i o n to the problem of determining c i r c u l a t i n g p l a t e l e t age d i s t r i b u t i o n i n f o r m a t i o n i n an i n v e s t i g a t i o n of p l a t e l e t heterogeneity. Aqueous two-polymer phase systems were shown to be charged, demonstrating, between the upper and lower phases, a p o t e n t i a l d i f f e r e n c e which decreased with decreasing phosphate and i n c r e a s i n g c h l o r i d e content. P l a t e l e t surface property d i s t r i b u t i o n s were heterogeneous based on the shape and spread o f the CCD curves f o r these blood elements i n comparison to expected t h e o r e t i c a l d i s t r i b u t i o n s f o r uniform p a r t i c l e s . i i i P l a t e l e t s from r a b b i t , monkey, and human demonstrated species s p e c i f i c d i f f e r e n c e s i n membrane sur f a c e p r o p e r t i e s as r e f l e c t e d by the s i n g l e step p a r t i t i o n measurement. The species s p e c i f i c d i f f e r e n c e s found by s i n g l e step p a r i t i t o n were a l s o confirmed by CCD. P l a t e l e t s , from r a b b i t s , monkeys, and humans, separated according to d e n s i t y which has been shown to be age r e l a t e d , were found to e x h i b i t d i f f e r e n c e s i n s i z e , and membrane surface p r o p e r t i e s by CCD. The p a r t i t i o n d i s t r i b u t i o n s of i n d i v i d u a l d e n s i t y separated p l a t e l e t subpopulations, low d e n s i t y , intermediate d e n s i t y , and high density p l a t e l e t s , were each heterogeneous and a l l overlapped ( p a r t i t i o n s c o i n c i d e d ) , i n d i c a t i n g t h a t w i t h i n each subpopulation there were p l a t e l e t s of d i f f e r e n t membrane surface p r o p e r t i e s , and t h a t each subpopulation was i n p a r t s i m i l a r to the ot h e r s . However, the r e l a t i v e p a r t i t i o n s o f these d e n s i t y separated p l a t e l e t s were a l s o d i f f e r e n t based on s t a t i s t i c a l comparisons of the CCD parameters. CCD and de n s i t y analyses 51 of i n v i t r o C r - l a b e l e d r a b b i t p l a t e l e t s revealed a p l a t e l e t subpopulation, found to be enriched i n high d e n s i t y p l a t e l e t s , which e x h i b i t e d enhanced uptake f o r ^ C r , but not f o r "'"''""''In, "^C- or 3 3 H-serotonin, nor f o r H-adenine. This p l a t e l e t subpopulation was of low p a r t i t i o n r e l a t i v e to the p l a t e l e t p o p u l a t i o n , and was no longer apparent a f t e r i n v i v o i n j e c t i o n and c i r c u l a t i o n f o r 24 to 48 hours. 35 Combinations of S-sulphate cohort l a b e l i n g i n v i v o , w i t h "^H-serotonin l a b e l i n g _in v i t r o , d e nsity gradient c e n t r i f ugation, and CCD, i n d i c a t e d that p l a t e l e t s which were newly re l e a s e d i n t o the c i r c u l a t i o n were of r e l a t i v e l y high density and low p a r t i t i o n . Although t h i s subpopulation was heterogeneous, i t s average d e n s i t y and surface p r o p e r t i e s changed with p l a t e l e t maturation and age i n the c i r c u l a t i o n . i v TABLE OF CONTENTS Page Chapter 1 INTRODUCTION 1 1.1 P l a t e l e t , O r i g i n , Production and Removal 1 1.2 Erythr o c y t e and P l a t e l e t Surface Charge 3 1.3 P l a t e l e t S u r v i v a l , Surface Charge, Density and Heterogeneity 4 1.4 Megakaryocyte and P l a t e l e t Heterogeneity 23 1.5 Membrane Surface P r o p e r t i e s by Aqueous Two-Phase P a r t i t i o n 27 1.6 P a r t i t i o n S t u d i e s of C e l l Surface P r o p e r t i e s During Ageing 32 1.7 C l i n i c a l and D i a g n o s t i c A p p l i c a t i o n of P a r t i t i o n Methodology 37 Chapter 2 MATERIALS AND METHODS 41 2.1 Blood Samples 41 2.2 Anticoagulant 41 2.3 Tyrode's S o l u t i o n s 42 2.4 Pr e p a r a t i o n s of P l a t e l e t s 43 2.5 P l a t e l e t Aggregation 43 2.6 P l a t e l e t F i x a t i o n 44 2.7 P l a t e l e t L a b e l i n g 44 2.8.1 S t r a c t a n I I Stock S o l u t i o n s 45 2.8.2 S t r a c t a n Density Gradient 46 2.9.1 Phase System Polymer Stock S o l u t i o n s 48 2.9.2 Phase System S a l t Stock S o l u t i o n s 49 2.9.3 Two-Polymer Aqueous Phase System Composition 49 2.10 S i n g l e Step P a r t i t i o n 51 2.11.1 Countercurrent D i s t r i b u t i o n (CCD) 51 2.11.2 CCD of Density Separated P l a t e l e t s 53 2.11.3 CCD of In V i t r o Labeled P l a t e l e t s Aged In Vivo 54 2.11.4 CCD of In Vivo Cohort Labeled P l a t e l e t s 54 V Page 2.12.1 P a r t i c l e Counting 55 2.12.2 P a r t i c l e S i z i n g 56 2.13 R a d i o a c t i v i t y Counting 58 2.14 G r a p h i c a l P r e s e n t a t i o n of R e s u l t s 58 2.15 C e n t r i f ugation 59 Chapter 3 RESULTS AND DISCUSSION 60 3.1 S i n g l e Step P l a t e l e t P a r t i t i o n and F e a s i b i l i t y o f P l a t e l e t CCD 60 3.2 CCD of Density Separated P l a t e l e t s 66 3.3 S i z e A n a l y s i s of Density Separated P l a t e l e t s 77 3.4 E l e c t r o p h o r e t i c M o b i l i t y (EPM) of Density Separated P l a t e l e t s 79 3.5 EPM of S i z e Separated P l a t e l e t s 79 3.6 CCD of 51-Cr-Labeled P l a t e l e t s , Aged In Vivo 81 3.7 CCD of P l a t e l e t s , 51-Cr-Labeled P l a t e l e t Enrichment...93 3.7.1 Experimental 95 3.7.2 R e s u l t s ...98 3.7.3 Di s c u s s i o n 104 3.8 CCD of In Vivo 35-S-Labeled P l a t e l e t s 110 3.8.1 Experimental I l l 3.8.2 R e s u l t s 113 3.8.3 D i s c u s s i o n 114 3.9 CCD of 35-S-Labeled LDP, IDP and HDP, Day Two and S i x , p . i 116 3.9.1 Experimental 117 3.9.2 R e s u l t s 119 3.9.3 D i s c u s s i o n 122 3.10 CCD of 35-S-Labeled LDP, IDP and HDP, Day Two and S i x , p . i 124 3.10.1 Experimental 125 3.10.2 R e s u l t s 126 v i Page 3.11 CCD of 35-S-Labeled P l a t e l e t s , Double Labeled with 3-H-Serotonin 127 3.11.1 Experimental 127 3.11.2 Results 128 3.11.3 Di s c u s s i o n 130 3.12 CCD of 35-S-Labeled P l a t e l e t s , Double Labeled with 3-H-Serotonin 131 3.12.1 Experimental 131 3.12.2 Re s u l t s 132 3.12.3 D i s c u s s i o n 132 3.13 S i n g l e Step P a r t i t i o n of P l a t e l e t s During Cancer Chemotherapy 135 3.13.1 Experimental 137 3.13.2 Re s u l t s 138 3.13.3 Di s c u s s i o n 140 GENERAL DISCUSSION AND CONCLUSION 150 Conclusion 152 REFERENCES 155 APPENDIX 176 v i i LIST OF TABLES Table Page 1.4.1 P l a t e l e t Heterogeneity O u t l i n e 26 2.8.2.1 S p e c i f i c G r a v i t y and Density of S t r a c t a n 47 2.9.3.1 Two-Polymer Phase System S a l t Concentrations and R a t i o s 50 2.9.3.2 Phase System E l e c t r o s t a t i c P o t e n t i a l D i f f e r e n c e s 50 3.1.1 Summary of Single-t u b e P a r t i t i o n of P l a t e l e t s 62 3.2.1 Summary of Rabbit, Human and Monkey, LDP, IDP and HDP 72 3.5.1 EPM of S i z e Separated, Fixed P l a t e l e t s , S t a t i s t i c s 80 3.6.1 Student t Test o f P l a t e l e t and R a d i o a c t i v i t y CCD 85 3.6.2 A n a l y s i s of Variance of the t S t a t i s t i c 86 3.6.3 S u b d i v i s i o n of CCD of 51-Cr-Labeled Rabbit P l a t e l e t s 87 3.6.4 Nonpaired and P a i r e d t Test S p e c i f i c A c t i v i t y of Regions I I and I I I , Normalized to the Mean at Ten Percent Combined E r r o r 90 3.8.2.1 Summary of 35-S-Sulphate Labeling K i n e t i c s 114 3.9.2.1 S t r a c t a n Concentration and Density 120 3.9.2.2 A n a l y s i s of 35-S S p e c i f i c A c t i v i t y Enrichment Slope 122 3.10.2.1 A n a l y s i s of 35-S S p e c i f i c A c t i v i t y Enrichment Slope 126 3.11.2.1 A n a l y s i s o f 35-S/3-H Ratio Slope, CCD Enrichment Day One, Three and F i v e , p . i 128 3.11.2.2 A n a l y s i s of 35-S/3-H Ra t i o Slope, CCD Enrichment Day Two, Four and S i x , p . i 128 3.12.2.1 A n a l y s i s of 35-S/3-H Ra t i o Slope, CCD Enrichment 132 3.13.2.1 Comparison of P l a t e l e t P a r t i t i o n s , Normal and MPD 139 v i i i LIST OF FIGURES Fig u r e Page 3.1.1.1 P o t e n t i a l D i f f e r e n c e s Between Upper PEG and Lower Dextran Phases , 61 3.1.1.2 Fixed Rabbit P l a t e l e t Two-Polymer Phase System P a r t i t i o n s 61 3.1.2.1 CCD of Fixed Rabbit P l a t e l e t s 65 3.1.2.2 R e d i s t r i b u t i o n of P l a t e l e t CCD F r a c t i o n s 65 3.2.1.1 Percent Human P l a t e l e t s Remaining on S t r a c t a n Steps...68 3.2.1.2 Human P l a t e l e t Density D i s t r i b u t i o n on S t r a c t a n 68 3.2.2.1 CCD of Density Separated Rabbit P l a t e l e t s 70 3.2.2.2 CCD of Density Separated Human P l a t e l e t s 70 3.3.1.1 S i z e D i s t r i b u t i o n of Density Separated Rabbit P l a t e l e t s 78 3.3.1.2 S i z e D i s t r i b i t o n o f Density Separated Human P l a t e l e t s 78 3.6.1.1 No Change i n Surface Property P a r t i t i o n with Time of C i r c u l a t i o n 82 3.6.1.2 Decreasing P a r i t i t i o n with Time i n C i r c u l a t i o n 82 3.6.2.1 CCD of 51-Cr-labeled P l a t e l e t s 84 3.6.2.2 51-Cr-labeled P l a t e l e t s , Two days of C i r c u l a t i o n 84 3.7.2.1.1 CCD of 51-Cr-labeled P l a t e l e t s 100 3.7.2.1.2 CCD of 111-In-labeled P l a t e l e t s 100 3.7.2.1.3 CCD of 51-Cr-labeled P l a t e l e t s 101 3.7.2.1.4 CCD of 111-In-labeled P l a t e l e t s 101 3.7.2.1.5 CCD of 51-Cr-labeled P l a t e l e t s 102 3.7.2.1.6 CCD of 111-In-labeled P l a t e l e t s 102 3.7.2.2.1 CCD of 111-In-Labeled, LDP, IDP, HDP, HHD 105 3.7.2.2.2 CCD of 51-Cr-Labeled, LDP, IDP, HDP, HHD 105 3.7.2.2.3 CCD of 51-Cr-Labeled LDP 106 3.7.2.2.4 CCD of 51-Cr-Labeled IDP 106 3.7.2.2.5 CCD of 51-Cr-Labeled HDP 107 3.7.2.2.6 CCD of 51-Cr-Labeled HHD, P l a t e l e t s of Density Greater than t h a t of 20% S t r a c t a n 107 3.9.2.1.1 CCD of Day Two 35-S-Labeled LDP, IDP, and HDP 121 3.9.2.1.2 CCD of Day S i x 35-S-Labeled LDP, IDP, and HDP 121 i z F i g u r e Page 3.11.2.1 CCD of Day One, Three, and F i v e , 35-S/3-H-Labeled P l a t e l e t s 129 3.11.2.1 CCD of Day Two, Four, and S i x , 35-S/3-H-Labeled P l a t e l e t s 129 3.12.2.1 CCD Leukocyte Contamination of Day Three 35-S/3-H-Labeled P l a t e l e t s 133 3.12.2.2 CCD Leukocyte Contamination and Day Three 35-S/3-H-Labeled P l a t e l e t s 133 3.12.2.3 CCD of Day Three 35-S/3-H-Labeled P l a t e l e t s 134 3.12.2.4 CCD of Day Three 35-S/3-H-Labeled P l a t e l e t s 134 x ABBREVIATIONS AAT aspartate amino t r a n s f e r a s e ACD a c i d c i t r a t e dextrose ADP adenosine diphosphate ag —18 attograms, 10" grams am —18 attomoles, 10 moles ATP adenosine triphosphate BTG beta thromboglobulin C c u r r e n t , Celloscope s e t t i n g CCD countercurrent d i s t r i b u t i o n (s) CCDC countercurrent d i s t r i b u t i o n c a v i t y ( i e s ) CCDF countercurrent d i s t r i b u t i o n f r a c t i o n (s) CE combined e r r o r CFC colony forming c e l l s Corr c o r r e l a t i o n c o e f f i c i e n t CPM cpm counts per minute D dextran DF df , degrees of ( f ) freedom DPM dpm, d i s i n t e g r a t i o n s per minute EPM e l e c t r o p h o r e t i c m o b i l i t y f femto, 10""'""' Fc c r y s t a l l i z a b l e fragment of immunoglobulin Fx f i x e d , glutaraldehyde f i x e d g 2 g r a v i t y , g f o r c e , a c c e l e r a t i o n g/cm , grams G Celloscope s e t t i n g g a i n , p a r t i t i o n c o e f f i c i e n t , x i GLx gradient load mixture Gm grand mean, r e f e r to t o t a l mean s p e c i f i c a c t i v i t y HDP high density p l a t e l e t s HHD den s i t y f r a c t i o n higher than HDP HHDP p l a t e l e t s of d e n s i t y equal to and higher than HDP Hr h r, hours 5HT s e r o t o n i n Hu human ITP i d o p a t h i c thrombocytopenia ( i c ) purpura Intcp i n t e r c e p t IDP intermediate d e n s i t y p l a t e l e t s IHHDP inte r m e d i a t e , high, high d e n s i t y p l a t e l e t s , a l l p l a t e l e t s o f de n s i t y equal or g r e a t e r than IDP K p a r t i t i o n c o e f f i c i e n t LDH l a c t i c a c i d dehydrogenase LDP low den s i t y p l a t e l e t s LIDP low, intermediate d e n s i t y p l a t e l e t s , lower d e n s i t y than HDP L i n l i n e a r Lx load mixture, CCD m mean, s p e c i f i c a c t i v i t y w i t h i n a l i m i t of CE MNC mononuclear c e l l Mo monkey MPD m y e l o p r o l i f e r a t i v e d i s o r d e r s NaPB sodium phosphate b u f f e r , pH 7.2 N, n number of t r a n s f e r s , experiments, p l o i d y number _9 ng nanograms, 10 grams _g nm nanomoles, 10 moles x i i p parametric s t a t i s t i c a l p r o b a b i l i t y P nonparametric p r o b a b i l i t y PEG polyethylene g l y c o l PF4 p l a t e l e t f a c t o r four p . i . post i n j e c t i o n P i t p l a t e l e t (s) PPP p l a t e l e t poor plasma PRP p l a t e l e t r i c h plasma PRT p l a t e l e t r i c h Tyrode's s o l u t i o n r peak or modal CCDC Ra r a b b i t RPM r e v o l u t i o n s per minute SA s p e c i f i c a c t i v i t y , s i a l i c a c i d SD standard d e v i a t i o n SDev standard d e v i a t i o n StR S t r a c t a n I I , S t. Regis T Celloscope t h r e s h o l d s e t t i n g , or time as appr o p r i a t e ug micrograms, 10 grams um micromoles, 10~^ moles Var variance WGA wheat germ a g g l u t i n i n SYMBOLS % percent, percentage, c o n c e n t r a t i o n w/v unless s t a t e d x i i i ACKNOWLEDGEMENTS The M.S. committee G.S. Ascher D.M. Carl b e r g B. Kazan and H. Walter i n a p p r e c i a t i o n f o r recommendation to Dr. D.E. Brooks g i v i n g opportunity. The Ph.D. committee D.E. Brooks J . F r o h l i c h S. Naiman R.H. Pearce and P.E. Reid. D.E. Brooks and the B r i t i s h Columbia Heart Foundation f o r personal support. Mom the unsung hero patron S a i n t Jude dad and brothers f o r moral support and encouragement. In fond memory of the wealth of f r i e n d s h i p i n us and Canada. - 1 -CHAPTER 1 INTRODUCTION 1.1 P l a t e l e t O r i g i n , P r o d u c t i o n , and Removal P l a t e l e t s are p e r i p h e r a l blood elements, not c e l l s but fragments or buds, seen by scanning e l e c t r o n microscopy as blebs ( D j a l d e t t i et a l . , 1979) of t i s s u e , which are d e r i v e d by a membrane r e o r g a n i z a t i o n process ( F u s i o n - F i s s i o n ; T a v a s s o l i , 1979) from h i g h l y d i f f e r e n t i a t e d , s p e c i a l i z e d bone marrow precursor c e l l s , megakaryocytes. These p o l y p l o i d c e l l s are formed when the cytoplasm remains undivided during k a r y o k i n e s i s . The process by which the megakaryocyte undergoes nuclear doublings without c e l l d i v i s i o n i s a l s o termed endomitoses. P l a t e l e t s , as anuclear blood elements, should be d i s t i n g u i s h e d from the nucleated "thrombocytes" of lower v e r t e b r a t e s ( T a v a s s o l i , 1980). They are s i m i l a r l y i n v o l v e d i n a v a r i e t y of complex mechanisms associated with hemostasis, p r i m a r i l y t h a t of blood c l o t t i n g and c o a g u l a t i o n ; but t h e i r o r i g i n , manner of production and r e l e a s e from t h e i r stem c e l l s , and l e v e l o f d i f f e r e n t i a t i o n i s d i f f e r e n t and heterogeneous. In order to maintain constant numbers of p l a t e l e t s , t h e i r p r e c u r s o r c e l l s , megakaryocytes, s t i m u l a t e d presumably by some surface modulated humoral feedback mechanism (Levin and E v a t t , 1979; McDonald, 1981; L e v i n , 1983) s p e c i f i c f o r megakaryocytes i n humans (Kawakita et a l . , 1983) produce "megathrombocytes" ( l a r g e aggregates of incompletely d i v i d e d megakaryocyte cytoplasm, " p o l y - p l a t e l e t s " ) which mature to become p l a t e l e t s , r e p l a c i n g those which are being removed by the r e t i c u l o e n d o t h e l i a l system. T h i s removal has been s a i d t o be due to ageing, because p l a t e l e t s l i k e red c e l l s show a defined mean - 2 -c i r c u l a t i o n ( s u r v i v a l ) time a f t e r which they are removed. The study of t h i s removal i n v o l v e s s e v e r a l questions. How are p l a t e l e t s formed, and during maturation and age, how do p l a t e l e t s change: s t r u c t u r a l l y , f u n c t i o n a l l y , e x t e r n a l l y , and i n t e r n a l l y ? S t u d i e s of p l a t e l e t ageing processes p a r a l l e l e d s i m i l a r s t u d i e s o f e r y t h r o c y t e ageing i n th a t d e n s i t y separation was combined with c e l l s u r face charge analyses, i n the form of e l e c t r o p h o r e t i c m o b i l i t y measurements, and biochemical analyses of s i a l i c a c i d and p r o t e i n . Generally i t i s f e l t by many t h a t p h y s i c a l p r o p e r t i e s , s i z e and d e n s i t y of both e r y t h r o c y t e s and p l a t e l e t s change during c i r c u l a t i o n , the e r y t h r o c y t e becoming smaller and more dense, the p l a t e l e t becoming sm a l l e r and l e s s dense. Theories developed i n i t i a l l y with regard to c e l l membrane changes a s s o c i a t e d with ageing and removal of e r y t h r o c y t e s (Danon and Marikowsky, 1961; Y a a r i , 1969; Seaman, 1973; B a l d u i n i et a l . , 1974 and 1977; Luner et a l . , 1977; Seaman et a l . , 1977; Lutz and Fehr, 1979; B o c c i , 1981) but which could apply to p l a t e l e t s , focussed on the a l t e r a t i o n or removal of t e r m i n a l c e l l surface s i a l i c a c i d . This was proposed to decrease c e l l s u r f a c e charge and enable the removal by the spleen of these o l d c e l l s , s i n c e s i a l i c a c i d has been shown to be a major charge bearing component o f both the e r y t h r o c y t e membrane (Cook et a l . , 1961; E y l a r et a l . , 1962) and the p l a t e l e t membrane (Madoff et a l . , 1964). Other t h e o r i e s invoke the exposure of age-related antigens, perhaps i n c l u d i n g galactose r e s i d u e s of i n t e g r a l membrane g l y c o p r o t e i n s (Alderman et a l . , 1981) or the exposure o f other normally c r y p t i c antigens (Kay, 1975 and 1978). In t h i s case the mere decrease i n surface charge i s f e l t s u f f i c i e n t to counter the normal charge - 3 -dependent r e p u l s i o n of c e l l s and endothelium. The exposure of normally hidden antigens could as w e l l f a c i l i t a t e antibody mediated removal by the r e t i c u l o e n d o t h e l i a l system. I t seems reasonable t h a t a l l of these processes might c o n t r i b u t e to the removal of both aged er y t h r o c y t e s and p l a t e l e t s . The t h e o r i e s are comparable but i n disagreement concerning the s i t e and mode of a c t i o n ( i . e . , removal of s i a l i c a c i d s , exposure of a n t i g e n s ) . In one case, i t i s thought t h a t e l i m i n a t i o n of the p h y s i c a l surface membrane charge-associated r e p u l s i o n conferred by t e r m i n a l s i a l i c a c i d i s s u f f i c i e n t t o cause c e l l u l a r removal. In the other cases c e l l u l a r removal i s thought t o be f a c i l i t a t e d through a combination of humoral and c e l l u l a r f a c t o r s . There i s a l s o another concept, s p e c i f i c with regard to the p l a t e l e t , which recognizes t h a t p l a t e l e t c i r c u l a t i o n might be l i m i t e d , but t h a t removal from the c i r c u l a t i o n might not be age r e l a t e d . No age r e l a t e d membrane change need occur i f p l a t e l e t s are removed randomly during c i r c u l a t i o n . In t h i s case, there could be ma t u r a t i o n - r e l a t e d p l a t e l e t surface changes ( l i k e n e d to a r e t i c u l o c y t e becoming a mature e r y t h r o c y t e ) . However there need not be age r e l a t e d membrane surface changes o c c u r r i n g i f , f o r i n s t a n c e , p l a t e l e t d i s s o l u t i o n a f t e r random removal i s proposed. 1.2 E r y t h r o c y t e and P l a t e l e t Surface Charge and P a r t i t i o n Comparing the e l e c t r o p h o r e t i c m o b i l i t y and the charge-associated p a r t i t i o n ( to be discussed) o f er y t h r o c y t e s and p l a t e l e t s from v a r i o u s species i s of i n t e r e s t t o t h i s work. Erythrocytes from the model system employed i n the present study, r a b b i t , e x h i b i t the lowest e l e c t r o p h o r -e t i c m o b i l i t y of a l l mammalian er y t h r o c y t e s t e s t e d whereas the _ 4 -e l e c t r o p h o r e t i c m o b i l i t i e s of rhesus monkey e r y t h r o c y t e s and human e r y t h r o c y t e s are s i m i l a r (Seaman, 1973). A comparison of the e l e c t r o p h o r e t i c m o b i l i t i e s of e r y t h r o c y t e s and p l a t e l e t s from these v a r i o u s mammalian species might provide i n s i g h t . I f the assumption i s made t h a t the e l e c t r o p h o r e t i c m o b i l i t i e s of p l a t e l e t s and e r y t h r o c y t e s from i n d i v i d u a l species p a r a l l e l one another (Seaman, 1967), then so should the charge-associated p a r t i t i o n c o e f f i c i e n t s . Rabbit p l a t e l e t s have a p a r t i t i o n s i m i l a r to t h a t of human p l a t e l e t s , both of which are markedly higher than that of Rhesus monkey p l a t e l e t s , yet r a b b i t e r y t h r o c y t e s p a r t i t i o n much lower than human and monkey (Chapter 3 ) . These are some exceptions t o a c o r r e l a t i o n of charge s e n s i t i v e p a r t i t i o n s of e r y t h r o c y t e s and p l a t e l e t s from given s p e c i e s . One must t h e r e f o r e be c a r e f u l when comparing b i o l o g i c a l phenomena from one c e l l type to another, and from d i f f e r e n t animals. There are not only d i f f e r e n c e s i n s u r v i v a l time but a l s o d i f f e r e n c e s i n the removal of r a b b i t e r y t h r o c y t e s and p l a t e l e t s : 55-90% of e r y t h r o c y t e s i n comparison to 26-28% of p l a t e l e t s are destroyed i n the bone marrow; the l i v e r and spleen sequestering the remainder ( H i l l - Z o b e l et a l . , 1983; and t h e r e i n , Hugh-Jones, 1961; T a v a s s o l i , 1977; T a v a s s o l i , 1974). 1.3 P l a t e l e t S u r v i v a l , Surface Charge, Density and Heterogeneity In 1964, methods f o r studying human 5 ^ C r - l a b e l e d p l a t e l e t s u r v i v a l and s e q u e s t r a t i o n of e f f e t e p l a t e l e t s i n the r e t i c u l o -e n d o t h e l i a l system were o u t l i n e d (Aster and J a n d l , 1964a) and the pathophysiology of p l a t e l e t removal and s e q u e s t r a t i o n induced by exposure of p l a t e l e t s t o i s o a n t i b o d i e s i n v i v o (e.g., I d i o p a t h i c Thrombocytopenic Purpura, ITP) was i n v e s t i g a t e d (Aster and J a n d l , - 5 -1964b). Using e x t e r n a l s c i n t i l l a t i o n scanning of the va r i o u s organs f o r d e p o s i t i o n o f r a d i o a c t i v e p l a t e l e t s , ethylenediamine t e t r a a c e t a t e (EDTA; an anticoag u l a n t ) was found to be i n j u r i o u s to normal human p l a t e l e t s , ("as suggested by ... Zucker and B o r r e l l i , 1954"). EDTA not only caused sphering, but f u r t h e r caused an i n i t i a l r a p i d ( l e s s than one h a l f - h o u r a f t e r i n j e c t i o n ) hepatic s e q u e s t r a t i o n of nearly a l l 51 t r a n s f u s e d C r - l a b e l e d p l a t e l e t s . These were then p a r t i a l l y destroyed i n the spleen ( s t a r t i n g four hours a f t e r i n j e c t i o n ) , l e a v i n g only o n e - t h i r d to c i r c u l a t e normally. The i n i t i a l h e p atic s e q u e s t r a t i o n d i d not occur with " ^ C r - l a b e l e d p l a t e l e t s harvested from blood a n t i c o a g u l a t e d with a c i d - c i t r a t e - d e x t r o s e (ACD). Of these p l a t e l e t s , g r e a t e r than two-thirds s u r v i v e d the spleen to c i r c u l a t e normally with a h a l f l i f e on the order of four days, the p l a t e l e t s being e v e n t u a l l y destroyed i n the l i v e r . Opsonization of p l a t e l e t s w i t h small amounts o f iso a n t i b o d y caused these p l a t e l e t s to be slowly destroyed by the spleen, s i m i l a r to the s i t u a t i o n i n p a t i e n t s with c h r o n i c ITP; whereas l a r g e amounts of isoantibody caused r a p i d d e s t r u c t i o n of p l a t e l e t s i n the l i v e r as i n p a t i e n t s w i t h acute ITP. Aster and J a n d l (1964) concluded t h a t m i l d l y damaged p l a t e l e t s are destroyed i n the spleen, and s e v e r e l y damaged p l a t e l e t s i n the l i v e r . Booyse et a l . (1968) separated human p l a t e l e t s by four step sucrose d e n s i t y gradient c e n t r i f u g a t i o n , comparing normal human p l a t e l e t s and p l a t e l e t s from diseased s t a t e s . Immunosuppression with a z a t h i o p r i n e (Immuram) therapy f o l l o w i n g r e n a l t r a n s p l a n t causes a depression of p l a t e l e t production. In p a t i e n t s with chronic thrombocytopenic purpura, p l a t e l e t production was induced with normal plasma; t h i s increased the numbers of heavy dense p l a t e l e t s . These heavy p l a t e l e t s were then - 6 -" t e n t a t i v e l y c h a r a c t e r i z i e d as newly formed young p l a t e l e t s " . Only a sm a l l percentage of these p l a t e l e t s were considered to be "young" and r i c h i n ribosome content. They were found to decrease i n de n s i t y when incubated i n s y n t h e t i c mixtures t o produce "a more mature p l a t e l e t " , with a corresponding decrease i n p r o t e i n s y n t h e s i z i n g a b i l i t y . A r d l i e et a l . (1970) demonstrated that p l a t e l e t s taken i n a c i d c i t r a t e dextrose and washed according to the method of Aster and J a n d l , could undergo adenosine diphosphate (ADP) induced aggregation and s u r v i v e normally i n the r a b b i t i n v i v o (Reimers et a l . , 1973a). Repeated thrombin treatment of r a b b i t p l a t e l e t s caused extensive p l a t e l e t granule r e l e a s e , evidenced by l o s s of s e r o t o n i n and adenine n u c l e o t i d e s . These p l a t e l e t s could s t i l l be aggregated under c e r t a i n c o n d i t i o n s , however, and s u r v i v e d normally a f t e r l a b e l i n g with ^ H - d i i s o p r o p y l p h o s p h o f l u o r i d e (DFP) i n v i t r o (Reimers et a l . , 1973b), 51 or a f t e r l a b e l i n g w i t h Cr-chromate (Reimers et a l . , 1976). Carty et a l . (1975) separated human, r a t and splenectomized r a t p l a t e l e t subpopulations by p r e p a r a t i v e sucrose d e n s i t y gradient e l e c t r o p h o r e s i s and demonstrated that the " f a s t e r " p l a t e l e t s , with the higher e l e c t r o p h o r e t i c m o b i l i t i e s , were l a r g e r , i n c o r p o r a t e d more ^Cr-chromate ( c o n s i s t e n t w i t h a subpopulation of p l a t e l e t s which aggregates more r e a d i l y i n the presence of ADP, as l a t e r reported by Gear, 1981; Haver and Gear, 1981; Haver and Gear, 1982), and contained more t o t a l s i a l i c a c i d , DNA, and RNA, than the "slower" p l a t e l e t s with lower e l e c t r o p h o r e t i c m o b i l i t i e s . However, the DNA found i n these p l a t e l e t preparations might be due to contaminating leukocytes because p l a t e l e t s have only t r a c e amounts of DNA i n t h e i r s m all and few mitochondria. - 7 -Isobe and Yamanaka (1976) a l s o s t u d i e d the c o r r e l a t i o n s of p l a t e l e t ageing, membrane surface charge, and d e n s i t y . The e l e c t r o p h o r e t i c m o b i l i t i e s of human p l a t e l e t s c o r r e l a t e d d i r e c t l y with p l a t e l e t d e n s i t i e s . The p l a t e l e t f r a c t i o n of highest d e n s i t y , c o n t a i n i n g young p l a t e l e t s , had the highest negative e l e c t r o p h o r e t i c m o b i l i t i e s . This decreased i n the lowest d e n s i t y f r a c t i o n to a value which was s i g n i f i c a n t l y l e s s negative. S i a l i c a c i d analyses of d e n s i t y separated r a b b i t p l a t e l e t s confirmed these f i n d i n g s , i n that the highest d e n s i t y p l a t e l e t s contained greater amounts of s i a l i c a c i d than d i d p l a t e l e t s of lower d e n s i t i e s . No d i s t i n c t i o n was made between t o t a l and c e l l s urface (neuraminidase-susceptible) s i a l i c a c i d , however. Isobe (1976) using p l a t e l e t s from normal s u b j e c t s , p a t i e n t s with acute and chronic ITP and p a t i e n t s a f t e r nephrectomy f o r Ba n t i ' s disease (a disease a s s o c i a t e d with pancytopenia and splenomegaly considered secondary to p o r t a l hypertension), f u r t h e r found that i n these p a t i e n t s , the p l a t e l e t e l e c t r o p h o r e t i c m o b i l i t i e s and s i a l i c a c i d l e v e l s were elevat e d r e l a t i v e to those of normals. At the molecular l e v e l , p l a t e l e t membrane s i a l i c a c i d and p r o t e i n analyses by polyacrylamide g e l e l e c t r o p h o r e s i s (SDS PAGE) a l s o revealed some i n t e r e s t i n g r e s u l t s , i n a d d i t i o n to those p r e v i o u s l y demonstrated by Booyse et a l . (1968). George et a l . (1976a and 1976b) used 125 125 d i a z o t i z e d I - d i i o d o s u l f a n i l i c a c i d (DD ISA; nonpenetrating and c o v a l e n t l y binding) to l a b e l exposed p r o t e i n on i n t a c t p l a t e l e t s . In the f i r s t paper they s t u d i e d t h i s l a b e l i n comparison t o l a c t o p e r oxidase-"''"5''"I l a b e l i n g of human p l a t e l e t s and found that DD^-ISA l a b e l e d p r i m a r i l y three polypeptides of 180,000, 125,000, and 9.2,000 da l t o n s , the g r e a t e s t q u a n t i t y of l a b e l being found i n the - 8 -180,000 d a l t o n and the l e a s t i n the 92,000 d a l t o n p o l y p e p t i d e s . These r e s u l t s are not u n l i k e those found by Bunting et a l . (1978). In the second paper r a b b i t p l a t e l e t s , double l a b e l e d w i t h i n t e r n a l i z e d 5"*"Cr-chromate and with D D ^ I S A attached to surface p r o t e i n s , were sub j e c t e d to s u r v i v a l analyses. The s u r v i v a l of 5 ^ C r ( T ^ 2 = 30 1015 w o hours) was nearly twice t h a t of DD ISA (T = 17 hours), but t h i s was not the case when s i m i l a r i l y l a b e l e d p l a t e l e t s were stored i n plasma f o r the same pe r i o d o f time. P l a t e l e t s harvested a f t e r 14-40 hours of c i r c u l a t i o n , and subjected to membrane p r o t e i n analyses by SDS 125 PAGE, a l l showed i d e n t i c a l DD ISA l a b e l i n g p a t t e r n s i n d i c a t i n g a symmetrical l o s s of p r o t e i n s . This supports the hypothesis that pieces of i n t a c t whole membranes or fragments are l o s t during the ageing process while the m a j o r i t y of cytoplasm remains w i t h i n the p l a t e l e t . Penington et a l . (1976a) separated p l a t e l e t s on p o l y v i n y l -p y r r o l i d o n e (PVP) coated s i l i c a g e l ( P e r c o l l ) d e n s i t y g r a d i e n t , and analyzed p l a t e l e t s i z e . A l l p l a t e l e t d e n s i t y subpopulations were heterogeneous i n s i z e , but the l i g h t p l a t e l e t s were on the average s m a l l e r . The most dense p l a t e l e t s ranged i n apparent s i z e from 3 f l , t o grea t e r than 21 f l i n man and r a t , while the l i g h t p l a t e l e t s contained p l a t e l e t s no gre a t e r than 13 f l i n both s p e c i e s . P l a t e l e t cohort l a b e l i n g with ^ S e (selenomethionine), using methods p r e v i o u s l y d e s c r i b e d by Charmatz and K a r p a t k i n (1974) who used 7 5Se-methionine i n r a b b i t s , showed no change i n buoyant density w i t h ageing. Their i n t e r p r e t a t i o n was th a t the g r e a t e r s p e c i f i c a c t i v i t y found i n the young p l a t e l e t s i n the most dense f r a c t i o n was r e l a t e d to the in c r e a s e i n p r o t e i n s y n t h e t i c a c t i v i t y shown i n v i t r o and that dense, in t e r m e d i a t e , and l i g h t p l a t e l e t s were released synchronously from - 9 -three d i f f e r e n t p l o i d y c l a s s e s of megakaryocyte p r e c u r s o r s . Boneu et a l . (1977) s t u d i e d the volume and d e n s i t y d i s t r i b u t i o n of human p l a t e l e t s i n d i f f e r e n t disease s t a t e s c h a r a c t e r i z e d by abnormal thrombopoietic s t a t e s . They found that i n thrombocythemia f o l l o w i n g splenectomy, i n s p i t e of a sudden p l a t e l e t i n c r e a s e i n the number of presumably "young" p l a t e l e t s , the p l a t e l e t volumes and d e n s i t i e s were w i t h i n the normal range. In ITP with macrothrombocytosis ( i . e . , i n c r e a s e i n p l a t e l e t volume) a f t e r splenectomy, the "young" p l a t e l e t s again demonstrated a normal volume and d e n s i t y . In consumption coagulopathy with evidence of macrothrombocytosis, there was a l s o an i n c r e a s e i n the l i g h t p l a t e l e t s . Therefore there are s i t u a t i o n s i n which " l a r g e , l i g h t p l a t e l e t s " occur. A d e n s i t y gradient of m a t e r i a l s i m i l a r to that used f o r e r y t h r o c y t e s e p a r a t i o n (Corash et a l . , 1974) was used to i n v e s t i g a t e the heterogeneity of human whole blood p l a t e l e t sub-populations (Corash et a l . , 1977). In the f i r s t d e s c r i p t i o n of a new supporting medium advantageous f o r d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , Corash et a l . (1974) separated human ery t h r o c y t e s according t o age on a s i m p l i f i e d d e n s i t y g r a d i e n t composed of v a r y i n g concentrations of S t r a c t a n I I , an arabinogalactan polymer, produced from Western Larch t r e e s ( L a r i x o c c i d e n t a l i s ) by a countercurrent e x t r a c t i o n process. E r y t h r o c y t e s were la y e r e d on a three step g r a d i e n t composed of d e n s i t i e s 1.087, 1.091, and 1.095 g/ml, at 20°C, and c e n t r i f u g e d at 20,000 rpm f o r 45 minutes (approximately 2.34 x 1 0 6 g x min, mean). Because p l a t e l e t s are l e s s dense than e r y t h r o c y t e s the gradient step d e n s i t i e s were decreased (1.062, 1.066, 1.071, 1.084, g/ml; 15%, 16%, 17% and 20%, S t r a c t a n , r e s p e c t i v e l y ) and again c e n t r i f u g a t i o n was f o r 50,000 g, but f o r only - 10 -30 minutes at 20°C (1.5 x 1 0 6 g x min, mean). The r e l a t i o n of p l a t e l e t buoyant d e n s i t y , c e l l volume and u l t r a s t r u c t u r e suggested t h a t d i f f e r e n c e s i n p l a t e l e t volume and granule content and d i f f e r e n c e s between l i g h t and heavy p l a t e l e t s might be due t o ageing. I t was found that p l a t e l e t s of low den s i t y are sm a l l e r on average and co n t a i n fewer granules than those of higher d e n s i t y but both low and high d e n s i t y p l a t e l e t s c o n t a i n comparable q u a n t i t i e s of mitochondria. Packham and Mustard (1977) emphasized the need f o r drugs which a f f e c t the i n t e r a c t i o n s of p l a t e l e t s and e n d o t h e l i a , t o prevent the development o f a t h e r o s c l e r o s i s and thromboembolism without i n t e r f e r i n g the hemostatic f u n c t i o n of p l a t e l e t s . This group a l s o stated that p l a t e l e t s u r v i v a l analyses might be u s e f u l i n c l i n i c a l s i t u a t i o n s i n which thrombosis occurs (Harker and Finch, 1969; Harker and S l i c h t e r , 1972; Harker and S l i c h t e r , 1974). Treatment of r a b b i t p l a t e l e t s with neuraminidase decreases the 51 membrane s i a l i c a c i d and the s u r v i v a l o f C r - l a b e l e d r a b b i t p l a t e l e t s , but has only a s l i g h t l y enhancing e f f e c t on p l a t e l e t aggregation (Greenberg et a l . , 1975). Greenberg et a l . (1977), using a three step S t r a c t a n g r a d i e n t f o r density s e p a r a t i o n of r a b b i t p l a t e l e t s , as p r e v i o u s l y d e s c r i b e d f o r the i n v e s t i g a t i o n of the ageing p r o p e r t i e s o f human e r y t h r o c y t e s and p l a t e l e t s , (Corash et a l . , 1974; Corash et a l . , 1977), r e p o r t e d that the most dense f r a c t i o n s were enriched i n the youngest p l a t e l e t s , as determined by l a b e l i n g with 51 35 Cr-chromate i n v i t r o and S-sulphate i n v i v o , while the l e a s t dense f r a c t i o n s were enriched i n o l d p l a t e l e t s . I t was concluded that average p l a t e l e t d e n s i t y decreases with i n c r e a s i n g age. T o t a l s i a l i c a c i d / p l a t e l e t and p r o t e i n / p l a t e l e t a l s o decrease such t h a t the s i a l i c - 11 -a c i d and p r o t e i n decrease i n c o n c e r t , i n d i c a t i n g a l o s s by p l a t e l e t s i n the form of s i a l o g l y c o p e p t i d e s , perhaps r e s u l t i n g i n t h e i r r e c o g n i t i o n as o l d and removal from the c i r c u l a t i o n . Tan et a l . (1978) showed th a t the u l t r a s t r u c t u r e o f the surface of normal human p l a t e l e t membranes does not change as a f u n c t i o n of d e n s i t y , and hence i n v i v o ageing. Corash et a l . (1978) used a sub-human primate model, Macaca mulata, to analyze the r e l a t i o n s h i p between p l a t e l e t d e n s i t y and p l a t e l e t age. A gradient of d e n s i t y steps s i m i l a r to that used i n the human study, was used and i t was shown th a t l i g h t p l a t e l e t s had a mean s u r v i v a l time of 74.6 hours compared to 51 313.6 hours f o r heavy p l a t e l e t s . They a l s o showed that C r - l a b e l e d l i g h t p l a t e l e t s were removed by the c i r c u l a t i o n and t h a t l a b e l e d dense p l a t e l e t s became l i g h t d u r i ng c i r c u l a t i o n . This confirmed the hypothesis t h a t , i n t h i s s p e c i e s , p l a t e l e t age c o r r e l a t e s i n v e r s e l y with d e n s i t y , and provided strong evidence supporting the use of "age-dependent" markers, s p e c i f i c a l l y d e n s i t y , to estimate p l a t e l e t turnover r a t e s . K a r p a t k i n c o r r e l a t e d human p l a t e l e t f u n c t i o n with volume. He compared the aggregation v e l o c i t y of p l a t e l e t s of d i f f e r e n t volumes, and proposed the term "megathrombocyte index," i . e . , l a r g e p l a t e l e t s as an index of p l a t e l e t production ( K a r p a t k i n , 1978). He a l s o s t a t e d that heavy p l a t e l e t s demonstrate 15 times greater monoamine oxidase a c t i v i t y (MOA), and 18 times more p r o t e i n ( F r i e d h o f f et a l . , 1978). Peerschke and Zucker (1978) found that ADP-induced human p l a t e l e t shape change d i d not expose a d d i t i o n a l s i a l i c a c i d , but that a c t u a l l y a decrease i n membrane s i a l i c a c i d was demonstrable, (Bunting et a l . , 1978). These r e p o r t s concur w i t h previous f i n d i n g s (Seaman and Vassar, - 12 -1966; Hampton and M i t c h e l l , 1966a and 1966b; Grottum, 1968; Seaman, 1976) of a decrease i n e l e c t r o p h o r e t i c m o b i l i t y during p l a t e l e t aggregation. However, Motamed et a l . , (1976) found an i n c r e a s e i n exposed p l a t e l e t membrane (neuraminidase-susceptible) s i a l i c a c i d during p l a t e l e t shape change, confirmed by recent f i n d i n g s of a member of our l a b o r a t o r y (B. Chiu, Ph.D. Thesis 1983) who, i n a d d i t i o n demonstrated a decrease i n e l e c t r o p h o r e t i c m o b i l i t y . These r e p o r t s focus on the thought t h a t p l a t e l e t s during c i r c u l a t i o n undergo encounters with endothelium, r e v e r s i b l e a c t i v a t i o n s and d e - a c t i v a t i o n s , aggregations and de-aggreagations. George et a l . (1978a) i n the t h i r d paper of a s e r i e s , showed th a t 125 the reported l o s s during c i r c u l a t i o n , of DD ISA-labeled membrane surf a c e p r o t e i n r e l a t i v e t o ^ C r - l a b e l e d cytoplasmic p r o t e i n i n r a b b i t p l a t e l e t s (George et a l . , 1976a), i s prevented by a s p i r i n -dipyridamole and a c c e l e r a t e d by thrombin ( i . e . , prevented by agents which i n h i b i t p l a t e l e t f u n c t i o n and a c c e l e r a t e d by agents which cause thrombosis). Later work of Hanson and Harker (1981) d i d not confirm these f i n d i n g s with dipyridamole and a s p i r i n . In a f o u r t h paper, George 125 et a l . (1978b) s t r e s s e d the q u a n t i t a t i v e nature of t h i s DD ISA l a b e l i n g procedure f o r analyses of human p l a t e l e t membrane g l y c o p r o t e i n s . A r e p o r t contrary t o the arguments f o r an i n v e r s e c o r r e l a t i o n of p l a t e l e t d e n s i t y and ageing was published by Leone et a l . (1979) who s t u d i e d p l a t e l e t s from i n d i v i d u a l s t r e a t e d with a s p i r i n . Separating these p l a t e l e t s i n t o dense and l i g h t subpopulations by repeated c e n t r i f u g a t i o n , a c o r r e l a t i o n was shown between d e n s i t y and s i z e . Even though the b a s a l a c t i v i t y of the enzyme cyclooxygenase was higher i n - 13 -the dense l a r g e p l a t e l e t s , t h i s a c t i v i t y a f t e r a s p i r i n i n g e s t i o n reappeared i n p a r a l l e l i n both l i g h t and heavy subpopulations of p l a t e l e t s , suggesting that although s i z e and de n s i t y c o r r e l a t e , these p r o p e r t i e s were not age r e l a t e d . I t was found i n the r a b b i t that when p l a t e l e t s were exposed to damaged endothelium (abdominal a o r t a , or c a r o t i d a r t e r y ) or f o r e i g n s u r f a c e s , p l a t e l e t turnover i n c r e a s e d . P l a t e l e t s i n t e r a c t e d with the surface and then c i r c u l a t e d i n a l e s s a c t i v e form (Buchanan et a l . , 1979). P l a t e l e t i n t e r a c t i o n s with b a l l o o n c a t h e t e r damaged r a b b i t aortae were t h e r e f o r e f u r t h e r i n v e s t i g a t e d (Groves et a l . , 1979). In these s i t u a t i o n s , although i n i t i a l l y a f t e r i n j u r y to the subendothelium 5"*"Cr-labeled r a b b i t p l a t e l e t s d i d adhere ( a l s o demonstrated by scanning e l e c t r o n microscopy), the p l a t e l e t s u r v i v a l time was not s i g n i f i c a n t l y reduced and thus was not considered t o be a r e l i a b l e estimate of e n d o t h e l i a l i n j u r y i n a l l circumstances. As the mechanisms r e s p o n s i b l e f o r shortened p l a t e l e t s u r v i v a l i n p a t i e n t s with thrombotic c o n d i t i o n s were s t i l l u n c l e a r , Packham et a l . (1979) i n v e s t i g a t e d the i n v i v o f a c t o r s that might i n f l u e n c e p l a t e l e t s u r v i v a l . Combining p l a t e l e t s u r v i v a l time and p l a t e l e t d e n s i t y analyses with s i t u a t i o n s i n which both e n d o t h e l i a l damage and thrombi had been induced they showed t h a t under these c o n d i t i o n s both p l a t e l e t s u r v i v a l and p l a t e l e t d e n s i t y decreased. Rabbit p l a t e l e t s t r e a t e d with ADP or thrombin i n v i t r o , e x h i b i t e d no change i n p l a t e l e t s u r v i v a l time but a f t e r plasmin treatment the s u r v i v a l time decreased. A f t e r a l l these treatments, the p l a t e l e t s i n the l i g h t e s t d e n s i t y f r a c t i o n were observed to increase p r o p o r t i o n a t e l y . They concluded that a change i n p l a t e l e t d e n s i t y i s not n e c e s s a r i l y a s s o c i a t e d with an a l t e r a t i o n i n - 14 -p l a t e l e t s u r v i v a l time and t h a t the decreased s u r v i v a l of plasmin t r e a t e d p l a t e l e t s might be due to a l t e r a t i o n s i n g l y c o p r o t e i n s I and I I i n r a b b i t p l a t e l e t s . Graf et a l . (1979) showed that blood p l a t e l e t s , i s o l a t e d with g r a d i e n t s of p o l y s a c c h a r i d e s , such as dextran T10 and S t r a c t a n suspended i n p r o t e i n f r e e b u f f e r s f u n c t i o n normally and might be used t o study s e r o t o n i n (5HT) uptake and receptor i n t e r a c t i o n i n a v a r i e t y of s p e c i e s (guinea p i g , r a b b i t and human). C i e s l a r et a l . (1979) using a density gradient of 13.5%, 15% and 18% S t r a c t a n , showed th a t normal r a b b i t p l a t e l e t s of higher density contained greater q u a n t i t i e s of s e r o t o n i n , adenine n u c l e o t i d e s , and h e p a r i n - n e u t r a l i z i n g a c t i v i t y than those of l i g h t e r d e n s i t i e s . They a l s o showed that p l a t e l e t s decreased t h e i r d e n s i t y upon treatment with thrombin and r e l e a s e of these c o n s t i t u e n t s . However, because p l a t e l e t c o n s t i t u e n t s d i f f e r e d only s l i g h t l y f o r p l a t e l e t s of d i f f e r e n t d e n s i t i e s , the d e n s i t y d i f f e r e n c e s were considered not t o be a t t r i b u t a b l e to l o s s of granule contents. These f i n d i n g s suggest that a decrease i n p l a t e l e t d e n s i t y might not be due to ageing alone but a l s o to r e v e r s i b l e encounters i n the c i r c u l a t i o n r e q u i r i n g f u n c t i o n a l a c t i v i t i e s , i . e . , p l a t e l e t r e l e a s e and shape change. Therefore, thrombin treatment was shown to decrease p l a t e l e t d ensity without a f f e c t i n g p l a t e l e t s u r v i v a l . They proposed that i t might be p o s s i b l e to use d e n s i t y gradient analyses to detect p l a t e l e t s t h a t have been exposed t o thrombin i n v i v o during thrombus formation, and which have subsequently returned t o the c i r c u l a t i o n when thrombi breakup. Analyses of p l a t e l e t s u r v i v a l a f t e r treatment with other surface modifying enzymes were pub l i s h e d by Greenberg et a l . (1979), f o l l o w i n g - 15 -Greenberg et a l . (1975 and 1977) and Packham et a l . (1979). Plasmin, chymotrypsin, t r y p s i n and neuraminidase a l l cause a l t e r a t i o n s i n p l a t e l e t membrane g l y c o p r o t e i n s ( p r i m a r i l y i n g l y c o p r o t e i n I , and some i n I I and I I I ) and p l a t e l e t s u r v i v a l ; only plasmin and t r y p s i n cause the p l a t e l e t r e l e a s e r e a c t i o n . The enzyme t r e a t e d p l a t e l e t s were sequestered at one hour, some p l a t e l e t s being removed ( e s p e c i a l l y i n the case of t r y p s i n and neuraminidase) by the l i v e r and not the spleen, and some p l a t e l e t s r e t u r n i n g to the c i r c u l a t i o n by four hours. Rand et a l . (1980) s t a t e d t h a t low concentrations of ADP (10 uM), i n s u f f i c i