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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., L o y o l a U n i v e r s i t y , 1972 B.S., C a l i f o r n i a S t a t e U n i v e r s i t y , 1976 M.S., C a l i f o r n i a S t a t e 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  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required  standard  UNIVERSITY OF BRITISH COLUMBIA May 1984 Timothy James Webber, 1984  In  presenting  requirements  this for  an  of  British  it  freely available  agree for  that  I  by  understood  that  his  that  or  be  her or  shall  of  the  University shall  and  study.  I  copying  granted  by  the  of  publication  not  be  allowed  Columbia  of  make  further this  head  representatives.  fCL  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  at  the  Library  permission.  Department  f u l f i l m e n t of  the  extensive  may  copying  f i n a n c i a l gain  degree  reference  for  purposes  or  partial  agree  for  permission  scholarly  in  advanced  Columbia,  department  for  thesis  It  this  without  thesis  of  my  i s  thesis my  written  ii ABSTRACT  P l a t e l e t s a r e heterogeneous w i t h r e s p e c t t o a v a r i e t y and  physical  properties,  Megakaryocytes c o n f e r which  then  analyses  change  as  a  upon t h e i r  during  of platelet  result  of  different  progeny d i f f e r e n t  circulation.  production  and  Since  of  phenomena.  platelet properties  platelet  platelet  functional  ageing  survival have  time  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 t o o b t a i n comparable information use  regarding  platelet  heterogeneity  through  the  o f 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 ,  density,  and membrane s u r f a c e  change  during  Platelet charge  the  membrane  survival  to  be  distribution  erythrocyte maturation  the  of  i n an  two-polymer  phase  and a g e i n g .  t h e upper  decreased  with  and  decreasing  during  here  the c i r c u l a t i o n .  because  cell  surface  circulation,  and  because  T h i s t e c h n i q u e , CCD i n phase systems  was t h e r e f o r e a s s e s s e d circulating  investigation systems  studied  within  are believed t o  o f d e t e c t i n g such membrane changes d u r i n g  determining  information  between  platelets  which  (CCD) i n two-polymer aqueous phase systems  t o membrane c h a r g e ,  problem  of  altered  been shown t o be c a p a b l e  sensitive  characteristics,  p r o p e r t i e s were  i s thought  countercurrent has  circulating  were  lower  platelet  of platelet shown  phases,  phosphate  t o be a  f o r application to age  distribution  heterogeneity. charged,  potential  Aqueous  demonstrating,  difference  and i n c r e a s i n g c h l o r i d e  which  content.  P l a t e l e t s u r f a c e p r o p e r t y d i s t r i b u t i o n s were heterogeneous based on t h e shape  and  spread  o f t h e CCD  c o m p a r i s o n t o expected  curves  f o r these  theoretical distributions  blood  elements  f o r uniform  in  particles.  iii Platelets  from r a b b i t ,  monkey, and human demonstrated s p e c i e s  d i f f e r e n c e s i n 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 step  p a r t i t i o n measurement. The  single  step  rabbits, been  parititon  were  and  related,  membrane  of individual  low  intermediate  density,  heterogeneous  indicating different in  part  these  confirmed  that  were  surface  distributions  each  also  specific  monkeys, and humans, s e p a r a t e d  shown t o be age  size,  species  found  a l l each  single  d i f f e r e n c e s found by CCD.  Platelets,  to exhibit  properties  density,  by t h e  from  a c c o r d i n g t o d e n s i t y which has  by  d e n s i t y separated  and  within  by  specific  and  CCD.  The  platelet  high  overlapped  differences i n  density  subpopulations, platelets,  (partitions  subpopulation  there  partition  were  were  coincided), platelets  membrane s u r f a c e p r o p e r t i e s , and t h a t each s u b p o p u l a t i o n similar  density  t o the others. separated  However,  platelets  the r e l a t i v e  were  also  was  partitions  different  based  s t a t i s t i c a l comparisons o f t h e CCD p a r a m e t e r s . CCD and d e n s i t y  of  of on  analyses  51 of  in  vitro  Cr-labeled  subpopulation,  found  rabbit  t o be e n r i c h e d  platelets i n high  revealed  density  a  platelet  platelets,  which  e x h i b i t e d enhanced uptake f o r ^ C r , but not f o r "'"''""''In, "^C- o r 3 3 H-serotonin, nor f o r H-adenine. T h i s p l a t e l e t s u b p o p u l a t i o n was o f low p a r t i t i o n r e l a t i v e t o t h e p l a t e l e t p o p u l a t i o n , and was no l o n g e r apparent  after  i n vivo  injection  and c i r c u l a t i o n  f o r 24 t o 48  hours.  35 Combinations "^H-serotonin CCD,  of  S-sulphate  labeling  indicated  that  _in v i t r o , platelets  cohort density which  labeling gradient  were  newly  in  vivo,  c e n t r i f ugation, released  into  c i r c u l a t i o n were o f r e l a t i v e l y h i g h d e n s i t y and low p a r t i t i o n . this  subpopulation  was h e t e r o g e n e o u s ,  i t s average d e n s i t y and  p r o p e r t i e s changed w i t h p l a t e l e t m a t u r a t i o n  with and the  Although surface  and age i n t h e c i r c u l a t i o n .  iv  TABLE OF CONTENTS Page Chapter  1  INTRODUCTION  1  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  1  1.2  E r y t h r o c y t e and P l a t e l e t S u r f a c e Charge  3  1.3  P l a t e l e t S u r v i v a l , S u r f a c e Charge, D e n s i t y and Heterogeneity  4  1.4  Megakaryocyte and P l a t e l e t H e t e r o g e n e i t y  23  1.5  Membrane S u r f a c e P r o p e r t i e s by Aqueous Two-Phase Partition  27  P a r t i t i o n S t u d i e s o f C e l l Surface P r o p e r t i e s During Ageing  32  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 o f P a r t i t i o n Methodology  37  MATERIALS AND METHODS  41  2.1  B l o o d Samples  41  2.2  Anticoagulant  41  2.3  Tyrode's S o l u t i o n s  42  2.4  Preparations 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  Platelet Fixation  44  2.7  Platelet Labeling  44  2.8.1 2.8.2  S t r a c t a n I I Stock S o l u t i o n s Stractan Density Gradient  45 46  2.9.1 2.9.2 2.9.3  Phase System Polymer Stock S o l u t i o n s Phase System S a l t S t o c k S o l u t i o n s Two-Polymer Aqueous Phase System C o m p o s i t i o n  48 49 49  S i n g l e Step P a r t i t i o n  51  C o u n t e r c u r r e n t D i s t r i b u t i o n (CCD) CCD o f D e n s i t y S e p a r a t e d P l a t e l e t s CCD o f In V i t r o L a b e l e d P l a t e l e t s Aged I n V i v o CCD o f In V i v o Cohort L a b e l e d P l a t e l e t s  51 53 54 54  1.6 1.7 Chapter  2  2.10 2.11.1 2.11.2 2.11.3 2.11.4  V  Page 2.12.1  P a r t i c l e Counting  55  2.12.2  Particle Sizing  56  2.13  Radioactivity  58  2.14  Graphical Presentation of Results  58  2.15  Centrif ugation  59  RESULTS AND DISCUSSION 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 o f D e n s i t y S e p a r a t e d P l a t e l e t s  66  3.3  S i z e A n a l y s i s o f D e n s i t y Separated P l a t e l e t s  77  3.4  Electrophoretic Mobility Platelets  79  Chapter  3 3.1  Counting  60  (EPM) o f D e n s i t y S e p a r a t e d  3.5  EPM o f S i z e S e p a r a t e d P l a t e l e t s  79  3.6  CCD o f 5 1 - 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  81  3.7  CCD o f 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 3.7.2  Experimental Results  3.7.3  Discussion  104  CCD o f I n V i v o 35-S-Labeled P l a t e l e t s  110  3.8 3.8.1 3.8.2 3.8.3 3.9 3.9.1 3.9.2 3.9.3 3.10 3.10.1 3.10.2  Experimental Results Discussion CCD o f 35-S-Labeled Six, p . i  95 ...98  I l l 113 114 LDP, IDP and HDP, Day Two and 116  Experimental Results Discussion  117 119 122  CCD o f 35-S-Labeled LDP, IDP and HDP, Day Two and Six, p . i  124  Experimental Results  125 126  vi  Page 3.11 3.11.1 3.11.2 3.11.3 3.12 3.12.1 3.12.2 3.12.3 3.13  3.13.1 3.13.2 3.13.3  CCD o f 35-S-Labeled w i t h 3-H-Serotonin  P l a t e l e t s , Double L a b e l e d 127  Experimental Results Discussion CCD o f 35-S-Labeled w i t h 3-H-Serotonin  127 128 130 P l a t e l e t s , Double L a b e l e d 131  Experimental Results Discussion S i n g l e Step P a r t i t i o n o f P l a t e l e t s Chemotherapy  131 132 132 During  Cancer 135  Experimental Results Discussion  137 138 140  GENERAL DISCUSSION AND CONCLUSION  150  Conclusion  152  REFERENCES  155  APPENDIX  176  vii LIST OF TABLES Table 1.4.1  Page P l a t e l e t Heterogeneity Outline  26  2.8.2.1  S p e c i f i c G r a v i t y and D e n s i t y  47  2.9.3.1  Two-Polymer Phase System S a l t and R a t i o s  2.9.3.2  of Stractan Concentrations  50  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 o f S i n g l e - t u b e  62  3.2.1  Summary o f R a b b i t , Human and Monkey, LDP, IDP and HDP EPM o f S i z e S e p a r a t e d , F i x e d P l a t e l e t s , S t a t i s t i c s Student t T e s t 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 Analysis of Variance of the t S t a t i s t i c S u b d i v i s i o n o f CCD o f 5 1 - C r - L a b e l e d R a b b i t Platelets Nonpaired and P a i r e d t T e s t S p e c i f i c A c t i v i t y o f Regions I I and I I I , N o r m a l i z e d t o t h e Mean a t Ten P e r c e n t Combined E r r o r  3.5.1 3.6.1 3.6.2 3.6.3 3.6.4  Partition of P l a t e l e t s  72 80 85 86 87 90  3.8.2.1  Summary o f 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  114  3.9.2.1 3.9.2.2  S t r a c t a n C o n c e n t r a t i o n and D e n s i t y A n a l y s i s o f 35-S S p e c i f i c A c t i v i t y Enrichment Slope  120 122  A n a l y s i s o f 35-S S p e c i f i c A c t i v i t y Enrichment Slope  126  3.10.2.1 3.11.2.1  o f 35-S/3-H R a t i o S l o p e , CCD Enrichment Three and F i v e , p . i o f 35-S/3-H R a t i o S l o p e , CCD Enrichment Four and S i x , p . i  128  3.12.2.1  A n a l y s i s o f 35-S/3-H R a t i o S l o p e , CCD Enrichment  132  3.13.2.1  Comparison o f P l a t e l e t P a r t i t i o n s , Normal and MPD  139  3.11.2.2  Analysis Day One, Analysis Day Two,  128  viii LIST OF FIGURES Figure 3.1.1.1 3.1.1.2  Page 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 , F i x e d R a b b i t P l a t e l e t Two-Polymer Phase System Partitions  61 61  3.1.2.1 3.1.2.2  CCD o f F i x e d R a b b i t P l a t e l e t s R e d i s t r i b u t i o n o f P l a t e l e t CCD F r a c t i o n s  3.2.1.1 3.2.1.2  P e r c e n t Human P l a t e l e t s Remaining on S t r a c t a n Steps...68 Human 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 on S t r a c t a n 68  3.2.2.1 3.2.2.2  CCD o f D e n s i t y S e p a r a t e d CCD o f D e n s i t y S e p a r a t e d  3.3.1.1  S i z e D i s t r i b u t i o n o f Density Separated Rabbit Platelets S i z e D i s t r i b i t o n o f D e n s i t y Separated Human Platelets  3.3.1.2 3.6.1.1  Rabbit P l a t e l e t s Human P l a t e l e t s  65 65  70 70  78 78  3.6.1.2  No Change i n S u r f a c e P r o p e r t y P a r t i t i o n w i t h Time o f C i r c u l a t i o n D e c r e a s i n g P a r i t i t i o n w i t h Time i n C i r c u l a t i o n  82 82  3.6.2.1 3.6.2.2  CCD o f 5 1 - C r - l a b e l e d P l a t e l e t s 5 1 - C r - l a b e l e d P l a t e l e t s , Two days o f C i r c u l a t i o n  84 84  3.7.2.1.1 3.7.2.1.2  CCD o f 5 1 - C r - l a b e l e d P l a t e l e t s CCD o f 1 1 1 - I n - l a b e l e d P l a t e l e t s  100 100  3.7.2.1.3 3.7.2.1.4  CCD o f 5 1 - C r - l a b e l e d P l a t e l e t s CCD o f 1 1 1 - I n - l a b e l e d P l a t e l e t s  101 101  3.7.2.1.5 3.7.2.1.6  CCD o f 5 1 - C r - l a b e l e d P l a t e l e t s CCD o f 1 1 1 - I n - l a b e l e d P l a t e l e t s  102 102  3.7.2.2.1 3.7.2.2.2  CCD o f 1 1 1 - I n - L a b e l e d , LDP, IDP, HDP, HHD CCD o f 5 1 - C r - L a b e l e d , LDP, IDP, HDP, HHD  105 105  3.7.2.2.3 3.7.2.2.4  CCD o f 5 1 - C r - L a b e l e d CCD o f 5 1 - C r - L a b e l e d  LDP IDP  106 106  3.7.2.2.5 3.7.2.2.6  CCD o f 5 1 - C r - L a b e l e d HDP CCD o f 5 1 - C r - L a b e l e d HHD, P l a t e l e t s o f D e n s i t y G r e a t e r than t h a t o f 2 0 % S t r a c t a n  107  CCD o f Day Two 35-S-Labeled CCD o f Day S i x 35-S-Labeled  121 121  3.9.2.1.1 3.9.2.1.2  LDP, IDP, and HDP LDP, IDP, and HDP  107  iz  Figure 3.11.2.1 3.11.2.1  3.12.2.1 3.12.2.2 3.12.2.3 3.12.2.4  Page CCD o f Day One, Three, and F i v e , 35-S/3-H-Labeled Platelets CCD o f Day Two, F o u r , and S i x , 35-S/3-H-Labeled Platelets CCD L e u k o c y t e C o n t a m i n a t i o n o f Day Three 35-S/3-H-Labeled P l a t e l e t s CCD L e u k o c y t e C o n t a m i n a t i o n and Day Three 35-S/3-H-Labeled P l a t e l e t s CCD o f Day Three 35-S/3-H-Labeled P l a t e l e t s CCD o f Day Three 35-S/3-H-Labeled P l a t e l e t s  129 129 133 133 134 134  x  ABBREVIATIONS  AAT  a s p a r t a t e amino t r a n s f e r a s e  ACD  acid c i t r a t e dextrose  ADP  adenosine  ag am  diphosphate  —18 10" grams —18 a t t o m o l e s , 10 moles attograms,  ATP  adenosine t r i p h o s p h a t e  BTG  beta  C  current, Celloscope  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  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  c o l o n y forming  Corr  correlation  CPM  cpm c o u n t s p e r minute  D  dextran  DF  d f , degrees o f ( f ) freedom  DPM  dpm, d i s i n t e g r a t i o n s p e r minute  EPM  electrophoretic mobility  f  femto, 10""'""'  Fc  c r y s t a l l i z a b l e fragment o f immunoglobulin  Fx  fixed, glutaraldehyde  g  gravity,  G  Celloscope setting gain, p a r t i t i o n  thromboglobulin setting  cavity(ies)  cells  coefficient  fixed 2  g f o r c e , a c c e l e r a t i o n g/cm  , grams coefficient  ,  xi  GLx  gradient load mixture  Gm  grand mean, r e f e r t o 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  HHD  d e n s i t y f r a c t i o n h i g h e r t h a n HDP  HHDP  p l a t e l e t s o f d e n s i t y e q u a l t o and h i g h e r t h a n HDP  Hr  h r , hours  5HT  serotonin  Hu  human  ITP  i d o p a t h i c thrombocytopenia ( i c ) purpura  Intcp  intercept  IDP  intermediate  IHHDP  intermediate,  platelets  density  platelets  high, high density p l a t e l e t s , a l l p l a t e l e t s o f  d e n s i t y e q u a l o r g r e a t e r than IDP K  partition coefficient  LDH  l a c t i c a c i d dehydrogenase  LDP  low d e n s i t y  LIDP  low, intermediate  Lin  linear  Lx  load mixture,  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 o f CE  MNC  mononuclear c e l l  Mo  monkey  MPD  myeloproliferative disorders  NaPB  sodium phosphate b u f f e r , pH 7.2  N, n  number o f t r a n s f e r s , e x p e r i m e n t s , p l o i d y number  ng nm  platelets d e n s i t y p l a t e l e t s , l o w e r d e n s i t y than HDP  CCD  _9 nanograms, 10 grams _g nanomoles, 10 moles  xii p  parametric s t a t i s t i c a l  probability  P  nonparametric  PEG  polyethylene glycol  PF4  platelet factor  p.i.  post i n j e c t i o n  Pit  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  r  peak o r modal CCDC  Ra  rabbit  RPM  r e v o l u t i o n s p e r minute  SA  specific activity, s i a l i c  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  C e l l o s c o p e t h r e s h o l d s e t t i n g , o r time as a p p r o p r i a t e  ug  micrograms, 10  um  micromoles,  Var  variance  WGA  wheat germ  probability  four  solution  acid  grams  10~^ moles  agglutinin  SYMBOLS  %  percent, percentage,  c o n c e n t r a t i o n w/v u n l e s s s t a t e d  xiii  ACKNOWLEDGEMENTS  The M.S. committee G.S. Ascher  D.M. C a r l b e r g  B. Kazan and H. W a l t e r  i n a p p r e c i a t i o n f o r recommendation t o Dr. D.E. Brooks giving opportunity.  The Ph.D. committee D.E. Brooks  J . Frohlich  S. Naiman  R.H. P e a r c e and P.E. R e i d .  D.E. Brooks and t h e B r i t i s h Columbia Heart F o u n d a t i o n for personal support.  Mom  t h e unsung hero  p a t r o n S a i n t Jude  dad and b r o t h e r s  f o r moral s u p p o r t and encouragement.  I n fond memory o f t h e w e a l t h o f 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 a r e p e r i p h e r a l b l o o d e l e m e n t s , not c e l l s but fragments o r buds, seen by s c a n n i n g e l e c t r o n m i c r o s c o p y as b l e b s ( D j a l d e t t i e t a l . , 1979) o f t i s s u e , which a r e d e r i v e d by a membrane r e o r g a n i z a t i o n p r o c e s s (Fusion-Fission; specialized polyploid  Tavassoli,  bone  cells  marrow  1979)  from  precursor  highly  cells,  differentiated,  megakaryocytes.  a r e formed when t h e c y t o p l a s m remains u n d i v i d e d  k a r y o k i n e s i s . The p r o c e s s by w h i c h t h e megakaryocyte doublings without c e l l  division  similarly  of  lower  involved  vertebrates  i n a variety  nuclear  Platelets,  from t h e n u c l e a t e d  (Tavassoli,  o f complex  during  undergoes  i s a l s o termed endomitoses.  as a n u c l e a r b l o o d e l e m e n t s , s h o u l d be d i s t i n g u i s h e d "thrombocytes"  These  1980).  They  are  mechanisms a s s o c i a t e d  with  h e m o s t a s i s , p r i m a r i l y t h a t o f b l o o d 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 origin,  manner o f p r o d u c t i o n  and  release  from  their  stem  cells,  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 o r d e r t o m a i n t a i n c o n s t a n t numbers o f p l a t e l e t s , t h e i r cells, humoral Levin, 1983) divided become  megakaryocytes, s t i m u l a t e d feedback 1983)  mechanism  specific  produce  megakaryocyte platelets,  because  (Levin  and  (large  cytoplasm,  replacing system.  platelets  like  1979;  i n humans  which  removal red  McDonald,  aggregates  are has  cells  of  show  1981;  incompletely  which  being been  modulated  (Kawakita et a l . ,  "poly-platelets")  those This  by some s u r f a c e  Evatt,  f o r megakaryocytes  "megathrombocytes"  reticuloendothelial ageing,  presumably  precursor  mature  removed  said a  to  by  t o be defined  the  due  to  mean  - 2 circulation this  ( s u r v i v a l ) t i m e a f t e r which they  removal i n v o l v e s s e v e r a l q u e s t i o n s .  during  maturation  and  age, how  do  a r e removed. The study o f  How a r e p l a t e l e t s  platelets  change:  formed, and structurally,  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 o f p l a t e l e t ageing erythrocyte surface  ageing  charge  measurements,  i n that  analyses,  processes  density i n the  and b i o c h e m i c a l  paralleled  separation form  of  analyses  similar  studies of  was combined w i t h  cell  electrophoretic mobility  of s i a l i c  acid  and p r o t e i n .  G e n e r a l l y 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  erythrocytes  erythrocyte  becoming  and  platelets  smaller  change  and more  during  dense,  circulation,  the p l a t e l e t  the  becoming  s m a l l e r and l e s s dense. Theories associated  developed with  initially  ageing  and  with  regard  removal  of  to c e l l  membrane changes  erythrocytes  (Danon  and  Marikowsky, 1961; Y a a r i , 1969; Seaman, 1973; B a l d u i n i e t a l . , 1974 and 1977;  Luner  Bocci,  e t a l . , 1977; Seaman e t a l . , 1977; L u t z  1981) b u t which  alteration  apply  to platelets,  o r removal o f t e r m i n a l c e l l  proposed t o d e c r e a s e c e l l spleen  could  o f these  focussed  sialic  on t h e  a c i d . This  was  s u r f a c e charge and e n a b l e t h e removal by t h e  old cells,  major c h a r g e b e a r i n g  surface  and Fehr, 1979;  since  sialic  a c i d has been shown t o be a  component o f both t h e e r y t h r o c y t e membrane (Cook  et a l . , 1961; E y l a r e t a l . , 1962) and t h e p l a t e l e t membrane (Madoff e t al.,  1964). Other t h e o r i e s i n v o k e  t h e exposure o f a g e - r e l a t e d  antigens,  perhaps i n c l u d i n g g a l a c t o s e r e s i d u e s o f i n t e g r a l membrane g l y c o p r o t e i n s (Alderman antigens surface  e t a l . , 1981) o r t h e exposure (Kay, 1975 and 1978). charge  i s felt  In t h i s  sufficient  to  o f other case  normally  t h e mere  counter  the  cryptic  decrease i n  normal  charge  - 3 dependent r e p u l s i o n o f c e l l s and e n d o t h e l i u m . The exposure o f n o r m a l l y hidden the  antigens  could  as w e l l f a c i l i t a t e  r e t i c u l o e n d o t h e l i a l system.  processes  antibody  mediated removal by  I t seems r e a s o n a b l e  that  a l l o f these  might c o n t r i b u t e t o t h e removal o f both aged e r y t h r o c y t e s and  platelets.  The t h e o r i e s a r e comparable but i n disagreement  concerning  the s i t e and mode o f a c t i o n ( i . e . , removal o f s i a l i c a c i d s , exposure o f a n t i g e n s ) . I n one c a s e , surface  membrane  i t i s thought t h a t e l i m i n a t i o n o f t h e p h y s i c a l  charge-associated  repulsion  conferred  by  terminal  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 r e m o v a l . I n t h e o t h e r cellular  removal i s thought t o be f a c i l i t a t e d  cases  through a combination  of  humoral and c e l l u l a r f a c t o r s . There  i s also  another  p l a t e l e t , which r e c o g n i z e s but  concept,  specific  that platelet  with  circulation  regard  t o the  might be l i m i t e d ,  t h a t removal from t h e c i r c u l a t i o n might not be age r e l a t e d . No age  related during  membrane change need circulation.  platelet  In t h i s  s u r f a c e changes  erythrocyte).  occur  i f platelets  case,  there  could  a r e removed be  randomly  maturation-related  ( l i k e n e d t o a r e t i c u l o c y t e becoming a mature  However t h e r e  need  not be age r e l a t e d membrane  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  dissolution  after  surface 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 S u r f a c e Charge and P a r t i t i o n Comparing partition  the e l e c t r o p h o r e t i c mobility  ( t o be d i s c u s s e d )  and t h e  charge-associated  o f e r y t h r o c y t e s and p l a t e l e t s  from  various  s p e c i e s i s o f i n t e r e s t t o t h i s work. E r y t h r o c y t e s from t h e model system employed i n t h e p r e s e n t etic  mobility  of  study,  r a b b i t , e x h i b i t the lowest  a l l mammalian  erythrocytes  tested  electrophorwhereas  the  _ 4 electrophoretic  mobilities  erythrocytes  are  electrophoretic various  similar  rhesus  (Seaman,  mobilities  of  monkey  erythrocytes  1973).  A  erythrocytes  electrophoretic  individual  should  the  species  mobilities  parallel  charge-associated  one  of  and  platelets I f the  platelets  another  and  comparison  mammalian s p e c i e s might p r o v i d e i n s i g h t .  made t h a t the from  of  human of  from  the these  assumption  and  erythrocytes  (Seaman, 1967), t h e n  partition coefficients.  Rabbit  higher  erythrocytes These  are  of  t h e r e f o r e be to  of  erythrocytes careful  in  Rhesus  lower  exceptions  another,  differences rabbit  that  p a r t i t i o n much some  partitions  type  than  to  than human and a  correlation  time  different  animals.  but  differences  also  d e s t r o y e d i n the  spleen  the  remainder  Hugh-Jones, 1961;  Tavassoli,  There  1964,  survival  methods  and  endothelial  pathophysiology exposure  of  for  sequestration system of  were  in  (Hill-Zobel 1977;  studying of  et  are  not  the  only  removal  of  to  Purpura,  and  isoantibodies ITP)  was  5  and  1983;  and and  1974).  ^Cr-labeled in  Jandl,  vivo  investigated  platelet  the  reticulo-  1964a)  sequestration in  liver  Heterogeneity  platelets  (Aster  removal  al.,  Tavassoli,  human  effete  outlined  platelet  platelets  Thrombocytopenic  sensitive  bone marrow; the  P l a t e l e t S u r v i v a l , S u r f a c e Charge, D e n s i t y and In  charge  3).  p l a t e l e t s : 55-90% o f e r y t h r o c y t e s i n comparison  t o 26-28% o f p l a t e l e t s are  1.3  (Chapter  cell  e r y t h r o c y t e s and  therein,  of  rabbit  when comparing b i o l o g i c a l phenomena from one from  platelets  monkey  yet  must  survival  sequestering  platelets,  are  from g i v e n s p e c i e s . One  and  and  monkey  so  platelets  have a p a r t i t i o n s i m i l a r t o t h a t o f human p l a t e l e t s , b o t h o f which markedly  is  (e.g., (Aster  and  the  induced  by  Idiopathic and  Jandl,  - 5 1964b). U s i n g e x t e r n a l s c i n t i l l a t i o n deposition (EDTA;  of  radioactive  an a n t i c o a g u l a n t )  platelets,  scanning  platelets,  was  found  o f t h e v a r i o u s organs f o r  ethylenediamine  t o be i n j u r i o u s t o normal  ("as suggested by ... Zucker and B o r r e l l i ,  only  caused  sphering,  one  half-hour  after  tetraacetate  but f u r t h e r caused  1954"). EDTA n o t  an i n i t i a l  i n j e c t i o n ) hepatic  human  rapid  sequestration  (less  than  of nearly a l l  51 transfused destroyed  Cr-labeled i n the spleen  platelets. (starting  These  were  f o u r hours a f t e r  only one-third t o c i r c u l a t e normally.  The i n i t i a l  did  platelets  not  occur  anticoagulated greater  destroyed  the  injection),  hepatic  Of  leaving  sequestration  harvested  a c i d - c i t r a t e - d e x t r o s e (ACD).  on t h e o r d e r  from  these  o f f o u r days, t h e p l a t e l e t s  i n the l i v e r . Opsonization  isoantibody  large  with  "^Cr-labeled  partially  blood  platelets,  than t w o - t h i r d s s u r v i v e d t h e s p l e e n t o c i r c u l a t e n o r m a l l y  a half l i f e  spleen,  with  then  caused  these  platelets  being  with  eventually  o f p l a t e l e t s w i t h s m a l l amounts o f t o be  slowly  destroyed  by t h e  s i m i l a r t o t h e s i t u a t i o n i n p a t i e n t s w i t h c h r o n i c ITP; whereas  amounts o f i s o a n t i b o d y liver  concluded  as that  i n patients  caused r a p i d d e s t r u c t i o n o f p l a t e l e t s i n with  acute  m i l d l y damaged p l a t e l e t s  ITP. A s t e r  and J a n d l  are destroyed  (1964)  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 t h e l i v e r . Booyse e t a l . (1968) s e p a r a t e d density  gradient  platelets  from  human p l a t e l e t s by f o u r s t e p  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 diseased  (Immuram) therapy  states.  Immunosuppression  following renal  transplant  causes  with  azathioprine  a depression  p l a t e l e t production. In p a t i e n t s with chronic thrombocytopenic platelet numbers  sucrose  production o f heavy  was i n d u c e d dense  of  purpura,  w i t h normal plasma; t h i s i n c r e a s e d t h e  platelets.  These  heavy  platelets  were  then  - 6 "tentatively  c h a r a c t e r i z i e d as newly  formed  young  platelets".  Only  s m a l l percentage of these  p l a t e l e t s were c o n s i d e r e d  rich  They were found t o d e c r e a s e i n d e n s i t y  i n ribosome c o n t e n t .  incubated  i n synthetic mixtures  with a corresponding Ardlie  could  undergo  survive Repeated  adenosine  granule  "a more mature  when  platelet",  treatment  of  These p l a t e l e t s  conditions,  however,  and  rabbit  taken  induced  could  i n acid  aggregation  (Reimers  et  platelets  still  caused  be a g g r e g a t e d  normally  (DFP) i n v i t r o  after  and  a l . , 1973a).  by l o s s o f s e r o t o n i n  survived  ^H-diisopropylphosphofluoride  platelets  (ADP)  i n vivo  r e l e a s e , evidenced  nucleotides.  that  t o t h e method o f A s t e r and J a n d l ,  diphosphate  i n the r a b b i t  thrombin  platelet  demonstrated  and washed a c c o r d i n g  normally  t o be "young" and  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 .  e t a l . (1970)  c i t r a t e dextrose  t o produce  a  extensive  and adenine  under c e r t a i n labeling  with  (Reimers e t a l . , 1973b),  51 or a f t e r l a b e l i n g with Carty platelet  Cr-chromate (Reimers e t a l . ,  e t a l . (1975) s e p a r a t e d subpopulations  by  human,  1976).  r a t and s p l e n e c t o m i z e d  preparative  sucrose  density  e l e c t r o p h o r e s i s and demonstrated t h a t t h e " f a s t e r " p l a t e l e t s , higher  electrophoretic  ^Cr-chromate  mobilities,  (consistent  a g g r e g a t e s more r e a d i l y  with  a  were  larger,  subpopulation  rat  gradient with the  incorporated of  platelets  i n t h e presence o f ADP, as l a t e r  more which  reported  by  Gear, 1981; Haver and Gear, 1981; Haver and Gear, 1982), and c o n t a i n e d more t o t a l s i a l i c lower  a c i d , DNA, and RNA, t h a n t h e " s l o w e r "  electrophoretic mobilities.  platelet platelets  However,  t h e DNA  p r e p a r a t i o n s might be due t o c o n t a m i n a t i n g have  mitochondria.  only  trace  amounts  of  DNA  platelets found  leukocytes  i n their  small  in  with these  because and  few  - 7 I s o b e and Yamanaka (1976) a l s o ageing,  membrane  mobilities  of  surface  human  charge,  fraction  o f highest  platelets,  had t h e h i g h e s t  decreased  i n the  negative  lowest  to  of  densities.  surface using ITP  platelets  associated portal  with  after  nephrectomy  pancytopenia  hypertension),  platelet  acid  acid,  from normal s u b j e c t s , p a t i e n t s  and p a t i e n t s  electrophoretic  value  than  with  found  that  mobilities  was  separated density  did platelets  total  and  cell  (1976)  a c u t e and c h r o n i c  disease  considered i n these  and  This  which  however. Isobe  f o r Banti's  and splenomegaly  further  a  was made between  (neuraminidase-susceptible) s i a l i c  young  mobilities.  i n that the highest  c o n t a i n e d g r e a t e r amounts o f s i a l i c distinction  platelet  analyses o f density  confirmed these f i n d i n g s ,  No  with  density, containing  fraction acid  of platelet  electrophoretic  directly  platelets lower  The  electrophoretic  density  less negative. S i a l i c  platelets  density.  correlated  The p l a t e l e t  rabbit  and  platelets  densities.  significantly  studied the c o r r e l a t i o n s  sialic  (a d i s e a s e secondary t o  patients,  acid  levels  the were  e l e v a t e d r e l a t i v e t o those o f normals. At  the molecular  level,  p l a t e l e t membrane s i a l i c  a n a l y s e s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s some i n t e r e s t i n g by  Booyse  et 125  diazotized covalently the  results,  a l . (1968).  I-diiodosulfanilic  paper  they  l a c t o p e r oxidase-"''" ''"I 5  and  George  binding) to l a b e l  first  DD^-ISA  i n addition  labeled  labeling  primarily  et  acid  of  three  and  (SDS PAGE) a l s o  t o those p r e v i o u s l y a l . (1976a  protein revealed  demonstrated  and  1976b)  125 (DD ISA; n o n p e n e t r a t i n g  exposed p r o t e i n  studied  acid  this human  on i n t a c t  label  platelets  polypeptides  9.2,000 d a l t o n s , t h e g r e a t e s t q u a n t i t y  in  of  of label  used and  p l a t e l e t s . In comparison  and  found  180,000, being  to that  125,000,  found i n t h e  - 8 180,000 d a l t o n and t h e l e a s t  i n t h e 92,000 d a l t o n p o l y p e p t i d e s .  results  a r e not u n l i k e those  second  paper  rabbit  "*"Cr-chromate  and  subjected  survival  5  to  found by B u n t i n g  platelets,  double  DD^ISA  attached  with  analyses.  The  labeled  was  nearly  twice  that  of  survival  DD  with  to surface of  w  1015 hours)  e t a l . (1978).  ISA  5  proteins, (T^  2  were =  30  o  (T  =  labeled platelets  plasma  Platelets  o f time.  In the  internalized  ^Cr  t h i s was not t h e case when s i m i l a r i l y f o r t h e same p e r i o d  These  17  hours),  but  were s t o r e d i n  harvested  after  14-40  hours o f c i r c u l a t i o n , and s u b j e c t e d t o membrane p r o t e i n a n a l y s e s by SDS 125 PAGE,  a l l showed  symmetrical of  DD  ISA  whole membranes  or  fragments  while the majority of cytoplasm  Penington pyrrolidone analyzed  labeling  l o s s o f p r o t e i n s . This supports  intact  process  identical  et  a l . (1976a)  (PVP) coated  platelet  heterogeneous  size.  i n size,  indicating  the hypothesis  are l o s t  during  a  that  pieces  the  ageing  remains w i t h i n t h e p l a t e l e t .  separated  silica  patterns  platelets  gel (Percoll)  A l l platelet  but t h e l i g h t  on  density  density platelets  polyvinyl-  gradient,  subpopulations were  on  and were  t h e 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 g r e a t e r than 21 f l i n man and r a t , w h i l e t h e l i g h t p l a t e l e t s platelets  no  labeling  with  described  by  in  rabbits,  greater ^Se  than  13  f l i n both  (selenomethionine),  Charmatz and K a r p a t k i n showed  interpretation  was  no  change  that  species. using  (1974) who  i n buoyant  the greater  specific  Platelet  methods used  density  protein  intermediate,  synthetic and  light  activity platelets  shown were  in  75  with  vitro  released  cohort  previously  Se-methionine ageing.  activity  young p l a t e l e t s i n t h e most dense f r a c t i o n was r e l a t e d in  contained  found  Their i n the  to the increase  and  that  synchronously  dense, from  - 9 t h r e e d i f f e r e n t p l o i d y c l a s s e s o f megakaryocyte p r e c u r s o r s . Boneu e t a l . (1977) s t u d i e d the volume and human p l a t e l e t s  in different  thrombopoietic  s t a t e s . They  splenectomy, i n s p i t e presumably within  again  found  normal  range.  in platelet  demonstrated  coagulopathy  with  increase  the  in  s t a t e s c h a r a c t e r i z e d by  that  in  thrombocythemia  o f a sudden p l a t e l e t  "young" p l a t e l e t s ,  the  increase  disease  the In  a  normal  evidence  ITP  light  and  which " l a r g e , l i g h t p l a t e l e t s "  number  Therefore  In  are  (i.e.,  platelets  consumption  there  there  of  d e n s i t i e s were  "young"  density.  macrothrombocytosis,  platelets.  abnormal  macrothrombocytosis  splenectomy, the  of  following  i n the  volumes and  with  volume  of  increase  platelet  volume) a f t e r  density d i s t r i b u t i o n  was  also  an  situations  in  occur.  A d e n s i t y g r a d i e n t o f m a t e r i a l s i m i l a r t o t h a t used f o r e r y t h r o c y t e separation  (Corash  et  al.,  1974)  heterogeneity  o f human whole b l o o d  al.,  In  1977).  the  first  was  used  platelet  to  investigate  sub-populations  d e s c r i p t i o n of  a  new  (Corash  supporting  the et  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 e r y t h r o c y t e s a c c o r d i n g  gradient  composed  arabinogalactan  of  varying  polymer,  t o age  on  concentrations  produced  from  a simplified of  Western  Stractan Larch  o c c i d e n t a l i s ) by a c o u n t e r c u r r e n t e x t r a c t i o n p r o c e s s . layered and  on  1.095  a three g/ml,  (approximately dense  than  (1.062,  step  gradient  a t 20°C, and 2.34  x  10  erythrocytes  1.066, 1.071,  r e s p e c t i v e l y ) and  again  6  g x the  II,  trees  an  (Larix  E r y t h r o c y t e s were  composed o f d e n s i t i e s 1.087, 1.091,  c e n t r i f u g e d a t 20,000 rpm min,  f o r 45  mean). Because p l a t e l e t s  gradient  1.084, g/ml;  density  step  15%,  c e n t r i f u g a t i o n was  d e n s i t i e s were  16%,  17%  and  20%,  f o r 50,000 g,  but  minutes are  less  decreased Stractan, f o r only  - 10 30  minutes  at  20°C  (1.5 x  10  p l a t e l e t buoyant d e n s i t y , c e l l differences  i n platelet  g  6  x  min, mean).  The  relation  volume and u l t r a s t r u c t u r e suggested  volume  and g r a n u l e  content  and  of that  differences  between l i g h t and heavy p l a t e l e t s might be due t o a g e i n g . I t was found t h a t p l a t e l e t s o f low d e n s i t y granules  a r e s m a l l e r on average and c o n t a i n  than t h o s e o f h i g h e r  density  but b o t h  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 o f Packham affect  and Mustard  (1977)  low and h i g h  emphasized  t h e need  f o r drugs  t h e i n t e r a c t i o n s o f p l a t e l e t s and e n d o t h e l i a ,  hemostatic  platelet  function  of platelets.  s u r v i v a l analyses  thrombosis occurs  1972;  H a r k e r and S l i c h t e r , 1974). Treatment  This  might be u s e f u l  which  ( H a r k e r and F i n c h ,  of rabbit  platelets  with  density  mitochondria.  group  which  t o prevent the  development o f a t h e r o s c l e r o s i s and thromboembolism w i t h o u t the  fewer  also  in clinical  interfering stated  that  situations i n  1969; H a r k e r and S l i c h t e r ,  neuraminidase  decreases the  51 membrane  sialic  acid  slightly  of  platelets,  as p r e v i o u s l y d e s c r i b e d  properties  o f human e r y t h r o c y t e s  Corash  e t a l . , 1977),  reported  f o r density  effect  separation  on  rabbit  (Greenberg e t a l . , 1975). Greenberg e t a l . (1977), u s i n g gradient  enhancing  Cr-labeled  aggregation  Stractan  a  survival  but  step  only  the  platelets,  three  has  and  platelet  of  a  rabbit  f o r the i n v e s t i g a t i o n o f t h e ageing and p l a t e l e t s , that  t h e most  (Corash e t a l . , 1974; dense  fractions  were  e n r i c h e d i n t h e youngest p l a t e l e t s , as determined by l a b e l i n g w i t h 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 e n r i c h e d average  p l a t e l e t density  acid/platelet  i n old platelets.  decreases  with  and p r o t e i n / p l a t e l e t a l s o  I t was c o n c l u d e d  increasing decrease  age. T o t a l  such  that  that sialic  the s i a l i c  - 11 a c i d and the  p r o t e i n decrease i n concert,  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  form o f 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  as o l d and Tan  removal from the  recognition  circulation.  et a l . (1978) showed t h a t the u l t r a s t r u c t u r e o f the s u r f a c e  normal  human  p l a t e l e t membranes  density,  and  hence  sub-human  primate  in  vivo  model,  between p l a t e l e t d e n s i t y  does  ageing.  Macaca and  not  change  Corash  mulata,  to  p l a t e l e t age.  et  as al.  analyze  used and  light  of  a  mean  s u r v i v a l time  function  (1978)  the  A gradient  s i m i l a r t o t h a t used i n t h e human s t u d y , was p l a t e l e t s had  a  of  used  a  relationship  of density i t was  74.6  of  steps  shown t h a t  h o u r s compared  to  51 313.6  hours  for  heavy  light  p l a t e l e t s were removed by  platelets  became  hypothesis  that,  with  density,  turnover  compared  an  in  this  also  showed  the c i r c u l a t i o n and  during  circulation.  species,  provided  markers,  They  specifically  evidence  that  This  p l a t e l e t age  strong  that  l a b e l e d dense confirmed  correlates  supporting  density,  Cr-labeled  to  the  estimate  the  correlated aggregation  human  platelet  v e l o c i t y of  function  p l a t e l e t s of  p r o p o s e d the term "megathrombocyte i n d e x , " index of p l a t e l e t production  (Karpatkin,  with  inversely use  of  platelet  volume.  (MOA), and  He  d i f f e r e n t volumes,  i . e . , l a r g e p l a t e l e t s as  1978). He  also stated  heavy p l a t e l e t s demonstrate 15 t i m e s g r e a t e r monoamine o x i d a s e  that  activity  18 t i m e s more p r o t e i n ( F r i e d h o f f e t a l . , 1978).  P e e r s c h k e and  Zucker  shape change d i d not decrease  the  rates.  Karpatkin  and  light  and  "age-dependent"  platelets.  in  membrane  (1978) found  that  ADP-induced human p l a t e l e t  expose a d d i t i o n a l s i a l i c a c i d , but sialic  acid  1978). These r e p o r t s c o n c u r w i t h  was  demonstrable,  previous  that a c t u a l l y a (Bunting  f i n d i n g s (Seaman and  et a l . , Vassar,  - 12 1966;  Hampton and  1976)  of  a  decrease  aggregation. exposed  in  However,  platelet  during of  Mitchell,  1966a  and  1966b;  electrophoretic  Motamed  membrane  et  laboratory  demonstrated focus  on  a  the  (B.  mobility  a l . , (1976)  1968;  Chiu,  decrease  in  thought  that  Ph.D.  by  found  recent  Thesis  an  platelet  increase sialic  in acid  f i n d i n g s o f a member  1983)  who,  in  electrophoretic mobility. platelets  Seaman,  during  (neuraminidase-susceptible)  p l a t e l e t shape change, c o n f i r m e d  our  Grottum,  during  addition  These  reports  circulation  undergo  e n c o u n t e r s w i t h e n d o t h e l i u m , 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 e t a l . (1978a) i n t h e  t h i r d paper o f a s e r i e s , showed t h a t 125  the  reported  surface  loss  protein  rabbit  thrombosis). these et  to  (George  and  which i n h i b i t  circulation,  relative  platelets  dipyridamole  during  et  Later  ^Cr-labeled  by  of  Hanson  f i n d i n g s with dipyridamole  al.  (1978b)  labeling  stressed  procedure  thrombin  f u n c t i o n and  work  the  for  DD  is  (i.e.,  Harker  aspirin.  quantitative  analyses  of  membrane  protein  prevented  a c c e l e r a t e d by and  and  ISA-labeled  cytoplasmic  a l . , 1976a),  accelerated  platelet  of  by  aspirin-  prevented  by  agents  a g e n t s which  (1981) d i d  in  not  cause confirm  In a f o u r t h paper, George 125 nature  human  of  this  platelet  DD  ISA  membrane  glycoproteins. A  report contrary  platelet  density  studied  platelets  these  platelets  centrifugation, though  and  the  to  ageing  from into  the  arguments f o r an  was  by  i n d i v i d u a l s treated dense  and  a c o r r e l a t i o n was  basal  published  activity  of  the  light  inverse  Leone e t with  correlation a l . (1979)  aspirin.  subpopulations  by  shown between d e n s i t y and enzyme c y c l o o x y g e n a s e was  of who  Separating repeated size. higher  Even in  - 13 the  dense  large  reappeared  platelets,  in parallel  platelets,  suggesting  this  i n both  that  activity light  although  and  after  aspirin  heavy  subpopulations  s i z e and d e n s i t y  ingestion of  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 . It  was  damaged  found  i n the r a b b i t  endothelium  surfaces,  (abdominal  p l a t e l e t turnover  that aorta,  and t h e n c i r c u l a t e d i n a l e s s  1979).  Platelet  interactions  were t h e r e f o r e  with  "*"Cr-labeled  scanning  electron  significantly estimate As  rabbit  further  platelets  and  thus  patients with  survival. analyses had  did  active  form  (Groves  platelet  foreign with  (Buchanan  catheter  adhere  the  et a l . ,  damaged  rabbit  e t a l . , 1979).  (also  platelet  was  not c o n s i d e r e d  In  demonstrated  survival  time  t o be  was  a  by not  reliable  circumstances.  f o r shortened  t h r o m b o t i c c o n d i t i o n s were s t i l l  Combining with  Platelets interacted  the  responsible  (1979) i n v e s t i g a t e d t h e i n v i v o  artery)  a f t e r i n j u r y t o t h e subendothelium  of endothelial i n j u r y i n a l l t h e mechanisms  or  investigated  microscopy),  reduced  or c a r o t i d  balloon  these s i t u a t i o n s , although i n i t i a l l y 5  exposed t o  increased.  surface  aortae  when p l a t e l e t s were  platelet survival in  unclear,  Packham  et a l .  f a c t o r s t h a t might i n f l u e n c e p l a t e l e t survival  time  and  platelet  density  s i t u a t i o n s i n which b o t h e n d o t h e l i a l damage and t h r o m b i  been i n d u c e d they showed t h a t under t h e s e c o n d i t i o n s both p l a t e l e t  survival  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 o r t h r o m b i n 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 t i m e but  after  plasmin  these treatments,  treatment  t h e s u r v i v a l time  the p l a t e l e t s i n the l i g h t e s t  observed  to increase  platelet  density  proportionately.  i s not n e c e s s a r i l y  decreased. density  They c o n c l u d e d associated  with  After a l l  f r a c t i o n were  that  a change i n  an a l t e r a t i o n i n  - 14 platelet  s u r v i v a l time  and  that  t r e a t e d p l a t e l e t s might be due  -  the  decreased  s u r v i v a l of  plasmin  t o 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  II  in rabbit platelets. Graf  et  gradients  al.  of  (1979)  showed  polysaccharides,  that  blood  such  as  platelets,  dextran  T10  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 n o r m a l l y to  study  serotonin  of species  receptor  human).  et a l . (1979) u s i n g  a density  Stractan,  contained  u p t a k e and  ( g u i n e a p i g , r a b b i t and  Cieslar 18%  (5HT)  showed  greater  that  normal  quantities  heparin-neutralizing  activity  of  isolated and  and  gradient  serotonin, those  Stractan  might be  of  13.5%, 15%  adenine  of  higher  nucleotides,  lighter  and  release  constituents densities,  of  differed the  these only  density  slightly  for  platelets  differences  were  considered  contents.  d e c r e a s e i n p l a t e l e t d e n s i t y might not to  reversible  activities, thrombin  encounters  i.e.,  treatment  in  platelet was  the  shown  to  of  with  platelet different  not  to  be  These f i n d i n g s suggest t h a t a  be due  t o a g e i n g a l o n e but  circulation  release  and  d e n s i t i e s . They  c o n s t i t u e n t s . However, because  a t t r i b u t a b l e to l o s s of granule  and  density  a l s o showed t h a t p l a t e l e t s d e c r e a s e d t h e i r d e n s i t y upon t r e a t m e n t thrombin  used  interaction in a variety  rabbit p l a t e l e t s of  than  with  and  decrease  also  requiring  functional  change.  Therefore,  shape  platelet  density  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 t h a t i t might be p o s s i b l e t o use  density  gradient  analyses  to  exposed t o t h r o m b i n i n v i v o d u r i n g  detect  p l a t e l e t s that  thrombus f o r m a t i o n ,  and  have  been  which  have  s u b s e q u e n t l y r e t u r n e d t o the c i r c u l a t i o n when t h r o m b i breakup. Analyses modifying  of  platelet  s u r v i v a l a f t e r treatment  enzymes were p u b l i s h e d  by  with  other  Greenberg e t a l . (1979),  surface  following  - 15 Greenberg e t a l . (1975 and 1977) and Packham e t a l . chymotrypsin,  trypsin  and  neuraminidase  p l a t e l e t membrane g l y c o p r o t e i n s in  I I and I I I )  the  platelet  sequestered  and p l a t e l e t release  reaction.  a t one hour,  a l l cause  (primarily  survival; The  Plasmin,  alterations  i n glycoprotein  only  treated  being  cause  platelets  removed  in  I , and some  p l a s m i n and t r y p s i n  enzyme  some p l a t e l e t s  (1979).  were  (especially i n  the c a s e o f t r y p s i n and n e u r a m i n i d a s e ) by t h e l i v e r and not t h e s p l e e n , and  some p l a t e l e t s r e t u r n i n g  t o t h e c i r c u l a t i o n by f o u r h o u r s .  Rand e t a l . (1980) s t a t e d insufficient alterations minutes), original  to  including a  al.,  granule  increase  s i z e by one h o u r ) ,  decrease  al.  alterations i n platelet  o f granule  human p l a t e l e t s reaction  platelets.  (reversible  i n five  (returning  t o the density  density  by t h r o m b i n associated  (Cieslar et  with  thrombo-  be a s s o c i a t e d w i t h t h e p l a t e l e t  these  sialoglycoproteins  release  ( e . g . , w i t h ADP). Packham e t  low c o n c e n t r a t i o n s  less  than  4%  Nor does ADP a l t e r In contrast,  from  of the s i a l i c  sialic  o f ADP do n o t cause  rabbit  acid  the i n vivo  thrombin releases  platelets  was  released  survival  (granule  a c i d o f human p l a t e l e t s , but s t i l l  i n v i v o s u r v i v a l time o f r a b b i t and  size  platelet  nor from  ( n o t even under c o n d i t i o n s wherein t h e p l a t e l e t  occurs:  platelets).  20%  induced  n o r an i n c r e a s e i n p l a t e l e t s i z e  (1980) showed t h a t  release  i n platelet  temporary  f o u r h o u r s . Thus i t was c o n c l u d e d t h a t , i n  embolism might n o t n e c e s s a r i l y reaction,  induced  and caused a d e c r e a s e i n p l a t e l e t  f o rat least  t o the p l a t e l e t  1979) a  release,  a g g r e g a t i o n and shape change  temporary  which p e r s i s t e d addition  cause  t h a t low c o n c e n t r a t i o n s o f ADP (10 uM),  platelets  time  release  from  human  of  rabbit  sialoglycoproteins) does not a l t e r t h e  (Reimers e t a l . , 1973a, 1973b  1979). Neuraminidase, on t h e o t h e r hand, c l e a v e s 4 0 % o f t h e t o t a l  - 16 platelet  sialic  clearance 1977  acid  of rabbit  from human p l a t e l e t platelets  from t h e c i r c u l a t i o n  and 1979). R e l e a s e o f s i a l i c  the  form  acid  membranes, and c a u s e s  acid  (Greenberg e t a l . ,  from i n t r a c e l l u l a r g r a n u l e s i n  o f s i a l o g l y c o p e p t i d e s , and c l e a v a g e  by n e u r a m i n i d a s e , produce very  rapid  of c e l l  surface  sialic  different  results  with  respect  to  protein  changes  associated  with  human p l a t e l e t a c t i v a t i o n , George e t a l . (1980) demonstrated t h a t  actin  platelet survival. Continuing  studies  of  membrane  becomes exposed d u r i n g t h i s p l a t e l e t shape change e v e n t . T h e i r  platelet  washing  was then  analyzed  membrane  proteins  with due  procedure  respect  f o r a n a l y s i s o f membrane p r o t e i n  to possible  to platelet  loss  of specific platelet  centrifugation  and/or p l a t e l e t  1981). The p r e v i o u s l y used p r o c e d u r e p l a t e l e t preparation of  the p l a t e l e t s ,  protein,  mostly  protein,  GPG.  subpopulations  due t o a  a recovery  of one-third  loss  demonstrated  o f membrane p r o t e i n s clearly  of their  no  difference  from  indicate  properly  glycoproteins  recovery  from  from  granule  glyco-  from  anti-platelet  small  dense  platelet  i n the concentration  washed, S t r a c t a n  a d i f f e r e n c e (e.g.,  a l . (1981)  membrane  these  glyco-  p e r p l a t e l e t . " However, t h e d e n s i t o m e t e r  to that o f the t o t a l p l a t e l e t population et  one-third  membrane  o f GPI and t h e major  membrane p r o t e i n s from p l a t e l e t s o f d i f f e r e n t  Blajchman  o f only  "Comparison o f t h e most dense and l e a s t  membrane g l y c o p r o t e i n s  platelets  a loss  (George e t a l . ,  (George e t a l . , 1978b) f o r human  was found t o p r o v i d e  with  damage  using large antibody  old platelets  density  an i n c r e a s e densities,  of  scans  separated i n GPG) i n  i n comparison  (George e t a l . , 1981).  rabbits, young  platelets  induced  obtained  compared  the  platelet  obtained  during  thrombocytopenia,  during  irradiation  with  induced  - 17 t h r o m b o c y t o p e n i a . The c h a r a c t e r young  (linear)  found  that  and o l d ( e x p o n e n t i a l )  there  glycoproteins.  were  However  glycoproteins  o f the s u r v i v a l  were  no  platelets  qualitative  and  were  f o r these  different.  differences  the concentrations  greater,  time curves  They  in  membrane  o f a l l s i x major  membrane  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 t o t h o s e i n t h e u n s e p a r a t e d , o r those in  the o l d , small  platelets.  membrane f r a g m e n t a t i o n to the d e - s i a l a t i o n  This  hypothesis  result  i s compatible  o f ageing  the  and r e m o v a l , and c o n t r a r y  theory.  Hanson and H a r k e r (1981) performed d o u b l e - l a b e l e d studies  with  platelet  i n baboons, s i m i l a r t o t h o s e performed w i t h r a b b i t s ,  survival 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  ageing but i n c o n t r a s t  in  DD^ISA  t o George's r e s u l t s  compared  to  t h i s could  "^Cr  during  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)  c o r r e l a t e s with with  platelet  platelet  reported density  and t h a t  age as r e f l e c t e d 35  cohort  label  ageing,  revealed  S-sulphate.  that  platelet  platelet  density  51 Cr-labeled  i n specific  activity  size  correlates  by t h e i n c o r p o r a t i o n _in v i v o  In_ v i t r o  an i n c r e a s e  i n rabbits,  of the  platelets,  after  o f 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 d e c r e a s e i n s p e c i f i c a c t i v i t y o f t h e most dense fraction. platelet  These r e s u l t s heterogeneity  were s t i l l  due t o a g e i n g  dense p l a t e l e t s c o u l d n o t be r u l e d al.,  1981b).  rabbit (1978),  Rand  et a l .  that  i n these  with  respect  because t h e s p e c i f i c  to the  removal o f  out (Greenberg e t a l . , 1977; Rand e t  (1981a) p u b l i s h e d  model system, analagous except  not c o n c l u s i v e  to that rabbit  further  demonstrated  studies  evidence by Corash  an i n v i v o  i n the et^ a l .  ^ S-sulphate 5  - 18 35 c o h o r t l a b e l was used. The S c o h o r t l a b e l i s t a k e n up by t h e megakaryocytes, l a b e l s newly s y n t h e s i z e d p r o t e o g l y c a n 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 s t e p s o f 15%, 17%, and 19% S t r a c t a n , and 15.5%, 17.5%, and 19% Stractan 1.076  (i.e.,  1.058,  1.067,  g/ml, r e s p e c t i v e l y ) .  and 1.976 g/ml; and 1.060,  This  enabled  the i s o l a t i o n  1.069 and  of  platelet  s u b p o p u l a t i o n s from t h e extremes o f t h e d e n s i t y d i s t r i b u t i o n , enhancing differences isolated shown  i n platelet  from  the t a i l s  that  the  significantly  density.  these  of the platelet  most  larger  Using  dense  density  platelet  amounts o f p r o t e i n  platelet  subpopulations  distribution  subpopulation  and t o t a l  i t was  contained  (not surface)  sialic  35 acid  per platelet  specific  activity  progressively  as w e l l  a s more  S cohort  label  p e r p l a t e l e t . The  peaked i n t h e most dense p l a t e l e t s  to  the  least  dense  and then  platelets.  The  shifted  most  dense  s u b p o p u l a t i o n reached i t s maximum s p e c i f i c  activity  between 48 and 72  hours  whereas  the  least  96 h o u r s .  This  was  and  then  subpopulation that  young  decreased  reached  platelets  gradually,  i t s maximum were  enriched  d e c r e a s e d i n average d e n s i t y a f t e r the  after  i n t h e most  dense  dense evidence  fraction  and  a g e i n g i n t h e c i r c u l a t i o n ; and t h a t  o l d p l a t e l e t s were e n r i c h e d i n t h e l e a s t dense f r a c t i o n . Mezzano e t a l .  (1981), a f t e r  results  o f human p l a t e l e t  density  gradient  survival  analyses of p l a t e l e t s  different,  new  Mezzano and A s t e r  density  centrifugation  interpretations  analysis methods,  separated from  decline  experiments and  published  with  Stractan  i n addition  according  their  (1979),  to size.  results,  i n specific  i.e.,  activity  reported They drew that  the 51  of  Cr  reason  f o r t h e more  rapid  labeled  light platelets  compared t o t h a t o f t h e heavy p l a t e l e t s  i s not  - 19 that  light  platelets  more r a p i d l y the  light  than the  in  By  both  heavy  and  centrifugation,  p l a t e l e t s was  found t o  the  light  are  removed  with  and  studying  differential  when  and  become h e a v i e r  some dense m a t e r i a l ,  platelets.  humans,  older  heavy young p l a t e l e t s , but  platelets  acquiring light  are  -  5  are  age  specific  remain c o n s t a n t or  platelets  lose  their  by  perhaps  newly  platelet  platelet  the  circulation  maturity,  replaced  "*"Cr-labeled  light  the  that a f t e r c i r c u l a t i o n  or  then  from  formed  survival  fractions activity  the  (decreasing  in  by  heavy  i n c r e a s e s l i g h t l y at  label  in  obtained of  by  a  time  specific  a c t i v i t y ) , s u g g e s t i n g a s h i f t i n d e n s i t y from l i g h t t o heavy p l a t e l e t s . G r a d i e n t m a t e r i a l s o t h e r t h a n S t r a c t a n do Shaw e t to  a l . (1981) a g a i n used P e r c o l l  isolate platelets  by  velocity  (as  p r o v i d e some advantages.  i n Penington et  s e d i m e n t a t i o n , and  t o d i s t r i b u t e p l a t e l e t s a c c o r d i n g t o d e n s i t y . The was  found  to  be  Gaussian  a  and  intragranule  molecules beta-thromboglobulin  the  studied,  cytoplasmic revealing  greater quantities  leakage  mode  distribution  and  r e l e a s e or  with  platelets  o f BTG,  linear  platelet  about  lactic from  w h i l e the  the  (BTG)  g/ml.  The  r e l e a s e ) of  and  serotonin  dehydrogenase denser  gradient  distribution  1.0645  (gradient induced  constituent  that  a  a l . , 1976a)  (5HT),  (LDH)  fractions  o t h e r markers c l o s e l y  the  were contain  parallel  the  p l a t e l e t count. Haver and  Gear (1981) d e v e l o p e d a method, f u n c t i o n a l  whereby p l a t e l e t s are aggregating (large) and  agent  reactive  contained  total  sialic  exposed t o low  after  which  platelets.  increased acid,  exhibited  c o n c e n t r a t i o n s (0.1-0.7 uM  they  were  centrifuged,  These more r e a c t i v e  amounts o f active  fractionation,  g l y c o g e n , ATP,  pelleting  platelets ADP,  chromate u p t a k e ,  1.9 and  ADP)  were  of the  larger  t i m e s more were  labeled  - 20 -  '  75 as young p l a t e l e t s ( i n r a t s ) by Using platelets (Rand e t said  ^Cr-  and  that  occur  5  be  as  a  than the  survival  platelets.  least  showed  the  most dense  that  ageing with  platelets  changes  were  either  hour  less  Data d i s c u s s e d 51 the  density  one  slightly  dense p l a t e l e t s .  a l . , 1983),  platelet  that  was  The  of  the  of p l a t e l e t s  except  platelets  "^Cr-labeled  platelets,  function  identical,  "'""'"'''In-labeled  et  "^In-labeled  a l . , 1981b). The  to  Se-methionine i n j e c t i o n .  followed label  recovery  than  that  survived  for  most  dense  was of for  longer  i n a l a t e r paper  Cr-labeled  of  (Rand  platelets  appeared t o s u r v i v e l o n g e r t h a n "'""'""''In-labeled most dense p l a t e l e t s . In " t o t a l agreement" w i t h Mezzano e t a l . (1981) but the  report  labeled  by  materials one  survival  arteritis  combination  with  of  the  Stractan,  injection  of  and  Gear, 1981  the  radioactivity before  observed. less it  and  a  decrease  Throughout  dense p l a t e l e t s  remained  results  the  constant  the  day  two,  there  radioactivity  platelet  in  the  There of  lifespan,  In the dense  was  the the  was  still a  On  an  in  increase  three  in  day  lag  platelets  was  radioactivity density  in  the  platelets  i t increased.  population".  day  Haver  These  suggested t h a t i n humans "young p l a t e l e t s are heterogeneous  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  II  obvious  found by an  dense  intermediate fractions  subjects)  (as  was  stage  findings.  there  o f dense p l a t e l e t s  Cr  various . gradient  novel  platelets  dense p l a t e l e t s . in  using  and  of Corash, i s 51  stable  normal  certain  (1979)  e x p e r i m e n t s , on with  and  analyses  revealed  decreased. and  limbs,  ^Cr-1abeled  1982); on of  (in patients  density  Aster  interpretation  whose c a r e f u l  lower  enrichment i n s p e c i f i c a c t i v i t y  phase  t o the  studies  platelet  including  after  contrary  Boneu et a l . (1982),  platelet  chronic  t h e work o f Mezzano and  and  - 21  -  In a n o t h e r p u b l i c a t i o n t h e method o f S t r a c t a n analyses  o f human p l a t e l e t s was  u s e f u l , and (e.g.,  to provide  assays of  discussed  aggregate r a t i o s  Stractan  distribution  found t o be  reproducible,  i n d e p e n d e n t i n f o r m a t i o n compared t o o t h e r and  BTG)  a c t i v a t i o n i n p a t i e n t s with thrombotic Using  and  density  density  for detection  disease  gradient  of  platelet  (van Oost e t a l . , 1982).  separation  as  a  technique  p r e p a r a t i v e i s o l a t i o n o f 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 be  of  clinical  diagnostic  value  in  patients  technique  t o d e t e r m i n e 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 o f s e c r e t a b l e  analyses  was  shown t o be  compartments,  evaluation  hematologic but  to  correlate  density  abnormal  disease.  stimulation  of  was  seconds,  I t was  shown  Burke,  again  1982). As  density  in  certain  size)  and  decrease and  1982)  in  with  Bessman, due  to  serotonin  and  reports  c o r r e l a t e with p l a t e l e t density Corash the  et  a l . , 1978), and  sub-human  size-separated  patients  average  platelet  the  degree  that  platelet  " p l a t e l e t s i z e does not  has  baboons  of  (Papio  of  induced  (occurring density  been  was  1983).  shown  s i z e (but  to  primate, not  in  centrifugation  cynocephalus)  c o r r e l a t e with p l a t e l e t  age  thrombin  sub-human  counterflow  with  Platelet  platelet  to c o r r e l a t e with  analyses from  age  in  1983).  secretion  ( s p e c i f i c a l l y i n the  density  primate), platelets  and  useful  shown t o i n c r e a s e a f t e r c a r d i o p u l m o n a r y bypass (van Oost et_ a l . , Although  a  gradient  be  to c o r r e l a t e with  (Levin  BTG  shown t o  production  platelet  to  which p a r a l l e l s and  s i z e was  shown not  thrombopoiesis  Gear  Stractan  platelet  (increasing  again  degranulation, in  of  (Corash,  with  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 e t a l . , 1982). P l a t e l e t the  destruction  of  in platelet  platelet  and  management  for  shown t o  disorders  non-secretable  production  the  tests  age"  revealed  (Thompson e t  - 22 al.,  1983b). The l a r g e r  platelets  survived longer  b u t were not younger  t h a n t h e s m a l l e r p l a t e l e t s . However, a d e c r e a s e i n p l a t e l e t a g e i n g c o u l d not be d i s p r o v e d . and  1983 a and b) s u p p o r t s  i s present  primarily  result of platelet In and  monkeys  subjected  size  with  The work o f Thompson e t a l . , (1981, 1982  the b e l i e f that p l a t e l e t s i z e  at the onset o f p l a t e l e t  heterogeneity  production  and i s not a  maturation.  (Macaca  fasicularis),  platelets  were l a b e l e d w i t h  to P e r c o l l density d i s t r i b u t i o n analyses  5  b e f o r e and  ^Cr  after  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 P e n i n g t o n , 1983).  No  significant  demonstrated  enrichment  for platelets  of  of higher  "*"Cr  uptake  5  density  in  (as was  vitro  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 t h e l a b e l e d platelet  density  density.  Considering  unlabeled, However,  distribution that  i t appeared  was  newly  found  produced  as i f t h e s e  i t was conceded  that  to  be  shifted  platelets  platelets  higher  were i n t h i s  were  "lighter platelets  to  o f lower  may  have a  case  density. slightly  s h o r t e r s u r v i v a l t i m e t h a n dense ones". Experimentally rat,  indicated  platelet  induced c h r o n i c  continuous  survival  vessel  shortened p l a t e l e t injury  produced  release sialic  interpreted 1983).  were  normal  characteristics) acid  and  required  and t h a t t h e r e s u l t a n t r e d u c t i o n  was n o t b a l a n c e d by p l a t e l e t p r o d u c t i o n . were  was  protein  as b e i n g  i n size  but were per  and  i n the  to  i n platelet  shorten numbers  However, t h o s e p l a t e l e t s function  of highest  platelet,  survival,  and  (by a g g r e g a t i o n  density,  o f lower  survived  longer  younger) t h a n normal p l a t e l e t s  which and mean (i.e.,  (Winocour e t a l . ,  - 23 1.4 Megakaryocyte and P l a t e l e t H e t e r o g e n e i t y Platelet  precursors,  themselves, (Paulus,  producing  1975;  megakaryocytes,  heterogeneous  P a u l u s e t a l . , 1979;  P e n i n g t o n e t a l . , 1976a and The  normal  population  of  are  heterogeneous  subpopulations P e n i n g t o n and  i n bone  platelets  Streatfield,  1976b; P e n i n g t o n , 1979;  megakaryocytes  of  in  1975;  P e n i n g t o n , 1981). marrow  consists  of  s u b p o p u l a t i o n s o f megakaryocytes which d i f f e r i n t h e i r p l o i d y number (4 N, the  1-2%;  8 N, 10-25%;  16 N, 46-76%; 32 N, 15-32%;  and 64 N,  1%)  with  16 N p l o i d y c l a s s p r e d o m i n a t i n g . 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 a r e l a r g e , have  many g r a n u l e s (dense and a l p h a ) and m i t o c h o n d r i a but have a low c o n t e n t of  d e m a r c a t i o n membrane system; whereas  (32 N, o r 64 N) deficient  in  produce s m a l l  granules  and  and  t h o s e which have a h i g h  less effective  mitochondrial  platelets,  content  but  ploidy  which a r e  have  a  high  q u a n t i t y o f d e m a r c a t i o n membrane system. Megakaryocytes  respond  to  thrombocytopenia  with,  of  course,  i n c r e a s e d p l a t e l e t p r o d u c t i o n , but t h i s response has been c h a r a c t e r i z e d as  being  specific  for  humans, u l t r a s t r u c t u r a l revealed  that  megakaryocyte "dilated"  individual  tubular  rats  situation  (Odell  et  platelet  volume,  demarcation  Acute t h r o m b o c y t o p e n i a was in  ploidy  s t u d i e s o f megakaryocytes  increased number,  megakaryocyte  and  classes.  