e n t to cause granule r e l e a s e , induced temporary p l a t e l e t a l t e r a t i o n s i n c l u d i n g aggregation and shape change ( r e v e r s i b l e i n f i v e minutes), a temporary i n c r e a s e i n p l a t e l e t s i z e ( r e t u r n i n g t o the o r i g i n a l s i z e by one hour), and caused a decrease i n p l a t e l e t d e n s i t y which p e r s i s t e d f o r at l e a s t f o u r hours. Thus i t was concluded t h a t , i n a d d i t i o n to the p l a t e l e t a l t e r a t i o n s induced by thrombin ( C i e s l a r et a l . , 1979) a decrease i n p l a t e l e t d ensity a s s o c i a t e d with thrombo-embolism might not n e c e s s a r i l y be ass o c i a t e d with the p l a t e l e t r e l e a s e r e a c t i o n , nor an in c r e a s e i n p l a t e l e t s i z e (e.g., with ADP). Packham et a l . (1980) showed that these low concentrations of ADP do not cause r e l e a s e of granule s i a l o g l y c o p r o t e i n s from r a b b i t p l a t e l e t s nor from human p l a t e l e t s (not even under c o n d i t i o n s wherein the p l a t e l e t r e l e a s e r e a c t i o n occurs: l e s s than 4% s i a l i c a c i d was rel e a s e d from human p l a t e l e t s ) . Nor does ADP a l t e r the i n v i v o s u r v i v a l time of r a b b i t p l a t e l e t s . In c o n t r a s t , thrombin r e l e a s e s (granule s i a l o g l y c o p r o t e i n s ) 20% of the s i a l i c a c i d of human p l a t e l e t s , but s t i l l does not a l t e r the i n v i v o s u r v i v a l time of r a b b i t p l a t e l e t s (Reimers et a l . , 1973a, 1973b and 1979). Neuraminidase, on the other hand, cleaves 40% of the t o t a l - 16 -p l a t e l e t s i a l i c a c i d from human p l a t e l e t membranes, and causes r a p i d c l e a r a n c e of r a b b i t p l a t e l e t s from the c i r c u l a t i o n (Greenberg et a l . , 1977 and 1979). Release of s i a l i c a c i d from i n t r a c e l l u l a r granules i n the form of s i a l o g l y c o p e p t i d e s , and cleavage of c e l l surface s i a l i c a c i d by neuraminidase, produce very d i f f e r e n t r e s u l t s with respect to p l a t e l e t s u r v i v a l . Continuing s t u d i e s of membrane p r o t e i n changes a s s o c i a t e d with human p l a t e l e t a c t i v a t i o n , George et a l . (1980) demonstrated t h a t a c t i n becomes exposed during t h i s p l a t e l e t shape change event. Their p l a t e l e t washing procedure f o r a n a l y s i s of membrane p r o t e i n was then analyzed w i t h respect to p o s s i b l e l o s s of s p e c i f i c p l a t e l e t membrane p r o t e i n s due to p l a t e l e t c e n t r i f u g a t i o n and/or p l a t e l e t damage (George et a l . , 1981). The p r e v i o u s l y used procedure (George et a l . , 1978b) f o r human p l a t e l e t p r e p a r a t i o n was found to provide a recovery o f only o n e - t h i r d of the p l a t e l e t s , w i t h a l o s s of o n e - t h i r d of t h e i r membrane g l y c o -p r o t e i n , mostly due t o a l o s s of GPI and the major granule g l y c o -p r o t e i n , GPG. "Comparison of the most dense and l e a s t dense p l a t e l e t subpopulations demonstrated no d i f f e r e n c e i n the con c e n t r a t i o n of membrane g l y c o p r o t e i n s per p l a t e l e t . " However, the densitometer scans of membrane p r o t e i n s from p r o p e r l y washed, S t r a c t a n d e n s i t y separated p l a t e l e t s c l e a r l y i n d i c a t e a d i f f e r e n c e (e.g., an increase i n GPG) i n membrane p r o t e i n s from p l a t e l e t s of d i f f e r e n t d e n s i t i e s , i n comparison to t h a t of the t o t a l p l a t e l e t p o p u l a t i o n (George et a l . , 1981). Blajchman et a l . (1981) using r a b b i t s , compared the p l a t e l e t membrane g l y c o p r o t e i n s from l a r g e young p l a t e l e t s obtained during recovery from a n t i - p l a t e l e t antibody induced thrombocytopenia, with these from small o l d p l a t e l e t s obtained during i r r a d i a t i o n induced - 17 -thrombocytopenia. The c h a r a c t e r of the s u r v i v a l time curves f o r these young ( l i n e a r ) and o l d (exponential) p l a t e l e t s were d i f f e r e n t . They found that there were no q u a l i t a t i v e d i f f e r e n c e s i n membrane g l y c o p r o t e i n s . However the co n c e n t r a t i o n s o f a l l s i x major membrane g l y c o p r o t e i n s were g r e a t e r , and the s u r v i v a l times longer, i n the young, l a r g e p l a t e l e t s , compared to those i n the unseparated, or those i n the o l d , small p l a t e l e t s . This r e s u l t i s compatible with the membrane fragmentation hypothesis of ageing and removal, and contrary to the d e - s i a l a t i o n theory. Hanson and Harker (1981) performed double-labeled p l a t e l e t s u r v i v a l s t u d i e s i n baboons, s i m i l a r t o those performed with r a b b i t s , by George (1976a and 1976b). P l a t e l e t s s i m i l a r l y l a b e l e d , a f t e r s u r v i v a l i n v i v o , again showed the decrease i n D D ^ I S A compared to "^Cr during ageing but i n c o n t r a s t to George's r e s u l t s t h i s could not be c o r r e c t e d with a s p i r i n nor dipyridamole. Rand et a l . (1981b) reported t h a t i n r a b b i t s , p l a t e l e t s i z e c o r r e l a t e s with p l a t e l e t d e n s i t y and th a t p l a t e l e t d e n s i t y c o r r e l a t e s with p l a t e l e t age as r e f l e c t e d by the i n c o r p o r a t i o n _in v i v o of the 35 51 cohort l a b e l S-sulphate. In_ v i t r o C r - l a b e l e d p l a t e l e t s , a f t e r ageing, revealed an in c r e a s e i n s p e c i f i c a c t i v i t y of the l e a s t dense p l a t e l e t f r a c t i o n and a decrease i n s p e c i f i c a c t i v i t y o f the most dense f r a c t i o n . These r e s u l t s were s t i l l not c o n c l u s i v e w i t h respect to the p l a t e l e t heterogeneity due to ageing because the s p e c i f i c removal of dense p l a t e l e t s could not be r u l e d out (Greenberg et a l . , 1977; Rand et a l . , 1981b). Rand et a l . (1981a) published f u r t h e r evidence i n the r a b b i t model system, analagous to that demonstrated by Corash et^ a l . (1978), except that i n these r a b b i t s t u d i e s an i n v i v o ^ 5 S - s u l p h a t e - 18 -35 cohort l a b e l was used. The S cohort l a b e l i s taken up by the megakaryocytes, l a b e l s newly synt h e s i z e d proteoglycan and i s used as a marker f o r young, newly produced p l a t e l e t s . They used S t r a c t a n density g r a d i e n t steps of 15%, 17%, and 19% S t r a c t a n , and 15.5%, 17.5%, and 19% S t r a c t a n ( i . e . , 1.058, 1.067, and 1.976 g/ml; and 1.060, 1.069 and 1.076 g/ml, r e s p e c t i v e l y ) . This enabled the i s o l a t i o n of p l a t e l e t subpopulations from the extremes of the den s i t y d i s t r i b u t i o n , enhancing d i f f e r e n c e s i n p l a t e l e t d e n s i t y . Using these p l a t e l e t subpopulations i s o l a t e d from the t a i l s of the p l a t e l e t d e n s i t y d i s t r i b u t i o n i t was shown th a t the most dense p l a t e l e t subpopulation contained s i g n i f i c a n t l y l a r g e r amounts o f p r o t e i n and t o t a l (not surface) s i a l i c 35 a c i d per p l a t e l e t as w e l l as more S cohort l a b e l per p l a t e l e t . The s p e c i f i c a c t i v i t y peaked i n the most dense p l a t e l e t s and then s h i f t e d p r o g r e s s i v e l y to the l e a s t dense p l a t e l e t s . The most dense subpopulation reached i t s maximum s p e c i f i c a c t i v i t y between 48 and 72 hours and then decreased g r a d u a l l y , whereas the l e a s t dense subpopulation reached i t s maximum a f t e r 96 hours. This was evidence that young p l a t e l e t s were enriched i n the most dense f r a c t i o n and decreased i n average d e n s i t y a f t e r ageing i n the c i r c u l a t i o n ; and that the o l d p l a t e l e t s were enriched i n the l e a s t dense f r a c t i o n . Mezzano et a l . (1981), a f t e r Mezzano and Aster (1979), published r e s u l t s of human p l a t e l e t d e n s i t y a n a l y s i s experiments with Stractan d e n s i t y gradient c e n t r i f u g a t i o n methods, and i n a d d i t i o n reported s u r v i v a l analyses of p l a t e l e t s separated according to s i z e . They drew d i f f e r e n t , new i n t e r p r e t a t i o n s from t h e i r r e s u l t s , i . e . , that the 51 reason f o r the more r a p i d d e c l i n e i n s p e c i f i c a c t i v i t y o f Cr la b e l e d l i g h t p l a t e l e t s compared to t h a t of the heavy p l a t e l e t s i s not - 19 -t h a t l i g h t p l a t e l e t s are o l d e r and are removed from the c i r c u l a t i o n more r a p i d l y than the heavy young p l a t e l e t s , but that a f t e r c i r c u l a t i o n the l i g h t p l a t e l e t s become heav i e r with age or m a t u r i t y , perhaps by a c q u i r i n g some dense m a t e r i a l , and are then replaced by newly formed l i g h t p l a t e l e t s . By studying 5"*"Cr-labeled p l a t e l e t s u r v i v a l i n humans, i n both heavy and l i g h t p l a t e l e t f r a c t i o n s obtained by d i f f e r e n t i a l c e n t r i f u g a t i o n , the s p e c i f i c a c t i v i t y of the heavy p l a t e l e t s was found to remain constant or i n c r e a s e s l i g h t l y at a time when the l i g h t p l a t e l e t s l o s e t h e i r l a b e l (decreasing i n s p e c i f i c a c t i v i t y ) , suggesting a s h i f t i n d ensity from l i g h t to heavy p l a t e l e t s . Gradient m a t e r i a l s other than S t r a c t a n do provide some advantages. Shaw et a l . (1981) again used P e r c o l l (as i n Penington et a l . , 1976a) to i s o l a t e p l a t e l e t s by v e l o c i t y sedimentation, and a l i n e a r gradient to d i s t r i b u t e p l a t e l e t s according to d e n s i t y . The p l a t e l e t d i s t r i b u t i o n was found to be Gaussian with a mode about 1.0645 g/ml. The d i s t r i b u t i o n and r e l e a s e or leakage (gradient induced r e l e a s e ) of the i n t r a g r a n u l e molecules beta-thromboglobulin (BTG) and s e r o t o n i n (5HT), and the cytoplasmic c o n s t i t u e n t l a c t i c dehydrogenase (LDH) were s t u d i e d , r e v e a l i n g that p l a t e l e t s from the denser f r a c t i o n s c o n t a i n g r e a t e r q u a n t i t i e s of BTG, w h i l e the other markers c l o s e l y p a r a l l e l the p l a t e l e t count. Haver and Gear (1981) developed a method, f u n c t i o n a l f r a c t i o n a t i o n , whereby p l a t e l e t s are exposed t o low c oncentrations (0.1-0.7 uM ADP) of aggregating agent a f t e r which they were c e n t r i f u g e d , p e l l e t i n g the ( l a r g e ) r e a c t i v e p l a t e l e t s . These more r e a c t i v e p l a t e l e t s were l a r g e r and contained increased amounts of glycogen, ATP, ADP, 1.9 times more t o t a l s i a l i c a c i d , e x h i b i t e d a c t i v e chromate uptake, and were l a b e l e d - 20 - ' 75 as young p l a t e l e t s ( i n r a t s ) by Se-methionine i n j e c t i o n . Using 5 ^ C r - and " ^ I n - l a b e l e d p l a t e l e t s , the d e n s i t y changes of p l a t e l e t s t h a t occur as a f u n c t i o n of p l a t e l e t ageing were followed (Rand et a l . , 1981b). The s u r v i v a l o f p l a t e l e t s with e i t h e r l a b e l was s a i d t o be i d e n t i c a l , except that the one hour recovery of "'""'"'''In-labeled p l a t e l e t s was s l i g h t l y l e s s than that f o r " ^ C r - l a b e l e d p l a t e l e t s . The most dense p l a t e l e t s s u r v i v e d f o r longer than the l e a s t dense p l a t e l e t s . Data discussed i n a l a t e r paper (Rand 51 et a l . , 1983), showed th a t the C r - l a b e l e d most dense p l a t e l e t s appeared to s u r v i v e longer than "'""'""''In-labeled most dense p l a t e l e t s . In " t o t a l agreement" with the work of Mezzano and Aster (1979) and Mezzano et a l . (1981) but c o n t r a r y t o the i n t e r p r e t a t i o n of Corash, i s 51 the r e p o r t by Boneu et a l . (1982), whose c a r e f u l experiments, on Cr l a b e l e d p l a t e l e t s u r v i v a l s t u d i e s ( i n p a t i e n t s with s t a b l e stage I I c h r o n i c a r t e r i t i s of the lower limbs, and normal subjects) i n combination with p l a t e l e t d e n s i t y analyses using various . gradient m a t e r i a l s i n c l u d i n g S t r a c t a n , revealed c e r t a i n novel f i n d i n g s . On day one a f t e r i n j e c t i o n of ^ C r - 1 a b e l e d p l a t e l e t s there was an obvious enrichment i n s p e c i f i c a c t i v i t y of dense p l a t e l e t s (as found by Haver and Gear, 1981 and 1982); on day two, there was s t i l l an i n c r e a s e i n the r a d i o a c t i v i t y of the dense p l a t e l e t s . There was a three day l a g phase before a decrease i n r a d i o a c t i v i t y of the dense p l a t e l e t s was observed. Throughout the p l a t e l e t l i f e s p a n , the r a d i o a c t i v i t y i n the l e s s dense p l a t e l e t s decreased. In the intermediate d e n s i t y p l a t e l e t s i t remained constant and i n the dense f r a c t i o n s i t increased. These r e s u l t s suggested t h a t i n humans "young p l a t e l e t s are heterogeneous and s l i g h t l y l e s s dense than the t o t a l p l a t e l e t p o p u l a t i o n " . - 21 -In another p u b l i c a t i o n the method of S t r a c t a n d e n s i t y d i s t r i b u t i o n analyses of human p l a t e l e t s was discussed and found to be r e p r o d u c i b l e , u s e f u l , and to provide independent i n f o r m a t i o n compared to other t e s t s (e.g., assays of aggregate r a t i o s and BTG) f o r d e t e c t i o n of p l a t e l e t a c t i v a t i o n i n p a t i e n t s w i t h thrombotic disease (van Oost et a l . , 1982). Using S t r a c t a n d e n s i t y g r a d i e n t separation as a technique f o r p r e p a r a t i v e i s o l a t i o n of p l a t e l e t s , p l a t e l e t s i z e a n a l y s i s was shown to be of c l i n i c a l d i a g n o s t i c value i n the management of p a t i e n t s with d i s o r d e r s i n p l a t e l e t production and d e s t r u c t i o n (Corash, 1982). As a technique to determine the d i f f e r e n t i a l d i s t r i b u t i o n s of s e c r e t a b l e and non-secretable p l a t e l e t compartments, S t r a c t a n d e n s i t y gradient analyses was shown to be a s e n s i t i v e i n d i c a t o r of p l a t e l e t a c t i v a t i o n (Corash et a l . , 1982). P l a t e l e t s i z e was again shown to be u s e f u l i n the e v a l u a t i o n of abnormal p l a t e l e t production i n p a t i e n t s w i t h hematologic disease. I t was shown not to c o r r e l a t e w i t h p l a t e l e t age but t o c o r r e l a t e ( i n c r e a s i n g p l a t e l e t s i z e ) with the degree of s t i m u l a t i o n of thrombopoiesis (Levin and Bessman, 1983). P l a t e l e t d e n s i t y was again shown to decrease due to thrombin induced d e g r a n u l a t i o n , which p a r a l l e l s BTG and s e r o t o n i n s e c r e t i o n ( o c c u r r i n g i n seconds, Gear and Burke, 1982) and average p l a t e l e t d e n s i t y was shown t o increase a f t e r cardiopulmonary bypass (van Oost et_ a l . , 1983). Although i n c e r t a i n r e p o r t s p l a t e l e t age has been shown to c o r r e l a t e with p l a t e l e t d e n s i t y ( s p e c i f i c a l l y i n the sub-human primate, Corash et a l . , 1978), and d e n s i t y to c o r r e l a t e with s i z e (but not i n the sub-human primate), analyses of counterflow c e n t r i f u g a t i o n s i z e - s e p a r a t e d p l a t e l e t s from baboons (Papio cynocephalus) revealed that " p l a t e l e t s i z e does not c o r r e l a t e with p l a t e l e t age" (Thompson et - 22 -a l . , 1983b). The l a r g e r p l a t e l e t s s urvived longer but were not younger than the smaller p l a t e l e t s . However, a decrease i n p l a t e l e t s i z e with ageing could not be disproved. The work of Thompson et a l . , (1981, 1982 and 1983 a and b) supports the b e l i e f that p l a t e l e t s i z e heterogeneity i s present p r i m a r i l y at the onset o f p l a t e l e t production and i s not a r e s u l t o f p l a t e l e t maturation. In monkeys (Macaca f a s i c u l a r i s ) , p l a t e l e t s were l a b e l e d with 5 ^ C r and subjected to P e r c o l l d e n s i t y d i s t r i b u t i o n analyses before and a f t e r i n j e c t i o n in_ v i v o and c i r c u l a t i o n f o r f i v e days (M a r t i n and Penington, 1983). No s i g n i f i c a n t enrichment of 5"*"Cr uptake i n v i t r o was demonstrated f o r p l a t e l e t s of higher d e n s i t y (as was demonstrated by Haver and Gear, 1981 and 1982). However, a f t e r c i r c u l a t i o n the l a b e l e d p l a t e l e t d ensity d i s t r i b u t i o n was found t o be s h i f t e d t o higher d e n s i t y . Considering t h a t newly produced p l a t e l e t s were i n t h i s case unlabeled, i t appeared as i f these p l a t e l e t s were of lower d e n s i t y . However, i t was conceded t h a t " l i g h t e r p l a t e l e t s may have a s l i g h t l y s h o r t e r s u r v i v a l time than dense ones". Experimentally induced c h r o n i c shortened p l a t e l e t s u r v i v a l , i n the r a t , i n d i c a t e d continuous v e s s e l i n j u r y was r e q u i r e d to shorten p l a t e l e t s u r v i v a l and t h a t the r e s u l t a n t r e d u c t i o n i n p l a t e l e t numbers was not balanced by p l a t e l e t p roduction. However, those p l a t e l e t s which were produced were normal i n s i z e and f u n c t i o n (by aggregation and r e l e a s e c h a r a c t e r i s t i c s ) but were of highest d e n s i t y , of lower mean s i a l i c a c i d and p r o t e i n per p l a t e l e t , and s u r v i v e d longer ( i . e . , i n t e r p r e t e d as being younger) than normal p l a t e l e t s (Winocour et a l . , 1983). - 23 -1.4 Megakaryocyte and P l a t e l e t Heterogeneity P l a t e l e t p r e c u r s o r s , megakaryocytes, are heterogeneous i n themselves, producing heterogeneous subpopulations of p l a t e l e t s (Paulus, 1975; Paulus et a l . , 1979; Penington and S t r e a t f i e l d , 1975; Penington et a l . , 1976a and 1976b; Penington, 1979; Penington, 1981). The normal pop u l a t i o n o f megakaryocytes i n bone marrow c o n s i s t s of subpopulations of megakaryocytes which d i f f e r i n t h e i r p l o i d y number (4 N, 1-2%; 8 N, 10-25%; 16 N, 46-76%; 32 N, 15-32%; and 64 N, 1%) with the 16 N p l o i d y c l a s s predominating. Those megakaryocytes which have a low p l o i d y number, 8 N or 16 N, produce p l a t e l e t s which are l a r g e , have many granules (dense and alpha) and mitochondria but have a low content of demarcation membrane system; whereas those which have a high p l o i d y (32 N, or 64 N) produce s m a l l and l e s s e f f e c t i v e p l a t e l e t s , which are d e f i c i e n t i n granules and m i t o c h o n d r i a l content but have a high q u a n t i t y of demarcation membrane system. Megakaryocytes respond t o thrombocytopenia w i t h , of course, increased p l a t e l e t p roduction, but t h i s response has been c h a r a c t e r i z e d as being s p e c i f i c f o r i n d i v i d u a l megakaryocyte p l o i d y c l a s s e s . In humans, u l t r a s t r u c t u r a l s t u d i e s of megakaryocytes i n p a t i e n t s with ITP revealed that increased p l a t e l e t production was r e l a t e d to megakaryocyte number, volume, and i n three cases, the content of " d i l a t e d " t u b u l a r demarcation membrane ( R i d e l l and Branehog, 1976). Acute thrombocytopenia was a l s o used to s t i m u l a t e megakaryocytopoiesis i n r a t s ( O d e l l et a l . , 1976; O d e l l and Shelton, 1979) producing a s i t u a t i o n i n which s p e c i f i c a l l y the d i p l o i d megakaryocyte precursor was s t i m u l a t e d to mature to a p l o i d y l e v e l one nuclear d i v i s i o n higher than normal with a p r o p o r t i o n a t e i n c r e a s e i n c e l l s i z e ; these c e l l s produced - 24 -more p l a t e l e t s at a f a s t e r r a t e . Furthermore, s i n c e megakaryocyte changes began to reverse before normal l e v e l s of c i r c u l a t i n g p l a t e l e t s were reached, these authors concluded t h a t the c e s s a t i o n of t h i s p l a t e l e t production was c o n t r o l l e d by something other than the t o t a l p l a t e l e t mass. This i s i n agreement with e a r l i e r f i n d i n g s ( K a r p a t k i n , 1974) t h a t the production i n h i b i t o r y e f f e c t may r a t h e r a r i s e from a c t i v i t y of an e a r l i e r c e l l i n the maturation sequence, or from a preponderance i n the c i r c u l a t i n g p l a t e l e t p o p u l a t i o n of young h i g h l y f u n c t i o n a l p l a t e l e t s , i . e . , "megathrombocytes being an index of p l a t e l e t production". In the guinea p i g and monkey, the 8 N p l o i d y c l a s s was found to predominate (Levine et a l . , 1980). In the guinea p i g and human, megakaryocyte heterogeneity was s a i d to be e s s e n t i a l l y due to a continuous process of p o l y p l o i d i z a t i o n (endomitosis), i n c r e a s i n g megakaryocyte s i z e with each nuclear doubling. In the guinea p i g p l a t e l e t production was more a c h a r a c t e r i s t i c of mature r a t h e r than of immature megakaryocytes (Levine et a l . , 1982). Study of r a t megakaryocytes i n steady-state marrow a l s o revealed heterogeneity of p l o i d y w i t h c e r t a i n q u a n t i t i e s of megakaryocytes a s c r i b e d to d i f f e r e n t p l o i d y c l a s s e s (33.4 +• 1.4%, 8 N; 40.8 +_ 1.4%, 16 N; and 17.9 + 1.3%, 32 N; and few 64 N) with the 16 N p l o i d y c l a s s predominating. By enumeration of megakaryocytes w i t h i n and between p l o i d y c l a s s e s i t was found t h a t there were r e l a t i v e l y l a r g e q u a n t i t i e s of c e l l s between the 2 N and 4 N, and between the 4 N and 8 N p l o i d y l e v e l s and few c e l l s between the 8 N and 16 N, and between the 16 N and 32 N. This was i n t e r p r e t e d t o mean that there was r a p i d turnover of the more immature 2 N and 4 N, compared to that of the 8 N and 16 N (Tanum - 25 -and Engeset, 1983). The heterogeneity o f megakaryocyte p l o i d y i s a l s o evident at another l e v e l , i n s o f t agar t i s s u e c u l t u r e of mouse marrow (Radley et a l . , 1980; Levin et a l . , 1981; L e v i n , 1983). The c o l o n i e s formed by megakaryocytes i n c u l t u r e are of two major types, the "big c e l l " and "heterogeneous" colony types. The " b i g c e l l " colony type cont a i n s l e s s than 30 c e l l s , most of which are l a r g e and about h a l f of which s t a i n s t r o n g l y f o r a c e t y l c h o l i n e s t e r a s e a c i t i v i t y . The average megakaryocyte p l o i d y i s 16.8 N (8 N, 30%; 16 N, 45%; 32 N, 20%), a p l o i d y d i s t r i b u t i o n c h a r a c t e r i s t i c o f mature bone marrow megakaryocytes. The "heterogeneous" colony type c o n t a i n s more than 40 c e l l s , of a l l s i z e s , with v a r y i n g a c e t y l c h o l i n e s t e r a s e a c t i v i t i e s , the s m a l l c e l l s being negative f o r t h i s a c t i v i t y . The average p l o i d y i s 6.8 N (2-4 N, 57%; 16-32 N, 17%), a p l o i d y d i s t r i b u t i o n c h a r a c t e r i s t i c of immature megakaryocytes and more p r i m i t i v e colony forming c e l l s (CFC). In response to acute thrombocytopenia, induced by i n j e c t i o n of r a b b i t anti-mouse p l a t e l e t serum, the p l o i d y d i s t r i b u t i o n of the heterogeneous colony type remains e s s e n t i a l l y unchanged, while that of the b i g c e l l colony s h i f t s to higher p l o i d y c l a s s e s , with an average p l o i d y of 21.5 N, i n c r e a s i n g the frequency o f 32 N and 64 N megakaryocytes (32 N, 30%; 64 N, 6%). This suggests t h a t there are at l e a s t two types of megakaryocyte CFC d i f f e r i n g i n response to p l a t e l e t d e p l e t i o n . Thus, d i f f e r e n t subpopulations of megakaryocytes might respond d i f f e r e n t l y to d i f f e r e n t s t i m u l i . Subpopulations of megakaryocytes i n the bone marrow could produce s m a l l p l a t e l e t s with low granule content which are s u f f i c i e n t l y e f f e c t i v e under c o n d i t i o n s of normal hemostasis; other megakaryocytes might produce l a r g e , more e f f e c t i v e p l a t e l e t s with - 26 -high granule content i n s t a t e s of a c t i v e thrombosis. As these p l a t e l e t s age and encounter r e v e r s i b l e (or not) adhesive r e a c t i o n s , they could become s m a l l e r , l o s i n g pieces of membrane, r e l e a s e granules and become l e s s dense. This i s another way i n which the p l a t e l e t population could become heterogeneous (by changing w i t h i n the c i r c u l a t i o n during ageing, to be d i s c u s s e d ) . In t h i s case, p l a t e l e t s from the low p l o i d y megakaryocyte c l a s s would come to s u p e r f i c i a l l y resemble those of the higher c l a s s e s . Table 1.4.1 P l a t e l e t Heterogeneity and C o n t r i b u t i n g F a c t o r s P l a t e l e t Heterogeneity P h y s i c a l P r o p e r t i e s : d e n s i t y , s i z e , s urface p r o p e r t i e s . S t r u c t u r a l Forms: d i s c o c y t e , echinocyte. Surface Receptors For: ADP, antibody fragment F c , c o l l a g e n , f i b r i n o g e n , f i b r o n e c t i n , thrombin, thrombospondin, von w i l l e b r a n d f a c t o r ( i n the absence of f i b r i n o g e n ) . S t r u c t u r a l Content: granules (dense and l i g h t , a l p h a ) , lysosomes, membrane c a n a l i c u l i , mitochondria. Molecular Content: ADP, ATP, beta-thromoglobulin, calcium, f i b r i n o g e n , glycogen, LDH, PDGF, PF4, s e r o t o n i n , 35-S-bearing proteoglycan, thrombospondin. Fac t o r s C o n t r i b u t i n g to P l a t e l e t Heterogeneity O r i g i n from Megakaryocyte P l o i d y C l a s s . P l a t e l e t Age. A c t i v a t i o n S t a t e : d i s c o c y t e - echinocyte. F u n c t i o n a l Purpose: bearing surface r e c e p t o r s , i n t e r n a l heterogeneity. Body Compartment: c i r c u l a t i n g p e r i p h e r a l blood, marginating ( w i t h i n organs), marrow. - 27 -Hence we have a p i c t u r e of heterogeneous pr o g e n i t o r c e l l s , producing heterogenous p o p u l a t i o n s of p l a t e l e t s (Table 1.4.1), a c t i v e and l e s s a c t i v e , with p l a t e l e t s as they age perhaps becoming l e s s a c t i v e . In t h i s manner the body could a l t e r the q u a l i t y and qua n t i t y of i t s c i r c u l a t i n g thrombotic elements. The Thrombon (used to describe i n t h i s case, the t o t a l mass of c i r c u l a t i n g p l a t e l e t s , t h e i r p r e c u r s o r s , and the q u a l i t y or p o t e n t i a l f o r a c t i v a t i o n and thrombosis, of t h i s mass) co u l d thus be c o n t r o l l e d not only i n number but a l s o i n q u a l i t y . 1.5 Membrane Surface P r o p e r t i e s Detected by Aqueous Two-Phase P a r t i t i o n When aqueous s o l u t i o n s of d i f f e r e n t polymers, e.g., dextran (D) and polye t h y l e n e g l y c o l (PEG), are mixed above c e r t a i n c o n c e n t r a t i o n s , they form i m m i s c i b l e two phase systems which can be buff e r e d and rendered i s o t o n i c . Such phase systems have been proven s u i t a b l e f o r the p r e p a r a t i v e and a n a l y t i c a l s e p a r a t i o n of macromolecules, s u b c e l l u l a r p a r t i c l e s , and c e l l s by t h e i r d i f f e r e n t i a l p a r t i t i o n w i t h i n the two polymer phases: upper, PEG-rich phase and lower, D-rich phase. B i o l o g i c a l p a r t i c a l s are u s u a l l y p a r t i t i o n e d between the D-PEG i n t e r f a c e and upper PEG phase. The d i s t r i b u t i o n of p a r t i c l e s w i t h i n the phases (the p a r t i t i o n ) depends on the surface p r o p e r t i e s of the p a r t i c l e s , and the p a r t i c u l a r type of phase system. The phase systems have been shown t o r e f l e c t a v a r i e t y of surface p r o p e r t i e s (e.g., charge-associated, hydrophobic, and b i o s p e c i f i c s urface p r o p e r t i e s ) , c o n t r o l l a b l e by v a r i a t i o n of the phase system composition. The e a r l y methodology of aqueous two-polymer phase system a n a l y s i s , most o f which i s s t i l l i n use today, was introduced and o u t l i n e d i n l a t e 1950 and e a r l y 1960 ( A l b e r t s s o n , 1960 and 1971; Alb e r t s s o n and - 28 -B a i r d , 1962; T i s e l i u s et a l . , 1963; Walter 1969). For more recent reviews see F i s h e r (1981), A l b e r t s s o n et a l . (1982), Walter (1982), A l b e r t s s o n (1983), and Brooks (1983). Polymer two-phase systems composed of dextran and polyethylene g l y c o l were shown to provide a m i l d environment f o r a v a r i e t y of b i o l o g i c a l c e l l s such t h a t , i n a d d i t i o n t o the p a r t i t i o n i n g o f s u b c e l l u l a r p a r t i c l e s and macromolecules ( A l b e r t s s o n , 1960), s e p a r a t i o n s of v i a b l e c e l l s was now be f e a s i b l e ( A l b e r t s s o n and B a i r d , 1962). R e s u l t s of countercurrent d i s t r i b u t i o n (CCD) of eukaryotic and p r o k a r y o t i c c e l l s were presented: e r y t h r o c y t e s of d i f f e r e n t species (dog, human, sheep, r a b b i t ) , algae ( C h l o r e l l a pyrenoidosa and e l i p s o i d e a ) yeast (Saccharomyces c a r l s b e r g e n s i s ) , and b a c t e r i a ( E s c h e r i c h i a c o l i ) . Not only could s i n g l e step p a r t i t i o n provide a species s p e c i f i c s e p a r a t i o n of c e l l s , and CCD i n a s i n g l e two-polymer phase system provide r e p r o d u c i b l e c h a r a c t e r i s t i c separations of d i f f e r e n t c e l l types, but a l s o a concept, new to aqueous polymer two-phase system a n a l y s i s was presented, namely the use of a s a l t g r a d i e n t of sodium c h l o r i d e and phosphate i n s p e c i f i c circumstances to f r a c t i o n a t e a mixture of c e l l s . Although at t h a t time the bases f o r these separations were s t i l l u n c l e a r , and i n s i m i l a r separations of c e r t a i n macromolecules and c e l l types, s i z e i s the major determinant, "the e x p l a n a t i o n which l i e s nearest t o hand i s t h a t i t i s d i f f e r e n c e s i n the surface p r o p e r t i e s . . . t h e presence of d i f f e r e n t chemical groupings on the surfaces of c e l l s " ( A l b e r t s s o n and B a i r d , 1962) which a f f e c t s p a r t i t i o n behavior. Ions were shown to p a r t i t i o n unequally between t h e ' phases, r e s u l t i n g i n an e l e c t r o s t a t i c p o t e n t i a l d i f f e r e n c e which a l s o a f f e c t e d - 29 -the p a r t i t i o n of substances i n c l u d e d i n the phase systems (Johansson, 1970) . Another major f a c t o r governing the p a r t i t i o n i n these phase systems was shown to be the i n t e r f a c i a l t e n s i o n between the D and PEG phases, dependent upon t h e i r concentrations (Ryden, and A l b e r t s s o n , 1971) . That surface charge a s s o c i a t e d p r o p e r t i e s are i n v o l v e d i n p a r t i t i o n of c e l l s was demonstrated by Brooks et a l . , (1971) who c o r r e l a t e d p a r t i t i o n of e r y t h r o c y t e s with e l e c t r o p h o r e t i c m o b i l i t y a f t e r CCD. The p o t e n t i a l d i f f e r e n c e between phases was a c t u a l l y measured, on the order of a few m i l l i v o l t s (Reitherman et a l . , 1973). Walter et a l . , (1976a) demonstrated that p a r t i t i o n i n two-polymer aqueous phase systems can d e t e c t membrane surface p r o p e r t i e s such as s u r f a c e charge; but by a p p r o p r i a t e manipulation of the two-phase system components, membrane surface p r o p e r t i e s other than charge can a l s o be s t u d i e d . They showed th a t with i n c r e a s i n g phosphate and polymer content the p o t e n t i a l d i f f e r e n c e between the two phases i n c r e a s e s , and w i t h i n c r e a s i n g c h l o r i d e the p o t e n t i a l decreases. A l s o the p a r t i t i o n c o e f f i c i e n t (K; number of c e l l s i n the upper phase, as a percentage of the t o t a l c e l l s added to the system) of e r y t h r o c y t e s (K = 54%) a f t e r removal of s i a l i c a c i d decreases to K = 8% when measured i n phase systems with high sodium phosphate content (e.g., 5/4:1), but not when measured i n phase systems with high sodium c h l o r i d e content (e.g., 5/3.5:5), i n which case p a r t i t i o n i s observed to i n c r e a s e to K = 92%. The equation which i l l u s t r a t e s mathematically what has been shown i n a general way e x p e r i m e n t a l l y , namely that p a r t i t i o n depends mainly on c e l l p r o p e r t i e s , one of which i s the surface charge and the other of which has to do with a non-charge i n t e r a c t i o n between the membrane and the phases (the c e l l / p h a s e i n t e r f a c i a l f r e e energy), brings out the - 30 -important p o i n t t h a t the p a r t i t i o n c o e f f i c i e n t depends e x p o n e n t i a l l y on the s u r f a c e p r o p e r t i e s of i n t e r e s t . This i m p l i e s t h a t s m a l l d i f f e r e n c e s i n s u r f a c e charge or i n the degree t o which the phases i n t e r a c t w i t h the membrane can cause l a r g e d i f f e r e n c e s i n p a r t i t i o n behavior (Brooks et a l . , 1971 and 1976). This i s i n c o n t r a s t to procedures such as e l e c t r o p h o r e s i s i n which the parameter which c h a r a c t e r i s e s the c e l l , the e l e c t r o p h o r e t i c m o b i l i t y , depends only l i n e a r l y on surface charge d e n s i t y . The strengths of the p a r t i t i o n approach to c e l l s u rface c h a r a c t e r i z a t i o n , then, are i t s extreme s e n s i t i v i t y to membrane surface charge and l i p i d composition and the c o n t r o l which the experimentor has over the c o n d i t i o n s which determine the f i n a l c e l l d i s t r i b u t i o n . Using species s p e c i f i c d i f f e r e n c e s i n l i p i d content of e r y t h r o c y t e membranes i t was demonstrated t h a t the p a r t i t i o n of e r y t h r o c y t e s from the d i f f e r e n t species i n phase systems bearing l i t t l e p o t e n t i a l d i f f e r e n c e between the phases increased as the r a t i o of poly/mono-unsaturated f a t t y a c i d s i n c r e a s e d , decreased with the sphingomyelin content and increased with the p h o s p h a t i d y l c h o l i n e content (as a percentage of the t o t a l l i p i d of the membrane). Thus i n systems with high c h l o r i d e content and w i t h the polymer c o n c e n t r a t i o n s c l o s e to the c r i t i c a l p o i n t , a surface property analogous t o " f l u i d i t y " or " l i p i d p acking" appeared to be detected. An age r e l a t e d CCD of i n v i v o 59 F e - l a b e l e d e r y t h r o c y t e s was obtained i n the r a t w i t h one of these high c h l o r i d e two-polymer phase system (Walter et a l . , 1976a). C e l l s e p arations are s a i d t o be based on the d i f f e r e n t i a l a f f i n i t y of the substances being p a r t i t i o n e d f o r the environments provided by one or the other phase system polymers, polyethylene g l y c o l being more hydrophobic than dextran. A c t u a l l y the polymers can be arranged i n a - 31 -hydrophobic s c a l e , and even polyphase systems of more than two polymers can be made ( A l b e r t s s o n , 1977). Changing the i o n i c environment of the phases changes the environment o f the polymers and hence p o s s i b l y the a t t r a c t i v e or r e p u l s i v e p r o p e r t i e s which the phase polymers provide. Based on experience of the type described above, three general types of phase systems are used ( A l b e r t s s o n , 1977; Walter, 1978). In the phase system c o n t a i n i n g high phosphate c o n c e n t r a t i o n , which i t s e l f p a r t i t i o n s to the lower d e x t r a n - r i c h phase c r e a t i n g an e l e c t r o s t a t i c p o t e n t i a l d i f f e r e n c e between the phases (upper phase, p o s i t i v e ) c e l l d i s t r i b u t i o n s are properly s a i d t o be membrane surface charge-associated, or c h a r g e - s e n s i t i v e . In the high c h l o r i d e phase system having n e g l i g i b l e p o t e n t i a l between the phases the d i f f e r e n c e between the i n t e r a c t i o n s of PEG and D with the membrane appears to be the governing f a c t o r . Based on the above c o r r e l a t i o n s , c e l l d i s t r i b u t i o n s are thought to be i n par t l i p i d - s e n s i t i v e . In the phase system of high c h l o r i d e content, by i n c o r p o r a t i n g a polymer l i g a n d , which may or may not be " b i o s p e c i f i c " , a c e l l s u r f a c e l i g a n d a f f i n i t y s e p a r a t i o n can be achieved (Walter, 1978). Thus a v a r i e t y o f c e l l s e p a r a t i o n s and analyses are p o s s i b l e , even t o the extent of using t h i s p a r t i t i o n i n g approach to q u a n t i t a t e weak i n t e r a c t i o n s between molecules ( A l b e r t s s o n , 1978) or to probe c e l l - c e l l i n t e r a c t i o n s (Walter, 1978). I t was a l s o recognized t h a t t h i s type of a n a l y s i s might be of use i n the study or d i a g n o s i s o f disease s t a t e s t h a t r e f l e c t themselves i n c e l l membrane surface changes (Walter, 1978). For the c e l l b i o l o g i s t and biochemist, t h i s type of c e l l s u r f a c e a n a l y s i s i n combination with separations based on other p r o p e r t i e s such as c e l l s i z e , shape, and/or d e n s i t y , should indeed be b e t t e r than the - 32 -use o f e i t h e r alone ( A l b e r t s s o n , 1978; J o v i n and A r n d t - J o v i n , 1980). 1.6 P a r t i t i o n Studies of C e l l Surface P r o p e r t i e s During Ageing E f f o r t s d i r e c t e d toward the e l u c i d a t i o n of c e l l age by counter-c u r r e n t d i s t r i b u t i o n i n two-polymer aqueous phase systems are centered i n one l a b o r a t o r y i n p a r t i c u l a r , that of Dr. H. Walter. This a b i l i t y o f CCD i n two-polymer aqueous phase systems to provide blood c e l l f r a c t i o n a t i o n s and d i s t r i b u t i o n s based on e r y t h r o c y t e c i r c u l a t i o n time, age, and length of time i n storage, has thus been e s t a b l i s h e d , but p r i m a r i l y with the r a t as a model system. 59 Using i n v i v o F e - l a b e l e d r a t e r y t h r o c y t e s , Walter and Selby (1966) f i r s t demonstrated t h a t CCD could d i s t i n g u i s h red blood c e l l s of s l i g h t l y d i f f e r e n t ages. A l s o i n the same year, Walter and A l b e r t s s o n reported the d e t e c t i o n of two populations o f r a t r e t i c u l o c y t e s i n phenylhydrazine induced anemia. Upon storage from 28 t o 48 days the p a r t i t i o n of human er y t h r o c y t e s i n c r e a s e s ; however there i s no change i n p a r t i t i o n w i t h storage i n ACD and adenosine (Walter et a l . , 1968). 59 Again, using the technique of i n v i v o Fe-cohort l a b e l i n g , a l t e r a t i o n s i n the surface p r o p e r t i e s during normal maturation of r a t e r y t h r o c y t e s were demonstrable by p a r t i t i o n ' . In a d d i t i o n , the innocuous and p r o t e c t i v e nature o f the polymers and the CCD technique was demonstrated, s i n c e e r y t h r o c y t e s harvested a f t e r CCD s u r v i v e d normally i n v i v o (Walter et a l . , 1971a and 1971b). Phenylhydrazine-induced r e t i c u l o c y t e s demonstrated an abnormal maturation p a t t e r n by CCD (Walter et a l . , 1972a); and the charge-a s s o c i a t e d (Walter et a l . , 1972b) membrane surface p r o p e r t i e s during - 33 -maturation of r e t i c u l o c y t e s e l i c i t e d by repeated bleeding were abnormal, being o f lower p a r t i t i o n and surface charge than normal r e t i c u l o c y t e s (Walter e t a l . , 1975). C e l l s other than blood c e l l s a l s o demonstrate membrane changes a s s o c i a t e d with d i f f e r e n t i a t i o n and maturation which can be detected by p a r t i t i o n . Rat l i v e r c e l l s d u r i ng regeneration can be observed to change t h e i r s urface p r o p e r t i e s as detected by p a r t i t i o n (Walter et a l . , 1973b). Rat i n t e s t i n a l e p i t h e l i a l c e l l s , as they mature and age, from c r y p t t o v i l l u s e x h i b i t a r a p i d i n c r e a s e i n p a r t i t i o n i n charged phase systems, and then a gradual decrease i n p a r t i t i o n , with ageing (Walter and Krob, 1975). Not only could p a r t i t i o n i n c h a r g e - s e n s i t i v e phase systems demonstrate t h a t e r y t h r o c y t e s change t h e i r surface p r o p e r t i e s during maturation and ageing; i t was a l s o found that phase systems known to r e f l e c t aspects of the c e l l membrane other than charge, such as the ch a r a c t e r of the hydrophobic l i p i d b i l a y e r , could a l s o demonstrate t h a t e r y t h r o c y t e s change during c i r c u l a t i o n (Walter et a l . , 1976a and 1976b). Most o f the s u c c e s s f u l CCD age d i s t r i b u t i o n s were obtained with r a t e r y t h r o c y t e s , although other animal models have been s t u d i e d b r i e f l y . I t i s hoped that human e r y t h r o c y t e s can be d i s t r i b u t e d according t o age by CCD. However, Walter et a l . (1980) reported that the r a t i s not a model f o r human red c e l l ageing. Erythrocyte age r e l a t e d changes detected by CCD of r a t red c e l l s could not be detected i n human e r y t h r o c y t e s , by CCD alone nor by c e l l e l e c t r o p h o r e s i s a f t e r c e l l s e p a r a t i o n by CCD. The e l e c t r o p h o r e t i c m o b i l i t i e s were the same f o r e r y t h r o c y t e s , r a t or human, young or o l d (obtained by the Murphy c e n t r i f u g a t i o n technique, 1973; er y t h r o c y t e d e n s i t y i n c r e a s e s with age - 34 -i n the c i r c u l a t i o n of both human and r a t ) . In the r a t , young mature e r y t h r o c y t e s have a higher p a r t i t i o n than o l d e r y t h r o c y t e s and the e l e c t r o p h o r e t i c m o b i l i t i e s o f er y t h r o c y t e s obtained a f t e r CCD in c r e a s e with i n c r e a s i n g p a r t i t i o n through the CCD d i s t r i b u t i o n s of e i t h e r young or o l d r a t e r y t h r o c y t e s . However there are no obvious d i f f e r e n c e s i n p a r t i t i o n nor e l e c t r o p h o r e t i c m o b i l i t y detected f o r human e r y t h r o y c t e s , young or o l d , before or a f t e r CCD, unless i s o t o p e methods are employed. Perhaps the e r y t h r o c y t e age d i s t r i b u t i o n through the density gradient d i f f e r s f o r r a t and human, so th a t the same age subpopulations are not r e a l l y being compared. Walter et a l . (1981) showed again that ageing of ery t h r o c y t e s r e s u l t s i n a l t e r e d red c e l l s u r f a c e p r o p e r t i e s i n the r a t model system but not i n the human. Using the elevated a s p a r t a t e amino t r a n s f e r a s e (AAT) l e v e l s of younger red c e l l s as a marker, young-mature e r y t h r o c y t e s of the human were found not to have the highest p a r t i t i o n c o e f f i c i e n t i n the red c e l l p o p u l a t i o n , as they do i n the r a t . In the r a t CCD, the AAT d i s t r i b u t i o n and the d i s t r i b u t i o n o f s i a l i c a c i d (SA) e x h i b i t two peaks of lower and higher p a r t i t i o n than the major p o r t i o n of the er y t h r o c y t e d i s t r i b u t i o n . In the human, AAT, SA, and r e t i c u l o c y t e s are enriched at t h e lower end of the d i s t r i b u t i o n . In the 59 dog, r e t i c u l o c y t e s , as demonstrated by F e - l a b e l i n g , are a l s o o f a lower p a r t i t i o n than the major e r y t h r o y c t e p o p u l a t i o n . Walter et a l . (1981) a l s o repeated e a r l i e r work of Walter et a l . (1980) i n v o l v i n g CCD of d e n s i t y separated young and o l d red c e l l s . Countercurrent d i s t r i b u t e d young and o l d red blood c e l l s of the r a t show increased e l e c t r o p h o r e t i c m o b i l i t y through the CCD t r a i n , but human red c e l l s do not d i s t r i b u t e based on charge a s s o c i a t e d p r o p e r t i e s - 35 -by t h i s method. Since f r a c t i o n s from e i t h e r s i d e of the middle of the d i s t r i b u t i o n r e d i s t r i b u t e as the o r i g i n a l p o p u l a t i o n , i t was concluded t h a t human red c e l l s do not p a r t i t i o n based on any sur f a c e parameter. I t was s t a t e d i n the paper, however that s m a l l e r q u a n t i t i e s of c e l l s can have d i f f e r e n t d i s t r i b u t i o n curves of a l t e r e d apparent p a r t i t i o n . This might be the case, t h a t excessive q u a n t i t i e s of c e l l s were being d i s t r i b u t e d i n t h i s work, thus hampering se p a r a t i o n and/or r e s o l u t i o n and r e s u l t i n g i n s i m i l a r p a r t i t i o n s f o r a l l populations being compared. I t was a l s o concluded t h a t dog red c e l l s , a f t e r a l l o w i n g 27 days o f c i r c u l a t i o n , y i e l d r e s u l t s s i m i l a r t o those found i n the human (with an e r y t h r o c y t e l i f e span of 120 days). However, as mentioned above, the data show c l e a r l y an i n c r e a s e i n the percentage of immature r e t i c u l o c y t e s , as w e l l as an enrichment i n AAT to the l e f t s i d e or lower p a r t i t i o n i n g p o r t i o n of the d i s t r i b u t i o n curves, f o r the human as w e l l as the r a t . Upon f u r t h e r experimentation and a n a l y s i s of sm a l l e r q u a n t i t i e s o f c e l l s a c l e a r cut d i f f e r e n c e might be demonstrated f o r age-density separated human red c e l l p o p u l a t i o n s , as has been found i n the r a t e r y t h r o c y t e model. A new method was r e c e n t l y described f o r d e t e c t i o n of surface d i f f e r e n c e s between two c l o s e l y r e l a t e d c e l l p o p u l a t i o n s , (Walter et a l . , 1983; Walter and Krob, 1983). This method i s a v a r i a t i o n o f the i n t e r n a l standard method, or more a p p r o p r i a t e l y an " i n t e r n a l sample" 59 method. I t i s s i m i l a r , i n p r i n c i p l e , t o the i n v i v o Fe cohort l a b e l i n g method of a n a l y s i s of ageing r a t e r y t h r o c y t e s used by Walter, wherein the c e l l p o p u l a t i o n under study i s l a b e l e d i n v i v o and compared i n the same.sample t o the unlabeled normal "standard" p o p u l a t i o n . The method d i f f e r s i n th a t c e l l s are i s o l a t e d and l a b e l e d i n v i t r o and then - 36 -mixed with normal, "standard" unlabeled c e l l s f o r comparison. An a d d i t i o n a l c o n s i d e r a t i o n f o r s t u d i e s using t h i s method i s t h a t of 51 Cr uptake v a r i a t i o n w i t h i n the l a b e l e d subpopulation, s i n c e enhanced l a b e l uptake has been reported f o r young e r y t h r o c y t e s from the r a t and human (Walter et a l . , 1962). P l a t e l e t s , as a c e l l u l a r p o p u l a t i o n i n blood, are second i n number only t o e r y t h r o c y t e s . Their stem c e l l s are r e l a t e d , and while p l a t e l e t s ( u n l i k e e r y t h r o c y t e s ) have been shown to be heterogeneous with respect to t h e i r megakaryocyte o r i g i n ( p l o i d y c l a s s ) , both p l a t e l e t s and e r y t h r o c y t e s are heterogeneous with respect to s i z e , d e n s i t y and s u r v i v a l time, or ageing. Analyses of blood c e l l membrane age-related h e t e r o g e n e i t i e s as r e f l e c t e d by p a r t i t i o n i n two-polymer aqueous phase systems have been d i r e c t e d , p r i m a r i l y and s u c c e s s f u l l y , toward the red blood c e l l . These methods of c e l l surface analyses, outside of t h i s t h e s i s , have not yet been a p p l i e d to the p l a t e l e t membrane heterogeneity r e p o r t e d l y a s s o c i a t e d with p l a t e l e t ageing. Analogies have been proposed between t h i s process and e r y t h r o c y t e ageing, namely i n the a l t e r a t i o n of membrane and r e d u c t i o n i n surface charge d e n s i t y . I t seems l o g i c a l to extend the a p p l i c a t i o n of these techniques t o p l a t e l e t age d i s t r i b u t i o n s and i n so doing, i n c r e a s e our l i m i t e d knowledge of p l a t e l e t membrane heterogeneity (Walter et a l . , 1969a and 1969b; Grant and Zucker, 1976 and 1978; Yarom et a l . , 1982). 