In  i n patients with  ITP  production in  three  membrane  was  cases,  (Ridell  and  related  the  content  Branehog,  to of  1976).  a l s o used t o s t i m u l a t e m e g a k a r y o c y t o p o i e s i s  a l . , 1976;  Odell  and  Shelton,  1979)  i n which s p e c i f i c a l l y t h e d i p l o i d megakaryocyte  producing a precursor  was  s t i m u l a t e d t o mature t o a p l o i d y l e v e l one n u c l e a r d i v i s i o n h i g h e r t h a n normal w i t h 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 ; t h e s e c e l l s produced  - 24 more  platelets  at  a  faster  rate.  Furthermore,  since  megakaryocyte  changes began t o r e v e r s e b e f o r e normal l e v e l s o f c i r c u l a t i n g were  reached,  these  authors  concluded  platelet  production  was  platelet  mass. T h i s  i s i n agreement w i t h  1974)  that  activity  the  of  production  an  preponderance functional  controlled  that  earlier  i n the  cell  in  the  i.e.,  cessation  something o t h e r  inhibitory  circulating  platelets,  by  the  earlier  effect  platelet  than  rather  this  the  total  arise  sequence,  population  "megathrombocytes  of  findings (Karpatkin,  may  maturation  platelets  of  being  or  from  young an  from a  highly  index  of  p l a t e l e t production". In  the  guinea  predominate  p i g and  (Levine  megakaryocyte continuous  et  monkey, the  a l . , 1980).  heterogeneity  process  megakaryocyte  of  size  In  said  to  each  nuclear  of  of  and  17.9  +  ploidy  predominating.  1.3%, By  with  N;  and  enumeration  p l o i d y c l a s s e s i t was of c e l l s  32  i n steady-state certain  In  the  few of  marrow  quantities  human,  due  to  4 N,  with  the  megakaryocytes  and  16  guinea  pig of  16 N,  revealed  megakaryocytes 40.8  +_ 1.4%,  16  N ploidy class  within  between the 4 N  and  between  large quantities and  8 N ploidy  and between the 16 N and  i n t e r p r e t e d t o mean t h a t t h e r e was  more immature 2 N and 4 N,  a  increasing  also  of  8 N;  +• 1.4%,  N)  64  few c e l l s between t h e 8 N and  32 N. T h i s was  (endomitosis),  found t h a t t h e r e were r e l a t i v e l y  between the 2 N and  l e v e l s and  essentially  and  to  1982).  a s c r i b e d t o d i f f e r e n t p l o i d y c l a s s e s (33.4 N;  be  pig  found  more a c h a r a c t e r i s t i c o f mature r a t h e r than  r a t megakaryocytes  heterogeneity  guinea  doubling.  immature megakaryocytes ( L e v i n e e t a l . , Study  the  polyploidization  with  p l a t e l e t p r o d u c t i o n was  was  8 N p l o i d y c l a s s was  r a p i d turnover of  compared t o t h a t o f the 8 N and  the  16 N (Tanum  - 25 and E n g e s e t , 1983). The  heterogeneity  another l e v e l , al.,  i n soft  1980; L e v i n  megakaryocytes  of  et a l . ,  "heterogeneous" c o l o n y  strongly  ploidy  1981; L e v i n ,  a r e o f two major  types,  t y p e s . The " b i g c e l l "  distribution  N  (8  N,  30%;  characteristic  16  N,  o f which  45%;  32  N,  varying  negative 16-32  for this  N,  17%), a  megakaryocytes response  activity.  and  to acute  ploidy more  and less stain  20%), a  ploidy  o f mature bone marrow megakaryocytes.  acetylcholinesterase  activities,  distribution  primitive  of a l l  the small  The average p l o i d y  i s 6.8  N  forming  t h r o m b o c y t o p e n i a , i n d u c e d by  The  sizes,  cells  being  (2-4 N, 57%;  characteristic  colony  by  megakaryocyte  "heterogeneous" c o l o n y t y p e c o n t a i n s more t h a n 40 c e l l s , with  formed  colony type contains  The a v e r a g e  at  (Radley e t  the "big c e l l "  most o f which a r e l a r g e and about h a l f  i s 16.8  evident  1983). The c o l o n i e s  for acetylcholinesterase a c i t i v i t y .  ploidy  i s also  agar t i s s u e c u l t u r e o f mouse marrow  i n culture  t h a n 30 c e l l s ,  megakaryocyte  of  cells  injection  immature (CFC). of  In  rabbit  anti-mouse p l a t e l e t serum, t h e p l o i d y d i s t r i b u t i o n o f t h e heterogeneous colony  t y p e remains e s s e n t i a l l y  colony s h i f t s t o higher ploidy  unchanged, w h i l e t h a t  of the b i g c e l l  c l a s s e s , w i t h an average p l o i d y o f 21.5  N, i n c r e a s i n g t h e f r e q u e n c y o f 32 N and 64 N megakaryocytes (32 N, 30%; 64  N,  6%).  This  suggests  that  there  are  at  least  two  types  of  megakaryocyte CFC d i f f e r i n g i n r e s p o n s e t o p l a t e l e t d e p l e t i o n . Thus, differently the  different  subpopulations of  to different  stimuli.  megakaryocytes  might  respond  S u b p o p u l a t i o n s o f megakaryocytes i n  bone marrow c o u l d produce s m a l l p l a t e l e t s w i t h low g r a n u l e c o n t e n t  which a r e 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 o f normal h e m o s t a s i s ; o t h e r 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 age  and  encounter  -  i n s t a t e s of a c t i v e thrombosis.  reversible  (or not)  adhesive  As these  r e a c t i o n s , they  become s m a l l e r , l o s i n g p i e c e s o f membrane, r e l e a s e g r a n u l e s l e s s dense. T h i s i s another  way  platelets  and  be  discussed).  In  this  become  i n which the p l a t e l e t p o p u l a t i o n c o u l d  become heterogeneous (by c h a n g i n g w i t h i n the c i r c u l a t i o n d u r i n g to  could  case,  platelets  megakaryocyte c l a s s would come t o s u p e r f i c i a l l y  from  the  low  resemble those  ageing, ploidy of  higher c l a s s e s .  Table 1.4.1 Platelet  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  Heterogeneity Physical Properties: density, size, surface properties. S t r u c t u r a l Forms: d i s c o c y t e , e c h i n o c y t e . S u r f a c e R e c e p t o r s F o r : ADP, a n t i b o d y fragment F , 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 t h e absence o f f i b r i n o g e n ) . S t r u c t u r a l Content: g r a n u l e s (dense and l i g h t , a l p h a ) , l y s o s o m e s , membrane c a n a l i c u l i , mitochondria. M o l e c u l a r Content: ADP, ATP, b e t a - t h r o m o g l o b u l i n , c a l c i u m , f i b r i n o g e n , g l y c o g e n , LDH, PDGF, PF4, s e r o t o n i n , 35-S-bearing p r o t e o g l y c a n , thrombospondin. c  Factors 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 State: discocyte - echinocyte. F u n c t i o n a l Purpose: b e a r i n g s u r f a c e 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 b l o o d , m a r g i n a t i n g ( w i t h i n o r g a n s ) , marrow.  the  - 27 Hence  we  have  a  producing  heterogenous  and  active,  less  active.  picture  populations  with  and  heterogeneous  of platelets  platelets  as they  progenitor  (Table  cells,  1.4.1),  age perhaps  active  becoming  less  I n t h i s manner t h e body c o u l d a l t e r t h e q u a l i t y and q u a n t i t y o f  i t s c i r c u l a t i n g thrombotic this  of  case,  e l e m e n t s . The Thrombon (used  t o describe i n  t h e t o t a l mass o f 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  the quality  or potential  for activation  precursors,  and t h r o m b o s i s ,  of this  mass) c o u l d t h u s be c o n t r o l l e d n o t o n l y i n number b u t a l s o i n q u a l i t y .  1.5 Membrane S u r f a c e P r o p e r t i e s Detected When aqueous s o l u t i o n s polyethylene glycol form  immiscible  isotonic.  particles, polymer  (D) and  (PEG), a r e mixed above c e r t a i n c o n c e n t r a t i o n s ,  phase  systems  and a n a l y t i c a l  have  upper,  particals  proven  o f macromolecules,  phase  usually  and  lower,  partitioned  and  suitable  differential partition  PEG-rich  are  been  separation  and c e l l s by t h e i r  phases:  Biological  o f d i f f e r e n t polymers, e.g., d e x t r a n  two phase systems which c a n be b u f f e r e d  Such  preparative  by Aqueous Two-Phase P a r t i t i o n  rendered f o r the  subcellular  within  t h e two  D-rich  between  they  phase.  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 o f p a r t i c l e s w i t h i n t h e phases  (the p a r t i t i o n )  depends  particles,  and t h e p a r t i c u l a r  have  shown  been  charge-associated,  to reflect  on  type a  hydrophobic,  the surface  early  most o f which  variety  of surface  and b i o s p e c i f i c  late  properties  surface  1950 and e a r l y  (e.g.,  properties),  composition.  methodology o f aqueous two-polymer phase system is still  of the  o f phase system. The phase systems  c o n t r o l l a b l e by v a r i a t i o n o f t h e phase system The  properties  i n use today, was i n t r o d u c e d  analysis,  and o u t l i n e d i n  1960 ( A l b e r t s s o n , 1960 and 1971; A l b e r t s s o n and  - 28 Baird,  1962;  reviews  see  Tiselius Fisher  et  a l . , 1963;  (1981),  Albertsson  A l b e r t s s o n (1983), and Brooks Polymer glycol  composed  shown  provide  a  to  subcellular  such  that,  particles  and  (dog,  human,  were  sheep,  elipsoidea)  yeast  (Escherichia  coli).  species  specific  phase  system cell  two-phase  system  gradient  "the in  a mixture were  of  the  surface  algae  Ions resulting  were  (1982),  also  still  a  unclear,  cell  ( A l b e r t s s o n and  Baird,  of  of  eukaryotic  different  pyrenoidosa and  and  bacteria  partition  provide  types,  nearest  properties...the  and size  a  i n a s i n g l e two-polymer  new  separations  to  namely  at  and  species  aqueous  the  use  of  polymer  of  a  salt  phosphate i n s p e c i f i c c i r c u m s t a n c e s Although  of  1960),  characteristic  presented,  of  (Albertsson,  step  concept,  variety  partitioning  (CCD)  CCD  polyethylene a  carlsbergensis),  and  g r o u p i n g s on the s u r f a c e s o f c e l l s " affects partition  recent  Walter  for  (Chlorella  of c e l l s ,  cells.  which l i e s  more  and  the  erythrocytes  single  was  macromolecules and  explanation  distribution  could  but  to  feasible  reproducible  analysis  separations  certain  only  o f sodium c h l o r i d e and  fractionate these  types,  dextran  environment  be  (Saccharomyces  provide  different  now  rabbit),  separation  of  addition  presented:  Not  For  a l . (1982),  macromolecules  countercurrent  cells  mild  in  s e p a r a t i o n s o f v i a b l e c e l l s was 1962). R e s u l t s o f  1969).  (1983).  systems  cells  prokaryotic  et  two-phase  were  biological  Walter  that  time  in similar i s the  the  bases  separations  major  to for of  determinant,  t o hand i s t h a t i t i s d i f f e r e n c e s presence  of  different  ( A l b e r t s s o n and  chemical  B a i r d , 1962)  which  behavior. shown  to  partition  unequally  between  t h e ' phases,  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  partition  1970) .  Another  systems was phases,  major  That  partition  factor  shown t o be  dependent  1971) .  cells  The  their  was  the  associated  in  Brooks  with  difference  (Ryden,  properties  by  erythrocytes  potential  partition  (Johansson, these  phase  t e n s i o n between the D and  demonstrated  of  phase systems  concentrations  charge  partition  CCD.  governing  the i n t e r f a c i a l  upon  surface  of  correlated after  o f s u b s t a n c e s i n c l u d e d i n the  and  Albertsson,  are  et  PEG  involved  in  a l . , (1971)  who  electrophoretic mobility  between  phases  was  actually  measured, on t h e o r d e r o f a few m i l l i v o l t s (Reitherman e t a l . , 1973). Walter  et  a l . , (1976a) demonstrated  aqueous phase systems can  detect  that  partition  membrane s u r f a c e  s u r f a c e c h a r g e ; but by a p p r o p r i a t e m a n i p u l a t i o n components, membrane s u r f a c e  p r o p e r t i e s other  properties  p o t e n t i a l d i f f e r e n c e between  increasing  chloride  coefficient  (K; number o f c e l l s  the  total  removal  cells  of  the  potential  added t o the  sialic  acid  the  two  t h a n c h a r g e can  in  phase  also  polymer  K  =  with  high  8%  and  Also  partition  the  when measured  chloride  in  equation  a general  way  which i l l u s t r a t e s experimentally,  on c e l l p r o p e r t i e s , one which has the  phases  t o do (the  mathematically  namely t h a t p a r t i t i o n  (e.g., 92%.  been shown  depends  o f which i s the s u r f a c e charge and  phase  not when  content  what has  of  after  in  5/3.5:5), i n which c a s e p a r t i t i o n i s observed t o i n c r e a s e t o K = The  with  (K = 54%)  ( e . g . , 5/4:1), but  sodium  be  increases,  system) o f e r y t h r o c y t e s to  as  content  i n t h e upper phase, as a p e r c e n t a g e  decreases  systems  phases  decreases.  systems w i t h h i g h sodium phosphate c o n t e n t measured  such  o f the two-phase system  s t u d i e d . They showed t h a t w i t h i n c r e a s i n g phosphate and the  i n two-polymer  mainly  the o t h e r  of  w i t h a non-charge i n t e r a c t i o n between the membrane and cell/phase  interfacial  free  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 surface 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 that small d i f f e r e n c e s in  s u r f a c e charge or i n t h e degree t o which t h e  t h e membrane can et  a l . , 1971  the  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 b e h a v i o r  and  electrophoresis  1976).  This  i n which  the  is in  contrast  parameter  which  e l e c t r o p h o r e t i c m o b i l i t y , depends o n l y  density.  The  phases i n t e r a c t  strengths  of  the  to  partition  (Brooks  procedures  such  c h a r a c t e r i s e s the  linearly  on  approach  with  as  cell,  surface  charge  cell  surface  to  c h a r a c t e r i z a t i o n , t h e n , a r e i t s extreme s e n s i t i v i t y t o membrane s u r f a c e charge and  lipid  composition  and  the c o n t r o l which the e x p e r i m e n t o r  o v e r t h e c o n d i t i o n s which d e t e r m i n e t h e f i n a l c e l l Using  species  membranes i t was the  different  specific  demonstrated t h a t the species  in  difference  between  the  unsaturated  fatty  acids  content  and  percentage of  differences i n l i p i d  phases  increased the  with lipid  and  the  of  the  decreased  little  ratio  with  from  potential  of  the  phosphatidylcholine  of the  erythrocyte  erythrocytes  bearing  as  of  poly/mono-  sphingomyelin content  (as  membrane). Thus i n systems  point, a  surface  packing"  appeared  to  property detected.  analogous t o An  age  "fluidity"  related  CCD  of  or  a  with  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 t o  critical  be  systems  increased  increased,  total  high c h l o r i d e content  phase  distribution. content  partition  has  the  "lipid  in  vivo  59 Fe-labeled  erythrocytes  was  obtained  i n the  rat with  h i g h c h l o r i d e two-polymer phase system ( W a l t e r e t a l . , Cell of one  the  s e p a r a t i o n s a r e s a i d t o be substances being  based on  p a r t i t i o n e d f o r the  A c t u a l l y the  environments  polymers can  of  these  1976a).  the d i f f e r e n t i a l  o r t h e o t h e r phase system p o l y m e r s , p o l y e t h y l e n e  hydrophobic than dextran.  one  affinity  provided  by  g l y c o l being  more  be  in  arranged  a  - 31 hydrophobic can  s c a l e , and even p o l y p h a s e systems o f more than two p o l y m e r s  be made  ( A l b e r t s s o n , 1977). Changing t h e i o n i c  environment o f t h e  phases changes t h e environment o f t h e polymers and hence p o s s i b l y t h e attractive  o r r e p u l s i v e p r o p e r t i e s which  t h e phase  polymers  provide.  Based on e x p e r i e n c e o f t h e t y p e d e s c r i b e d above, t h r e e g e n e r a l t y p e s o f phase systems a r e used ( A l b e r t s s o n , 1977; W a l t e r , In  1978).  t h e phase system c o n t a i n i n g h i g h phosphate c o n c e n t r a t i o n , which  itself  partitions  electrostatic positive)  the  potential  cell  having  lower  dextran-rich  d i f f e r e n c e between  distributions  charge-associated, system  to  negligible  potential  creating  t h e phases  are properly said  or charge-sensitive.  phase  (upper  an  phase,  t o be membrane s u r f a c e  In the high  chloride  between t h e phases  phase  the d i f f e r e n c e  between t h e i n t e r a c t i o n s o f PEG and D w i t h t h e membrane appears t o be the  governing  distributions system  factor.  Based  on  o f high  c h l o r i d e content,  s e p a r a t i o n can be a c h i e v e d Thus a v a r i e t y o f c e l l the extent  of using  (Walter,  reflect  a cell  surface  s e p a r a t i o n s and a n a l y s e s  this  I n t h e phase  ligand a f f i n i t y  partitioning  a r e p o s s i b l e , even  approach  t o q u a n t i t a t e weak  ( A l b e r t s s o n , 1978) o r t o probe  1978). I t was a l s o r e c o g n i z e d  themselves  in cell  or diagnosis  membrane  as c e l l  cell-cell  t h a t t h i s type o f o f disease  surface  changes  1978). F o r t h e c e l l b i o l o g i s t and b i o c h e m i s t , t h i s t y p e o f c e l l a n a l y s i s i n combination  cell  by i n c o r p o r a t i n g a polymer l i g a n d ,  a n a l y s i s might be o f use i n t h e study that  correlations,  ( W a l t e r , 1978).  i n t e r a c t i o n s between m o l e c u l e s interactions  above  a r e thought t o be i n p a r t l i p i d - s e n s i t i v e .  which may o r may n o t be " b i o s p e c i f i c " ,  to  the  states (Walter, surface  w i t h s e p a r a t i o n s based on o t h e r p r o p e r t i e s such  s i z e , shape, and/or d e n s i t y , s h o u l d  indeed  be b e t t e r than t h e  - 32 use o f e i t h e r a l o n e ( 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 S t u d i e s o f C e l l S u r f a c e P r o p e r t i e s D u r i n g Efforts  d i r e c t e d toward  current d i s t r i b u t i o n  the elucidation  of c e l l  Ageing age by  counter-  i n two-polymer aqueous phase systems a r e c e n t e r e d  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 , t h a t o f Dr. H. W a l t e r . T h i s a b i l i t y o f CCD  blood  cell  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,  in  two-polymer  and l e n g t h  aqueous  o f time  phase  systems  i n storage,  to  has thus  provide  been  e s t a b l i s h e d , but  p r i m a r i l y w i t h t h e r a t a s a model system. 59 Using  i n vivo  Fe-labeled  r a t erythrocytes,  Walter  and  Selby  (1966) f i r s t demonstrated t h a t CCD c o u l d d i s t i n g u i s h r e d b l o o d c e l l s o f slightly  different  ages. A l s o i n t h e same y e a r , W a l t e r  reported  the d e t e c t i o n  phenylhydrazine induced  o f two  populations  and A l b e r t s s o n  of r a t reticulocytes i n  anemia.  Upon s t o r a g e from 28 t o 48 days t h e p a r t i t i o n o f human e r y t h r o c y t e s i n c r e a s e s ; however t h e r e i s no change i n p a r t i t i o n w i t h s t o r a g e i n ACD and adenosine  (Walter e t a l . ,  1968). 59  Again, alterations  using  the  technique  of  i n vivo  Fe-cohort  labeling,  i n t h e s u r f a c e p r o p e r t i e s d u r i n g normal m a t u r a t i o n  of r a t  e r y t h r o c y t e s were d e m o n s t r a b l e by p a r t i t i o n ' . I n a d d i t i o n , t h e innocuous and  p r o t e c t i v e nature  of  t h e polymers  demonstrated, s i n c e e r y t h r o c y t e s harvested  and  t h e CCD  technique  was  a f t e r CCD s u r v i v e d n o r m a l l y  i n v i v o ( W a l t e r e t a l . , 1971a and 1971b). Phenylhydrazine-induced  reticulocytes (Walter  demonstrated  maturation  pattern  by CCD  e t a l . , 1972a);  associated  (Walter  e t a l . , 1972b) membrane s u r f a c e  an  and t h e  abnormal charge-  properties during  - 33 maturation  of  reticulocytes  abnormal,  being  o f lower  elicited  partition  by  and  repeated  surface  bleeding  charge  were  than  normal  r e t i c u l o c y t e s ( W a l t e r e t a l . , 1975). Cells  other  than  blood  cells  also  demonstrate  a s s o c i a t e d w i t h d i f f e r e n t i a t i o n and m a t u r a t i o n partition. change al.,  Rat l i v e r  their  cells  surface  regeneration  epithelial  exhibit  phase systems, and then  a rapid  a gradual  changes  which c a n be d e t e c t e d by  p r o p e r t i e s as d e t e c t e d  1973b). Rat i n t e s t i n a l  from c r y p t t o v i l l u s  during  membrane  can be  observed  to  (Walter  et  by p a r t i t i o n  cells,  a s they  mature and age,  increase i n p a r t i t i o n  decrease i n p a r t i t i o n ,  i n charged with  ageing  ( W a l t e r and K r o b , 1975). Not  only  demonstrate maturation reflect  could  that  erythrocytes  and a g e i n g ;  aspects  partition  in  change  i t was a l s o  of the c e l l  charge-sensitive their found  surface  phase  systems  properties  during  t h a t phase systems known t o  membrane o t h e r  than  charge,  such  as t h e  c h a r a c t e r o f t h e h y d r o p h o b i c l i p i d b i l a y e r , c o u l d a l s o demonstrate t h a t e r y t h r o c y t e s change d u r i n g c i r c u l a t i o n ( W a l t e r e t a l . , 1976a and 1976b). Most o f t h e 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 o b t a i n e d erythrocytes,  although  other  animal  models have been s t u d i e d  with r a t briefly.  I t i s hoped t h a t 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 a c c o r d i n g t o age by  CCD. However, W a l t e r  model  f o r human  detected  by CCD  erythrocytes, separation  by  red c e l l  ageing.  of r a t red c e l l s CCD  by CCD.  erythrocytes,  e t a l . (1980) r e p o r t e d  alone  n o r by  t h a t t h e r a t i s not a  Erythrocyte could cell  n o t be  c e n t r i f u g a t i o n technique,  young  related  detected  electrophoresis  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  r a t o r human,  age  were  or o l d (obtained  changes i n human  after  cell  t h e same f o r by  t h e Murphy  1973; e r 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 w i t h age  - 34 in  the c i r c u l a t i o n  erythrocytes  have  o f b o t h human and r a t ) . I n t h e r a t , young a  higher  partition  than  electrophoretic m o b i l i t i e s of erythrocytes  o l d erythrocytes  obtained  mature and t h e  a f t e r CCD  increase  w i t h i n c r e a s i n g p a r t i t i o n t h r o u g h t h e CCD d i s t r i b u t i o n s o f e i t h e r young or  o l d r a t erythrocytes.  However t h e r e  a r e no o b v i o u s 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 young o r o l d , b e f o r e  o r a f t e r CCD, u n l e s s i s o t o p e methods a r e employed.  Perhaps t h e e r y t h r o c y t e differs  the density  gradient  f o r r a t and human, so t h a t t h e same age s u b p o p u l a t i o n s  are not  r e a l l y being Walter  age d i s t r i b u t i o n  e t a l . (1981)  showed  levels  erythrocytes  of  younger  that  the elevated red  ageing  of  erythrocytes  cells  aspartate as  a  amino  marker,  transferase young-mature  o f t h e human were found not t o have t h e h i g h e s t  c o e f f i c i e n t i n the red c e l l rat  again  s u r f a c e p r o p e r t i e s i n t h e r a t model system  n o t i n t h e human. U s i n g  (AAT)  through  compared.  r e s u l t s i n altered red c e l l but  f o r human e r y t h r o y c t e s ,  population,  partition  as they do i n t h e r a t . I n t h e  CCD, t h e AAT d i s t r i b u t i o n and t h e 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 o f lower and h i g h e r  p a r t i t i o n t h a n t h e major p o r t i o n  of  In  the  erythrocyte  distribution.  r e t i c u l o c y t e s are enriched  the  human,  AAT,  SA,  a t t h e l o w e r end o f t h e d i s t r i b u t i o n .  and  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 also  l o w e r p a r t i t i o n t h a n t h e major e r y t h r o y c t e W a l t e r e t a l . (1981) a l s o (1980)  i n v o l v i n g CCD  Countercurrent show  increased  repeated  of density  d i s t r i b u t e d young electrophoretic  population.  earlier  separated  work o f W a l t e r e t a l .  young  and o l d r e d c e l l s .  and o l d r e d b l o o d mobility  of a  through  cells  t h e CCD  human r e d c e l l s do n o t d i s t r i b u t e based on charge a s s o c i a t e d  of the r a t train,  but  properties  - 35 by t h i s method. S i n c e distribution  redistribute  t h a t human r e d c e l l s It  was s t a t e d  can  f r a c t i o n s from e i t h e r s i d e o f t h e m i d d l e o f t h e  do not p a r t i t i o n  based on any s u r f a c e  concluded parameter.  i n t h e paper, however t h a t s m a l l e r q u a n t i t i e s o f c e l l s  have d i f f e r e n t  distribution  T h i s might be t h e c a s e , distributed  as t h e o r i g i n a l p o p u l a t i o n , i t was  i n this  curves  o f a l t e r e d apparent  that excessive  partition.  q u a n t i t i e s o f c e l l s were  work, t h u s hampering  separation  being  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 p o p u l a t i o n s b e i n g compared. I t was a l s o c o n c l u d e d  t h a t dog r e d 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 erythrocyte data  show  life  clearly  reticulocytes, lower well  span o f 120 d a y s ) . an  as w e l l  increase  found i n t h e human ( w i t h an  However, as mentioned in  the  as an enrichment  percentage  a s t h e r a t . Upon f u r t h e r e x p e r i m e n t a t i o n  age-density  of cells  of  immature  i n AAT t o t h e l e f t  p a r t i t i o n i n g p o r t i o n o f the d i s t r i b u t i o n curves,  quantities  above, t h e  side or  f o r t h e human as  and a n a l y s i s o f s m a l l e r  a c l e a r c u t d i f f e r e n c e might be demonstrated f o r  separated  human r e d c e l l  p o p u l a t i o n s , as has been found i n  t h e r a t e r y t h r o c y t e model. A  new  differences al.,  method  recently  between two c l o s e l y  1983; W a l t e r  internal  was  standard  described  f o r detection  related c e l l  populations,  of  (Walter  and Krob, 1983). T h i s method i s a v a r i a t i o n method, o r more a p p r o p r i a t e l y  surface  an " i n t e r n a l  et  o f the sample"  59 method.  I t i s similar,  i n principle,  l a b e l i n g method o f a n a l y s i s o f ageing wherein t h e c e l l p o p u l a t i o n under study in  t h e same.sample t o t h e u n l a b e l e d  t o the i n vivo  Fe  cohort  r a t e r y t h r o c y t e s used by  Walter,  i s l a b e l e d i n v i v o and compared  normal " s t a n d a r d "  p o p u l a t i o n . The  method d i f f e r s i n t h a t c e l l s a r e 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,  additional  "standard"  consideration  for  -  unlabeled studies  cells  using  for  this  comparison.  method  is  that  An of  51 Cr  uptake  variation  within  the  labeled  enhanced l a b e l uptake has been r e p o r t e d  subpopulation,  since  f o r young e r y t h r o c y t e s from  the  r a t and human ( W a l t e r 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 b l o o d ,  a r e second i n number  o n l y t o e r y t h r o c y t e s . T h e i r stem c e l l s are r e l a t e d , (unlike to  megakaryocyte  erythrocytes survival  are  time,  origin  or a g e i n g .  thesis,  reflected  by  heterogeneity  not  yet  reportedly  respect  platelets  the  It  seems  cell  been  and  platelet  age  to  associated  extend  distributions  platelets  and  density  and  size, membrane  age-related  analyses,  to  with  the  platelet  and  toward the outside  of  red this  platelet  membrane  ageing.  Analogies  e r y t h r o c y t e ageing,  namely  r e d u c t i o n i n s u r f a c e charge d e n s i t y .  the and  successfully,  application in  so  and 1978;  of  doing,  knowledge o f p l a t e l e t membrane h e t e r o g e n e i t y 1969b; G r a n t and Z u c k e r , 1976  to  cell  surface  applied  a l t e r a t i o n o f membrane and logical  both  respect  p a r t i t i o n i n two-polymer aqueous phase  have been proposed between t h i s p r o c e s s in  class),  of blood  primarily  These methods o f  have  with  Analyses  systems have been d i r e c t e d , cell.  (ploidy  heterogeneous  h e t e r o g e n e i t i e s as  1.7  while  e r y t h r o c y t e s ) have been shown t o be heterogeneous w i t h  their  blood  and  these  techniques  increase  (Walter  our  to  limited  e t a l . , 1969a  and  Yarom e t a l . , 1982).  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 o f P a r t i t i o n Methodology The  determiniation of  prognostic ranging  value  in a  platelet  variety  from t h r o m b o c y t o s i s  of  survival  time i s of d i a g n o s t i c  situations:  in clinical  t o t h r o m b o c y t o p e n i a and  and  conditions  thrombocytemia, i n  - 37 c a r d i o v a s c u l a r d i s e a s e s and c o a g u l o p a t h i e s , as w e l l as i n the judgement of  the  efficacy  of  implants  and  other  surgical  t h r o m b o s i s might o c c u r ( H a r k e r and F i n c h 1969;  procedures  wherein  Harker and S l i c h t e r  1972  and 1974). However e v i d e n c e t o t h e c o n t r a r y has a l s o been p r e s e n t e d . In  a  clinical  patient's then  platelets,  sampling  phlebotomies fraction data  setting  of  are  labeling  (as  are  measurement them  platelets  involved)  injected then  the  to  or  5  graphical  10 to  i n the  analyses  the  reinjecting,  days,  times  remaining  isolating  "^In,  about  appropriate  radioactivity  subjected  w i t h "^Cr  survive  at  i s made by  numerous  determine  bloodstream. and  the The  computations,  p r o d u c i n g f i n a l l y t h e mean p l a t e l e t s u r v i v a l t i m e . There  are s e v e r a l  problems  standard s u r v i v a l time t e s t . labeling when  should  platelets  be are  a s s o c i a t e d w i t h t h e performance  The  procedures  standardized. Cellular prepared  for this  test  for platelet  isolation  contamination may  cause  o f the and  encountered  alterations  in  apparent s u r v i v a l t i m e s i n c e r e d b l o o d c e l l s and w h i t e b l o o d c e l l s when present  also  become l a b e l e d  t h a t o f p l a t e l e t s . T h i s may platelet  survival  l a b e l e d and of  time,  injected,  generally  sequestration  from  or  and  have a  survival  disappearance  of  the  i n v a r i o u s organs,  lungs. P l a t e l e t s injected  total  curve.  to  several  from  platelets  are  l o s s w i t h i n the f i r s t  hour  Once  injected  f o r example  the  platelets  platelet  different  increased  from  to and  inactive  not s u r v i v e n o r m a l l y .  i n the c i r c u l a t i n g labeled reasons,  due  spleen, l i v e r  i n t h i s manner a r e a l s o f u n c t i o n a l l y  (based on a g g r e g a t i o n ) f o r one day, and t h u s may Variation  different  cause changes i n t h e apparent shape o f t h e  t h e r e i s an immediate  50-60%  time  c o n c e n t r a t i o n i s due production  and/or  d e c r e a s e d u t i l i z a t i o n c a u s i n g an i n c r e a s e i n p l a t e l e t c o n c e n t r a t i o n , t o  - 38 d e c r e a s e d p r o d u c t i o n and/or i n c r e a s e d u t i l i z a t i o n c a u s i n g a d e c r e a s e i n platelet  concentration.  I f i t were p o s s i b l e  to differentiate  young and o l d p l a t e l e t s , t h e s e s i t u a t i o n s  could  standard  platelet  survival  detailed  analysis,  usually  provides  platelet with  population  calculation  test, only  (Belcher  o f mean  without  survival  time  some t y p e o f e x p o n e n t i a l o r c u r v i - l i n e a r platelet  survival  time  u n c o m f o r t a b l e and c h a l l e n g i n g the  patient,  test  complex  There  be g a i n e d  fitting  and  f o r the entire  are also  problems  i t i s questionable  t o the standard l i n e a r , or  i s therefore  time  consuming,  exposure t o r a d i o a c t i v i t y ) f o r f o r the c l i n i c i a n  c e n t e r s i t i s n o t p e r f o r m e d , even i n s i t u a t i o n s to  The  function.  (requiring  and s u f f i c i e n t l y  curve  time  i n that  whether o r n o t t h e p o i n t s s h o u l d be f i t t e d  This  be d i s t i n g u i s h e d .  a mean s u r v i v a l  e t a l . , 1977).  between  would be p a r t i c u l a r l y u s e f u l .  that  i n many  where t h e i n f o r m a t i o n  Substantial  benefits  would  a c c r u e i f t h e t e s t c o u l d be improved. In  order  radioactivity, have  been  to eliminate different  introduced.  t h e exposure  methods o f p l a t e l e t  Aspirin  arachidonate-prostaglandin cyclooxygenase unaffected such  platelets.  a s BTG  because  activity  their  determination  provide loss  o f the patient  ingestion  pathway,  used  only  a  indicates  of platelet  and  the  used  stimulation.  to block the  reappearance  f o r example  i n hematological  changes a r e o f t e n r e f l e c t e d  at the c e l l  surface,  contents age,  The p r o c e d u r e f o r  and s i n c e  disease  of  o f new,  of platelet  degranulated  p l a t e l e t s s u r v i v e n o r m a l l y t h i s might p r o v i d e s p u r i o u s r e s u l t s Since,  to  estimation  of platelet  estimate  i s insensitive  time  of production  analyses  qualitative platelet  BTG  has been  as an i n d i c a t o r  Methods i n v o l v i n g  survival  and p u b l i c  also.  states,  pathologic  initially  as a cause  - 39 or  eventually  as  a  result  of  the  disease,  analyses  of  blood  s u r f a c e p r o p e r t i e s might be o f d i a g n o s t i c and p r o g n o s t i c v a l u e  cell  (Walter,  1978). The  p o t e n t i a l o f p a r t i t i o n i n two-polymer aqueous phase systems  to  changes  detect  in  blood  cell  (and  subcellular  particle)  p r o p e r t i e s t h a t o c c u r as a f u n c t i o n o f d i f f e r e n t i a t i o n , age  as w e l l as t h o s e  that occur  surface  maturation  i n d i s e a s e o r f o l l o w i n g i n v i v o and i n  v i t r o t r e a t m e n t s , has a l r e a d y been e s t a b l i s h e d on an e x p e r i m e n t a l for  certain  cells  (the  platelet  not  being  one  p a r t i t i o n has seldom been a p p l i e d a t a c l i n i c a l The  c h a r g e - s e n s i t i v e , and  as o r i g i n a l l y Walter  (1969, 1977,  employed. applied (of  The  varying and  still  Further  and  polymer  and  However,  level.  phase systems are s t i l l  (1960, 1971,  1977,  1978)  used  and  by  and a r e the phase systems most commonly  lipid-sensitive  salt  them).  basis  phase  systems  concentrations).  could  be  phase system  sets  specific  lipid  The  s u r f a c e r e c e p t o r a f f i n i t y two-phase systems (many o f which can  f u r t h e r these  variations in  present  Albertsson  of  i f made a v a i l a b l e i n s t a n d a r d i z e d  system c o m p o s i t i o n at  hydrophobic  1982)  experimental)  investigate possible  1978,  charge-  routinely  affinity are  d e s c r i b e d by  and  by  (and  be  applied  specific cell partitions  where  warranted  in  induced  determining  to  s u r f a c e p r o p e r t i e s . Because o f by  differences  t e c h n i q u e ) , the phase systems a r e  accurately  order  the  polymer  in  phase  standardized  concentrations.  improvements a r e w o r t h 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 p r o v i d e  a specific  p a r t i c l e with a constant  partition  partition,  p l a t e l e t s , or polystyrene p a r t i c l e s .  coefficient  (K)  f o r example f i x e d During  CCD  for a  standard  red c e l l s ,  the e x p e r i m e n t a l  t h e sample under i n v e s t i g a t i o n c o u l d be compared t o t h a t o f a  fixed K  for  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 t h e phase system.  - 40 When o n l y subpopulation individually repetition resolve  slight  deviations  heterogeneity  from  i s suspected,  or simultaneously, of the p a r t i t i o n i n g  subtle  normalcy  differences  properties.  This  t h e s i s i s an  methodology  f o r the purposes  t o CCD.  samples  This  p r o c e d u r e and and/or  must be  increases i s often  heterogeneities  assessment of  are detected,  p l a t e l e t s , and an i n v e s t i g a t i o n o f p l a t e l e t  an  age  r e s o l u t i o n by sufficient in  to  surface o f such  distribution  heterogeneity.  when  subjected,  of the a p p l i c a t i o n  obtaining  or  with  - 41 -  CHAPTER 2  2.1  Blood  MATERIALS AND METHODS  Samples  New Zealand Unit  white  male r a b b i t s were o b t a i n e d  a t the U n i v e r s i t y of B r i t i s h  large  plastic  syringe  attached  C a n n u l a , Sherwood M e d i c a l into  a  central  Research  Columbia.  Monkeys  Pentobarbitol Human b l o o d male  t o a cannula  Rhesus  anesthetized  was o b t a i n e d , personnel  with  3.8%  Ketamine with  a t t h e Acute  Care  prior  study.  using  a  Medicut  U.S.A.) i n s e r t e d taken  at the  Vancouver,  British  (5-25 mg/kg) and  needle  n e e d l e and s y r i n g e ,  (MPD), p r i m a r i l y a c u t e  a portion of this  hours  with  Argyle  was  Hospital,  t o bleeding  Cancer p a t i e n t s , a t Vancouver G e n e r a l  for  blood  and s y r i n g e .  from normal  adult  Hospital, University of  Columbia, and t h e C a n a d i a n Red C r o s s B l o o d  disorders  was drawn  (Sterile  monkey  at S t . Paul's  (25 mg/kg) p r i o r  laboratory  British  Unit were  Blood  I n d u s t r i e s , S t . L o u i s , MO,  ear a r t e r y .  Pulmonary  Columbia.  from t h e Animal Care  Bank i n Vancouver.  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  myeloblastic  leukemia,  donated  blood  These specimens were c o l l e c t e d (about 4  t o experiments) w i t h  needle  and s y r i n g e ,  and mixed  with  sodium c i t r a t e (9 ml b l o o d p l u s 1 ml c i t r a t e ) .  2.2 A n t i c o a g u l a n t 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  solutions Jandl,  below  1964a).  were  essentially  The a n t i c o a g u l a n t  as p r e v i o u s l y was prepared  described  ( A s t e r and  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 o f 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 o f c i t r i c monohydrate, and 2.0 g o f d e x t r o s e  acid  p e r f i n a l volume o f 100 ml i n water.  I t was f r o z e n a t -20°C i n 25 ml a l i q u o t s , and used a t a d i l u t i o n o f 1  - 42 part  per  were  6 parts  Certified  manufacturing sodium  Reagent  division,  citrate,  acidified  of blood, immediately a f t e r  was  Grade Fair  also  t o pH 6.5  (Fisher  Lawn, NJ.  used.  w i t h 0.11  A  drawing.  Scientific similar  Blood, taken M citric  Co.,  Chemical  anticoagulant,  into  acid  A l l chemicals  sodium  ( o r ACD,  3.8%  citrate  was  lml/lOml blood)  prior to centrifugation. 2.3 Tyrode's S o l u t i o n s Tyrode's I  s o l u t i o n s were p r e p a r e d from t h r e e s t o c k s o l u t i o n s .  c o n t a i n e d 16 g o f N a C l , 0.4  of  NaH P0 .1H 0, 2  A  contained  2.03  Scientific 2.19  g,  2  1.47  in  water  of  MgCl .6H 0  g  Co.,  Fair g,  was  Nos.  2  or  1.11 ml  final  i n 100  g  of  water.  288,  or  U.S.A.; o r B o v i n e Albumin Sigma C h e m i c a l Co.,  3  water. (6H 0,  2  of  100  Magnesium  ml  CaCl  2  Miles  Stock 2H 0, 2  V,  MO)  II  Fisher  I I I contained or  anhydrous,  platelet  (Pentex B o v i n e Albumin  Fraction  Stock  g  washing  o f 5 ml o f S t o c k I t o 90 ml o f  Laboratories  St. Louis,  ml.  Chloride,  C a l c i u m - f r e e Tyrode's  g of albumin  363,  g o f NaHC0 , and 0.116  volume  (Reagent  2  p r e p a r e d by t h e a d d i t i o n  water c o n t a i n i n g 0.35 Lot  per  Lawn, NJ)  r e s p e c t i v e l y ) i n 100 solution  g o f KCI, 2.0  Stock  and  Inc.  Sigma No 0.1  Fraction  Research  V,  Products,  A-4503, L o t 78C-0202,  g o f d e x t r o s e (anhydrous,  F i s h e r S c i e n t i f i c Co., C h e m i c a l M a n u f a c t u r i n g D i v i s i o n , F a i r Lawn, N J ) . A 2 ml the  volume o f s t o c k I I was  volume  made  up  to  100  then added, ml  with  the pH a d j u s t e d t o 6.5  water.  Tyrode's  solution  and for  r e s u s p e n s i o n o f p l a t e l e t s was p r e p a r e d by the a d d i t i o n o f 5 ml o f s t o c k I t o 80 ml o f water c o n t a i n i n g 0.35 A  1 ml  added,  volume o f t h e pH  s t o c k I I and  a d j u s t e d t o 7.35  g o f a l b u m i n and 0.1  a 2 ml and  g of dextrose.  volume o f s t o c k I I I was  the volume made up  t o 100  water. The f i n a l o s m o l a r i t y o f t h e s e s o l u t i o n s was 290 mOsM.  ml  then with  - 43 A variation making  larger  addition  of  quantities less  MgCl .6H 0, 2  o f t h e above p r o c e d u r e was o f t e n  albumin,  0.04%,  2  of  and  buffer, 0.2%  EGTA  usually  (Zucker  used  one  and  (Ethylene-glycol  which  liter,  Grant,  involved with  1978),  the solid  b i s beta-amino-ethyl-  e t h e r N, N' t e t r a a c e t i c a c i d , MW 380.4, L o t No. B4X, Eastman Kodak Co., R o c h e s t e r , NY), 0.02-0.04% ( Z u c k e r and G r a n t , 1978), t h e same r a t i o o f s t o c k I s o l u t i o n t o w a t e r , and t h e adjustment o f pH w i t h HC1 o r 0.11 M citric  acid.  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 Platelet of  rich  anticoagulated  temperature, blood.  The  with pellet  plasma  (PRP) was u s u a l l y h a r v e s t e d by c e n t r i f u g a t i o n  ( a c i d i f i e d ) b l o o d a t 275 g f o r 15-20 minutes a t room the force was  being varied  diluted  c o n t a i n i n g 0.35% b o v i n e serum centrifugation diluted poor  (PPP) was  solution  a l b u m i n and 0.02% EGTA a t pH 6.5 and t h e added  t o t h e PRP. The  a t 2000 g f o r 30 minutes and t h e p l a t e l e t  removed.  i n modified  on t h e volume o f  C a l c i u m - f r e e Tyrode's  r e p e a t e d . The s u p e r n a t a n t was  PRP was c e n t r i f u g e d  plasma  with  depending  pH  The  6.5  platelets  Tyrode's  were  solution.  resuspended  and  Platelets  were  washed  twice  finally  resuspended i n Tyrode's s o l u t i o n pH 7.35 p r i o r t o 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 t o p a r t i t i o n . 2.5 P l a t e l e t A g g r e g a t i o n An A g g r e g a t i o n and Shape Change M o n i t o r , B o r n / M i c h a l , was used w i t h a  Thermomix  studies  (Triplett  platelets/ml Tyrode's. ten  1420, B. Braun, Melsungen  was  e t a l . , 1978) a c o n c e n t r a t i o n used.  An a l i q u o t  seconds  AG, W Germany. F o r a g g r e g a t i o n g  Platelets  were  washed  o f 0.8-1.0 ml sample  i n the cuvette  holder  with  o f 0.5 and  t o 1 x 10  resuspended  in  was i n c u b a t e d a t 37°C f o r stir  bar, after  which  ADP  - 44 (adenosine  diphosphate,  MW 427.2, Sigma C h e m i c a l  Co.) was added. ADP  _2 was made as a s t o c k  solution  concentrations ranging  o f 10  M which  was d i l u t e d  from 0.06 t o 25 x 1 0 ~ M. A g g r e g a t i o n 6  as a c o n t r o l t o examine t o f u n c t i o n and v i a b i l i t y washing by  for final  procedures p r i o r  to fixation  and/or  was used  of platelets  surface property  after  analyses  partition.  2.6 P l a t e l e t F i x a t i o n Platelet (1973c)  f i x a t i o n by a m o d i f i c a t i o n o f t h e method o f W a l t e r  involved  the a d d i t i o n  erythrocytes) into ml  vials  Bell,  platelet  suspension  t e n volumes o f 1.85% g l u t a r a l d e h y d e  a t 70%, Ladd  Vancouver  of a  B.C.)  Research  I n d u s t r i e s , obtained  i n 300 mOsM  sodium  ( o r packed  (obtained from  phosphate  and a d j u s t e d  not n e c e s s a r y . last  washing  buffer  pH  7.4  The pH  t o pH 7.2-7.4 each day b u t t h i s was u s u a l l y  When p l a t e l e t s were f i x e d , albumin was o m i t t e d and  in 2  Ingram and  (composed from phase system s a l t s t o c k s o l u t i o n s ) , w i t h m i x i n g . was c h e c k e d  et a l .  resuspension  solution.  Platelets  from t h e  were  finally  resuspended i n Tyrode's w i t h g l u t a r a l d e h y d e . 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 a n 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 m i n u t e s i n a water  bath  with  at  37°C,  "^H-serotonin, and  "^H-adenine;  the  various  see  ( Cr, 5 l  Experimental  Discussion) radioactive nuclide(s),  m  i n , C1 A  Sections  and washed  within  and Results  ( u s u a l l y t w i c e was  sufficient) until  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 a n a l y s e s  (e.g.,  injection  i n vivo  of  "^Cr-labeled  platelets  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 except  when  density  gradient  separation  for survival  surface properties),  of labeled platelets  was t o  - 45 precede  these  analyses.  Platelets  were  also  labeled  in  vivo  by  35 injection course  of a s i n g l e cohort  of  incorporation  label  of  S-sulphate  radioactivity  movement o f l a b e l e d p l a t e l e t s i n t o and p o o l was  into  out  compared  to  a  entire labeled  40%  solution  grade, S t . Regis volume  of  given time.  platelet  at  platelet  I f the c o h o r t was  population,  the  various  dilutions  (BDH  was  a  second  the  to  in  be  vitro  used.  osmolarity  of  of  analyses  the  this  (Appendix  make  an  until  was  Table  tonicity  of  checked  by  al.,  by  of  the  Stractan,  i n comparison t o t h a t  the  was  with resin  measured index  solution  as and  point  the  partial  et a l . , 1974), s u f f i c i e n t  Tyrode's  stock  solution  1979). The  isotonic  examined  equal  freezing  Considering  i n other  of  20%  deionized  Stractan  in  osmolarity  (Appendix T a b l e  A.2),  Tyrode's s o l u t i o n .  Stractan  volume  of  concentration,  s t o c k , were r e - c h e c k e d  final  observation  in  same r e f r a c t i v e stock  and t h e g r a d i e n t s t e p s were d i l u t e d i n pH 6.5 The  change  Stractan  A.l).  an  England),  w a t e r were added t o the o r i g i n a l  isoosmotic  final  no  research  using  Ltd., Poole,  deionized  (Corash  Tyrode's s o l u t i o n ( C i e s l a r et density of t h i s  deionized  Stractan concentration  stock  volume o f S t r a c t a n  to  was  Chemicals  repeating  arabinogalactan;  c a l c u l a t e d assuming the  sodium c h l o r i d e , and  solution  (StR;  Tacoma, WA)  4-5°C;  and  sucrose;  salts,  MB-3  II  ( u s u a l l y t w i c e ) . The  refractometry  depression  Stractan  Paper Co.,  mixing  observed  specific  of  Amberlite  continuous  and  the  S t r a c t a n I I Stock S o l u t i o n A  by  time  i.e.,  o f the c i r c u l a t i n g  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  platelets,  The  f o l l o w e d , i n o r d e r t o d e t e r m i n e the age c h a r a c t e r i s t i c s o f  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 t any  2.8.1  (1-2.5 mCi).  stock  packed  solutions  was  erythrocytes  known i s o t o n i c  media.  in  Equal  - 46 volumes  (0.2 ml) o f washed  packed  human e r y t h r o c y t e 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 t u b e s , MB  microhematocrit  microcapillary  centrifuge,  reader  centrifuged with  and t h e h e m a t o c r i t  (Damon/IEC  Division,  i n the various  was  Needham  read  an IEC with  Heights,  a  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  Tyrode's  solution,  (average  saline,  o f three  plasma,  and  measurements  10%,  14%,  each) f o r  and  18% StR,  respectively. To  observe  hematocrit rabbit salt, 1160  osmotic  pressure,  erythrocytes  mOsM, from  measurements  erythrocyte  volume, o r  the hematocrit  i n various  known  of  washed  concentrations  s t o c k s o l u t i o n o f 4 x i s o t o n i c , 0.616 M  which  dilutions  (Appendix  immediately  range o f t h e s e  was measured  (using a s t a r t i n g  tonicities  in  the s e n s i t i v i t y  Table  were made) c o r r e s p o n d i n g  A.3).  a f t e r resuspension  The  hematocrit  of  NaCl,  to various  was  measured  o f e r y t h r o c y t e s , s i n c e a f t e r 10 minutes  c e r t a i n samples t h e h e m a t o c r i t  was observed  t o change,  presumeably  due t o l y s i s a t 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 In  order  Gradient  t o s e l e c t the appropriate  step  d e n s i t i e s which  provide  the desired p l a t e l e t  density  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 o f  PRP  on  single  concentrations platelets percentage  ranging  recovered  of  on  each  of  gradient  the steps  platelets,  F o r example  from b l o o d t a k e n  Stractan  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  solution  at  various  from 10-20%, i n i n c r e m e n t s o f 0.5% S t r a c t a n . The  of the t o t a l  concentration. PRP,  steps  population  gradient  were  and t a b u l a t e d  (Appendix  i n sodium c i t r a t e  Table  determined  as  a  with  the Stractan  A . 4 ) , human  platelets i n  (11/23,29) o r EDTA (11/27) were  - A7 layered  in  1-2  concentrations  ml and  volumes  on  -  single  centrifuged  at  steps  4000-6000  of g  Stractan f o r one  work o f 1979; the  others  density determinations  (Corash  et a l . , 1974,  Appendix Table A.5),  1977,  and and  of  the  platelet  subpopulations  of  low  density  density (5-10%,  The used  g r a d i e n t s t e p volumes and  for density gradient  14.5-16%,  3  ml  of  the  17.5-18%,  species.  intermediate The  steps  density  of  and  were these  R e g i s Paper Co.); 1977  and  (**)  designed  3  of  varied  with  gradient weighing  et a l . ,  to  by  the  harvest  isolating density  HDP).  were: 1 ml  ml  to  intermediate  20%.  of  the  steps  10%,  These  S e c t i o n s o f Chapter 3. The  measured a t room t e m p e r a t u r e by (*), o r were c a l c u l a t e d  Cieslar  ranges o f S t r a c t a n c o n t e n t ,  centrifugation  d e s c r i b e d i n the E x p e r i m e n t a l of  1978;  LDP),  room  RC5.  reference  distribution  (80-90%, IDP) and h i g h d e n s t i y p l a t e l e t s (5-10%,  at  by  a f o u r s t e p g r a d i e n t was  extremes  various  hour  t e m p e r a t u r e (11/23,27) and a t 5-10°C (11/29), i n a S o r v a l l From such p l a t e l e t  of  are  3  ml  of  further  concentrations  platelet (Table  actually  sample  2.8.2.1)  and were  a measured volume o f S t r a c t a n  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 .  and compared t o v a l u e s o f o t h e r s  (Corash  e t a l . 1974,  1978).  T a b l e 2.8.2.1 S p e c i f i c G r a v i t y and D e n s i t y o f S t r a c t a n StR Cone (%)  D e n s i t y * (g/ml)  StR Cone (%)  0.00 8.68 10.00 20.00 21.18 30.00 33.33  0.9475 1.000 1.010 1.072 1.080 1.140 1.160  0.00 13.5 15.0 16.0 17.0 18.0 20.0  Density**(g/ml) 0.995 1.055 1.062 1.066 1.071 1.075 1.084  - 48 PRP  obtained  from 30-40 ml  of blood  g r a d i e n t s , and p l a t e l e t s were s e p a r a t e d 1-2  hours  at  20-25°C, on  isosmotic  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,  was  l a y e r e d on  eight  by c e n t r i f u g a t i o n a t 4000 g f o r  density  IDP  four to  and  step-gradients  HDP.  isolating  They were p i p e t t e d from  t h e g r a d i e n t s t e p i n t e r f a c e s , d i l u t e d w i t h an e q u a l volume o f Tyrode's (pH 6.5) and  s o l u t i o n , washed (by c e n t r i f u g a t i o n a t 2600 g f o r 60  sampled  or  examination, surface  resuspended  platelet  property  for  further  and/or  sizing,  partition  in  counting  studies  by  analyses:  microscopic  radioactivity  two-polymer  minutes),  counting,  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 S t o c k S o l u t i o n s Dextran  T500  (D,  Pharmacia, Uppsala, were  composed  dextran  per  polarimetry path and  the  100  g  addition of total).  final  reading  and  dextran  factor of  (range  Reuter  factor neglected  gm/ml  a  6.256 was  Lot.  solution  derived  Polyethylene  concentrations  to  was  used  percent  20%  were  this  glycol  (PEG),  (6,000-8,000 d a l t o n s , Union C a r b i d e ,  new  (22  checked 4731;  to  convert  the  dextran;  dextran  density.  g by  100  accordingly to provide  (w/w)  20°C). A  (w/w)  mm  s t o c k ) t o 50 g w a t e r ,  the density of dextran at  FD16027,  water  P o l a r i m e t e r , GmbH, SR6  considering  by a d d i t i o n o f 150 g PEG  deionized  o f 4 g (20% d e x t r a n  6.2814  3.0-3.5)  20%  and  c o n c e n t r a t i o n i n the phase system.  conversion for  7830  to  phase system r e c i p e s were a d j u s t e d  conversion  (w/w)  Lot.  dextran  Dextran  l e n g t h ) , at a d i l u t i o n the  daltons,  Sweden) s t o c k s o l u t i o n s o f a p p r o x i m a t e l y  (Dr. Steeg  appropriate a  by  500,000  Stock  Carbowax Pitscathaway,  t o a f i n a l o f 500 g water.  the  Initially  polarimeter  however,  this  (approximately  1.08  conversion  factor  s o l u t i o n s of  6000 NJ),  renamed  30% 8000  were composed  - 49 Because t h e s e p o l y m e r s ,  d e x t r a n and  PEG,  were r e l a t i v e l y  ion  free  when o b t a i n e d from t h e 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 t o t h e t o n i c i t y  of  a s o l u t i o n was  of  s m a l l a t low c o n c e n t r a t i o n s . In t h e c a s e  200,000-275,000 d a l t o n s , t h e o s m o t i c was  calculated  tonicity  pressure at a given concentration  from d a t a o f LeNeveu e t a l . (1977). As  contribution of dextran  of dextran  calculated,  (200,000-275,000 d a l t o n s ) was  t o t h a t f o r d e x t r a n (500,000 d a l t o n s ) when measured by  the  similar  freezing  point.  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 , t h e t o n i c i t y d e i o n i z e d S t r a c t a n I I ( a p p r o x i m a t e l y 30,000 d a l t o n s ) , was  only  of  slightly  h i g h e r t h a n d e x t r a n (Appendix T a b l e A.6). 2.9.2  Phase System S a l t S t o c k S o l u t i o n s Concentrated  was a  sodium c h l o r i d e  (usually  0.6  M,  composed by weight by t h e a d d i t i o n o f 17.53 total  of  (NaPB)  pH  g  g  7.2  isotonic) 5.8  500  was  of  of  solution.  (0.218  M  composed  NaH P0^  by  to  2  made  by  weight  the  water  d i b a s i c - s o d i u m phosphate, were  2  and  A  addition for  a  as  they  were  M  21.67  g  of  700  total  Sigma Chemical Co., and  sodium  0.069 of  isotonic)  g o f NaCl t o water f o r  Concentrated  Na HP0  f o u r times  phosphate  buffer  Nah^PO^,  twice  of  Na HP0  4  and  g  (mono-  and  2  S t . L o u i s , MO).  Solutions  approximations  to  molar  c o n c e n t r a t i o n s , they a r e r e f e r r e d t o as such. 2.9.3  Two-polymer Aqueous Phase System C o m p o s i t i o n  Phase systems were composed by weight (w/w)  dextran,  30%  (w/w)  from  polyethylene glycol,  stock s o l u t i o n s of and  s o l u t i o n s . U s i n g a d e x t r a n s t o c k . s o l u t i o n o f 21.73% systems were p r e p a r e d , 5%  D and  (5/4:A  4%  PEG,  through  and  each  consisting  with s a l t  concentrated  salt  (w/w), 50 g phase  o f the polymer c o n c e n t r a t i o n s ,  composition designations A  5/4:G, r e s p e c t i v e l y ) ,  20%  as d e s i g n e d  by  J.M.  through  Van  G  Alstine  - 50 and D.E.  Brooks (Appendix T a b l e A . 7 ) , a f t e r  t h e method o f 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; w i t h 2x s t o c k  salt  s o l u t i o n s ) o f t h e s e phase systems a r e as l i s t e d i n T a b l e 2.9.3.1 ( e . g . , phase  system  5/4:A;  5%  D,  4%  PEG,  0.109  M  Na HP0 , 2  0.0345  4  M  NaHLPO., and a s a l t r a t i o o f 1 0 / 0 ) .  T a b l e 2.9.3.1 Two-Polymer  Phase System S a l t C o n c e n t r a t i o n s and R a t i o s  5/4:  B  A  Na HP04 (M) NaH P04 (M) NaCl (M) NaPB/NaCl  0.109 0.0345 0.0 10/0  2  2  0.0910 0.0288 0.025 8.4/1.7  C  E  D  0.073 0.023 0.0498 6.7/3.3  0.055 0.0174 0.075 5.1/5.0  F  0.037 0.012 0.099 3.4/6.7  G  0 .021 0 .0067 0 .125 1 .9/8.3  0.0095 0.003 0.15 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 t h e upper and l o w e r phases o f t h e two-polymer phase systems ( i . e . , PEG,  but with  concentration  varying and  buffer  increasing  o f c o n s t a n t polymer c o n t e n t s , 5% and  salt;  sodium  D e s i g n a t i o n A t h r o u g h G) were measured microcapillary millivolt  pipets,  meter  (mV) between  Ag-AgCl  decreasing  chloride  electrodes  and l o w e r phases  was  phosphate  concentration,  using s a l t bridges,  (Reitherman e t a l . , 1973).  t h e upper  sodium  and  a  D/4%  from  agar-filled  high  impedance  The p o t e n t i a l  difference  found  t o decrease  with  d e c r e a s i n g phosphate and i n c r e a s i n g c h l o r i d e ( T a b l e 2.9.3.2).  T a b l e 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: P.D. (mV) + S.Dev. N  A  B  1.0656 0.9831 0.2103 0.1519 8 14  C  D  E  F  0.9420 0.6010 0.4767 0.2784 0.2528 0.0622 0.0655 0.0438 9 12 14 12  G 0.1756 0.0327 9  - 51 An  alternate  recipe  f o r phase  (Appendix T a b l e A.8). T h i s phase "salt  ratio",  sodium  several  phase  c o m p o s i t i o n was  system r e c i p e was  with twice i s o t o n i c  when p r e p a r i n g  system  salt  solutions.  systems, d i f f e r i n g  also  used  f o r c o m p o s i t i o n by I t was  only  of  advantage  i n the r a t i o  of  phosphate t o sodium c h l o r i d e , u s i n g a s i n g l e 2x polymer m i x t u r e  o f 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 w i t h w a t e r . 2.10 S i n g l e Step P a r t i t i o n Single  tube  partition  experiments  were  performed  essentially  as  previously described  ( W a l t e r , e t a l . 1969a and 1969b; Grant and Z u c k e r ,  1978)  for  and  modified  small  samples  (e.g.,  of  fixed  or  labeled  p l a t e l e t s , and i n c e r t a i n c a s e s e r y t h r o c y t e s ) . P l a t e l e t samples (20 u l , 10  platelets),  7  systems  suspended  in  Tyrode's  solution,  are  added  to  phase  (0.5-1.5 ml upper- and 0.5-1.5 ml l o w e r p h a s e ) . The phases were  mixed  by  inversion  hour,  after  which  twenty the  t i m e s , and  upper  phases  allowed were  to  settle  sampled  f o r 0.5-1.0  (0.3-0.5  ml)  and  c o u n t e d 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 o f p a r t i c l e s (or  radioactivity)  compared  to  coefficients  the  which  total  had  partitioned  added  (as a  into  the  p e r c e n t ) and  upper thus  phases  the  were  partition  (K) were c a l c u l a t e d . A v a r i e t y o f systems were examined i n  t h i s manner f o r p l a t e l e t  partition.  2.11.1 C o u n t e r c u r r e n t D i s t r i b u t i o n Multiple partition carried  out u s i n g  Nuclear  Chicago  plates  (CCD  Corp.,  Midland  s t e p s by c o u n t e r c u r r e n t d i s t r i b u t i o n  a CCD-1200 a p p a r a t u s ( B u c h l e r Corp.,  NJ).  I t consisted  of  with  120  cavities  two  (CCDC)  were  Instruments D i v i s i o n , circular  1250C, 71014), s e a l e d w i t h s i l i c o n e l u b r i c a n t MI),  (CCD)  arranged  plexiglas  (Dow  Corning  around  the  - 52 p e r i p h e r y o f b o t h t h e t o p and bottom p l a t e s . on  a  mechanical, e l e c t r o n i c a l l y  timed  shaking,  contained machine  settling  timing  control  controlled  and t h e r o t a t i o n  t h e upper phase) w i t h  These p l a t e s were mounted base,  which  facilitated  o f the top plate  (which  respect  t o t h e bottom. The a u t o m a t i c  was checked w i t h  a s t o p w a t c h p e r i o d i c a l l y and  w i t h each change o f c o n t r o l . F o r t h e s e e x p e r i m e n t s , a l a r g e volume was  mixed a t 5-7°C, f i l t e r e d  Company, D i v i s i o n  (250-300 m l ) o f phase system  (Nalgene F i l t e r  U n i t , 0.45 m i c r o n , Nalge  o f Sybron/Nalge C o r p o r a t i o n , R o c h e s t e r , NY), a l l o w e d  t o s e t t l e o v e r n i g h t i n a s e p a r a t o r y f u n n e l , and t h e upper P E G - r i c h and lower D - r i c h phases were t h e n c o l l e c t e d The upper  phase,  Cornwall ml)  platelet  into  samples  separately.  t o be p a r t i t i o n e d  by CCD were suspended i n  and 0.9 ml o f t h e s e m i x t u r e s  pipetor, each  Becton-Dickinson  of a  few CCDC  number; 3 CCDC l o a d e d  were  Co.) a l o n g  (less  than  loaded  with  (using  bottom  a B-D  phase (0.4  10% o f t h e t o t a l  transfer  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 m l ) , and l o w e r phase (0.5 ml) were l o a d e d i n t o t h e r e m a i n i n g CCDC. T h i s p r o v i d e d f o r a t l e a s t 0.1 ml o f s t a t i o n a r y l o w e r phase. F i r s t , upper  phase,  and l a s t l y  a l l l o w e r phase was l o a d e d , t h e n a l l  t h e samples  i n upper  phase  (load  mixtures).  B e f o r e each CCD r u n began, t h e phases w i t h i n a l l c a v i t i e s were mixed by one m i n u t e o f s h a k i n g on manual c o n t r o l . Then, t h e machine was s w i t c h e d t o a u t o m a t i c which i n i t i a t e d  a preset  thirty  seven minute s e t t l i n g c y c l e  followed  three  t o t h e bottom  degrees w i t h  platelet  respect  s a m p l e ( s ) were  repetition of this cycle.  exposed  second s h a k i n g  by t h e r o t a t i o n plate.  t o a number  cycle,  a  of the top plate  In t h i s  of partition  manner t h e steps  by  - 53 In  order  studied just An  to  was  determine  i f the  b e i n g s e p a r a t e d by  randomly  distributed,  experiment  of  this  a re-partition  sort  required  since with  change  properties  necessary might  surface  t o use  have  system  cell  sample  properties,  experiment  was  exposure  of  reflected  different  cells  by  by  CCD.  fractions  (CCDF)  Platelets, obtained  pooled  from  from  different  of 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  a r e a from which they were s e l e c t e d  CCD  i t  fixed  CCDC  (left,  by a second CCD  was  cells  properties  adjacent areas  out.  were known t o  though  surface  be not  to  partition,  even  membrane  and  cells  time of storage c e l l s as  to  carried  In t h i s c a s e , 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  transfers  right)  blood  the  glutaraldehyde fixed  demonstrably  unfixed c e l l s .  of  t h e phase  c o n d i t i o n s t w i c e , and their  type  from  over  116  into  CCD  middle,  and  t o t h e same  (see Chapter 3.1).  2.11.2 CCD o f D e n s i t y S e p a r a t e d P l a t e l e t s The  CCD  separated  unit  was  platelet  loaded with populations  both (LDP,  phases IDP  and  and  HDP)  l o a d e d i n CCDC 1-3, 41-43, and 81-83, r e s p e c t i v e l y . involving  37-40  completed  within  cycles about  of five  o f Tyrode's washing s o l u t i o n collected  and  p o o l e d by  These CCDF were sampled, f o r each o f t h e LDP,  mixing, hours  were  separately  The CCD p r o c e d u r e s , a  transfer  were ml  was added t o each CCDC and the phases were  (2-4) a d j a c e n t CCDC, t o c o m p r i s e counted  IDP, and HDP  d i s t r i b u t i o n parameters Peak,  and/or s t a n d a r d d e v i a t i o n  and  three density-  a t 5-7°C. Upon c o m p l e t i o n , 0.7  for platelets, were p l o t t e d ,  o f t h e peak p l a t e l e t CCDF. The g r a p h i c a l the  settling  the  and  the  10-20  CCDF.  distributions  n o r m a l i z e d as a p e r c e n t  d a t a was t h e n c h a r a c t e r i z e d  by  Mode, M i d d l e (and/or M e d i a n ) , spread  (see Chapter 3.2).  - 54 2.11.3 CCD o f I n V i t r o L a b e l e d P l a t e l e t s Aged I n V i v o Platelets  from  centrifugation  30  ml  of  rabbit  blood  were  concentrated  by  and l a b e l e d i n 5 ml o f plasma by t h e a d d i t i o n o f 0.54 mC  51 of  Cr as sodium chromate ( I C N , I r v i n e ,  minutes acid  CA) and i n c u b a t i o n f o r 30-60  a t 37°C (about 1 mCi/10 ml o f PRP). Next, 0.4 ml o f a s c o r b i c  (250 mg/ml; n o t always u s e d ) i n Tyrode's s o l u t i o n  incubation platelet  mixture,  counts.  resuspended  washed  The  in a  radioactivity  and 5 1  samples  Cr-labeled  small  volume  and p l a t e l e t s ,  erythrocytes  were  taken  o f PPP, counted  and i n j e c t e d  came. F o r p a r t i t i o n s t u d i e s n o t i n v o l v i n g  and  for radioactivity  and  p l a t e l e t s were washed t w i c e i n PPP, a  second  ( i n certain  from t h e sample) i n t o  sample, t h e p l a t e l e t s  was added t o t h e  the rabbit re-injection  time f o r  instances  with  from which  they  of the p l a t e l e t  were washed as above b u t i n Tyrode's  solution,  resuspended i n t h e upper phase. B l o o d samples were t a k e n a t v a r i o u s  t i m e s p o s t i n j e c t i o n ( p . i . ) o f t h e l a b e l e d p l a t e l e t sample. These aged, labeled, by  platelet  samples were h a r v e s t e d , washed, and s u b f r a c t i o n a t e d  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 specific  activities  (CPM/platelet)  were  calculated  f o r each  p o i n t (CCDF) o f t h e d i s t r i b u t i o n . 2.11.4 CCD o f I n V i v o C o h o r t L a b e l e d P l a t e l e t s 35 Sulfuric England 10~  6  acid,  Nuclear,  moles/Ci).  