1.7 C l i n i c a l and Diagnostic A p p l i c a t i o n of P a r t i t i o n Methodology The d e t e r m i n i a t i o n of p l a t e l e t s u r v i v a l time i s of d i a g n o s t i c and p r o g n o s t i c value i n a v a r i e t y of s i t u a t i o n s : i n c l i n i c a l c o n d i t i o n s ranging from thrombocytosis to thrombocytopenia and thrombocytemia, i n - 37 -c a r d i o v a s c u l a r diseases and coagulopathies, as w e l l as i n the judgement of the e f f i c a c y of implants and other s u r g i c a l procedures wherein thrombosis might occur (Harker and Finch 1969; Harker and S l i c h t e r 1972 and 1974). However evidence to the contrary has a l s o been presented. In a c l i n i c a l s e t t i n g the measurement i s made by i s o l a t i n g the p a t i e n t ' s p l a t e l e t s , l a b e l i n g them with 5"^Cr or " ^ I n , r e i n j e c t i n g , then sampling (as p l a t e l e t s s u r v i v e about 10 days, numerous phlebotomies are involved) at appropriate times to determine the f r a c t i o n of i n j e c t e d r a d i o a c t i v i t y remaining i n the bloodstream. The data are then subjected to g r a p h i c a l analyses and computations, producing f i n a l l y the mean p l a t e l e t s u r v i v a l time. There are s e v e r a l problems a s s o c i a t e d with the performance of the standard s u r v i v a l time t e s t . The procedures f o r p l a t e l e t i s o l a t i o n and l a b e l i n g should be s t a n d a r d i z e d . C e l l u l a r contamination encountered when p l a t e l e t s are prepared f o r t h i s t e s t may cause a l t e r a t i o n s i n apparent s u r v i v a l time s i n c e red blood c e l l s and white blood c e l l s when present a l s o become l a b e l e d and have a s u r v i v a l time d i f f e r e n t from t h a t of p l a t e l e t s . This may cause changes i n the apparent shape of the p l a t e l e t s u r v i v a l time, or disappearance curve. Once p l a t e l e t s are l a b e l e d and i n j e c t e d , there i s an immediate l o s s w i t h i n the f i r s t hour of g e n e r a l l y from 50-60% of the t o t a l i n j e c t e d p l a t e l e t s due to s e q u e s t r a t i o n i n v a r i o u s organs, f o r example the spleen, l i v e r and lungs. P l a t e l e t s i n j e c t e d i n t h i s manner are a l s o f u n c t i o n a l l y i n a c t i v e (based on aggregation) f o r one day, and thus may not s u r v i v e normally. V a r i a t i o n i n the c i r c u l a t i n g l a b e l e d p l a t e l e t c o n c e n t r a t i o n i s due t o s e v e r a l d i f f e r e n t reasons, from increased production and/or decreased u t i l i z a t i o n causing an i n c rease i n p l a t e l e t c o n c e n t r a t i o n , to - 38 -decreased production and/or in c r e a s e d u t i l i z a t i o n causing a decrease i n p l a t e l e t c o n c e n t r a t i o n . I f i t were p o s s i b l e to d i f f e r e n t i a t e between young and o l d p l a t e l e t s , these s i t u a t i o n s could be d i s t i n g u i s h e d . The standard p l a t e l e t s u r v i v a l t e s t , without d e t a i l e d curve f i t t i n g and a n a l y s i s , u s u a l l y provides only a mean s u r v i v a l time f o r the e n t i r e p l a t e l e t p o p u l a t i o n (Belcher et a l . , 1977). There are a l s o problems with c a l c u l a t i o n of mean s u r v i v a l time i n t h a t i t i s questionable whether or not the p o i n t s should be f i t t e d to the standard l i n e a r , or some type of exponential or c u r v i - l i n e a r f u n c t i o n . T h i s p l a t e l e t s u r v i v a l time t e s t i s t h e r e f o r e time consuming, uncomfortable and c h a l l e n g i n g ( r e q u i r i n g exposure to r a d i o a c t i v i t y ) f o r the p a t i e n t , and s u f f i c i e n t l y complex f o r the c l i n i c i a n that i n many center s i t i s not performed, even i n s i t u a t i o n s where the i n f o r m a t i o n to be gained would be p a r t i c u l a r l y u s e f u l . S u b s t a n t i a l b e n e f i t s would accrue i f the t e s t could be improved. In order to e l i m i n a t e the exposure of the p a t i e n t and p u b l i c to r a d i o a c t i v i t y , d i f f e r e n t methods of p l a t e l e t s u r v i v a l time e s t i m a t i o n have been introduced. A s p i r i n i n g e s t i o n has been used to block the arachidonate-prostaglandin pathway, and the reappearance o f cyclooxygenase a c t i v i t y used as an i n d i c a t o r of production of new, unaffec t e d p l a t e l e t s . Methods i n v o l v i n g analyses of p l a t e l e t contents such as BTG provide only a q u a l i t a t i v e estimate of p l a t e l e t age, because t h e i r l o s s i n d i c a t e s p l a t e l e t s t i m u l a t i o n . The procedure f o r determination of p l a t e l e t BTG i s i n s e n s i t i v e and s i n c e degranulated p l a t e l e t s s u r v i v e normally t h i s might provide spurious r e s u l t s a l s o . S i n c e , f o r example i n hematological disease s t a t e s , p a t h o l o g i c changes are o f t e n r e f l e c t e d at the c e l l s u r f a c e , i n i t i a l l y as a cause - 39 -or e v e n t u a l l y as a r e s u l t o f the d i s e a s e , analyses of blood c e l l s u r f a c e p r o p e r t i e s might be of d i a g n o s t i c and p r o g n o s t i c value (Walter, 1978). The p o t e n t i a l of p a r t i t i o n i n two-polymer aqueous phase systems to d e t e c t changes i n blood c e l l (and s u b c e l l u l a r p a r t i c l e ) surface p r o p e r t i e s that occur as a f u n c t i o n of d i f f e r e n t i a t i o n , maturation and age as w e l l as those t h a t occur i n disease or f o l l o w i n g i n v i v o and i n  v i t r o treatments, has already been e s t a b l i s h e d on an experimental b a s i s f o r c e r t a i n c e l l s (the p l a t e l e t not being one of them). However, p a r t i t i o n has seldom been a p p l i e d at a c l i n i c a l l e v e l . The c h a r g e - s e n s i t i v e , and hydrophobic phase systems are s t i l l used as o r i g i n a l l y described by A l b e r t s s o n (1960, 1971, 1977, 1978) and by Walter (1969, 1977, 1978, 1982) and are the phase systems most commonly employed. The charge- and l i p i d - s e n s i t i v e phase systems could be a p p l i e d r o u t i n e l y i f made a v a i l a b l e i n standardized phase system se t s (of v a r y i n g polymer and s a l t c o n c e n t r a t i o n s ) . The s p e c i f i c l i p i d a f f i n i t y and surface receptor a f f i n i t y two-phase systems (many of which are s t i l l experimental) can be a p p l i e d where warranted i n order to i n v e s t i g a t e f u r t h e r these s p e c i f i c c e l l surface p r o p e r t i e s . Because of p o s s i b l e v a r i a t i o n s i n p a r t i t i o n s induced by d i f f e r e n c e s i n phase system composition (and technique), the phase systems are standardized at present by a c c u r a t e l y determining the polymer c o n c e n t r a t i o n s . Further improvements are worth c o n s i d e r i n g such as a d j u s t i n g the phase system t o provide a s p e c i f i c p a r t i t i o n c o e f f i c i e n t (K) f o r a standard p a r t i c l e with a constant p a r t i t i o n , f o r example f i x e d red c e l l s , f i x e d p l a t e l e t s , or polystyrene p a r t i c l e s . During CCD the experimental K f o r the sample under i n v e s t i g a t i o n could be compared to t h a t of a standard (perhaps r a d i o a c t i v e l y l a b e l e d ) , p a r t i c l e i n c l u d e d i n the phase system. - 40 -When only s l i g h t d e v i a t i o n s from normalcy are detected, or when subpopulation heterogeneity i s suspected, samples must be subjected, i n d i v i d u a l l y or simultaneously, to CCD. This i n c r e a s e s r e s o l u t i o n by r e p e t i t i o n of the p a r t i t i o n i n g procedure and i s o f t e n s u f f i c i e n t to r e s o l v e s u b t l e d i f f e r e n c e s and/or h e t e r o g e n e i t i e s i n surface p r o p e r t i e s . This t h e s i s i s an assessment of the a p p l i c a t i o n of such methodology f o r the purposes of o b t a i n i n g an age d i s t r i b u t i o n with p l a t e l e t s , and an i n v e s t i g a t i o n of p l a t e l e t heterogeneity. - 41 -CHAPTER 2 MATERIALS AND METHODS 2.1 Blood Samples New Zealand white male r a b b i t s were obtained from the Animal Care Unit at the U n i v e r s i t y of B r i t i s h Columbia. Blood was drawn using a l a r g e p l a s t i c s y r i nge attached to a cannula ( S t e r i l e Argyle Medicut Cannula, Sherwood Medical I n d u s t r i e s , S t . Lo u i s , MO, U.S.A.) i n s e r t e d i n t o a c e n t r a l ear a r t e r y . Rhesus monkey blood was taken at the Pulmonary Research Unit at S t . Paul's H o s p i t a l , Vancouver, B r i t i s h Columbia. Monkeys were an e s t h e t i z e d with Ketamine (5-25 mg/kg) and P e n t o b a r b i t o l (25 mg/kg) p r i o r to bleeding with needle and s y r i n g e . Human blood was obtained, w i t h needle and s y r i n g e , from normal ad u l t male l a b o r a t o r y personnel at the Acute Care H o s p i t a l , U n i v e r s i t y of B r i t i s h Columbia, and the Canadian Red Cross Blood Bank i n Vancouver. Cancer p a t i e n t s , at Vancouver General H o s p i t a l , with m y e l o p r o l i f e r a t i c e d i s o r d e r s (MPD), p r i m a r i l y acute m y e l o b l a s t i c leukemia, donated blood f o r a p o r t i o n o f t h i s study. These specimens were c o l l e c t e d (about 4 hours p r i o r to experiments) w i t h needle and s y r i n g e , and mixed with 3.8% sodium c i t r a t e (9 ml blood p l u s 1 ml c i t r a t e ) . 2.2 Anticoagulant The a n t i c o a g u l a n t , a c i d - c i t r a t e - d e x t r o s e (ACD) and p l a t e l e t washing s o l u t i o n s below were e s s e n t i a l l y as p r e v i o u s l y described (Aster and J a n d l , 1964a). The ant i c o a g u l a n t was prepared i n l a r g e q u a n t i t i e s by the a d d i t i o n of 2.5 g t r i s o d i u m c i t r a t e d i h y d r a t e , 1.5 g of c i t r i c a c i d monohydrate, and 2.0 g of dextrose per f i n a l volume of 100 ml i n water. I t was frozen at -20°C i n 25 ml a l i q u o t s , and used at a d i l u t i o n of 1 - 42 -pa r t per 6 p a r t s of blood, immediately a f t e r drawing. A l l chemicals were C e r t i f i e d Reagent Grade (Fisher S c i e n t i f i c Co., Chemical manufacturing d i v i s i o n , F a i r Lawn, NJ. A s i m i l a r a n t i c o a g u l a n t , 3.8% sodium c i t r a t e , was a l s o used. Blood, taken i n t o sodium c i t r a t e was a c i d i f i e d t o pH 6.5 with 0.11 M c i t r i c a c i d (or ACD, lml/lOml blood) p r i o r t o c e n t r i f u g a t i o n . 2.3 Tyrode's S o l u t i o n s Tyrode's s o l u t i o n s were prepared from three stock s o l u t i o n s . Stock I contained 16 g of NaCl, 0.4 g of KCI, 2.0 g of NaHC0 3, and 0.116 g of NaH 2P0 A.1H 20, i n water per f i n a l volume of 100 ml. Stock I I contained 2.03 g o f MgCl 2.6H 20 (Reagent Magnesium C h l o r i d e , F i s h e r S c i e n t i f i c Co., F a i r Lawn, NJ) i n 100 ml water. Stock I I I contained 2.19 g, 1.47 g, or 1.11 g of C a C l 2 (6H 20, 2H 20, or anhydrous, r e s p e c t i v e l y ) i n 100 ml water. Calcium-free Tyrode's p l a t e l e t washing s o l u t i o n was prepared by the a d d i t i o n of 5 ml of Stock I to 90 ml of water c o n t a i n i n g 0.35 g of albumin (Pentex Bovine Albumin F r a c t i o n V, Lot Nos. 288, or 363, M i l e s L a b o r a t o r i e s Inc. Research Products, U.S.A.; or Bovine Albumin F r a c t i o n V, Sigma No A-4503, Lot 78C-0202, Sigma Chemical Co., S t . L o u i s , MO) and 0.1 g of dextrose (anhydrous, F i s h e r S c i e n t i f i c Co., Chemical Manufacturing D i v i s i o n , F a i r Lawn, NJ). A 2 ml volume of stock I I was then added, the pH adjusted to 6.5 and the volume made up t o 100 ml with water. Tyrode's s o l u t i o n f o r resuspension of p l a t e l e t s was prepared by the a d d i t i o n of 5 ml of stock I to 80 ml of water c o n t a i n i n g 0.35 g of albumin and 0.1 g of dextrose. A 1 ml volume of stock I I and a 2 ml volume of stock I I I was then added, the pH adjusted t o 7.35 and the volume made up to 100 ml with water. The f i n a l o s m o l a r i t y of these s o l u t i o n s was 290 mOsM. - 43 -A v a r i a t i o n o f the above procedure was o f t e n used which i n v o l v e d making l a r g e r q u a n t i t i e s of b u f f e r , u s u a l l y one l i t e r , w i t h the a d d i t i o n of l e s s albumin, 0.2% (Zucker and Grant, 1978), s o l i d MgCl 2.6H 20, 0.04%, and EGTA ( E t h y l e n e - g l y c o l b i s beta-amino-ethyl-ether N, N' t e t r a a c e t i c a c i d , MW 380.4, Lot No. B4X, Eastman Kodak Co., Rochester, NY), 0.02-0.04% (Zucker and Grant, 1978), the same r a t i o of stock I s o l u t i o n to water, and the adjustment of pH with HC1 or 0.11 M c i t r i c a c i d . 2.4 P r e p a r a t i o n s o f P l a t e l e t s P l a t e l e t r i c h plasma (PRP) was u s u a l l y harvested by c e n t r i f u g a t i o n of a n t i c o a g u l a t e d ( a c i d i f i e d ) blood at 275 g f o r 15-20 minutes at room temperature, with the force being v a r i e d depending on the volume o f blood. The p e l l e t was d i l u t e d with Calcium-free Tyrode's s o l u t i o n c o n t a i n i n g 0.35% bovine serum albumin and 0.02% EGTA at pH 6.5 and the c e n t r i f u g a t i o n repeated. The supernatant was added t o the PRP. The d i l u t e d PRP was c e n t r i f u g e d at 2000 g f o r 30 minutes and the p l a t e l e t poor plasma (PPP) was removed. The p l a t e l e t s were resuspended and washed twice i n modified pH 6.5 Tyrode's s o l u t i o n . P l a t e l e t s were f i n a l l y resuspended i n Tyrode's s o l u t i o n pH 7.35 p r i o r to f i x a t i o n , and resuspended i n upper phase o f a two-phase system, p r i o r to p a r t i t i o n . 2.5 P l a t e l e t Aggregation An Aggregation and Shape Change Monitor, Born/Michal, was used with a Thermomix 1420, B. Braun, Melsungen AG, W Germany. For aggregation g s t u d i e s ( T r i p l e t t et a l . , 1978) a co n c e n t r a t i o n of 0.5 to 1 x 10 p l a t e l e t s / m l was used. P l a t e l e t s were washed and resuspended i n Tyrode's. An a l i q u o t of 0.8-1.0 ml sample was incubated at 37°C f o r ten seconds i n the cuvette holder with s t i r bar, a f t e r which ADP - 44 -(adenosine diphosphate, MW 427.2, Sigma Chemical Co.) was added. ADP _2 was made as a stock s o l u t i o n o f 10 M which was d i l u t e d f o r f i n a l c o n c e n t r a t i o n s ranging from 0.06 t o 25 x 10~ 6 M. Aggregation was used as a c o n t r o l to examine to f u n c t i o n and v i a b i l i t y of p l a t e l e t s a f t e r washing procedures p r i o r to f i x a t i o n and/or surface property analyses by p a r t i t i o n . 2.6 P l a t e l e t F i x a t i o n P l a t e l e t f i x a t i o n by a m o d i f i c a t i o n o f the method of Walter et a l . (1973c) i n v o l v e d the a d d i t i o n of a p l a t e l e t suspension (or packed e r y t h r o c y t e s ) i n t o ten volumes of 1.85% glutaraldehyde (obtained i n 2 ml v i a l s at 70%, Ladd Research I n d u s t r i e s , obtained from Ingram and B e l l , Vancouver B.C.) i n 300 mOsM sodium phosphate b u f f e r pH 7.4 (composed from phase system s a l t stock s o l u t i o n s ) , with mixing. The pH was checked and adjusted to pH 7.2-7.4 each day but t h i s was u s u a l l y not necessary. When p l a t e l e t s were f i x e d , albumin was omitted from the l a s t washing and resuspension s o l u t i o n . P l a t e l e t s were f i n a l l y resuspended i n Tyrode's with glutaraldehyde. 2.7 P l a t e l e t L a b e l i n g P l a t e l e t s concentrated i n an t i c o a g u l a t e d plasma (PRP), were l a b e l e d (or double-labeled) i n v i t r o by i n c u b a t i o n f o r 15-60 minutes i n a water bath at 37°C, with the various ( 5 l C r , m i n , 1 A C - and "^H-serotonin, "^H-adenine; see Experimental S e c t i o n s w i t h i n Results and D i s c u s s i o n ) r a d i o a c t i v e n u c l i d e ( s ) , and washed ( u s u a l l y twice was s u f f i c i e n t ) u n t i l f r e e l a b e l was n e g l i g i b l e , p r i o r t o f u r t h e r analyses (e.g., i n v i v o i n j e c t i o n of " ^ C r - l a b e l e d p l a t e l e t s f o r s u r v i v a l a n a l y s i s , and/or CCD a n a l y s i s o f l a b e l e d p l a t e l e t s u r f a c e p r o p e r t i e s ) , except when d e n s i t y gradient separation of l a b e l e d p l a t e l e t s was to - 45 -precede these analyses. P l a t e l e t s were a l s o l a b e l e d i n v i v o by 35 i n j e c t i o n of a s i n g l e cohort l a b e l S-sulphate (1-2.5 mCi). The time course of i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o p l a t e l e t s , i . e . , the movement of l a b e l e d p l a t e l e t s i n t o and out of the c i r c u l a t i n g p l a t e l e t pool was f o l l o w e d , i n order to determine the age c h a r a c t e r i s t i c s of the l a b e l e d p l a t e l e t p o p u l a t i o n at any given time. I f the cohort was to be compared to a e n t i r e l a b e l e d p l a t e l e t p o p u l a t i o n , a second i n v i t r o l a b e l i n g procedure ( u s u a l l y w i t h ^ H - s e r o t o n i n ) was used. 2.8.1 S t r a c t a n I I Stock S o l u t i o n A 40% s o l u t i o n of S t r a c t a n I I (StR; arabinogalactan; research grade, S t . Regis Paper Co., Tacoma, WA) was d e i o n i z e d using an equal volume of Amberlite MB-3 (BDH Chemicals L t d . , Poole, England), with continuous mixing at 4-5°C; repeating u n t i l no change i n the r e s i n was observed ( u s u a l l y t w i c e ) . The S t r a c t a n c o n c e n t r a t i o n was measured by refractometry and c a l c u l a t e d assuming the same r e f r a c t i v e index as sucrose; the osmolarity of the d e i o n i z e d S t r a c t a n stock s o l u t i o n and v a r i o u s d i l u t i o n s of t h i s stock was checked by f r e e z i n g p o i n t depression analyses (Appendix Table A . l ) . Considering the p a r t i a l s p e c i f i c volume of S t r a c t a n (Corash et a l . , 1974), s u f f i c i e n t Tyrode's s a l t s , sodium c h l o r i d e , and water were added to the o r i g i n a l d e i o n i z e d s o l u t i o n to make an isoosmotic stock s o l u t i o n of 20% S t r a c t a n i n Tyrode's s o l u t i o n ( C i e s l a r et al., 1979). The c o n c e n t r a t i o n , osmolarity and d e n s i t y of t h i s f i n a l s t o c k , were re-checked (Appendix Table A.2), and the gradient steps were d i l u t e d i n pH 6.5 Tyrode's s o l u t i o n . The t o n i c i t y of f i n a l i s o t o n i c S t r a c t a n stock s o l u t i o n s was examined by observation of the volume of packed e r y t h r o c y t e s i n S t r a c t a n , i n comparison to t h a t i n other known i s o t o n i c media. Equal - 46 -volumes (0.2 ml) of washed packed human e r y t h r o c y t e s i n the v a r i o u s s o l u t i o n s were added t o m i c r o c a p i l l a r y tubes, c e n t r i f u g e d with an IEC MB microhematocrit c e n t r i f u g e , and the hematocrit was read with a m i c r o c a p i l l a r y reader (Damon/IEC D i v i s i o n , Needham Heights, MA). T y p i c a l r e s u l t s were: 43.3 + 0.3, 41.3 + 0.3, 42.7 + 0.3, 42.2 + 0.3, 42.3 +_ 0.3, 41.8 +_ 0.1 (average of three measurements each) f o r Tyrode's s o l u t i o n , s a l i n e , plasma, and 10%, 14%, and 18% StR, r e s p e c t i v e l y . To observe the s e n s i t i v i t y range of these e r y t h r o c y t e volume, or hematocrit osmotic pressure, measurements the hematocrit of washed r a b b i t e r y t h r o c y t e s was measured i n va r i o u s known concentrations of s a l t , (using a s t a r t i n g stock s o l u t i o n of 4 x i s o t o n i c , 0.616 M NaCl, 1160 mOsM, from which d i l u t i o n s were made) corresponding to va r i o u s t o n i c i t i e s (Appendix Table A.3). The hematocrit was measured immediately a f t e r resuspension of e r y t h r o c y t e s , s i n c e a f t e r 10 minutes i n c e r t a i n samples the hematocrit was observed to change, presumeably due to l y s i s at low s a l t c o n c e n t r a t i o n . 2.8.2 S t r a c t a n Density Gradient In order t o s e l e c t the appr o p r i a t e gradient step d e n s i t i e s which provide the d e s i r e d p l a t e l e t p o p u l a t i o n s u b f r a c t i o n a t i o n , the p l a t e l e t d e n s i t y d i s t r i b u t i o n s were e m p e r i c a l l y .determined by c e n t r i f u g a t i o n of PRP on s i n g l e steps of S t r a c t a n gradient s o l u t i o n at va r i o u s c o n c e n t r a t i o n s ranging from 10-20%, i n increments o f 0.5% S t r a c t a n . The p l a t e l e t s recovered on each of the steps were determined as a percentage o f the t o t a l p l a t e l e t s , and ta b u l a t e d w i t h the S t r a c t a n c o n c e n t r a t i o n . For example (Appendix Table A.4), human p l a t e l e t s i n PRP, from blood taken i n sodium c i t r a t e (11/23,29) or EDTA (11/27) were - A7 -l a y e r e d i n 1-2 ml volumes on s i n g l e steps of S t r a c t a n of v a r i o u s c o n c e n t r a t i o n s and c e n t r i f u g e d at 4000-6000 g f o r one hour at room temperature (11/23,27) and at 5-10°C (11/29), i n a S o r v a l l RC5. From such p l a t e l e t d e n s i t y determinations and by reference to the work of others (Corash et a l . , 1974, 1977, and 1978; C i e s l a r et a l . , 1979; Appendix Table A.5), a four step gradient was designed t o harvest the extremes of the p l a t e l e t d ensity d i s t r i b u t i o n by i s o l a t i n g subpopulations of low d e n s i t y (5-10%, LDP), intermediate d e n s i t y (80-90%, IDP) and high d e n s t i y p l a t e l e t s (5-10%, HDP). The gradient step volumes and ranges of S t r a c t a n content, a c t u a l l y used f o r d e n s i t y gradient c e n t r i f u g a t i o n were: 1 ml of 10%, 3 ml of 14.5-16%, 3 ml of 17.5-18%, and 3 ml of 20%. These are f u r t h e r d e s c r i b e d i n the Experimental S e c t i o n s of Chapter 3. The concentrations of the intermediate steps were v a r i e d with the p l a t e l e t sample and s p e c i e s . The d e n s i t y of these gradient steps (Table 2.8.2.1) were measured at room temperature by weighing a measured volume of S t r a c t a n (*), or were c a l c u l a t e d (**) from the s p e c i f i c g r a v i t y o f S t r a c t a n ( S t . Regis Paper Co.); and compared t o values of others (Corash et a l . 1974, 1977 and 1978). Table 2.8.2.1 S p e c i f i c G r a v i t y and Density of S t r a c t a n StR Cone (%) D e n s i t y * (g/ml) StR Cone (%) Density**(g/ml) 0.00 0.9475 0.00 0.995 8.68 1.000 13.5 1.055 10.00 1.010 15.0 1.062 20.00 1.072 16.0 1.066 21.18 1.080 17.0 1.071 30.00 1.140 18.0 1.075 33.33 1.160 20.0 1.084 - 48 -PRP obtained from 30-40 ml of blood was l a y e r e d on four to e i g h t g r a d i e n t s , and p l a t e l e t s were separated by c e n t r i f u g a t i o n at 4000 g f o r 1-2 hours at 20-25°C, on i s o s m o t i c d e n s i t y s t e p - g r a d i e n t s i s o l a t i n g d i f f e r e n t p l a t e l e t d e n s i t i e s LDP, IDP and HDP. They were p i p e t t e d from the g r a d i e n t step i n t e r f a c e s , d i l u t e d with an equal volume of Tyrode's (pH 6.5) s o l u t i o n , washed (by c e n t r i f u g a t i o n at 2600 g f o r 60 minutes), and sampled or resuspended f o r f u r t h e r analyses: microscopic examination, p l a t e l e t counting and/or s i z i n g , r a d i o a c t i v i t y counting, s u r f a c e property s t u d i e s by p a r t i t i o n i n two-polymer aqueous phase systems and/or e l e c t r o p h o r e t i c m o b i l i t y . 2.9.1 Phase System Polymer Stock S o l u t i o n s Dextran T500 (D, 500,000 d a l t o n s , Lot. 7830 and Lot. FD16027, Pharmacia, Uppsala, Sweden) stock s o l u t i o n s of approximately 20% (w/w) were composed by the a d d i t i o n of dextran to d e i o n i z e d water (22 g dextran per 100 g t o t a l ) . Dextran concentrations were checked by p o l a r i m e t r y (Dr. Steeg and Reuter P o l a r i m e t e r , GmbH, SR6 4731; 100 mm path l e n g t h ) , at a d i l u t i o n of 4 g (20% dextran stock) t o 50 g water, and the phase system r e c i p e s were adjusted a c c o r d i n g l y to provide the a p p r o p r i a t e f i n a l dextran c o n c e n t r a t i o n i n the phase system. I n i t i a l l y a conversion f a c t o r of 6.2814 was used to convert the p o l a r i m e t e r reading (range 3.0-3.5) t o percent (w/w) dextran; however, t h i s c o n v e r s i o n f a c t o r neglected the d e n s i t y of dextran (approximately 1.08 gm/ml f o r a 20% s o l u t i o n at 20°C). A new dextran conversion f a c t o r 6.256 was derived c o n s i d e r i n g t h i s d e n s i t y . Stock s o l u t i o n s of 30% (w/w) Polyethylene g l y c o l (PEG), Carbowax 6000 renamed 8000 (6,000-8,000 d a l t o n s , Union Carbide, Pitscathaway, NJ), were composed by a d d i t i o n of 150 g PEG to a f i n a l of 500 g water. - 49 -Because these polymers, dextran and PEG, were r e l a t i v e l y i o n f r e e when obtained from the s u p p l i e r s , t h e i r c o n t r i b u t i o n to the t o n i c i t y o f a s o l u t i o n was s m a l l at low c o n c e n t r a t i o n s . In the case of dextran of 200,000-275,000 d a l t o n s , the osmotic pressure at a given c o n c e n t r a t i o n was c a l c u l a t e d from data of LeNeveu et a l . (1977). As c a l c u l a t e d , the t o n i c i t y c o n t r i b u t i o n of dextran (200,000-275,000 d a l t o n s ) was s i m i l a r to t h a t f o r dextran (500,000 d a l t o n s ) when measured by f r e e z i n g p o i n t . When compared w i t h i n the c o n c e n t r a t i o n ranges u t i l i z e d , the t o n i c i t y of d e i o n i z e d S t r a c t a n I I (approximately 30,000 d a l t o n s ) , was only s l i g h t l y higher than dextran (Appendix Table A.6). 2.9.2 Phase System S a l t Stock S o l u t i o n s Concentrated sodium c h l o r i d e ( u s u a l l y 0.6 M, four times i s o t o n i c ) was composed by weight by the a d d i t i o n of 17.53 g of NaCl to water f o r a t o t a l of 500 g of s o l u t i o n . Concentrated sodium phosphate b u f f e r (NaPB) pH 7.2 (0.218 M Na 2HP0 A and 0.069 M Nah^PO^, twice i s o t o n i c ) was composed by the a d d i t i o n of 21.67 g of Na 2HP0 4 and 5.8 g of NaH 2P0^ t o water f o r a t o t a l of 700 g (mono- and dibasic-sodium phosphate, Sigma Chemical Co., St. L o u i s , MO). S o l u t i o n s were made by weight and as they were approximations to molar c o n c e n t r a t i o n s , they are r e f e r r e d to as such. 2.9.3 Two-polymer Aqueous Phase System Composition Phase systems were composed by weight from stock s o l u t i o n s of 20% (w/w) dextran, 30% (w/w) polyethylene g l y c o l , and concentrated s a l t s o l u t i o n s . Using a dextran stock . s o l u t i o n of 21.73% (w/w), 50 g phase systems were prepared, each c o n s i s t i n g of the polymer c o n c e n t r a t i o n s , 5% D and 4% PEG, and with s a l t composition d e s i g n a t i o n s A through G (5/4:A through 5/4:G, r e s p e c t i v e l y ) , as designed by J.M. Van A l s t i n e - 50 -and D.E. Brooks (Appendix Table A.7), a f t e r the method of H. Walter. The f i n a l c o n c e n t r a t i o n s and s a l t r a t i o s (NaPB/NaCl; with 2x stock s a l t s o l u t i o n s ) o f these phase systems are as l i s t e d i n Table 2.9.3.1 (e.g., phase system 5/4:A; 5% D, 4% PEG, 0.109 M Na 2HP0 4, 0.0345 M NaHLPO., and a s a l t r a t i o o f 10/0). Table 2.9.3.1 Two-Polymer Phase System S a l t Concentrations and R a t i o s 5/4: A B C D E F G Na 2HP04 (M) 0.109 0.0910 0.073 0.055 0.037 0 .021 0.0095 NaH 2P04 (M) 0.0345 0.0288 0.023 0.0174 0.012 0 .0067 0.003 NaCl (M) 0.0 0.025 0.0498 0.075 0.099 0 .125 0.15 NaPB/NaCl 10/0 8.4/1.7 6.7/3.3 5.1/5.0 3.4/6.7 1 .9/8.3 0.9/10 The p o t e n t i a l d i f f e r e n c e s between the upper and lower phases of the two-polymer phase systems ( i . e . , of constant polymer contents, 5% D/4% PEG, but with varying b u f f e r and s a l t ; decreasing sodium phosphate c o n c e n t r a t i o n and i n c r e a s i n g sodium c h l o r i d e c o n c e n t r a t i o n , from Designation A through G) were measured using s a l t b r i d g e s , a g a r - f i l l e d m i c r o c a p i l l a r y p i p e t s , Ag-AgCl e l e c t r o d e s and a high impedance m i l l i v o l t meter (Reitherman et a l . , 1973). The p o t e n t i a l d i f f e r e n c e (mV) between the upper and lower phases was found t o decrease with decreasing phosphate and i n c r e a s i n g c h l o r i d e (Table 2.9.3.2). Table 2.9.3.2 Phase System E l e c t r o s t a t i c P o t e n t i a l D i f f e r e n c e s 5/4: A B C D E F G P.D. (mV) 1.0656 0.9831 0.9420 0.6010 0.4767 0.2784 0.1756 + S.Dev. 0.2103 0.1519 0.2528 0.0622 0.0655 0.0438 0.0327 N 8 14 9 12 14 12 9 - 51 -An a l t e r n a t e r e c i p e f o r phase system composition was a l s o used (Appendix Table A.8). This phase system r e c i p e was f o r composition by " s a l t r a t i o " , with twice i s o t o n i c s a l t s o l u t i o n s . I t was of advantage when preparing s e v e r a l phase systems, d i f f e r i n g only i n the r a t i o o f sodium phosphate to sodium c h l o r i d e , using a s i n g l e 2x polymer mixture of D and PEG (e.g., 10% and 8% (w/w), r e s p e c t i v e l y ) . The phase system volume, was completed with water. 2.10 S i n g l e Step P a r t i t i o n S i n g l e tube p a r t i t i o n experiments were performed e s s e n t i a l l y as p r e v i o u s l y described (Walter, et a l . 1969a and 1969b; Grant and Zucker, 1978) and modified f o r s m a l l samples (e.g., o f f i x e d or l a b e l e d p l a t e l e t s , and i n c e r t a i n cases e r y t h r o c y t e s ) . P l a t e l e t samples (20 u l , 1 0 7 p l a t e l e t s ) , suspended i n Tyrode's s o l u t i o n , are added to phase systems (0.5-1.5 ml upper- and 0.5-1.5 ml lower phase). The phases were mixed by i n v e r s i o n twenty times, and allowed to s e t t l e f o r 0.5-1.0 hour, a f t e r which the upper phases were sampled (0.3-0.5 ml) and counted f o r p a r t i c l e s and/or r a d i o a c t i v i t y . The q u a n t i t i e s of p a r t i c l e s (or r a d i o a c t i v i t y ) which had p a r t i t i o n e d i n t o the upper phases were compared to the t o t a l added (as a percent) and thus the p a r t i t i o n c o e f f i c i e n t s (K) were c a l c u l a t e d . A v a r i e t y of systems were examined i n t h i s manner f o r p l a t e l e t p a r t i t i o n . 2.11.1 Countercurrent D i s t r i b u t i o n M u l t i p l e p a r t i t i o n steps by countercurrent d i s t r i b u t i o n (CCD) were c a r r i e d out using a CCD-1200 apparatus (Buchler Instruments D i v i s i o n , Nuclear Chicago Corp., NJ). I t c o n s i s t e d of two c i r c u l a r p l e x i g l a s p l a t e s (CCD 1250C, 71014), s e a l e d with s i l i c o n e l u b r i c a n t (Dow Corning Corp., Midland MI), with 120 c a v i t i e s (CCDC) arranged around the - 52 -periphery o f both the top and bottom p l a t e s . These p l a t e s were mounted on a mechanical, e l e c t r o n i c a l l y c o n t r o l l e d base, which f a c i l i t a t e d timed shaking, s e t t l i n g and the r o t a t i o n o f the top p l a t e (which contained the upper phase) with respect to the bottom. The automatic machine timing c o n t r o l was checked with a stopwatch p e r i o d i c a l l y and with each change of c o n t r o l . For these experiments, a l a r g e volume (250-300 ml) of phase system was mixed at 5-7°C, f i l t e r e d (Nalgene F i l t e r U n i t , 0.45 micron, Nalge Company, D i v i s i o n of Sybron/Nalge Corporation, Rochester, NY), allowed t o s e t t l e overnight i n a separatory f u n n e l , and the upper PEG-rich and lower D-rich phases were then c o l l e c t e d s e p a r a t e l y . The p l a t e l e t samples to be p a r t i t i o n e d by CCD were suspended i n upper phase, and 0.9 ml of these mixtures were loaded (using a B-D Cornwall p i p e t o r , Becton-Dickinson Co.) along with bottom phase (0.4 ml) i n t o each o f a few CCDC ( l e s s than 10% of the t o t a l t r a n s f e r number; 3 CCDC loaded f o r 40 t r a n s f e r s , 4 CCDC f o r 60 t r a n s f e r s and 6 CCDC f o r 120 t r a n s f e r s ) . Upper phase (0.8 ml), and lower phase (0.5 ml) were loaded i n t o the remaining CCDC. This provided f o r at l e a s t 0.1 ml of s t a t i o n a r y lower phase. F i r s t , a l l lower phase was loaded, then a l l upper phase, and l a s t l y the samples i n upper phase (load m i x t u r e s ) . Before each CCD run began, the phases w i t h i n a l l c a v i t i e s were mixed by one minute of shaking on manual c o n t r o l . Then, the machine was switched to automatic which i n i t i a t e d a preset t h i r t y second shaking c y c l e , a seven minute s e t t l i n g c y c l e followed by the r o t a t i o n of the top p l a t e three degrees with respect to the bottom p l a t e . In t h i s manner the p l a t e l e t sample(s) were exposed to a number of p a r t i t i o n steps by r e p e t i t i o n of t h i s c y c l e . - 53 -In order to determine i f the type of blood c e l l sample to be s t u d i e d was being separated by the phase system p r o p e r t i e s , and not j u s t randomly d i s t r i b u t e d , a r e - p a r t i t i o n experiment was c a r r i e d out. An experiment of t h i s s o r t r e q u i r e d the exposure o f c e l l s to CCD c o n d i t i o n s t w i c e , and s i n c e w i t h time of storage c e l l s were known to change t h e i r surface p r o p e r t i e s as r e f l e c t e d by p a r t i t i o n , i t was necessary t o use glutaraldehyde f i x e d c e l l s even though f i x e d c e l l s might have demonstrably d i f f e r e n t membrane surface p r o p e r t i e s from u n f i x e d c e l l s . In t h i s case, f i x e d p l a t e l e t s were d i s t r i b u t e d over 116 t r a n s f e r s by CCD. P l a t e l e t s , pooled from adjacent CCDC i n t o CCD f r a c t i o n s (CCDF) obtained from d i f f e r e n t areas ( l e f t , middle, and r i g h t ) o f the d i s t r i b u t i o n , r e - p a r t i t i o n e d by a second CCD t o the same area from which they were s e l e c t e d (see Chapter 3.1). 2.11.2 CCD of Density Separated P l a t e l e t s The CCD u n i t was loaded w i t h both phases and the three d e n s i t y -separated p l a t e l e t p o p u l a t i o n s (LDP, IDP and HDP) were separately loaded i n CCDC 1-3, 41-43, and 81-83, r e s p e c t i v e l y . The CCD procedures, i n v o l v i n g 37-40 c y c l e s of mixing, s e t t l i n g and a t r a n s f e r were completed w i t h i n about f i v e hours at 5-7°C. Upon completion, 0.7 ml of Tyrode's washing s o l u t i o n was added to each CCDC and the phases were c o l l e c t e d and pooled by (2-4) adjacent CCDC, to comprise 10-20 CCDF. These CCDF were sampled, counted f o r p l a t e l e t s , and the d i s t r i b u t i o n s f o r each of the LDP, IDP, and HDP were p l o t t e d , normalized as a percent of the peak p l a t e l e t CCDF. The g r a p h i c a l data was then c h a r a c t e r i z e d by the d i s t r i b u t i o n parameters Peak, Mode, Middle (and/or Median), spread and/or standard d e v i a t i o n (see Chapter 3.2). - 54 -2.11.3 CCD of In V i t r o Labeled P l a t e l e t s Aged In Vivo P l a t e l e t s from 30 ml of r a b b i t blood were concentrated by c e n t r i f u g a t i o n and l a b e l e d i n 5 ml of plasma by the a d d i t i o n o f 0.54 mC 51 of Cr as sodium chromate (ICN, I r v i n e , CA) and i n c u b a t i o n f o r 30-60 minutes at 37°C (about 1 mCi/10 ml of PRP). Next, 0.4 ml of a s c o r b i c a c i d (250 mg/ml; not always used) i n Tyrode's s o l u t i o n was added to the i n c u b a t i o n mixture, and samples were taken f o r r a d i o a c t i v i t y and p l a t e l e t counts. The 5 1 C r - l a b e l e d p l a t e l e t s were washed twice i n PPP, resuspended i n a small volume o f PPP, counted a second time f o r r a d i o a c t i v i t y and p l a t e l e t s , and i n j e c t e d ( i n c e r t a i n i n s tances with washed e r y t h r o c y t e s from the sample) i n t o the r a b b i t from which they came. For p a r t i t i o n s t u d i e s not i n v o l v i n g r e - i n j e c t i o n of the p l a t e l e t sample, the p l a t e l e t s were washed as above but i n Tyrode's s o l u t i o n , and resuspended i n the upper phase. Blood samples were taken at v a r i o u s times post i n j e c t i o n ( p . i . ) of the l a b e l e d p l a t e l e t sample. These aged, l a b e l e d , p l a t e l e t samples were harvested, washed, and s u b f r a c t i o n a t e d by CCD. The d i s t r i b u t i o n s were counted f o r p l a t e l e t s and r a d i o a c t i v i t y , and the s p e c i f i c a c t i v i t i e s (CPM/platelet) were c a l c u l a t e d f o r each p o i n t (CCDF) of the d i s t r i b u t i o n . 2.11.4 CCD of In Vivo Cohort Labeled P l a t e l e t s 35 S u l f u r i c a c i d , H 2 SO^, i n water was obtained from New England Nuclear, Boston, MA (NEX-042, 15 mCi/ml, 43 Ci/mg, 46.5 x 10~ 6 moles/Ci). Lot 094394 was d i l u t e d to 7.5 ml f o r 2 mCi/ml; l o t 239419 was d i l u t e d to to 6.0 ml f o r 2.5 mCi/ml; and l o t 307321 was not d i l u t e d . These r a d i o a c t i v e compounds were used as i n v i v o p l a t e l e t cohort l a b e l s and were i n j e c t e d d i r e c t l y (or s l i g h t l y d i l u t e d ) i n t o r a b b i t s . P l a t e l e t s , at v a r i o u s times post i n j e c t i o n , p . i . , were d e n s i t y - 55 -separated and subjected t o CCD. D e t a i l s are provided as Experimental i n s e c t i o n s 3.8 through 3.12. 2.12.1 P a r t i c l e Counting An Electrozone C e l l o s c o p e , (Model 112 CLTH/RWP, P a r t i c l e Data Inc., Elmhurst, IL) was used. I n i t i a l l y p l a t e l e t s had been counted on the " P l a t e l e t Mode", which provided preset c u r r e n t ( C), gain (G) and th r e s h o l d (T) s e t t i n g s s p e c i f i c a l l y designed f o r human p l a t e l e t s . L a t e r , appropriate s e t t i n g s f o r counting i n the " L i n / D e l t a Mode" were expe r i m e n t a l l y determined us i n g standard p a r t i c l e s , and r a b b i t and human f i x e d p l a t e l e t s . The s e t t i n g s used f o r p l a t e l e t s were then set by 1/2 the operator (C 2(2) , G 96, T 19-90, f o r a 76 um diameter o r i f i c e and C 1, G 68, T 15-90, f o r a 48 um diameter o r i f i c e ) . The L i n / D e l t a Mode d i s p l a y e d counts w i t h i n a s p e c i f i e d s i z e range, separate from those o f greater s i z e . The l i n e a r i t y and coincidence of counts with these s e t t i n g s , were s t u d i e d (Appendix Table A.9). P a r t i c l e s i n s o l u t i o n were d i l u t e d with a z i d e - f r e e s a l i n e " D i l u i d " (J.T. Baker D i a g n o s t i c s , Bethlehem, PA.) such t h a t coincidence was n e g l i g i b l e and e l e c t r o n i c counts c o r r e l a t e d with v i s u a l hemocytometer counts. A stock p l a t e l e t s o l u t i o n was added t o 9.84 ml of D i l u i d s a l i n e at v a r i o u s d i l u t i o n s which were c a l c u l a t e d from the o r i g i n a l stock s o l u t i o n . These samples were counted and compared both t o hemocytometer 8 counts using a phase c o n t r a s t microscope (1.05 _+ 0.1 x 10 o p l a t e l e t s / m l ) and to c a l c u l a t e d t h e o r e t i c a l counts (1.03 + 0.28 x 10 p l a t e l e t s / m l ) based on the p o r t i o n of counts w i t h i n the range of l i n e a r c o u n t i n g . Celloscope counts were performed using a 76 um o r i f i c e with a 1 /? 255 u l v o l u m e t r i c , on s e t t i n g s , C 2(2) , G 96, L i n / D e l t a Mode, T 10-90. With these counting c o n d i t i o n s , the counts were l i n e a r up to a - 56 -counting r a t e o f AO,000-50,000 counts. The Percent-Countless (Appendix Table A.9) was c a l c u l a t e d u s i n g the equation,% countless = 0.13 (N)(D)' 5; where, N = observed counts/ml; D = o r i f i c e diameter i n mm; under the above c o n d i t i o n s , 2.2A x I O - 4 = % C/(N/255 u l ) . 2.12.2 P a r t i c l e S i z i n g Volume c a l i b r a t i o n (Appendix Table A.10) f o r the Electrozone Celloscope was c a r r i e d out with standard p o l y s t y r e n e p a r t i c l e s ( P a r t i c l e Information S e r v i c e , Grants Pass, OR), and f i x e d blood c e l l s . Standard s p h e r i c a l p a r t i c l e s w i t h f i x e d diameters, 2.02 um ( p a r t i c l e mode at T = 32.5), 3.05 um ( p a r t i c l e mode at T = 65.0), f i x e d r a b b i t p l a t e l e t s ( p l a t e l e t mode at T = 25.0), and f i x e d human e r y t h r o c y t e s ( o f f s c a l e ) were s i z e d with the same machine s e t t i n g s but by varying the T s e t t i n g s from 0 t o 100, i n d i f f e r e n t increments. R e l a t i v e volume d i s t r i b u t i o n s were obtained by manually a l t e r i n g the T s e t t i n g s f o r s e q u e n t i a l counts, i n order t o accumulate counts o f p a r t i c l e s producing s u c c e s s i v e l y l a r g e r r e s i s t i v e p u l s e s . Since the c a l i b r a t i o n was c a r r i e d out w i t h s p h e r i c a l p a r t i c l e s and e l e c t r o n i c s i z i n g of t h i s type was a f u n c t i o n of both shape and volume, the r e s u l t s were expressed a r b i t r a r i l y i n terms o f percent t h r e s h o l d or percent window s e t t i n g s , as w e l l as by the apparent c a l c u l a t e d volumes. The expected volumes and diameters a t the va r i o u s t h r e s h o l d (T) s e t t i n g s were c a l c u l a t e d by the 3 3 equation, v = (pi/6) d = 1.25 um = K (T/IG); where v = volume; d = diameter; K = o r i f i c e constant ( f o r 76 um o r i f i c e , A2); T = t h r e s h o l d ; I = c u r r e n t ; G = g a i n . S i m i l a r l y , f i x e d human p l a t e l e t standards (Dade P l a t e l e t C o n t r o l s , s t a b i l i z e d human p l a t e l e t suspensions i n l e s s than 0.1% sodium a z i d e , Dade D i v i s i o n of American H o s p i t a l Supply C o r p o r a t i o n , Miami, F l . ) - 57 -were used t o c a l i b r a t e the instrument (Electrozone Celloscope) f o r counting, using a 48 um diameter o r i f i c e and a 100 u l v o l u m e t r i c , w i t h standard modes at 15, 25, and 55, f o r s e t t i n g s : constant G 68, and v a r i e d C 1/2, 1, and 2, r e s p e c t i v e l y . A l l experimental counts and s i z i n g s were performed, immediately, on Tyrode's washed blood c e l l s and elements washed once a f t e r S t r a c t a n d e n s i t y gradient c e n t r i f u g a t i o n and/or CCD, resuspended and d i l u t e d i n a z i d e - f r e e s a l i n e ( D i l u i d , J.T. Baker D i a g n o s t i c s , Bethlehem, PA). P a r t i c l e c o ncentrations were adjusted such t h a t c o i n c i d e n c e counts were n e g l i g i b l e and e l e c t r o n i c and hemocytometer counts c o i n c i d e d . P l a t e l e t s i n s o l u t i o n were " s i z e d " using the Electrozone C e l l o s c o p e , with 76 um diameter o r i f i c e by o b t a i n i n g counts at various t h r e s h o l d s e t t i n g s (T: 5-10, 10-15, 15-20, e t c . ) . These counts were p l o t t e d as a percentage o f the p l a t e l e t peak channel (T s e t t i n g ) , versus the T s e t t i n g s at which they were obtained. Both' standards and p l a t e l e t s were i n the ap p r o p r i a t e c a l c u l a t e d range (Appendix Table A.10); d e n s i t y separated p l a t e l e t s were s i z e d i n t h i s manner. A C o u l t e r Counter, Channelyzer and X,Y recorder ( C o u l t e r E l e c t r o n i c s Inc., H i a l e a h , FL) with a 70 um o r i f i c e and 100 u l v o l u m e t r i c , was a l s o used to s i z e p l a t e l e t s of d i f f e r e n t f r a c t i o n s a f t e r CCD (not shown) and t o s i z e f r a c t i o n s o f f i x e d p l a t e l e t s which were separated by ( s i z e ) sedimentation i n s a l i n e at u n i t g r a v i t y . In c e r t a i n i n s t a n c e s , e.g., d e n s i t y separated p l a t e l e t s and p l a t e l e t s from cancer p a t i e n t s were s i z e d w i t h an Ortho D i a g n o s t i c s l a s e r l i g h t s c a t t e r i n g c e l l counter (data were provided by C l i n i c a l Laboratory Medicine at H.S.C.H., Vancouver BC). - 58 -2.13 R a d i o a c t i v i t y Counting For beta s c i n t i l l a t i o n counting a Beckman LS-233 L i q u i d S c i n t i l l a t i o n counter, Beckman Instruments Inc., F u l l e r t o n , CA, S c i e n t i f i c Instruments D i v i s i o n , I r v i n e , CA; and a P h i l i p s PW4700 L i q u i d S c i n t i l l a t i o n Counter, P h i l i p s S c i e n t i f i c & I n d u s t r i a l Equipment D i v i s i o n , N.V. P h i l i p s ' Gloeilampenfabrieken, Eindhoven, Netherlands, were used. L i q u i d f l u o r was Atomlight NEN-968, NEN, Boston, MA. For gamma counting a microcomputer c o n t r o l l e d CompuGamma counter model 1282-002 gamma counter, LKB Wallac Oy, Turku, F i n l a n d was used. A Nuclear Chicago Model 4216, s i n g l e channel automatic gamma s c i n t i l l a t i o n 100 sample c a p a c i t y Model 1085 was a l s o used i n e a r l i e r experiments. 