H  SO^,  2  Boston,  MA  L o t 094394  in  water  (NEX-042,  was  obtained  15 mCi/ml,  was d i l u t e d  43  from  Ci/mg,  New  46.5 x  t o 7.5 ml f o r 2 mCi/ml; l o t  239419 was d i l u t e d t o t o 6.0 ml f o r 2.5 mCi/ml; and l o t 307321 was n o t diluted. cohort rabbits.  These labels  radioactive and were  compounds  injected  were  directly  used  as i n v i v o  (or s l i g h t l y  platelet  diluted)  into  P l a t e l e t s , a t v a r i o u s t i m e s p o s t i n j e c t i o n , p . i . , were d e n s i t y  - 55 separated  and s u b j e c t e d t o CCD. D e t a i l s a r e p r o v i d e d  as E x p e r i m e n t a l i n  s e c t i o n s 3.8 t h r o u g h 3.12. 2.12.1 P a r t i c l e An  Counting  Electrozone Celloscope,  (Model 112 CLTH/RWP, P a r t i c l e Data I n c . ,  Elmhurst,  I L ) was used.  Initially  "Platelet  Mode",  provided  threshold  (T) s e t t i n g s  Later,  which  appropriate  experimentally  platelets preset  specifically  using  current  designed  s e t t i n g s f o r counting  determined  had been counted (C), gain  f o r human  on t h e (G) and  platelets.  i n t h e " L i n / D e l t a Mode" were  standard  particles,  and  rabbit  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 s e t by 1/2 the  operator  and  C 1, G 68, T 15-90, f o r a 48 um d i a m e t e r o r i f i c e ) .  Mode  (C 2 ( 2 )  displayed  those  , G 96, T 19-90, f o r a 76 um d i a m e t e r  counts  o f greater  within  size.  a  specified  The l i n e a r i t y  size  The L i n / D e l t a  range,  and c o i n c i d e n c e  orifice  separate  from  o f counts  with  t h e s e s e t t i n g s , were s t u d i e d (Appendix T a b l e 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 (J.T.  Baker  negligible counts. at  Diagnostics,  Bethlehem,  with azide-free s a l i n e " D i l u i d " PA.) such  that  and e l e c t r o n i c c o u n t s c o r r e l a t e d w i t h  coincidence  visual  was  hemocytometer  A s t o c k p l a t e l e t s o l u t i o n was added t o 9.84 ml o f D i l u i d s a l i n e  various  dilutions  which  were  calculated  from  the o r i g i n a l  stock  s o l u t i o n . These samples were c o u n t e d and compared b o t h t o hemocytometer 8 counts using a phase contrast microscope (1.05 _+ 0.1 x 10 o  p l a t e l e t s / m l ) and t o 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 t h e p o r t i o n o f c o u n t s w i t h i n t h e range o f l i n e a r c o u n t i n g . C e l l o s c o p e c o u n t s were performed u s i n g a 76 um o r i f i c e w i t h a 1 /? 255  u l volumetric,  10-90. W i t h t h e s e  on s e t t i n g s , C 2 ( 2 )  counting  , G 96, L i n / D e l t a Mode, T  c o n d i t i o n s , t h e c o u n t s were l i n e a r  up t o a  - 56 c o u n t i n g r a t e o f AO,000-50,000 c o u n t s . The P e r c e n t - C o u n t l e s s Table  A.9)  (N)(D)' ; 5  was  calculated  using  where, N = observed  the equation,%  counts/ml;  under t h e above c o n d i t i o n s , 2.2A x I O 2.12.2 P a r t i c l e Volume  countless  D = orifice  =  diameter  0.13  i n mm;  = % C/(N/255 u l ) .  Sizing  calibration  Celloscope  - 4  (Appendix  was  (Appendix  carried  out  Table  with  A.10)  standard  f o r the Electrozone polystyrene  particles  ( P a r t i c l e I n f o r m a t i o n S e r v i c e , G r a n t s P a s s , OR), and f i x e d b l o o d c e l l s . Standard  spherical  particles  mode a t T = 3 2 . 5 ) , 3.05 um platelets (off  (platelet  mode  s c a l e ) were s i z e d  with  fixed  d i a m e t e r s , 2.02 um  (particle  ( p a r t i c l e mode a t T = 6 5 . 0 ) , f i x e d  a t T = 25.0),  and f i x e d  rabbit  human e r y t h r o c y t e s  w i t h t h e same machine s e t t i n g s but by v a r y i n g  t h e 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 i n c r e m e n t s . R e l a t i v e volume distributions  were  o b t a i n e d by manually  s e q u e n t i a l c o u n t s , i n o r d e r t o accumulate  altering  the T settings f o r  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 . S i n c e t h e 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 function  of  arbitrarily  both  shape  and e l e c t r o n i c  and  volume,  i n terms o f p e r c e n t t h r e s h o l d  sizing  the  of this  results  or percent  type was a  were  expressed  window  settings,  as w e l l as by t h e apparent c a l c u l a t e d volumes. The e x p e c t e d volumes and diameters a t the various threshold 3 equation, =  v = (pi/6) d  diameter;  K  =  (T) s e t t i n g s were c a l c u l a t e d by t h e 3  = 1.25 um  orifice  constant  = K  ( T / I G ) ; where v = volume; d  ( f o r 76  um  orifice,  A2); T  =  threshold; I = current; G = gain. Similarly, stabilized Dade  f i x e d human p l a t e l e t  human p l a t e l e t  Division  standards  (Dade P l a t e l e t  Controls,  s u s p e n s i o n s i n l e s s than 0.1% sodium  o f American  Hospital  Supply  azide,  C o r p o r a t i o n , Miami, F l . )  - 57 were  used  to calibrate  the  instrument  (Electrozone  Celloscope)  for  c o u n t i n g , u s i n g a 48 um d i a m e t e r 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 a t 15, 25, and  v a r i e d C 1/2, 1, and 2, A l l experimental Tyrode's density  washed  55, f o r s e t t i n g s :  c o u n t s and s i z i n g s were p e r f o r m e d , i m m e d i a t e l y ,  blood  saline  G 68, and  respectively.  cells  and  gradient centrifugation  azide-free  constant  (Diluid,  elements washed  and/or CCD,  J.T.  Baker  once a f t e r  on  Stractan  resuspended and d i l u t e d i n  Diagnostics,  Bethlehem,  P a r t i c l e c o n c e n t r a t i o n s were a d j u s t e d such t h a t c o i n c i d e n c e  PA).  c o u n t s were  n e g l i g i b l e and e l e c t r o n i c and hemocytometer c o u n t s c o i n c i d e d . Platelets  in  solution  were  "sized"  using  the  Electrozone  C e l l o s c o p e , w i t h 76 um d i a m e t e r o r i f i c e by o b t a i n i n g c o u n t s a t v a r i o u s threshold plotted versus  settings as  a  (T:  percentage  the T settings  platelets  5-10,  were  the p l a t e l e t  a t which they  i n the appropriate  A.10); d e n s i t y s e p a r a t e d Counter,  of  10-15, 15-20, e t c . ) .  Channelyzer  and  X,Y  size platelets of different  size  fractions  sedimentation density  of  i n saline  separated  sized  with  (data  were  fixed  an  provided  Vancouver BC).  calculated  (T  setting),  Both' s t a n d a r d s  range  (Appendix  and Table  (Coulter  fractions  at u n i t and  Electronics  Inc.,  and 100 u l v o l u m e t r i c , was a l s o used a f t e r CCD  which  gravity. platelets  Diagnostics by  were o b t a i n e d .  recorder  platelets  platelets  Ortho  channel  were  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  H i a l e a h , F L ) w i t h a 70 um o r i f i c e to  peak  These c o u n t s  Clinical  laser  were  ( n o t shown) and t o  separated  In c e r t a i n from  light  Laboratory  by  (size)  i n s t a n c e s , e.g.,  cancer  scattering Medicine  patients cell at  were  counter  H.S.C.H.,  - 58 2.13  Radioactivity For  beta  scintillation  Scintillation Scientific  Counting  counter,  Beckman  Instruments  Division,  L i q u i d S c i n t i l l a t i o n Counter, Division,  N.V.  were used. gamma  Philips'  Liquid  counting  gamma c o u n t e r ,  Nuclear  Chicago  scintillation  Beckman  Inc., Fullerton,  Irvine,  and  CA;  a  Philips  LKB W a l l a c  Eindhoven,  Oy,  CompuGamma  Turku,  single  PW4700  Netherlands, MA. F o r  counter  F i n l a n d was  channel  CA,  Equipment  NEN-968, NEN, B o s t o n ,  controlled  4216,  Liquid  Instruments  was A t o m l i g h t  Model  LS-233  Philips Scientific & Industrial  microcomputer  1282-002  a  Gloeilampenfabrieken,  fluor  a  counting  model  used.  automatic  A  gamma  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  Graphical Presentation of Results A  "Specific  calculator  Activity"  and  9872A  program  plotter  r u n on  (Hewlett  a  Hewlett  Packard,  Packard  Vancouver,  B.C.),  developed  by o u r l a b o r a t o r y (K.A. Sharp and J . Janzen) was used,  expressed  results  (solid  line,  line,  right  9815A  which  ( u n l e s s o t h e r w i s e s t a t e d ) i n terms o f : p l a t e l e t s / C C D C  left  ordinate);  ordinate);  ((cpm/platelet)/CCDC),  radioactivity  and  specific  i . e . , the r a t i o  dpm  o r cpm/CCDC  activity  (dotted  (dashed  (S.A./CCDC)  line,  plotted  or  either  w i t h t h e mean S.A. a t one h a l f t h e maximum i n s i d e t h e l e f t o r d i n a t e , o r by  normalizing  labeled 10 ). 6  error  with Data  t h e peak  t h e power were  a t t h e maximum).  The  and t h e s i g n o f t h e exponent  accepted  (CE, e x p r e s s e d  S.A.  by  placing  a  limit  on  ordinates  were  ( e g . , E+06 f o r  combined  counting  as a p e r c e n t ) o f 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 o r CCDF, u s i n g t h e b e s t  estimate of the standard  deviation,  - 59 -  i.e (PL)l/2 +  CE  (PB)l/2  (CPM) +  PL PB CPM CB  where  2.15  = = = =  +  (CB) / 1  2  (CPM - CB)  (PL - PB)  100  1 / 2  p l a t e l e t count p l a t e l e t count background p l a t e l e t cpm cpm background  Centrifugation For  platelet isolation,  done u s i n g  IEC Model SBV  B o s t o n , MA. done u s i n g  High speed various  centrifugations  temperature  were  and IEC Model 2K, I n t e r n a t i o n a l Equipment  Co.,  rotors  (HB-4, r a d i u s  No. 363 c u s h i o n s ) i n t h e S o r v a l l RC-5  Newtown, CT.  Du  Pont  room  c e n t r i f u g a t i o n s a t c o n t r o l l e d t e m p e r a t u r e s were 5.75  s w i n g i n g bucket r o t o r w i t h 50 ml c a p a c i t y  Centrifuge,  at  Co.,  Instrument  i n . , 14.61  cm,  a 4-place  each, o r 15 ml c a p a c i t y  A u t o m a t i c Superspeed Products,  with  Refrigerated  Biomedical  Division,  - 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 o f P l a t e l e t CCD Glutaraldehyde 22-23°C, salts,  rabbit  i n phase  systems  designations  5/4:  differences, and  fixed  platelets  o f 5% A  dextran,  through  G  were 4%  partitioned  PEG,  (Chapter  with  at  buffer  2.9.3).  and  Potential  measured between t h e upper and lower phases ( T a b l e 2.9.3.2  F i g u r e 3,1.1.1) d e c r e a s e d , as t h e p a r t i t i o n s o f t h e s e f i x e d  rabbit  p l a t e l e t s ( F i g u r e 3.1.1.2; T a b l e 3.1.1). 51 R a b b i t , human, and monkey p l a t e l e t s at  a concentration  washed in  and  these  22-23°C, rabbit  also  phase  o f 0.2 mCi/ml  partitioned systems  platelets  had  a  3.1.1).  phase  higher  with  Cr i n PRP  f o r 1 hour a t 37°C. P l a t e l e t s  a t 22-23°C  (Table  i n charge-sensitive  were l a b e l e d  and  Single  systems  partition  4-5°C  (CCD  tube  conditions)  partitions,  (5/4:A) showed than  unfixed,  5  that  platelet  partitions,  a t 22-23°C, were v e r y  at  fixed  "*"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 . human  were  similar  R a b b i t and (with  human  o n l y s l i g h t l y h i g h e r t h a n r a b b i t ) , w h i l e t h a t o f monkey was much l o w e r ; K = 31.8 + 8.4%, 33.8 + 6.3%, and 21.1 + 3.4%, f o r r a b b i t , monkey, r e s p e c t i v e l y . human  platelets  monkey  platelets  At 4-5°C, t h e (5/4:A) p a r t i t i o n s  were h i g h e r was  lower.  than The  a t 22-23°C,  while  low t e m p e r a t u r e  p l a t e l e t s was a g a i n l o w e r t h a n t h a t o f both r a b b i t at  this  temperature  that of the rabbit;  the p a r t i t i o n  o f human  human, and  of rabbit  and  the p a r t i t i o n of  partition  o f monkey  and human, however,  platelets  was lower  than  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,  respectively.  - 61 F i g u r e 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 d e x t r a n P h a s e s : (means and S.Dev. i n T a b l e 2.9.3.2); i n c r e m e n t s o f 0.70 mV P.D. ( 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 d e c r e a s i n g t o 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 t o 0.15 M ( a b s c i s s a ) ; phases 5/4:A t h r o u g h G.  1. 40- 1.  SB--  TWO-PHASE P O T E N T I A L D I F F . IN 5 % D / 4 % PEG WITH V A R Y I N G N A C L AND N A P  1. 12- •  •  0_ Vj,  a. ev a-saa. 28 a. 14 B.7B+  0.42  108.0-  Figure 3.1.1.2 Fixed Rabbit Platelet Two-Polymer Phase System Partitions: (means and S.Dev. i n T a b l e 3.1.1); i n c r e m e n t s o f 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 d e c r e a s i n g t o 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 t o 0.15 M ( a b s c i s s a ) ; phases 5/4:A t h r o u g h G.  - 62 T a b l e 3.1.1 Summary o f S i n g l e - t u b e P a r t i t i o n o f P l a t e l e t s 5/4:  A  B  F i x e d R a b b i t P l a t e l e t s a t 25°C Mean 85.9 + S.Dev. 14.9 n  74.9 16.7  28.4 7.7 3  12.3 10.7 3  3.4 2.5 3  1.4 0.5 3  21.75 4.35 4  13.5 0.58 4  15.75 6.65 4  9.0 1.15 4  9.75 0.5 4  24.38 3.07  18.0 2.83 4  18.13 6.10 8  17.5 5.42 8  14.4 5.31 5  15.2 2.9 10  12.8 3.3 10  10.1 2.5 10  11.0 2.45 8  9.5 1.72 10  30.88 3.64 8  19.13 2.42 8  11.0 1.26 6  4.33 2.94 6  3.57 2.76 7  32.0 30.25 0.82(4) 2.06 4 4 4  29.67 3.21 3  18.0 0.0 2  13.3 0.58 3  19.75 0.5 4  5.75 2.55 8  4.63 2.2 8  3  64.5 15.3 3  3  R a b b i t P l a t e l e t s a t 25°C Mean 31.75 + S.Dev. 8.42 n 4  31.0 2.58 4  Human P l a t e l e t s a t 25°C Mean 33.75 + S.Dev. 6.27 n  30.75 2.49 8  8  8  Monkey P l a t e l e t s a t 25°C Mean 21.13 + S.Dev. 3.44 n 8  17.6 4.6 10  R a b b i t P l a t e l e t s a t 5°C Mean 53.75 + S.Dev. 3.54 n  42.38 3.38 8  8  Human P l a t e l e t s a t 5°C Mean 43.5 + S.Dev. 5.26 n  Monkey P l a t e l e t s a t 5°C Mean 11.38 + S.Dev. 1.6 n  9.75 1.49 8  5.0 0.53 8  8  4.5 1.05 6  5.88 1.55 8  - 63 Suitable T500,  4%  phase systems f o r p l a t e l e t p a r t i t i o n work c o n t a i n e d : 5%  PEG  8,000,  isotonic  sodium  Contrary  t o the  charge  on  and  different  chloride fact  that  macromolecules  and  sodium  ions  ( e . g . , phosphates)  such  t h e s e phase systems e x h i b i t phosphate  i s rejected  by  resulting i n a difference 1984;  Brooks  the  PEG  1976  varied  phosphate/chloride  phase  ranging  to  3.1.1.1). rabbit,  0.1  The  from  mV  for a  partitions  monkey, and highest  1984).  ratio, respect  mV,  for a  1.0  system  of  o f washed, f i x e d  by  3.1.1.1  and  and  fraction  decreasing  Table  1969),  phase,  of  polymer  potential  of  chloride  rabbit  total  3.1.1).  high  specific  content  (Figure  and  native  i n t h e s e systems,  platelets  in  the  system  potential  with  the  potential  difference  The  high  phosphate  containing,  charge-associated monkey.  The  whereas  that  partitions increasing,  differences  partitions  partitions  of  were rabbit  o f monkey p l a t e l e t s  changed and  differently  monkey  in  and was  with  decreasing  platelet  found  in  (Figure charge  the  rabbit,  different.  temperature;  with  decreasing  studies.  membrane  human p l a t e l e t s very  and  phosphate  s e n s i t i v e phase system was most o f t e n used t h r o u g h o u t t h e s e CCD Species  top,  phase,  platelets  varied  systems  i n the  bottom  system  et a l . ,  differences  a p p e a r i n g i n t o p phase) i n t h a t system w i t h the h i g h e s t phase difference,  electric  D-rich  potential  t o the  likewise  7.2).  Sterling,  buffered  high  of  due t o i o n r e j e c t i o n ; than  the  with  human p l a t e l e t s  (largest  (pH  i n d u c e an  and  The  these i s o t o n i c  positive  i n magnitude  content,  being  being  within  and  phases  PEG-rich  more  ratios)  buffers  (Sieh  differences  phase  (or  i n phase i o n c o n c e n t r a t i o n (Bamberger  between t h e two the  phosphate  as g l y c o g e n  potential  e t a l . , 1971,  with  concentrations  D  surface  human  and  were  similar,  These  platelet  rabbit  and  temperature  human from  - 64 25°C  to  5°C,  but  the  relative  d i f f e r e n c e s , r a b b i t being  human, and h i g h e r than monkey, were m a i n t a i n e d Although humans,  a l l three  were  used  distributions r a b b i t was property major  for  comparison  platelets  of  analyses  by  system  CCD, for  in  was  vivo  the  rabbit  relative  instances  later ageing  feasibility  platelets  to  age-dependent  used  d i s t r i b u t i o n was (low,  over  the  72  distribution  obtained  property the  surface  most  of  the these  i t was in  hours o f  via  reported,  subjected  assuming  random  high see  and  to  membrane  surface  the  experiment.  over  in  i n these  120  Figure  plate.  studies  the  original  cell  factors  a broad  solid  (Figure  t r a n s f e r s each,  loaded  expected.  from t h e f i r s t  distribution  3.1.2.2).  and then  been t a k e n  platelet  of  were  s u r f a c e p r o p e r t i e s , and  amount o f d i s t r i b u t i o n o v e r l a p i s as  dotted,  the  fixation.  These samples r e - p a r t i t i o n e d t o had  It  certain  CCDC, and  3.1.2.1)  o f 40  use  f i x a t i o n does not  However  partition;  t o a second CCD  from where they  the b a s i s of i n t r i n s i c primarily  and  respectively,  same r e l a t i v e a r e a s expected,  CCD.  necessary  ( F i g u r e 3.1.2.1). Samples from t h r e e areas  on t h r e e s e c t o r s o f the CCD  of  and  t h e s i s as  p l a t e l e t p a r t i t i o n s i n c r e a s e d upon p l a t e l e t  intermediate,  lines,  simultaneously  as  in this  changes  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  dashed  surface  s t u d i e s . For  study  prevent  a l t e r n a t e f i n d i n g s are  charge-associated  CCD  monkeys,  d e n s i t i e s ( S e c t i o n 3.2),  been shown w i t h e r y t h r o c y t e s t h a t g l u t a r a l d e h y d e  obliterate  the  rabbits,  o f p l a t e l e t membrane s u r 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  p r o p e r t i e s which might o c c u r has  of  different  and  used, however f o r t h i s CCD fixed  systems,  to  temperature.  used here t o demonstrate the f e a s i b i l i t y o f p l a t e l e t  model  analyses  of  experimental  at e i t h e r  similar  not  However,  had  been  the CCD, on  the r e s u l t a n t the  certain  - 65 F i g u r e 3.1.2.1 CCD o f F i x e d R a b b i t P l a t e l e t s : ( 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 .  p l a t e l e t s % peak /CCDC  F i g u r e 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, i n t e r m e d i a t e , and h i g h p a r t i t i o n , ( d o t t e d , 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 of Density Separated  E f f o r t s o f groups headed by been  d i r e c t e d toward  based  on  platelet  p l a t e l e t s are  Corash and  obtaining  size  and  platelet  density,  l a r g e r , more dense and  have become e f f e t e such s t u d i e s  -  w i t h i n the  (Corash e t  Platelets  by Packham and age  the  distribution  suggestion  active  Mustard have information  being  that  young  than t h o s e p l a t e l e t s  which  c i r c u l a t i o n . In t h e  most c o m p e l l i n g  a l . , 1978), e v i d e n c e i s p r e s e n t e d  that i n  of the  51 Rhesus  monkey,  circulation  more r a p i d l y  platelets, and  Cr-labeled  on  the  above  hypothesis  1981a, 1983; cohort  labeling,  platelets  while  t h a n the  remains  the  from  similar Shafer,  the  least acid  amount  heterogeneous due  as  work  in  of  1982). U s i n g  constant).  acid  with  3 3  S  per-  Platelets  were  with  i n the  rabbit  Oost e t a l . , 1983), which c o u l d  size  and  (Leone e t a l . , 1979; In platelet  order  to  membrane  density  changes r e l a t e d  information  surface  p a r t i t i o n s of p l a t e l e t s  platelet young  old platelets, density  platelet  protein to  and  the  human  of d i f f e r e n t  (van age  interpretations  t o human p l a t e l e t  ageing  Boneu e t a l . , 1982).  concerning  properties,  be  i n a decrease i n  o c c u r as a f u n c t i o n o f c i r c u l a t i o n  Mezzano e t a l . , 1981;  obtain  et a l . ,  found  as w e l l . However, o t h e r groups have developed d i f f e r e n t of p l a t e l e t  the  and  also  ( C i e s l a r e t a l ^ . , 1979)  (Rand  age  of  t o degree o f t h r o m b o s i s , e v i d e n c e d  for  contained  contained  gram  time i n v i v o ,  i n vivo  subpopulation  the  density  Support  rabbits  p l a t e l e t decreasing  sialic  from  a whole; h i g h  population.  dense s u b p o p u l a t i o n per  disappear  i n density  t h e most dense p l a t e l e t  roughly  their density  population  mean f o r the  C o r a s h and  t h e amount o f s i a l i c (although  platelets  hand, d e c r e a s e  comes  and  density  than the  other  s u r v i v e longer  low  and  the  relative  age-related  heterogeneity  of  charge-sensitive  characteristics,  this  - 67 series  of  between  CCD  experiments  platelet  age  and  took  advantage  density.  The  of  reported  methods  of  correlations  Stractan  g r a d i e n t p r e p a r a t i o n o f C i e s l a r e t a l . (1979), m o d i f i e d  density  from t h e method  f o r e r y t h r o c y t e s by Corash e t a l . (1974) and f o r p l a t e l e t s by Corash e t al.,  (1977  populations  and  1978)  were  from r a b b i t s ,  modified  to  subfractionate  monkeys and humans, i n t o  three  subfractions  c o n t a i n i n g 5-10% o f t h e p l a t e l e t s  o f low d e n s i t y  p l a t e l e t s of intermediate  ( I D P ) , and 5-10% o f t h e h i g h  platelets  (HDP).  gradient  centrifugation  population 3.2.1.1  The d e n s i t i e s  density  and  density  of the i n d i v i d u a l  were  determined  distributions  3.2.1.2).  Several  analyses platelet  (see a l s o ,  modified  80-90% o f t h e  Stractan  empirically  by  i n concentration  from  density  1.075-1.084 ( g / m l ) ,  t h e 10% s t e p , s e p a r a t i n g them from  ranges,  namely  1.042-1.066,  subpopulations  1.066-1.075,  to achieve  the desired f r a c t i o n a t i o n .  t o study  platelet  from a n i m a l  also  related  populations  t o animal  to variations  solutions,  This density gradient from r a b b i t s ,  i n the gradient allow  and a t h i g h e r  system  was  inconsistency  environment.  f o r the freezing concentration  t h e s e polymers e x e r t g r e a t e r o s m o t i c p r e s s u r e s .  experiments  humans and monkeys.  v a r i a t i o n , density separation  p o i n t osmometry does not c o r r e c t l y arabinogalactan  and  respectively.  As d e s c r i b e d , minor a d j u s t m e n t s were made t o i n d i v i d u a l  is  10% t o  van Oost e t a l . , 1982). These d a t a were used t o d e s i g n  a l l p l a t e l e t s penetrated  various  Apart  (Figure  were c e n t r i f u g e d  plasma p r o t e i n s , w h i l e t h e o t h e r s t e p s i s o l a t e d p l a t e l e t  used  platelet  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 .  Generally,  of  density  steps f o r  f o r human p l a t e l e t s preparations  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 r a n g i n g 20%  (LDP),  platelet  Freezing  k i n e t i c s of  (i.e.,  density)  - 68 F i g u r e 3.2.1.1 P e r c e n t Human P l a t e l e t s Remaining on S t r a c t a n S t e p s : t h e number o f p l a t e l e t s r e c o v e r e d on d e n s i t y s t e p s (from 12.5% t o 2 0 % S t r a c t a n , a b s c i s s a ) , e x p r e s s e d as a percentage o f t h e t o t a l p l a t e l e t s added t o t h e tube ( i n c r e m e n t s o f 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 o f p l a t e l e t s from t h r e e a n i m a l s a r e shown w i t h t h e i r s t a n d a r d deviations. PERCENT  HUMAN  P L A T E L E T S REMAINING ON  STRACTAN-STEP  in  yUJ  _l  UJ  t< _J  0_  UJ L) OH hi a.  DENSITY  10.  0" 0-  ia.  a- •  20.  CO r-  LU _J  UJ  f-  DISTRIBUTION  HUMAN  P L A T E L E T S ON  STRACTAN-STEP  14.0  <  iz. e- •  Q_  10. B •  _J  1— 2 ' LU U  8.0  CH  B. Z- •  Q_  4.  UJ  0'  2.0  0.01 ti  Figure 3.2.1.2 Human P l a t e l e t Density Distribution on S t r a c t a n S o l u t i o n s o f Various Concentrations: p l a t e l e t s % t o t a l (increments o f 1%, o r d i n a t e ) ; % S t r a c t a n (from 12% t o 20%, a b s c i s s a ) ; from above.  - 69 The 20.0  total  + 15.6%  rabbit LDP,  platelet  62.5  g r a d i e n t s o f 10%,  populations  + 17.6%  IDP,  HDP,  Stractan;  6.7  IDP,  u s i n g d e n s i t y g r a d i e n t s o f 10%,  15.5  + 0.5%,  18 %, and  2%  of  HDP,  the  platelets  +_ 3.0%  rhesus  sialic  40  monkey,  transfer  decreasing  have  on  the  and  76.7  human  IDP,  CCD  HDP)  LDP,  15%,  platelets  procedures,  been  reported  basis  in  to  age,  20%  Monkey  18%,  IDP,  and  20%  individually  intermediate  compare  d e n s i t y and  older  17.3  + 8.4%  and  and  subjected  their  to  relative  membrane s u r f a c e  instances  i t would seem t h a t CCD  of  20%.  were  (low,  certain  and  t h e 20% S t r a c t a n .  were h a r v e s t e d  Because p l a t e l e t  w i t h i n v i v o age,  distribute  + 8.8%  three density fractions  distributions.  acid  the  of the p l a t e l e t s penetrated  h i g h d e n s i t y p l a t e l e t s ; LDP, simultaneous  14.7  penetrated  u s i n g d e n s i t y g r a d i e n t s o f 10%,  i n each c a s e ,  partition  using density  + 17.3%  S t r a c t a n ; o n l y 4.0  analyzed;  HDP,  62.6  + 3.0%  Rabbit,  + 9.7%  into  LDP,  about  + 12.9%  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 and  18.0  subfractionated  1 5 . 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 + 10.5%  and  were  to  should  platelets  change,  be  having  able a  to  lower  p a r t i t i o n t h a n the b u l k o f the p l a t e l e t p o p u l a t i o n . T h i s appeared t o be t h e c a s e f o r the 3.2.2.1 was  and  partitions  3.2.2.2).  different;  fractions  CCD  i n the  concentration  among  p a r t i t i o n behavior  5%  D,  them  from 4%  and  The  e x h i b i t e d very  differences  of r a b b i t  4%  PEG  phase  human p l a t e l e t s of  the  system,  monkey a l l three  low  partition coefficients,  to  be  t o 3.3%  distinguished.  provided  too  low  Decreasing  only minimal r e s o l u t i o n  t h e i n t e r m e d i a t e d e n s i t y p o p u l a t i o n and the LDP  and  HDP.  (Figure platelets density for the  any PEG  between  - 70 F i g u r e 3.2.2.1 CCD o f D e n s i t y S e p a r a t e d R a b b i t 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% w i t h i n c r e m e n t s 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 o f D e n s i t y S e p a r a t e d 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% w i t h i n c r e m e n t s o f 5%, o r d i n a t e ) ; CCDC 1-40 ( a b s c i s s a ) .  - 71 In  order  density  t o compare a l l o f t h e p a r t i t i o n  separated p l a t e l e t  subpopulations  distributions  of  Stractan  each to  isolated  of  the nearest  (LDP,  subpopulation recovered),  the pertinent  IDP,  0.1%),  and  (expressed  HDP),  as  countercurrent  experiments, types  a  of  numbers  percentage  distribution  gradients  platelet of of  were t a b u l a t e d :  platelets the  cavity  the  n o r m a l i z e d by e x p r e s s i n g t h e c a v i t y  number  of transfers  containing CCDC  t h e peak number  (Mode),  width  (Appendix  middle  at half  distribution  Tables  of platelets  o f t h e CCD  or  of  total  each  platelet  cavities  (CCDC)  parameters  number as a f r a c t i o n o f  A.11, A.12 and A.13): (Peak),  distribution  t h e peak h e i g h t ) , and spread  (the f u l l  used ( i n %  subpopulations  l o a d e d , and number o f CCDC t r a n s f e r s ( n ) . These d i s t r i b u t i o n were t h e n  the  from each a n i m a l system, t h e  g r 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 dates  for  the estimated  CCDC modal  (Mid, mid-point  of the  (Spd) o r b r e a d t h  of the  w i d t h , i n CCDC, a t one h a l f  the height o f the 1/2  peak n o r m a l i z e d  by t h e square  r o o t o f t h e number o f t r a n s f e r s ,  The n o r m a l i z e d d i s t r i b u t i o n parameters averaged, c a l c u l a t i n g Partition terms  ratios  similar  transfers) calculated  from  to  f o r each s u b p o p u l a t i o n were t h e n  (K) f o r t h e CCD parameters  that  which  of  an  R^,,  related  the d i s t r i b u t i o n  to  were c a l c u l a t e d ( i n t h e number  coefficients  of  CCD  (G) c o u l d  be  u s i n g t h e e q u a t i o n s : K = r / n , and G = r / ( n - r ) , where r I s  number o f t r a n s f e r s  into  ).  t h e mean and s t a n d a r d d e v i a t i o n ( T a b l e 3.2.1).  t h e CCD d i s t r i b u t i o n parameter  Ideally,  n  t o which  (peak, mode, m i d d l e ) the platelet  and n i s t h e t o t a l  subpopulation i s subjected.  K s h o u l d e q u a l t h e f r a c t i o n o f t h e p o p u l a t i o n which  t h e t o p phase i n a s i n g l e  distribution  partitions  s t e p and G would be t h e  r a t i o o f c e l l numbers i n t h e two phases i n t h a t s t e p .  - 72 T a b l e 3.2.1 Summary o f R a b b i t , Human, and Monkey, LDP, IDP, and HDP Peak  Mode  Mid  CCDC/n  CCDC/n  CCDC/n  Spd  CCDC/Cn) 1 / 2  R a b b i t (12) 0.487 + 0.098 LDP IDP 0.578 + 0.10 HDP 0.564 + 0.10  0.498 + 0.096 0.567 + 0.092 0.537 + 0.098  0.479 + 0.095 0.552 + 0.081 0.518 + 0.089  1.727 + 0.541 1.752 + 0.388 1.902 + 0.842  Human (11) LDP 0.475 + 0.136 IDP 0.526 + 0.092 HDP 0.549 + 0.092  0.471 + 0.123 0.512 + 0.073 0.572 + 0.094  0.463 + 0.112 0.510 + 0.072 0.550 + 0.082  2.186 1.789 2.412  Monkey (4) LDP 0.188 + 0.05 IDP 0.263 + 0.129 HDP 0.238 + 0.096  0.184 + 0.086 0.244 + 0.108 0.234 + 0.093  0.180 + 0.073 0.239 + 0.113 0.228 + 0.094  1.235 + 0.317 1.376 + 0.380 1.831 + 0.199  In  + 0.548 + 0.736 + 0.940  c a s e s where more than one CCDC was l o a d e d , t h e K v a l u e s were  c a l c u l a t e d assuming (e.g.,  the o r i g i n  a s t h e m i d d l e o f t h e l o a d m i x t u r e CCDC  f o r CCDC 1-3, CCDC 2 was t h e o r i g i n ,  and t h u s 2 was s u b t r a c t e d  from t h e peak, mode, and m i d d l e CCDC, p r i o r t o c a l c u l a t i o n ; f o r CCDC 1, t h e o r i g i n was CCDC 1, and t h u s 1 was s u b t r a c t e d from CCD p a r a m e t e r s ) . The  sample  X-^/(X +X ), 1  2  sample 1960;  partition  where  X  ratios  refers  were e s t i m a t e s o f t h e i r  t o the equilibrium  concentrations  ( i . e . , t h e numbers o f p l a t e l e t s ) i n phases Snyder,  1968).  K, e q u a l t o  1 and 2  of a  (Albertsson,  As t h e n ( i n t h i s case t h e number o f t r a n s f e r s )  i n c r e a s e d , t h e d i s t r i b u t i o n s approached normalcy and c o u l d be d e s c r i b e d by  the c o e f f i c i e n t s  of  the binomial  expansion  (x  +  y ) . Thus, n  1/2 distribution  spreads  were  also  normalized with  respect to n  , as  w e l l a s t o n. With i n c r e a s i n g n, t h e bandwidth ( o r s p r e a d ) narrowed. The  G value expresses the d i f f e r e n t i a l  f o r t h e two phase 1,  expresses  system p o l y m e r s  equal  affinity  affinity  o f the population  (Appendix T a b l e 1 4 ) . A v a l u e o f G =  of a c e l l  population  f o r either  o f the  - 73 polymer  phases,  interface. tendency less  i . e . , the  Values  t o be  than  g r e a t e r than  transfered with  one  stationary  upper  indicate  with  the  an  lower  phase  and  indicate  the  upper P E G - r i c h  affinity D-rich  zero  G=n/(n-n);  and  f o r , and phase.  G=0/(n-0), t o t h e  these  extreme  an  lower  one  v a l u e s c o u l d range from z e r o t o i n f i n i t y ; equals  the  affinity  the  Thus,  plus  the  f o r , and  the  phase w h i l e  values  tendency  remain  to  theoretically  the  G  from t h e s i t u a t i o n i n which r  situation  situations  phase  i n which  are  seldom  r equals  useful  n  and  for  cell  s e p a r a t i o n or a n a l y s i s . The  partition  or  G value  to  the  of K =  r/n  parameter w i t h which t o c h a r a c t e r i z e a CCD,  as  surface p r o p e r t i e s of the c e l l was  t h e more c o n v e n i e n t  it  permited  the  distribution,  averaging  i t  is  the  i s i n part exponentially related p o p u l a t i o n . Although  of  data  value  of  and G  which  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 By  comparison  after  the  these  to  theoretical  subpopulations  the  were  visualization  is  directly  of  related  f o r uniform  distribution  indicative  of  particles for a l l  membrane  surface  The  relative  s i m i l a r i t i e s o f 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 b e h a v i o r , and  obtained  from  by  that  CCD,  of  monkey  were a g a i n  which compared c l o s e l y  Packard  t  and  ( d i f f e r e n c e of  S t a t i s t i c s V o l . 1 P a r t No.  comparing LDP, A. 16  tests  IDP,  A.17).  and HDP  evident  with those  s t e p p a r t i t i o n (5/4:A a t 5°C)'of unseparated Student  means/S.E.),  t  obtained  using  09815-15000 Rev.  these  i n the  their  partitions by  single  p l a t e l e t s ( S e c t i o n 3.1).  from t h e a l l a n i m a l s  A p p l i c a t i o n s of  to  spreads  p r o p e r t y h e t e r o g e n e i t y w i t h i n each p l a t e l e t s u b p o p u l a t i o n .  difference  the  behavior.  distributions  same number o f t r a n s f e r s ,  density  rapid  t h e use  test  C,  the  Hewlett  were performed  (Appendix  Tables  statistics  A.15,  indicated  - 74 certain  correlations  platelet level  density.  The  significant  of uncertainty,  were o f l o w e r than  between c h a r g e - a s s o c i a t e d s u r f a c e p r o p e r t i e s  IDP;  were as f o l l o w s :  partition  and  of  differences  (based  lower  at the p =  LDP p l a t e l e t s  on average  partition  found,  CCD  (based  from  CCD  0.05  the r a b b i t  peak, mode and  on  and  peak)  middle)  than  HDP  p l a t e l e t s , which were l o w e r t h a n IDP (based on CCD mode). LDP p l a t e l e t s from t h e monkey were o f l o w e r p a r t i t i o n than IDP, based mode and m i d d l e . LDP p l a t e l e t s (based  on t h e average  CCD  (based  on mode and m i d d l e )  on average  from t h e human were o f l o w e r  middle) than  than HDP  IDP, were  platelets,  partition  o f lower  which  partition  were o f h i g h e r  p a r t i t i o n than IDP (based on CCD mode). I n t h e human t h e average of  t h e CCD d i s t r i b u t i o n  f o r HDP  A  nonparametric  applied.  statistical  method  and Cochran,  1974) t h e d a t a  that  than IDP. of  data  analysis  U s i n g t h e W i l c o x o n Matched P a i r s D i f f e r e n c e S i g n e d  (Snedecor  spread  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  t h i s s u b p o p u l a t i o n s was more heterogeneous  CCD  f o r some o f t h e  was  also  Rank  Test  statistically  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 s u b p o p u l a t i o n s , as judged by t h e t t e s t , (again  with  t h e peak  K  for rabbit  as an example) were  found  t o be  significant. Attempts  t o compare  distributions  of density  were c o m p l i c a t e d by two f a c t o r s :  strictly  positions  not have  (i.e., of  on CCD  plates  should  t h e need f o r i n t e r n a l  separated  distributions been  from  compared  s t a n d a r d s ) ; and unequal  platelets different  this  closely  platelet  numbers  t h e t h r e e d e n s i t y f r a c t i o n s were l o a d e d ( a l t h o u g h i n c e r t a i n samples  when e q u a l q a u n t i t i e s were l o a d e d , d i f f e r e n c e s s t i l l To  better  comparisons  standardize  such  CCD  analyses,  persisted). and  allow  absolute  o f d i s t r i b u t i o n s among a s e r i e s o f e x p e r i m e n t s , an i n t e r n a l  - 75 standard  c o u l d have been i n c l u d e d i n samples, so t h a t the  parameters could peak.  be  r e f e r r e d t o the  Glutaraldehyde-fixed  concentration  2-5  x  although  were  not  they  stable  partition  composition For  human  10 ),  erythrocytes  for  6  used  example,  and  thus  standard  proved  minor  to  at  be  fixed  low  suitable,  cells  differences  exhibit in  phase  c o n d i t i o n s c o u l d be a l l o w e d f o r .  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,  comparison  particle  (included  r o u t i n e l y , because  behavior,  or CCD  l o c a t i o n o f the  distribution  w i t h i n an  individual  CCD  experiment,  an  i . e . , for better internal  standard  t y p e o f r a d i o a c t i v e l a b e l i n g p r o c e d u r e c o u l d a l s o have been used  (e.g.,  as i n t h e l a t e r p a r t o f t h i s w o r k ) ; l a b e l i n g the e n t i r e p o p u l a t i o n one  label  labeled  and  with  comparing a  a  different  to  CCD  CCD  Webber and  The in  analyses  subpopulations  r e c e n t l y presented  with  density  separated  this cell  platelet  s e p a r a t i o n and  Walter  (Walter,  Walter  and  1983; 1975,  Selby,  of  1977,  1966;  1981,  1982,  1965, 1983).  and  by  prior  Brooks and  Webber,  1983). were a n a l y z e d  to assess to provide  the  by  CCD  ability  of  a separation  of  r e l a t e d p r o p e r t i e s , as i s found i n  1982;  (Chapter Walter  1.6),  and  A l b e r t s s o n , 1971;  1968, In  also  platelets  ^C-serotonin,  et a l . ,  i n order  ageing  1978,  Walter  W a l t e r e t a l . , 1962, 1980,  cell  or  B r o o k s , 1981;  a n a l y s i s technique  model 1969,  ^H-  separated  subpopulations  t h e p l a t e l e t p o p u l a t i o n based on age erythroycyte  within i t  This type of s t a n d a r d i z a t i o n f o r  and W a l t e r  c h a r g e s e n s i t i v e phase s y s t e m s ,  the  of density  (Webber and  B r o o k s , 1983;  population  S i m i l a r standardization could  o f the combined s u b p o p u l a t i o n s .  was  1982;  different  subpopulation  isotope.  have been a p p l i e d t o t h e s e labeling  second  with  1971a  this  and  developed  Albertsson, Walter  and  b, 1972a, 1973a,  model, t h e  by  1966; Krob, 1974,  r e t i c u l o c y t e has  a  - 76 lower  partition  surface  than  the  erythrocyte  properties rapidly  mature e r y t h r o c y t e erythrocyte properties  has  population.  change w i t h i n two  the h i g h e s t  distribution. gradually  -  With  change  As  i t matures, i t s  days, so  that the  partition coefficient  of t h e  erythrocyte  cell  such  that  ageing,  the  partition  which i s l o w e r than t h e e r y t h r o c y t e  Even  though  heterogeneities  possible are  non-age-related destruction), density cell  superimposed  sources the  upon  (e.g.,  progressively  platelets  i f no  can  distinction  monkey. Both the  density  platelets  or  of  found  than IDP,  i n those p l a t e l e t s partition  same  (containing associated  to  old) partition  made  platelets  have a  mean  platelets than  and  age.  The  CCD  platelets partition  curves of  of  the  relatively  coefficients.  IDP  other  and  the  CCD  curve  LDP  appear  distribution of  the  HDP  high  subpopulations, partition enriched higher  insignificant), IDP.  lower  width  and  the  population  the  Thus,  surface which  be  but is  LDP  charge  demonstrate  distributions. to  red  human,  w h i l e always o f  than a  these  t o the  associated  subpopulations,  homogeneous The  of  reticulocyte  lower ( u s u a l l y  partition  other random  a p o r t i o n of  HDP,  n e a r l y t h e same mean p a r t i t i o n , but d i f f e r e n t the  the  charge  demonstrate  the  to  ploidy,  young p l a t e l e t  lower  are o f s l i g h t l y  higher  due  distributions  between  surface  f o r a l l animals t e s t e d , r a b b i t ,  i n o l d and  of intermediate  or  heterogeneities  t h e m a j o r i t y o f the p l a t e l e t  t h a n the LDP,  the  density  (enriched  r e s p e c t i v e l y ) were coefficient  low  membrane  i n t e r p r e t e d analogously  is  e r y t h r o y c t e . Trends were s i m i l a r and  platelet  partition be  partition  distribution.  megakaryocyte  countercurrent  separated  model  age-related  entire surface  d e c r e a s e s u n t i l once a g a i n the o l d e r y t h r o y c t e d e m o n s t r a t e s a coeficient  young,  composed of  Both of  different  heterogeneous,  - 77 indicative  perhaps  partition,  and another  higher  than  o f two  platelet  with  a  subpopulations,  partition  one  coefficient  t h a t o f t h e IDP. T h i s i s a l s o suggested  with  a low  similar  t o , or  by t h e spread  of  t h e HDP i n case o f t h e human. These r e s u l t s a r e found i n t h e r a b b i t and human p l a t e l e t systems. These system  demonstrate  i s capable  membrane density  that  of distributing  surface  characteristics  and s i z e .  However, t h e r e  spread, among  results  indicating a l l platelet  CCD  platelets which  charge-sensitive  phase  on t h e b a s i s o f p l a t e l e t  are related  are considerable  heterogeneity) density  in a  i n surface  subpopulations  to platelet  overlaps  property studied,  age,  (and g r e a t  distributions as  are  there  o v e r l a p s i n d e n s i t y and s i z e .  3.3 S i z e A n a l y s i s o f D e n s i t y S e p a r a t e d P l a t e l e t s i z e analyses separated confirmed  platelet  (England  e t a l . , 1982) o f t h e t h r e e d e n s i t y  subpopulations  the c o r r e l a t i o n s  Platelets  (Figures  of increasing size  3.3.1.1 with  and  density  3.3.1.2) found f o r  human (Corash e t a l . , 1977), and r a b b i t p l a t e l e t s (Rand e t a l . , 1981a). Minimal platelets  cellular were  contamination  enumerated  by  d i d not a f f e c t  particle  e l i m i n a t e d p a r t i c l e s o f g r e a t e r than  counting  the results at  the p l a t e l e t s i z e .  because  settings  which  In a d d i t i o n t o  overlap i n s i z e d i s t r i b u t i o n s o f a l l p l a t e l e t density subpopulations all  animals  were l a r g e r A.18,  t e s t e d , HDP than  ( R a ) , human  IDP, which were a l s o l a r g e r  A.19, A.20).  density separated  from r a b b i t  However,  no  subpopulations  Corash e t a l . , 1978.  ( H u ) , and a r a t  than LDP (Appendix  differences i n size  were  of (Rt)  Tables  found f o r  o f monkey p l a t e l e t s , i n agreement w i t h  - 78 F i g u r e 3.3.1.1 S i z e D i s t r i b u t i o n o f D e n s i t y S e p a r a t e d R a b b i t 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%, i n c r e m e n t s o f 5%, o r d i n a t e ) ; % window, t h r e s h o l d (T 0-100, i n c r e m e n t s o f 5, a b s c i s s a ) .  •»  i  i  i  i  i  i  i  i  i  i  F i g u r e 3.3.1.2 S i z e D i s t r i b i t o n o f D e n s i t y S e p a r a t e d 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%, i n c r e m e n t s o f 5%, o r d i n a t e ) ; % window, t h r e s h o l d (T 0-100, i n c r e m e n t s o f 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 o f D e n s i t y S e p a r a t e d P l a t e l e t s Platelet 20%  s u b p o p u l a t i o n s , h a r v e s t e d from g r a d i e n t s o f 15%, 18%, and  Stractan,  solution. sec "''  x  -  were  The  washed  and  electrophoretic  V"*"  x  -  cm)  (HuRBC) s t a n d a r d s  of  were  resuspended mobilities  platelet  measured  in  (EPM;  samples,  (Seaman  Tyrode's  and  or  expressed  and  human  Brooks,  saline  as  um  x  erythroycte  1977).  Readings  were t a k e n a t 40 V and 1.78 mA,  and t i m e ( t ) was c o n v e r t e d t o m o b i l i t y  using  V a l u e s were c o n s i d e r e d v a l i d  5.4785/t (B. C h i u , 1983).  significant for  f i g u r e s and numbers p r e s e n t e d i n t h e appendix were c a r r i e d  computations  partition platelets and HDP  to three  and c o m p a r i s o n .  (Chapter  3.1  and  t o human and r a b b i t  The  3.2)  differences observed  platelets,  i n charge  when  sensitive  comparing  and when comparing  monkey LDP,  IDP  were not apparent i n t h e s e l i m i t e d EPM r e s u l t s (Appendix T a b l e  A.21). There was l i t t l e d i f f e r e n c e i n t h e 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 monkey  platelets  (MoPlt)  ( H u P l t ) and r a b b i t  i n comparison  to  that  of  both  the  human  ( 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 s e p a r a t e d p l a t e l e t s u b p o p u l a t i o n s (Appendix T a b l e 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 S e p a r a t e d P l a t e l e t s Glutaraldehyde (FxHuPlt)  0.05%  and  sodium  distance  fixed  monkey  platelets  (FxMoPlt) 9  were  from  rabbit  suspended  a z i d e (1-5 x 10 , t o t a l ) ,  i n saline  containing  o f 25-30 cm i n l o n g g l a s s columns (30-50 ml) f o r n e a r l y  column. P l a t e l e t s were washed and suspended locate  platelet  human  and were a l l o w e d t o s e t t l e  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  To  (FxRaPlt),  column f r a c t i o n s  sizes,  platelets  containing  were c o l l e c t e d ,  a  one  from t h e t o p o f t h e  i n 0.5 ml o f s a l i n e . the g r e a t e s t d i f f e r e n c e s i n counted  and s i z e d  (Appendix  - 80 T a b l e A.23) u s i n g C o u l t e r E l e c t r o n i c s with  a 50 u o r i f i c e  amplification 3-50,  ( H i a l e a h , F L ) Counter  Model ZB1,  and 100 u l v o l u m e t r i c ( a p e r a t u r e c u r r e n t 1/2, and  1/2), w i t h C o u l t e r C h a n n e l y z e r  s e t t o accumulate  4000  counts  BCR  (window w i d t h BCT-WW  i n t h e peak  channel)  and  X,Y  R e c o r d e r . The s i z e d i s t r i b u t i o n s o b t a i n e d w i t h t h e C o u l t e r Counter were also confirmed especially  (both t h e s i z e  d u r i n g , and c o r r e l a t e d w i t h , t h e r i s e i n p l a t e l e t c o u n t ) by  Electrozone sedimented  Celloscope  sizing  analyses  platelets,  from  sedimented  fixed  platelets  were determined  measurements i n d i c a t e d  fractions  were counted  of  The of  data  were  mobility  However t h e p a r t i t i o n s  also versus  for total  and t h e p l a t e l e t  (Appendix T a b l e A.24).  a difference  0.01 t o 0.0005) i n t h e s u r f a c e charge  analyses  on  were a d j u s t e d t o e q u a l number i n s a l i n e ,  electrophoretic mobilities  sizes.  performed  platelets.  Fractions  mobility  and t h e s p r e a d i s o b s e r v e d t o i n c r e a s e ,  (p v a l u e s  These  ranging  from  of fixed platelets of different  significant  based  sedimentation  on  linear  distance  regression  (Table  3.5.1).  (5/4:A) f o r F x H u P l t 94.9 + 2.4% ( 2 ) , f o r FxMoPlt  92.4 + 3.9% ( 9 ) , and f o r F x R a P l t 89.8 + 3.8% ( 9 ) , were a l l so h i g h t h a t no d i f f e r e n c e s c o u l d be d e t e c t e d f o r s i z e s e p a r a t e d f i x e d p l a t e l e t s .  T a b l e 3.5.1 EPM o f S i z e S e p a r a t e d , F i x e d P l a t e l e t s ,  Intcpt Slope Corr p  Statistics  F x H u P l t 12/19  F x H u P l t 1/22  FxMoPlt  FxRaPlt  1.352 + 0.0183 0.005 + 0.0013 0.713 0.01 t o 0.005  1.368 + 0.0089 0.004 + 0.0011 0.840 0.005 t o 0.003  1.394 + 0.0168 0.002 + 0.0006 0.693 0.005 t o 0.001  1.273 + 0.0046 0.002 + 0.0002 0.908 0.0005  - 81 3.6  C o u n t e r c u r r e n t D i s t r i b u t i o n o f 51-Cr-Labeled In  P l a t e l e t s , Aged i n V i v o  o r d e r t o d e f i n e a p l a t e l e t p o p u l a t i o n a t a g i v e n t i m e , a "whole 51  population"  label,  Cr-chromate  i s t a k e n up e v e n l y by platelet was  ages. With  compared  platelet  with  ageing,  the e n t i r e  and  the  total  no  change  i n vivo,  occurs  CCD  radioactivity  Theoretically  t h i s labeled  population  radioactivity  partition)  of  platelet  shown)  points;  distributions  and  thus  exhibiting  the  distributions  platelet  should  t h e s e models p r e d i c t  what was  a  activity  no change i n s l o p e w i t h ( e . g . , a decrease  in  shift  to  and  the  negative  slope.  the  left  specific  However,  (lower activity  neither  of  t o be d i s c u s s e d , u s u a l l y of rabbits  30 ml t o 50 ml o f b l o o d  weighing  i n e x c e s s o f A.5  kg,  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 d e s c r i b e d . F o l l o w i n g p l a t e l e t  incubation for  specific  be  found e x p e r i m e n t a l l y .  were drawn from an ear a r t e r y and  should  distribution,  exhibit  In t h e experiments  should  F i g u r e 3.6.1.2; Model 2 ) , t h e n p r o g r e s s i v e l y  distributions,  the  I f , during  ( F i g u r e 3.6.1.1; Model 1 ) , t h e  t i m e . I f , d u r i n g p l a t e l e t a g e i n g , changes do o c c u r  the  population  present.  platelet  of a l l  surface  (not  in  label  membrane  s h o u l d be h o r i z o n t a l ,  membrane s u r f a c e charge;  this  platelet population consisting  platelet  i d e n t i c a l i n shape a t a l l t i m e distributions  used.  c i r c u l a t i o n time  p r o p e r t i e s as d e t e c t e d by particle  was  S2,  0.5  designating platelets, donor  with  51  Cr-chromate  mCi/10  ml  survival  The  S3,  experiment  washed and  animal.  for  (0.5  mCi/5 ml and  O.A  PRP mCi/6  number), washed  f o r S l , 0.2 ml red  v a r i o u s t i m e s , from one  survival  curve  was  for  SA;  cells  and  labeled  determined,  t o s i x days o f i n v i v o c i r c u l a t i o n ,  were i s o l a t e d and s u b j e c t e d t o  CCD.  ml  PRP  resuspended i n plasma, were r e i n j e c t e d  platelet  mCi/5  S#  into  the  and  at  platelets  - 82 F i g u r e 3.6.1.1 No Change i n S u r f a c e P r o p e r t i e s , w i t h C i r c u l a t i o n : Model 1, p l a t e l e t c o u n t s (ordinate), and C r specific activity h o r i z o n t a l on a l l days; CCDC ( a b s c i s s a ) . 5  DECREASING  PARTITION  WITH PLATELET AGE  1  PLATELET COUNTS  SPECIFIC ACTIVITY CURVES (CPM/PLATELET) DAY 0  F i g u r e 3.6.1.2 D e c r e a s i n g P a r t i t i o n w i t h 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 ageing  example CCD  ( S l ) o f one  of  these  studies i s presented.  i n vivo  At T=0  5 1  days,  Cr-labeled  following  platelet  labeling  but 51  prior  to injection  uptake, the  ( o f two  as e v i d e n c e d  specific  relative  there  i n the r a d i o a c t i v i t y  activity  t o bulk  animals)  of  distributions, the p l a t e l e t  was an enrichment  distributions  i n platelets  distribution  in  as w e l l  of  low  (Figure  Cr  as i n  partition  3.6.2.1  and  Appendix F i g u r e A . l ) . At T=2 days, c i r c u l a t i o n t i m e , t h e ageing l a b e l e d platelet  populations  were  (Figures  3.6.2.2  Appendix  (Mendenhall  and  and  identical  Scheaffer,  to  Figure  1973)  can  the  platelet  distributions  A . 2 ) . Nonparametric be  rapidly  statistics  applied  to  analyze  t h e s e 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 d a t a . A p p l y i n g t h e "Runs T e s t " t o the  specific  activity  distributions  of  0.008  t o 0.01)  days.  However,  randomly  CCD  ^Cr  were  associated after  two  with  was  found  (significantly  the p l a t e l e t  that  distributions,  days o f c i r c u l a t i o n  t o analyze  data  characterized  that as h a v i n g  p a r a m e t e r s (CCD m i d d l e )  radioactivity  two peaks (examples, of the p l a t e l e t  was The  days.  platelet  CCDs were p l o t t e d  distributions  =  zero  distributions.  (as p r e v i o u s l y d e s c r i b e d Chapter  the  at 5  a l l^Cr-labeled  and r a d i o a c t i v i t y  p a r a m e t e r s were n o r m a l i z e d exception  from  P  i n v i v o , the ^ C r  5  order  the  nonrandom,  by CCD o f "*"Cr a t T=0 was no l o n g e r p r e s e n t a t T=2  s t u d i e s , the p l a t e l e t  the  unevenly  i t  (P = 1.0) a s s o c i a t e d w i t h t h e CCD p l a t e l e t  enrichment In  5  distributions,  ageing and t h e  3.2), with were  often  Appendix Table A.25). The  and r a d i o a c t i v i t y  distributions  were compared a t each time p o i n t , u s i n g p a i r e d and n o n p a i r e d  student t  tests  (HP S t a t i s t i c s Volume  0.05)  found  only  at  T=0,  whereas  1 ) . A d i f f e r e n c e (p l e s s after  circulation,  than  the d i s t r i b u t i o n s  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 ( T a b l e 3.6.1).  was of  - 84 F i g u r e 3.6.2.1 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t ordinate; cpm/platelet p e r CCDC ( d o t t e d l i n e ) w i t h t h e mean s p e c i f i c a c t i v i t y a t one h a l f o r d i n a t e h e i g h t ; CCDC 1-60, a b s c i s s a . 5 1  3.09E+03  1. 75E+07-,  1.90E-04  0. 00E+00-  •0. 00E+00  1.7BE+08n  •1.90E+02  1.23E-06  0. 00E+00-  0.00E+00  F i g u r e 3.6.2.2 CCD o f 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 p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; cpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h t h e mean s p e c i f i c a c t i v i t y a t one h a l f o r d i n a t e h e i g h t ; CCDC 1-60, a b s c i s s a . 5 1  - 85 T a b l e 3.6.1 S t u d e n t t T e s t 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 Time Days  Number o f Exp  T=0 T=0  (3) (8)  0.078/0.019 0.071/0.009  3.99 7.89  0.029 0.00005  2.13 1.96  0.050 0.035  T=l T=2 T=3 T=4  (2) (3) (3) (1)  0.009/0.009 -0.004/0.004 0.010/0.012 0.003/0.055  1.0 1.0 0.83 0.06  0.25 0.21 0.25  0.16 0.03 0.65  0.444 0.489 0.275  In  Mid (Plt-CPM) /S.E.  addition , using  paired t  P  grouped s t a t i s t i c a l  platelet  number  t  nonpaired P  a n a l y s e s , w i t h the  CCDC as  and r a d i o a c t i v e c o u n t s per  CCDC as  the  group, and the  the  f r e q u e n c y , t h e median CCDC and s t a n d a r d e r r o r  f o r t h e 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 a t each t i m e , T=0 t h r o u g h 6 days of  (examples, Appendix T a b l e A.26). On days T=4 and T=6, when one  the distributions  approaches  zero  with  being  compared  increasing  approached' z e r o  survival  time),  while  distribution  remained r e l a t i v e l y c o n s t a n t , t h e v a l i d i t y  statistical  comparison  groups c o n t a i n i n g calculation  decreased.  To  circumvent  (radioactivity  this  the  other  o f t h e grouped problem  only  s i g n i f i c a n t numbers o f c o u n t s were c o n s i d e r e d i n t h e  ( i . e . , t h e l a s t 20 CCDF o f both S4T=4, and S4T=6). With t h e  median CCDC and t h e s t a n d a r d e r r o r , t h e 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  CCD  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  distributions  (Appendix T a b l e A.26). Using different  a one way a n a l y s i s times:  o f variance  (T=0 f o r group  of the t s t a t i s t i c  1; T = l , group 2; T=2, group  at the 3; T=3,  group 4; T=4, group 5; T=6, group 6 ) , t h e f r a t i o was 2.489, and p = 0.133 ( T a b l e 3.6.2).  - 86 T a b l e 3.6.2 A n a l y s i s o f V a r i a n c e o f t h e t S t a t i s t i c Source  D.F.  Sum Sqs  Mean Sqs  F Ratio  F Prob  Between groups W i t h i n groups T o t a l groups  5 7 12  4954.07 2786.39 7740.46  990.81 398.06  2.489  0.133  The  Duncan procedure gave, f o r t h e p = 0.05 l e v e l ,  ranges o f 3.34,  3.48,  3.55, 3.58, and 3.60, w i t h one s u b s e t (group 6, 2.02; 5, 2.60; 3,  4.59;  4, 20.1; 2, 35.8; 1, 5 3 . 2 ) . T h i s i n d i c a t e d no d i f f e r e n c e a t t h i s  level  of  significance  distributions level,  between  the  platelet  a t any o f t h e t i m e p o i n t s .  and  The ranges  radioactivity  f o r t h e p = 0.10  were 2.68, 2.79, 2.83, 2.87, 2.88, w i t h two s u b s e t s (groups 6,  5, 3, 4, 2, and 2, 1 ) . T h i s different  from  indicated  that  T=0  and T=l days,  the r e s t . Analyses o f variance tested  f o r a t r e n d over  t h e e n t i r e p e r i o d and a l l o w e d f o r a n i m a l t o a n i m a l v a r i a t i o n , missing  data.  Given  variation  and  limited  time  were  and f o r  dependence  of the  observed d i f f e r e n c e , t h e s i g n i f i c a n c e o f t h e t r e n d was o n l y a t a l e v e l of  p  = 0.1, b u t i n t h e same  during the i n i t i a l  animal  the difference  was  significant  period. 51  Each  CCD  curve  for  Cr-labeled  r a b b i t was a l s o o p e r a t i o n a l l y  platelets  sub-divided into  aged  i n vivo  four regions  i n the  (I,II,III,  51 and  IV) f o r f u r t h e r  activity  distributions  portion  of  coefficient, region  the  data  and d i s c u s s i o n  ( T a b l e 3.6.3).  platelet  of large  variation  distribution  occurred  in  in  Cr  of  Cr  specific  S t a r t i n g a t t h e extreme l e f t , o r with  (few t o n e g l i g i b l e p l a t e l e t s , 51  e r r o r , CE; S e c t i o n 2.14). present,  analyses  specific  a  very  Region  low  partition  I ) , there  activity  (high  was  a  combined  Red and w h i t e b l o o d c e l l c o n t a m i n a t i o n , when  Region  I  although  these  preparations  were  - 87 virtually  free  of c e l l u l a r contamination. In t h i s  region  region  IV ( t o be d i s c u s s e d ) , problems w i t h background  since  very  low  numbers  of  platelets  and  and a l s o i n  were encountered  radioactive  counts  were  p r e s e n t . T h e r e f o r e , when e i t h e r o f t h e two numbers which were combined to  provide the s p e c i f i c  activity  (cpm/plt)  approached  or decreased  below z e r o when background was s u b t r a c t e d , h i g h v a r i a t i o n o c c u r r e d such t h a t t h e 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 o f t h e s p e c i f i c a c t i v i t y o f the  platelets  i n this  region  and t h u s  was judged  unreliable  as an  e s t i m a t e o f s u c h . I n t h i s work, i f t h e CE due t o low v a l u e s o f e i t h e r the used  platelet  count  f o r analysis.  o r cpm exceeded  10% t h e s p e c i f i c  a c t i v i t y was not  T h i s l i m i t was used t o d i v i d e t h e LEFT o f t h e CCD  i n t o R e g i o n s I and I I , and t h e RIGHT i n t o Regions I I I and IV.  T a b l e 3.6.3 S u b d i v i s i o n o f CCD o f C r - L a b e l e d R a b b i t 5 l  Platelets  Region  Characteristics  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 t o v e r y low numbers p l a t e l e t and r a d i o a c t i v i t y c o u n t s . B. P o s s i b l e c o n t a m i n a t i o n ( e r y t h r o y c t e and l e u k o c y t e ) .  of  Region I I .  I n t e r m e d i a t e 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 o f t h e d i s t r i b u t i o n . B. Sub-population of high specific activity r e l a t i v e t o t h e mean, a t T=0 t o 1 days, and low p a r t i t i o n r e l a t i v e t o t h e 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 t o t h e portion i n the t h i r d region. C. P o s s i b l e c o n t a m i n a t i o n ( 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 o f t h e d i s t r i b u t i o n . B. Lower 51Q s p e c i f i c a c t i v i t y , a t T=0 t o 1 days, r e l a t i v e t o t h e 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 t o t h e p o r t i o n i n t h e second r e g i o n . T  Region I V .  Very h i g h 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 p l a t e l e t and r a d i o a c t i v i t y c o u n t s .  very  low  numbers  of  - 88 R e g i o n I I , a l s o o f low p a r t i t i o n  coefficient  platelet  population, c h a r a c t e r i s t i c a l l y  specific  activity.  throughout  the  evidenced both  by  was  distribution  important,  distribution,  position  this  Region  Cr,  I I I ( d i v i d e d by t h e CCD the  T=0  I I , the  was  distribution,  of  slope  the  away  low  a l s o have been due  circulation  f o r unknown  s u b p o p u l a t i o n by  distribution  under t h e RIGHT  or c e n t r a l p o r t i o n  activity  distribution from one  side  with  time  platelets  from  of  the  of  which  platelet  circulation,  of a s h i f t  i n the  Region  across  II  in  i n c r e a s i n g i n numbers i n Therefore  exhibited  relative  The  to  circulation  t h e r e was  a  uptake  of  enhanced total  was  the  platelet  r e p l a c e d by  newly  T h i s might a l s o have been i n t e r p r e t e d as  partition to  regions  major d i s t r i b u t i o n .  partition  a  these  partition,  which d u r i n g p l a t e l e t  from  as  t h a t t h e newly r e l e a s e d p l a t e l e t s ,  low  platelets,  unlabeled p l a t e l e t s .  could  middle  t o be i n d i c a t i v e  labeled  were o f  low  in  thought  released shift  over  distribution  activity  specific  decreased  suggested  LEFT o f of  carried  c u r v e t o t h e o t h e r were d e t e c t e d here.  days,  radioactively  unlabeled,  and  slope of the  z e r o . T h i s was  subpopulation(s) 5 1  specific  CCD  platelet  curve.  Region I I I and  case  activity  p o r t i o n of the  s l o p e of the  activity  at  of  major  distribution  specific  approaching  in specific  t o t h e major  of high  since subtle s h i f t s of labeled platelets  o f the p l a t e l e t negative  a region  i n Region I I I , t h e p o r t i o n o f p l a t e l e t s  distribution),  through  the  the negative  In Regions I I and of the  enrichment  LEFT o f  h e r e and  o f t h e CCD  This  was  relative  the  reasons,  with  platelet  removal o f t h i s e.g.,  circulation  subpopulation  sequestration of  It  during  this  t h e r e t i c u l o e n d o t h e l i a l system, i n t h e s p l e e n  and K a r p a t k i n , 1975b) o r t h e l u n g s (Trowbridge  time.  "young" (Freeman  and M a r t i n , 1982).  - 89 Region  IV  characterized relative  demarcated  the  end  by t h e few p l a t e l e t s  t o the majority  of  the d i s t r i b u t i o n  which were o f v e r y  of the platelet  high  population.  a c t i v i t y o f t h i s r e g i o n was a g a i n h i g h l y v a r i a b l e ,  which  was  partition  The  specific  r e l a t i v e t o t h e mean  i n R e g i o n s I I and I I I . Analyses o f 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 different  ( I I and I I I ; LEFT and RIGHT) o f t h e d i s t r i b u t i o n s vivo  ^Cr-labeled  mean s p e c i f i c platelet the  platelet  activity  distribution,  provided  of  ageing  distribution).  In  the  errors  times,  less  significant.  than  platelets  (i.e.,  calculations  inherent  10%  of  mean o f t h e p o p u l a t i o n . correlation  of  i n counting  platelet  specific  both  specific  The s l o p e s  activity  activities,  platelet  activity  numbers  the and  i n which t h e CE were  considered  activity,  after Table  v . s . CCDC) and  activity  distributions  a c t i v i t i e s i n Regions I I and I I I o f  various times o f i n v i v o A.31) w i t h  considering  respect  a l lplatelets  w i t h t h e 10% CE l i m i t a p p l i e d .  I I I , at the d i f f e r e n t  activity  of the s p e c i f i c  The s p e c i f i c  (Appendix  f o r Regions I I and I I I and t h e  (specific  were used t o compare t h e n o r m a l i z e d and  specific  specific  c o u n t s p e r CCDC, and were n o r m a l i z e d r e l a t i v e activity  by  and o n l y d a t a  as c a l c u l a t e d  distributions  were n o r m a l i z e d (Gm)  the  coefficients  were a l s o c a l c u l a t e d . the  to the  Data were e x p r e s s e d as s p e c i f i c a c t i v i t y (cpm p e r p l a t e l e t  w i t h i n t h e 10% CE l i m i t ) ,  the  and comparison  a n o t h e r means o f d e s c r i b i n g changes i n  r a d i o a c t i v e e v e n t s were a l s o c a l c u l a t e d were  different i n  (Appendix T a b l e s A.27 t h r o u g h A.31) t h r o u g h t h e  partition  statistical  circulation  after  Regions  t o t h e grand and  mean  radioactive  t o t h e mean (m) s p e c i f i c  Paired  specific  circulation  and n o n p a i r e d  t  tests  a c t i v i t i e s o f Regions I I  t i m e p o i n t s ( T a b l e 3.6.A).  - 90 T a b l e 3.6.4 Nonpaired and P a i r e d t T e s t S p e c i f i c A c t i v i t y o f Regions I I and I I I , N o r m a l i z e d t o t h e Mean a t Ten P e r c e n t Combined E r r o r T  II Mean +_ S.D.(n)  III Mean _+ S.D.(n)  II-III Mean +_ S.D. + S.E.  T==0 1.498 + 0.234(3) 0.351 + 0.206(3)  nonpaired p paired p  0.00155 1.148 + 0.135 + 0.078 0.00229 0.00001 1.228 + 0.715 + 0.253 0.00092 0.12146 0.328 + 0.392 + 0.278 0.22375 0.11641 0.219 + 0.349 + 0.201 0.19542 0.08272 0.236 + 0.337 + 0.194 0.17456  1.637 + 0.454(8) 0.409 + 0.313(8) T==1 1.137 + 0.158(2) 0.810 + 0.234(2) T==2 1.065 + 0.097(3) 0.846 + 0.251(3) T==3 1.090 + 0.147(3) 0.855 + 0.191(3)  The mean s p e c i f i c a c t i v i t i e s o f t h e major d i s t r i b u t i o n decreased  with  t i m e , as e x p e c t e d , i n a s i m i l a r  normal s u r v i v a l  curve. Labeled p l a t e l e t s  to  and  four  days,  circulation (most  f o r up  pronounced  specific  and  i n Region  to that  remained i n c i r c u l a t i o n  radioactivity (not shown).  remained There  T = l , as e v i d e n c e d by  related  distributions circulation. partition  of ^ C r - l a b e l e d These  region  Changes  of  the  occurred  platelet  became o b v i o u s platelet  the  enrichments  activity  of  circulation.  a c t i v i t y was c o n s i d e r e d t o be an  5  characteristic.  f o r up  the slope of the  I I , T a b l e 3.6.4) i n s p e c i f i c  i n "*"Cr s p e c i f i c  of a  within  were  o f l o w e r p a r t i t i o n , w h i c h changed w i t h p l a t e l e t  T h i s enrichment age  case,  t o s i x days  a t T=0,  activity  platelets  i n one  manner  of platelets  by  within  the  partition  populations following comparison  distribution.  i n vivo  o f t h e low t o h i g h When  platelets  were  51 labeled  with  specific which  was  Cr i n v i t r o , t h e r e was an enrichment  activity 4.3  (^Cr/platelet)  times  labeled platelets  that  in  i n Region  were i n j e c t e d  into  the  low  ( a t T=0  partition  I I I ( T a b l e 3.6.4).  