2.14 G r a p h i c a l P r e s e n t a t i o n of R e s u l t s A " S p e c i f i c A c t i v i t y " program run on a Hewlett Packard 9815A c a l c u l a t o r and 9872A p l o t t e r (Hewlett Packard, Vancouver, B.C.), developed by our l a b o r a t o r y (K.A. Sharp and J . Janzen) was used, which expressed r e s u l t s (unless otherwise stated) i n terms o f : platelets/CCDC ( s o l i d l i n e , l e f t o r d i n a t e ) ; r a d i o a c t i v i t y dpm or cpm/CCDC (dashed l i n e , r i g h t o r d i n a t e ) ; and s p e c i f i c a c t i v i t y (S.A./CCDC) or ((cpm/platelet)/CCDC), i . e . , the r a t i o (dotted l i n e , p l o t t e d e i t h e r with the mean S.A. at one h a l f the maximum i n s i d e the l e f t o r d i n a t e , or by n o r m a l i z i n g the peak S.A. at the maximum). The o r d i n a t e s were l a b e l e d with the power and the s i g n of the exponent (eg., E+06 f o r 1 0 6 ) . Data were accepted by p l a c i n g a l i m i t on combined counting e r r o r (CE, expressed as a percent) of s p e c i f i c a c t i v i t y , c a l c u l a t e d f o r each CCDC or CCDF, using the best estimate of the standard d e v i a t i o n , - 59 -i .e CE ( P L ) l / 2 + (PB)l/2 ( C P M ) 1 / 2 + ( C B ) 1 / 2 + 100 (PL - PB) (CPM - CB) where PL = p l a t e l e t count PB = p l a t e l e t count background CPM = p l a t e l e t cpm CB = cpm background 2.15 C e n t r i f u g a t i o n For p l a t e l e t i s o l a t i o n , c e n t r i f u g a t i o n s at room temperature were done using IEC Model SBV and IEC Model 2K, I n t e r n a t i o n a l Equipment Co., Boston, MA. High speed c e n t r i f u g a t i o n s at c o n t r o l l e d temperatures were done usi n g v a r i o u s r o t o r s (HB-4, r a d i u s 5.75 i n . , 14.61 cm, a 4-place swinging bucket r o t o r with 50 ml c a p a c i t y each, or 15 ml c a p a c i t y with No. 363 cushions) i n the S o r v a l l RC-5 Automatic Superspeed R e f r i g e r a t e d C e n t r i f u g e , Du Pont Co., Instrument Products, Biomedical D i v i s i o n , Newtown, CT. - 60 -CHAPTER 3 RESULTS AND DISCUSSION 3.1 S i n g l e Step P l a t e l e t P a r t i t i o n , and F e a s i b i l i t y of P l a t e l e t CCD Glutaraldehyde f i x e d r a b b i t p l a t e l e t s were p a r t i t i o n e d at 22-23°C, i n phase systems of 5% dextran, 4% PEG, with b u f f e r and s a l t s , d esignations 5/4: A through G (Chapter 2.9.3). P o t e n t i a l d i f f e r e n c e s , measured between the upper and lower phases (Table 2.9.3.2 and F i g u r e 3,1.1.1) decreased, as the p a r t i t i o n s of these f i x e d r a b b i t p l a t e l e t s (Figure 3.1.1.2; Table 3.1.1). 51 R a b b i t , human, and monkey p l a t e l e t s were l a b e l e d with Cr i n PRP at a c o n c e n t r a t i o n of 0.2 mCi/ml f o r 1 hour at 37°C. P l a t e l e t s were washed and a l s o p a r t i t i o n e d at 22-23°C and 4-5°C (CCD c o n d i t i o n s ) i n these phase systems (Table 3.1.1). S i n g l e tube p a r t i t i o n s , at 22-23°C, i n c h a r g e - s e n s i t i v e phase systems (5/4:A) showed that f i x e d r a b b i t p l a t e l e t s had a higher p a r t i t i o n than u n f i x e d , 5"*"Cr-labeled p l a t e l e t s , K = 85.9 + 14.9% and 31.8 + 8.4%, r e s p e c t i v e l y . Rabbit and human p l a t e l e t p a r t i t i o n s , a t 22-23°C, were very s i m i l a r (with human only s l i g h t l y higher than r a b b i t ) , while t h a t of monkey was much lower; K = 31.8 + 8.4%, 33.8 + 6.3%, and 21.1 + 3.4%, f o r r a b b i t , human, and monkey, r e s p e c t i v e l y . At 4-5°C, the (5/4:A) p a r t i t i o n s of r a b b i t and human p l a t e l e t s were higher than at 22-23°C, while the p a r t i t i o n of monkey p l a t e l e t s was lower. The low temperature p a r t i t i o n of monkey p l a t e l e t s was again lower than t h a t of both r a b b i t and human, however, at t h i s temperature the p a r t i t i o n of human p l a t e l e t s was lower than that of the r a b b i t ; K = 53.8 + 3.5%, 43.5 + 5.3%, and 11.4 + 1.6%, f o r r a b b i t , human, and monkey, r e s p e c t i v e l y . - 61 -Figure 3.1.1.1 P o t e n t i a l D i f f e r e n c e s Between Upper PEG and Lower dextran Phases: (means and S.Dev. i n Table 2.9.3.2); increments of 0.70 mV P.D. ( o r d i n a t e ) ; NaPB co n c e n t r a t i o n 0.14 M decreasing to 0.013 M ( a b s c i s s a ) ; NaCl c o n c e n t r a t i o n 0.00 M i n c r e a s i n g to 0.15 M ( a b s c i s s a ) ; phases 5/4:A through G. 1. 40- -1. SB--1. 12- • • 0_ a. ev Vj, B . 7 B + a-sa-0 . 4 2 TWO-PHASE P O T E N T I A L D I F F . IN 5 % D / 4 % P E G WITH V A R Y I N G NACL AND NAP a. 28 a. 14 108.0-Figure 3.1.1.2 Fixed Rabbit P l a t e l e t Two-Polymer Phase System P a r t i t i o n s : (means and S.Dev. i n Table 3.1.1); increments of 5% p a r t i t i o n ( o r d i n a t e ) ; NaPB c o n c e n t r a t i o n 0.14 M decreasing to 0.013 M ( a b s c i s s a ) ; NaCl c o n c e n t r a t i o n 0.00 M i n c r e a s i n g to 0.15 M ( a b s c i s s a ) ; phases 5/4:A through G. - 62 -Table 3.1.1 Summary of Sin g l e - t u b e P a r t i t i o n of P l a t e l e t s 5/4: A B Fixed Rabbit P l a t e l e t s at 25°C Mean 85.9 74.9 64.5 28.4 12.3 3.4 1.4 + S.Dev. 14.9 16.7 15.3 7.7 10.7 2.5 0.5 n 3 3 3 3 3 3 3 Rabbit P l a t e l e t s at 25°C Mean 31.75 31.0 21.75 13.5 15.75 9.0 9.75 + S.Dev. 8.42 2.58 4.35 0.58 6.65 1.15 0.5 n 4 4 4 4 4 4 4 Human P l a t e l e t s at 25°C Mean 33.75 30.75 24.38 18.0 18.13 17.5 14.4 + S.Dev. 6.27 2.49 3.07 2.83 6.10 5.42 5.31 n 8 8 8 4 8 8 5 Monkey P l a t e l e t s at 25°C Mean 21.13 17.6 15.2 12.8 10.1 11.0 9.5 + S.Dev. 3.44 4.6 2.9 3.3 2.5 2.45 1.72 n 8 10 10 10 10 8 10 Rabbit P l a t e l e t s at 5°C Mean 53.75 42.38 30.88 19.13 11.0 4.33 3.57 + S.Dev. 3.54 3.38 3.64 2.42 1.26 2.94 2.76 n 8 8 8 8 6 6 7 Human P l a t e l e t s at 5°C Mean 43.5 32.0 30.25 29.67 18.0 13.3 19.75 + S.Dev. 5.26 0.82(4) 2.06 3.21 0.0 0.58 0.5 n 4 4 4 3 2 3 4 Monkey P l a t e l e t s at 5°C Mean 11.38 9.75 5.0 4.5 5.88 5.75 4.63 + S.Dev. 1.6 1.49 0.53 1.05 1.55 2.55 2.2 n 8 8 8 6 8 8 8 - 63 -S u i t a b l e phase systems f o r p l a t e l e t p a r t i t i o n work contained: 5% D T500, 4% PEG 8,000, and d i f f e r e n t c o n c e n t r a t i o n s (or r a t i o s ) o f i s o t o n i c sodium c h l o r i d e and sodium phosphate b u f f e r s (pH 7.2). Contrary to the f a c t that ions (e.g., phosphates) induce an e l e c t r i c charge on macromolecules such as glycogen (Sieh and S t e r l i n g , 1969), these phase systems e x h i b i t p o t e n t i a l d i f f e r e n c e s due t o i o n r e j e c t i o n ; phosphate i s r e j e c t e d by the PEG phase more than by D-rich phase, r e s u l t i n g i n a d i f f e r e n c e i n phase i o n c o n c e n t r a t i o n (Bamberger et a l . , 1984; Brooks et a l . , 1971, 1976 and 1984). The p o t e n t i a l d i f f e r e n c e s between the two phases w i t h i n these i s o t o n i c b u f f e r e d polymer systems v a r i e d with the phosphate/chloride r a t i o , the p o t e n t i a l i n the top, PEG-rich phase being p o s i t i v e w i t h respect t o the bottom phase, and ranging i n magnitude from 1.0 mV, f o r a system of high phosphate content, to 0.1 mV f o r a system of high c h l o r i d e content (Figure 3.1.1.1). The p a r t i t i o n s of washed, f i x e d r a b b i t p l a t e l e t s and n a t i v e r a b b i t , monkey, and human p l a t e l e t s l i k e w i s e v a r i e d i n these systems, being highest ( l a r g e s t f r a c t i o n of t o t a l p l a t e l e t s i n the system appearing i n top phase) i n t h a t system with the highest phase p o t e n t i a l d i f f e r e n c e , and decreasing wi t h the p o t e n t i a l d i f f e r e n c e (Figure 3.1.1.1 and Table 3.1.1). The high phosphate c o n t a i n i n g , charge s e n s i t i v e phase system was most o f t e n used throughout these CCD s t u d i e s . Species s p e c i f i c d i f f e r e n c e s i n p l a t e l e t membrane surface charge-associated p a r t i t i o n s were found i n the r a b b i t , human and monkey. The p a r t i t i o n s o f r a b b i t and human p l a t e l e t s were s i m i l a r , whereas that of monkey p l a t e l e t s was very d i f f e r e n t . These p l a t e l e t p a r t i t i o n s changed d i f f e r e n t l y with temperature; r a b b i t and human i n c r e a s i n g , and monkey decreasing with decreasing temperature from - 64 -25°C to 5°C, but the r e l a t i v e d i f f e r e n c e s , r a b b i t being s i m i l a r to human, and higher than monkey, were maintained at e i t h e r temperature. Although a l l three experimental systems, r a b b i t s , monkeys, and humans, were used f o r comparison of the r e l a t i v e s urface property d i s t r i b u t i o n s of p l a t e l e t s of d i f f e r e n t d e n s i t i e s ( S e c t i o n 3.2), the r a b b i t was used here to demonstrate the f e a s i b i l i t y of p l a t e l e t surface property analyses by CCD, and was l a t e r used i n t h i s t h e s i s as the major model system f o r i n v i v o ageing s t u d i e s . For most of these analyses of p l a t e l e t membrane sur f a c e p r o p e r t i e s , n a t i v e p l a t e l e t s were used, however f o r t h i s CCD f e a s i b i l i t y study i t was necessary to use f i x e d r a b b i t p l a t e l e t s to prevent changes i n membrane surface p r o p e r t i e s which might occur over the 72 hours of the experiment. I t has been shown with e r y t h r o c y t e s that glutaraldehyde f i x a t i o n does not o b l i t e r a t e age-dependent d i s t r i b u t i o n v i a CCD. However i n c e r t a i n i n s t a n c e s a l t e r n a t e f i n d i n g s are reported, and i n these s t u d i e s the charge-associated p l a t e l e t p a r t i t i o n s increased upon p l a t e l e t f i x a t i o n . F i x e d r a b b i t p l a t e l e t s were d i s t r i b u t e d over 120 CCDC, and a broad d i s t r i b u t i o n was obtained (Figure 3.1.2.1). Samples from three areas of the CCD (low, i n t e r m e d i a t e , and high p a r t i t i o n ; d otted, s o l i d and dashed l i n e s , r e s p e c t i v e l y , see Figure 3.1.2.1) were then simultaneously subjected to a second CCD of 40 t r a n s f e r s each, loaded on three s e c t o r s of the CCD p l a t e . These samples r e - p a r t i t i o n e d to the same r e l a t i v e areas from where they had been taken from the f i r s t CCD, as expected, assuming the o r i g i n a l p l a t e l e t d i s t r i b u t i o n had been on the b a s i s of i n t r i n s i c c e l l s u rface p r o p e r t i e s , and not the r e s u l t a n t of p r i m a r i l y random f a c t o r s (Figure 3.1.2.2). However, the c e r t a i n amount of d i s t r i b u t i o n overlap i s as expected. - 65 -Figure 3.1.2.1 CCD of Fixed Rabbit P l a t e l e t s : p l a t e l e t s % peak /CCDC ( s o l i d l i n e , o r d i n a t e ) ; CCDC 1-120 a b s c i s s a . Figure 3.1.2.2 R e d i s t r i b u t i o n o f P l a t e l e t F r a c t i o n s Taken from Above CCD: f i x e d p l a t e l e t s % peak, o f low, intermediate, and high p a r t i t i o n , ( dotted, s o l i d , and dashed l i n e , o r d i n a t e ) ; CCDC 1-40 a b s c i s s a . - 66 -3.2 Countercurrent D i s t r i b u t i o n o f Density Separated P l a t e l e t s E f f o r t s of groups headed by Corash and by Packham and Mustard have been d i r e c t e d toward o b t a i n i n g p l a t e l e t age d i s t r i b u t i o n i n f o r m a t i o n based on p l a t e l e t s i z e and d e n s i t y , the suggestion being t h a t young p l a t e l e t s are l a r g e r , more dense and a c t i v e than those p l a t e l e t s which have become e f f e t e w i t h i n the c i r c u l a t i o n . In the most compelling of such s t u d i e s (Corash et a l . , 1978), evidence i s presented that i n the 51 Rhesus monkey, C r - l a b e l e d low density p l a t e l e t s disappear from the c i r c u l a t i o n more r a p i d l y than the population as a whole; high d e n s i t y p l a t e l e t s , on the other hand, decrease i n d e n s i t y w i t h time i n v i v o , and s u r v i v e longer than the mean f o r the p o p u l a t i o n . Support f o r the above hypothesis comes from s i m i l a r work i n r a b b i t s (Rand et a l . , 1981a, 1983; and Corash and Shafer, 1982). Using 3 3 S i n v i v o p l a t e l e t cohort l a b e l i n g , the most dense p l a t e l e t subpopulation contained young p l a t e l e t s while the l e a s t dense subpopulation contained o l d p l a t e l e t s , the amount of s i a l i c a c i d per p l a t e l e t decreasing with age and d e n s i t y (although the amount of s i a l i c a c i d per- gram of p l a t e l e t p r o t e i n remains roughly c o n s t a n t ) . P l a t e l e t s were a l s o found t o be heterogeneous due to degree of thrombosis, evidenced i n a decrease i n t h e i r d e n s i t y i n the r a b b i t ( C i e s l a r et a l ^ . , 1979) and the human (van Oost et a l . , 1983), which could occur as a f u n c t i o n of c i r c u l a t i o n age as w e l l . However, other groups have developed d i f f e r e n t i n t e r p r e t a t i o n s of p l a t e l e t s i z e and d e n s i t y changes r e l a t e d t o human p l a t e l e t ageing (Leone et a l . , 1979; Mezzano et a l . , 1981; Boneu et a l . , 1982). In order to o b t a i n i n f o r m a t i o n concerning the heterogeneity of p l a t e l e t membrane surface p r o p e r t i e s , and r e l a t i v e c h a r g e - s e n s i t i v e p a r t i t i o n s of p l a t e l e t s of d i f f e r e n t age-related c h a r a c t e r i s t i c s , t h i s - 67 -s e r i e s of CCD experiments took advantage of reported c o r r e l a t i o n s between p l a t e l e t age and d e n s i t y . The methods of St r a c t a n d e n s i t y g r a d i e n t p r e p a r a t i o n of C i e s l a r et a l . (1979), modified from the method f o r e r y t h r o c y t e s by Corash et a l . (1974) and f o r p l a t e l e t s by Corash et a l . , (1977 and 1978) were modified to s u b f r a c t i o n a t e p l a t e l e t p o p u l a t i o n s from r a b b i t s , monkeys and humans, i n t o three s u b f r a c t i o n s c o n t a i n i n g 5-10% of the p l a t e l e t s of low density (LDP), 80-90% of the p l a t e l e t s of intermediate d e n s i t y (IDP), and 5-10% of the high d e n s i t y p l a t e l e t s (HDP). The d e n s i t i e s of the i n d i v i d u a l S t r a c t a n steps f o r gr a d i e n t c e n t r i f u g a t i o n were determined e m p i r i c a l l y by p l a t e l e t p o p u l a t i o n d e n s i t y d i s t r i b u t i o n s analyses f o r human p l a t e l e t s (Figure 3.2.1.1 and 3.2.1.2). S e v e r a l p l a t e l e t preparations were c e n t r i f u g e d s e p a r a t e l y on S t r a c t a n s o l u t i o n s ranging i n c o n c e n t r a t i o n from 10% to 20% (see a l s o , van Oost et a l . , 1982). These data were used to design modified d e n s i t y g r a d i e n t s , most o f t e n 10%, 16%, 18%, and 20% S t r a c t a n . G e n e r a l l y , a l l p l a t e l e t s penetrated the 10% step, s e p a r a t i n g them from plasma p r o t e i n s , while the other steps i s o l a t e d p l a t e l e t subpopulations of v a r i o u s density ranges, namely 1.042-1.066, 1.066-1.075, and 1.075-1.084 (g/ml), r e s p e c t i v e l y . As de s c r i b e d , minor adjustments were made t o i n d i v i d u a l experiments to achieve the d e s i r e d f r a c t i o n a t i o n . This d e n s i t y g r a d i e n t system was used t o study p l a t e l e t p o p u l a t i o n s from r a b b i t s , humans and monkeys. Apart from animal to animal v a r i a t i o n , d ensity s e p a r a t i o n i n c o n s i s t e n c y i s a l s o r e l a t e d to v a r i a t i o n s i n the gradient environment. Freezing p o i n t osmometry does not c o r r e c t l y allow f o r the f r e e z i n g k i n e t i c s of arabinogalactan s o l u t i o n s , and at higher c o n c e n t r a t i o n ( i . e . , d e n s i t y ) these polymers exert greater osmotic pressures. - 68 -Figure 3.2.1.1 Percent Human P l a t e l e t s Remaining on Str a c t a n Steps: the number of p l a t e l e t s recovered on density steps (from 12.5% to 20% S t r a c t a n , a b s c i s s a ) , expressed as a percentage of the t o t a l p l a t e l e t s added t o the tube (increments of 5% p l a t e l e t s , o r d i n a t e ) . The mean r e s u l t s of p l a t e l e t s from three animals are shown with t h e i r standard d e v i a t i o n s . PERCENT HUMAN PLATELETS REMAINING ON STRACTAN-STEP i n y-UJ _ l UJ t-< _J 0_ UJ L) OH hi a. DENSITY DISTRIBUTION HUMAN PLATELETS ON STRACTAN-STEP 20. 0" 10. 0-CO ia. a- • r-LU _J 14.0 UJ f -< iz. e- • _J Q_ 10. B • 1— 2 ' 8.0 LU U CH B. Z- • UJ Q_ 4. 0' 2.0 0.01 ti Figure 3.2.1.2 Human P l a t e l e t Density D i s t r i b u t i o n on St r a c t a n S o l u t i o n s of Various Concentrations: p l a t e l e t s % t o t a l (increments of 1%, o r d i n a t e ) ; % S t r a c t a n (from 12% to 20%, a b s c i s s a ) ; from above. - 69 -The t o t a l r a b b i t p l a t e l e t populations were s u b f r a c t i o n a t e d i n t o 20.0 + 15.6% LDP, 62.5 + 17.6% IDP, and 18.0 + 9.7% HDP, using density g r a d i e n t s of 10%, 15.7+0.7%, 17.9%, and 20% S t r a c t a n . Human p l a t e l e t s were s u b f r a c t i o n a t e d i n t o 20.0 + 12.9% LDP, 62.6 + 17.3% IDP, and 17.3 + 10.5% HDP, using d e n s i t y g r a d i e n t s of 10%, 15.5 + 0.5%, 18 %, and 20% S t r a c t a n ; about 2% of the p l a t e l e t s penetrated the 20%. Monkey p l a t e l e t s were s u b f r a c t i o n a t e d i n t o 14.7 + 8.8% LDP, 76.7 + 8.4% IDP, and 6.7 + 3.0% HDP, using d e n s i t y gradients of 10%, 15%, 18%, and 20% S t r a c t a n ; only 4.0 +_ 3.0% of the p l a t e l e t s penetrated the 20% S t r a c t a n . Rabbit, rhesus monkey, and human p l a t e l e t s were i n d i v i d u a l l y analyzed; i n each case, three d e n s i t y f r a c t i o n s (low, intermediate and high d e n s i t y p l a t e l e t s ; LDP, IDP, HDP) were harvested and subjected to simultaneous 40 t r a n s f e r CCD procedures, to compare t h e i r r e l a t i v e p a r t i t i o n d i s t r i b u t i o n s . Because p l a t e l e t d e n s i t y and membrane sur f a c e s i a l i c a c i d have been reported i n c e r t a i n i n s t a n c e s to change, decreasing with i n v i v o age, i t would seem that CCD should be able to d i s t r i b u t e on the b a s i s of age, o l d e r p l a t e l e t s having a lower p a r t i t i o n than the bulk of the p l a t e l e t p o p u l a t i o n . This appeared to be the case f o r the CCD p a r t i t i o n s of r a b b i t and human p l a t e l e t s (Figure 3.2.2.1 and 3.2.2.2). The p a r t i t i o n behavior of the monkey p l a t e l e t s was d i f f e r e n t ; i n the 5% D, 4% PEG phase system, a l l three d e n s i t y f r a c t i o n s e x h i b i t e d very low p a r t i t i o n c o e f f i c i e n t s , too low f o r any d i f f e r e n c e s among them to be d i s t i n g u i s h e d . Decreasing the PEG c o n c e n t r a t i o n from 4% to 3.3% provided only minimal r e s o l u t i o n between the intermediate d e n s i t y p o p u l a t i o n and the LDP and HDP. - 70 -Figure 3.2.2.1 CCD of Density Separated Rabbit P l a t e l e t s : LDP, IDP, and HDP% peak p l a t e l e t number (0-100% with increments o f 5%, o r d i n a t e ) ; CCDC 1-40 ( a b s c i s s a ) . CCD DENSITY SEPARATED RABBIT PLATELETS F i g u r e 3.2.2.2 CCD of Density Separated Human P l a t e l e t s : LDP, IDP, and and HDP% peak p l a t e l e t number (0-100% with increments of 5%, o r d i n a t e ) ; CCDC 1-40 ( a b s c i s s a ) . - 71 -In order to compare a l l o f the p a r t i t i o n d i s t r i b u t i o n s f o r the d e n s i t y separated p l a t e l e t subpopulations from each animal system, the gr a d i e n t and CCD d i s t r i b u t i o n c o n d i t i o n s and parameters were t a b u l a t e d : dates o f each of the p e r t i n e n t experiments, g r a d i e n t s used ( i n % S t r a c t a n to the nearest 0.1%), types of p l a t e l e t subpopulations i s o l a t e d (LDP, IDP, and HDP), numbers of p l a t e l e t s of each subpopulation (expressed as a percentage o f the t o t a l p l a t e l e t r e covered), countercurrent d i s t r i b u t i o n c a v i t y or c a v i t i e s (CCDC) loaded, and number of CCDC t r a n s f e r s (n). These d i s t r i b u t i o n parameters were then normalized by expressing the c a v i t y number as a f r a c t i o n o f the number of t r a n s f e r s (Appendix Tables A.11, A.12 and A.13): CCDC c o n t a i n i n g the peak number of p l a t e l e t s (Peak), the estimated modal CCDC (Mode), middle of the CCD d i s t r i b u t i o n (Mid, mid-point o f the width at h a l f the peak h e i g h t ) , and spread (Spd) or breadth of the d i s t r i b u t i o n (the f u l l width, i n CCDC, at one h a l f the height of the 1/2 peak normalized by the square root of the number of t r a n s f e r s , n ). The normalized d i s t r i b u t i o n parameters f o r each subpopulation were then averaged, c a l c u l a t i n g the mean and standard d e v i a t i o n (Table 3.2.1). P a r t i t i o n r a t i o s (K) f o r the CCD parameters were c a l c u l a t e d ( i n terms s i m i l a r to that of an R^ ,, r e l a t e d to the number o f CCD t r a n s f e r s ) from which the d i s t r i b u t i o n c o e f f i c i e n t s (G) could be c a l c u l a t e d using the equations: K = r/n, and G = r / ( n - r ) , where r I s the CCD d i s t r i b u t i o n parameter (peak, mode, middle) and n i s the t o t a l number o f t r a n s f e r s to which the p l a t e l e t subpopulation i s subjected. I d e a l l y , K should equal the f r a c t i o n of the po p u l a t i o n which p a r t i t i o n s i n t o the top phase i n a s i n g l e d i s t r i b u t i o n step and G would be the r a t i o of c e l l numbers i n the two phases i n that step. - 72 -Table 3.2.1 Summary of Rabbit, Human, and Monkey, LDP, IDP, and HDP Peak Mode Mid Spd CCDC/n CCDC/n CCDC/n CCDC/Cn)1/2 Rabbit (12) LDP 0.487 + 0.098 0.498 + 0.096 0.479 + 0.095 1.727 + 0.541 IDP 0.578 + 0.10 0.567 + 0.092 0.552 + 0.081 1.752 + 0.388 HDP 0.564 + 0.10 0.537 + 0.098 0.518 + 0.089 1.902 + 0.842 Human (11) LDP 0.475 + 0.136 0.471 + 0.123 0.463 + 0.112 2.186 + 0.548 IDP 0.526 + 0.092 0.512 + 0.073 0.510 + 0.072 1.789 + 0.736 HDP 0.549 + 0.092 0.572 + 0.094 0.550 + 0.082 2.412 + 0.940 Monkey (4) LDP 0.188 + 0.05 0.184 + 0.086 0.180 + 0.073 1.235 + 0.317 IDP 0.263 + 0.129 0.244 + 0.108 0.239 + 0.113 1.376 + 0.380 HDP 0.238 + 0.096 0.234 + 0.093 0.228 + 0.094 1.831 + 0.199 In cases where more than one CCDC was loaded, the K values were c a l c u l a t e d assuming the o r i g i n as the middle o f the load mixture CCDC (e.g., f o r CCDC 1-3, CCDC 2 was the o r i g i n , and thus 2 was subtracted from the peak, mode, and middle CCDC, p r i o r to c a l c u l a t i o n ; f o r CCDC 1, the o r i g i n was CCDC 1, and thus 1 was subtracted from CCD parameters). The sample p a r t i t i o n r a t i o s were estimates of t h e i r K, equal t o X-^/(X 1+X 2), where X r e f e r s to the e q u i l i b r i u m c oncentrations o f a sample ( i . e . , the numbers of p l a t e l e t s ) i n phases 1 and 2 (A l b e r t s s o n , 1960; Snyder, 1968). As the n ( i n t h i s case the number o f t r a n s f e r s ) i n c r e a s e d , the d i s t r i b u t i o n s approached normalcy and cou l d be described by the c o e f f i c i e n t s of the binomial expansion (x + y ) n . Thus, 1/2 d i s t r i b u t i o n spreads were a l s o normalized with respect to n , as w e l l as t o n. With i n c r e a s i n g n, the bandwidth (or spread) narrowed. The G value expresses the d i f f e r e n t i a l a f f i n i t y o f the population f o r the two phase system polymers (Appendix Table 14). A value of G = 1, expresses equal a f f i n i t y of a c e l l p o p u l a t i o n f o r e i t h e r of the - 73 -polymer phases, i . e . , the upper phase and the lower phase pl u s the i n t e r f a c e . Values g r e a t e r than one i n d i c a t e an a f f i n i t y f o r , and the tendency to be t r a n s f e r e d with the upper PEG-rich phase while values l e s s than one i n d i c a t e an a f f i n i t y f o r , and the tendency to remain s t a t i o n a r y with the lower D-rich phase. Thus, t h e o r e t i c a l l y the G values could range from zero t o i n f i n i t y ; from the s i t u a t i o n i n which r equals zero and G=0/(n-0), t o the s i t u a t i o n i n which r equals n and G=n/(n-n); these extreme s i t u a t i o n s are seldom u s e f u l f o r c e l l s e p a r a t i o n or a n a l y s i s . The p a r t i t i o n or G value i s i n p a r t e x p o n e n t i a l l y r e l a t e d to the surface p r o p e r t i e s of the c e l l p o p u l a t i o n . Although the use of K = r/n was the more convenient parameter with which t o c h a r a c t e r i z e a CCD, as i t permited the averaging of data and r a p i d v i s u a l i z a t i o n of the d i s t r i b u t i o n , i t i s the value of G which i s d i r e c t l y r e l a t e d to c e l l u l a r p r o p e r t i e s determining p a r t i t i o n behavior. By comparison to t h e o r e t i c a l d i s t r i b u t i o n s f o r uniform p a r t i c l e s a f t e r the same number of t r a n s f e r s , the d i s t r i b u t i o n spreads f o r a l l these d e n s i t y subpopulations were i n d i c a t i v e o f membrane surface property heterogeneity w i t h i n each p l a t e l e t subpopulation. The r e l a t i v e s i m i l a r i t i e s of r a b b i t and human p l a t e l e t p a r t i t i o n behavior, and t h e i r d i f f e r e n c e from that of monkey were again evident i n the p a r t i t i o n s obtained by CCD, which compared c l o s e l y with those obtained by s i n g l e step p a r t i t i o n (5/4:A at 5°C)'of unseparated p l a t e l e t s (Section 3.1). Student t t e s t s ( d i f f e r e n c e of means/S.E.), using the Hewlett Packard S t a t i s t i c s V o l . 1 P a r t No. 09815-15000 Rev. C, were performed comparing LDP, IDP, and HDP from the a l l animals (Appendix Tables A.15, A. 16 and A.17). A p p l i c a t i o n s of these t t e s t s t a t i s t i c s i n d i c a t e d - 74 -c e r t a i n c o r r e l a t i o n s between charge-associated surface p r o p e r t i e s and p l a t e l e t d e n s i t y . The s i g n i f i c a n t d i f f e r e n c e s found, at the p = 0.05 l e v e l of u n c e r t a i n t y , were as f o l l o w s : LDP p l a t e l e t s from the r a b b i t were of lower p a r t i t i o n (based on average CCD peak, mode and middle) than IDP; and of lower p a r t i t i o n (based on CCD peak) than HDP p l a t e l e t s , which were lower than IDP (based on CCD mode). LDP p l a t e l e t s from the monkey were of lower p a r t i t i o n than IDP, based on average CCD mode and middle. LDP p l a t e l e t s from the human were o f lower p a r t i t i o n (based on the average CCD middle) than IDP, were of lower p a r t i t i o n (based on mode and middle) than HDP p l a t e l e t s , which were of higher p a r t i t i o n than IDP (based on CCD mode). In the human the average spread of the CCD d i s t r i b u t i o n f o r HDP p l a t e l e t s was g r e a t e r , i n d i c a t i n g that t h i s subpopulations was more heterogeneous than IDP. A nonparametric s t a t i s t i c a l method of data a n a l y s i s was a l s o a p p l i e d . Using the Wilcoxon Matched P a i r s D i f f e r e n c e Signed Rank Test (Snedecor and Cochran, 1974) the data f o r some of the s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n t p l a t e l e t subpopulations, as judged by the t t e s t , (again with the peak K f o r r a b b i t as an example) were found t o be s i g n i f i c a n t . Attempts to compare d i s t r i b u t i o n s of d e n s i t y separated p l a t e l e t s were complicated by two f a c t o r s : s t r i c t l y d i s t r i b u t i o n s from d i f f e r e n t p o s i t i o n s on CCD p l a t e s should not have been compared t h i s c l o s e l y ( i . e . , the need f o r i n t e r n a l standards); and unequal p l a t e l e t numbers of the three d e n s i t y f r a c t i o n s were loaded (although i n c e r t a i n samples when equal q a u n t i t i e s were loaded, d i f f e r e n c e s s t i l l p e r s i s t e d ) . To b e t t e r standardize such CCD analyses, and allow absolute comparisons of d i s t r i b u t i o n s among a s e r i e s of experiments, an i n t e r n a l - 75 -standard could have been i n c l u d e d i n samples, so t h a t the d i s t r i b u t i o n parameters could be r e f e r r e d t o the l o c a t i o n of the standard p a r t i c l e peak. G l u t a r a l d e h y d e - f i x e d human ery t h r o c y t e s ( i n c l u d e d at low c o n c e n t r a t i o n 2-5 x 1 0 6 ) , f o r example, proved to be s u i t a b l e , although they were not used r o u t i n e l y , because f i x e d c e l l s e x h i b i t s t a b l e p a r t i t i o n behavior, and thus minor d i f f e r e n c e s i n phase composition or CCD c o n d i t i o n s c o u l d be allowed f o r . For s t a n d a r d i z a t i o n purposes of a d i f f e r e n t type, i . e . , f o r b e t t e r comparison w i t h i n an i n d i v i d u a l CCD experiment, an i n t e r n a l standard type of r a d i o a c t i v e l a b e l i n g procedure could a l s o have been used (e.g., as i n the l a t e r p a r t of t h i s work); l a b e l i n g the e n t i r e population with one l a b e l and comparing a second subpopulation p o p u l a t i o n w i t h i n i t l a b e l e d with a d i f f e r e n t i s o t o p e . S i m i l a r s t a n d a r d i z a t i o n could a l s o have been a p p l i e d t o these analyses of d e n s i t y separated p l a t e l e t s by l a b e l i n g d i f f e r e n t subpopulations w i t h ^H- or ^ C - s e r o t o n i n , p r i o r to CCD o f the combined subpopulations. This type of s t a n d a r d i z a t i o n f o r CCD was r e c e n t l y presented (Webber and Brooks, 1981; Brooks and Webber, 1982; Webber and Brooks, 1983; and Walter et a l . , 1983). The d e n s i t y separated p l a t e l e t subpopulations were analyzed by CCD i n charge s e n s i t i v e phase systems, i n order to assess the a b i l i t y of t h i s c e l l s e p aration and a n a l y s i s technique to provide a s e p a r a t i o n of the p l a t e l e t p o p u l a t i o n based on age r e l a t e d p r o p e r t i e s , as i s found i n the e r y t h r o y c y t e model of c e l l ageing (Chapter 1.6), developed by Walter (Walter, 1969, 1977, 1978, 1982; Walter and A l b e r t s s o n , 1966; Walter and Selby, 1966; Walter and A l b e r t s s o n , 1971; Walter and Krob, 1983; Walter et a l . , 1962, 1965, 1968, 1971a and b, 1972a, 1973a, 1974, 1975, 1980, 1981, 1982, 1983). In t h i s model, the r e t i c u l o c y t e has a - 76 -lower p a r t i t i o n than the e r y t h r o c y t e p o p u l a t i o n . As i t matures, i t s s u r f a c e p r o p e r t i e s r a p i d l y change w i t h i n two days, so that the young, mature e r y t h r o c y t e has the h i g h e s t p a r t i t i o n c o e f f i c i e n t of the e n t i r e e r y t h r o c y t e d i s t r i b u t i o n . With erythrocyte ageing, c e l l s u rface p r o p e r t i e s g r a d u a l l y change such that the p a r t i t i o n p r o g r e s s i v e l y decreases u n t i l once again the o l d e r y t h r o y c t e demonstrates a p a r t i t i o n c o e f i c i e n t which i s lower than the e r y t h r o c y t e d i s t r i b u t i o n . Even though p o s s i b l e age-related p l a t e l e t membrane surface h e t e r o g e n e i t i e s are superimposed upon h e t e r o g e n e i t i e s due to other non-age-related sources (e.g., megakaryocyte p l o i d y , random d e s t r u c t i o n ) , the countercurrent p a r t i t i o n d i s t r i b u t i o n s of these d e n s i t y separated p l a t e l e t s can be i n t e r p r e t e d analogously to the red c e l l model i f no d i s t i n c t i o n i s made between the r e t i c u l o c y t e and e r y t h r o y c t e . Trends were s i m i l a r f o r a l l animals t e s t e d , r a b b i t , human, and monkey. Both the low d e n s i t y p l a t e l e t s and a p o r t i o n of the high d e n s i t y p l a t e l e t s (enriched i n o l d and young p l a t e l e t subpopulations, r e s p e c t i v e l y ) were found to have a lower charge a s s o c i a t e d p a r t i t i o n c o e f f i c i e n t than IDP, the m a j o r i t y of the p l a t e l e t p o p u l a t i o n enriched i n those p l a t e l e t s of i n t e r m e d i a t e age. The HDP, while always of higher p a r t i t i o n than the LDP, are of s l i g h t l y lower ( u s u a l l y i n s i g n i f i c a n t ) , or of the same or higher mean p a r t i t i o n than the IDP. Thus, LDP ( c o n t a i n i n g old) p l a t e l e t s demonstrate a lower surface charge a s s o c i a t e d p a r t i t i o n than the other subpopulations, which demonstrate ne a r l y the same mean p a r t i t i o n , but d i f f e r e n t width d i s t r i b u t i o n s . Both the CCD curves of the IDP and the LDP appear to be composed of p l a t e l e t s of r e l a t i v e l y homogeneous d i s t r i b u t i o n but of d i f f e r e n t p a r t i t i o n c o e f f i c i e n t s . The CCD curve of the HDP i s heterogeneous, - 77 -i n d i c a t i v e perhaps of two p l a t e l e t subpopulations, one with a low p a r t i t i o n , and another with a p a r t i t i o n c o e f f i c i e n t s i m i l a r t o , or higher than that of the IDP. This i s a l s o suggested by the spread of the HDP i n case of the human. These r e s u l t s are found i n the r a b b i t and human p l a t e l e t systems. These r e s u l t s demonstrate that CCD i n a c h a r g e - s e n s i t i v e phase system i s capable of d i s t r i b u t i n g p l a t e l e t s on the b a s i s of p l a t e l e t membrane surface c h a r a c t e r i s t i c s which are r e l a t e d t o p l a t e l e t age, de n s i t y and s i z e . However, there are consi d e r a b l e overlaps (and great spread, i n d i c a t i n g heterogeneity) i n surface property d i s t r i b u t i o n s among a l l p l a t e l e t d e n s i t y subpopulations s t u d i e d , as are there overlaps i n den s i t y and s i z e . 3.3 S i z e A n a l y s i s of Density Separated P l a t e l e t s P l a t e l e t s i z e analyses (England et a l . , 1982) of the three density separated p l a t e l e t subpopulations (Figures 3.3.1.1 and 3.3.1.2) confirmed the c o r r e l a t i o n s o f i n c r e a s i n g s i z e with d e n s i t y found f o r human (Corash et a l . , 1977), and r a b b i t p l a t e l e t s (Rand et a l . , 1981a). Minimal c e l l u l a r contamination d i d not a f f e c t the r e s u l t s because p l a t e l e t s were enumerated by p a r t i c l e counting at s e t t i n g s which e l i m i n a t e d p a r t i c l e s of g r e a t e r than the p l a t e l e t s i z e . In a d d i t i o n to overlap i n s i z e d i s t r i b u t i o n s of a l l p l a t e l e t d e n s i t y subpopulations of a l l animals t e s t e d , HDP from r a b b i t (Ra), human (Hu), and a r a t (Rt) were l a r g e r than IDP, which were a l s o l a r g e r than LDP (Appendix Tables A.18, A.19, A.20). However, no d i f f e r e n c e s i n s i z e were found f o r de n s i t y separated subpopulations of monkey p l a t e l e t s , i n agreement with Corash et a l . , 1978. - 78 -Figure 3.3.1.1 S i z e D i s t r i b u t i o n of Density Separated Rabbit P l a t e l e t s : LDP, IDP and HDP% peak p l a t e l e t number (0-100%, increments o f 5%, o r d i n a t e ) ; % window, t h r e s h o l d (T 0-100, increments o f 5, a b s c i s s a ) . •» i i i i i i i i i i Figure 3.3.1.2 S i z e D i s t r i b i t o n o f Density Separated Human P l a t e l e t s : LDP, IDP and HDP% peak p l a t e l e t number (0-100%, increments of 5%, o r d i n a t e ) ; % window, t h r e s h o l d (T 0-100, increments of 5, a b s c i s s a ) . - 79 -3.4 E l e c t r o p h o r e t i c M o b i l i t y of Density Separated P l a t e l e t s P l a t e l e t subpopulations, harvested from g r a d i e n t s of 15%, 18%, and 20% S t r a c t a n , were washed and resuspended i n Tyrode's or s a l i n e s o l u t i o n . The e l e c t r o p h o r e t i c m o b i l i t i e s (EPM; expressed as um x sec-"'' x V-"*" x cm) of p l a t e l e t samples, and human er y t h r o y c t e (HuRBC) standards were measured (Seaman and Brooks, 1977). Readings were taken at 40 V and 1.78 mA, and time ( t ) was converted to m o b i l i t y using 5.4785/t (B. Chiu, 1983). Values were considered v a l i d to three s i g n i f i c a n t f i g u r e s and numbers presented i n the appendix were c a r r i e d f o r computations and comparison. The d i f f e r e n c e s i n charge s e n s i t i v e p a r t i t i o n (Chapter 3.1 and 3.2) observed when comparing monkey p l a t e l e t s to human and r a b b i t p l a t e l e t s , and when comparing LDP, IDP and HDP were not apparent i n these l i m i t e d EPM r e s u l t s (Appendix Table A.21). There was l i t t l e d i f f e r e n c e i n the e l e c t r o p h o r e t i c m o b i l i t i e s of monkey p l a t e l e t s (MoPlt) i n comparison to th a t of both the human (HuPlt) and r a b b i t ( R a P l t ) , and no d i f f e r e n c e s i n m o b i l i t i e s were found f o r d e n s i t y separated p l a t e l e t subpopulations (Appendix Table A.22). 3.5 E l e c t r o p h o r e t i c M o b i l i t y o f S i z e Separated P l a t e l e t s Glutaraldehyde f i x e d p l a t e l e t s from r a b b i t ( F x R a P l t ) , human (FxHuPlt) and monkey (FxMoPlt) were suspended i n s a l i n e c o n t a i n i n g 9 0.05% sodium azide (1-5 x 10 , t o t a l ) , and were allowed to s e t t l e a d i s t a n c e of 25-30 cm i n long g l a s s columns (30-50 ml) f o r nearly one month, a f t e r which 1-2 ml f r a c t i o n s were c o l l e c t e d from the top of the column. P l a t e l e t s were washed and suspended i n 0.5 ml of s a l i n e . To l o c a t e column f r a c t i o n s c o n t a i n i n g the g r e a t e s t d i f f e r e n c e s i n p l a t e l e t s i z e s , p l a t e l e t s were c o l l e c t e d , counted and s i z e d (Appendix - 80 -Table A.23) using C o u l t e r E l e c t r o n i c s (Hialeah, FL) Counter Model ZB1, with a 50 u o r i f i c e and 100 u l volumetric (aperature current 1/2, and a m p l i f i c a t i o n 1/2), with C o u l t e r Channelyzer BCR (window width BCT-WW 3-50, set to accumulate 4000 counts i n the peak channel) and X,Y Recorder. The s i z e d i s t r i b u t i o n s obtained with the C o u l t e r Counter were a l s o confirmed (both the s i z e and the spread i s observed to i n c r e a s e , e s p e c i a l l y d u r i n g , and c o r r e l a t e d w i t h , the r i s e i n p l a t e l e t count) by Ele c t r o z o n e Celloscope s i z i n g analyses performed on f r a c t i o n s of sedimented p l a t e l e t s . F r a c t i o n s from sedimented f i x e d p l a t e l e t s were counted f o r t o t a l p l a t e l e t s , were adjusted to equal number i n s a l i n e , and the p l a t e l e t e l e c t r o p h o r e t i c m o b i l i t i e s were determined (Appendix Table A.24). These m o b i l i t y measurements i n d i c a t e d a d i f f e r e n c e (p values ranging from 0.01 to 0.0005) i n the su r f a c e charge o f f i x e d p l a t e l e t s of d i f f e r e n t s i z e s . The data were a l s o s i g n i f i c a n t based on l i n e a r r e g r e s s i o n analyses of m o b i l i t y versus sedimentation d i s t a n c e (Table 3.5.1). However the p a r t i t i o n s (5/4:A) f o r FxHuPlt 94.9 + 2.4% (2 ) , f o r FxMoPlt 92.4 + 3.9% ( 9 ) , and f o r FxRaPlt 89.8 + 3.8% ( 9 ) , were a l l so high t h a t no d i f f e r e n c e s could be detected f o r s i z e separated f i x e d p l a t e l e t s . Table 3.5.1 EPM of S i z e Separated, Fixed P l a t e l e t s , S t a t i s t i c s FxHuPlt 12/19 FxHuPlt 1/22 FxMoPlt FxRaPlt I n t c p t 1.352 + 0.0183 1.368 + 0.0089 1.394 + 0.0168 1.273 + 0.0046 Slope 0.005 + 0.0013 0.004 + 0.0011 0.002 + 0.0006 0.002 + 0.0002 Corr 0.713 0.840 0.693 0.908 p 0.01 to 0.005 0.005 to 0.003 0.005 to 0.001 0.0005 - 81 -3.6 Countercurrent D i s t r i b u t i o n of 51-Cr-Labeled P l a t e l e t s , Aged i n Vivo In order to d e f i n e a p l a t e l e t p opulation at a given time, a "whole 51 p o p u l a t i o n " l a b e l , Cr-chromate was used. T h e o r e t i c a l l y t h i s l a b e l i s taken up evenly by the e n t i r e p l a t e l e t p o p u l a t i o n c o n s i s t i n g of a l l p l a t e l e t ages. With c i r c u l a t i o n time i n v i v o , t h i s l a b e l e d p opulation was compared with the t o t a l p l a t e l e t p o p u l a t i o n present. I f , during p l a t e l e t ageing, no change occurs i n p l a t e l e t membrane surface p r o p e r t i e s as detected by CCD (Figure 3.6.1.1; Model 1 ) , the p l a t e l e t p a r t i c l e and r a d i o a c t i v i t y (not shown) d i s t r i b u t i o n s should be i d e n t i c a l i n shape at a l l time p o i n t s ; and thus the s p e c i f i c a c t i v i t y d i s t r i b u t i o n s should be h o r i z o n t a l , e x h i b i t i n g no change i n slope with time. I f , during p l a t e l e t ageing, changes do occur (e.g., a decrease i n membrane surface charge; F i g u r e 3.6.1.2; Model 2 ) , then p r o g r e s s i v e l y the r a d i o a c t i v i t y d i s t r i b u t i o n s , should s h i f t to the l e f t (lower p a r t i t i o n ) of the p l a t e l e t d i s t r i b u t i o n , and the s p e c i f i c a c t i v i t y d i s t r i b u t i o n s should e x h i b i t a negative s l o p e . However, n e i t h e r of these models p r e d i c t what was found e x p e r i m e n t a l l y . In the experiments t o be d i s c u s s e d , u s u a l l y 30 ml t o 50 ml of blood were drawn from an ear a r t e r y of r a b b i t s weighing i n excess of A.5 kg, and p l a t e l e t s were i s o l a t e d as p r e v i o u s l y described. F o l l o w i n g p l a t e l e t i n c u b a t i o n with 5 1Cr-chromate (0.5 mCi/5 ml PRP f o r S l , 0.2 mCi/5 ml f o r S2, 0.5 mCi/10 ml f o r S3, and O.A mCi/6 ml PRP f o r SA; S# d e s i g n a t i n g s u r v i v a l experiment number), washed red c e l l s and l a b e l e d p l a t e l e t s , washed and resuspended i n plasma, were r e i n j e c t e d i n t o the donor animal. The p l a t e l e t s u r v i v a l curve was determined, and at various times, from one t o s i x days of i n v i v o c i r c u l a t i o n , p l a t e l e t s were i s o l a t e d and subjected to CCD. - 82 -Figu r e 3.6.1.1 No Change i n Surface P r o p e r t i e s , with C i r c u l a t i o n : Model 1, p l a t e l e t counts ( o r d i n a t e ) , and 5 1 C r s p e c i f i c a c t i v i t y h o r i z o n t a l on a l l days; CCDC ( a b s c i s s a ) . DECREASING PARTITION WITH PLATELET AGE PLATELET COUNTS SPECIFIC ACTIVITY CURVES (CPM/PLATELET) DAY 0 Figure 3.6.1.2 Decreasing P a r t i t i o n with Time i n C i r c u l a t i o n : Model 2, p l a t e l e t counts ( o r d i n a t e ) , and ^1-Cr s p e c i f i c a c t i v i t y changing on a l l days; CCDC ( a b s c i s s a ) . - 83 -An example ( S l ) of one of these i n v i v o 5 1 C r - l a b e l e d p l a t e l e t ageing CCD s t u d i e s i s presented. At T=0 days, f o l l o w i n g l a b e l i n g but 51 p r i o r t o i n j e c t i o n (of two animals) there was an enrichment i n Cr uptake, as evidenced i n the r a d i o a c t i v i t y d i s t r i b u t i o n s as w e l l as i n the s p e c i f i c a c t i v i t y d i s t r i b u t i o n s , i n p l a t e l e t s of low p a r t i t i o n r e l a t i v e to bulk of the p l a t e l e t d i s t r i b u t i o n ( F i g u r e 3.6.2.1 and Appendix Figure A . l ) . At T=2 days, c i r c u l a t i o n time, the ageing l a b e l e d p l a t e l e t populations were i d e n t i c a l to the p l a t e l e t d i s t r i b u t i o n s ( F i g u r e s 3.6.2.2 and Appendix Figure A.2). Nonparametric s t a t i s t i c s (Mendenhall and S c h e a f f e r , 1973) can be r a p i d l y a p p l i e d t o analyze these CCD s p e c i f i c a c t i v i t y g r a p h i c a l data. Applying the "Runs Test" to the s p e c i f i c a c t i v i t y d i s t r i b u t i o n s , i t was found that the d i s t r i b u t i o n s of 5 ^ C r were unevenly ( s i g n i f i c a n t l y nonrandom, P = 0.008 t o 0.01) as s o c i a t e d w i t h the p l a t e l e t d i s t r i b u t i o n s , at zero days. However, a f t e r two days of c i r c u l a t i o n i n v i v o , the 5 ^ C r was randomly (P = 1.0) a s s o c i a t e d w i t h the CCD p l a t e l e t d i s t r i b u t i o n s . The enrichment by CCD of 5"*"Cr at T=0 was no longer present at T=2 days. In order t o analyze data from a l l ^ C r - l a b e l e d p l a t e l e t ageing CCD s t u d i e s , the p l a t e l e t and r a d i o a c t i v i t y CCDs were p l o t t e d and the parameters were normalized (as p r e v i o u s l y described Chapter 3.2), with the exception t h a t the r a d i o a c t i v i t y d i s t r i b u t i o n s were of t e n c h a r a c t e r i z e d as having two peaks (examples, Appendix Table A.25). The parameters (CCD middle) of the p l a t e l e t and r a d i o a c t i v i t y d i s t r i b u t i o n s were compared at each time p o i n t , using paired and nonpaired student t t e s t s (HP S t a t i s t i c s Volume 1 ) . A d i f f e r e n c e (p l e s s than 0.05) was found only at T=0, whereas a f t e r c i r c u l a t i o n , the d i s t r i b u t i o n s of p l a t e l e t s and r a d i o a c t i v i t y were i d e n t i c a l (Table 3.6.1). - 84 -Figure 3.6.2.1 CCD of 5 1 C r - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with the mean s p e c i f i c a c t i v i t y at one h a l f o r d i n a t e height; CCDC 1-60, a b s c i s s a . 1. 75E+07-, 0. 00E+00-1.90E-04 1.7BE+08n 0. 00E+00-3.09E+03 •0. 00E+00 1.23E-06 •1.90E+02 0.00E+00 Figure 3.6.2.2 CCD of 5 1 C r - l a b e l e d P l a t e l e t s , Two days C i r c u l a t i o n : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with the mean s p e c i f i c a c t i v i t y at one h a l f o r d i n a t e height; CCDC 1-60, a b s c i s s a . - 85 -Table 3.6.1 Student t Test of P l a t e l e t and R a d i o a c t i v i t y CCD Time Number Mid (Plt-CPM) p a i r e d nonpaired Days of Exp /S.E. t P t P T=0 (3) 0.078/0.019 3.99 0.029 2.13 0.050 T=0 (8) 0.071/0.009 7.89 0.00005 1.96 0.035 T=l (2) 0.009/0.009 1.0 0.25 0.16 0.444 T=2 (3) -0.004/0.004 1.0 0.21 0.03 0.489 T=3 (3) 0.010/0.012 0.83 0.25 0.65 0.275 T=4 (1) 0.003/0.055 0.06 In a d d i t i o n , using grouped s t a t i s t i c a l analyses , with the CCDC as the group, and the p l a t e l e t number and r a d i o a c t i v e counts per CCDC as the frequency, the median CCDC and standard e r r o r f o r the p l a t e l e t and r a d i o a c t i v e CCD d i s t r i b u t i o n s were c a l c u l a t e d at each time, T=0 through 6 days (examples, Appendix Table A.26). On days T=4 and T=6, when one of the d i s t r i b u t i o n s being compared approached' zero ( r a d i o a c t i v i t y approaches zero with i n c r e a s i n g s u r v i v a l t i m e ) , while the other d i s t r i b u t i o n remained r e l a t i v e l y constant, the v a l i d i t y of the grouped s t a t i s t i c a l comparison decreased. To circumvent t h i s problem only groups c o n t a i n i n g s i g n i f i c a n t numbers of counts were considered i n the c a l c u l a t i o n ( i . e . , the l a s t 20 CCDF of both S4T=4, and S4T=6). With the median CCDC and the standard e r r o r , the t s t a t i s t i c was c a l c u l a t e d f o r the comparison of the p l a t e l e t and r a d i o a c t i v i t y CCD d i s t r i b u t i o n s (Appendix Table A.26). Using a one way a n a l y s i s of variance of the t s t a t i s t i c at the d i f f e r e n t times: (T=0 f o r group 1; T=l, group 2; T=2, group 3; T=3, group 4; T=4, group 5; T=6, group 6 ) , the f r a t i o was 2.489, and p = 0.133 (Table 3.6.2). - 86 -Table 3.6.2 A n a l y s i s of Variance o f the t S t a t i s t i c Source D.F. Sum Sqs Mean Sqs F R a t i o F Prob Between groups 5 4954.07 990.81 2.489 0.133 Within groups 7 2786.39 398.06 T o t a l groups 12 7740.46 The Duncan procedure gave, f o r the p = 0.05 l e v e l , ranges of 3.34, 3.48, 3.55, 3.58, and 3.60, w i t h one subset (group 6, 2.02; 5, 2.60; 3, 4.59; 4, 20.1; 2, 35.8; 1, 53.2). This i n d i c a t e d no d i f f e r e n c e at t h i s l e v e l of s i g n i f i c a n c e between the p l a t e l e t and r a d i o a c t i v i t y d i s t r i b u t i o n s at any of the time p o i n t s . The ranges f o r the p = 0.10 l e v e l , were 2.68, 2.79, 2.83, 2.87, 2.88, with two subsets (groups 6, 5, 3, 4, 2, and 2, 1). This i n d i c a t e d t h a t T=0 and T=l days, were d i f f e r e n t from the r e s t . Analyses o f variance t e s t e d f o r a tren d over the e n t i r e period and allowed f o r animal to animal v a r i a t i o n , and f o r missing data. Given v a r i a t i o n and l i m i t e d time dependence of the observed d i f f e r e n c e , the s i g n i f i c a n c e of the trend was only at a l e v e l of p = 0.1, but i n the same animal the d i f f e r e n c e was s i g n i f i c a n t during the i n i t i a l p e r i o d . 51 Each CCD curve f o r C r - l a b e l e d p l a t e l e t s aged i n v i v o i n the r a b b i t was a l s o o p e r a t i o n a l l y sub-divided i n t o four regions ( I , I I , I I I , 51 and IV) f o r f u r t h e r data analyses and d i s c u s s i o n o f Cr s p e c i f i c a c t i v i t y d i s t r i b u t i o n s (Table 3.6.3). S t a r t i n g at the extreme l e f t , or p o r t i o n of the p l a t e l e t d i s t r i b u t i o n with a very low p a r t i t i o n c o e f f i c i e n t , (few t o n e g l i g i b l e p l a t e l e t s , Region I ) , there was a 51 region of l a r g e v a r i a t i o n i n Cr s p e c i f i c a c t i v i t y (high combined e r r o r , CE; S e c t i o n 2.14). Red and white blood c e l l contamination, when present, occurred i n Region I although these preparations were - 87 -v i r t u a l l y f r e e of c e l l u l a r contamination. In t h i s r e g i o n and a l s o i n region IV (to be d i s c u s s e d ) , problems with background were encountered s i n c e very low numbers of p l a t e l e t s and r a d i o a c t i v e counts were present. Therefore, when e i t h e r o f the two numbers which were combined to provide the s p e c i f i c a c t i v i t y (cpm/plt) approached or decreased below zero when background was subtracted, high v a r i a t i o n occurred such th a t the f u n c t i o n was not a t r u e r e f l e c t i o n of the s p e c i f i c a c t i v i t y of the p l a t e l e t s i n t h i s region and thus was judged u n r e l i a b l e as an estimate of such. In t h i s work, i f the CE due to low values of e i t h e r the p l a t e l e t count or cpm exceeded 10% the s p e c i f i c a c t i v i t y was not used f o r a n a l y s i s . This l i m i t was used to d i v i d e the LEFT of the CCD i n t o Regions I and I I , and the RIGHT i n t o Regions I I I and IV. Table 3.6.3 S u b d i v i s i o n of CCD of 5 l C r - L a b e l e d Rabbit P l a t e l e t s Region C h a r a c t e r i s t i c s Region I . Very low p a r t i t i o n c o e f f i c i e n t . A. High combined e r r o r due to very low numbers of p l a t e l e t and r a d i o a c t i v i t y counts. B. P o s s i b l e contamination ( e r y t h r o y c t e and l e u k o c y t e ) . Region I I . Intermediate low p a r t i t i o n c o e f f i c i e n t . A. Major LEFT p o r t i o n of the d i s t r i b u t i o n . B. Sub-population of high s p e c i f i c a c t i v i t y r e l a t i v e t o the mean, at T=0 to 1 days, and low p a r t i t i o n r e l a t i v e to the t o t a l p l a t e l e t d i s t r i b u t i o n , and to the p o r t i o n i n the t h i r d r e g i o n . C. P o s s i b l e contamination ( l e u k o c y t e ) . Region I I I . Intermediate high p a r i t i t i o n c o e f f i c i e n t . A. Major RIGHT p o r t i o n of the d i s t r i b u t i o n . B. Lower 51QT s p e c i f i c a c t i v i t y , at T=0 to 1 days, r e l a t i v e to the t o t a l p l a t e l e t d i s t r i b u t i o n and to the p o r t i o n i n the second r e g i o n . Region IV. Very high p a r t i t i o n c o e f f i c i e n t A. High combined e r r o r due t o very low numbers of p l a t e l e t and r a d i o a c t i v i t y counts. - 88 -Region I I , a l s o of low p a r t i t i o n c o e f f i c i e n t r e l a t i v e to the major p l a t e l e t p o p u l a t i o n , c h a r a c t e r i s t i c a l l y was a region of high p l a t e l e t s p e c i f i c a c t i v i t y . This enrichment i n s p e c i f i c a c t i v i t y c a r r i e d over throughout the LEFT of the major p o r t i o n of the CCD d i s t r i b u t i o n as evidenced by the negative slope of the s p e c i f i c a c t i v i t y d i s t r i b u t i o n both here and i n Region I I I , the p o r t i o n of p l a t e l e t s under the RIGHT of the CCD d i s t r i b u t i o n curve. In Regions I I and I I I ( d i v i d e d by the CCD middle or c e n t r a l p o r t i o n of the d i s t r i b u t i o n ) , the slope of the s p e c i f i c a c t i v i t y d i s t r i b u t i o n was important, s i n c e s u b t l e s h i f t s o f l a b e l e d p l a t e l e t s from one s i d e of the p l a t e l e t d i s t r i b u t i o n curve to the other were detected here. The negative s p e c i f i c a c t i v i t y slope i n these regions of the p l a t e l e t d i s t r i b u t i o n , at T=0 days, decreased with time of c i r c u l a t i o n , approaching zero. This was thought to be i n d i c a t i v e o f a s h i f t i n the p o s i t i o n of r a d i o a c t i v e l y l a b e l e d p l a t e l e t s from Region I I across through Region I I I and suggested that the newly re l e a s e d p l a t e l e t s , i n t h i s case unlabeled, were of low p a r t i t i o n , i n c r e a s i n g i n numbers i n Region I I , the LEFT of the major d i s t r i b u t i o n . Therefore there was a subpopulation(s) of p l a t e l e t s , which e x h i b i t e d enhanced uptake of 5 1 C r , and was of low p a r t i t i o n r e l a t i v e to t o t a l the p l a t e l e t d i s t r i b u t i o n , which during p l a t e l e t c i r c u l a t i o n was replaced by newly r e l e a s e d unlabeled p l a t e l e t s . This might a l s o have been i n t e r p r e t e d as a s h i f t away from low p a r t i t i o n with p l a t e l e t c i r c u l a t i o n time. I t could a l s o have been due t o the removal of t h i s subpopulation during c i r c u l a t i o n f o r unknown reasons, e.g., s e q u e s t r a t i o n of t h i s "young" subpopulation by the r e t i c u l o e n d o t h e l i a l system, i n the spleen (Freeman and K a r p a t k i n , 1975b) or the lungs (Trowbridge and M a r t i n , 1982). - 89 -Region IV demarcated the end of the d i s t r i b u t i o n which was c h a r a c t e r i z e d by the few p l a t e l e t s which were of very high p a r t i t i o n r e l a t i v e to the ma j o r i t y of the p l a t e l e t p o p u l a t i o n . The s p e c i f i c a c t i v i t y of t h i s r e gion was again h i g h l y v a r i a b l e , r e l a t i v e to the mean i n Regions I I and I I I . Analyses of s p e c i f i c a c t i v i t y of p l a t e l e t s i n the d i f f e r e n t Regions ( I I and I I I ; LEFT and RIGHT) of the d i s t r i b u t i o n s a f t e r d i f f e r e n t i n v i v o ^ C r - l a b e l e d p l a t e l e t c i r c u l a t i o n times, and comparison to the mean s p e c i f i c a c t i v i t y (Appendix Tables A.27 through A.31) through the p l a t e l e t d i s t r i b u t i o n , provided another means of d e s c r i b i n g changes i n the p a r t i t i o n of ageing p l a t e l e t s ( i . e . , by s p e c i f i c a c t i v i t y d i s t r i b u t i o n ) . In the c a l c u l a t i o n s of s p e c i f i c a c t i v i t i e s , the s t a t i s t i c a l e r r o r s inherent i n counting both p l a t e l e t numbers and r a d i o a c t i v e events were a l s o c a l c u l a t e d and only data i n which the CE were l e s s than 10% of the s p e c i f i c a c t i v i t y were considered s i g n i f i c a n t . Data were expressed as s p e c i f i c a c t i v i t y (cpm per p l a t e l e t w i t h i n the 10% CE l i m i t ) , as c a l c u l a t e d f o r Regions I I and I I I and the mean of the po p u l a t i o n . The slopes ( s p e c i f i c a c t i v i t y v.s. CCDC) and the c o r r e l a t i o n c o e f f i c i e n t s of the s p e c i f i c a c t i v i t y d i s t r i b u t i o n s were a l s o c a l c u l a t e d . The s p e c i f i c a c t i v i t i e s i n Regions I I and I I I of the p l a t e l e t d i s t r i b u t i o n s a f t e r v a r i ous times of i n v i v o c i r c u l a t i o n were normalized (Appendix Table A.31) with respect t o the grand mean (Gm) s p e c i f i c a c t i v i t y , c o n s i d e r i n g a l l p l a t e l e t s and r a d i o a c t i v e counts per CCDC, and were normalized r e l a t i v e to the mean (m) s p e c i f i c a c t i v i t y with the 10% CE l i m i t a p p l i e d . P a i r e d and nonpaired t t e s t s were used to compare the normalized s p e c i f i c a c t i v i t i e s of Regions I I and I I I , at the d i f f e r e n t time p o i n t s (Table 3.6.A). - 90 -Table 3.6.4 Nonpaired and P a i r e d t Test S p e c i f i c A c t i v i t y of Regions I I and I I I , Normalized to the Mean at Ten Percent Combined E r r o r T I I I I I I I - I I I nonpaired p Mean +_ S.D.(n) Mean _+ S.D.(n) Mean +_ S.D. + S.E. pa i r e d p T= =0 1.498 + 0.234(3) 0.351 + 0.206(3) 0.00155 1.148 + 0.135 + 0.078 0.00229 1.637 + 0.454(8) 0.409 + 0.313(8) 0.00001 1.228 + 0.715 + 0.253 0.00092 T= =1 1.137 + 0.158(2) 0.810 + 0.234(2) 0.12146 0.328 + 0.392 + 0.278 0.22375 T= =2 1.065 + 0.097(3) 0.846 + 0.251(3) 0.11641 0.219 + 0.349 + 0.201 0.19542 T= =3 1.090 + 0.147(3) 0.855 + 0.191(3) 0.08272 0.236 + 0.337 + 0.194 0.17456 The mean s p e c i f i c a c t i v i t i e s of the major d i s t r i b u t i o n of p l a t e l e t s decreased with time, as expected, i n a s i m i l a r manner to that of a normal s u r v i v a l curve. Labeled p l a t e l e t s remained i n c i r c u l a t i o n f o r up to four days, and i n one case, r a d i o a c t i v i t y remained w i t h i n the c i r c u l a t i o n f o r up t o s i x days (not shown). There were enrichments (most pronounced at T=0, and T=l, as evidenced by the slope of the s p e c i f i c a c t i v i t y i n Region I I , Table 3.6.4) i n s p e c i f i c a c t i v i t y of p l a t e l e t s of lower p a r t i t i o n , which changed with p l a t e l e t c i r c u l a t i o n . T h is enrichment i n 5"*"Cr s p e c i f i c a c t i v i t y was considered to be an age r e l a t e d c h a r a c t e r i s t i c . Changes occurred w i t h i n the p a r t i t i o n d i s t r i b u t i o n s of ^ C r - l a b e l e d p l a t e l e t populations f o l l o w i n g i n v i v o c i r c u l a t i o n . These became obvious by comparison of the low t o high p a r t i t i o n region of the p l a t e l e t d i s t r i b u t i o n . When p l a t e l e t s were 51 l a b e l e d w i t h Cr i n v i t r o , t h e re was an enrichment (at T=0 days) i n s p e c i f i c a c t i v i t y ( ^ C r / p l a t e l e t ) i n the low p a r t i t i o n Region I I , which was 4.3 times t h a t i n Region I I I (Table 3.6.4). A f t e r these l a b e l e d p l a t e l e t s were i n j e c t e d i n t o a r a b b i t and allowed t o c i r c u l a t e - 91 -f o r v a r i o u s times, the r e l a t i v e enrichment r a p i d l y decreased. This decrease i n r e l a t i v e enrichment of region I I compared t o region I I I was most pronounced w i t h i n the f i r s t 24 hours, region I I decreasing to 1.4 times the s p e c i f i c a c t i v i t y of region I I I by day one, and to that of 1.3 by days two and three (Table 3.6.4). A f t e r four days, and even as long as s i x days, there was d e t e c t a b l e l a b e l p a r a l l e l i n g the p l a t e l e t d i s t r i b u t i o n . However the a c t i v i t i e s became so low as to be i n s u f f i c i e n t f o r a s t a t i s t i c a l l y s i g n i f i c a n t comparisons to the p l a t e l e t d i s t r i b u t i o n s . 51 * The use of C r - l a b e l i n g , which t h e o r e t i c a l l y d e l i n e a t e s o l d p l a t e l e t s a f t e r ageing i n v i v o , was t h e r e f o r e considered i n s u f f i c i e n t f o r these purposes. The low p a r t i t i o n subpopulation c h a r a c t e r i z e d by the highest s p e c i f i c a c t i v i t y o f ^ C r was presumably that which has been shown to be more f u n c t i o n a l l y a c t i v e i n terms of aggregation as d e s c r i b e d by another method (Haver and Gear, 1981). Here i t was shown to have d i s t i n c t membrane s u r f a c e p r o p e r t i e s , which apparently changed with c i r c u l a t i o n time. A p a r t i a l f u n c t i o n a l f r a c t i o n a t i o n process might be o c c u r r i n g during CCD, s i n c e p l a t e l e t s of a s i z e g r e a t e r than average were a l s o found to have a s l i g h t l y lower (the s i g n i f i c a n c e of which has not been tested) p a r t i t i o n than the d i s t r i b u t i o n of normal s i z e 51 p l a t e l e t s . These changes i n Cr s p e c i f i c a c t i v i t y d i s t r i b u t i o n s c ould have been due to the s p e c i f i c s e q u e s t r a t i o n of the p l a t e l e t s of lower p a r t i t i o n , which e x h i b i t e d enhanced "^Cr uptake, or have been due t o a s h i f t i n the p a r t i t i o n of these l a b e l e d p l a t e l e t s , being r e p l a c e d by newly r e l e a s e d unlabeled p l a t e l e t s . C e l l u l a r contamination of p l a t e l e t p r eparations was checked by m i c r o s c o p i c examination. P l a t e l e t suspensions to be subjected to CCD, - 92 -and i n c e r t a i n i n s t a n c e s a f t e r CCD, were washed and resuspended i n Tyrode's s o l u t i o n c o n t a i n i n g 2% albumin. Smears or C y t o c e n t r i f u g e s l i d e s were prepared, which were Wright's s t a i n e d and observed f o r c e l l u l a r contamination. The suspensions i n general contained l e s s than 1 c e l l per 1 0 5 p l a t e l e t s . T h i s was determined by enumerating the number of p l a t e l e t s per microscope f i e l d and repeatedly scanning s e v e r a l f i e l d s u n t i l a c e l l ( e i t h e r red blood c e l l or white blood c e l l ) was observed. Thus, unless otherwise s t a t e d , c e l l u l a r contamination i n these p r e p a r a t i o n s was n e g l i g i b l e . In one experiment, a f t e r three weeks post i n j e c t i o n of the " ^ C r - l a b e l e d p l a t e l e t p o p u l a t i o n , a blood sample was taken and p l a t e l e t r i c h plasma prepared i n an unusual manner, such t h a t i t contained white blood c e l l and red blood c e l l contamination (0.02% WBC and 4.5% RBC, as a percentage of the t o t a l blood c e l l s ) . This sample was subjected to CCD t o determine the p a r t i t i o n s of contaminating c e l l s , and to determine i f r e i n c o r p o r a t i o n of the ^"''Cr-label ( o r i g i n a l l y p l a t e l e t bound) i n t o any of the other c e l l s had occurred, which i t had. The r e l a t i v e p a r t i t i o n s of contaminating c e l l u l a r types were as p r e v i o u s l y o u t l i n e d i n Table 3.6.3. In c onjunction w i t h the p l a t e l e t ageing CCD s t u d i e s , other (T=0) experiments were performed i n v o l v i n g analyses of p l a t e l e t s l a b e l e d with 5 1 C r s e p a r a t e l y , and together with other r a d i o a c t i v e l a b e l s (Section 3.7). The CCD was analyzed and i n a d d i t i o n samples were c e n t r i f u g e d and the supernatants are counted f o r f r e e l a b e l i n response t o a v a r i e t y of c o n d i t i o n s . The q u a n t i t y of f r e e r a d i o a c t i v e l a b e l r eleased at 4°C i n t o the phases during the s e p a r a t i o n procedure was n e g l i g i b l e , and the p a r t i t i o n c o e f f i c i e n t f o r f r e e 5 1 C r was K=0.5 (G=1.0) i n d i c a t i n g that - 93 -i t d i s t r i b u t e s evenly between the two phases. These s t u d i e s support the e x i s t e n c e of a p l a t e l e t subpopulation which e x h i b i t e d enhanced uptake 51 s p e c i f i c a l l y f o r Cr-chromate (not f r e e l a b e l ) , a p l a t e l e t subpopulation which was concentrated by CCD to a confined area of low p a r t i t i o n under the p l a t e l e t d i s t r i b u t i o n curve. Rabbit p l a t e l e t s (and a l i m i t e d number of human p l a t e l e t samples), l a b e l e d with ^Cr-chromate and/or ^ C - s e r o t o n i n (simultaneously or s e p a r a t e l y ) and d i s t r i b u t e d soon t h e r e a f t e r (T=0) by CCD, produced d i s t r i b u t i o n s which were d i f f e r e n t i n terms of the s p e c i f i c a c t i v i t y of "^Cr and "^C. As mentioned above, there was an enrichment i n s p e c i f i c a c i t i v i t y of p l a t e l e t s of lower p a r t i t i o n when p l a t e l e t s were l a b e l e d with *^Cr, but the s p e c i f i c a c t i v i t y was r e l a t i v e l y constant f o r a l l p l a t e l e t s when l a b e l e d with " ^ - s e r o t o n i n ( t h i s homogeneity of p l a t e l e t l a b e l i n g by s e r o t o n i n was subsequently e x p l o i t e d i n use of t h i s l a b e l as a standard). P r e l i m i n a r y CCD experiments i n d i c a t e d p l a t e l e t heterogeneity by the c h a r a c t e r i s t i c s of post CCD p l a t e l e t 14 C-serotonin r e l e a s e i n response to p l a t e l e t a c t i v a t i n g agents. These f i n d i n g s could be evidence of p l a t e l e t s which are heterogeneous not only with respect to 5"*"Cr uptake but a l s o with respect t o q u a l i t y o f g r a n u l a r contents (Caranobe et a l . , 1982), there being r e l e a s a b l e and non-releasable compartments of s e r o t o n i n i n human p l a t e l e t s (Valdorf-Hansen and Zucker, 1971; Costa et a l . , 1982). 3.7 Countercurrent D i s t r i b u t i o n o f P l a t e l e t s , Chromium Enrichment 51 Because an enrichment i n Cr uptake, as i n d i c a t e d by a higher p l a t e l e t s p e c i f i c a c t i v i t y a f t e r l a b e l i n g in_ v i t r o at T=0, was found i n a subpopulation of p l a t e l e t s which could be d i s t i n g u i s h e d by i t s - 94 -c h a r a c t e r i s t i c low p a r t i t i o n r e l a t i v e to the major p l a t e t e t s u r f a c e property d i s t r i b u t i o n , the p l a t e l e t p opulation was described as being heterogeneous with respect t o both 5"*"Cr uptake and with respect to membrane surface p r o p e r t i e s . Furthermore, s i n c e t h i s enrichment i n 51 Cr s p e c i f i c a c t i v i t y decreased r a p i d l y with time i n the c i r c u l a t i o n , t h i s subpopulation demonstrated age r e l a t e d changes i n surface p r o p e r t i e s . P l a t e l e t d o u b l e - l a b e l i n g , d e n s i t y c e n t r i f u g a t i o n and CCD experiments were designed to a s c e r t a i n whether t h i s subpopulation was s i m i l a r t o the subpopulation of p l a t e l e t s which e x h i b i t e d a c t i v e uptake f o r ^ C r , was of high d e n s i t y (Haver and Gear, 1981; evident a l s o i n the density analyses o f Boneu et a l . , 1982), and was young (Tsukada et a l . , 1971; Haver and Gear, 1981). These enabled the d e f i n i t i o n o f t h i s subpopulation of p l a t e l e t s by i t s p r o p e r t i e s of enhanced 5 ^ C r uptake ( r e l a t i v e to another r a d i o a c t i v e l a b e l which l a b e l e d the p l a t e l e t population more evenly, e.g., " ^ I n , as determined by CCD), i t s high density and c h a r a c t e r i s t i c low p a r t i t i o n i n phase systems s e n s i t i v e to membrane sur f a c e charge. 51 P l a t e l e t s were double l a b e l e d , with Cr at conc e n t r a t i o n s at which enrichment was both evident and l e s s obvious, and with '^"'"In at va r i o u s concentrations f o r which no such s i m i l a r a c t i v e uptake has been observed. These double l a b e l e d p l a t e l e t s were subjected to CCD i n c h a r g e - s e n s i t i v e two-polymer phase systems and the d i s t r i b u t i o n s of p l a t e l e t s and r a d i o a c t i v e l a b e l s were determined. They not only 51 confirmed the enrichment i n Cr uptake by the subpopulation o f p l a t e l e t s with a low p a r t i t i o n which had been p r e v i o u s l y found at T=0 i n the s u r v i v a l experiments, but a l s o demonstrated that t h i s subpopulation e x h i b i t e d no such s i m i l a r p r e f e r e n t i a l uptake f o r ^ I n . - 95 -The r e s u l t s of these experiments were c o n s i s t e n t with the i n t e r p r e t a t i o n that dense ( p o s s i b l y young) p l a t e l e t s contained a subpopulation which p r e f e r e n t i a l l y takes up ^ C r , and that these p l a t e l e t s have surface p r o p e r t i e s uniquely c h a r a c t e r i z e d by a low p a r t i t i o n c o e f f i c i e n t ( i n phase systems capable of d e t e c t i n g surface charge d i f f e r e n c e s ) r e l a t i v e not only to the m a j o r i t y of the p l a t e l e t p o p u l a t i o n , but a l s o lower than that of the HDP (young) p l a t e l e t subpopulation i t s e l f . Although t h i s 5 J"Cr " a c t i v e uptake" p l a t e l e t subpopulation was enriched i n HDP, i t was s t i l l of c h a r a c t e r i s t i c a l l y lower p a r t i t i o n than t h a t of the HDP, and the subpopulation separated by CCD was t h e r e f o r e a s p e c i f i c subpopulation which c o n s t i t u t e d only a p o r t i o n of that separated by d e n s i t y c e n t r i f u g a t i o n . 51 In review (of S e c t i o n 3.6), as the Cr l a b e l e d p l a t e l e t s aged i n v i v o , the s p e c i f i c a c t i v i t y o f t h i s subpopulation decreased, more r a p i d l y than that of the r e s t of the p o p u l a t i o n , as evidenced by a change i n the slope of the s p e c i f i c a c t i v i t y through the d i s t r i b u t i o n i n Region I I compared to Region I I I . This r e s u l t supported the idea that newly released p l a t e l e t s (unlabeled) were of low p a r t i t i o n ( i n Region I I ) , and suggested e i t h e r that t h e i r surface p r o p e r t i e s changed, i n c r e a s i n g i n p a r t i t i o n with maturation or that t h i s subpopulation was s p e c i f i c a l l y sequestered s h o r t l y a f t e r i n j e c t i o n . 3.7.1 Experimental P l a t e l e t P r e p a r a t i o n : Blood, 80 ml from one r a b b i t , was taken i n ACD (8 ml i n each of two 50 ml Corning c e n t r i f u g e t u b e s ) , s p l i t i n two p a r t s , about 40 ml blood and anticoagulant each, and c e n t r i f u g e d at (750-1000 RPM, IEC SBV) 145 g f o r 15 minutes at room temperature. In t o t a l there were approximately 45 ml of packed RBC p l u s r e s i d u a l PRP, . - 96 -and 35 ml of PRP harvested. The PRP was s p l i t i n t o three p a r t s , c e n t r i f u g e d at l,000g f o r 15 minutes and each part was resuspended i n 2 ml of PPP. The remainder of the PPP was saved. P l a t e l e t Double L a b e l i n g : ^ C r-CrO^ (NEN) was used at an o r i g i n a l stock s o l u t i o n c o n c e n t r a t i o n of 1 mCi/ml. In-hydroxy-q u i n o l i n e (Amersham) was used at an o r i g i n a l stock s o l u t i o n c o n c e n t r a t i o n of 1 mCi/ml. The three i d e n t i c a l preparations of p l a t e l e t s were each l a b e l e d s e p a r a t e l y with d i f f e r e n t q u a n t i t i e s of "'"'"Cr and ^ ^ I n , and with d i f f e r e n t times of i n c u b a t i o n f o r the g p a r t i c u l a r l a b e l . P l a t e l e t concentrates at 4 x 10 /ml i n 2 ml PRP were l a b e l e d with 5"*"Cr (stock s o l u t i o n at 2-5 ug Cr/ml and 3.9-9.8 x —8 111 10~ moles Cr/ml) and In, at three l a b e l i n g c o n c e n t r a t i o n s : 5 1 C r at 1 mCi/ml, 100, 500, and 1000 u l ; n i I n at 1 mCi/ml, 100, 200, and 500 u l ; ( f o r r e l a t i v e l e v e l s of 5 1 C r of 1, 5, and 10 and f o r " ^ I n of 1, 2, and 5 ) . The l a b e l i n g c oncentrations were c a l c u l a t e d (Appendix Table A.32) c o n s i d e r i n g the stock c o n c e n t r a t i o n ranges (e.g., 51 R f o r Cr, 0.2-0.5 ug/2 per ml, or 0.4-1.0 x 10 moles/2 per ml). F i r s t , 5 1 C r was added, 0.1, 0.5, and 1.0 ml each to one of the three p l a t e l e t concentrate p r e p a r t a i o n s . The preparations were incubated at 37°C i n a water bath f o r 15 minutes, then they were gently mixed and the second l a b e l , '"'""''"'"In was added, 0.1, 0.2, and 0.5 ml r e s p e c t i v e l y as above, and i n c u b a t i o n continued f o r another 15 minutes. Each l a b e l e d PRP concentrate was d i l u t e d t o 10 ml with the remaining PPP and was c e n t r i f u g e d at 1,000 g f o r 15 minutes at room temperature. The e f f i c i e n c y o f l a b e l i n g was determined by sampling before and a f t e r c e n t r i f u g a t i o n . The p l a t e l e t p e l l e t s were each then resuspended i n 10 ml of Tyrode's s o l u t i o n ( c o n t a i n i n g albumin, EGTA, MgCl 2) and were - 97 -c e n t r i f u g e d again as above. The wash was repeated and each p r e p a r a t i o n was f i n a l l y resuspended i n 4.5 ml of upper phase of the two-phase system at 5°C. The s p e c i f i c a c t i v i t i e s of p l a t e l e t s , w i t h i n these load mixtures (Lx) were compared with those of p l a t e l e t s a f t e r CCD (Appendix Table A.33). Countercurrent D i s t r i b u t i o n : The two-polymer system designated as 5/4:B was prepared from stock s o l u t i o n s by the admixture of 171.39 g of D T500 ( l o t 7830), 99.99 g of PEG 8,000, 313.2 g of NaPB, 31.2 g of NaCl, and 134.21 g of water f o r a t o t a l of 750 g of phase system. The CCD apparatus was loaded with 0.4 ml lower phase and 0.9 ml of load mixture i n the load c a v i t i e s (Ld CCDC); and 0.5 ml lower phase and 0.8 ml of upper phase i n the t r a n s f e r c a v i t i e s (Tr CCDC). The three, double l a b e l e d p l a t e l e t p reparations were loaded i n t o four c a v i t i e s each, and 36 t r a n s f e r s were completed, with a mixing time of 30 seconds and a s e t t l i n g time set at 8.5, or 7 minutes. Upon completion of the CCD run, the contents of the 120 CCDC were d i l u t e d with 0.7 ml of Tyrode's s o l u t i o n , mixed a u t o m a t i c a l l y , c o l l e c t e d , pooled by two adjacent CCDC f o r a t o t a l of 60 CCDF, which were sampled f o r p a r t i c l e counting ( d i l u t i o n 247/1) and then counted d i r e c t l y f o r ^ C r and ^'4n using the LKB Compugamma. These CCDF were analyzed at room temperature but a sample of each was c e n t r i f u g e d i n the c o l d f o r determination of f r e e l a b e l , and leakage due to p o s s i b l e p l a t e l e t damage. Samples, 0.5 ml, of the 60 CCDF were taken and pooled by two adjacent CCDF i n t o Eppendorf c e n t r i f u g e tubes f o r a separate set of 30 CCDF samples of 1.0 ml each. These samples were then c e n t r i f u g e d at top speed f o r 6 minutes at 5°C. The 30 supernatants were sampled, 0.5 ml each, and counted f o r f r e e l a b e l along with the other 60 CCDF. - 98 -C y t o c e n t r i f u g e s l i d e s of the CCDF were prepared and s t a i n e d with Wright's s t a i n . The percentage o f c e l l u a r contamination was determined by f i r s t counting p l a t e l e t s on o i l immersion (lOOx l O x ) , e s t i m a t i n g an average number of p l a t e l e t s per f i e l d , and then because of the s p a r s i t y of c e l l u l a r contamination, counting (repeatedly) and recording the number of f i e l d s before a s i n g l e c e l l (WBC or RBC) was observed. The number of p l a t e l e t s per f i e l d v a r i e d and ranged i n some instances from a number which was a c c u r a t e l y countable t o , at times, a complete monolayer. An estimated 5,000-10,000 p l a t e l e t s per f i e l d were present. A t o t a l of 6 WBC were counted over 29 f i e l d s (1/4.83), and a t o t a l of 2 RBC over a t o t a l of 16 f i e l d s (1/8). Based on 5,000-10,000 p l a t e l e t s per f i e l d , the WBC contamination was 0.004-0.002% and the RBC contamination was 0.0025-0.00125%. 3.7.2 R e s u l t s Countercurrent D i s t r i b u t i o n A n a l y s i s : The d i s t r i b u t i o n s of p a r t i c l e s of a s i z e greater than t h a t of the p l a t e l e t (not shown), resembled those of p l a t e l e t s . However they were c o n s i s t e n t l y s h i f t e d s l i g h t l y to the l e f t , e x h i b i t i n g a lower K than p l a t e l e t s . The percentage of p a r t i c l e s of a s i z e greater than t h a t o f p l a t e l e t s , r e p r e s e n t i n g mostly aggregates, counting coincidence, and due i n pa r t to the s e l e c t i o n of window s e t t i n g s , and a very minor q u a n t i t y of c e l l u l a r contamination, was of the order of 5.72% of the t o t a l p a r t i c l e s , based on (average o v e r a l l ) recovery a f t e r CCD, and 6.65%, 9.28%, and 7.28%, r e s p e c t i v e l y f o r the load mixture p r e p a r a t i o n s . 51 P l a t e l e t s , double-labeled with Cr (at three d i f f e r e n t c o n c e n t r a t i o n s ) and "'"''""'"In, subjected to CCD a n a l y s i s , e x h i b i t e d a modal p a r t i t i o n f o r the major p l a t e l e t d i s t r i b u t i o n of K=0.394 or - 99 -G=0.66, c o n s i s t e n t l y f o r a l l three i d e n t i c a l p o p u l a t i o n s l a b e l e d w i t h v a r i o u s concentrations of the l a b e l s (Figure 3.7.2.1.1 through 3.7.2.1.6). The K value was c a l c u l a t e d f o r the peak or modal CCDC ( i n t h i s case equal to the mean), d i v i d e d by the number of t r a n s f e r s or p a r t i t i o n steps to which the popu l a t i o n was subjected (as i n S e c t i o n 3.2). Since each p l a t e l e t p r e p a r a t i o n was loaded i n four CCDC ( i . e . , CCDC 1 through 4, 41 through 44, and 81 through 84), the load CCDC, or o r i g i n , was taken as "CCDC 2.5" (the middle of CCDC 1,2,3,4). The number o f CCDC t r a n s f e r r e d by the p l a t e l e t p o p u l a t i o n was 14.17 i n each case: the peaks (taken from the graphs, see Figure s 3.7.1.) of the d i s t r i b u t i o n s were at a d i s t a n c e of 16.67 CCDC from CCDC 1, but the a c t u a l o r i g i n s were at CCDC 2.5; t h e r e f o r e , the t o t a l number o f CCDC t r a n s f e r r e d by the p l a t e l e t p o p u l a t i o n was 2.5 l e s s than 16.67, or 14.17. Taking t h i s number, 14.17 d i v i d e d by 36 t r a n s f e r s r e s u l t e d i n a K value of 0.3935. The K value would be 0.41 f o r an o r i g i n of 1. The ^•'"In r a d i o a c t i v i t y d i s t r i b u t i o n s , at a l l l e v e l s of p l a t e l e t l a b e l i n g , c l o s e l y p a r a l l e l e d the p l a t e l e t d i s t r i b u t i o n s ( F i g u r e s 3.7.2.1.2, 3.7.2.1.4, and 3.7.2.1.6). However, there were two peaks i n ^CT r a d i o a c t i v i t y d i s t r i b u t i o n s , one comparable to the p a r t i t i o n of p l a t e l e t s and one which was of lower r e l a t i v e p a r t i t i o n . The a c t i v e nature of t h i s 5"*"Cr uptake was suggested i n th a t i t became saturated 51 at p r o g r e s s i v e l y higher c o n c e n t r a t i o n s of Cr (Figures 3.7.2.1.1, 3.7.2.1.3, and 3.7.2.1.5). This enrichment by CCD of p l a t e l e t s which e x h i b i t e d enhanced uptake f o r 5 ^ C r , but not f o r " ^ I n , was a l s o 51 evidenced i n the magnitude o f the Cr s p e c i f i c a c t i v i t y slopes through CCD regions I I and I I I f o r p l a t e l e t s l a b e l e d w i t h s u c c e s s i v e l y higher (A, B, and C) co n c e n t r a t i o n s of 5 J-Cr (Appendix Table A.30). - 100 -Figu r e 3.7.2.1.1 CCD of 5 1 C r - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-40, a b s c i s s a . R A B B I T P L A T E L E T S (^CR, SD^CI / tL AND m l N , SO^Ci / UJ 0 2 0 10 Figure 3.7.2.1.2 CCD of m I n - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-40, a b s c i s s a . - 101 -Figure 3.7.2.1.3 CCD of 5 1 C r - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 41-80, a b s c i s s a . RABBIT P L A T E L E T S <5]CR, 250 «CI / ML AND m l N , lOCUCi / tO R A B B I T P L A T E L E T S C 5 1 ^, 250^1 / ML AND m l N , IDO^Ci / ri) 4 0 6 0 8 0 Figure 3.7.2.1.4 CCD of i ; L 1 I n - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) w i t h mean at one h a l f the graph height; CCDC 41-80, a b s c i s s a . - 102 -Figu r e 3.7.2.1.5 CCD of 5 1 C r - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 81-120, a b s c i s s a . R A B B I T P L A T E L E T S SOO^Ci / rL AND m l N , 250^Ci / iD 8 0 1 0 0 1 2 0 Figure 3.7.2.1.6 CCD of m I n - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 81-120, a b s c i s s a . - 103 -Further CCD analyses ( F i g u r e s 3.7.2.2.1 through 3.7.2.2.6; and Appendix Figures A.3 and A.4) were conducted on r a b b i t p l a t e l e t s , 51 double l a b e l e d with Cr (at the lowest c o n c e n t r a t i o n , demonstrating the highest p r e f e r e n t i a l uptake) and * ^ I n , and separated i n t o d e n s i t y subpopulations (LDP, IDP, HDP, and HHD; LDP and IHHD; LIDP and HHD). LDP were p l a t e l e t s of d e n s i t y l e s s than that of 16% S t r a c t a n . IDP were of greater d e n s i t y than 16%, but l e s s than 18% S t r a c t a n . HDP were of g r e a t e r d e n s i t y than 18%, but l e s s than 20% S t r a c t a n . The subpopulations of unusual d e s i g n a t i o n s were e i t h e r combinations of the above subpopulations (e.g., LIDP were a combination of LDP and IDP; i . e . , of d e n s i t y l e s s than that of 18% S t r a c t a n ) , or other subpopulations obtained from the g r a d i e n t . HHD were (and IHHD contained) subpopulations of p l a t e l e t s which penetrated the lowermost 20% S t r a c t a n d e n s i t y step, and were p l a t e l e t s of d e n s i t y even higher than HDP. These f r a c t i o n s of very high d e n s i t y might c o n t a i n c e l l u l a r contamination which was confined to given regions of the CCD, e.g., 51 e r y t h r o c y t e s i n Region I evidenced by a sharp peak i n Cr s p e c i f i c a c t i v i t y (Figure 3.7.2.2.2, CCDC 90-95), but absent i n the m i n s p e c i f i c a c t i v i t y d i s t r i b u t i o n s (Figure 3.7.2.2.1, CCDC 90-95). CCD r e s u l t s from these ^ C r - , "'""'"'''In-double l a b e l e d , d e n s i t y separated p l a t e l e t s were s i m i l a r to previous f i n d i n g s of S e c t i o n 3.2. A low p a r t i t i o n was c o n s i s t e n t l y a s s o c i a t e d with p l a t e l e t s of low d e n s i t y , LDP (Figure 3.7.2.2.3), compared to that of IDP (Figure 3.7.2.2.4), HDP (Figure 3.7.2.2.5), and HHD (Figure 3.7.2.2.6). The v a r i a b i l i t y of the p a r t i t i o n of p l a t e l e t s of high d e n s i t y was again apparent. In one case, the p a r t i t i o n of HDP (Figure 3.7.2.2.5) was equal t o that of p l a t e l e t s o f lower density IDP (Figure 3.7.2.2.4), but - 104 -s t i l l of higher p a r t i t i o n than p l a t e l e t s of even higher density HHD (Figure 3.1.2.2.6). A c o r r e l a t i o n of p l a t e l e t p a r t i t i o n and density was again found i n another experiment (Appendix Figures A.3 and A.4). While enrichment by CCD ( i n Region I I ) of a p l a t e l e t subpopulation 51 which e x h i b i t s enhanced uptake f o r Cr was more pronounced i n p l a t e l e t s of high d e n s i t y ( F i g u r e s 3.7.2.2.2, CCDC 65-80, CCDC 95-105; and Appendix Figure A.4, CCDC 35-45, CCDC 95-105) i t was a l s o present i n the other subpopulations o f lower d e n s i t y (e.g., LDP, Appendix Figure A.4, CCDC 5-15). Density dependence, and enrichment by CCD, d i d not occur as s t r o n g l y f o r " ^ I n l a b e l e d p l a t l e t s ( Figures 3.7.2.2.1 and Appendix Figure A.3). 3.7.3 D i s c u s s i o n A subpopulation c o n t a i n i n g l a r g e , dense, p o s s i b l y young p l a t e l e t s 51 e x h i b i t s s a t u r a b l e , perhaps a c t i v e uptake f o r Cr-chromate. At con c e n t r a t i o n s of l e s s than 1.5 uCi/ml, enhanced uptake of chromate occurs; at concentrations g r e a t e r than t h i s both non-saturating and s a t u r a t i n g uptake occur. By comparing the CCD d i s t r i b u t i o n s of p l a t e l e t p o p u l a t i o n s l a b e l e d with d i f f e r e n t c oncentrations of "^'"Cr and ^ " ^ I n , i t i s p o s s i b l e to d i s t i n g u i s h a p l a t e l e t subpopulation which e x h i b i t s enhanced uptake f o r 5 1 C r but not f o r 1" L" 1"In. The subpopulation which s t r o n g l y takes up ^ C r i s c h a r a c t e r i z e d by a r e l a t i v e l y low p a r t i t i o n i n a two-phase system s e n s i t i v e t o sur f a c e charge. In these same ^ C r - l a b e l e d p l a t e l e t p r e p a r a t i o n s , the "'""'""'"In CCD d i s t r i b u t i o n s matched c l o s e l y the p l a t e l e t d i s t r i b u t i o n s . Therefore, ^ I n , l i k e "^C-or-^H-serotonin, i s considered to l a b e l p l a t e l e t s r e l a t i v e l y homogeneously, and to be u s e f u l as a standard f o r comparisons. - 105 -Fig u r e 3.7.2.2.1 CCD of i : L 1 I n - L a b e l e d , LDP CCDC 1-30, IDP CCDC 31-60, HDP CCDC 61-90, HHD CCDC 91-120: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f graph height; CCDC 1-120, a b s c i s s a . 0 10 20 30 40 50 60 70 60 90 100 110 120 F i g u r e 3.7.2.2.2 CCD of 5 1 C r - L a b e l e d , LDP, IDP, HDP, HHD: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120, a b s c i s s a . - 106 -Fig u r e 3.7.2.2.3 CCD of 5 1 C r - L a b e l e d LDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f graph height; CCDC 1-30, a b s c i s s a . Figure 3.7.2.2.4 CCD of 5 1 C r - L a b e l e d IDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f the graph h e i g h t ; CCDC 31-60, a b s c i s s a . - 107 -Fig u r e 3.7.2.2.5 CCD of 5 1 C r - L a b e l e d HDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f graph height; CCDC 61-90, a b s c i s s a . Figure 3.7.2.2.6 CCD of 5 1 C r - L a b e l e d HHD, P l a t e l e t s of Density Greater than that of 20% S t r a c t a n : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with mean at one h a l f graph height; CCDC 91-120, a b s c i s s a . - 108 -"''"''"''Indium, by v i r t u r e of i t s complexing with hydroxyquinoline, i s taken up through the hydrophobic b i l a y e r and i s f i n a l l y l o c a t e d predominantly (80%) i n the p l a t e l e t c y t o s o l a s s o c i a t e d with a 50,000 d a l t o n molecule (Hudson et a l . , 1979 and 1981; Baker et a l . , 1982). 51 Cr i s l i k e w i s e found predominately i n the c y t o s o l ( S t e i n e r and B a l d i n i , 1970). " ^ I n i s considered a b e t t e r l a b e l f o r p l a t e l e t s u r v i v a l time analyses (Thakur et a l . , 1976; S c h e f f e l et a l . , 1977 and 1979; Born, 1980) than i s 5 * C r , f o r a v a r i e t y of reasons: short h a l f l i f e , s u p e r i o r gamma camera imaging (Riba et a l . , 1979; Davis et a l . , 1978; Heaton et a l . , 1979; Heyns et a l . , 1980 and 1982; L o t t e r et a l . , 1980; L i n d e n f e l d and S t e e l e , 1982), b e t t e r p l a t e l e t recovery i n v i v o (Schmidt et a l . , 1983; H i l l - Z o b e l et a l . , 1983), and normal p l a t e l e t f u n c t i o n ( S c h e f f e l et a l . , 1977; Schmidt and Rasmussen, 1979; Schmidt et a l . , 1982a and b ) . In a d d i t i o n , there i s f u r t h e r evidence, presented here, f o r preference of 1 1"'"In i n t h a t i t more evenly l a b e l s the e n t i r e p l a t e l e t p opulation whereas "'"'"Cr can p r e f e r e n t i a l l y l a b e l a p o r t i o n of the p l a t e l e t p o p u l a t i o n with d i s t i n c t membrane surface p r o p e r t i e s and g r e a t e r s p e c i f i c a c t i v i t y than the r e s t of the p o p u l a t i o n (Haver and Gear, 1981; Gear, 1981). This p o p u l a t i o n i s s a i d to be young, more a c t i v e i n terms of aggregation p o t e n t i a l , more dense and e x h i b i t s a longer c i r c u l a t i o n time than the normal p l a t e l e t 51 p o p u l a t i o n d i s t r i b u t i o n . Thus, use of Cr as a p l a t e l e t l a b e l at low c o n c e n t r a t i o n s would r e s u l t i n anomalous s u r v i v a l times. At very high c o n c e n t r a t i o n s t h i s s p e c i f i c uptake appears to be l e s s dramatic, r e l a t i v e to the major p o p u l a t i o n of p l a t e l e t s , but p l a t e l e t s l a b e l e d at 51 these high concentrations of Cr are a l s o u n s u i t a b l e f o r s u r v i v a l a n a l y s i s and are not recommended f o r the p a t i e n t (Belcher et a l . , 1977). - 109 -Because "''"'""'"In i s a l s o known t o l a b e l leukocytes which d i s t r i b u t e i n Regions I and I I with p a r t i t i o n s lower than the mean of normal p l a t e l e t d i s t r i b u t i o n s , the s p e c i f i c a c t i v i t y i n terms of " ^ I n per p l a t e l e t , being h o r i z o n t a l throughout the p l a t e l e t d i s t r i b u t i o n , could be evidence of the l e u k o c y t e - f r e e nature of these p l a t e l e t preparations and/or an i n d i c a t i o n of a g r e a t e r preference f o r p l a t e l e t l a b e l i n g by ^ * I n r e l a t i v e to ^ C r . In the l a t t e r case, ^"^In i s seen as more p l a t e l e t s p e c i f i c , and again more u s e f u l i n assessing p l a t e l e t s u r v i v a l time. The even d i s t r i b u t i o n of " ^ I n might a l s o i n d i c a t e that t h i s enrichment i n l a b e l i s not due to (perhaps CCD induced) p l a t e l e t aggregation ( i f one considers the d i s t r i b u t i o n of p a r t i c l e s of a s i z e g r e a t e r than p l a t e l e t s to be an i n d i c a t i o n of such), which i s more pronounced i n the low p a r t i t i o n r e gion I I of the p l a t e l e t d i s t r i b u t i o n . The p l a t e l e t subpopulation which e x h i b i t s enhanced uptake f o r 51 51 Cr i s apparent when the d i s t r i b u t i o n of Cr i s compared to other r a d i o a c t i v e p l a t e l e t l a b e l s such as ^ C - s e r o t o n i n , or ^"*"In, as w e l l as when compared to the p l a t e l e t number. Taking advantage of the a c t i v e uptake f o r ^ C r by a young subpopulation of p l a t e l e t s , and the apparently homogeneous l a b e l i n g o f p l a t e l e t s by ^"''In, p l a t e l e t s double l a b e l e d with these two n u c l i d e s ( f o r example) and subjected to CCD might provide means f o r q u a l i t a t i v e l y assessing p a t i e n t s ' p l a t e l e t s i n terms of t h e i r c i r c u l a t i o n - a g e c h a r a c t e r i s t i c s , s i n c e the p l a t e l e t s which e x h i b i t an a c t i v e uptake f o r 5"*"Cr demonstrate a c h a r a c t e r i s t i c 51 lower p a r t i t i o n than normal. That the Cr enrichment obtained by CCD i s apparent i n an e n t i r e p l a t e l e t p o p u l a t i o n and i s not a s i n g u l a r c h a r a c t e r i s t i c o f the HDP, i n d i c a t e s that p a r t i t i o n provides more c h a r a c t e r i s t i c s e p arations of t h i s p l a t e l e t subpopulation. - 110 -3.8 Countercurrent D i s t r i b u t i o n of In Vivo 35-S-Labeled P l a t e l e t s The cohort l a b e l S-sulphate, when i n j e c t e d i n t o a r a b b i t i s taken up i n v i v o by the megakaryocyte i n the bone marrow and i s i n c o r p o r a t e d i n t o p l a t e l e t s which are then r e l e a s e d i n t o the c i r c u l a t i o n . This " ^ S - l a b e l e d p l a t e l e t cohort, when harvested during the r i s e of p l a t e l e t i n c o r p o r a t e d r a d i o a c t i v i t y found i n the c i r c u l a t i o n from one to four days a f t e r i n j e c t i o n ( p . i . ) , depending on 35 the q u a n t i t y of S i n j e c t e d , i s considered to be i n d i c a t i v e of a 35 young p l a t e l e t subpopulation. This S cohort l a b e l e d , newly r e l e a s e d , young p l a t e l e t subpopulation r e l a t i v e l y enriched i n the high d e n s i t y f r a c t i o n but i s c h a r a c t e r i z e d as having a low p a r t i t i o n i n a l l p l a t e l e t subpopulations r e g a r d l e s s of d e n s i t y . This i n d i c a t e s not only t h a t CCD might be more s e l e c t i v e than d e n s i t y gradient c e n t r i f u g a t i o n f o r s e p a r a t i o n of t h i s . young p l a t e l e t cohort, but a l s o t h a t the combination of d e n s i t y and surface property-based c e l l separations provides greater s e p a r a t i o n of young p l a t e l e t s than does de n s i t y alone. 