days) i n  Region I I , After  these  a r a b b i t and a l l o w e d t o c i r c u l a t e  - 91 for  various  times,  the  relative  enrichment  rapidly  decreased.  This  d e c r e a s e i n r e l a t i v e enrichment o f r e g i o n I I compared t o r e g i o n I I I was most pronounced w i t h i n the times 1.3  the  by  long  specific  days two  as  insufficient platelet  24 h o u r s , r e g i o n I I d e c r e a s i n g  a c t i v i t y of region  and  three  s i x days, t h e r e  distribution.  first  However for  day  ( T a b l e 3.6.4). A f t e r  was  detectable  the  a  I I I by  label  activities  statistically  one,  and  to  to that  f o u r d a y s , and paralleling  became  so  significant  1.4  the  low  even  of as  platelet  as  to  comparisons  be  to  the  delineates  old  distributions.  The  use  platelets  after  for  these  the  highest  51  of  ageing  purposes.  by  low  of  subpopulation  ^Cr  more f u n c t i o n a l l y  was  active  (Haver and  t o have d i s t i n c t membrane s u r f a c e with c i r c u l a t i o n  therefore considered  partition  activity  a n o t h e r method  * theoretically  which  i n v i v o , was  The  specific  been shown t o be described  Cr-labeling,  characterized  presumably  that  i n terms o f  which  p r o p e r t i e s , which a p p a r e n t l y  since platelets  fractionation  changed  process  might  o f a s i z e g r e a t e r than average (the s i g n i f i c a n c e  o f which  not  distribution  normal  tested)  partition  than  the  as  shown  were a l s o found t o have a s l i g h t l y lower been  by has  aggregation  Gear, 1981). Here i t was  time. A p a r t i a l f u n c t i o n a l  be o c c u r r i n g d u r i n g CCD,  insufficient  of  has size  51 platelets.  These  changes  c o u l d have been due lower due  partition, to  a  shift  t o the  which in  the  in  Cr  specific  exhibited partition  microscopic  contamination examination.  of  of  "^Cr  these  the  uptake,  labeled  distributions platelets or  have  platelets,  of been  being  platelets.  platelet  Platelet  activity  sequestration of  enhanced  r e p l a c e d by newly r e l e a s e d u n l a b e l e d Cellular  specific  preparations  suspensions  to  be  was  checked  subjected  to  by CCD,  - 92 and  i n certain  Tyrode's slides  solution  were  cellular 1  prepared,  per of  10  5  after  containing which  contamination.  cell  number  instances  were  per  This  washed  albumin.  Smears  Wright's  stained  The s u s p e n s i o n s was  field  resuspended i n  or  Cytocentrifuge  and  by  and  observed  than  enumerating  the  scanning  or white blood  stated, cellular  for  less  repeatedly  ( e i t h e r red blood c e l l  Thus, u n l e s s o t h e r w i s e  and  i n general contained determined  microscope  several fields u n t i l a c e l l was o b s e r v e d .  2% were  platelets.  platelets  CCD,  cell)  contamination i n  t h e s e 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,  "^Cr-labeled platelet  platelet  rich  plasma  after  population, prepared  contained white blood c e l l and  subjected  cells,  and  (originally which  t o CCD to  a  post  blood unusual  to  o f the t o t a l  determine i f  bound) i n t o  i t had. The r e l a t i v e  injection  sample  and r e d b l o o d c e l l  determine  platelet  weeks  i n an  4.5% RBC, as a p e r c e n t a g e  was  three  was  manner,  such  cells).  the p a r t i t i o n s  reincorporation  of  of  partitions  of contaminating  and  that i t (0.02%  WBC  T h i s sample contaminating  the  any o f t h e o t h e r c e l l s  the  taken  contamination  blood  of  ^"''Cr-label  had o c c u r r e d , cellular  types  s t u d i e s , other  (T=0)  were as p r e v i o u s l y o u t l i n e d i n T a b l e 3.6.3. In  conjunction with  experiments 5 1  Cr  the p l a t e l e t  ageing  CCD  were performed i n v o l v i n g a n a l y s e s o f p l a t e l e t s l a b e l e d w i t h  s e p a r a t e l y , and t o g e t h e r  with  other  radioactive labels  (Section  3.7). The CCD was a n a l y z e d 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  a r e counted  conditions.  quantity  The  of  f o r f r e e l a b e l i n response free  radioactive label  i n t o t h e phases d u r i n g t h e s e p a r a t i o n procedure partition  coefficient  for free  5 1  to a variety of released  at  4°C  was n e g l i g i b l e , and t h e  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 e v e n l y between t h e two phases. These s t u d i e s s u p p o r t t h e existence of a platelet  subpopulation  which  e x h i b i t e d enhanced  uptake  51 specifically  for  Cr-chromate  (not  s u b p o p u l a t i o n which was c o n c e n t r a t e d  free  label),  a  platelet  by CCD t o a c o n f i n e d a r e a o f low  p a r t i t i o n under t h e p l a t e l e t d i s t r i b u t i o n c u r v e . Rabbit p l a t e l e t s labeled  with  separately)  (and a l i m i t e d number o f human p l a t e l e t  ^Cr-chromate  and/or  and d i s t r i b u t e d  soon  ^C-serotonin thereafter  samples),  (simultaneously  (T=0) by  CCD,  or  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 o f t h e s p e c i f i c a c t i v i t y o f "^Cr  and  "^C.  As  specific acitivity labeled for  with  mentioned  platelet  was  an  enrichment  * ^ C r , b u t t h e 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 when  of p l a t e l e t l a b e l i n g label  there  o f p l a t e l e t s o f lower p a r t i t i o n when p l a t e l e t s  a l l platelets  this  above,  as  a  labeled with  "^-serotonin  by s e r o t o n i n was s u b s e q u e n t l y standard).  heterogeneity  by  Preliminary  CCD  (this  were  constant  homogeneity  e x p l o i t e d i n use o f  experiments  the characteristics  in  o f post  indicated  CCD  platelet  activating  agents.  14 C-serotonin  release  in  response  These f i n d i n g s c o u l d be e v i d e n c e not  platelet  of platelets  which  a r e heterogeneous  o n l y w i t h r e s p e c t t o "*"Cr uptake b u t a l s o w i t h r e s p e c t t o q u a l i t y 5  of granular contents and  to  non-releasable  (Valdorf-Hansen  (Caranobe e t a l . , 1982), compartments  of  there  serotonin  in  being  releasable  human  platelets  and Z u c k e r , 1971; C o s t a e t a l . , 1982).  3.7 C o u n t e r c u r r e n t 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  in  C r uptake,  platelet specific activity after labeling a  subpopulation  of platelets  which  as i n d i c a t e d  by  a  higher  in_ v i t r o a t T=0, was found i n  could  be  d i s t i n g u i s h e d by i t s  - 94 characteristic property  low p a r t i t i o n  distribution,  heterogeneous membrane  with  surface  relative  the p l a t e l e t  respect  t o t h e major  population  was d e s c r i b e d  t o both "*"Cr uptake  and w i t h  5  properties.  Furthermore,  platetet  since  this  surface as b e i n g  respect  enrichment  to in  51 Cr  specific  circulation, surface and  this  subpopulation  experiments  subpopulation exhibited  1982),  decreased  properties. Platelet  CCD  Gear,  activity  was  active  were  similar uptake  1981; e v i d e n t  demonstrated  with  designed  to  ascertain  o f high  i n the density  (Tsukada  label as  o f enhanced  which  5  labeled the p l a t e l e t  determined  by  CCD),  which  (Haver  o f Boneu  and  et a l . ,  and Gear,  1981).  o f p l a t e l e t s by i t s  t o another r a d i o a c t i v e  p o p u l a t i o n more e v e n l y , density  in  of platelets  analyses  (relative  i t s high  changes  this  e t a l . , 1971; Haver  ^ C r uptake  the  whether  density  These e n a b l e d t h e d e f i n i t i o n o f t h i s s u b p o p u l a t i o n properties  in  density centrifugation  the subpopulation  f o r ^ C r , was  time  age r e l a t e d  double-labeling,  to  also  and was young  rapidly  and  e.g.,  "^In,  characteristic  low  p a r t i t i o n i n phase systems s e n s i t i v e t o membrane s u r f a c e c h a r g e . 51 Platelets which  were  double  labeled,  enrichment was b o t h e v i d e n t  with  Cr  at concentrations  and l e s s o b v i o u s ,  at  and w i t h '^"'"In a t  v a r i o u s c o n c e n t r a t i o n s f o r which no such s i m i l a r a c t i v e uptake has been o b s e r v e d . These double l a b e l e d p l a t e l e t s were s u b j e c t e d t o CCD i n c h a r g e - s e n s i t i v e two-polymer phase systems and t h e d i s t r i b u t i o n s o f platelets and r a d i o a c t i v e l a b e l s were d e t e r m i n e d . They not o n l y 51 confirmed  t h e enrichment  in  p l a t e l e t s w i t h a low p a r t i t i o n in  the  survival  Cr  uptake  by  the  subpopulation  of  which had been p r e v i o u s l y found a t T=0  experiments,  but  also  demonstrated  that  this  s u b p o p u l a t i o n 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  results  interpretation subpopulation  of  these  that  dense  which  experiments (possibly  preferentially  platelets  have  surface  partition  coefficient  ( i n phase  population,  than  subpopulation  itself.  subpopulation  was  lower p a r t i t i o n by CCD  was  lower  this  i n HDP,  than t h a t o f  therefore a s p e c i f i c  p o r t i o n of that separated  HDP,  of  "active still  and  the  subpopulation  that a  low  surface  o f the  platelet  (young)  platelet  uptake"  of  a  these  by  detecting  HDP  the  contained  and  majority the  "Cr  i t was  the  ^Cr,  capable  of  5J  platelets  with  characterized  t o the  that  consistent  up  uniquely  only  Although  enriched  young)  systems  not  also  were  takes  properties  charge d i f f e r e n c e s ) r e l a t i v e but  -  platelet  characteristically  subpopulation  separated  which c o n s t i t u t e d o n l y  a  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  vivo,  the  rapidly  ( o f S e c t i o n 3.6),  specific  than  that  change i n the in  Region  that  slope  activity of  the  newly  released  R e g i o n I I ) , and  to  of  rest  of the  I I compared  as  the this  of  the  specific Region  population,  I I I . This  p l a t e l e t s (unlabeled)  decreased, as  evidenced  t h r o u g h the  of  low  by  a  distribution  r e s u l t supported were  more  the  idea  partition  (in  suggested e i t h e r t h a t t h e i r s u r f a c e p r o p e r t i e s changed,  s p e c i f i c a l l y sequestered  or t h a t t h i s  subpopulation  was  shortly after injection.  Experimental Platelet  ACD  l a b e l e d p l a t e l e t s aged i n  subpopulation  activity  i n c r e a s i n g i n p a r t i t i o n with maturation  3.7.1  Cr  (8 ml  parts,  Preparation:  i n each o f two  about 40  (750-1000 RPM, total  Blood,  there  ml IEC  50 ml  blood SBV)  and  145  g  were a p p r o x i m a t e l y  80  ml  Corning  from one  centrifuge tubes),  anticoagulant f o r 15 45  ml  r a b b i t , was  each,  minutes a t  split  in  in two  centrifuged  at  room t e m p e r a t u r e .  In  o f packed RBC  and  taken  plus residual  PRP,  . and  35  ml  o f PRP  harvested.  c e n t r i f u g e d a t l,000g  - 96 The  PRP  was  split  into  three  parts,  f o r 15 minutes and each p a r t was resuspended i n 2  ml o f PPP. The remainder o f t h e PPP was saved. Platelet original  Double  stock  quinoline  Labeling:  solution  (Amersham)  concentration  of  1  were  "'"'"Cr  ^ ^ I n , and  and  particular  each  label.  concentration  was  used  mCi/ml.  platelets  ^Cr-CrO^  The  labeled  different  5 1  Cr  200,  Cr/ml)  a t 1 mCi/ml,  and  111  In,  "^In  o f 1, 2,  and 5 ) . The  of  at 4  solution  preparations  incubation  f o r the ml PRP  a t 2-5 ug Cr/ml and 3.9-9.8 x  at  three  labeling  levels of labeling  5 1  n  I n  i  concentrations:  a t 1 mCi/ml, 100,  C r o f 1, 5, and 10 and f o r  concentrations  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 t h e s t o c k c o n c e n t r a t i o n ranges for  51  First,  C r , 0.2-0.5 5 1  platelet  ug/2 p e r m l , o r 0.4-1.0  concentrate  prepartaions.  10  R  second  moles/2 p e r m l ) .  they  were i n c u b a t e d  at  were g e n t l y mixed and  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  PRP c o n c e n t r a t e  was d i l u t e d  centrifuged  1,000  efficiency  The p r e p a r a t i o n s  f o r 15 m i n u t e s , then  as above, and i n c u b a t i o n c o n t i n u e d  at  g  of labeling  centrifugation. ml  x  (e.g.,  C r was added, 0.1, 0.5, and 1.0 ml each t o one o f t h e t h r e e  37°C i n a water b a t h the  of  quantities of  g 10 /ml i n 2  x  an  In-hydroxy-  different  times  at  stock  identical  100, 500, and 1000 u l ;  and 500 u l ; ( f o r r e l a t i v e  used  mCi/ml.  with  concentrates  was  original  three  5  moles  1  an  were l a b e l e d w i t h "*"Cr ( s t o c k s o l u t i o n —8 10~  of  separately  with  Platelet  at  (NEN)  o f Tyrode's  The p l a t e l e t solution  t o 10 ml w i t h  f o r 15 was  f o r another  minutes  determined  by  15 m i n u t e s . Each l a b e l e d  the remaining at  room  sampling  p e l l e t s were each t h e n  (containing  albumin,  EGTA,  PPP and was  temperature. before  and  resuspended MgCl ) 2  The after i n 10  and were  - 97 c e n t r i f u g e d a g a i n as above. was  finally  system load  at  resuspended 5°C.  mixtures  The  The  wash was  i n 4.5  specific  ( L x ) were  ml  r e p e a t e d and each  of  upper  activities  compared  with  phase  of  of  the  platelets,  those  of  preparation two-phase  within  platelets  these  after  CCD  (Appendix Table A.33). Countercurrent 5/4:B D  Distribution:  The  two-polymer  ( l o t 7830), 99.99 g o f PEG  8,000, 313.2  N a C l , and 134.21 g o f water f o r a t o t a l a p p a r a t u s was  loaded with  mixture i n the load c a v i t i e s  0.4  ml  labeled  lower phase  (Ld CCDC); and 0.5  were c o m p l e t e d , w i t h  and  0.9  contents of  solution, for  a  mixed  total  (dilution  the  120  CCDC  automatically,  of  60  CCDF,  247/1) and  diluted  collected,  which  then c o u n t e d  were  0.8  (Tr CCDC). The t h r e e , double four c a v i t i e s  a m i x i n g t i m e o f 30  were  The  and  each,  seconds  s e t t l i n g t i m e s e t a t 8.5, o r 7 m i n u t e s . Upon c o m p l e t i o n o f t h e CCD the  g of  ml o f l o a d  ml l o w e r phase  p l a t e l e t p r e p a r a t i o n s were l o a d e d i n t o  transfers  g o f NaPB, 31.2  o f 750 g o f phase system.  ml o f upper phase i n t h e t r a n s f e r c a v i t i e s  36  d e s i g n a t e d as  was p r e p a r e d from s t o c k s o l u t i o n s by t h e a d m i x t u r e o f 171.39 g o f  T500  CCD  system  with  0.7  p o o l e d by  sampled  directly  ml  two  of  and  a  run,  Tyrode's  a d j a c e n t CCDC  for particle  for ^ C r  and  and  counting  ^'4n  using  t h e LKB Compugamma. These CCDF were a n a l y z e d a t room t e m p e r a t u r e but a sample  o f each  was  centrifuged  i n the c o l d  for determination of  free  l a b e l , and l e a k a g e due t o p o s s i b l e p l a t e l e t damage. Samples, 0.5 ml, o f t h e 60  CCDF were t a k e n and  p o o l e d by two a d j a c e n t CCDF i n t o  c e n t r i f u g e t u b e s f o r a s e p a r a t e s e t o f 30 CCDF samples These 5°C.  samples The  30  were  then  centrifuged  s u p e r n a t a n t s were  at  sampled,  f r e e l a b e l a l o n g w i t h t h e o t h e r 60 CCDF.  top 0.5  speed ml  o f 1.0  for 6  each,  and  Eppendorf ml  each.  minutes  at  counted f o r  - 98 Cytocentrifuge slides Wright's by  o f t h e CCDF were p r e p a r e d  s t a i n . The p e r c e n t a g e  of celluar  and s t a i n e d w i t h  c o n t a m i n a t i o n was  f i r s t c o u n t i n g p l a t e l e t s on o i l immersion  determined  (lOOx l O x ) , e s t i m a t i n g an  a v e r a g e number o f p l a t e l e t s p e r f i e l d ,  and t h e n because o f t h e s p a r s i t y  of  (repeatedly)  cellular  number  contamination,  of f i e l d s before  counting  a single  number o f p l a t e l e t s p e r f i e l d a  number  which  was  cell  varied  accurately  (WBC  recording  the  o r RBC) was observed.  The  and ranged  countable  A t o t a l o f 6 WBC were counted RBC o v e r per  the  WBC  times,  per f i e l d  a  complete  were p r e s e n t .  o v e r 29 f i e l d s ( 1 / 4 . 8 3 ) , and a t o t a l o f 2  a t o t a l o f 16 f i e l d s  field,  i n some i n s t a n c e s from  t o , at  monolayer. An e s t i m a t e d 5,000-10,000 p l a t e l e t s  and  ( 1 / 8 ) . Based  contamination  was  on 5,000-10,000  0.004-0.002%  platelets  and  the  RBC  c o n t a m i n a t i o n was 0.0025-0.00125%. 3.7.2 R e s u l t s Countercurrent  Distribution  particles  of a size  resembled  those  slightly  to  percentage  the  cellular particles,  left,  mostly  the selection  of a  greater  was  (average  lower  K  than  than  of  ( n o t shown),  were c o n s i s t e n t l y  a  size  distributions  of the p l a t e l e t  exhibiting  o f window  on  that  The  However they  aggregates,  contamination, based  than  of platelets.  of particles  representing to  greater  Analysis:  shifted  platelets.  that  of  The  platelets,  c o u n t i n g c o i n c i d e n c e , and due i n p a r t  settings, of  the  overall)  9.28%, and 7.28%, r e s p e c t i v e l y  and  a  order  very of  recovery  minor  5.72% after  of CCD,  quantity of the  total  and 6.65%,  f o r the load mixture p r e p a r a t i o n s . 51  Platelets, concentrations) modal  partition  double-labeled and  "'"''""'"In,  with  subjected  f o r t h e major  Cr to  platelet  CCD  (at  three  analysis,  distribution  different exhibited  a  o f 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 t h r e e various  concentrations  of  the  identical labels  populations  (Figure  labeled  3.7.2.1.1  with  through  3.7.2.1.6). The K v a l u e was c a l c u l a t e d f o r t h e peak o r modal CCDC ( i n this  case  partition 3.2).  equal steps  Since  t o which  the population  each p l a t e l e t  CCDC 1 through origin,  t o t h e mean), d i v i d e d by t h e number o f t r a n s f e r s o r  p r e p a r a t i o n was loaded  4, 41 through  was t a k e n  was s u b j e c t e d  44, and 81 through  as "CCDC  2.5" ( t h e middle  (as i n S e c t i o n  i n f o u r CCDC  (i.e.,  8 4 ) , t h e l o a d CCDC, o r o f CCDC  1,2,3,4). The  number o f CCDC t r a n s f e r r e d by t h e 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:  t h e peaks  distributions actual  origins  transferred  (taken  from  t h e graphs,  were a t a d i s t a n c e o f 16.67 CCDC from  3.7.1.) o f t h e CCDC 1, but t h e  were a t CCDC 2.5; t h e r e f o r e , t h e t o t a l  by t h e p l a t e l e t  14.17. Taking  see F i g u r e s  population  was 2.5 l e s s  number o f CCDC than  16.67, o r  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 v a l u e o f 0.3935. The K v a l u e would be 0.41 f o r an o r i g i n o f 1. The  ^•'"In r a d i o a c t i v i t y  labeling,  closely  distributions,  paralleled  the  at a l l levels  platelet  of platelet  distributions  (Figures  3.7.2.1.2, 3.7.2.1.4, and 3.7.2.1.6). However, t h e r e were two peaks i n ^CT  radioactivity  platelets nature  distributions,  and one which  one comparable  was o f lower  relative  o f t h i s "*"Cr uptake was suggested  to the p a r t i t i o n of partition.  The a c t i v e  i n t h a t i t became s a t u r a t e d  5  51 at  progressively  higher  3.7.2.1.3, and 3.7.2.1.5).  concentrations This  exhibited  enhanced  evidenced  i n t h e magnitude  through higher  CCD r e g i o n s  uptake  of  enrichment  Cr  (Figures  by CCD o f p l a t e l e t s  f o r ^ C r , but n o t f o r " ^ I n , 51 5  of  the  Cr  3.7.2.1.1,  specific  was  activity  which also  slopes  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  (A, B, and C) c o n c e n t r a t i o n s o f - C r (Appendix Table A.30). 5J  - 100 F i g u r e 3.7.2.1.1 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-40, a b s c i s s a . 5 1  RABBIT  (^CR, SD^CI / tL  PLATELETS  0  20  AND l N , SO^Ci / UJ m  10  F i g u r e 3.7.2.1.2 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-40, a b s c i s s a . m  - 101 F i g u r e 3.7.2.1.3 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 41-80, a b s c i s s a . 5 1  RABBIT  RABBIT  PLATELETS  PLATELETS  < CR, 250 «CI / ML AND m l N , lOCUCi / tO 5]  C ^ , 250^1 / ML AND m l N , IDO^Ci / ri) 51  40  60  80  F i g u r e 3.7.2.1.4 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 41-80, a b s c i s s a . i ; L 1  - 102 F i g u r e 3.7.2.1.5 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 81-120, a b s c i s s a . 5 1  RABBIT  SOO^Ci / rL AND l N , 250^Ci / iD  PLATELETS  m  80  100  120  F i g u r e 3.7.2.1.6 CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 81-120, a b s c i s s a . m  - 103 Further Appendix  CCD  analyses  Figures  A.3  (Figures  and  A.4)  -  3.7.2.2.1  were  through  conducted  on  3.7.2.2.6;  rabbit  and  platelets,  51 double the  labeled  highest  with  Cr  preferential  density subpopulations HHD).  LDP  (at  the  lowest  uptake)  (LDP,  IDP,  and  HDP,  but  density  subpopulations  than  density  subpopulations  (e.g., less  than  obtained  Stractan  less  density  of  step,  were a  the  20%  combination  of  18%  were  Stractan.  of  LDP  The  HHD  were  which p e n e t r a t e d  were p l a t e l e t s  of  IDP;  or  other  (and  IHHD  the  density  the  and  Stractan),  gradient.  platelets and  than  S t r a c t a n . HDP  were e i t h e r c o m b i n a t i o n s o f  that  from  contained) subpopulations 20%  LIDP  and  l e s s than 18%  of unusual d e s i g n a t i o n s  subpopulations of  18%,  LDP  into  IDP  greater  HHD;  separated  were p l a t e l e t s o f d e n s i t y l e s s than t h a t o f 16% S t r a c t a n .  of  and  and  and  but  i.e.,  *^In,  demonstrating  IHHD; LIDP  were o f g r e a t e r d e n s i t y than 16%,  above  concentration,  lowermost  even  higher  t h a n HDP. These f r a c t i o n s o f v e r y h i g h 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 c o n f i n e d t o g i v e n r e g i o n s o f the CCD, e.g., 51 erythrocytes activity  i n Region  (Figure  I  evidenced  3.7.2.2.2,  CCDC  by  a sharp  90-95),  but  peak  in  Cr  absent  in  specific the  i n  m  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 ( F i g u r e 3.7.2.2.1, CCDC 90-95). CCD separated low  results  was  LDP  ^Cr-,  HDP  variability  of In  consistently  (Figure  3.7.2.2.4),  apparent.  these  "'""'"'''In-double  p l a t e l e t s were s i m i l a r t o p r e v i o u s  partition  density,  from  3.7.2.2.3),  (Figure the  one  partition case,  compared  the  of  and  platelets  partition  of  density  f i n d i n g s o f S e c t i o n 3.2.  associated  3.7.2.2.5),  labeled,  with to  HHD of HDP  e q u a l t o t h a t o f p l a t e l e t s o f l o w e r d e n s i t y IDP  platelets  that  of  (Figure high  IDP  low  (Figure  3.7.2.2.6).  density  (Figure  of  was  A  The again  3.7.2.2.5)  was  ( F i g u r e 3.7.2.2.4),  but  - 104 still  of higher  partition  than  platelets  o f even h i g h e r  density  HHD  ( F i g u r e 3.1.2.2.6). A c o r r e l a t i o n o f p l a t e l e t p a r t i t i o n and d e n s i t y was a g a i n found i n another  e x p e r i m e n t (Appendix F i g u r e s A.3 and A.4).  W h i l e enrichment by CCD ( i n Region I I ) o f a p l a t e l e t  subpopulation  51 which  exhibits  enhanced  uptake  p l a t e l e t s of high density and in  for  Cr  was  more  pronounced  ( F i g u r e s 3.7.2.2.2, CCDC 65-80, CCDC 95-105;  Appendix F i g u r e A.4, CCDC 35-45, CCDC 95-105) i t was a l s o the other  subpopulations  o f lower  F i g u r e A.4, CCDC 5-15). D e n s i t y not  occur  as s t r o n g l y  and Appendix F i g u r e 3.7.3  in  density  (e.g.,  present  LDP, Appendix  dependence, and enrichment by CCD, d i d  f o r " ^ I n labeled p l a t l e t s  ( F i g u r e s 3.7.2.2.1  A.3).  Discussion A subpopulation  exhibits  saturable,  concentrations occurs;  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 51 perhaps  of less  than  at concentrations  active  uptake  1.5 u C i / m l ,  greater  for  enhanced  than  this  Cr-chromate. uptake  both  platelets At  o f chromate  n o n - s a t u r a t i n g and  s a t u r a t i n g uptake o c c u r . By comparing t h e CCD d i s t r i b u t i o n s o f p l a t e l e t populations ^"^In,  labeled  enhanced  subpopulation relatively charge.  concentrations  5 1  Cr  takes  same ^ C r - l a b e l e d matched  relatively  f o r comparisons.  a platelet  up  i n a two-phase  ^ I n , like  platelets  for  strongly  low p a r t i t i o n  distributions  standard  uptake  which  I n these  Therefore, label  different  i t i s possible to distinguish  exhibits  CCD  with  closely  of  ^"'"Cr  subpopulation  but  not  ^Cr  i s characterized  system  platelet the  1  L  sensitive  preparations, platelet  "^C-or-^H-serotonin, homogeneously,  f o r " " "In.  and  is  1  and which The by a  t o surface t h e "'""'""'"In  distributions. considered  t o be  useful  to as  a  - 105 F i g u r e 3.7.2.2.1 CCD o f In-Labeled, 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f graph h e i g h t ; CCDC 1-120, a b s c i s s a . i : L 1  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 o f C r - L a b e l e d , LDP, IDP, HDP, HHD: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120, a b s c i s s a . 5 1  - 106 F i g u r e 3.7.2.2.3 CCD o f C r - L a b e l e d LDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f graph h e i g h t ; CCDC 1-30, a b s c i s s a . 5 1  F i g u r e 3.7.2.2.4 CCD o f C r - L a b e l e d IDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 31-60, a b s c i s s a . 5 1  - 107 F i g u r e 3.7.2.2.5 CCD o f C r - L a b e l e d HDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f graph h e i g h t ; CCDC 61-90, a b s c i s s a . 5 1  Figure 3.7.2.2.6 CCD o f C r - L a b e l e d HHD, P l a t e l e t s o f D e n s i t y G r e a t e r than t h a t o f 2 0 % S t r a c t a n : p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f graph h e i g h t ; CCDC 91-120, a b s c i s s a . 5 1  - 108 "''"''"''Indium, by taken  up  through  predominantly dalton  virture the  i t s complexing  with hydroxyquinoline,  hydrophobic  bilayer  and  the  platelet  cytosol  associated  (Hudson e t  a l . , 1979  (80%)  molecule  of  -  in  and  1981;  is  finally  located  with  Baker  et  is  a  50,000  a l . , 1982).  51 Cr  is  likewise  Baldini,  found  1970).  predominately  "^In  is  considered  s u r v i v a l t i m e a n a l y s e s (Thakur e t 1980)  than  in  the  a  better  a l . , 1976;  Born,  life,  s u p e r i o r gamma camera i m a g i n g  (Riba et  1978;  Heaton et  a l . , 1980  1980;  Lindenfeld  a l . , 1979; and  (Schmidt  et  function  (Scheffel  a l . , 1983;  e t a l . , 1982a and for  entire  of  properties population t o be and  Hill-Zobel a l . , 1977;  et  of  11  population  in  whereas  population  and  greater  specific  Gear, 1981;  exhibits  a  longer  it  Schmidt  evidence, presented  more  evenly  labels  preferentially  with  distinct  activity  than  label  membrane the  rest  the a  surface the  Gear, 1981). T h i s p o p u l a t i o n i s  said  circulation  time 51  of  Cr  than as  the  i n anomalous s u r v i v a l  specific  major p o p u l a t i o n o f p l a t e l e t s , but 51  concentrations  of  uptake  Cr  are  appears  also  recommended f o r the p a t i e n t  normal  a platelet  r e l a t i v e t o the  are not  vivo  Rasmussen, 1979;  this  and  in  platelet  concentrations  analysis  et a l . ,  i n terms o f a g g r e g a t i o n p o t e n t i a l , more dense  c o n c e n t r a t i o n s would r e s u l t  high  Lotter  of  p o p u l a t i o n d i s t r i b u t i o n . Thus, use  these  half  Davis et a l . ,  recovery  and  and  normal  "'"'"Cr can  platelet  young, more a c t i v e  a l . , 1983),  that  e t a l . , 1977  1982;  there i s further  the  (Haver and  and  and  platelet  reasons: short  platelet  Schmidt and  "'"In  of  for  a l . , 1979;  1982), b e t t e r  In a d d i t i o n ,  preference  platelet  portion  b).  a variety  Heyns e t  Steele,  et  for  5  (Steiner  label  Scheffel  1979;  here,  is *Cr,  cytosol  to  times. be  platelet  l a b e l at At  less  very  for  high  dramatic,  p l a t e l e t s labeled  unsuitable  low  at  survival  ( B e l c h e r e t a l . , 1977).  - 109 Because in  "''"'""'"In i s a l s o  Regions  platelet  I and  known t o l a b e l  I I with  distributions,  partitions  the s p e c i f i c  p l a t e l e t , being h o r i z o n t a l  l e u k o c y t e s which  lower  than  activity  distribute  t h e mean o f  i n terms  throughout the p l a t e l e t  normal  o f " ^ I n per  distribution,  could  be e v i d e n c e o f t h e l e u k o c y t e - f r e e n a t u r e o f t h e s e p l a t e l e t  preparations  and/or  labeling  an i n d i c a t i o n  ^*In  relative  platelet time.  t o ^ C r . In t h e l a t t e r  s p e c i f i c , and a g a i n more u s e f u l  The  even  distribution  enrichment i n aggregation greater  label  case,  platelets  as more  i n assessing p l a t e l e t  survival  o f " ^ I n might  also  indicate  an i n d i c a t i o n  of p a r t i c l e s  subpopulation  which  exhibits  51  of a  size  i s more  distribution.  enhanced  uptake  for  51 Cr  i s apparent  radioactive well  when  platelet  the d i s t r i b u t i o n labels  such  as  as when compared t o t h e p l a t e l e t  active  of  Cr i s compared  ^C-serotonin,  or  to other ^"*"In,  number. T a k i n g advantage  uptake f o r ^ C r by a young s u b p o p u l a t i o n o f p l a t e l e t s ,  apparently  homogeneous  double l a b e l e d  with  labeling  of  t h e s e two n u c l i d e s  platelets  by  ^"''In,  i n terms o f t h e i r c i r c u l a t i o n - a g e which  exhibit  an a c t i v e  lower p a r t i t i o n apparent  characteristic  characteristics,  of  platelet  t h e HDP,  5  the  a  Cr enrichment  population  indicates  of the and t h e  platelets  that  platelets  since the p l a t e l e t s  f o r "*"Cr demonstrate 51  than normal. That  i n an e n t i r e  characteristic  uptake  as  ( f o r example) and s u b j e c t e d t o  CCD might p r o v i d e means f o r q u a l i t a t i v e l y a s s e s s i n g p a t i e n t s '  is  this  platelet  o f s u c h ) , which  i n t h e low p a r t i t i o n r e g i o n I I o f t h e p l a t e l e t  platelet  that  i s n o t due t o (perhaps CCD i n d u c e d )  t o be  by  ^ " ^ I n i s seen  ( i f one c o n s i d e r s t h e d i s t r i b u t i o n  than  pronounced The  of a greater preference f o r p l a t e l e t  characteristic o b t a i n e d by CCD  and i s not a partition  separations of t h i s p l a t e l e t subpopulation.  singular  p r o v i d e s more  - 110 3.8  Countercurrent The  taken  cohort up  in  circulation  the  which  "^S-labeled  platelet  from one  when  megakaryocyte  platelets  This of  o f In V i v o 35-S-Labeled  S-sulphate,  by  into  circulation. rise  label  vivo  incorporated  the  Distribution  -  injected in  the  are  platelet  into bone  then  rabbit  marrow  injection  is  into  found  (p.i.),  is  and  when h a r v e s t e d  radioactivity  t o f o u r days a f t e r  a  released  cohort,  incorporated  Platelets  the during  in  the  depending  on  35 the  quantity  young  of  S  platelet  injected,  subpopulation.  r e l e a s e d , young p l a t e l e t density  fraction  platelet  but  CCD  might be  for  separation  combination  subpopulation  S  be  indicative  cohort  labeled,  r e l a t i v e l y enriched a low  of  than  t h i s . young  platelet  i n the  density  and  surface  but  property-based  high  partition in a l l  density gradient cohort,  a  newly  r e g a r d l e s s o f d e n s i t y . T h i s i n d i c a t e s not  more s e l e c t i v e  of  of  This  to  i s c h a r a c t e r i z e d as h a v i n g  subpopulations  that  i s considered 35  only  centrifugation also  that  cell  the  separations  p r o v i d e s g r e a t e r s e p a r a t i o n o f young p l a t e l e t s than does d e n s i t y a l o n e . 35 These analyses of newly released S-cohort-labeled platelets, Chapters 3.8 through 3.12, involve eight (not all presented) experiments  i n two  general  c a t e g o r i e s . Four  experiments  involved  the  35 isolation  of  S-labeled  (day  and  day  two  platelets,  primarily  s i x ) , harvesting  a t two  platelet  time  density  points p . i .  subpopulations  35 (  S-LDP,  platelets,  -IDP, and  and CCD;  -HDP) a  using  total  of  Stractan 21  gradient  distributions.  centrifugation Four  of  experiments  35 involved (daily) second 22  the  isolation  p . i . , from label  day  ( C r , or 5 1  distributions.  