35 These analyses of newly r e l e a s e d S-cohort-labeled p l a t e l e t s , Chapters 3.8 through 3.12, i n v o l v e e i g h t (not a l l presented) experiments i n two general c a t e g o r i e s . Four experiments i n v o l v e d the 35 i s o l a t i o n of S-labeled p l a t e l e t s , p r i m a r i l y at two time p o i n t s p . i . (day two and day s i x ) , h a r v e s t i n g p l a t e l e t d e n s i t y subpopulations 35 ( S-LDP, -IDP, and -HDP) u s i n g S t r a c t a n gradient c e n t r i f u g a t i o n of p l a t e l e t s , and CCD; a t o t a l of 21 d i s t r i b u t i o n s . Four experiments 35 i n v o l v e d the i s o l a t i o n of S-labeled p l a t e l e t s at s h o r t e r i n t e r v a l s ( d a i l y ) p . i . , from day one through day s i x , d o u b l e - l a b e l i n g with a second l a b e l ( 5 1 C r , or 3 H - s e r o t o n i n ) i n v i t r o , and CCD, a t o t a l of 22 d i s t r i b u t i o n s . - I l l -In the experiment presented i n S e c t i o n 3.8, newly r e l e a s e d p l a t e l e t subpopulations were double-labeled, f i r s t i n v i v o by staggered 35 i n j e c t i o n s of low l e v e l s o f S-sulphate. Blood samples from a l l animals were drawn. P l a t e l e t s were harvested and l a b e l e d i n v i t r o with low l e v e l s of 5"*"Cr-chromate which l a b e l e d the whole p l a t e l e t p o p u l a t i o n (however unevenly, as p r e v i o u s l y d i s c u s s e d ) . These p l a t e l e t s were washed and simultaneously subjected to CCD. In a d d i t i o n , the time 35 course ( k i n e t i c s ) of i n v i v o , S-cohort l a b e l e d p l a t e l e t r e l e a s e , 35 was determined by f o l l o w i n g the S a c t i v i t y i n v a r i o u s f r a c t i o n s obtained from the blood samples. 3.8.1 Experimental Sulphate I n j e c t i o n : At, T=0 (day of experiment), the cohort l a b e l , -9 35 0.5 ml (200 uCi; 9.3 x 10 moles) of S-sulphate stock s o l u t i o n ( l o t no 094394) and 0.2 ml of s a l i n e were i n j e c t e d using a t u b e r c u l i n s y r i n g e and needle, i n t o the l e f t marginal ear vein of r a b b i t (R) 22 (5.5 k g ) . Other r a b b i t i n j e c t i o n s were staggered (Appendix Table A.34). Blood Samples: On T=4, day of experiment, 30 ml of blood were taken from each of the four r a b b i t s . Using s t e r i l e Argyle Medicuts Cannulas and 30 ml s y r i n g e s , the blood samples were placed i n separate 50 ml s t e r i l e Corning polypropylene c e n t r i f u g e tubes c o n t a i n i n g 6 ml of ACD each, and weighed. The weighed recovery of blood from r a b b i t number 26 was 38.07 g; 24, 38.7 g; 23, 42.0 g; and 22, 39.0 g (Appendix Table A.34). A d d i t i o n a l blood was taken from number 23, and t h e r e f o r e , 1 ml more of ACD was added at the time of draw. P l a t e l e t P r e p a r a t i o n : Tyrode's s o l u t i o n was prepared with 0.02% M g C l 2 > 0.02% EGTA, and with albumin and dextrose. An attempt to f o l l o w p r e v i o u s l y described p l a t e l e t p r e p a r a t i o n methods by - 112 -c e n t r i f u g a t i o n at 250 g f o r 10 minutes f a i l e d , most probably due t o d i f f e r e n c e s i n the volume of blood being c e n t r i f u g e d , and t h e r e f o r e the time of c e n t r i f u g a t i o n was extended t o 15-20 minutes with 20 minutes being r e q u i r e d to completely sediment a l l e r y t h r o y c t e s . Blood samples were c e n t r i f u g e d at 10-1100 RPM i n the IEC SBV f l o o r c e n t r i f u g e at room temperature f o r 10 minutes, and f o r 10 minutes again. From each blood sample, approximately 15 ml of PRP was recovered. The remainder of blood samples were resuspended to 50 ml each with Tyrode's and c e n t r i f u g e d again as above. The p l a t e l e t r i c h Tyrode's (PRT) supernatants were harvested, concentrated i n one ml and added to the PRPs, which were then weighed ( r a b b i t number 26, 16.12 g; 24, 18.5 g; 23, 14.7 g; and 22, 14.6 g ) , sampled, and counted f o r recovery of p l a t e l e t s (Appendix Table A.35) and r a d i o a c t i v i t y (Appendix Table A.36). Chromium L a b e l i n g : P l a t e l e t samples are l a b e l e d with 5 1 C r at low c o n c e n t r a t i o n . "^Cr-CrO^ ( l o t 23/1BA; at 1 mCi/ml) was added to the p l a t e l e t s i n PRP, at a f i n a l c o n c e n t r a t i o n of approximately 16.7 uCi/ml. Incubation was f o r one hour i n a 37°C water bath. Labeled p l a t e l e t samples were d i l u t e d t o 50 ml with Tyrode's s o l u t i o n , c e n t r i f u g e d , washed t w i c e , and resuspended i n 3 ml of upper phase each. Countercurrent D i s t r i b u t i o n : The phase system was 5/4:A. P l a t e l e t s i n the CCD load mixtures (Lx) were adjusted to equal c o n c e n t r a t i o n (2.8 9 x 10 /ml) i n upper phase. The t r a n s f e r CCDC were loaded with 0.5 ml of lower phase and 0.8 ml of upper phase. The load c a v i t i e s , CCDC 1-3, 31-33, 61-63 and 91-93, were loaded with 0.5 ml of lower phase and 0.9 ml of lo a d mixture (Lx) each from samples R26, R24, R23 and R22, r e s p e c t i v e l y . Twenty-seven t r a n s f e r s were completed, at 4-5°c, with a 30 second mixing time and a s e t t l i n g time of 7 minutes. - 113 -A n a l y s i s of P l a t e l e t s and R a d i o a c t i v i t y : Upon completion, 0.7 ml of Tyrode's s o l u t i o n was added t o each of the CCDC, the contents were mixed, c o l l e c t e d , and each CCDC was sampled and counted f o r p l a t e l e t p a r t i c l e s (Celloscope, 48 um diameter o r i f i c e , 100 u l volumetric tube, 35 s e t t i n g s C 1, G 68, T 15-95, l i n / d e l t a mode); and assayed f o r S and 51 Cr r a d i o a c t i v i t y of 1 ml i n 10 ml of A t o m l i t e , using a P h i l i p s 4700 s c i n t i l l a t i o n counter. In p a r a l l e l , samples of blood, PRP, PPP, and the load mixtures were a l s o counted (Appendix Table A.36). Approximately 1 ml samples of the CCDC were a l s o counted f o r "'"'"Cr r a d i o a c t i v i t y using a LKB Compugamma gamma counter with automatic quench c o r r e c t i o n . Since the l e v e l of i n v i t r o ^ C r - l a b e l i n g was high ( r e l a t i v e to i n vivo 35 S l a b e l i n g ) , the r a d i o a c t i v e CCDC samples were again counted when three h a l f l i v e s (Tl/2) of 5 1 C r and one Tl/2 of 3 5 S had passed. 3.8.2 R e s u l t s P l a t e l e t Recovery: On average, from 33.35 +_ 1.11 (4) g of blood, there were recovered 15.08 + 1.88 (8) g of PRP, c o n t a i n i n g a t o t a l o f 8.49 + 1.9 x 10 9 (8) p l a t e l e t s , at a PRP con c e n t r a t i o n of 5.58 + 0.67 x 10 (4) p l a t e l e t s per ml, and t h e r e f o r e a blood c o n c e n t r a t i o n of 2.55 +_ 0.6 x 10 (8) p l a t e l e t s per ml (Appendix Table A.35). Based on p a r t i c l e counting there were 1.53 + 0.27% (4) p a r t i c l e s of a s i z e g r e a t e r than that of a p l a t e l e t ; however, not a l l of t h i s was c e l l u l a r contamination, based on microscopic examination, and most was probably r e p r e s e n t a t i v e of very l a r g e p l a t e l e t s and p l a t e l e t aggregates. R a d i o a c t i v i t y Counting: The P h i l i p s 4700 S c i n t i l l a t i o n Counter was 51 35 stand a r d i z e d with Cr and S, quenched with v a r y i n g q u a n t i t i e s of phase system ranging from 0.5 ml to 5.0 ml, with equal q u a n t i t i e s of r a d i o a c t i v i t y and s c i n t i l l a t i o n f l u i d , 10 ml A t o m l i t e . The e x t e r n a l - 114 -standard r a t i o (ESR) f o r these standards v a r i e d only s l i g h t l y w i t h i n t h i s l a r g e range of v i s i b l e quenching, and q u a n t i t i e s of phase system: average ESR 1.564 + 0.042 (10), range 1.51-1.62 f o r 5 1 C r ; and ESR 1.559 + 0.032 (10), range 1.51-1.61 f o r 3 5 S . The quenching of the CCDC samples was constant, and the ESR d i d not vary (from 1.55-1.56) throughout the e n t i r e CCD. Dual Channel (CH) Program 14 was used (CHI, 0.4-6.0; CH2, 15-80), 51 35 st a n d a r d i z e d with Cr and S. Samples of blood, PRP, PPP, and CCD load mixtures were counted once f o r I minute (Appendix Table A.36). Samples were recounted f o r 5 minutes each, twice (Appendix Table A.36). Again program 14 was used (CHI, 0.4-8.0; CH2, 30-1700), standardized with 3H and 1 4 C , as major part of the 5 1 C r r a d i o a c t i v i t y d i s t r i b u t i o n o f decay can be counted i n the 3H channel and that of 3 5 S w i t h i n the ^ C channel. The combined r a d i o a c t i v i t i e s , normalized as a percentage of day one a c t i v i t y , were c a l c u l a t e d and are presented as an average of s i x measurements (Table 3.8.2.1). Table 3.8.2.1 Summary of 3 5 S - S u l p h a t e L a b e l i n g K i n e t i c s Time Blood PRP PPP CCD P i t Lx Day Hour A c t i v i t y % T=l A c t i v i t y % T=l A c t i v i t y % T=l A c t i v i t y % T=l 1 21.0 100 100 100 100 2 45.5 38.31 + 2.91 51.75 + 8.05 53.25 + 4.55 123.30 + 12.92 3 68.25 16.68 + 4.75 28.05 + 6.52 28.53 + 6.74 130.11 + 9.94 4 93.5 19.23 + 3.83 27.96 +6.52 30.27 + 7.44 87.89 + 19.17 3.8.3 D i s c u s s i o n 51 Sulphate L a b e l i n g K i n e t i c s : Since the Cr i n v i t r o l a b e l i n g was much higher (more e f f i c i e n t ) than the 3 5 S i n v i v o l a b e l i n g , these samples were stored and l a t e r counted again. The time course of - 115 -35 S-sulphate l a b e l i n g k i n e t i c s w i t h i n the blood, PRP, PPP, and p l a t e l e t s was r e v e a l i n g . The i n c o r p o r a t i o n of l a b e l i n t o the megakaryocyte, r e l e a s e of l a b e l e d p l a t e l e t s i n t o the c i r c u l a t i o n and t h e i r removal must be followed i n order to make i n f e r e n c e s about the age c h a r a c t e r of the cohort l a b e l e d p l a t e l e t s . P l a t e l e t s , harvested during the r i s e o f p l a t e l e t i n c o r p o r a t e d r a d i o a c t i v i t y i n the plasma, were taken t o be newly r e l e a s e d young p l a t e l e t s u n t i l the peak and d e c l i n e i n t h i s p l a t e l e t i n c o r p o r a t e d r a d i o a c t i v i t y was observed (Table 35 3.8.2.1). These data were presented as S counts as a percentage of day one a c t i v i t y . The blood r a d i o a c t i v i t y per ml decreased from day one u n t i l day three, a f t e r which i t was l e v e l . In p a r a l l e l , the PRP and PPP a c t i v i t i e s a l s o decreased, but were higher than the blood, s i n c e the i n c o r p o r a t i o n of t h i s l a b e l i n t o the er y t h r o c y t e s was n e g l i g i b l e at a l l time p o i n t s measured. The PPP a c t i v i t y was found to be only s l i g h t l y higher at a l l time p o i n t s than the PRP, i n d i c a t i n g t h a t much of the 35 S a c t i v i t y was a l s o i n the plasma, and perhaps t h a t the volume of p l a t e l e t s i n PRP was not n e g l i g i b l e . The p l a t e l e t a c t i v i t i e s on days one, two, and three were s u c c e s s i v e l y higher, but on day four d e c l i n e d , 35 i n d i c a t i n g that the peak entry of i n vivo S-labeled p l a t e l e t s i n t o the c i r c u l a t i o n was reached, i n t h i s experiment, between days two and four , as was found by others ( H i r s h et a l . , 1968). Countercurrent D i s t r i b u t i o n : The CCD curves f o r these experiments 35 51 are not shown but are di s c u s s e d . On day one p . i . , the S/ Cr r a t i o through the d i s t r i b u t i o n was skewed s l i g h t l y to the r i g h t of the p l a t e l e t p o p u l a t i o n , suggesting that p l a t e l e t s which had been l a b e l e d 35 i n v i v o with S, were of higher p a r t i t i o n than p l a t e l e t s l a b e l e d w i t h 5 1 C r i n v i t r o ; (but are of lower p a r t i t i o n than the d i s t r i b u t i o n - 116 -o f p l a t e l e t p a r t i c l e s and the d i s t r i b u t i o n of ^H-serotonin; to be shown). This might a l s o be an i n d i c a t i o n o f the r e l a t i v e enrichment o f 51 Cr uptake i n the low p a r t i t i o n area of the p l a t e l e t d i s t r i b u t i o n . On day two p . i . , the ^ S / ^ C r r a t i o was h o r i z o n t a l , d i s t r i b u t e d 35 evenly through the p l a t e l e t d i s t r i b u t i o n , i n d i c a t i n g that the S 51 l a b e l i n v i v o on day two approaches that o f Cr uptake i n v i t r o . On 35 51 day three the S/ Cr r a t i o was s h i f t e d , enriched to the l e f t , 35 i n d i c a t i n g t h a t at t h i s time, s t i l l during the r i s e of S-labeled 51 p l a t e l e t r e l e a s e , the cohort i s of lower p a r t i t i o n than Cr l a b e l e d p l a t e l e t s , which are lower than the p l a t e l e t d i s t r i b u t i o n as a whole. 3.9 CCD of 35-S-Labeled LDP, IDP, and HDP, Day Two and S i x , p . i . Newly released p l a t e l e t cohorts were l a b e l e d i n v i v o with " 5 5S-sulphate, harvested on days two and day s i x p . i . , separated by S t r a c t a n density gradient c e n t r i f u g a t i o n and analyzed by countercurrent d i s t r i b u t i o n . These times, day two and s i x , had been s e l e c t e d s i n c e they represented p l a t e l e t cohorts of d i f f e r e n t age c h a r a c t e r , day two being young and during the r i s e of l a b e l e d p l a t e l e t entry i n t o the c i r c u l a t i o n , and day s i x being more mature, during the d e c l i n e of p l a t e l e t r a d i o a c t i v i t y ( P l t L x , Table 3.8.2.1 and D i s c u s s i o n 3.8.3). 35 On day two p . i . , the S-sulphate l a b e l e d p l a t e l e t cohort was found t o be enriched i n the high density p l a t e l e t (HDP) f r a c t i o n ; the opposite was found i n the low de n s i t y p l a t e l e t (LDP) f r a c t i o n . On day 35 s i x p . i . , t h i s d i f f e r e n c e i n S - s p e c i f i c a c t i v i t y i n the three d e n s i t y f r a c t i o n s was l e s s dramatic. These f i n d i n g s i n d i c a t e d t h a t p l a t e l e t s decreased t h e i r mean den s i t y as a f u n c t i o n of maturation i n c i r c u l a t i o n , the newly re l e a s e d p l a t e l e t cohort being enriched i n HDP. - 117 -35 Membrane surface property CCD analyses of these S-labeled, d e n s i t y separated p l a t e l e t subpopulations i n d i c a t e d t h a t the p l a t e l e t cohort not only had d i f f e r e n t density c h a r a c t e r i s t i c s but a l s o had d i s t i n c t i v e surface p r o p e r t i e s which were more pronounced on day two i n 35 comparison to day s i x . The s p e c i f i c a c t i v i t y of S-sulphate was enriched i n the low p a r t i t i o n CCD p l a t e l e t region ( I I ) , on day two p . i . , an enrichment which decreased on day s i x . 3.9.1 Experimental Sulphate I n j e c t i o n : At 4:35 PM, T=0 days, Rabbit 27 (4.95 kg) was i n j e c t e d w i t h 5 ml, 1.67 mCi, of 3 5 S - s u l p h a t e stock s o l u t i o n . Blood Sample and P l a t e l e t P r e p a r a t i o n : At 3:30 PM, day two (71 hr) p . i . , approximately 45 ml of blood was taken and mixed i n two 50 ml Corning polypropylene c e n t r i f u g e tubes with a t o t a l of 10.5 ml ACD. The q u a n t i t y of blood and ACD was measured to be 27.5 ml p l u s 30 ml, and weighed f o r a t o t a l of 60.78 g from which one ml was sampled. The blood i n each tube was d i l u t e d t o 50 ml with Tyrode's and c e n t r i f u g e d at (1,200-1,400) RPM f o r 15 minutes. A t o t a l of 80 ml of d i l u t e d PRP supernatant was recovered. The blood p e l l e t s were resuspended to 50 ml wit h Tyrode's, and c e n t r i f u g e d again as above with s i m i l a r recovery of d i l u t e d p l a t e l e t r i c h Tyrode's (PRT). The four p l a t e l e t p r e p a r a t i o n s , two of PRP, and two of PRT, were d i l u t e d to 50 ml with Tyrode's and c e n t r i f u g e d at 2,200 RPM f o r 15 minutes (IEC SBV). PRT supernatant contained n e g l i g a b l e p l a t e l e t s , e r y t h roycte contamination, and was d i s c a r d e d . P l a t e l e t s were f i n a l l y resuspended i n 8.6 ml of Tyrode's from which one ml was sampled. On day s i x p . i . , at 5:00 PM, blood (40-45 ml) was again taken, mixed with ACD (6 ml) i n two tubes, measured (52-52.5 ml) and weighed (54.46 g ) . The blood was d i l u t e d w i t h - 118 -Tyrode's (to a t o t a l weight o f 106.12 g; 50 ml each, approximate 1/2 d i l u t i o n ) which was sampled. P l a t e l e t s were prepared as on day two. Density Gradient Separation: On day two, and again on day s i x , the p l a t e l e t concentrates (8.5 ml) were layered e q u a l l y (about 2 ml) on four S t r a c t a n d e n s i t y 4-step g r a d i e n t s of compositions 1 ml 10%, 3 ml 16%, 3 ml 18%, and 3 ml 20% S t r a c t a n . The gra d i e n t s were balanced and c e n t r i f u g e d f o r 2 hours at 10,000-11,000 RPM, 23,000 g, at room temperature i n the S o r v a l RC5, using the swinging bucket r o t o r . The gr a d i e n t s were harvested from the top; LDP from the f i r s t 5 ml, IDP from the next 5 ml, and HDP from the next 3 ml, l e a v i n g the remainder of the grad i e n t . Each p l a t e l e t subpopulation was washed i n 40 ml of Tyrode's by c e n t r i f u g a t i o n at 10,000 RPM f o r 10 minutes, resuspended i n 5 ml of Tyrode's, and sampled (0.1 ml) f o r p l a t e l e t number and r a d i o a c t i v i t y analyses. The subpopulations were again washed i n 40 ml Tyrode's as above, and each f i n a l l y resuspended i n 3.1 ml of upper phase (load mixture) f o r CCD a n a l y s i s . Countercurrent D i s t r i b u t i o n : The phase system 5/4:A was used. Three CCDC each were loaded with 0.9 ml load mixtures (CCDC 1-3, LDP; 41-43, IDP; 81-83; HDP) and 0.5 ml lower phase. The t r a n s f e r CCDC re c e i v e d 0.5 ml lower and 0.8 ml upper phase. Thirty-seven c y c l e s were completed with 30 seconds shaking time, and 7 minutes s e t t l i n g time. Upon completion, the contents df each CCDC were d i l u t e d with 0.7 ml Tyrode's s o l u t i o n , mixed, harvested and analyzed f o r p l a t e l e t s and r a d i o a c t i v i t y . P l a t e l e t and R a d i o a c t i v i t y Counting: Samples from the CCDC were i n i t i a l l y d i l u t e d , 1/247 i n D i l u i d (and again where necessary to avoid coincidence e r r o r s ) . LDP, IDP, and HDP load mixtures, samples from the - 119 -g r a d i e n t LDP, IDP, and HDP, the blood, and the unseparated p l a t e l e t p o p u l a t i o n , were a l s o d i l u t e d and counted using the Celloscope (C 1, G 68, T 15-95, l i n / d e l t a , 48 um diameter o r i f i c e , and 100 u l v o l u m e t r i c ) . Samples from each CCDC (1.5 ml) i n 10 ml of A t o m l i t e were counted 35 ( 1 , 5, and 10 minutes) f o r S r a d i o a c t i v i t y using a P h i l i p s 4700 counter (Program 9 CHI 0.4-130.0, CH2 0.4-2). The samples from day two were saved and analyzed c o n c u r r e n t l y with those of day s i x ; and again using a double l a b e l program (14 CHI 0.4-8.0, CH2 30-1700). 3.9.2 R e s u l t s S t r a c t a n Density Gradient: The S t r a c t a n (StR) concentrations and d e n s i t i e s (g/ml) of the g r a d i e n t steps were measured, and compared to the r e s u l t s of others. The gradient c o n d i t i o n s and S t r a c t a n Q c o n c e n t r a t i o n s of Rand et a l . (1981a) were: 3.5 x 10 p l a t e l e t s / m l 9 l a y e r e d on gradient 15.5%, 17.5%, 19% S t r a c t a n ; 2 x 10 p l a t e l e t s / m l l a y e r e d on 15%, 17%, 19% S t r a c t a n (Appendix Table A.37). The d e n s i t i e s and S t r a c t a n concentrations a f t e r the method of Corash and Shafer (1982) were a l s o t a b u l a t e d (Appendix Table A.38). Based on these data, the s p e c i f i c d e n s i t y of S t r a c t a n , i n terms of grams per% Stractan was 0.003949 + 0.0000695 ( 9 ) , or 0.003946 + 0.0000597 (12). Based on d e n s i t y measurments of the S t r a c t a n s o l u t i o n s used i n t h i s study, the s p e c i f i c d e n s i t y of S t r a c t a n was 0.004113 + 0.0002473 (4 ) . The s p e c i f i c d e n s i t y of S t r a c t a n could be used to c a l c u l a t e the d e n s i t y of a given g r a d i e n t step, Table 3.9.2.1. Density-^ was c a l c u l a t e d based on the average g /% S t r a c t a n as c a l c u l a t e d from the combined data (Appendix Tables A.37 and A.38). D e n s i t y 2 was c a l c u l a t e d based on the average estimate of 0.004 g /% S t r a c t a n , obtained from measurements of S t r a c t a n used i n t h i s study. - 120 -Table 3.9.2.1 S t r a c t a n Concentration and Density % StR (g/ml) Density^ (g/ml) Density2 (g/ml) % StR (g/ml) Density^ (g/ml) Density2 (g/ml) 10 1.039 1.040 16 1.063 1.064 11 1.043 1.044 17 1.067 1.068 12 1.047 1.048 18 1.071 1.072 13 1.051 1.052 19 1.075 1.076 14 1.055 1.056 20 1.079 1.080 15 1.059 1.060 21 1.083 1.084 P l a t e l e t Recoveries: The r e c o v e r i e s of p l a t e l e t s and of p a r t i c l e s o f a s i z e greater t h a t of p l a t e l e t s were c a l c u l a t e d throughout the experiment (Appendix A.39). 35 Countercurrent D i s t r i b u t i o n : The CCD d i s t r i b u t i o n s of S-cohort l a b e l e d LDP, IDP, and HDP are shown (CCDC 1-40, 41-80, 81-120, r e s p e c t i v e l y ; Day Two p . i . , Figure 3.9.2.1.1 and Day S i x , Figure 3.9.2.1.2). The e n t i r e CCD i s given f o r comparison of r e l a t i v e q u a n t i t i e s o f p l a t e l e t s and r a d i o a c t i v i t y and f o r p a r t i t i o n comparison. 35 The r a t i o of r a d i o a c t i v i t y , i n t h i s case S per p l a t e l e t , i s p l o t t e d (dotted l i n e ) with the mean s p e c i f i c a c t i v i t y (noted i n s i d e the l e f t o r d i n a t e ) at one h a l f the height of the o r d i n a t e . 35 The enrichments by CCD of the S-labeled cohort w i t h i n each subpopulation were compared by c a l c u l a t i o n of the average s p e c i f i c a c t i v i t y and slope of the p l o t of s p e c i f i c a c t i v i t y versus CCDC, and analyzed by the nonparametric (P) "Runs Test" (Table 3.9.2.2). On day two p . i . , the mean s p e c i f i c a c t i v i t y , and the magnitude of the slope were g r e a t e s t f o r HDP i n comparison to IDP, and LDP ( i n s u f f i c i e n t data w i t h i n 10% CE f o r complete c a l c u l a t i o n s ) . On Day s i x p . i . , the s p e c i f i c a c t i v i t y of a l l the subpopulations was e s s e n t i a l l y equal (IDP s l i g h t l y l e s s ) , and the d i s t r i b u t i o n of s p e c i f i c a c t i v i t y was random. - 121 -Fig u r e 3.9.2.1.1 CCD of Day Two 3 3 S - L a b e l e d LDP CCDC 1-40, IDP CCDC 41-80, and HDP CCDC 81-120: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt at 10% CE (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . F i g u r e 3.9.2.1.2 CCD of Day S i x 3 5 S - L a b e l e d LDP, IDP, and HDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt at 10% CE (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . - 122 -Table 3.9.2.2 A n a l y s i s of S p e c i f i c A c t i v i t y Enrichment Slope Day Two CE% Mean + S.Dev. n Corr Slope Intcp P LDP CPM/Plt 10 IDP CPM/Plt 10 HDP CPM/Plt 10 1.25 x 10-6 2.82 x IO" 8 2 1.89 x IO" 6 3.15 x IO" 7 8 2.51 x 10-6 A.90 x IO" 7 6 -0.987 -0.941 -1.27 x IO" 7 2.33 x IO" 6 0.029 -2.46 x IO" 7 3.13 x IO" 6 0.10 Day S i x CE% Mean + S.Dev. 100 1.65 x IO" 6 5.32 x IO" 8 4 10 1.22 x IO" 6 6.55 x IO" 8 7 10 1.56 x IO" 6 8.22 x IO" 8 4 n 3.9.3 Di s c u s s i o n The i n j e c t i o n of a r a d i o a c t i v e compound to l a b e l a cohort of newly re l e a s e d p l a t e l e t s i s oft e n used (Amorosi et a l . , 1971; Charmatz and K a r p a t k i n , 1974; K a r p a t k i n , 1978; McDonald, 1976, 1978, 1980 and 1981; C l i f t and MacDonald, 1979; McDonald et a l . , 1979; Dassin e t a l . , 1979; Rand et a l . , 1981a; Corash and Shafer, 1982). On day two, during the r i s e of " ^ S - p l a t e l e t r e l e a s e i n t o the c i r c u l a t i o n and when the g r e a t e s t d i f f e r e n c e i s found i n r e l a t i v e s p e c i f i c a c t i v i t y between the most dense and the l e a s t dense p l a t e l e t subpopulations (Rand et a l . , 1981a; Corash and Shafer, 1982), the HDP CCD subpopulation c o n t a i n s more 3 5 S per p l a t e l e t . However, there i s d e t e c t a b l e a c t i v i t y i n a l l three density f r a c t i o n s . Since r a b b i t p l a t e l e t s u r v i v a l time i s approximately four days, and 51 p l a t e l e t s do s u r v i v e up to s i x days (Se c t i o n 3.6, C r - p l a t e l e t s u r v i v a l ) , r a d i o a c t i v i t y i n LDP at two days might be due to incomplete c o r r e l a t i o n of den s i t y and p l a t e l e t age, i n c o n s i s t e n c y of den s i t y gradient separation (Corry et a l . , 1981; Mar t i n and Trowbridge, 1982b), - 123 -p l a t e l e t a l t e r a t i o n by procedure and gradient ( B o l i n et a l . , 1981; Holme and Murphy, 1983), or to young p l a t e l e t s being the l e a s t dense (Mezzano et a l . , 1981). However, i n a l l p l a t e l e t d e n s i t y subpopulations analyzed by CCD on day two p . i . , the i n v i v o "^S-sulphate p l a t e l e t cohort i s s t r o n g l y and c o n s i s t e n t l y enriched at the lower end of the p l a t e l e t CCD. On day s i x , the l e v e l s of "^S-sulphate a c t i v i t y throughout the CCD are more equal, i . e . , the l e v e l of a c t i v i t y of the the LDP r e l a t i v e to the HDP has i n c r e a s e d , suggesting that HDP become l i g h t e r with maturation. In t h i s experiment (with t h i s q u a n t i t y of i n j e c t e d c o h o r t ) , on day s i x the l e v e l o f a c t i v i t y through the CCD of LDP, IDP, or HDP i s more evenly d i s t r i b u t e d ( h o r i z o n t a l ) . 35 Therefore, not only does t h i s S-sulphate cohort c o r r e l a t e with a dense young p l a t e l e t subpopulation, but a l s o with a subpopulation of p l a t e l e t s w i t h s p e c i f i c membrane surface p r o p e r t i e s , thus f a r d i s t i n g u i s h a b l e only by CCD i n charge s e n s i t i v e two-polymer phase systems, and c h a r a c t e r i z e d as having a low (surface charge s e n s i t i v e ) p a r t i t i o n r e l a t i v e to the p l a t e l e t p o p u l a t i o n c o n s i s t i n g of a l l ages. These f i n d i n g s a l s o i n d i c a t e that surface property analyses of "^S-sulphate l a b e l e d p l a t e l e t cohorts are more s e l e c t i v e than d e n s i t y analyses. More c o n s i s t e n t and b e t t e r r e s o l u t i o n of t h i s p l a t e l e t cohort 35 i s apparent i n t h a t the S-sulphate i n v i v o l a b e l e d p l a t e l e t s are present i n a l l d e n s i t y subpopulations on day two, but can s t i l l be separated w i t h i n each by CCD, surface property heterogeneity w i t h i n each being a l s o c o n s i d e r a b l e . On both day two and day s i x , i n a l l d e n s i t y f r a c t i o n a t e d subpopulations the percentage of p a r t i c l e s o f s i z e greater than the - 124 -p l a t e l e t (10% t o t a l p a r t i c l e s ) i s to the l e f t , or of lower p a r t i t i o n than the p l a t e l e t CCD. The f i n d i n g s of enrichments i n the r a t i o s of 5 * C r per p l a t e l e t , 5 1 C r per "^C-serotonin, ^ C r per 1 1 1 I n , 35 35 3 S per p l a t e l e t , and S per H-serotonin ( to be shown), being c h a r a c t e r i s t i c s o f p l a t e l e t s o f low p a r t i t i o n r e l a t i v e to the p l a t e l e t CCD, can be i n t e r p r e t e d i n two ways. In the most dramatic i n t e r p r e t a t i o n the enrichment i s due to younger, l a r g e r p l a t e l e t s with a lower net negative s u r f a c e charge and t h e r e f o r e lower charge s e n s i t i v e p a r t i t i o n . The r e s u l t s a l s o might be due t o p l a t e l e t aggregates, o c c u r r i n g and/or sedimenting during CCD (escaping l a t e r p l a t e l e t count), or leucko c y t e s . Leukocyte contamination, although i n the same area where these enrichments are found, i s i n s i g n i f i c a n t , r e q u i r i n g unreasonably high s p e c i f i c a c t i v i t y to change the p l a t e l e t r a d i o a c t i v i t y CCD, even c o n s i d e r i n g greater l a b e l uptake (DPM/ug) f o r leukocytes (Corash and Shafer, 1982). Arguments against leukocyte and/or p l a t e l e t aggregates c o n t r i b u t i n g to cohort enrichment by CCD r e l y a l s o on evidence from CCD of double l a b e l e d p l a t e l e t s ; leukocytes and p l a t e l e t s , clumped or not, would c o n t a i n both 5 ^ C r and " ^ I n . 3.10 CCD of 35-S-Labeled LDP, IDP, and HDP, Day Two and S i x p . i . A second s e r i e s of measurements of t h i s general type was c a r r i e d out. Newly released p l a t e l e t c o h o r t s , l a b e l e d i n v i v o with a high l e v e l 35 of S-sulphate, were again separated according to de n s i t y and subjected t o CCD i n . c h a r g e - s e n s i t i v e phase systems, e s s e n t i a l l y reproducing r e s u l t s of S e c t i o n 3.9, with the exception that on day s i x p . i . , an enrichment of cohort p e r s i s t e d i n HDP and i n the low p a r t i t i o n areas o f a l l d e n s i t y subpopulations, however s t i l l l e s s than on day two. - 125 -3.10.1 Experimental Sulphate I n j e c t i o n : On T=0 days, 1 ml c o n t a i n i n g 2 mCi of 35 S-sulphate was i n j e c t e d i n t o a r a b b i t (R32). Blood Samples and P l a t e l e t P r e p a r a t i o n : On day two (as on day s i x ) , p . i . , blood (40-45 ml) and ACD (7.5) was d i l u t e d to 100 ml with Tyrode's s o l u t i o n i n two s t e r i l e Corning tubes and c e n t r i f u g e d at 1,000-1,300 RPM f o r 15-20 minutes. The PRP was harvested, c e n t r i f u g e d ( S o r v a l RC5) at 5,000 RPM f o r 10 minutes, and the p l a t e l e t s were resuspended i n 8-8.5 ml Tyrode's s o l u t i o n , gradient load mixture (GLx). Density Gradient C e n t r i f u g a t i o n : A sample was taken f o r p l a t e l e t and r a d i o a c t i v i t y counts (80 u l of the GLx) and the remainder was layer e d onto four S t r a c t a n 4-step d e n s i t y g r a d i e n t s , and c e n t r i f u g e d f o r 2 hours at 13,000 RPM. The g r a d i e n t , as p r e v i o u s l y described ( S e c t i o n 3.9.1), was prepared by d i l u t i o n of S t r a c t a n with Tyrode's s o l u t i o n (Appendix Table A.40). The stock 22% S t r a c t a n , and gradient-step c o n c e n t r a t i o n s were rechecked with a B r i x 0-30° Refractometer, and the d e n s i t i e s were measured by weighing approximately 5 ml volumes i n 10 ml graduated c y l i n d e r s at room temperature (Appendix Table A.41). The d e n s i t y subpopulations, LDP, IDP, and HDP were harvested, washed t w i c e , resuspended i n 50 ml Tyrode's s o l u t i o n , c e n t r i f u g e d at 5,000 RPM (So r v a l ) f o r 10 minutes, and f i n a l l y resuspended i n 3 ml top phase f o r CCD analyses. Countercurrent D i s t r i b u t i o n : The phase system was again 5/4:A. S u f f i c i e n t phase system f o r two CCD experiments (500-550 ml) was f i l t e r e d and allowed to e q u i l i b r a t e at 5°C overnight i n two separatory funnels. The lower phases were c o l l e c t e d from each of the separatory funnels and pooled; the upper phases were l i k e w i s e - 126 -c o l l e c t e d . The CCD procedures were as p r e v i o u s l y described ( S e c t i o n 3.9.1). CCDF were i n i c e water during analyses. The recovery from the IEC CCDC c o l l e c t i o n tubes was 1.81 + 0.06 g (10) of a t o t a l of 2.1 ml added, a phase f l u i d y i e l d o f 86.2%. 3.10.2 Re s u l t s The r e c o v e r i e s of p l a t e l e t s and of p a r t i c l e s of a s i z e greater than p l a t e l e t s were again ta b u l a t e d f o r t h i s s e r i e s o f experiments i n v o l v i n g 35 CCD of i n v i v o S-sulphate l a b e l e d , density separated p l a t e l e t s (Appendix Table A.42). 35 CCD of the S-cohort i n v i v o l a b e l e d LDP, IDP, and HDP were again p l o t t e d (Appendix F i g u r e s A.5 through A.8) and the d i s t r i b u t i o n s o f s p e c i f i c a c t i v i t y were compared (Table 3.10.2.1). Table 3.10.2.1 A n a l y s i s of ^ 5S S p e c i f i c A c t i v i t y Enrichment Slope Day Two LDP CPM/Plt IDP CPM/Plt HDP CPM/Plt CE% 10 10 10 Mean 8.93 x IO" 7 1.34 x IO" 6 1.59 x 10~ 6 + S.Dev. 3.08 x IO" 7 3.19 x 10~ 7 5.82 x IO" 7 n 5 8 6 Corr -0.913 -0.928 -0.911 Slope -1.78 x 10~ 7 -1.21 x IO" 7 -2.83 x IO" 7 I n t 1.25 x 10~ 6 1.76 x 10~ 6 2.30 x IO" 6 P 0.5 0.040 0.20 Day S i x CE% 100 100 100 Mean 1.35 x IO" 6 1.59 x IO" 6 1.85 x IO" 6 +_ S.Dev. 1.92 x IO" 7 2.92 x IO" 7 1.21 x IO" 7 n 4 9 3 The recovery on the i n d i v i d u a l d e n s i t y gradient steps of p l a t e l e t s (Appendix Table A.43) and p l a t e l e t bound r a d i o a c t i v i t i e s (Appendix Table A.44) were tabu l a t e d f o r day two p . i . , o f another s e r i e s of experiments i n v o l v i n g CCD of ^H-thymidine i n v i v o and -^S-sulphate - 127 -i n v i v o double-labeled, d e n s i t y separated p l a t e l e t s . This s e r i e s of experiments was intended as an a d d i t i o n a l t e s t f o r the presence and l o c a t i o n of c e l l u l a r contamination w i t h i n p l a t e l e t p reparations subjected to d e n s i t y f r a c t i o n a t i o n and CCD. 3.11 CCD of 35-S-Labeled P l a t e l e t s , Double Labeled with 3-H-Serotonin In t h i s s e r i e s , p l a t e l e t cohorts were double-labeled, i n v i v o with 35 a high l e v e l of S-sulphate, and i n v i t r o with a low l e v e l of 3 H - s e r o t o n i n , and subjected to countercurrent d i s t r i b u t i o n . To improve the s e n s i t i v i t y of the method, i n a d d i t i o n t o CCD analyses of the cohort l a b e l r e l a t i v e to the p l a t e l e t p a r t i c l e number, 35 the in_ v i v o S-sulphate age-labeled p l a t e l e t s (Days 1,3,5 and 2,4,6 p . i . ) were a l s o l a b e l e d with 3 H - s e r o t o n i n i n v i t r o , p r i o r t o CCD analyses. Because s e r o t o n i n l a b e l i n g was l e v e l throughout the d i s t r i b u t i o n ( i . e . , p l a t e l e t s o f a l l p a r t i t i o n c o e f f i c i e n t s w i t h i n the d i s t r i b u t i o n take up s e r o t o n i n e q u a l l y ) s e r o t o n i n was considered to be a b e t t e r i n d i c a t o r of p l a t e l e t s than was e l e c t r o n i c p a r t i c l e counting, c o n s i d e r i n g the problems of counting coincidence and aggregation i n 35 3 polymer s o l u t i o n s . The d i s t r i b u t i o n of t h i s 5/ H r a t i o i n d i c a t e d t h a t CCD (without p r i o r p l a t e l e t d e n s i t y s u b f r a c t i o n a t i o n ) was capable of d i f f e r e n t i a t i n g p l a t e l e t age c h a r a c t e r i s t i c s . 3.11.1 Experimental 35 Sulphate I n j e c t i o n : Rabbits were i n j e c t e d with S-sulphate (Appendix Table A.45), and blood samples were taken from each of the animals on days 5 and 6 of the experiment. Samples were a l s o taken and s t u d i e d on days 11,13,15 and 12,14,16 (not shown). P l a t e l e t s (harvested on two successive days) from three r a b b i t s - 128 -3 3 were l a b e l e d by i n c u b a t i o n with H-serotonin (5-Hydroxy G- H tryptamine, c r e a t i n i n e s u l p h a t e , 500 mCi/mmole, 1 ml, 0.1 mCi, TRA .223, Batch 66, Amersham), washed with Tyrode's, and subjected to CCD. P l a t e l e t s and r a d i o a c t i v i t y ( P h i l i p s 4700 counter, 3H and ^ C program) were determined. 3.11.2 Re s u l t s 35 CCD d i s t r i b u t i o n s of the i n vivo staggered S-cohorts, l a b e l e d i n v i t r o with "^H-serotonin were p l o t t e d (Appendix F i g u r e s A.9 through A.12; and Figures 3.11.2.1 and 3.11.2.2), and the d i s t r i b u t i o n s of 35 3 S/ H r a t i o s which e x h i b i t e d l e s s than e i t h e r 3% or 10% combined e r r o r (CE) compared (Tables 3.11.2.1 and 3.11.2.2). Table 3.11.2.1 A n a l y s i s of 3 5 S / 3 H Ra t i o Slope, CCD Enrichment 10% CE Day One Ra t i o Day Three R a t i o Day F i v e R a t i o Mean 2.33 x IO" 3 5.93 x IO"-3 6.42 x 10~ 3 + S.Dev. 3.65 x 10-4 8.57 x IO" 4 6.91 x IO" 4 n 9 11 11 3% CE Mean 2.24 x IO" 3 5.50 x IO" 3 6.10 x IO" 3 + S.Dev. 1.54 x IO" 4 2.70 x I O - 4 2.23 x 10~ 4 n 4 7 7 Corr -0.815 -0.929 0.176 Slope -9.70 x 10~ 5 -1.16 x I O - 4 -1.82 x 10~ 5 Table 3. 11.2.2 A n a l y s i s of 3 5 S / 3 H Ra t i o Slope, CCD Enrichment 10% CE Day Two R a t i o Day Four R a t i o Day S i x Ra t i o Mean 3.66 x 10~ 3 6.66 x 10"^ 4.81 x 10~ 3 +_ S.Dev. • 2.51 x IO" 4 3.52 x IO" 3 2.45 x IO" 4 n 11 15 11 3% CE Mean 3.74 x IO" 3 4.56 x IO" 3 4.75 x IO" 3 + S.Dev. 1.63 x IO" 4 1.34 x IO" 4 1.33 x 10~ 4 n 6 7 8 Corr -0.810 -0.844 0.542 Slope -7.06 x IO" 5 -5.25 x IO" 5 2.94 x IO" 5 - 129 -Fig u r e 3.11.2.1 CCD of Day One, Three, and F i v e , 3 5 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 5 S / 3 H at 3% CE per CCDC (dotted l i n e ) with mean at one h a l f the graph h e i g h t ; CCDC 1-120 a b s c i s s a . Figure 3.11.2.2 CCD of Day Two, Four, and S i x , 3 3 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 5 S / 3 H at 3% CE per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . - 130 -3.11.3 D i s c u s s i o n 35 In v i v o S-sulphate age-labeled p l a t e l e t s harvested on days 1,3,5, and 2,4,6, p . i . were a l s o l a b e l e d with ^H-serotonin i n v i t r o , and subjected to simultaneous CCD analyses. Comparing the r a t i o of a p l a t e l e t in_ v i v o cohort l a b e l to another i n v i t r o p l a t e l e t p o p u l a t i o n l a b e l was more s e n s i t i v e than a comparison to p l a t e l e t p a r t i c l e number, as p r e v i o u s l y discussed. 35 3 On day one the slope of S/ H r a t i o through the CCD p l a t e l e t d i s t r i b u t i o n was negative, i n d i c a t i n g that the f i r s t p l a t e l e t s , l a b e l e d 35 i n the bone marrow a f t e r i n j e c t i o n of the S-sulphate cohort l a b e l , and newly re l e a s e d i n t o the blood c i r c u l a t i o n were of low p a r t i t i o n r e l a t i v e to the normal ageing p l a t e l e t p opulation d i s t r i b u t i o n , a l l o f which take up s e r o t o n i n , r e l a t i v e l y evenly. On day two, and on day 35 3 three e s p e c i a l l y (P l e s s than 0.03), t h i s S/ H enrichment of cohort l a b e l e d p l a t e l e t s w i t h a low p a r t i t i o n , became i n c r e a s i n g l y apparent, and on day four the slope was s t i l l n u m e r i c a l l y negative, however s l i g h t l y l e s s than on day three and was not s t a t i s t i c a l l y s i g n i f i c a n t . On day f i v e , the ^S/^H slope was zero, or h o r i z o n t a l and on day s i x t h i s slope became p o s i t i v e , i n d i c a t i n g a s h i f t i n the p a r t i t i o n o f the cohort l a b e l e d p l a t e l e t s . At t h i s time, the c i r c u l a t i n g p l a t e l e t 35 S-sulphate r a d i o a c t i v i t y neared i t s peak (see F i g u r e s 3.11.2.1 and 3.11.2.2), i n d i c a t i n g that cohort l a b e l e d p l a t e l e t s were both maturing ageing, and being removed from the c i r c u l a t i o n , while s t i l l being newly released from the bone marrow. 35 3 The presence, and s h i f t of the S/ H slope through the CCD p l a t e l e t d i s t r i b u t i o n were i n d i c a t i o n s of the f a c t t h a t the enrichment - 131 -35 of S-sulphate cohort l a b e l e d p l a t e l e t , to the l e f t of the d i s t r i b u t i o n was an enrichment of young p l a t e l e t s , newly released i n t o the c i r c u l a t i o n , and was a b s o l u t e l y not due to the presence of p l a t e l e t aggregates. That these s h i f t s i n the 3 5 S / 3 H slopes were observed f o r d i f f e r e n t p l a t e l e t p o p u l a t i o n s being compared simultaneously w i t h i n a s i n g l e CCD, and were repeated the day a f t e r , a l s o lend great v a l i d i t y to t h i s approach and these i n t e r p r e t a t i o n s . 3.12 CCD of 35-S-Labeled P l a t e l e t s , Double Labeled with 3-H-Serotonin To r e t e s t the f i n d i n g s of the previous S e c t i o n (3.11; i . e . , the young " 5 5 S - l a b e l e d p l a t e l e t cohort was of lower p a r t i t i o n than the 35 p l a t e l e t p o p u l a t i o n ) , another r a b b i t was i n j e c t e d with S-sulphate, and blood was taken on day three p . i . the time during which the 35 3 magnitude of S/ H slope was the g r e a t e s t , and t h e r e f o r e the enrichment of young p l a t e l e t s was most pronounced. P l a t e l e t s were harvested, l a b e l e d i n v i t r o with ^H-serotonin, and subjected to 120 t r a n s f e r CCD p r o v i d i n g the greatest p o s s i b l e r e s o l u t i o n without i n t r o d u c i n g p o p u l a t i o n o v e r l a p . 3.12.1 Experimental Approximately 60 ml of blood ( i n ACD) were taken from one r a b b i t , t h r ee days p . i . of 3 3 S i r i v i v o . P l a t e l e t s (2.38 x l O " ^ p l a t e l e t s ; 98.75% p l a t e l e t s , and 1.25% p a r t i c l e s of greater s i z e ) were harvested from 30 ml of PRP, l a b e l e d i n v i t r o , and subjected to CCD. A t o t a l of 1.49 x 1 0 1 0 p l a t e l e t s (90.7% p l a t e l e t s , and 9.27% p a r t i c l e s o f greater s i z e , counted i n the CCDLx) were d i s t r i b u t e d , and analyzed ( S e c t i o n 3.11.1). C y t o c e n t r i f u g e s l i d e s were prepared f o r each CCDF, and the d i s t r i b u t i o n and l e v e l o f c e l l u l a r contamination was determined. - 132 -3.12.2 R e s u l t s From data obtained by enumeration of c e l l u l a r content w i t h i n each CCDF, the t o t a l leukocytes was determined to be 6.17 x 10 5, assuming f u l l recovery i n the CCD which was loaded with 1.49 x 10"*"° p l a t e l e t s (a l e v e l of 0.004% contamination). The d i s t r i b u t i o n of leukocytes was found t o be predominately ( r e g i o n I I ) of lower p a r t i t i o n than p l a t e l e t s ( F i g u r e s 3.12.2.1 and 3.12.2.2). The CCD r a d i o a c t i v i t y d i s t r i b u t i o n s were p l o t t e d (Figures 3.12.2.3 35 3 and 3.12.2.4). The S/ H r a t i o slope was h i g h l y s i g n i f i c a n t (P = 0.002); and of greater magnitude (Table 3.12.2.1; Figures 3.12.2.4) a f t e r 120 t r a n s f e r CCD, than a f t e r 40 t r a n s f e r s (Table 3.11.2.1). Table 3.12.2.1 A n a l y s i s of 3 5 S / 3 H R a t i o Slope, CCD Enrichment Day Three R a t i o 10% CE Mean 7.39 x I O - 3 + S.Dev. 1.22 x I O - 3 n 19 Corr -0.909 Slope -1.97 x IO" 4 P 0.002 3.12.3 D i s c u s s i o n 35 P l a t e l e t s , three days p . i . S-sulphate, demonstrated the g r e a t e s t CCD enrichment, by comparison to a p o p u l a t i o n l a b e l , 3 H - s e r o t o n i n . CCD was capable of s u b f r a c t i o n a t i n g p l a t e l e t s i n t o d i f f e r e n t ages, c r e a t i n g at l e a s t a p a r t i a l p l a t e l e t age d i s t r i b u t i o n based on surface p r o p e r t i e s . Since these two-polymer phase systems are s e n s i t i v e to membrane surface charge, i t was concluded that changes i n surface p r o p e r t i e s were a s s o c i a t e d with p l a t e l e t s of d i f f e r e n t age. - 133 - -Figure 3.12.2.1 CCD Leukocyte Contamination of Day Three 3 5 S / 3 H - L a b e l e d P l a t e l e t s : leukocytes ( s o l i d l i n e histogram) per t o t a l CCDF, l e f t o r d i n a t e ; CCDC a b s c i s s a . 3.40E+04 r3. 45E+04 T" • I 0. 00E+00 100 110 120 Figure 3.12.2.2 CCD Leukocyte 3 5 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t per CCDC, l e f t o r d i n a t e ; leukocytes CCDF, r i g h t o r d i n a t e ; CCDC a b s c i s s a . Contamination and Day Three 3 H - s e r o t o n i n ( s o l i d l i n e ) dpm (dashed l i n e histogram) per t o t a l - 134 -Fi g u r e 3.12.2.3 CCD of Day Three 3 5 S / 3 H - L a b e l e d P l a t e l e t s : 3 H - s e r o t o n i n ( s o l i d l i n e ) dpm per CCDC, l e f t o r d i n a t e ; cohort 3 5 S (dashed l i n e ) , dpm per CCDC, r i g h t o r d i n a t e ; 3 5 S / 3 H r a t i o (dotted l i n e ) , mean r a t i o at h a l f graph height; CCDC a b s c i s s a . 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 F i g u r e 3.12.2.4 CCD of Day Three 3 5 S / 3 H - L a b e l e d P l a t e l e t s : 3 H - s e r o t o n i n ( s o l i d l i n e ) dpm per CCDC, l e f t o r d i n a t e ; cohort 3 5 S (dashed l i n e ) , dpm per CCDC, r i g h t o r d i n a t e ; 3 5 S / 3 H r a t i o at 10% CE (dotted l i n e ) , peak r a t i o at h a l f graph height; CCDC a b s c i s s a . - 135 -3.13 S i n g l e Step P a r t i t i o n of P l a t e l e t s During Cancer Chemotherapy P a t i e n t s being t r e a t e d f o r bone marrow cancer were studied as a model system to analyze the. membrane charge-associated surface p r o p e r t i e s of p l a t e l e t s as they aged w i t h i n the human c i r c u l a t i o n . . The p e r i p h e r a l blood p l a t e l e t count of p a t i e n t s undergoing chemotherapy and/or r a d i a t i o n therapy f o r a c t i v e myelogenous leukemia v a r i e s due to successive bouts of bone marrow suppression and regeneration brought on by the therapy and recovery process. By f o l l o w i n g the p l a t e l e t count, together with previous experience of the course of treatment and recovery, r e l a t i v e l y young and o l d p l a t e l e t subpopulations can be s e p a r a t e l y s e l e c t e d . Upon p r e s e n t a t i o n , the p e r i p h e r a l blood c e l l p i c t u r e i s one of l e u k o c y t o s i s , anemia, and thrombocytopenia. Due to common precursors, p r o l i f e r a t i o n from the white blood c e l l compartment causes "crowding out" or depression of the e r y t h r o c y t e and p l a t e l e t compartments w i t h i n the bone marrow. I n i t i a t i o n of treatment, which s t r a t e g i c a l l y and e f f e c t i v e l y i n h i b i t s r a p i d l y d i v i d i n g c e l l s , the megakaryocyte i n c l u d e d , causes p l a t e l e t production to d e c l i n e , and the c i r c u l a t i n g p l a t e l e t count decreases even f u r t h e r . For a p e r i o d of time, s i n c e no new p l a t e l e t s are being produced and those remaining i n the c i r c u l a t i o n are being removed by the r e t i c u l o e n d o t h e l i a l system, presumably as they age, only ageing p l a t e l e t s are present. Thus, with knowledge of the length of p l a t e l e t s u r v i v a l , about ten days i n humans, a f t e r an appropriate p e r i o d of time (e.g., seven days) post agent a d m i n i s t r a t i o n , one can o b t a i n a subpopulation s i g n i f i c a n t l y enriched i n i s o l o g o u s l y aged p l a t e l e t s and d e f i c i e n t i n newly produced p l a t e l e t s . E ventually there i s a p e r i o d of time, t r a n s i e n t however, during which there are very few - 136 -t o n e g l i g i b l e c i r c u l a t i n g p l a t e l e t s , and homologous p l a t e l e t t r a n s f u s i o n i s required to maintain b a s a l p e r i p h e r a l blood l e v e l s necessary to prevent hemorrhage. However there i s a l a g of approximately one day i n the f u n c t i o n i n g of these t r a n s f u s e d p l a t e l e t s . Again, i f a n t i n e o p l a s t i c therapy i s continued and i f one waits the a p p r o p r i a t e period of time post t r a n s f u s i o n , one can harvest a subpopulation of homologously aged p l a t e l e t s s i m i l i a r t o those above. Upon c e s s a t i o n of treatment and recovery from leukemia, bone marrow c e l l s begin to regenerate. U s u a l l y the normal prec u r s o r s stem c e l l s have p r e f e r e n t i a l c a p a b i l i t y over t h e i r leukemic c e l l counterparts and young p l a t e l e t production again ensues, r e s t o r i n g t h e i r c i r c u l a t i n g p e r i p h e r a l blood numbers. The p l a t e l e t count i s observed to s t e a d i l y i n c r e a s e and by sampling p e r i p h e r a l blood at t h i s time, a subpopulation enriched i n newly produced young p l a t e l e t s can be obtained. The p l a t e l e t count approaches normal w i t h i n a few days and even surpasses t h i s normal l e v e l s i n c e f o r some reason the bone marrow precursor c e l l s tend to overcompensate. Young and o l d p l a t e l e t subpopulations were harvested from p e r i p h e r a l blood taken at v a r i o u s stages through t h i s therapy and recovery c y c l e . These p l a t e l e t s were s i z e d , s i n c e p l a t e l e t s i z e has been shown to be and i n d i c a t i o n of p l a t e l e t age, heterogeneity, and abnormality (O'Brien and Jamieson, 1974; Goldschmidt and Sorland, 1976; Bessman et a l . , 1981a and b, 1982; Thompson et a l . , 1981a and b, 1982a and b, 1983a and b ) . P l a t e l e t s were a l s o examined f o r t h e i r r e l a t i v e charge-associated membrane s u r f a c e p r o p e r t i e s as r e f l e c t e d by t h e i r d i f f e r e n t i a l p a r t i t i o n i n charged two-polymer aqueous phase systems, s i n c e p l a t e l e t age has been shown to a f f e c t t h e i r p a r t i t i o n i n r a b b i t s , - 137 -and s i n c e p l a t e l e t s from malignant diseases have been shown i n one study t o be reduced by 24% i n membrane n-acetylneuraminic a c i d (Martin et a l . , 1982). P l a t e l e t membrane phospholipids have a l s o been shown to be abnormal i n m y e l o p r o l i f e r a t i v e d i s o r d e r s (Breuer et a l . , 1981; C a s t a l d i et a l . , 1982). D i f f e r e n c e s were detected both i n p l a t e l e t s i z e by l a s e r l i g h t s c a t t e r i n g , and i n membrane surface p r o p e r t i e s as r e f l e c t e d by a v a r i e t y o f two-phase systems with varying s a l t compositions and p o t e n t i a l d i f f e r e n c e s . 3.13.1 Experimental Specimens: Blood from p a t i e n t s undergoing chemotherapy f o r a c t i v e myelogenous leukemia (AML), was obtained from Vancouver General H o s p i t a l , Department of Hematology, taken by venepuncture and mixed with 3.8% sodium c i t r a t e (1 part/9 p a r t s b l o o d ) . Blood was a l s o taken i n a s i m i l a r manner from presumably normal l a b o r a t o r y personnel. P l a t e l e t P r e p a r a t i o n : Only p l a s t i c equipment was used throughout. Blood i n 10 ml volumes was c e n t r i f u g e d at 150 g f o r 20 minutes at room temperature. PRP, a f t e r being separated from the c e l l s , was a c i d i f i e d with 0.5-1.0 ml of ACD, c e n t r i f u g e d at 3000 g f o r 20 minutes and resuspended i n 0.5 ml Tyrode's s o l u t i o n . These resuspended p l a t e l e t s were used immediately f o r analyses of surface p r o p e r t i e s by p a r t i t i o n . S i n g l e Step P a r t i t i o n : Phase systems were composed by weight from stock s o l u t i o n s o f 20% w/w D T500 ( l o t 7830), 30% w/w PEG, and t w i c e - i s o t o n i c s o l u t i o n s of pH 7.2 sodium phosphate b u f f e r , and sodium c h l o r i d e . A set of f i v e phase systems (5/4:A-l through 5/4:E-l) with varying s a l t compositions was made by f i r s t preparing a s o l u t i o n of 10% D and 8% PEG (10/8) and then d i l u t i n g with d i f f e r e n t r a t i o s of twice i s o t o n i c s a l t s o l u t i o n s (Appendix Table A.8). - 138 -A s m a l l sample of p l a t e l e t s , 50 u l , of known p l a t e l e t c o n c e n t r a t i o n was added to each tube (Falcon p l a s t i c disposable tubes) of a set of phase systems, each composed of one ml PEG r i c h upper phase and one ml D r i c h lower phase, of v a r i o u s s a l t compostions. Mixing was by i n v e r s i o n , and s e t t l i n g was f o r one hour, a f t e r which the systems were assayed f o r p l a t e l e t s i n the upper phase by sampling (200 u l i n t o 10 ml D i l u i d ) and counting with the Electrozone Celloscope Model 112 CLTH/RWP (48 um diameter o r i f i c e , 100 u l v o l u m e t r i c , C 1, G 68, T 12.5-90). P l a t e l e t S i z i n g : P l a t e l e t s from normals and m y e l o p r o l i f e r a t i v e d i s o r d e r s (MPD) were s i z e d i n D i l u i d s a l i n e , using a l a s e r l i g h t s c a t t e r i n g (LS) c e l l counter (Ortho D i a g n o s t i c s ) with histogram p r i n t o u t . Histogram s i z i n g p l o t s were t a b u l a t e d , expressed as p l a t e t e t s o f each s i z e accumulated ( i n mm from the x a x i s ) versus% window. 3.13.2 R e s u l t s S t a t i s t i c a l Comparison of P a r t i t i o n s : The p a r t i t i o n s were expressed as the q u a n t i t y of p l a t e l e t s i n the upper phase as a percentage of the t o t a l p l a t e l e t s added. P l a t e l e t s from p a t i e n t s and normals were p a r t i t i o n e d and compared c o n c u r r e n t l y (Appendix Table A.47). P l a t e l e t s from a group of age/sex matched (to p a t i e n t s ) normals are a l s o p a r t i t i o n e d (Appendix Table A.48). The p a r t i t i o n s were then organized: MPD w i t h p l a t e l e t count d e c l i n i n g (PCD), MPD with p l a t e l e t count r i s i n g (PCR), normal p l a t e l e t s p a r t i t i o n e d c o n c u r r e n t l y with MPD, and p l a t e l e t s from matched normals p a r t i t i o n e d s e p a r a t e l y (Appendix Table A.49). Student t t e s t s (HP S t a t i s t i c s Volume 1 P a r t No. 09815-15000, Rev. C) were used to compare the p a r t i t i o n s of p l a t e l e t from normal s u b j e c t s , f o r which 22 p l a t e l e t p a r t i t i o n measurements were c o l l e c t e d i n phase systems A - l through D-1 (*only 18 i n phase system E-1) and the - 139 -p l a t e l e t p a r t i t i o n s of p a t i e n t s with MPD (10), (Table 3.13.2.1). In a d d i t i o n , MPD p a t i e n t s were d i v i d e d i n t o two c a t e g o r i e s based on the p l a t e l e t s u r v i v a l dynamics; e i t h e r with c i r c u l a t i n g p l a t e l e t count d e c l i n i n g (PCD; old) ageing a f t e r chemotherapy, or with p l a t e l e t count r i s i n g (PCR; young) during p l a t e l e t regeneration. At 95% l e v e l of s i g n i f i c a n c e (p = 0.05), there were no d i f f e r e n c e s i n p a r t i t i o n i n any of the phase systems f o r normals compared t o a l l of the p l a t e l e t s from MPD i f p l a t e l e t s u r v i v a l dynamics were disregarded (PCD and PCR). However, when p l a t e l e t s from MPD were subdivided i n t o PCD or PCR, there were d i f f e r e n c e s , compared to normals, found f o r MPD PCD using phase system 5/4:A-l; and d i f f e r e n c e s found f o r MPD PCR using phase systems 5/4:B-1 through 5/4:E-l. Table 3.13.2.1 Comparison of P l a t e l e t P a r t i t i o n s , Normal and MPD 5741 A ^ i BZJ (>1 f > l E^T Normal (22) * 55.7 + 14.9 47.0 + 14.5 43.2 + 18.0 29.9 + 13.5 19.8 + 10.4 MPD (10) 62.9 + 17.4 42.8 + 18.2 36.9 + 17.4 24.8 + 12.5 16.4 + 9.28 t 1.21 0.706 0.9377 1.0142 0.8545 df 30 30 30 30 26 P 0.1179 0.2428 0.1779 0.1593 0.2003 88.2% 75.7% 82.2% 84.1% 80.0% PCD (7) 72.0 + 10.9 52.6 + 10.1 45.1 + 13.4 30.6 + 10.2 20.9 + 7.2 t 2.66 0.943 0.254 0.111 0.251 df 27 27 27 27 23 P 0.0065 0.1771 0.4009 0.4562 0.402 99.4% 88.3% 59.9% 54.4% 59.8% PCR (3) 41.7 + .6.3 19.9 + 7.6 17.6 + 6.41 11.4 + 2.9 6.0 + 0.26 t 1.5784 3.1372 2.4093 2.322 2.2448 df 23 23 23 23 19 P 0.0641 0.0023 0.0122 0.0147 0.0184 93.6% 99.8% 98.8% 98.5% 98.2% - 140 -S t a t i s t i c a l Comparison of S i z e s : The mean modal s i z e of 6.40 _+ 1.14 (5) % window found f o r normal p l a t e l e t s , and a s i z e of 5.38 + 0.58 (8) % window f o r MPD p l a t e l e t s , was a s i g n i f i c a n t (p = 0.03) d i f f e r e n c e . 3.13.3 S p e c i f i c D i s c u s s i o n P a r t i t i o n s t u d i e s on p l a t e l e t s from cancer p a t i e n t s with various diagnoses (acute myelogenous leukemia, acute leukemia, leukemia-acute leukemia, leukemia, and lymphoma) whose p l a t e l e t counts had been d r a s t i c a l l y reduced by chemotherapy have shown th a t d i f f e r e n c e s i n s u r f a c e p r o p e r t i e s were evident between normal c o n t r o l s , p l a t e l e t s taken when the count i s low and d e c l i n i n g , and those taken when the count i s i n c r e a s i n g due to renewed production. The treatment, a combination of thioguanine, c y t o s i n e a r a b i n o s i d e and daunorubicin (TAD/TRAMCO), e r a d i c a t e s p l a t e l e t s y n t h e s i s i n the bone marrow f o r v a r y i n g l e n g t h s of time ( s e v e r a l days to weeks), although the drugs are c l e a r e d much more r a p i d l y (Young et a l . , 1981). F o l l o w i n g therapy, no new p l a t e l e t s are formed; the p l a t e l e t s i n the c i r c u l a t i o n become p r o g r e s s i v e l y o l d e r with time and are removed, decreasing the c i r c u l a t i n g p l a t e l e t count. About seven days post-therapy, a l l p l a t e l e t s i n the c i r c u l a t i o n are s u b s t a n t i a l l y aged. I f the p l a t e l e t count i s observed to r i s e , i t i s an i n d i c a t i o n t h a t p l a t e l e t production has resumed, r e l e a s i n g new p l a t e l e t s i n t o the c i r c u l a t i o n . Samples taken e a r l y i n t h i s process w i l l be enriched i n young p l a t e l e t s . S i n g l e tube p a r t i t i o n s were performed i n a s e r i e s of 5% D, 4% PEG systems c o n t a i n i n g varying i s o t o n i c r a t i o s of phosphate to c h l o r i d e , with v a r y i n g p o t e n t a l d i f f e r e n c e s between the upper and lower phases. High phosphate c o n t a i n i n g systems have been shown to be s e n s i t i v e t o - 141 -c e l l s u r f a c e charge, whereas the p a r t i t i o n i n c h l o r i d e - r i c h systems has been c o r r e l a t e d with membrane l i p i d a s s o c iated p r o p e r t i e s i n red c e l l s ( h y d r o p h o b i c i t y , l i p i d p a c k i n g ) . P l a t e l e t s were taken at v a r i o u s stages through a chemotherapy and recovery c y c l e i n order t o c h a r a c t e r i z e t h e i r r e l a t i v e membrane charge-associated surface p r o p e r t i e s . "Old" p l a t e l e t s , obtained from p a t i e n t s whose p l a t e l e t count was d e c l i n i n g (PCD) as a r e s u l t of therapy, e x h i b i t e d a p a r t i t i o n c o e f f i c i e n t s l i g h t l y higher than normal p l a t e l e t s i n one phase system (72% compared t o 63%, i n 5/4:A-l, r e s p e c t i v e l y . The few "young" p l a t e l e t subpopulations obtained during bone marrow regeneration and the r i s i n g p l a t e l e t count (PCR), the recovery phase, e x h i b i t e d lower than normal p a r t i t i o n c o e f f i c i e n t s i n four o f the f i v e phase systems t e s t e d (20% compared t o 47%, i n 5/4:B-l; 18% compared to 43%, i n 5/4-.C-1; 11% compared t o 30%, i n 5/4:D-l; and 6% compared to 20%, i n 5 / 4 : E - l ) . When the p a r t i t i o n c o e f f i c i e n t s from a l l MPD, PCD and PCR were pooled and t e s t e d together against the p a r t i t i o n s of normals, no s i g n i f i c a n t d i f f e r n c e s were found i n any of these phase systems. That p l a t e l e t s from p a t i e n t s with MPD, taken from the r i s i n g phase during recovery from chemotherapy, demonstrated a lower surface charge a s s o c i a t e d p a r t i t i o n c o e f f i c i e n t than normal p l a t e l e t s , suggests that lower membrane surface charge i s ass o c i a t e d with young p l a t e l e t s . However there are other e x p l a n a t i o n s : p l a t e l e t s and megakaryocytes from cancer p a t i e n t s have been shown to be a t y p i c a l (Rodman et a l . , 1978; Cooper et a l . , 1978; Gerwitz et a l . , 1983). P l a t e l e t s produced i n response to t h i s extreme c o n d i t i o n of thrombocytopenia are analogous to s.tress-induced r e t i c u l o c y t e s i n hemopoiesis which have been shown to - 142 -d i f f e r i n t h e i r surface p r o p e r t i e s , as detected by p a r t i t i o n , from r e t i c u l o c y t e s produced normally. The chemotherapeutic agents themselves (Young e_t a l . , 1981), through metabolites or i n d i r e c t e f f e c t s , might be r e s p o n s i b l e f o r the observed behavior. Tumor c e l l s and leukemic b l a s t s are a l s o known t o a f f e c t p l a t e l e t surface p r o p e r t i e s , a f f e c t i n g aggregation (Gasic et a l . , 1977; Zawilska et a l . , 1981). The r e s u l t s are of c o n s i d e r a b l e i n t e r e s t because i f production o f new p l a t e l e t s can be detected at an e a r l y stage, using t h i s p a r t i t i o n approach or p l a t e l e t s i z i n g ( p l a t e l e t s from the MPD p a t i e n t s are sm a l l e r than normal), i t would provide c l i n i c a l i n f o r m a t i o n which would be u s e f u l i n the management of these p a t i e n t s . - 143 -GENERAL DISCUSSION AND CONCLUSION The p o t e n t i a l of p a r t i t i o n i n g and countercurrent d i s t r i b u t i o n (CCD) of p l a t e l e t s i n two-polymer aqueous phase systems to d i s t r i b u t e p l a t e l e t s according to age was demonstrated. In a d d i t i o n , evidence was presented which i n d i c a t e s t h a t the p l a t e l e t p o p u l a t i o n i s heterogeneous based on analyses of membrane surface p r o p e r t i e s . The method, s e l e c t e d i n t h i s case to separate p l a t e l e t s on the b a s i s of membrane charge-associated p r o p e r t i e s , has been shown to d i s t r i b u t e other c e l l s on the b a s i s of i n v i v o age ( S e c t i o n 1.6). In order t o assess t h i s CCD technique f o r p l a t e l e t age d i s t r i b u t i o n a n a l y s i s , s e v e r a l means of l a b e l i n g and o b t a i n i n g p l a t e l e t s of v a r i o u s ages were examined. Since p l a t e l e t s of v a r y i n g density have been shown to have d i f f e r e n t age-related c h a r a c t e r i s t i c s ( S e c t i o n 1.3) t h e i r p a r t i t i o n behavior was i n v e s t i g a t e d by comparative CCD of d e n s i t y separated p l a t e l e t s using specimens from r a b b i t , man, and monkey. The r e s u l t s ( S e c t i o n 3.2) i n two-polymer phase systems known to be s e n s i t i v e to charge-associated membrane p r o p e r t i e s showed that f o r human and r a b b i t , low d e n s i t y p l a t e l e t f r a c t i o n s ( c o n t a i n i n g o l d p l a t e l e t s ) were of lower p a r t i t i o n than intermediate d e n s i t y p l a t e l e t f r a c t i o n s ( c o n t a i n i n g p l a t e l e t s of mean age), while high d e n s i t y p l a t e l e t s were of v a r i a b l e p a r t i t i o n . The CCD d i s t r i b u t i o n s of a l l three d e n s i t y subpopulations overlapped and were i n d i c a t i v e of surface property heterogeneity w i t h i n the i n d i v i d u a l subpopulations, e s p e c i a l l y evident i n the high d e n s i t y p l a t e l e t s . Examination of p l a t e l e t s i z e ( S e c t i o n 3.3) showed that the modal volumes, of p l a t e l e t s from both r a b b i t s and humans, increased - 144 -with i n c r e a s i n g d e n s i t y . Rhesus monkey p l a t e l e t s were an exception. Rabbit p l a t e l e t s , l a b e l e d i n v i t r o with ^ C r , were stud i e d as a f u n c t i o n of s u r v i v a l time i n v i v o , a f t e r i n j e c t i o n . This n u c l i d e i s used i n the standard c l i n i c a l s u r v i v a l time t e s t and f o r experimental p l a t e l e t s u r v i v a l s t u d i e s ( S e c t i o n 1.3 and 1.7). In v i t r o , 5 * C r l a b e l i n g permits the i d e n t i f i c a t i o n of the e n t i r e p l a t e l e t p o p u l a t i o n at a s p e c i f i c time p o i n t , and i t should be p o s s i b l e t o f o l l o w changes i n the surface p r o p e r t i e s of t h i s population as a f u n c t i o n of c i r c u l a t i o n time, or i n v i v o ageing, s i n c e p l a t e l e t s newly released from the marrow should be unlabeled. CCD of 5 1 C r l a b e l e d r a b b i t p l a t e l e t s , at v a r i o u s i n v i v o ageing times a f t e r intravenous i n j e c t i o n , were s t u d i e d to compare the surface p r o p e r t i e s of aged, l a b e l e d p l a t e l e t s , with those of the t o t a l normal unlabeled p l a t e l e t p o p u l a t i o n ( S e c t i o n 3.6). Immediately a f t e r i n v i t r o 5 ^ C r p l a t e l e t l a b e l i n g , the CCD d i s t r i b u t i o n revealed heterogeneity i n uptake of r a d i o a c t i v i t y by p l a t e l e t s of a s p e c i f i c p a r t i t i o n , lower than the mean f o r the 51 p o p u l a t i o n . This heterogeneity i n uptake of Cr by a subpopulation of p l a t e l e t s of s p e c i f i c p a r t i t i o n was demonstrable f o r r a b b i t and human p l a t e l e t s , and was confirmed by d o u b l e - l a b e l i n g . A f t e r i n j e c t i o n and in_ v i v o c i r c u l a t i o n i n the r a b b i t t h i s p l a t e l e t p o p u l a t i o n 51 51 e x h i b i t i n g enhanced uptake f o r Cr r a p i d l y decreased. As the Cr l a b e l e d p l a t e l e t p o p u l a t i o n aged, the t o t a l r a d i o a c t i v i t y became p r o g r e s s i v e l y lower, such t h a t the d e t a i l s of the CCD r a d i o a c t i v i t y d i s t r i b u t i o n could not be d i s t i n g u i s h e d . I t has been shown that the l a r g e dense p l a t e l e t s are most a c t i v e i n terms of aggregation a b i l i t y , and can be p r e f e r e n t i a l l y l a b e l e d at very low c o n c e n t r a t i o n s of ^ C r ( i . e . , greater uptake and s p e c i f i c - 145 -a c t i v i t y than the m a j o r i t y of the p l a t e l e t p o p u l a t i o n ) but not at s u c c e s s i v e l y higher c o n c e n t r a t i o n s (Section 1.3). I t appeared that t h i s subpopulation e x h i b i t s a c t i v e uptake f o r "^Cr at these low c o n c e n t r a t i o n s . The CCD p a t t e r n s and s p e c i f i c a c t i v i t y d i s t r i b u t i o n s of r a b b i t p l a t e l e t s , double l a b e l e d with 5 ^ C r and "'""'""'"In at various 51 c o n c e n t r a t i o n s of l a b e l i n c l u d i n g those where a c t i v e uptake f o r Cr had been reported to occur ( S e c t i o n 3.7), showed th a t a subpopulation with d i s t i n c t surface p r o p e r t i e s c h a r a c t e r i z e d by a r e l a t i v e l y low p a r t i t i o n i n a charged two-phase system, was s t r o n g l y enriched i n 5"*"Cr but not i n '^"''In r e l a t i v e to the p o p u l a t i o n average s p e c i f i c a c t i v i t y . The enrichment was s a t u r a b l e , as p r o g r e s s i v e l y higher l e v e l s o f ^ C r reduce the enrichment, a phenomenon which was not observed with " ^ I n nor with "^C-serotonin (used as standards f o r p l a t e l e t CCD), nor f o r 3H-adenine (not shown). The c h a r a c t e r i s t i c s of t h i s dense p l a t e l e t subpopulation which 51 e x h i b i t e d " a c t i v e " uptake f o r Cr and demonstrated a low (charge-a s s o c i a t e d ) p a r t i t i o n c o e f f i c i e n t was f u r t h e r i n v e s t i g a t e d . Rabbit 51 p l a t e l e t s were l a b e l e d with Cr, at low concentrations at which a c t i v e uptake occurs, and w i t h "''"'""''In and "^C s e r o t o n i n f o r which t h i s phenomenon had not been reported. These p l a t e l e t s were separated i n t o subpopulations according t o d e n s i t y and then analyzed by CCD i n two-polymer aqueous phase systems. In these cases, a d i r e c t c o r r e l a t i o n of i n c r e a s i n g modal (s u r f a c e charge s e n s i t i v e ) p a r t i t i o n with p l a t e l e t d e n s i t y was found. However, the dense p l a t e l e t subpopulation which e x h i b i t e d enhanced uptake f o r *^Cr, but not f o r " ^ I n nor " ^ C - s e r o t o n i n , was again found to have a low p a r t i t i o n , unexpected according to the young dense p l a t e l e t model. - 146 -Using 3 5 S - s u l p h a t e intravenous i n j e c t i o n i n t o r a b b i t s , a young cohort of p l a t e l e t s was l a b e l e d (Sections 3.8 through 3.12). Sulphate i s i n c o r p o r a t e d i n t o the megakaryocyte w i t h i n the bone marrow and only p l a t e l e t s which are newly r e l e a s e d i n t o the c i r c u l a t i o n appear l a b e l e d , while ageing p l a t e l e t s remain unlabeled and are being removed (Se c t i o n 3.8). Both of these processes occur simultaneously and e v e n t u a l l y the 35 l a b e l e d cohort ages. Various combinations of t h i s i n v i v o S cohort l a b e l i n g technique with i n v i t r o whole p o p u l a t i o n l a b e l i n g ( i . e . , with "^H-serotonin as a standard f o r comparison), d e n s i t y s e p a r a t i o n , and p l a t e l e t membrane surface property a n a l y s i s provided i n s i g h t s i n t o the i n t e r r e l a t i o n s of p l a t e l e t age, d e n s i t y , and membrane p r o p e r t i e s . On day two post i n j e c t i o n ( p . i . ) , the i n v i v o "^S-sulphate l a b e l e d young p l a t e l e t cohort was found to be enriched i n the high d e n s i t y p l a t e l e t (HDP) f r a c t i o n obtained by S t r a c t a n d e n s i t y gradient c e n t r i f u g a t i o n ; the opposite was found i n the low density p l a t e l e t (LDP) f r a c t i o n . On day s i x p . i . , t h i s d i f f e r e n c e i n density of 35 S-sulphate l a b e l e d p l a t e l e t s was l e s s dramatic ( S e c t i o n 3.9 and 3.10). These f i n d i n g s were i n t e r p r e t e d to mean that p l a t e l e t s decreased t h e i r mean den s i t y as a f u n c t i o n of age i n the c i r c u l a t i o n , the young being enriched i n HDP and the o l d c o n s i s t i n g predominately of LDP. P l a t e l e t membrane surface property analyses by CCD of both d e n s i t y separated and unseparated "^S-sulphate l a b e l e d p l a t e l e t s i n d i c a t e d that the young p l a t e l e t cohort not only had d i f f e r e n t d e n s i t y c h a r a c t e r i s t i c s but a l s o had d i s t i n c t i v e surface p r o p e r t i e s which were more pronounced on day two i n comparison to day s i x . The s p e c i f i c a c t i v i t y was c o n s i s t e n t l y enriched i n the low p a r t i t i o n region of CCD d i s t r i b u t i o n curves of p l a t e l e t s (even without p r i o r d e n s i t y - 147 -f r a c t i o n a t i o n ) when drawn two or three days p . i . , an enrichment which decreased on subsequent days (Se c t i o n s 3.11 and 3.12). P l a t e l e t s from cancer p a t i e n t s ( S e c t i o n 3.13) were studied as a model o f p l a t e l e t ageing i n v i v o . The p e r i p h e r a l blood p l a t e l e t count of p a t i e n t s undergoing chemotherapy f o r leukemia v a r i e s due t o suc c e s s i v e bouts of bone marrow suppression and regeneration brought on by the therapy and recovery process. I n i t i a t i o n of treatment e r a d i c a t e s p l a t e l e t s y n t h e s i s and f o r a p e r i o d o f time only ageing p l a t e l e t s are present and are g r a d u a l l y removed from the c i r c u l a t i o n . Cessation of treatment permits the production of young p l a t e l e t s and t h e i r p e r i p h e r a l blood numbers are once again r e s t o r e d . P l a t e l e t s taken at va r i o u s stages i n the therapy and recovery c y c l e were examined by s i n g l e tube p a r t i t i o n i n order to c h a r a c t e r i z e t h e i r r e l a t i v e charge-a s s o c i a t e d surface p r o p e r t i e s of human p l a t e l e t s of d i f f e r e n t ages. Knowledge of an a l t e r a t i o n i n p l a t e l e t membrane surface charge with p l a t e l e t ageing, as examined i n the work of othe r s , i s l i m i t e d to analyses of p l a t e l e t membrane s i a l i c a c i d cleaved by enzymes and to e l e c t r o p h o r e t i c m o b i l i t y measurements. Studies have been c a r r i e d out on p l a t e l e t s separated according t o other p h y s i c a l p r o p e r t i e s such as d e n s i t y and s i z e , combinations of density and s i z e , and combinations of de n s i t y and e l e c t r o p h o r e s i s , a l l of which are thought to provide at l e a s t p a r t i a l p l a t e l e t age based separations. C e r t a i n of the f i n d i n g s do c o n f l i c t and not only because of the d i f f e r e n c e s i n t o t a l experimental systems; there are admittedly technique (e.g., g r a d i e n t ) i n c o n s i s t e n c i e s and p o s s i b l e technique (e.g., g r a d i e n t ) induced changes i n p l a t e l e t s . Also most of the c o r r e l a t i o n s which do e x i s t between p l a t e l e t age and p l a t e l e t d e n s t i y are incomplete, i n that there i s a - 148 -young p l a t e l e t d e n s i t y d i s t r i b u t i o n which i s only s l i g h t l y s h i f t e d i n the d i r e c t i o n of higher d e n s i t y , i . e . , there are p l a t e l e t s which are young and of low d e n s i t y , but with the mean d e n s i t y f o r young p l a t e l e t s being higher. The megakaryocyte-produced p l a t e l e t heterogeneity i s a l s o i n t e r e s t i n g and worth c o n s i d e r i n g as a p a r t i a l e x p l a i n a t i o n of the overlaps of p l a t e l e t d e n s i t y , s i z e and surface p r o p e r t i e s as demonstrated i n t h i s t h e s i s . The evidence t h u s f a r , based on data found i n a b s t r a c t s (Carty et a l . , 1975; Isobe, 1976; Isobe and Yamanaka, 1976) and i n a t h e s i s (Rand, Ph.D. 1983), i n d i c a t e s that t o t a l p l a t e l e t s urface charge and surface charge d e n s i t y , assayed by p l a t e l e t e l e c t r o p h o r e s i s and neuraminidase cleavage of s i a l i c a c i d , should c o r r e l a t e d i r e c t l y with p l a t e l e t d e n s i t y which i s thought to be i n v e r s e l y r e l a t e d to p l a t e l e t age. Therefore, p r o v i d i n g the p r o p e r t i e s of HDP do r e f l e c t those of young p l a t e l e t s , HDP ( c o n t a i n i n g young p l a t e l e t s ) should be of higher net n e g a t i v e , higher t o t a l s u r f a c e charge and surface charge per u n i t area. The opposite should be t r u e f o r LDP ( c o n t a i n i n g o l d p l a t e l e t s ) r e l a t i v e to the mean f o r the p l a t e l e t p o p u l a t i o n . P l a t e l e t s upon s t i m u l a t i o n with thrombin have been shown t o decrease i n d e n s i t y ( C i e s l a r et a l . , 1979). A f t e r s t i m u l a t i o n with ADP, they demonstrate an in c r e a s e i n neuraminidase-susceptible s i a l i c a c i d , i n d i c a t i n g an in c r e a s e i n t o t a l surface charge (Chiu, 1983). I t i s p o s s i b l e that such i n v i t r o s t i m u l a t i o n does not r e f l e c t p l a t e l e t -e n d o t h e l i a l - p l a t e l e t encounters w i t h i n the c i r c u l a t i o n , but i f i t does then i n v i v o s t i m u l a t e d p l a t e l e t s could demonstrate lower mean d e n s i t y and higher r e l a t i v e s urface charge than unstimulated p l a t e l e t s . A l l analyses of p l a t e l e t s and den s i t y separated p l a t e l e t - 149 -subpopulations r e l y on the assumptions that the p l a t e l e t p r e p a r a t i o n s were f r e e of aggregates and c e l l contamination. These assumptions are o f t e n i n v a l i d as great care must be taken to o b t a i n pure p l a t e l e t p r e p a r a t i o n s . The d i f f e r e n c e s observed i n the p r o p e r t i e s of p l a t e l e t s (e.g., o f d i f f e r e n t d e n s i t y ) are small and could o f t e n be explained or a l t e r e d by the presence of contamination (e.g., p l a t e l e t aggregates and/or leukocytes) of higher d e n s i t y than p l a t e l e t s . Separations of blood c e l l s according t o s i z e by d i f f e r e n t i a l c e n t r i f u g a t i o n i s seldom s u f f i c i e n t f o r a complete separation o f p l a t e l e t s from contaminants and v i s u a l e s t i m a t i o n of p u r i t y at best i s only an estimate. However, a c e r t a i n degree of accuracy can be obtained using the methods o u t l i n e d h e r e i n ( S e c t i o n 3.6 and 3.12). I t i s wise to monitor f o r the presence of c e l l u l a r contamination throughout a l l c e l l s e p a r a t i o n procedures, although i t s presence may be i n s i g n i f i c a n t r e l a t i v e to the phenomenon sought. This was the case f o r r a b b i t e r y t h r o c y t e contamination i n the present work, i t being of such low p a r t i t i o n as not to i n t e r f e r e w i t h any CCD r e s u l t regarding p l a t e l e t s . However, t h i s was not the case f o r leukocytes, which were monitored c a r e f u l l y by microscopy. In a d d i t i o n to these, other methods were used h e r e i n t o d e f i n e the "pure p l a t e l e t " p o p u l a t i o n f o r comparison with the age - r e l a t e d subpopulation, p a r t i c u l a r l y double l a b e l i n g techniques using one r e l a t i v e l y s p e c i f i c p l a t e l e t l a b e l and one more general l a b e l . That p a r t i t i o n i s s e n s i t i v e to c e l l membrane s u r f a c e charge i s w e l l documented ( S e c t i o n 1.5 through 1.7). However, the p r e c i s e character of the s u r f a c e charge detected by p a r t i t i o n may be d i f f e r e n t from that detected by e l e c t r o p h o r e s i s or surface s i a l i c a c i d analyses (Walter and Seaman, 1971). The e l e c t r o p h o r e t i c m o b i l i t y depends i n a complex way on - 150 -the d i s t r i b u t i o n of charge on membrane g l y c o p r o t e i n s and g l y c o l i p i d s (Seaman and Brooks 1977; Levine, et a l . , 1983) and no d e t a i l e d a n a l y s i s of the r e l a t i o n between the two has been c a r r i e d out f o r p l a t e l e t s . Membrane s i a l i c a c i d analyses by enzymatic cleavage assays the r e l e a s e of t e r m i n a l s i a l i c a c i d , predominately from i n t e g r a l membrane g l y c o p r o t e i n s . A l a r g e f r a c t i o n of membrane sur f a c e charge appears to d e r i v e from groups other than s i a l i c a c i d , however (Seaman, 1973, 1976). P a r t i t i o n i s i n p r i n c i p l e s e n s i t i v e to the t o t a l surface charge at a l l depths i n the membrane, and hence d i f f e r s from e i t h e r of the other two methods mentioned (D.E. Brooks, personal communication). Also c e r t a i n f i n d i n g s i n d i c a t e t h a t c h a r g e - s e n s i t i v e phase systems are s e n s i t i v e t o a l t e r a t i o n s i n membrane charge r e s u l t i n g from changes ( s h i f t s ) i n the l i p i d environment of the membrane (Walter, 1982). The obs e r v a t i o n that newly r e l e a s e d p l a t e l e t s have a low p a r t i t i o n i n charge s e n s i t i v e systems i s not n e c e s s a r i l y i n c o n f l i c t with the l i t e r a t u r e , t h e r e f o r e . The evidence presented here i n d i c a t e s that p l a t e l e t s i n the HDP f r a c t i o n are no higher i n p a r t i t i o n than the bulk of the popu l a t i o n and are very heterogeneous i n systems which r e f l e c t membrane surface charge a s s o c i a t e d p r o p e r t i e s . This cannot be i n t e r p r e t e d s p e c i f i c a l l y w i t h regard t o the t o t a l surface charge or surface charge d e n s i t y of p l a t e l e t s s i n c e a d e t a i l e d r e l a t i o n s h i p has not been e s t a b l i s h e d with t h i s c e l l type. However, a reasonable i n t e r p r e t a t i o n of the data would be t h a t , a l l other membrane parameters being the same f o r a l l p l a t e l e t s w i t h i n the po p u l a t i o n s t u d i e d ( i . e . , i f surface charge i s the only d i f f e r e n c e , r e g a r d l e s s o f s p a c i a l expression of such), then HDP ( c o n t a i n i n g young p l a t e l e t s ) are not of higher average t o t a l surface - 151 -. charge than the m a j o r i t y of the po p u l a t i o n . The f a c t t h a t the CCD d i s t r i b u t i o n of HDP p a r t i c u l a r l y i s heterogeneous i n d i c a t e s that a c e r t a i n f r a c t i o n o f the p l a t e l e t s contained w i t h i n HDP are a l s o of p a r t i t i o n equal to or higher than IDP, as was found i n c e r t a i n cases with the r a b b i t and i n the case of human den s i t y separated p l a t e l e t s . That LDP i s of lower p a r t i t i o n c o r r e l a t e s with other assays of c e l l s u r face charge. The present r e s u l t s on p l a t e l e t s are i n a sense analogous to the s i t u a t i o n found when the maturation of er y t h r o c y t e s ( f o r which evidence ranging from a c o r r e l a t i o n of decreasing surface charge with age, to no c o r r e l a t i o n at a l l have been reported) i s analyzed by CCD i n charge s e n s i t i v e phase systems. Here the r e t i c u l o c y t e s are of low p a r t i t i o n , two day "mature" e r y t h r o c y t e s are of high p a r t i t i o n , and 48 day o l d e r y t h r o c y t e s are again of low p a r t i t i o n s i m i l a r t o the youngest c e l l s , r e t i c u l o c y t e s . There i s some precedent t o the thought that there might be a " r e t i c u l o c y t e - l i k e " counterpart i n the megakaryocyte-platelet system i n the megathrombocyte c o n s i s t i n g of l a r g e f u n c t i o n a l l y a c t i v e p l a t e l e t s or p o l y p l a t e l e t s r eleased from the megakaryocyte which then change i n the c i r c u l a t i o n ( K a r p a t k i n , 1974, 1977, 1978a; K a r p a t k i n and Charmatz, 1970; D j a l d e t t i , 1979; Obrien and Jamieson, 1974; Corash, 1982). However, a s i m i l a r maturation-ageing sequence has not been documented f o r the p l a t e l e t as has been f o r the e r y t h r o c y t e . Based on CCD experiments analogies can be drawn between the p a r t i t i o n behavior of er y t h r o c y t e s and p l a t e l e t s , i n that the data i n d i c a t e that young p l a t e l e t s demonstrate low s u r f a c e c h a r g e - s e n s i t i v e p a r t i t i o n r e l a t i v e t o the p l a t e l e t p o p u l a t i o n . The f a c t t h a t t h i s a ge-related p l a t e l e t - 152 -subpopulation was e n r i c h e d . i n a confined area of the CCD i n d i c a t e s that p l a t e l e t s of other ages present d i f f e r e n t membrane p r o p e r t i e s upon maturation or ageing, i . e . t h e i r p a r t i t i o n behavior becomes more heterogeneous. However, LDP ( c o n t a i n i n g o l d p l a t e l e t s ) are of lower p a r t i t i o n , i n agreement with other methods of s u r f a c e charge analyses and analogous to the e r y t h r o c y t e model as w e l l . Conclusion The purpose of t h i s t h e s i s was e s s e n t i a l l y t w o - f o l d : to examine the heterogeneity of p l a t e l e t age d i s t r i b u t i o n s by countercurrent d i s t r i b u t i o n i n two-polymer aqueous phase systems, g a i n i n g i n f o r m a t i o n on the p l a t e l e t ageing process at the membrane l e v e l ; and to assess the p o s s i b i l i t y of e v e n t u a l l y developing a r e l a t i v e l y r a p i d , t e c h n i c a l l y simple, inexpensive, a l t e r n a t e method f o r p l a t e l e t s u r v i v a l a n a l y s i s of c l i n i c a l specimens. This would provide an estimate of the p l a t e l e t age d i s t r i b u t i o n i n a s i n g l e experiment, enabling the d e t e c t i o n of newly-released, young p l a t e l e t s i n the c i r c u l a t i o n . Whether the processes a s s o c i a t e d with p l a t e l e t removal from the c i r c u l a t i o n are random or age r e l a t e d , and whether these processes are r e g u l a t e d by the immune system, perhaps by b i n d i n g of n a t u r a l l y o c c u r r i n g immunoglobulins due t o the appearance of a new age r e l a t e d a n t i g e n , have not yet been proven. In g e n e r a l , the aspects of the ageing c e l l ( p l a t e l e t ) s urface are of i n t e r e s t i n themselves. Since o l d c e l l s are removed from the c i r c u l a t i o n by the r e t i c u l o e n d o t h e l i a l system, i t has been proposed that c e l l senescence i n v o l v e s a t r a n s f o r m a t i o n which takes p l a c e at the c e l l membrane s u r f a c e , enabling the microenvironment w i t h i n the system t o recognize the c e l l as being - 153 -o l d , and e l i m i n a t e i t from the c i r c u l a t i o n . To examine and e l u c i d a t e such membrane age r e l a t e d molecular t r a n s f o r m a t i o n s would indeed advance our understanding of the i n t e r a c t i o n s of p l a t e l e t s w i t h i n t h e i r environment i n terms of the surface to surface communications a s s o c i a t e d with p l a t e l e t r e l e a s e , maturation, ageing, a r r e s t and removal. Moreover, the i n f o r m a t i o n might provide some means of p h y s i c a l l y h a r v e s t i n g p l a t e l e t s of d i f f e r i n g s u r v i v a l c a p a c i t y and/or b i o c h e m i c a l l y manipulating p l a t e l e t l o n g e v i t y . At present no method i s a v a i l a b l e to determine an unequivocal age d i s t r i b u t i o n of a p a t i e n t ' s p l a t e l e t p o p u l a t i o n . Determination of p l a t e l e t s u r v i v a l time by l a b e l i n g with I n d i u m - I l l or Chromium-51 has s e v e r a l drawbacks s i n c e the procedure i s hazardous, the c a l c u l a t i o n complex, and the i n f o r m a t i o n gained incomplete, p r o v i d i n g only a mean age f o r the p a t i e n t s ' p l a t e l e t p o p u l a t i o n . Since attempts to analyze p l a t e l e t age d i s t r i b u t i o n a f t e r d e n s i t y and s i z e based separations provide p a r t i a l and sometimes c o n f l i c t i n g c o r r e l a t i o n s , a c e l l ( p l a t e l e t ) s e p a r a t i o n / a n a l y s i s procedure i s needed which i s more s e n s i t i v e to c h a r a c t e r i s t i c s which change during c e l l u l a r ageing, e.g., membrane surface p r o p e r t i e s . One such c e l l s u rface membrane a n a l y s i s technique i s countercurrent d i s t r i b u t i o n i n two-polymer aqueous phase systems. This technique separates on the b a s i s of a c o n t r o l l a b l e v a r i e t y of surface p r o p e r t i e s , and has been shown to be capable of separating mammalian e r y t h r o c y t e s on the b a s i s of i n v i v o maturation and ageing. I t was a p p l i e d i n t h i s t h e s i s to d i s t r i b u t e p l a t e l e t s according to membrane surface p r o p e r t i e s which are shown to be r e l a t e d , at l e a s t i n p a r t , to the p l a t e l e t age d i s t r i b u t i o n . These blood elements, while being heterogeneous due t o - 154 -v a r i o u s age and non-age r e l a t e d sources, are d i s t r i b u t e d based on su r f a c e p r o p e r t i e s which are d i f f e r e n t f o r p l a t e l e t subpopulations of d i f f e r e n t ageing c h a r a c t e r i s t i c s . 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(1983) Changes of p l a t e l e t c e l l volumes i n hypotonic s o l u t i o n . Thrombos. Res. 31:13-21. Webber, T.J., and Brooks, D.E. (1981) P a r t i t i o n s t u d i e s o f p l a t e l e t s aged i n v i v o . Second I n t e r n a t . Conf. on P a r t i t i o n i n g , S h e f f i e l d , England (abst. 16). Webber, T.J., and Brooks, D.E. (1983) Surface p r o p e r t i e s of density separated p l a t e l e t s as detected by p a r t i t i o n i n two-phase aqueous polymer systems ( i n p r e p a r a t i o n ) . Webber, T.J., and Brooks, D.E. (1983) CCD s t u d i e s o f p l a t e l e t s aged i n v i v o . T h i r d I n t e r n a t . Conf. on P a r t i t i o n i n g , Vancouver, B.C. Canada. W i l k i n s o n , A.R., Hawker, R.J., and Hawker, L.M. (1979) The i n f l u e n c e of a n t i p l a t e l e t drugs on p l a t e l e t s u r v i v a l a f t e r a o r t i c damage or i m p l a n t a t i o n of a dacron a r t e r i a l p r o t h e s i s . Thrombos. Res. 15:181-189. Winocour, P.D., Kinlough-Rathbone, R.L., P e r r y , D.W., Rand, M.L., Packham, M.A., and Mustard, J.F. (1983) Changes i n the p r o p e r t i e s of p l a t e l e t s from r a t s with experimentally induced shortened p l a t e l e t s u r v i v a l . J . Lab. C l i n . Med. 101:175-182. Y a a r i , A. (1969) M o b i l i t y of human blood c e l l s of d i f f e r e n t age groups i n an e l e c t r i c f i e l d . Blood 33:159-163. Yamazaki, H., Motomiya, T., Watanabe, C , Miyagawa, N., Yahara, Y., Okawa, Y., and Onozawa, Y. (1980) Consumption of l a r g e r p l a t e l e t s w i t h decrease i n adenine n u c l e o t i d e content i n thrombosis, disseminated i n t r a v a s c u l a r c o a g u l a t i o n and post o p e r a t i v e s t a t e . Thrombos. Res. 18:77-88. Yarom, R., Meyer, S., and More, R. (1982) Negative s u r f a c e charge on p l a t e l e t s of p a t i e n t s w i t h i d i o p a t h i c s c o l i o s i s . Haemostas. 12:289-292. 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Blood 52:505-514. Zucker, M.B., Jerushalmy, Z., and Grant, R. (1967) K i n e t i c s t u d i e s of p l a t e l e t aggregation induced by adenosine diphosphate and i t s i n h i b i t i o n by c h e l a t i n g agents, guanidino compounds, and adenosine. Thrombos Diat h . Haemorrh. 18:713-725. Zucker, M.B., and Peterson, J. (1968) I n h i b i t i o n of adenosine diphosphate-induced secondary aggregation and other p l a t e l e t f u n c t i o n s by a c e t y l s a l i c y l i c a c i d i n g e s t i o n . Proc. Soc. E x p t l . B i o l . Med. 127:547-551. - 176 -APPENDIX - 177 -Table A . l Freezing P o i n t Depression A n a l y s i s of Deionized S t r a c t a n T o n i c i t y (mOsm, mean + S.Dev.) StRSB 0.00 8.06 10.75 16.13 32.26 + 0.16 (5) Std 100 Std 500 3.50 27.67 33.60 50.67 160.67 100.0 502.5 2.29 (3) 0.57 (3) 1.15 2.08 8.0 (3) (3) (3) 0.00 5.66 + 1.1 (3) 6.18 28.33 + 1.2 (3) 7.42 33.00 + 1.0 (3) 9.28 36.00 + 1.0 (3) 12.37 45.00 + 1.0 (3) 18.55 73.00 + 3.6 (3) 37.09 + 0.38 (4) 219.67 + 5.5 (3) Std 100 130.33 + 5.1 (3) Std 500 524.67 + 3.1 (3) 2.25 2.8 + 0.4 (3) . 4.55 5.5 + 0.8 (3) 9.00 14.3 + 1.2 (3) 18.0 46.8 + 1.4 (3) 36.0 + 0.73 (4) Std 100 89.3 + 1.2 (3) Std 500 457.3 + 2.1 (3) 1.2 1.50 + 2.12 (2) 2.4 2.50 + 0.71 (2) 4.8 6.75 + 2.47 (2) 9.6 13.70 + 0.42 (2) 19.10 + 0.14 (2) 27.75 + 1.77 (2) 40 80.50 + 0.71 (2) Std 100 101 Std 500 512 Refer t o S e c t i o n 2.8 Table A.2 Density of S t r a c t a n Stock S o l u t i o n StR% Density (g/ml, mean _+ S.Dev.) 20.0 10.0 5.0 2.5 1.25 0.00 1.08250 1.04575 1.00743 0.98931 0.99686 0.9997 + 0.0025 + 0.0042 + 0.0019 + 0.0019 + 0.0017 (3T (4) (3) (2) (2) 2.8 - 178 -Table A.3 T o n i c i t y by Er y t h r o c y t e Volume, Osmolarity v.s. Hematocrit S a l t Hmct (%) Hmct (%) S a l t Hmct (%) Hmct (%) mOsM @ 10 min mOsM @ 10 min 235 46.5 42 295 44.0 43.5 240 46.0 41 300 44.0 43 245 47.5 38 305 43.5 43 250 47.0 43 310 43.0 43 255 44.5 44 315 42.5 42.5 260 45.5 43 320 42.5 42 265 43.5 42.5 325 42.0 42 270 45.0 42 330 41.5 41.5 275 45.5 43.5 335 41.5 41.5 280 45.0 43 340 40.5 38.5 285 44.5 43 345 40.0 40.0 290 44.0 43.5 350 2.8 Table A.4 Density D i s t r i b u t i o n of Human P l a t e l e t s on S t r a c t a n StR HuPlt HuPlt HuPlt (%) 11/23 (% Tot) 11/27 (% Tot) 11/29 (% Tot) 12.5 0.00 0.00 6.28 13.0 3.26 1.47 4.20 13.5 7.87 5.88 0.00 14.0 4.34 6.67 5.94 14.5 11.58 22.79 4.77 15.0 13.73 38.97 4.77 15.5 22.91 24.51 5.89 16.0 31.42 44.61 9.33 16.5 52.45 51.72 13.31 17.0 65.10 60.05 27.65 17.5 77.68 71.81 • 40.25 18.0 87.93 99.26 55.95 18.5 92.99 100 69.65 19.0 97.95 91.42 84.68 19.5 95.90 95.1 98.16 20.0 100 82.35 100 2.8 - 179 -Table A.5 Blood C e l l Recovery from S t r a c t a n Density Gradient StR% StR (g/ml) RaPlt (%) ApePlt (%) HuPlt (%) Hu MNC (%) 13.5 1.055 6.0 1.061 11.7 15.0 1.062 32.0 14.7+2.3 17.42+6.68 1.065 18.5 16.0 1.066 43.3+4.2 38.61+7.05 1.070 33.8 17.0 1.071 29.3+3.1 29.95+6.67 1.074 27.1 18.0 1.075 62.0 19.0 1.080 20.0 1.084 12.7+3.1 14.03+3.58 9.0 2.8 Table A.6 S t r a c t a n and Dextran, Concentration and Osmolarity S t r a c t a n % (g/ml) T o n i c i t y mOsm % Tonic % 290 mOsm Dextran% (g/ml) T o n i c i t y mOsm % Tonic % 290 mOsm 14.0 44 15.2 14.0 17.0 5.9 16.0 50 17.2 16.0 22.5 7.8 18.0 64 22.1 18.0 29.5 10.2 32.0 160 55.2 32.0 200 69.0 2.9 Table A.7 Two-Polymer Phase System Composition 5/4: A B C D E F G Dextran (g) 1X3 IT75 1775 LT75 LT75 I D iTTT" PEG (g) , 6.67 6.67 6.67 6.67 6.67 6.67 6.67 NaCl (g) 2.08 4.15 6.25 8.33 10.43 12.5 NaPB (g) 25.0 20.88 16.75 12.63 8.48 4.83 2.18 H 20 (g) 6.83 8.87 10.93 12.95 15.02 16.57 17.15 2.9 - 180 -Table A.8 A l t e r n a t e S a l t Concentration and Recipe by S a l t R a t i o 5/4: A - l B-2 C-3 D-4 E-5 F-6 Na 2HP04 (M) °~^9 0.0872 0.0654 0.0436 0.0218 0.0 NaH 2P0 4 (M) 0.0345 0.0276 0.0207 0.0138 0.0069 0.0 NaCl (M) 0.0 0.03 0.06 0.09 0.12 0.15 NaPB/NaCl 10/0 8/2 6/4 4/6 2/8 0/10 2.9 Table A.9 L i n e a r i t y of Cel l o s c o p e Counts Using 76 Micron O r i f i c e Stock Counts Counts Counts Counts Count Less V o l ( u l ) /255 u l /255 u l /255ul /255 u l Rate/255 u l Count(%) 1.25 4149 2.50 8658 8254 9062 3.75 11981 5.0 16369 15964 15526 7.5 22343 22343 10.0 28729 28916 28542 15.0 40769 39873 20.0 52697 40.0 88616 60.0 115794 80.0 132834 100.0 146273 10000 2.24 17616 20000 4.48 41665 40000 8.96 52697 60000 13.43 80000 17.90 100000 22.38 140000 31.33 160000 35.81 180000 40.28 2.12 Table A.10 Volume and Diameter v.s. Threshold, with 76 um O r i f i c e Window Volume Diameter Standards Fixed C e l l s S e t t i n g (um 3) (um) (um diameter) 5 1.55 1.44 10 3.09 1.81 20 6.19 2.28 30 9.28 2.61 40 12.37 2.87 50 15.47 3.09 60 18.56 3.28 70 21.66 3.46 80 24.75 3.62 90 27.84 3.76 100 30.94 3.89 FxRaPlt 2.02 3.05 FxHuRBC 2.12 - 181 -Table A.11 Normalized Data Summary, Rabbit LDP, IDP, and HDP CCD Exp Grad P i t P i t Load Tr Peak Mod Mid Spd Spd CCDC MDY %StR Pop % CCDC n CCDC/n CCDC/n CCDC/n CCDC/n / ( n ) 1 / 2 11/ 16 LDP 28.2 1--3 40 0.5789 0.5921 0.5721 0.297 1.875 15/ 17. 5 IDP 35.5 41--43 0.5789 0.5921 0.5797 0.317 2.006 79 20 HDP 36.4 81--83 0.5789 0.5921 0.5829 0.317 2.006 1/ 16 LDP 62.1 1--3 40 0.6316 0.6447 0.6105 0.305 1.929 5/ 18 IDP 26.6 41--43 0.7368 0.6447 0.6074 0.317 2.003 80 20 HDP 11.2 81--83 0.6316 0.6447 0.6105 0.288 1.820 1/ 14 LDP 4.1 1--3 40 0.3816 0.4342 0.3889 0.501 3.170 28/ 17.5 IDP 81.8 41--43 0.5921 0.5921 0.6082 0.359 2.269 80 20 HDP 14.1 81--83 0.6974 0.5921 0.4945 0.697 4.405 10/ 14. 8 LDP 16.3 81--83 40 0.6184 0.6447 0.6000 0.309 1.951 7/ 17. 8 IDP 73.7 41--43 0.5658 0.5921 0.5650 0.335 2.119 80 20 HDP 10.0 1--3 0.5658 0.5395 0.5471 0.280 1.796 1/ 16 LDP 5.2 1 29 0.3214 0.3393 0.3214 0.268 1.445 12/ 18 IDP 45.6 31 0.7500 0.7500 0.6775 0.323 1.740 82 20 HDP 32.4 61 0.7143 0.7500 0.6804 0.268 1.445 1/ 28/ 82 16 18 LDP IHHD LIDP 26.5 78.7 78.6 1 31 61 29 0.5536 0.5536 0.5536 0.5357 0.5714 0.5357 0.5443 0.5725 0.5443 0.306 0.339 0.339 1.649 1.827 1.827 8/ 16 LDP 14.1 1--3 37 0.5143 0.5429 0.5351 0.246 1.496 21/ 18 IDP 68.6 41--43 0.5714 0.5571 0.5443 0.240 1.462 83 20 HDP 17.3 81--83 0.6000 0.5429 0.5443 0.246 1.496 8/ 16 LDP 9.0 1--3 37 0.4286 0.4571 0.4171 0.309 1.879 27/ 18 IDP 64.0 41--43 0.4571 0.4571 0.4294 0.343 2.087 83 20 HDP 27.0 81--83 0.5143 0.4571 0.4323 0.360 2.191 11/ 16 LDP 14.9 1--3 37 0.4571 0.4571 0.4571 0.200 1.218 17/ 18 IDP 74.2 41--43 0.5714 0.5286 0.5200 0.200 1.218 83 20 HDP 10.8 81--83 0.4857 0.4857 0.4837 0.212 1.287 11/ 16 LDP 24.5 1--3 37 0.5143 0.5000 0.4957 0.177 1.078 23/ 18 IDP 67.6 41--43 0.6571 0.6429 0.6454 0.166 1.009 83 20 HDP 7.9 81--83 0.4286 0.4429 0.4263 0.189 1.148 4/ 16 LDP 15.4 1--3 37 0.4000 0.4000 0.3763 0.215 1.305 23/ 18 IDP 67.7 41--43 0.4571 0.4286 0.4263 0.240 1.462 83 20 HDP 16.9 81--83 0.5429 0.4571 0.4443 0.275 1.670 4/ 16 LDP 29.5 1--3 37 0.4429 0.4286 0.4306 0.283 1.723 29/ 18 IDP 50.2 41--43 0.4429 0.4571 0.4580 0.300 1.826 83 20 HDP 20.3 81--83 0.3857 0.4000 0.3797 0.275 1.670 3.2 - 182 -Table A.12 Normalized Data Summary, Human LDP, IDP, and HDP CCD Exp Grad MDY %StR P i t Pop P i t % Load CCDC Tr n Peak CCDC/n Mod CCDC/n Mid CCDC/n Spd CCDC/n Spd CCDC / ( n ) l / 2 11/ 16 2/ 18 79 20 LDP IDP HDP 30.5 63.6 5.9 1-3 41-43 81-83 40 40 40 0.4125 0.5125 0.5125 0.4125 0.4625 0.5375 0.4049 0.4867 0.4432 0.1451 0.3086 0.5644 0.918 1.952 3.569 11/ 16 LDP 34.1 1--3 40 0 .3125 0.300 0.3298 0.3376 2.135 5/ 18 IDP 54.0 41--43 40 0 .4125 0.425 0.401 0.1372 0.867 79 20 HDP 11.9 81--83 40 0 .6125 0.5375 0.5329 0.3112 1.968 11/ 16 LDP 29.9 1--3 40 0.6125 0.525 0. 5408 0.4220 2.669 8/ 18 IDP 38.3 41--43 40 0.525 0.525 0. 5118 0.3482 2.202 79 20 HDP 31.8 81--83 40 0.525 0.5625 0. 5448 0.2559 1.618 11/ 16 LDP 42.9 1--3 40 0.4125 0.450 0.431 0.449 2.837 9/ 18 IDP 33.0 41--43 40 0.4125 0.4375 0.441 0.285 1.801 79 20 HDP 24.1 81--83 40 0.5125 0.575 0.509 0.485 3.069 1/ 15.5 LDP 16.3 1--3 40 0.50 0.50 0.498 0.375 2.372 8/ 18 IDP 60.8 41--43 40 0.50 0.50 0.508 0.180 1.140 80 20 HDP 22.9 81--93 40 0.60 0.675 0.603 0.334 2.114 1/ 15 LDP 5.5 1--3 40 0.21 0.225 0.210 0.361 2.285 10/ 18 IDP 82.5 31--33 40 0.51 0.463 0.462 0.264 1.668 80 20 HDP 12.0 81--83 40 0.61 0.60 0.623 0.390 2.469 1/ 15 LDP 16.5 1--3 40 0.51 0.525 0.507 0.301 1.902 24/ 18 IDP 69.8 41--43 40 0.61 0.575 0.558 0.277 1.752 80 20 HDP 13.7 81--83 40 0.6125 0.650 0.661 0.277 1.752 1/ 15 LDP 10.0 1--3 40 0.4125 0.450 0.492 0.441 2.787 31/ 18 IDP 84.0 41--43 40 0.4125 0.45 0.450 0.240 1.518 80 20 HDP 6.0 81--83 40 0.3125 0.325 0.426 0.557 3.520 2/ 15 LDP 13.0 1--3 40 0 .6125 0.5625 0.545 0.377 2.385 6/ 18 IDP 83.6 41--43 40 0 .6125 0.5625 0.618 0.586 3.703 80 20 HDP 3.4 81--83 40 0 .5125 0.575 0.464 0.607 3.837 11/ 15.5 LDP 1.5 1--3 40 0.5875 0.6125 0 .593 0.314 1.983 12/ 18 IDP 65.6 41--43 40 0.5875 0.575 0 .551 0.271 1.726 81 20 HDP 31.3 81--83 40 0.5875 0.60 0 .596 0.205 1.299 HDP* 1.6 11/ 16 LDP 20.0 1--3 37 0.6351 0.6216 0.5389 0.2922 1.777 18/ 18 IDP 53.0 41--43 37 0.6891 0.6530 0.6238 0.2221 1.351 81 20 HDP 27.0 81--83 37 0.6351 0.6530 0.6443 0.2162 1.315 3.2 - 183 -Table A.13 Normalized Data Summary, Monkey LDP, IDP, and HDP CCD Exp Grad P i t P i t Load Tr Peak Mod Mid Spd Spd CCDC MDY %StR Pop % CCDC n CCDC/n CCDC/n CCDC/n CCDC/n / ( n ) 1 / 2 Phase System 5/4:A 2/ 15 LDP 10.1 1--3 40 0.1125 0.0625 0.08188 0.1899 1.201 11/ 18 IDP 81.3 41--43 40 0.1125 0.1125 0.10025 0.1424 0.9008 80 20 HDP 7.3 81--83 40 0.1125 0.1125 0.11475 0.1556 0.9842 HHD 1.3 Phase System 5/3.5:A 2/ 15 LDP 26.5 1--3 40 0.2125 0.2000 0.1845 0.2058 1.3013 19/ 18 IDP 67.2 41--43 40 0.2125 0.2250 0.2255 0.1978 1.2507 80 20 HDP 2.3 81--83 40 0.3125 0.3000 0.3035 0.4430 2.8018 HHD 4.0 Phase System 5/3.3:A 3/ 15 LDP 16.1 1--3 40 0.2125 0.2125 0.1953 0.1319 0.8341 10/ 18 IDP 72.5 41--43 40 0.3125 0.2625 0.2560 0.2532 1.6014 80 20 HDP 8.8 81--83 40 0.2125 0.2125 0.1861 0.2242 1.418 HHD 2.6 Phase System 4.85/3.3:A 3/ 15 LDP 6.1 1--3 40 0.2125 0.2625 0.2586 0.2532 1.6014 11/ 18 IDP 85.7 41--43 40 0.4125 0.3750 0.3760 0.2770 1.7516 80 20 HDP 8.2 81--83 40 0.3125 0.3125 0.3074 0.3350 2.1186 3.2 Table A.14 P a r t i t i o n C o e f f i c i e n t s K and G as a Function of CCDC CCDC K = r/n G = r / ( n - r ) CCDC K = r/n G = r / ( n - r ) 0 5 0.05 0.052632 55 0.55 1.22222 10 0.10 0.111111 60 0.60 1.50000 15 0.15 0.176471 65 0.65 1.85714 20 0.20 0.250000 70 0.70 2.33333 25 0.25 0.333333 75 0.75 3.00000 30 0.30 0.428571 80 0.80 4.00000 35 0.35 0.538462 85 0.85 5.66661 40 0.40 0.666667 90 0.90 9.00000 45 0.45 0.818182 95 0.95 19.00000 50 0.50 1.000000 100 3.2 - 184 -Table A.15 Student t Test of Rabbit LDP, IDP, and HDP CCD Parameters Peak LDP 0.487 + 0.098 - IDP 0 .578 + 0.10 = - 0.091 p a i r e d t = 2.441 df = 11, P = 0.016 98.4% nonpaired t = 2.257 , df = 22, P = 0.017 98.3% IDP 0.578 + 0.10 - HDP 0 .564 + 0.10 = 0.014 p a i r e d t = 0.495 , df = 11, P = 0.315 68.5% nonpaired t = 0.336 df = 22, P = 0.370 63.0% LDP 0.487 + 0.098 - HDP 0 .564 + 0.100 = - 0.077 pa i r e d t = 1.825 df = 11, P = 0.048 95.2% nonpaired t = 1.918 df = 22, P = 0.034, 96.6% Mode LDP 0.498 + 0.096 - IDP 0 .567 + 0.092 = - 0.069 p a i r e d t = 1.914 df — 11, P = 0.041, 95.9% nonpaired t = 1.780 df = 22, P = 0.044 95.6% IDP 0.567 + 0.092 - HDP 0 .537 + 0.098 = 0.030 p a i r e d t = 1.810 df = 11, P = 0.049 95.1% nonpaired t = 0.616 df — 22, P = 0.272, 73.0% LDP 0.498 + 0.096 - HDP 0 .543 + 0.10 = - 0.045 p a i r e d t = 1.162 df = 11, P = 0.135, 86.5% nonpaired t = 1.109 df = 22, P = 0.140 86.0% Middle LDP 0.479 + 0.095 - IDP 0.552 + 0.081 = - 0.073 p a i r e d t = 2.203 df = 11, P = 0.025, 97.5% nonpaired t = 2.016 df = 22, P = 0.028, 97.2% IDP 0.552 + 0.081 - HDP 0 .518 + 0.089 = 0.034 p a i r e d t = 1.658 df = 11, P = 0.063, 93.7% nonpaired t = 0.954, df = 22, P = 0.175, 82.5% LDP 0.479 + 0.095 - HDP 0 .517 + 0.089 = - 0.038 p a i r e d t = 1.180, df = 11, P = 0.131, 86.9% nonpaired t = 1.079 df — 22, P = 0.146, 85.4% Spread LDP 0.285 + 0.082 - IDP 0.290 + 0.063 = - 0.005 p a i r e d t = 0.364, df = 11, P = 0.361, 63.9% nonpaired t = 0.312, df 23, P = 0.379, 62.1% IDP 0.290 + 0.063 - HDP 0.313 + 0.131 = - 0.023 p a i r e d t = 0.776 df = 11, P = 0.227, 77.3% nonpaired t = 0.481, df = 23, P = 0.318, 68.2% LDP 0.285 + 0.082 - HDP 0.313 + 0.131 = - 0.028 p a i r e d t = 1.659, df = 11, P = 0.063, 93.7% nonpaired t = 0.638, df = 22, P = 0.265, 73.5% Spread* = Spd x ( n ) 1 ' ^ LDP 1.727 + 0.541 - IDP 1 752 + 0.388 = - 0.026 p a i r e d t = 0.289, df = 11, P = 0.390, 61.1% nonpaired t = 0.134, df - 22, P = 0.447, 55.3% IDP 1.752 + 0.388 - HDP 1 902 + 0.842 = - 0.149 p a i r e d t = 0.798, df = 11, P = 0.221, 77.9% nonpaired t = 0.558, df = 22, P = 0.291, 70.9% LDP 1.727 + 0.541 - HDP 1 902 + 0.842 = - 0.175 p a i r e d t = 1.639, df = 11, P = 0.065, 93.5% nonpaired t = 0.607, df = 22, P = 0.275, 72.5% + 0.129, S.E. + 0.096, S.E. + 0.147, S.E. + 0.125, S.E. + 0.059, S.E. + 0.133, S.E. + 0.114, S.E. + 0.071, S.E. + 0.113, S.E. + 0.050, S.E. + 0.103, S.E. + 0.059, S.E. + 0.310, S.E. + 0.649, S.E. + 0.370, S.E. 0.037 (12) 0.028 (12) 0.042 (12) 0.036 (12) 0.017 (12) 0.038 (12) 0.033 (12) 0.021 (12) 0.033 (12) 0.014 (12) 0.030 (12) 0.017 (12) 0.090 (12) 0.187 (12) 0.107 (12) 3.2 - 185 -Table A.16 Student t Test of Human LDP, IDP, and HDP CCD Parameters Peak LDP 0.475 + 0.136 - IDP 0.526 + 0.092 = - 0.052 + 0.101, S.E. 0.030 ( I D p a i r e d t = 1.696, df = 10, P = 0.060, 94.0% nonpaired t = 1.039, df = 20, P = 0.156, 84.4% IDP 0.526 + 0.092 - HDP 0.549 + 0.092 = - 0.022 + 0.094, S.E. 0.028 ( I D p a i r e d t = 0.793, df = 10, P = 0.223, 77.7% nonpaired t = 0.570, df = 20, P = 0.287, 71.3% LDP 0.475 + 0.136 - HDP 0.549 + 0.092 = - 0.074 + 0.161, S.E. 0.048 (11) p a i r e d t = 1.526, df = 10, P = 0.079, 92.1% nonpaired t = 1.493, df = 20, P = 0.076, 92.5% Mode LDP 0.471 + 0.123 - IDP 0.512 + 0.073 = - 0.040 + 0.079, S.E. 0.024 (11) p a i r e d t = 1.704, df = 10, P = 0.060, 94.0% nonpaired t = 0.936, df = 20, P = 0.180, 82.0% IDP 0.512 + 0.073 - HDP 0.572 + 0.094 = - 0.060 + 0.084, S.E. 0.025 (11) p a i r e d t = 2.383, df = 10, P = 0.019, 98.1% nonpaired t = 1.683, df = 20, P = 0.054, 94.6% LDP 0.471 + 0.123 - HDP 0.572 + 0.094 = - 0.101 + 0.135, S.E. 0.041 ( I D p a i r e d r = 2.476, df = 10, P = 0.016, 98.4% nonpaired t = 2.153, df = 20, P = 0.022, 97.8% Middle LDP 0.463 + 0.112 - IDP 0.510 + 0.072 = - 0.047 + 0.084, S.E. 0.025 (11) p a i r e d t = 1.873, df = 10, P = 0.045, 95.5% nonpaired t = 1.180, df = 20, P = 0.126, 87.4% IDP 0.510 + 0.072 - HDP 0.550 + 0.082 = - 0.040 + 0.090, S.E. 0.027 (11) p a i r e d t = 1.469, df = 10, P = 0.086, 91.4% nonpaired t = 1.203, df = 20, P = 0.122, 87.9% LDP 0.463 + 0.112 - HDP 0.550 + 0.082 = - 0.087 + 0.139, S.E. 0.042 ( I D p a i r e d t = 2.077, df = 10, P = 0.032, 96.8% nonpaired t = 2.075, df = 20, P = 0.026, 97.5% Spread LDP 0.347 + 0.086 - IDP 0.284 + 0.116 = 0.063 + 0.139, S.E. 0.042 (11) p a i r e d t = 1.501, df = 10, P = 0.082, 91.8% nonpaired t = 1.449, df = 20, P = 0.081, 91.9% IDP 0.284 + 0.116 - HDP 0.382 + 0.148 = - 0.098 + 0.135, S.E. 0.041 (11) p a i r e d t = 2.410, df = 10, P = 0.018, 98.2% nonpaired t = 1.736, df = 20, P = 0.049, 95.1% LDP 0.347 + 0.086 - HDP 0.382 + 0.148 = - 0.035 + 0.168, S.E. 0.051 ( I D p a i r e d t = 0.686, df = 10, P = 0.254, 74.6% nonpaired t = 1.684, df = 20, P = 0.251, 74.9% -Spread* = Spd x ( n ) 1 ^ LDP 2.186 + 0.548 - IDP 1.789 + 0.736 = 0.397 + 0.880, S.E. 0.265 (11) p a i r e d t = 1.497, df = 10, P = 0.083, 91.7% nonpaired t = 1.431, df = 20, P = 0.084, 91.6% IDP 1.789 + 0.736 - HDP 2.412 + 0.940 = - 0.623 + 0.855, S.E. 0.258 ( I D p a i r e d t = 2.416, df = 10, P = 0.018, 98.2% nonpaired t = 1.731, df = 20, P = 0.049, 95.1% LDP 2.186 + 0.548 - HDP 2.029 + 0.908 = 0.157 + 0.527, S.E. 0.159 (11) p a i r e d t = 0.991, df = 10, P = 0.173, 82.8% nonpaired t = 0.688, df = 20, P = 0.250, 75.0% 3.2 - 186 -Table A.17 Student t Test of Monkey LDP, IDP, and HDP CCD Parameters Peak LDP 0.188 + 0.050 - IDP p a i r e d t = 1.567 , df nonpaired t = 1.083 , df IDP 0.263 + 0.129 - HDP p a i r e d t = 0.522 , df nonpaired t = 0.310 , df LDP 0.188 + 0.050 - HDP p a i r e d t = 1.732 , df nonpaired t = 0.926 , df Mode LDP 0.184 + 0.086 - IDP p a i r e d t = 3.181 df nonpaired t = 0.860 , df IDP 0.244 + 0.108 - HDP p a i r e d t = 0.301 i df nonpaired t = 0.132 df LDP 0.184 + 0.086 - HDP p a i r e d t = 2.450 df nonpaired t = 0.793 df Middle LDP 0.180 + 0.073 - IDP p a i r e d t = 2.791 df nonpaired t = 0.881 df IDP 0.239 + 0.113 - HDP p a i r e d t = 0.317 df nonpaired t = 0.156 df LDP 0.180 + 0.073 - HDP p a i r e d t = 1.794 df nonpaired t = 0.803 df Spread LDP 0.195 + 0.050 - IDP p a i r e d t = 0.621, df nonpaired t = 0.573 df IDP 0.218 + 0.060 - HDP p a i r e d t = 1.189, df nonpaired t = 1.028, df LDP 0.195 + 0.050 - HDP p a i r e d t = 1.695, df nonpaired t = 1.388, df Spread* = Spd x ( n ) 1 ^ LDP 1.235 + 0.317 - IDP p a i r e d t = 0.621, df nonpaired t = 0.523, df IDP 1.376 + 0.380 - HDP p a i r e d t = 1.189, df nonpaired t = 1.028, df LDP 1.235 + 0.317 - HDP p a i r e d t = 1.695, df nonpaired t = 1.388, df 0.263 + 0.129 = - 0.075 = 3, p = . 0.108, 89.2% = 6, p = : 0.160, 84.0% 0.238 + 0.096 = 0.025 = 3, p = : 0.319, 68.1% = 6, p = = 0.384, 61.7% 0.238 + 0.096 = - 0.050 = 3, p = : 0.091, 90.9% = 6, p = : 0.195, 80.5% 0.244 + 0.108 = - 0.059 = 3, p = 0.025, 97.5% = 6, p = : 0.211, 78.9% 0.234 + 0.093 = 0.009 = 3, p = 0.392, 60.8% = 6, p = 0.450, 55.0% 0.234 + 0.093 = - 0.050 = 3, p = 0.046, 85.4% = 6, p = 0.229, 77.1% 0.239 + 0.113 = - 0.059 = 3, p = 0.034, 96.6% = 6, p = 0.206, 79.4% 0.228 + 0.094 = 0.011 = 3, p = 0.386, 61.4% = 6, p = 0.441, 56.0% 0.228 + 0.094 = - 0.048 = 3, p = 0.085, 91.5% = 6, p = 0.226, 77.4% 0.218 + 0.060 = - 0.022 = 3, p = 0.289, 71.1% = 6, p = 0.294, 70.6% 0.290 + 0.126 = - 0.072 = 3, p = 0.160, 84.0% = 6, p = 0.172, 82.8% 0.290 + 0.126 = - 0.094 = 3, p = 0.094, 90.6% = 6, p = 0.107, 89.3% 1.376 + 0.380 = - 0.142 = 3, p = 0.289, 71.1% = 6, p = 0.291, 70.6% 1.831 + 0.799 = - 0.455 = 3, p = 0.160, 84.0% =• 6, p = 0.172, 82.8% 1.831 + 0.799 = - 0.596 = 3, p = 0.094, 90.6% = 6, p = 0.107, 89.3% + 0.096, S.E. 0.048 (4) + 0.096, S.E. 0.048 (4) + 0.058, S.E. 0.029 (4) + 0.037, S.E. 0.019 (4) + 0.062, S.E. 0.031 (4) + 0.041, S.E. 0.020 (4) + 0.043, S.E. 0.021 (4) + 0.072, S.E. 0.036 (4) + 0.053, S.E. 0.027 (4) + 0.072, S.E. 0.036 (4) + 0.121, S.E. 0.061 (4) + 0.111, S.E. 0.056 (4) + 0.456, S.E. 0.228 (4) + 0.765, S.E. 0.382 (4) + 0.704, S.E. 0.352 (4) 3.2 - 187 -Table A.18 S i z e A n a l y s i s of Rabbit LDP, IDP, and HDP (Celloscope) Exp P i t Subpop Peak% W Mode% W Mid% W Spd% W 1 7 LDP 26725 26 26792 53783 28/ IDP 33.75 30 37.47 74.93 80 HDP 42.5 40 43.54 87.08 PRP 31.25 30 40.90 81.79 3.3 Table A.19 Rabbit and Rat LDP, IDP, and HDP, S i z e by Laser S c a t t e r i n g Wn RaPRP RaPRP RaLDP RalDP RaHDP RtLDP RtlDP RtHDP % 10/8/80 10/16/80 10/8/80 2/25/81 2 0.25 3 0.25 1.75 1.45 4.5 0.25 0.25 4 1.95 8.95 6.5 2.65 1.65 1.45 1.45 1.0 5 2.3 8.1 4.95 3.5 2.15 1.45 1.45 0.75 6 2.15 6.75 3.15 3.5 2.3 0.95 0.75 2.1 7 1.6 3.15 1.8 2.65 1.95 0.75 0.95 1.6 8 1.45 1.75 1.25 2.15 1.45 0.6 0.75 1.45 9 0.95 1.45 0.6 1.25 0.95 0.45 0.75 0.6 10 0.95 0.95 0.25 1.1 0.95 0.45 0.6 0.6 11 0.6 0.6 0.25 0.75 0.75 0.25 0.25 0.45 12 0.25 0.6 0.55 0.6 0.45 0.25 13 0.25 0.45 0.4 0.25 0.45 14 0.25 0.45 0.25 0.25 0.25 15 0.25 0.25 0.25 16 0.25 17 0.25 0.25 18 0.25 19 0.25 20 0.25 0.25 21-•26 0.25 3.3 P l a t e l e t s were a l s o s i z e d using an Ortho D i a g n o s t i c s l a s e r l i g h t s c a t t e r i n g c e l l counter. P l a t e l e t s i z e histograms were tabulated i n r e l a t i v e u n i t s , i . e . , d i s t a n c e from x a x i s ( i n cm). The r e l a t i v e p l a t e l e t c o ncentrations were: (10/8/80) LDP (57), IDP (52), HDP (35), RaPRP (33), (10/16/80) RaPRP (343), WBC (5.9), RBC (5.56); and f o r the f o l l o w i n g t a b l e , (10/23/80) HuPlt (228), HuRBC (4.37) 33% W, HuWBC (8.7) 40% W, (10/8/80) HuPlt (505). - 188 -Table A.20 Human LDP, IDP, and HDP, S i z e by Laser S c a t t e r i n g % w HuPRP HuPRP HuLDP HuIDP HuHDP HuLDP HuIDP HuHDP 10/23/80 10/8/80 11/12/81 11/18/81 3 0.25 1.25 4.5 2.5 4 1.1 5.75 6.0 2.3 0.75 0.75 1.6 1.1 5 1.6 7.05 7.5 3.65 0.95 0.95 2.45 1.75 6 1.95 7.6 6.0 3.45 1.1 0.75 3.1 1.45 7 2.1 7.6 4.5 2.95 0.95 0.75 2.6 1.75 8 2.25 7.05 4.5 2.95 1.25 0.75 2.6 1.6 9 2.45 6.25 4.5 2.95 1.1 0.6 2.1 1.6 10 2.1 5.2 2.5 2.45 1.45 0.45 2.1 1.6 11 1.95 4.35 2.5 2.3 1.1 0.45 1.95 1.45 12 1.6 4.35 2.5 2.15 0.75 0.25 1.6 1.6 13 1.95 3.6 1.95 0.95 0.45 1.6 1.45 14 1.6 2.45 1.75 0.75 0.25 1.45 1.25 15 1.45 2.1 1.45 0.6 1.25 1.45 16 1.6 1.75 1.1 0.45 0.25 1.1 0.95 17 0.95 1.6 0.95 0.6 0.95 1.1 18 1.1 1.45 0.6 0.45 0.95 0.75 19 1.1 1.1 0.75 0.25 0.95 0.75 20 0.95 1.25 0.6 0.25 0.75 0.45 21 0.6 0.75 0.6 0.25 0.75 0.45 22 0.95 0.75 0.45 0.25 0.6 0.45 23 0.75 0.55 0.25 0.45 0.6 24 0.75 0.55 0.25 0.25 0.6 0.6 25 0.45 0.55 0.25 0.25 0.45 0.45 26 0.6 0.45 0.25 0.25 0.45 27 0.6 0.25 0.25 0.45 28 0.45 0.45 0.25 0.45 29 0.6 0.25 0.25 0.25 0.25 30 0.45 0.45 0.25 0.25 31 0.25 0.25 0.25 0.25 32 0.45 0.45 0.25 0.25 33 0.45 0.25 0.25 34 0.45 0.25 0.25 35 0.45 0.25 0.25 0.25 36 0.25 0.25 0.25 37 0.25 0.25 39 0.25 40 0.25 41 0.25 43 0.25 44 0.25 3.3 - 189 -Table A.21 E l e c t r o p h o r e t i c M o b i l i t y of P l a t e l e t s and RBC Standards Sample E l e c t r o p h o r e t i c M o b i l i t y (3/10/80) Monkey P i t i n PRP 1.021 + 0.301 (23) Monkey P i t i n PRP 1.047 + 0.113 (40) Human P i t i n PRP 0.939 + 0.058 (40) Human RBC (HuRBC) 1.127 + 0.108 (19) (12/19/80) Monkey P i t (MoPlt) 1.408 + 0.082 (20) Monkey P i t 1.413 + 0.078 (20) Rabbit P i t ( R a P l t ) 1.214 + 0.062 (20) Rabbit P i t 1.231 + 0.061 (20) (1/22/81) Human RBC 1.087 + 0.025 (10) (2/25/81) Human RBC 1.096 + 0.037 (10) 3.4 Table A.22 E l e c t r o p h o r e t i c M o b i l i t y of Density Separated P l a t e l e t s Sample LDP EPM IDP EPM HDP EPM MoPlt 0.988 + 0.170 (21) 1.043 + 0.144 (23) 1.081 + 0.120 (23) MoPlt 1.089 + 0.143 (40) 1.076 + 0.065 (40) 1.083 + 0.098 (40) HuPlt 1.094 + 0.026 (10) 1.081 + 0.046 (10) 1.087 + 0.037 (10) -1.078 + 0.024 (10) F x R t P l t 1.323 + 0.043 (10) 1.320 + 0.046 (10) 1.330 + 0.033 (10) 3.4 Table A.23 Separation of Fixed Rabbit P l a t e l e t s by S i z e Sedimentation Sed Distance FxRaPlt S i z e % W Fr # Vol (ml) Cone x l O - 7 Mode Mid Spd 1 10 0.167 18 18 36 2 15 0.436 19 19 35 3 20 1.76 25 30 39 4 25 3.09 30 35 50 5 30 3.46 30 37 52 6 35 3.77 30 36 53 7 40 3.81 30 39 55 8 45 4.15 30 38 56 9 50 4.13 30 38 55 10 55 4.12 30 37 58 11 57.5 3.93 30 38 53 12 60 17.4 30 38 55 2.02 Std 46 46 6 4.04 Std 100 100 6(2) 3.5 - 190 -Table A. 24 EPM of S i z e Separated, Fixed P l a t e l e t s Sed FxHuPlt FxHuPlt FxMoPlt FxRaPlt Fr # 12/19/80 1/22/81 1/26/81 1/27/81 1 1.376 + 0.0654 2 1.378 + 0.0389 1.275 + 0.0343 3 4 1.371 + 0.0428 5 1.400 + 0.0626 1.278 + 0.0355 6 1.362+0.0744 1.410+0.0432 7 1.370 + 0.0789 1.424 + 0.0400 8 1.373+0.0890 1.388+0.0577 1.425+0.0532 1.282+0.0522 9 1.409 + 0.0774 1.394 + 0.0480 1.432 + 0.0457 10 1.409+0.0754 1.406+0.0443 1.435+0.0386 1.290+0.0328 11 1.403 + 0.0764 1.404 + 0.0411 1.435 + 0.0386 1.290 + 0.0436 12 1.416 + 0.0762 1.432 + 0.0420 1.436 + 0.0510 1.303 + 0.0507 13 1.417 + 0.0873 1.421 + 0.0435 1.294 + 0.0479 14 1.449+0.0676 1.416+0.0530 1.424+0.0315 1.290+0.0328 15 1.408 + 0.0668 1.423 + 0.0519 16 1.417 + 0.0710 1.431 + 0.0311 1.300 + 0.0491 17 1.395 + 0.0604 18 1.436 + 0.0734 1.429 + 0.0363 1.306 + 0.0518 19 20 1.435 + 0.0432 1.306 + 0.0430 21 22 1.428 + 0.0463 1.303 + 0.0442 23 1.425 + 0.0563 3.5 - 191 -Fig u r e A . l CCD of 5 l C r - l a b e l e d P l a t e l e t s : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) with the mean s p e c i f i c a c t i v i t y at one h a l f o r d i n a t e h e i g h t ; CCDC 61-120, a b s c i s s a . 3.6 5. 31E+06-0.00E+00 1.52E-04 ~ i i i — — i i i \— 8.10E+02 •0. 00E+00 1.73E+08 0.00E+00 2.20E+02 0.00E+00 F i g u r e A.2 CCD of 5 1 C r - l a b e l e d P l a t e l e t s , Two days C i r c u l a t i o n : p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet per CCDC (dotted l i n e ) , with the mean s p e c i f i c a c t i v i t y at h a l f o r d i n a t e height; CCDC 61-120, a b s c i s s a . 3.6 - 192 -Table A.25 Normalized CCD of 5 l C r - L a b e l e d P l a t e l e t s , Aged i n vivo Time Sample Ld Tr Peak Mod Mid Spd Spd CCDC Days CCD CCDC n CCDC/n CCDC/n CCDC/n CCDC/n / ( n ) l / 2 S l T=0 P i t 1-3 60 0.620 0.575 0.598 0.280 2.17 CPM 0.522 0.522 0.497 0.283 2.19 S l T=0 P i t 61-63 60 0.522 0.575 0.540 0.352 2.72 CPM 0.422 0.470 0.447 0.310 2.40 S l T=2 P i t 1-3 60 0.422 0.470 0.508 0.310 2.40 CPM 0.517 0.475 0.508 0.323 2.50 S l T=2 P i t 61-63 60 0.623 0.575 0.515 0.283 2.19 CPM 0.522 0.575 0.515 0.283 2.19 S2 T=0 P i t 1-3 120 0.463 0.463 0.483 0.181 1.65 CPM 0.463 0.432 0.444 0.181 1.65 S2 T=3 P i t 1-3 120 0.712 0.735 0.723 0.269 2.46 CPM 0.712 0.735 0.723 0.250 2.28 T=0 P i t 1-3 57 0.418 0.446 0.449 0.167 1.258 CPM 0.418 0.416 0.381 0.257 2.080 CPM 0.296 0.298 T=0 P i t 1-4 36 0.528 0.494 0.450 0.406 2.43 CPM 0.422 0.458 0.414 0.353 2.12 T=0 P i t 1-4 36 0.408 0.369 0.386 0.256 1.53 CPM 0.353 0.369 0.286 0.447 2.68 CPM 0.131 0.147 T=0 P i t 41-44 36 0.406 0.383 0.392 0.233 1.40 CPM 0.353 0.369 0.308 0.383 2.30 CPM 0.131 0.142 T=0 P i t 81-84 36 0.408 0.400 0.411 0.272 1.63 CPM 0.408 0.369 0.356 0.344 2.07 CPM 0.128 0.106 3.6 - 193 -Table A. 26 Grouped S t a t i s t i c s , -Labeled P l a t e l e t s Aged i n vi v o S t a t i s t i c P l a t e l e t s R a d i o a c t i v i t y S t a t i s t i c Comparison S3T=1 (40 CCDF/120 CCDC) 10 3 Ef 4.023 x 10 8 3.989 X x-x 7.481 x l O " 1 Exf 1.123 x i o i o 1.084 X 105 E r r 9.365 x l O " 2 Ex2f 3.233 x i o n 3.084 X 10 6 t 7.988 XCCDF 2.792 x i o i 2.717 X i o i S 2 2.430 x i o i 3.498 X i o i S3T=3 (40 CCDF/120 CCDC) Ef 4.012 x 10* 5.863 Exf 1.149 x i o n 1.641 Ex2f 3.339 x 1012 4.736 XCCDF 2.864 x i o i 2.799 S2 1.213 x i o i 2.453 X 10 3 X-X 6.519 x 10" 1 X l O 3 E r r 6.469 x l O " 2 X 10 6 t 10.079 X 10 1 X 10 1 S4T=1 (40 CCDF/120 CCDC) Ef 8.988 x 108 6.489 Exf 2.853 x i o i o 1.979 Ex2f 9.186 x i o n 6.199 XCCDF 3.174 x i o i 3.051 S2 1.437 x i o i 2.449 X 10* X-X 1.236 X 10 6 E r r 1.942 X lO? t 63.594 X i o i X i o i S4T=3 (40 CCDF/120 CCDC) Ef 1.110 x 10 9 1.801 x 10 4 X-X 2.054 Exf 3.228 x 1 0 1 0 4.867 x 10 5 E r r 4.825 x 10" Ex2f 9.639 x 1 0 1 1 1.391 x 1 0 7 t 42.560 XCCDF 2.908 x 10 1 S 2 2.285 x 10 1 X lO^ X 05 r r X 0 7  2.702 X i o i 4.193 X i o i S4T=6* (40 CCDF/120 CCDC) Ef 9.506 x 1 0 8 5.131 Exf 1.272 x 1 0 1 0 6.342 Ex2f 1.758 x 1 0 1 1 8.462 XCCDF(-20) 1.338 x 10 1 1.237 S 2 5.795 1.237 X i o i X-X 1.022 X 10 2 E r r 4.959 X 10 3 t 2.017 X 10 1 X i o i S4T=2 (40 CCDF/120 CCDC) lO^ Ef 1.797 x 10* 2.203 X X-X Exf 5.804 x i o i o 7.045 X 105 E r r Ex2f 1.904 x l O * 2 2.301 X 1 0 7 t XCCDF 3.230 x i o i 3.197 X i o i S2 16.111 2.207 X 10 1 3.296 x 10" 1 3.164 x l O " 2 10.413 S4T=4* (40 CCDF/120 CCDC) Ef 2.035 x 10 9 4.330 Exf 2.694 x 1 0 1 0 5.585 Ex2f 3.664 x 1 0 1 1 7.530 XCCDF(-20) 1.332 x 10 1 1.328 S 2 4.739 7.530 X 10 2 X-X 3.426 x i o - i X 10 3 E r r 1.320 x i o - i X 10^ t 2.595 X 10 1 3.6 - 194 -Table A.27 S p e c i f i c A c t i v i t y of 5 1 C r - L a b e l e d P l a t e l e t s w i t h i n CCD Regions I I and I I I i n Comparison t o the E n t i r e D i s t r i b u t i o n CCD Mean I I I I I CPM/Plt CPM/Plt CPM/Plt S1T=0 1-60 Gm 1.90 x IO" 4 m 3.15 x 10-4 4.78 x IO" 4 7.02 x 10-5 _+ S.Dev. 2.56 x 10-4 1.94 x 10-4 3.49 x 10-5 n 10 6 4 Corr -0.952 -0.904 -0.857 Slope -8.05 x IO" 5 -9.36 x IO" 5 -2.32 x IO" 5 Intcp 6.77 x IO" 4 7.12 x IO" 4 1.05 x IO" 4 S1T=0 61-120 Gm 1.52 x IO" 4 m 2.07 x 10-4 2.60 x 10-4 4.86 x IO" 5 + S.Dev. 1.22 x IO" 4 8.49 x IO" 5 2.18 x IO" 5 n 8 6 2 Corr -0.910 -0.774 -1.0 Slope -4.52 x IO" 5 -3.51 x IO" 5 -3.08 x IO" 5 Intcp 3.65 x IO" 4 3.48 x IO" 4 6.40 x 10-5 S1T=2 1-60 Gm 1.23 x 10-6 m 1.19 x IO" 6 1.19 x IO" 6 1.18 x IO" 6 + S.Dev. 1.23 x IO" 7 1.98 x IO" 7 1.03 x IO" 7 n 5 2 3 Corr -0.129 -0.999 0.292 Slope -1.00 x 10~ 8 -2.80 x IO" 7 3.00 x IO" 8 Intcp 1.21 x IO" 6 1.04 x IO" 5 1.15 x IO" 6 S1T=2 61-120 Gm 1.30 x IO" 6 m 1.30 x IO" 6 1.32 x IO" 6 1.28 x IO" 6 + S.Dev. 5.20 x IO" 8 4.95 x IO" 8 6.36 x IO" 8 n 4 2 2 Corr 0.0 1.0 1.0 Slope 0.0 7.00 x IO" 8 9.00 x IO" 8 Intcp 1.30 x IO" 6 1.28 x IO" 6 1.23 x 10~ 6 3.6 - 195 -Table A.28 S p e c i f i c A c t i v i t y of 5 1 C r - l _ a b e l e d P l a t e l e t s w i t h i n CCD Regions I I and I I I i n Comparison t o the E n t i r e D i s t r i b u t i o n CCD Mean I I • I I I CPM/Plt CPM/Plt CPM/Plt S2T=0 1-120 Gm 3.93 x 10-^ m 3.62 x 10- A 6.24 x 10-4 2.13 x 10-4 +_ S.Dev. 2.44 x 10-4 1.42 x l O " 4 1.34 x l O " 4 n 11 4 7 Corr -0.984 -0.986 -0.971 Slope -7.25 x l O " 5 -1.08 x l O " 4 -6.01 x 10~ 5 Intcp 7.25 x 10-4 7.86 x 10-4 3.93 x l O " 4 S2T=3 1-120 Gm 3.94 x 10~ 7 m 3.71 x l O " 7 3.71 x l O " 7 3.72 x l O " 7 +_ S.Dev. 1.93 x l O " 8 2.73 x l O " 8 1.41 x l O " 9 n 5 3 2 Corr -0.394 -0.979 -1.0 Slope -4.82 x 10" 9 -2.67 x 10" 8 -2.00 x 10~ 9 Intcp 3.81 x l O " 7 3.97 x l O " 7 3.74 x l O " 7 S3T=1 1-120 Gm 9.86 x 10" 6 m 9.31 x 10~ 6 9.54 x 10" 6 9.08 x 10~ 6 _+ S.Dev. 9.85 x l O " 7 9.83 x l O " 7 1.04 x 10~ 6 n 10 5 5 Corr -0.203 -0.888 0.890 Slope -6.60 x 10" 8 -5.52 x l O " 7 5.85 x l O " 7 Intcp 9.61 x 10" 6 1.06 x 10" 5 7.91 x 10" 6 S3T=3 1-120 Gm 1.45 x 10" 6 m 1.44 x 10~ 6 1.46 x 10" 6 1.42 x 10~ 6 +_ S.Dev. 7.23 x 10" 8 8.12 x 10" 8 6.81 x 10" 8 n 8 4 4 Corr 0.0117 0.677 0.407 Slope 3.45 x 1 0 " 1 0 4.26 x 10" 8 2.15 x 10~ 8 Intcp 1.44 x 10" 6 1.39 x 10" 6 1.39 x 10" 6 3.6 - 196 -Table A.29 S p e c i f i c A c t i v i t y of 5 1 C r - L a b e l e d P l a t e l e t s w i t h i n CCD Regions I I and I I I i n Comparison to the E n t i r e D i s t r i b u t i o n CCD Mean I I I I I CPM/Plt CPM/Plt CPM/Plt S4T=1 1-120 Gm 7.25 x IO" 5 m 9.29 x IO" 5 1.16 x IO"4 5.98 x 10-5 + S.Dev. 5.79 x IO" 5 6.69 x IO" 5 4.69 x IO"6 n 17 10 7 Corr -0.734 -0.824 0.684 Slope -8.40 x IO" 6 -1.82 x IO" 5 1.48 x 10~ 6 Intcp 1.60 x 10" A 1.98 x IO"4 5.54 x 10-5 S4T=3 1-120 Gm 1.62 x 10-5 m 2.11 x 10-5 2.65 x IO"3 1.28 x 10-5 + S.Dev. 1.06 x 10-5 1.04 x 10-5 1.90 x IO"6 n 25 15 10 Corr -0.864 -0.944 0.619 Slope -1.24 x IO"6 -2.19 x IO"6 3.89 x io - 7 Intcp 3.59 x IO" 5 4.19 x IO" 5 1.11 X 10-5 S4T=2 1-120 Gm 1.23 x IO" 5 m 1.89 x IO" 5 2.23 x IO" 5 1.05 x 10-5 + S.Dev. 1.20 x IO" 5 1.25 x IO"3 4.57 x IO" 6 n 28 20 8 Corr -0.559 -0.577 -0.835 Slope -8.41 x IO"7 -1.22 x 10~ 6 -1.56 x IO"6 Intcp 2.96 x IO" 5 3.39 x IO" 5 1.60 x 10-5 S4T=4 1-120 Gm 2.10 x IO" 7 2.03 x 10~ 7 IO"7 m 2.07 x 10~ 7 2.12 x + S.Dev. 1.65 x IO"8 7.12 x IO"9 2.27 x 10-8 n 8 4 4 Corr 0.367 -0.276 0.365 Slope 2.47 x IO"9 -1.52 x 10~ 9 6.43 x 10-9 Intcp 1.99 x IO"7 2.05 x 10~ 7 2.03 x io- 7 3.6 - 197 -Table A.30 S p e c i f i c A c t i v i t y of 5 1 C r - L a b e l l e d P l a t e l e t s w i t h i n CCD Regions I I and I I I i n Comparison to the E n t i r e D i s t r i b u t i o n CCD Mean CPM/Plt I I CPM/Plt I I I CPM/Plt 5/5/81 Gm 7.01 x IO" 3 m 1.77 x IO" 4 1.90 x 10-4 1.63 x 10-4 + S.Dev. 1.11 x IO" 4 1.67 x 10-4 1.79 x 10-5 n 8 4 4 Corr -0.281 -0.397 -0.886 Slope -1.27 x IO" 5 -5.15 x 10~ 5 -1.23 x IO" 5 Intcp 2.21 x IO" 4 2.68 x 10-4 1.82 x 10-4 1/21/82 Gm 3.05 x 10~ 5 m A.04 x-10-5 4.69 x IO" 3 3.18 x 10-5 + S.Dev. 2.13 x IO" 5 2.16 x 10-5 1.94 x 10-5 n 14 8 6 Corr -0.252 0.648 -0.932 Slope -1.29 x 10~ 6 5.70 x IO" 6 -9.65 x IO" 6 Intcp 4.88 x IO" 5 2.70 x 10-5 5.59 x 10-5 12/10/81A Gm 8.53 x IO" 5 m 5.16 x IO" 4 1.17 x IO" 3 5.58 x 10-5 _+ S.Dev. 1.03 x IO" 3 1.40 x IO" 3 9.88 x 10~ 6 n 17 7 10 Corr -0.665 -0.896 0.572 Slope -1.35 x 10-4 -5.81 x 10-4 1.87 x 10~ 6 Intcp 1.60 x IO" 3 2.92 x i o - 3 4.74 x 10-5 12/10/81B Gm 3.23 x IO" 4 m 1.33 x IO" 3 2.72 x i o - 3 3.23 x 10-4 + S.Dev. 2.22 x IO" 3 2.98 x IO" 3 1.11 X 10-4 n 19 8 11 Corr -0.583 -0.567 0.898 Slope -2.30 x 10-4 -6.91 x IO" 4 3.00 x 10-5 Intcp 3.41 x IO" 3 5.14 x IO" 3 1.72 x IO" 4 12/10/81C Gm 7.20 x IO" 4 m 4.25 x IO" 3 8.72 x IO" 3 6.71 x IO" 4 +_ S.Dev. 7.30 x IO" 3 9.39 x i o - 3 1.87 x IO" 4 n 18 8 10 Corr -0.639 -0.652 0.777 Slope -8.73 x IO" 4 -2.50 x : 10" 3 4.80 x 10-5 Intcp 1.17 x IO" 2 1.75 x IO" 2 4.55 x IO" 4 3.6 - 198 -Table A.31 S p e c i f i c A c t i v i t y of Regions I I and I I I , Normalized w i t h Respect to the Grand Mean, and the Mean at Ten Percent Combined E r r o r SA/Gm T=0 I I / I I I T=l I I / I I I T=2 I I / I I I T=3 I I / I I I T=4 I I / I I I SI 1.656/0.369 0.967/0.962 1.710/0.320 1.012/0.981 S2 1.589/0.5A2 1.003/0.946 S3 0.968/0.921 1.003/0.977 SA ' 1.599/0.825 1.636/0.791 1.816/0.855 0.964/1.012 5/5/81 2.6A1/2.266 1/21/82 1.538/1.0A3 12/10/81A 13.72/0.65A 12/10/81B 8.A21/1.000 12/10/81C 12.11/0.932 SA/m T=0 I I / I I I T=l I I / I I I T=2 I I / I I I T=3 I I / I I I T=4 I I / I I I SI 1.518/0.229 1.003/0.998 1.255/0.235 1.015/0.985 S2 1.722/0.588 0.998/0.941 S3 1.025/0.975 1.013/0.987 SA 1.2A9/0.6AA 1.260/0.636 1.177/0.556 0.976/1.024 5/5/81 1.076/0.92A 1/21/81 1.161/0.787 12/10/81A 2.267/0.108 12/10/81B 2.0A5/0.2A3 12/10/81C 2.052/0.158 3.6 Table A.32 Concentrations of Chromium and Indium i n L a b e l i n g Mixtures CCDC 51-Cr Concentration Range Concentration Range Stock ml ug nmoles ag amoles 1-40 0.1 0.1-0.25 2-5 25-625 0.5-1.25 41-80 0.5 0.5-1.25 10-25 125-312.5 1.25-3.125 81-120 1.0 1.0-2.5 20-50 250-625 5-12.5 CCDC 111-In Maximum Concentration Maximum Concentration Stock ml ng nmoles ag amoles 1-40 0.1 5 45 1.25 11.25 1-80 0.2 10 90.1 2.5 22.53 81-120 0.5 25 225 6.25 56.25 3.7 - 199 -Table A. 33 S p e c i f i c A c t i v i t y of 5 1 C r and inin Labeled P l a t e l e t s Sample 111-In CPM/Plt 51-Cr CPM/Plt x l O 4 x l O A L x l 0.9875 1.0556 Lx2 1.861 3.3523 Lx3 12.08 7.5379 CCDC 1-40 0.684 0.853 CCDC 41-80 1.61 3.23 CCDC 81-120 9.99 7.20 3.7 - 200 -Fi g u r e A. 3 CCD of i ; L 1 I n - L a b e l e d LDP CCDC 1-30, IHHD CCDC 31-60, LIDP 61-90, and HHD CCDC 91-120: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet at 10% CE per CCDC (dotted l i n e ) with mean at one h a l f graph h e i g h t ; CCDC 1-120, a b s c i s s a . 3.7 : 0 10 20 30 40 50 60 70 60 90 100 110 120 F i g u r e A.A CCD of 5 1 C r - L a b e l e d LDP, IHHD, LIDP, and HHD: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y cpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; cpm/platelet at 10% CE per CCDC (dotted l i n e ) with mean at one h a l f graph h e i g h t ; CCDC 1-120, a b s c i s s a . 3.7 - 201 -Table A. 34 Sulphate I n j e c t i o n Schedule Date Day of Time of Rabbit Rabbit Days Old ( p . i . 35-S) on Aug Expt I n j e c t i o n Number Weight T 17 W 18 T 19 F 20 S 21 F 13 0 2:05 PM 22 5.5 4 5 6 7 8 S 14 1 3:15 PM 23 4.2 3 4 5 6 7 S 15 2 2:00 PM 24 4.5 2 3 4 5 6 M 16 3 2:30 PM 26 4.55 1 2 3 4 5 3.8 Table A.35 P l a t e l e t Recovery Rabbit Blood PRP Blood P i t / m l PRP P i t / m l P i t Recovered No. ml (g) ml (g) x l O " 8 x l 0 ~ 8 x l O " 9 R26 32.7 16.12 2.55 5.18 8.35 14.8 2.35 7.66 R24 32.7 18.5 3.70 6.54 12.1 16.23 3.25 10.62 R23 35.0 14.7 2.32 5.53 8.13 12.48 1.97 6.90 R22 33.0 14.6 2.25 5.08 7.42 13.24 2.04 6.73 3.8 - 202 -Table A. 36 3 3 S - S u l p h a t e L a b e l i n g K i n e t i c s Time Blood PRP PPP CCD P i t Lx ~ Day CPM/ml DPM/ml CPM/ml DPM/ml CPM/ml DPM/ml CPM/ml DPM/ml x l O " 3 xlO-4 x l O " 3 x l 0 ~ 4 x l O " 3 x l O - 4 x l 0 ~ 3 x l 0 ~ 3 1 4.08 1.62 2 1.49 0.62 3 0.58 0.24 4 0.74 0.34 6.62 1.046 7.52 3.96 0.634 4.18 1.90 0.304 1.96 1.94 0.302 2.30 1.202 2.54 5.45 0.656 2.97 6.54 0.312 3.24 6.95 0.370 1.60* 3.67* A c t i v i t y % 1=1 A c t i v i t y % T=l A c t i v i t y % 1=1 A c t i v i t y % 1=1 36.52 38.27 59.82 60.61 55.59 54.58 116.93 120.00 3 14.22 14.81 28.70 29.06 26.06 26.96 127.56 127.52 4 18.14 20.99 29.31 28.87 30.59 30.78 62.99 67.34 CPM/ml DPM/ml CPM/ml DPM/ml CPM/ml DPM/ml CPM/ml DPM/ml x l O " 3 x l O " 3 x l O " 3 x l O " 3 x l O " 3 x l O " 3 x l O " 3 x l O " 3 1 2.93 6.40 3.42 5.94 3.28 5.76 0.39 1.14 3.01 6.63 3.52 6.14 3.36 5.92 0.40 1.15 2 1.06 2.70 1.48 3.18 1.54 3.28 0.47 1.25 1.06 2.75 1.46 3.18 1.62 3.40 0.59 1.44 3 3.70 1.45 0.74 2.14 0.76 2.16 0.57 1.39 3.90 1.51 0.68 2.06 0.74 2.16 0.55 1.38 4 4.00 1.40 0.70 2.08 0.92 1.94 0.35 1.23 5.00 1.60 0.70 2.10 0.60 2.42 0.36 1.24 A c t i v i t y % T=l A c t i v i t y % T=l A c t i v i t y % 1=1 2 36.18 42.19 43.27 53.53 46.95 56.94 120.51 35.22 41.48 41.48 51.79 48.21 57.43 147.50 3 12.63 22.66 21.64 36.03 23.17 37.50 146.15 12.96 22.78 19.32 33.55 22.02 36.49 137.50 4 13.65 21.88 20.47 35.02 18.29 33.68 91.28 16.61 24.13 19.89 34.20 27.38 40.88 90.50 A c t i v i t y % 1=1 109.65 125.22 121.93 120.00 107.72 107.48 Day Hr A c t i v i t y % T=l A c t i v i t y ' % 1=1 A c t i v i t y % 1=1 A c t i v i t y % T=l 1 21 100 100 100 100 2 46 35.97 + 0.67 48.19 + 10.11 50.25 + 4.67 128.3 + 16.71 3 68 13.27 + 0.83 23.22 + 4.49 23.75 + 2.08 137.1 + 9.30 4 94 16.13 + 2.28 23.22 + 5.28 25.42 + 6.38 81.92 + 16.40 Based on three samples 1 21 100 100 100 100 2 46 40.65 + 2.08 55.31 + 4.67 56.32 + 1.52 118.29 + 7.92 3 68 20.08 + 4.57 32.88 + 3.53 33.32 + 6.39 123.15 + 3.91 4 94 22.33 + 1.62 32.70 + 3.33 35.11 + 5.20 94.18 + 23.24 Based on three samples 1 21 100 100 100 100 2 46 38.31 + 2.91 51.75 + 8.05 53.25 + 4.55 123.30 + 12.92 3 68 16.68 + 4.75 28.05 + 6.52 28.53 + 6.74 130.11 + 9.94 4 94 19.23 + 3.83 27.96 + 6.52 30.27 + 7.44 87.89 + 19.17 Based on s i x samples 3.8 - 203 -Table A.37 St r a c t a n Concentration and Density, Rand et a l . (1981) % StR Density Density Density (g/ml) (g/ml) (g/SBStR) (g/ % StR) 15 1.058 0.003867 15.5 1.060 0.003871 16 1.063 0.003938 0.003958 17 1.067 0.003941 0.003922 17.5 1.069 0.003943 19 1.076 0.004 Average 0.003929 + 0.0000441 (8) 3.9 Table A.38 S t r a c t a n Concentration and Density, Corash and Shafer (1982) % StR Density Density Density (g/ml) (g/ml) ( g / % StR) (g/% StR) 15 1.059 0.003933 16 1.063 0.003938 0.003958 17 1.067 0.003941 0.003922 20 1.082 0.0041 Average 0.003965 + 0.0000671 (6) 3.9 Table A.39 P l a t e l e t Recovery from Gradient, Day Two and Day S i x Day Two P l a t e l e t s x IO" 9 P l a t e l e t s % Recovered Greater x IO" 9 Greater % Recovered Greater % Sample Bloody 8.424 Blood2 8.727 Blood3 9.225 PRT 11.781 0.6414 GLx 11.644 0.6339 LDPT 1.893 15.4 0.0381 5.2 1.60 LDPLxi 1.699 0.0633 3.59 LDPL.X2 2.085 0.0932 4.28 IDPT 7.281 59.1 0.1406 23.5 1.89 IDPLxj_ 4.173 0.5539 11.72 IDPLx 2 8.385 0.5572 6.23 HDPT 2.866 23.3 0.0840 14.1 2.85 HDPLxj_ 2.194 0.2929 11.78 HDPLx 2 3.080 0.3482 10.16 HHDT 0.281 2.3 0.3426 57.3 54.91 T o t a l 12.32 100 0.5981 100 - 204 -Table A.39-cont. P l a t e l e t Recovery from Gradient, Day Two and Day S i x Day S i x P l a t e l e t s P l a t e l e t s Greater Greater Greater x 10-9 % Recovered x l O " 9 % Recovered % Sample Bloodi 13.21 Blood2 13.334 Blood3 13.83 PRT 9.797 1.0375 GLx 9.683 1.049 LDPT 2.097 15.4 0.2379 17.2 LDPLxi 0.813 0.0834 LDPLx 2 I DPT 7.504 55.0 0.5794 41.9 IDPLxi 7.133 0.4335 IDPLx 2 HDPT 3.849 28.2 0.3661 26.5 HDPLxi 2.314 0.1612 HDPLx 2 2.824 0.2084 HHDT 0.182 1.3 0.1989 14.4 0.207 0.4658 T o t a l 13.633 100 1.3824 100 3.9 Day Two: The blood and ACD p l a t e l e t count was 1.52 x 1 0 8 p i t / m l , range 1.36-1.68 x 1 0 8 p i t / m l . Blood]_ was based on 55.5 ml; Blood2 was based on 57.5 ml; Blood3 was based on 60.8 g. P l a t e l e t s , harvested from blood and resuspended i n 8.6 ml of Tyrode's (PRT) s o l u t i o n were r e p r e s e n t a t i v e o f 127% recovery from blood. The gradient load mixture (GLx) was suspended i n 8.5 ml and gradi e n t f r a c t i o n s LDPT, IDPT, HDPT and HHDT were suspended i n 5 ml of Tyrode's (T). Lx\ was the CCD load mixture (3.1 ml) counted with the CCDF. L x 2 was counted again two days a f t e r CCD a n a l y s i s . HHD was a small subpopulation of p l a t e l e t s of de n s i t y even g r e a t e r than that of the HDP, and p e l l e t s through the l a s t step of 20% S t r a c t a n , along with the majo r i t y of the c e l l u l a r contamination. The t o t a l p l a t e l e t recovery from the gradient represented 105.81% recovery o f the p l a t e l e t s , and 94.35% of those p a r t i c l e s o f a s i z e greater than the normal p l a t e l e t based on the den s i t y gradient load mixture (GLx) counts. Day S i x : The blood and ACD p l a t e l e t count was 2.54 x 1 0 8 p i t / m l . Bloodj_ was based on 52.0 ml; Blood2 was based on 52.5 ml; Blood3 was based on 54.5 g. P l a t e l e t s harvested from blood and resuspended i n 8.6 ml of Tyrode's (PRT) s o l u t i o n , were r e p r e s e n t a t i v e of 73.4% recovery from blood. The t o t a l p l a t e l e t recovery from the gradient represented 140% recovery o f the p l a t e l e t s , and 133% of those p a r t i c l e s of a s i z e greater than the normal p l a t e l e t , based on the de n s i t y g r a d i e n t load mixture (GLx) counts; and 102% p l a t e l e t recovery, based on the blood p l a t e l e t count. - 205 -Table A.40 S t r a c t a n Gradient Composition StR% 22% Stock StR Tyrode•s F i n a l (ml) (ml) 10 2.2 2.64 16 21.82 8.18 18 24.55 5.45 20 27.27 2.73 3.10 Table A.41 S t r a c t a n Concentration and Density StR% StR% Density Density Density (w/v) (g/ml) g/% StR) (g/ % StR) Measured* - Based on M* Tyrode•s 1.45 10 11.4 1.04127 0.004127 0.003620 16 16.6 1.05102 0.003189 0.003073 18 18.5 1.08095 0.004497 0.004376 20 21.0 1.10652 0.005326 0.005072 22 22.2 1.08595 0.003907 0.003872 Average 0.004209 0.004003 3TTo Table A.42 P l a t e l e t Recovery from Gradient, Day Two and Day Six P l a t e l e t P l a t e l e t Greater Greater Greater P a r t i c l e s P a r t i c l e s P a r t i c l e s P a r t i c l e s P a r t i c l e s x 1 0 - 9 % Recovered x 1 0 - 8 % Recovered % Sample Day Two GLx 7.675 LDPLx 1.624 IDPLx 6.834 HDPLx 1.791 T o t a l 10.251 Day S i x GLx 14.6 LDPLx 3.744 IDPLx 8.259 HDPLx 1.716 HHD 0.193 T o t a l 10.391 6.07 15.85 0.426 66.67 4.041 17.48 1.199 100 5.667 5.284 26.91 4.265 59.37 9.925 12.33 2.857 1.38 0.739 100 17.78 7.33 7.52 2.54 71.3 17.91 21.17 6.70 100 5.24 3.49 23.98 10.23 55.8 10.73 16.06 14.27 4.15 27.27 100 11.34 3.10 On day two p . i . , the t o t a l p l a t e l e t s recovered represented 133% of the p l a t e l e t s loaded on the g r a d i e n t (GLx) i n 8-8.73 ml; on day s i x the t o t a l p l a t e l e t s recovered represented 95% of GLx. - 206 -Table A. 43 P l a t e l e t Recovery from Gradient, Day Two P l a t e l e t P l a t e l e t Greater Greater Greater P a r t i c l e s P a r t i c l e s P a r t i c l e s P a r t i c l e s P a r t i c l e s x 10-9 % Recovered x l O " 8 % Recovered % Sample GLx 8.189 2.6997 3.19 LDPLx 2.594 25.93 1.717 16.41 6.21 IDPLx 5.342 53.40 5.765 55.1 9.74 HDPLx 1.767 17.66 2.735 26.14 13.40 HHD 0.301 3.01 0.246 2.35 7.56 T o t a l 10.004 100 10.463 100 9.47 3.10 A l l samples were resuspended i n 3.1 ml except that o f the GLx which was resuspended i n 9.46 g, of which 9.2 g was a p p l i e d t o the g r a d i e n t s . The p l a t e l e t s and p a r t i c l e s of s i z e greater than p l a t e l e t s (Greater P a r t i c l e s ) , were expressed as counts, c a l c u l a t e d f o r the e n t i r e sample. The t o t a l p l a t e l e t s recovered represented 122% of the p l a t e l e t s loaded on the gradient i n 9.2 ml (GLx); whereas the t o t a l Greater P a r t i c l e s recovered represented 388% of that which was loaded. The number of Greater P a r t i c l e s recovered was c o n s i s t a n t l y higher i n the IDP, but was v a r i a b l e w i t h i n each d e n s i t y subpopulation. Table A.44 Recovery of R a d i o a c t i v i t y from Gradient, Day Two 3-H 3-H 35-S 35-S 3-H 35-S DPM % T o t a l DPM % T o t a l DPM/Plt DPM/Plt x 10" 3 Recovered x 10" 3 Recovered x 1 0 6 x 10 6 Glx 48.116 20.332 5.88 2.48 LDPLx 0.465 18.75 1.674 10.24 0.179 0.645 IDPLx 0.806 32.5 8.773 53.70 0.151 1.64 HDPLx 0.775 31.25 5.332 32.64 0.439 3.02 HHD 0.434 17.5 0.558 3.42 1.44 1.85 T o t a l 2.48 100 16.337 100 0.248 1.63 3.10 DPM ( 3 5 S , or 3H) The r a d i o a c t i v i t y , expressed i n was th a t c a l c u l a t e d f o r the e n t i r e sample. The t o t a l r a d i o a c t i v i t y recovered was represented by 5.15% of the 3H and 80.35% of the 3 5 S loaded onto the g r a d i e n t . Increased 3H-thymidine uptake ( i n a d d i t i o n to 3 5 S - ) , might a l s o be c h a r a c t e r i s t i c o f p l a t e l e t s of higher d e n s i t y . - 207 -Fig u r e A.5 CCD of Day Two 3 5 S - LDP CCDC 1-40, IDP CCDC 41-80, and HDP CCDC 81-120: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt per CCDC (dotted l i n e ) mean at h a l f graph height; CCDC 1-120 a b s c i s s a . 3.10 0 10 20 30 40 50 60 70 80 90 100 110 120 F i g u r e A.6 CCD of Day Two 3 5 S - LDP, IDP, and HDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt at 10% CE per CCDC (dotted l i n e ) mean at h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 - 208 -Fi g u r e A.7 CCD of Day S i x 3 5 S - LDP CCDC 1-40, IDP CCDC 41-80, and HDP CCDC 81-120: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t ordinate;* r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt at 200% CE per CCDC (dotted l i n e ) mean at h a l f graph height; CCDC 1-120 a b s c i s s a . 3.10 F i g u r e A.8 CCD of Day S i x 3 5 S - LDP, IDP, and HDP: p l a t e l e t s per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; r a d i o a c t i v i t y per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt at 100% CE per CCDC (dotted l i n e ) mean at h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 - 209 -Table A.45 Sulphate I n j e c t i o n Schedule Date Jan Day of Expt Time of I n j e c t i o n Rabbit Number Rabbit Days Old ( p . i . Wt (kg) W 12 T 13 F , 35-S) on 14 S 15 S16 F 7 0 1:00 PM 40 3.65 5 6 7 8 9 S 9 2 3:00 PM 39 4.15 3 4 5 6 7 T 11 4 1:00 PM 37 4.16 1 2 3 4 5 3.11 Table A.46 C e l l u l a r E s t i m a t i o n Using the C y t o c e n t r i f u g e Lens F i e l d F i e l d Scan Scan Power Diameter Area Area Cytospot mm mm2 mm2 % T o t a l 4x 4.4 15.205 30.8 80.03 lOx 1.8 2.545 12.6 32.74 40x 0.46 0.166 3.22 8.37 lOOx 0.19 0.028 1.33 3.46 3.12 The area of the c e l l u l a r specimen on the c y t o c e n t r i f u g e s l i d e (Appendix Table A.46; Cytospot area) was 38.48 mm2 based on 7 mm diameter. The number of c e l l s i n the t o t a l specimen was c a l c u l a t e d from Scan% T o t a l . - 210 -Figure A. 9 CCD of Day One CCDC 1-40, Three CCDC 41-80, and F i v e CCDC 81-120, 3 5 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 5 S / 3 H per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . 3.11 Fig u r e A. 10 CCD of Day One, Three, and F i v e , 3 3S/ 3H-l_abeled P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 5 S / 3 H at 10% CE per CCDC (dotted l i n e ) w i t h mean at one h a l f the graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.11 - 211 -Figure A. 11 CCD of Day Two CCDC 1-40, Four CCDC 41-80, and S i x CCDC 81-120, 3 5 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 3 S / 3 H per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . 3.11 Figure A. 12 CCD of Day Two, Four, and S i x , 3 3 S / 3 H - L a b e l e d P l a t e l e t s : p l a t e l e t s e r o t o n i n r a d i o a c t i v i t y dpm per CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; sulphate dpm per CCDC (dashed l i n e ) r i g h t o r d i n a t e ; 3 5 S / 3 H at 10% CE per CCDC (dotted l i n e ) with mean at one h a l f the graph height; CCDC 1-120 a b s c i s s a . 3.11 - 212 -Table A.47 P l a t e l e t s of M y e l o p r o l i f e r a t i v e Disorders Sample P i t 1 Ct A - l B-1 C - l D-l E - l IW F30 PCD 72.9 47.1 38.6 25.2 20.4 AER F42 PCD 66.3 43.3 30.7 25.4 10.1 WB M61 PCR 39.2 11.4 10.4 8.0 5.9 Normal 41.9 28.8 24.1 17.4 10.7 IW F30 PCR 48.9 22.3 19.7 13.2 5.8 Normal 66.2 66.3 60.7 41.9 22.1 WB M61 PCD 61.0 53.0 52.0 39.1 26.4 EVB F48 PCD 71.2 65.7 62.2 48.4 31.3 Normal 88.1 85.5 76.0 55.4 38.2 LHS M61 PCD 91.5 65.0 58.8 32.4 24.8 GEM M53 PCD 61.3 39.8 28.0 18.4 15.8 NKC F19 PCD 79.9 54.2 45.4 25.0 17.5 Normal 71.8 64.1 51.3 24.7 13.7 JM M47 PCR 37.1 26.0 22.7 13.0 6.3 Normal 68.8 59.7 53.9 28.9 16.4 3.13 Table A.48 P l a t e l e t s of Age, Sex Matched Normal C o n t r o l s CRCBBNormal A - l B-1 C - l D-l E - l NCB F20 A+ 39.3 31.9 16.8 15.3 JAM F32 A- 48.2 39.02 33.5 20.9 29.6 DJS F24 B+ 45.7 39.2 31.8 26.1 10.9 BH F44 0+ 49.8 37.9 27.9 18.8 JBB F58 0+ 44.1 42.4 37.1 52.3 RJR M34 A+ 40.9 35.0 24.2 16.7 11.8 ES M52 A+ 87.2 44.5 46.0 28.7 23.0 EDO M56 A+ 59.2 49.4 45.3 28.0 23.0 OGP M42 A- 60.9 59.9 87.2 57.4 48.0 TT M40 AB+ 51.4 47.1 42.0 21.2 7.9 L J M58 0+ 59.4 53.4 54.4 24.4 18.9 JAN M30 0- 41.2 35.5 28.4 28.6 13.0 DL M40 0- 62.0 64.2 58.8 36.1 24.1 JED M59 0- 63.6 47.9 53.2 54.1 20.7 PMS 34 0+ 37.3 29.5 27.1 18.3 13.2 JC M38 61.2 36.7 47.7 23.1 TP M52 37.3 36.2 23.5 20.3 11.5 3.13 - 213 -Table A.49 P l a t e l e t s of M y e l o p r o l i f e r a t i v e Disorders and Normals Sample P i t Ct A - l B-1 C-1 D-1 E-1 IW F30 PCD 7279 4771 3876 2572" 2075 AER F42- PCD 66.3 43.3 30.7 25.4 . 10.1 WB M61 PCD 61.0 53.0 52.0 39.1 26.4 EVB F48 PCD 71.2 65.7 62.2 48.4 31.3 LHS M61 PCD 91.5 65.0 58.8 32.4 24.8 GEM M53 PCD 61.3 39.8 28.0 18.4 15.8 NKC F19 PCD 79.9 54.2 45.4 25.0 17.5 JM M47 PCR 37.1 26.0 22.7 13.0 6.3 WB M61 PCR 39.2 11.4 10.4 8.0 5.9 IW F30 PCR 48.9 22.3 19.7 13.2 5.8 Normal 66.2 66.3 60.7 41.9 22.1 Normal 41.9 28.8 24.1 17.4 10.7 Normal 88.1 85.5 76.0 55.4 38.2 Normal 71.8 64.1 51.3 24.7 13.7 Normal 68.8 59.7 53.9 28.9 16.4 _____ : A d d i t i o n a l specimens from MPD p a t i e n t s EEB M40, MLF F31, DMA F39, VMS F45 were not in c l u d e d i n the p a r t i t i o n s t u d i e s , however s i z i n g analyses were performed. - 214 -Table A.50 S i z e Analyses of P l a t e l e t s from Normals and MPD %~W NorTW NorBb NorBb MPD.EEB MPD,MLF MPD,PMA MPD,VSM 2 0.25 0.25 3 0.6 0.25 0.75 4 5.85 1.45 4.85 5 7.55 1.95 7.55 6 8.25 2.15 10.3 7 7.55 1.95 10.65 8 6.7 1.75 10.8 9 6.05 1.45 10.45 10 5.45 1.45 10.1 11 5.45 1.25 9.05 12 4.35 0.75 7.75 13 4.7 0.95 7.9 14 3.5 0.75 6.05 15 3.3 0.6 6.4 16 2.95 0.45 5.2 17 2.45 0.45 4.2 18 2.15 0.25 3.65 19 1.95 0.25 3.5 20 1.75 0.25 2.8 21 1.45 0.45 2.75 22 1.45 0.25 1.95 23 1.25 0.25 1.95 24 0.75 1.8 25 0.55 0.25 1.6 26 0.95 1.45 27 0.75 1.25 28 0.75 1.1 29 0.6 1.1 30 0.6 1.1 31 0.6 1.1 32 0.45 0.75 33 0.45 0.25 0.75 34 0.25 0.6 35 0.25 0.6 36 0.25 0.6 37 0.25 0.6 38 0.25 0.45 39 0.45 40 0.25 0.45 41 0.25 0.25 42 0.45 43 0.45 44 0.25 46 0.25 47 0.25 49 0.25 51 0.25 0.25 0.25 0.95 0.25 0.45 0.6 6.7 2.95 2.8 4.55 7.55 3.3 3.15 4.9 7.75 3.15 3.65 4.9 7.35 3.3 3.65 4.7 6.7 2.45 2.95 4.2 6.05 2.3 2.95 3.15 5.7 1.95 2.65 3.15 4.35 1.95 2.15 2.25 3.95 1.45 1.95 2.1 3.1 1.45 1.65 1.6 2.95 1.1 1.3 1.75 2.45 1.25 1.15 1.45 1.95 0.95 0.75 1.1 1.95 0.75 0.95 0.95 1.6 0.75 0.95 0.95 1.6 0.75 0.45 0.95 1.25 0.45 0.6 0.6 0.95 0.45 0.6 0.45 0.75 0.45 0.45 0.6 0.95 0.45 0.25 0.45 0.6 0.45 0.25 0.45 0.45 0.45 0.45 0.75 0.45 0.25 0.45 0.6 0.45 0.45 0.45 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.45 0.25 0.25 0.25 0.25 0.25 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 3.13 - 215 -Table A.51 S i z e Analyses of P l a t e l e t s from Normals and MPD % w NorPS NorBB MPD,NKC MPD,GEM MPD,LHS MPD, JM 2 0.18 0.17 0.34 0.34 3 0.35 0.51 0.35 0.68 1.0 0.18 4 2.9 4.3 1.5 5.5 6.9 1.2 5 3.7 6.4 1.9 6.0 8.3 1.4 6 3.4 5.9 1.7 5.2 7.7 1.0 7 4.5 5.7 1.4 5.0 7.2 1.0 8 3.4 4.3 1.2 4.2 5.4 0.69 9 3.4 4.5 0.69 3.4 4.7 0.87 10 3.2 3.6 0.87 2.4 4.2 0.69 11 3.1 3.2 0.69 2.4 3.2 0.87 12 2.0 2.4 0.51 1.9 2.5 0.51 13 2.4 2.2 0.51 2.0 2.4 0.51 14 1.7 1.5 0.34 1.7 1.9 0.35 15 1.7 1.7 0.34 1.0 1.4 0.35 16 1.7 1.4 0.34 1.0 1.4 0.18 17 1.5 1.5 0.18 1.2 1.0 0.18 18 1.2 1.0 0.85 1.0 0.18 19 1.2 0.85 0.85 0.51 0.35 20 0.85 0.68 0.18 0.51 0.51 0.18 21 0.68 0.68 0.32 0.51 0.18 22 0.85 0.51 0.18 0.32 0.51 0.18 23 0.69 0.51 i 0.50 0.18 0.18 24 0.51 0.35 0.32 0.35 0.18 25 0.51 0.50 0.17 0.18 0.18 26 0.51 0.34 0.17 0.18 27 0.51 0.34 0.17 0.18 28 0.34 0.34 0.17 0.18 29 0.34 0.17 0.35 30 0.17 0.17 0.16 31 0.17 0.17 0.16 32 0.17 0.17 0.16 33 0.17 0.16 0.18 34 0.34 0.17 35 0.18 0.17 36 0.18 0.16 37 0.35 38 0.17 3.13 The r e l a t i v e p l a t e l e t c o n c e n t r a t i o n s during s i z i n g analyses were: NorTW, 287; NorBb, 55; NorBb, 417; MPD,EEB, 235; MPD,MLF, 98; MPD,DMA, 97 ® 1/6 d i l u t i o n of 649; MPD,VSM, 137; NorPS, 146; NorBb, 162; MPD,NKC, 34; MPD,GEM, 144; MPD,LHS, 182; MPD,JM, 31. 

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