of one 3  S-labeled through  H-serotonin)  platelets day  at  shorter  intervals  s i x , double-labeling with  in vitro,  and  CCD,  a  total  a of  - Ill In t h e experiment subpopulations  presented  were  -  i n S e c t i o n 3.8,  double-labeled,  newly r e l e a s e d p l a t e l e t  first  in  vivo  by  staggered  35 injections  of  low  levels  of  S-sulphate.  Blood  samples  a n i m a l s were drawn. P l a t e l e t s were h a r v e s t e d and  labeled  low  the  levels  of  5  "*"Cr-chromate  p o p u l a t i o n (however u n e v e n l y ,  which  labeled  from a l l  i n v i t r o with  whole  platelet  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  s i m u l t a n e o u s l y s u b j e c t e d t o CCD. In a d d i t i o n , t h e time 35 (kinetics) of i n vivo, 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  course was  determined  by  following  o b t a i n e d from t h e b l o o d 3.8.1  the  S  activity  in  various  samples.  Experimental  Sulphate 0.5  ml  Injection:  (200  uCi;  A t , T=0 -9  9.3  x  ( l o t no 094394) and  0.2  syringe  into  and  needle,  10  (day o f e x p e r i m e n t ) , 35 moles)  of  S-sulphate  ml o f s a l i n e were i n j e c t e d the  left  the cohort  marginal  stock  using a  B l o o d Samples: On from  each o f t h e  and  30  ml  sterile each,  A.34).  2 >  follow  sterile  c e n t r i f u g e tubes  42.0  taken  g; from  and  22  Table A.34).  Argyle Medicuts  samples were p l a c e d  g; 23,  (R)  Cannulas  i n separate  c o n t a i n i n g 6 ml  22,  from  39.0  number 23,  50 of  ml ACD  r a b b i t number 26  g and  (Appendix  Table  t h e r e f o r e , 1 ml  was added a t t h e t i m e o f draw.  Platelet MgCl  38.7  Using  tuberculin  30 ml o f b l o o d were taken  weighed r e c o v e r y o f b l o o d  A d d i t i o n a l b l o o d was  more o f ACD  o f experiment,  blood  polypropylene  weighed. The  38.07 g; 24,  day  four r a b b i t s .  s y r i n g e s , the  Corning  and  T=4,  label,  solution  ear v e i n o f r a b b i t  (5.5 k g ) . Other r a b b i t i n j e c t i o n s were s t a g g e r e d (Appendix  was  fractions  0.02%  P r e p a r a t i o n : Tyrode's EGTA,  previously  and  with  described  solution  albumin  and  platelet  was  prepared  dextrose. preparation  An  with attempt methods  0.02% to by  - 112 centrifugation  a t 250  g f o r 10  minutes  failed,  most p r o b a b l y due  to  d i f f e r e n c e s i n t h e volume o f b l o o d b e i n g c e n t r i f u g e d , and t h e r e f o r e t h e time o f c e n t r i f u g a t i o n being  required  was  extended  t o 15-20  t o c o m p l e t e l y sediment  were c e n t r i f u g e d a t 10-1100 RPM  minutes  w i t h 20  minutes  a l l e r y t h r o y c t e s . Blood  samples  i n t h e IEC SBV  f l o o r c e n t r i f u g e a t room  t e m p e r a t u r e f o r 10 m i n u t e s , and  f o r 10 minutes a g a i n . From each b l o o d  sample,  of  PRP  was  r e c o v e r e d . The  remainder  resuspended  to  50  ml  Tyrode's  blood  a p p r o x i m a t e l y 15 samples  centrifuged  were  again  ml  as  above.  The  each  platelet  rich  s u p e r n a t a n t s were h a r v e s t e d , c o n c e n t r a t e d i n one PRPs, which were then weighed 23,  14.7  g;  and  22,  14.6  with  ml  Tyrode's and  ( r a b b i t number 26, 16.12  g ) , sampled,  and  counted  added  g; 24,  of and  (PRT) to the 18.5  g;  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 : concentration. platelets  in  Platelet  "^Cr-CrO^ PRP,  at  uCi/ml.  Incubation  was  platelet  samples  were  samples  are l a b e l e d  with  ( l o t 23/1BA; a t 1 mCi/ml) was a  final  concentration  f o r one  hour  diluted  to  in a 50  ml  of  Cr  at  added  low  t o the  approximately  37°C water with  5 1  16.7  bath. Labeled  Tyrode's  solution,  c e n t r i f u g e d , washed t w i c e , and resuspended i n 3 ml o f 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 t h e CCD 9  l o a d m i x t u r e s (Lx) were a d j u s t e d t o e q u a l c o n c e n t r a t i o n (2.8  x  i n upper  10 /ml)  phase.  The  transfer  CCDC were l o a d e d w i t h 0.5  o f l o w e r phase and 0.8 ml o f upper phase. The 31-33,  61-63  ml  load  of  respectively.  ml  l o a d c a v i t i e s , CCDC 1-3,  and 91-93, were l o a d e d w i t h 0.5 ml o f l o w e r phase and mixture  (Lx)  Twenty-seven  each  from  transfers  samples  R26,  R24,  R23  and  0.9 R22,  were c o m p l e t e d , a t 4-5°c, w i t h  30 second m i x i n g t i m e and a s e t t l i n g t i m e o f 7 m i n u t e s .  a  - 113 A n a l y s i 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 : Upon c o m p l e t i o n , Tyrode's mixed,  s o l u t i o n was added  collected,  particles  t o each  0.7 ml o f  o f t h e CCDC, t h e c o n t e n t s  and each CCDC was sampled  and counted  ( C e l l o s c o p e , 48 um d i a m e t e r o r i f i c e ,  were  for platelet  100 u l v o l u m e t r i c  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 a s s a y e d f o r  S and  51 Cr  radioactivity  o f 1 ml i n 10 ml o f A t o m l i t e , u s i n g  s c i n t i l l a t i o n counter. load mixtures ml  I n p a r a l l e l , samples o f b l o o d , PRP, PPP, and t h e  were a l s o c o u n t e d  (Appendix T a b l e A.36). A p p r o x i m a t e l y  samples o f t h e CCDC were a l s o counted  a LKB Compugamma gamma c o u n t e r the  level  a P h i l i p s 4700  of i n vitro  f o r "'"'"Cr r a d i o a c t i v i t y  with automatic  ^ C r - labeling  1  using  quench c o r r e c t i o n . S i n c e  was h i g h  (relative  to i n vivo  35 S  labeling),  t h e r a d i o a c t i v e CCDC samples  three h a l f l i v e s (Tl/2) of  5 1  3.8.2  Recovery:  t h e r e were r e c o v e r e d 8.49  + 1.9 x 1 0  10  2.55  counted  when  C r and one T l / 2 o f S had passed. 3 5  Results Platelet  x  were a g a i n  9  particle greater  counting  15.08 + 1.88 (8) g o f PRP, c o n t a i n i n g a t o t a l o f  p e r m l , and t h e r e f o r e  a blood  there  were  Counting: 51  with  phase system r a n g i n g radioactivity  of a  size  however, n o t a l l o f t h i s was c e l l u l a r examination,  and most was  r e p r e s e n t a t i v e o f very l a r g e p l a t e l e t s and p l a t e l e t  standardized  concentration of  1.53 + 0.27% (4) p a r t i c l e s  based on m i c r o s c o p i c  Radioactivity  o f 5.58 + 0.67  ( 8 ) p l a t e l e t s p e r ml (Appendix Table A.35). Based on  than t h a t o f a p l a t e l e t ;  contamination,  from 33.35 +_ 1.11 (4) g o f b l o o d ,  (8) p l a t e l e t s , a t a PRP c o n c e n t r a t i o n  (4) p l a t e l e t s +_ 0.6 x 10  On a v e r a g e ,  probably  aggregates.  The P h i l i p s 4700 S c i n t i l l a t i o n  Counter was  35 C r and  S, quenched  with  varying  from 0.5 ml t o 5.0 m l , w i t h  and s c i n t i l l a t i o n  fluid,  equal  10 ml A t o m l i t e .  quantities of quantities of The e x t e r n a l  - 114 standard  slightly  within  t h i s l a r g e range o f v i s i b l e q u e n c h i n g , and q u a n t i t i e s o f phase  system:  average 1.559  ratio  ESR +  (ESR)  1.564  0.032  CCDC samples  f o r these standards v a r i e d  +  0.042  ( 1 0 ) , range  was  D u a l Channel  (CH)  with  1.51-1.62  for  t h e ESR  3 5  S.  for  The  d i d not  5 1  Cr;  and  ESR  of  the  quenching  vary  (from 1.55-1.56)  CCD. Program  51  load  1.51-1.61  c o n s t a n t , and  t h r o u g h o u t the e n t i r e  standardized  ( 1 0 ) , range  only  14 was  used  (CHI, 0.4-6.0; CH2,  15-80),  35 Cr  and  S.  m i x t u r e s were c o u n t e d  once  Samples for I  o f b l o o d , PRP, minute  PPP,  (Appendix  and  Table  CCD  A.36).  Samples were r e c o u n t e d f o r 5 m i n u t e s each, t w i c e (Appendix T a b l e A.36). Again with  program 3  H  and  distribution 3 5  S  within  14 1 4  was C,  used as  o f decay the  (CHI, major  can ^C  be  0.4-8.0; CH2, part  of  counted  channel.  30-1700), s t a n d a r d i z e d  the  i n the H 3  The  5 1  Cr  radioactivity  channel  combined  and  that  of  radioactivities,  n o r m a l i z e d as a p e r c e n t a g e o f day one a c t i v i t y , were c a l c u l a t e d and a r e p r e s e n t e d as an average o f s i x measurements ( T a b l e 3.8.2.1). T a b l e 3.8.2.1 Summary o f  3 5  S-Sulphate Labeling Kinetics  Time Day Hour  Blood A c t i v i t y % T=l  PRP A c t i v i t y % T=l  PPP A c t i v i t y % T=l  CCD P i t Lx A c t i v i t y % T=l  1 2 3 4  100 38.31 + 16.68 + 19.23 +  100 51.75 + 8.05 28.05 + 6.52 27.96 + 6 . 5 2  100 53.25 + 28.53 + 30.27 +  100 123.30 + 12.92 130.11 + 9.94 87.89 + 19.17  21.0 45.5 68.25 93.5  3.8.3  2.91 4.75 3.83  4.55 6.74 7.44  Discussion 51  Sulphate much  higher  samples  were  Labeling (more  Kinetics:  efficient)  stored  and  S i n c e the than  later  the  counted  3 5  Cr S  in vitro  i n vivo  again.  The  labeling  labeling, time  was  these  course  of  - 115 35  S-sulphate  platelets  labeling was  megakaryocyte, their age  kinetics  revealing. release  The  during were  of the cohort  the r i s e of p l a t e l e t taken  t o be newly  the  blood,  incorporation  of labeled  removal must be f o l l o w e d character  within  platelets  i n order labeled  into  young  label  PPP,  and  into  the  t h e c i r c u l a t i o n and  t o make i n f e r e n c e s  platelets.  incorporated  released  of  PRP,  about t h e  Platelets,  radioactivity platelets  harvested  i n t h e plasma,  until  t h e 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 d a t a  were p r e s e n t e d  as  day one a c t i v i t y . The b l o o d r a d i o a c t i v i t y  S c o u n t s as a p e r c e n t a g e o f p e r ml d e c r e a s e d from day one  u n t i l day t h r e e , a f t e r which i t was l e v e l . I n p a r a l l e l , t h e PRP and PPP activities  a l s o decreased,  b u t were h i g h e r  than  the blood,  since the  i n c o r p o r a t i o n o f t h i s l a b e l i n t o t h e e r y t h r o c y t e s was n e g l i g i b l e t i m e p o i n t s measured. The PPP a c t i v i t y was found higher  a t a l l time p o i n t s  than  t o be o n l y  t h e PRP, i n d i c a t i n g  that  at a l l  slightly  much o f t h e  35 S a c t i v i t y was a l s o i n t h e plasma, and perhaps t h a t t h e volume o f p l a t e l e t s i n PRP was n o t 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 t h r e e were s u c c e s s i v e l y h i g h e r , b u t on day f o u r d e c l i n e d , 35  indicating the  that  t h e peak e n t r y  of i n vivo  S-labeled  platelets  into  c i r c u l a t i o n was r e a c h e d , i n t h i s e x p e r i m e n t , between days two and  f o u r , a s was found by o t h e r s Countercurrent  ( H i r s h e t a l . , 1968).  experiments 35 51 a r e n o t shown but a r e d i s c u s s e d . On day one p . i . , t h e S/ Cr r a t i o t h r o u g h t h e d i s t r i b u t i o n was skewed s l i g h t l y t o t h e r i g h t o f t h e platelet  Distribution:  population,  suggesting  The CCD c u r v e s  that  platelets  f o r these  which  had been l a b e l e d  35 in with  vivo 5 1  with  S,  C r i n vitro;  were  of higher  partition  ( b u t a r e o f lower  than  platelets  labeled  p a r t i t i o n than the d i s t r i b u t i o n  - 116 of  platelet  particles  and  the d i s t r i b u t i o n  shown). T h i s might a l s o be an i n d i c a t i o n  of ^H-serotonin;  t o be  o f t h e r e l a t i v e enrichment o f  51 Cr On  uptake day  i n t h e low p a r t i t i o n  two  area  p . i . , the ^ S / ^ C r  of the p l a t e l e t  ratio  was  distribution.  horizontal,  distributed 35  evenly label  through  the p l a t e l e t  indicating 51  i n v i v o on day two approaches t h a t o f 35  day  distribution,  three  the  indicating  that  platelet  that  the  S  Cr u p t a k e i n v i t r o .  On  51 S/  Cr  ratio  was  shifted,  at t h i s  time,  still  during  r e l e a s e , the cohort  i s o f lower  enriched the r i s e  partition  to  the l e f t , 35 of S-labeled 51  than  Cr l a b e l e d  p l a t e l e t s , which a r e l o w e r than t h e 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 o f 35-S-Labeled LDP, IDP, and HDP, Day Two and S i x , p . i . Newly  released  " S-sulphate, 55  platelet  harvested  cohorts  on days  were  they  These t i m e s ,  represented  being  young  circulation,  platelet  and d u r i n g and day  and a n a l y z e d  cohorts the r i s e  vivo  with  of different  selected  since  age c h a r a c t e r , day two  of labeled platelet more  by  by c o u n t e r c u r r e n t  day two and s i x , had been  s i x being  platelet radioactivity  in  two and day s i x p . i . , s e p a r a t e d  Stractan density gradient centrifugation distribution.  labeled  mature,  entry  during  into the  the d e c l i n e o f  ( P l t L x , T a b l e 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  found t o be e n r i c h e d opposite  S-sulphate  i n the high  labeled  density p l a t e l e t  was found i n t h e low d e n s i t y p l a t e l e t  platelet  cohort  was  (HDP) f r a c t i o n ; t h e  (LDP) f r a c t i o n .  On day  35 six  p . i . , this  density platelets  difference  fractions decreased  circulation,  was  less  their  in  S-specific  dramatic.  These  activity  i n the  three  findings  indicated  that  mean d e n s i t y as a f u n c t i o n o f m a t u r a t i o n i n  t h e newly r e l e a s e d p l a t e l e t  cohort being  enriched  i n HDP.  - 117 Membrane density cohort  surface  separated not  property  platelet  only  had  CCD  analyses  subpopulations  different  of  indicated  density  35  these that  characteristics  S-labeled,  the  platelet  but  also  had  d i s t i n c t i v e s u r f a c e p r o p e r t i e s which were more pronounced on day two i n 35 comparison enriched  to  day  i n the  s i x . The  low  p . i . , an enrichment 3.9.1  specific  partition  CCD  activity  platelet  of  S-sulphate  region  ( I I ) , on  two  kg)  was  (71  hr)  which d e c r e a s e d on day s i x .  Injection:  At 4:35  i n j e c t e d w i t h 5 ml, 1.67 B l o o d Sample and p.i.,  PM,  mCi, o f  3 5  T=0  days,  Rabbit  S - s u l p h a t e stock  45  ml  of  blood  was  taken  (4.95  solution.  P l a t e l e t P r e p a r a t i o n : At 3:30  approximately  27  PM,  and  day  mixed  two i n two  C o r n i n g p o l y p r o p y l e n e c e n t r i f u g e tubes w i t h a t o t a l o f 10.5 quantity  o f b l o o d and  ACD  was  measured t o be  27.5  weighed f o r a t o t a l o f 60.78 g from which one ml was each  tube  was  (1,200-1,400) RPM supernatant  was  w i t h Tyrode's, diluted two  day  Experimental Sulphate  in  was  centrifuged contained  and at  to  for  minutes.  and  of  80  and  sampled. The and ml  blood  c e n t r i f u g e d at of  diluted  two  o f PRT,  2,200 RPM  one  (PRT).  were  for  15  minutes  PRP  sampled.  four p l a t e l e t preparations, t o 50  ml  (IEC  SBV).  erythroycte  finally  taken,  The  were d i l u t e d  platelets,  was  again  b l o o d p e l l e t s were resuspended  Tyrode's  negligable  was  total  p l u s 30 ml,  of  from  ml)  A  Tyrode's  The  c e n t r i f u g e d a g a i n as above w i t h s i m i l a r r e c o v e r y  Platelets  (40-45  with  ml ACD.  ml  rich  ml  ml  ml  t o 50  discarded. which  15  50  r e c o v e r e d . The  platelet  o f PRP,  diluted  ml  50  mixed  day  i n 8.6  s i x p . i . , at  with  Tyrode's  PRT  ACD  measured (52-52.5 ml) and weighed (54.46 g ) . The  (6  ml)  ml  b l o o d was  and  of  5:00 in  and  supernatant  contamination,  resuspended  On  with  was  Tyrode's PM,  two  blood tubes,  diluted  with  - 118 Tyrode's  (to a total  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 p r e p a r e d as on day two. D e n s i t y G r a d i e n t S e p a r a t i o n : On day two, and a g a i n on day s i x , t h e platelet  c o n c e n t r a t e s (8.5 m l ) were  layered  equally  (about 2 ml) on  f o u r 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 o f c o m p o s i t i o n s 1 ml 10%, 3 ml 16%,  3 ml 18%, and 3 ml 2 0 % S t r a c t a n . The g r a d i e n t s were b a l a n c e d and  centrifuged  for 2  temperature  i n the Sorval  gradients  hours  at  10,000-11,000  RC5, u s i n g  were h a r v e s t e d from  RPM,  23,000  g,  t h e s w i n g i n g bucket  t h e t o p ; LDP from t h e f i r s t  at  room  rotor.  The  5 m l , IDP  from t h e next 5 m l , and HDP from t h e next 3 m l , l e a v i n g t h e remainder of the gradient. Each  platelet  centrifugation  s u b p o p u l a t i o n was washed  i n 40 ml o f Tyrode's  a t 10,000 RPM f o r 10 m i n u t e s ,  Tyrode's,  and sampled  analyses.  The s u b p o p u l a t i o n s were a g a i n washed  above,  and each  (0.1 ml) f o r p l a t e l e t  resuspended  finally  resuspended  by  i n 5 ml o f  number and r a d i o a c t i v i t y i n 40 ml Tyrode's  i n 3.1 ml o f upper  phase  as  (load  m i x t u r e ) 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 l o a d e d w i t h 0.9 ml l o a d m i x t u r e s (CCDC 1-3, LDP; 41-43, IDP; 81-83; HDP) and 0.5 ml l o w e r phase. The t r a n s f e r ml  lower  with  30  and 0.8 ml upper seconds  shaking  phase. time,  Thirty-seven  and 7  minutes  CCDC r e c e i v e d 0.5  cycles  were  settling  completed  time.  Upon  c o m p l e t i o n , t h e c o n t e n t s d f each CCDC were d i l u t e d w i t h 0.7 ml Tyrode's s o l u t i o n , mixed, h a r v e s t e d and a n a l y z e d 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 . Platelet initially  and R a d i o a c t i v i t y  diluted,  C o u n t i n g : Samples from  1/247 i n D i l u i d  coincidence errors).  t h e CCDC  were  (and a g a i n where n e c e s s a r y t o a v o i d  LDP, IDP, and HDP l o a d m i x t u r e s , samples  from t h e  - 119 gradient  LDP,  IDP,  and  HDP,  -  the  blood,  and  counted u s i n g  p o p u l a t i o n , were a l s o d i l u t e d  and  the  unseparated  the C e l l o s c o p e  68, T 15-95, l i n / d e l t a , 48 um d i a m e t e r o r i f i c e , and Samples from each CCDC (1.5  ml)  platelet  100  (C 1,  G  u l volumetric).  i n 10 ml o f A t o m l i t e were counted  35 (1,  5,  and  counter  10  minutes)  (Program 9 CHI  were saved and  for  S  radioactivity  0.4-130.0, CH2  analyzed  concurrently  using  0.4-2). The with  Philips  4700  samples from day  t h o s e o f day  u s i n g a double l a b e l program (14 CHI 0.4-8.0, CH2 3.9.2  a  two  s i x ; and  again  30-1700).  Results Stractan  densities the  Density  Gradient:  (g/ml) o f t h e  results  of  The  gradient  others.  Stractan  steps  The  (StR)  concentrations  were measured, and  gradient  conditions  and  compared  and  to  Stractan  Q  concentrations  of  Rand  et  a l . (1981a) were:  3.5  x  10  platelets/ml 9  layered  on  l a y e r e d on and  gradient 15%,  Stractan  15.5%, 17.5%, 19%  17%,  19%  Stractan  concentrations  (1982) were a l s o t a b u l a t e d the  s p e c i f i c density  0.003949 density  +  or  of Stractan  gradient  step,  average g Tables estimate  /%  A.37  could  Table  and  A.38).  as  study.  platelets/ml  method  of  Corash  densities and  be  +  0.0000597  (12).  s o l u t i o n s used i n t h i s  Shafer  study,  Density-^  was  c a l c u l a t e d from the 2  was  calculated combined  c a l c u l a t e d based  based data  on  the  from measurements o f  was on the  specific  used t o c a l c u l a t e the d e n s i t y o f a  Density  data,  Based  0.004113 + 0.0002473 ( 4 ) . The  o f 0.004 g /% S t r a c t a n , o b t a i n e d  used i n t h i s  10  i n terms of grams p e r % S t r a c t a n  Stractan  3.9.2.1.  Stractan  the  0.003946  s p e c i f i c d e n s i t y o f S t r a c t a n was density  x  (Appendix T a b l e A.38). Based on t h e s e  (9),  measurments o f t h e  2  (Appendix Table A.37). The  after  of Stractan,  0.0000695  Stractan;  on  given the  (Appendix average Stractan  - 120 T a b l e 3.9.2.1 S t r a c t a n C o n c e n t r a t i o n and D e n s i t y % StR (g/ml)  Density^ (g/ml)  Density2 (g/ml)  % StR (g/ml)  Density^ (g/ml)  Density2 (g/ml)  10 11 12 13 14 15  1.039 1.043 1.047 1.051 1.055 1.059  1.040 1.044 1.048 1.052 1.056 1.060  16 17 18 19 20 21  1.063 1.067 1.071 1.075 1.079 1.083  1.064 1.068 1.072 1.076 1.080 1.084  Platelet of  a size  experiment  Recoveries: greater  (Appendix  that  The r e c o v e r i e s o f p l a t e l e t s of platelets  were  and o f p a r t i c l e s  calculated  throughout  the  A.39). 35  Countercurrent labeled  LDP,  respectively; 3.9.2.1.2). quantities  Distribution:  IDP, and Day  The  Two entire  HDP  The CCD  a r e shown  p . i . , Figure CCD  distributions (CCDC  3.9.2.1.1  i s given  and  S-cohort  41-80,  81-120,  Day  f o r comparison  S i x , Figure of  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 35  The r a t i o o f r a d i o a c t i v i t y , i n t h i s case (dotted  1-40,  of  l i n e ) with  t h e mean s p e c i f i c  relative comparison.  S per p l a t e l e t , i s p l o t t e d  activity  (noted  inside  the l e f t  o r d i n a t e ) a t one h a l f t h e h e i g h t o f t h e o r d i n a t e . 35 The  enrichments  subpopulation  were  by  CCD  compared  activity  and s l o p e  analyzed  by t h e n o n p a r a m e t r i c  o f the  S-labeled  by c a l c u l a t i o n  o f the p l o t  cohort  within  o f t h e average  o f s p e c i f i c a c t i v i t y versus  ( P ) "Runs T e s t "  each  specific CCDC, and  ( T a b l e 3.9.2.2). On day  two p . i . , t h e mean s p e c i f i c a c t i v i t y , and t h e magnitude o f t h e s l o p e were g r e a t e s t f o r HDP i n c o m p a r i s o n t o IDP, and LDP ( i n s u f f i c i e n t d a t a w i t h i n 10% CE f o r complete c a l c u l a t i o n s ) . activity  o f a l l the subpopulations  On Day s i x p . i . , t h e s p e c i f i c  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 t h e d i s t r i b u t i o n o f s p e c i f i c a c t i v i t y was random.  - 121 F i g u r e 3.9.2.1.1 CCD o f Day Two 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 p e r CCDC ( s o l i d l i n e ) left o r d i n a t e ; r a d i o a c t i v i t y p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt a t 10% CE ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3 3  F i g u r e 3.9.2.1.2 CCD o f Day S i x S - L a b e l e d LDP, IDP, and HDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt a t 10% CE ( d o t t e d l i n e ) w i t h mean at one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3 5  - 122 T a b l e 3.9.2.2 A n a l y s i s o f Day Two CE% Mean + S.Dev. n Corr Slope Intcp P  S p e c i f i c A c t i v i t y Enrichment S l o p e  LDP CPM/Plt 10 1.25 x 10-6 2.82 x I O " 2  IDP CPM/Plt 10 1.89 x I O " 3.15 x I O " 8 -0.987 -1.27 x I O " 2.33 x I O " 0.029  HDP CPM/Plt 10 2.51 x 10-6 A.90 x I O " 6 -0.941 -2.46 x I O " 3.13 x I O " 0.10  6  8  7  7  7  6  Day S i x CE% Mean + S.Dev. n  100 1.65 x I O " 5.32 x I O " 4  10 1.22 x I O " 6.55 x I O " 7  6 8  7  6  10 1.56 x I O " 8.22 x I O " 4  6 8  6 8  3.9.3 D i s c u s s i o n The  i n j e c t i o n o f a r a d i o a c t i v e compound t o l a b e l a c o h o r t  released  platelets  Karpatkin, Clift  i s often  used  1974; K a r p a t k i n ,  (Amorosi e t a l . , 1971; Charmatz and  1978; McDonald, 1976, 1978, 1980 and 1981;  and MacDonald, 1979; McDonald e t a l . , 1979; D a s s i n  Rand e t a l . , 1981a; Corash and S h a f e r , On  day  circulation specific  two, d u r i n g and when  CCD  the r i s e  of "^S-platelet  the greatest  difference  contains  more  3 5  S  i s found  per p l a t e l e t .  detectable a c t i v i t y i n a l l three density Since  release  (Rand e t a l . , 1981a; Corash and S h a f e r ,  subpopulation  rabbit  e t a l . , 1979;  1982).  a c t i v i t y between t h e most dense and t h e l e a s t  subpopulations  o f newly  into  the  i n relative  dense  platelet  1982), t h e HDP  However,  there i s  fractions.  p l a t e l e t s u r v i v a l time i s approximately  f o u r days, and 51  platelets  do  survive  up  to  s i x days  (Section  3.6,  Cr-platelet  s u r v i v a l ) , r a d i o a c t i v i t y i n LDP a t two days might be due t o i n c o m p l e t e correlation gradient  of density  separation  and  platelet  age, i n c o n s i s t e n c y  of  density  ( C o r r y e t a l . , 1981; M a r t i n and Trowbridge,  1982b),  - 123 platelet  alteration  by  procedure  Holme and Murphy, 1983), (Mezzano e t a l . ,  (Bolin  o r t o young p l a t e l e t s b e i n g  e t a l . , 1981; the least  dense  1981).  However, i n a l l p l a t e l e t day  and g r a d i e n t  d e n s i t y s u b p o p u l a t i o n s a n a l y z e d by CCD on  two p . i . , t h e i n v i v o " ^ S - s u l p h a t e  platelet  cohort  i s strongly  and c o n s i s t e n t l y e n r i c h e d a t t h e lower end o f t h e p l a t e l e t CCD. On day six,  the levels  of "^S-sulphate  activity  equal, i . e . , the l e v e l o f a c t i v i t y has  increased, suggesting  t h i s experiment level  t h e CCD a r e more  o f t h e t h e LDP r e l a t i v e  t h a t HDP become l i g h t e r  t o t h e HDP  w i t h maturation. In  ( w i t h t h i s q u a n t i t y o f i n j e c t e d c o h o r t ) , on day s i x t h e  of activity  distributed  throughout  through  t h e CCD o f LDP, IDP, o r HDP i s more e v e n l y  (horizontal). 35  Therefore,  not o n l y  a dense young p l a t e l e t platelets  with  distinguishable systems,  S-sulphate  relative  findings  "^S-sulphate  by  membrane CCD  also  indicate platelet  surface  i n charge  to the p l a t e l e t  labeled  cohort  correlate  properties,  sensitive  two-polymer  a low ( s u r f a c e charge  population consisting  that  surface  thus  property  c o h o r t s a r e more s e l e c t i v e  apparent  present  i n that  the  S-sulphate  i n a l l density subpopulations  separated  within  each  by CCD,  surface  i n vivo  labeled  far phase  sensitive)  o f a l l ages. analyses  cohort  p l a t e l e t s are  on day two, b u t c a n s t i l l property  of  than d e n s i t y  a n a l y s e s . 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 o f t h i s p l a t e l e t 35 is  with  s u b p o p u l a t i o n , but a l s o w i t h a s u b p o p u l a t i o n o f  specific only  this  and c h a r a c t e r i z e d as h a v i n g  partition These  does  heterogeneity  be  within  each b e i n g a l s o c o n s i d e r a b l e . On  both  subpopulations  day  two  and  t h e percentage  day  six, in  of p a r t i c l e s  a l l density of size  fractionated  greater  than t h e  - 124 platelet than 5  (10% t o t a l  the platelet  *Cr  per  particles) CCD.  platelet,  i s t o the l e f t ,  5 1  Cr  per  "^C-serotonin,  35 per p l a t e l e t ,  partition  The f i n d i n g s o f e n r i c h m e n t s i n t h e r a t i o s  35 S  o r o f lower  and  ^Cr  per  of  In,  1 1 1  3 S  per  H-serotonin  ( t o be  shown),  being  characteristics  of platelets  o f low p a r t i t i o n r e l a t i v e t o t h e p l a t e l e t  CCD,  interpreted  in  can  be  two  ways.  In  the  most  dramatic  interpretation  t h e enrichment i s due t o younger, l a r g e r p l a t e l e t s  a  negative  lower  net  sensitive  partition.  aggregates, platelet the  requiring  area  unreasonably  high  leukocytes  also  sedimenting  or leuckocytes. these  charge  results  and/or  where  radioactivity  and/or  The  occurring  count),  same  surface  and  might  platelet  and  aggregates  r e l y a l s o on e v i d e n c e  due  CCD  lower to  specific  a r e found,  activity  1982).  platelet later  although i n  i s insignificant,  t o change  label  charge  (escaping  Leukocyte c o n t a m i n a t i o n ,  enrichments  Shafer,  be  during  CCD, even c o n s i d e r i n g g r e a t e r  (Corash  therefore  with  uptake  Arguments  c o n t r i b u t i n g t o cohort  the p l a t e l e t (DPM/ug) f o r  against  leukocyte  enrichment  from CCD o f d o u b l e l a b e l e d p l a t e l e t s ;  by  CCD  leukocytes  and p l a t e l e t s , clumped o r n o t , would c o n t a i n both ^ C r and " ^ I n . 5  3.10 CCD o f 35-S-Labeled LDP, IDP, and HDP, Day Two and S i x p . i . A second  s e r i e s o f measurements o f t h i s  general  type  was  carried  o u t . Newly r e l e a s e d 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 w i t h a h i g h  level  35 of  S-sulphate,  subjected reproducing  to  CCD  results  were  again  separated  i n . charge-sensitive  according phase  to  systems,  density  and  essentially  o f S e c t i o n 3.9, w i t h t h e e x c e p t i o n t h a t on day s i x  p . i . , an enrichment o f c o h o r t p e r s i s t e d i n HDP and i n t h e low p a r t i t i o n areas o f a l l d e n s i t y s u b p o p u l a t i o n s , however s t i l l l e s s than on day two.  - 125 3.10.1 E x p e r i m e n t a l Sulphate  Injection:  On  T=0  days,  1  ml  containing  2  mCi  of  35 S - s u l p h a t e was i n j e c t e d i n t o a r a b b i t (R32). B l o o d Samples and P l a t e l e t P r e p a r a t i o n : On day two ( a s on day s i x ) , p.i.,  blood  Tyrode's  (40-45  solution  1,000-1,300 RPM (Sorval  RC5)  ml)  and  i n two  ACD  (7.5) was  sterile  Corning  diluted tubes  t o 100 ml  and  centrifuged  with at  f o r 15-20 m i n u t e s . The PRP was h a r v e s t e d , c e n t r i f u g e d  a t 5,000  RPM  f o r 10 m i n u t e s ,  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 , g r a d i e n t l o a d m i x t u r e (GLx). Density and  radioactivity  layered for  Gradient C e n t r i f u g a t i o n :  2  onto  counts  four  hours  a t 13,000  3.9.1),  solution  (Appendix  step  4-step  RPM.  gradients,  gradient,  by d i l u t i o n  Table A.40). were  density  The  was p r e p a r e d  concentrations  was t a k e n  for platelet  (80 u l o f t h e GLx) and t h e remainder  Stractan  (Section  A sample  as  and c e n t r i f u g e d  previously  of Stractan  with  a Brix  described  with  The s t o c k 22% S t r a c t a n ,  rechecked  was  Tyrode's  and g r a d i e n t -  0-30° R e f r a c t o m e t e r ,  and t h e d e n s i t i e s were measured by w e i g h i n g a p p r o x i m a t e l y 5 ml  volumes  i n 10 ml g r a d u a t e d c y l i n d e r s a t room t e m p e r a t u r e (Appendix Table A.41). The washed  density twice,  s u b p o p u l a t i o n s , LDP,  resuspended  IDP, and  i n 50 ml Tyrode's  5,000 RPM ( S o r v a l ) f o r 10 m i n u t e s , and f i n a l l y  HDP  were  solution,  harvested,  centrifuged at  resuspended  i n 3 ml t o p  phase f o r CCD a n a l y s e s . Countercurrent Sufficient filtered  phase and  Distribution: system  allowed  separatory  funnels.  separatory  funnels  f o r two to  The CCD  equilibrate  The l o w e r phases and  phase  pooled;  system  experiments at  5°C  were c o l l e c t e d  the  upper  was  again  5/4:A.  (500-550  ml)  was  overnight  in  two  from each  phases  were  of the likewise  - 126 collected. 3.9.1).  The CCD  procedures  CCDF were i n i c e water  were  as p r e v i o u s l y  described  (Section  d u r i n g a n a l y s e s . The r e c o v e r y from t h e  IEC CCDC c o l l e c t i o n t u b e s was 1.81 + 0.06 g (10) o f a t o t a l  o f 2.1 ml  added, a phase f l u i d y i e l d o f 86.2%. 3.10.2 R e s u l t s The r e c o v e r i e s o f p l a t e l e t s and o f p a r t i c l e s o f a s i z e g r e a t e r than p l a t e l e t s were a g a i n t a b u l a t e d f o r t h i s s e r i e s o f e x p e r i m e n t s  involving  35 CCD  of  i n vivo  S-sulphate  labeled,  density  separated  platelets  (Appendix T a b l e A.42). 35 CCD  of the  again p l o t t e d  S-cohort  i n vivo  labeled  LDP,  IDP, and HDP  were  (Appendix F i g u r e s A.5 t h r o u g h A.8) and t h e 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 ( T a b l e 3.10.2.1). T a b l e 3.10.2.1 A n a l y s i s o f ^ S S p e c i f i c A c t i v i t y Enrichment S l o p e 5  Day Two CE% Mean + S.Dev. n Corr Slope Int P  LDP CPM/Plt 10 8.93 x I O " 3.08 x I O " 5 -0.913 -1.78 x 1 0 ~ 1.25 x 1 0 ~ 0.5  Day S i x CE% Mean +_ S.Dev. n  100 1.35 x I O " 1.92 x I O " 4  The  7  HDP CPM/Plt 10 1.59 x 1 0 ~ 5.82 x I O " 6 -0.911 -2.83 x I O " 2.30 x I O " 0.20  6  7  6  7  7  6  6 7  7  7  6  100 1.59 x I O " 2.92 x I O " 9  7  6  100 1.85 x I O " 1.21 x I O " 3  6 7  6 7  r e c o v e r y on t h e i n d i v i d u a l d e n s i t y g r a d i e n t s t e p s o f p l a t e l e t s  (Appendix Table  IDP CPM/Plt 10 1.34 x I O " 3.19 x 1 0 ~ 8 -0.928 -1.21 x I O " 1.76 x 1 0 ~ 0.040  Table  A.44) were  experiments  A.43)  and  tabulated  involving  CCD  platelet  bound  radioactivities  f o r day two p . i . , o f a n o t h e r o f ^H-thymidine  i n vivo  (Appendix series  of  and - ^ S - s u l p h a t e  - 127 in  vivo  double-labeled,  experiments location  was  of  density  intended cellular  as  separated  an  CCD  platelets.  additional  contamination  s u b j e c t e d t o d e n s i t y f r a c t i o n a t i o n and  3.11  -  test  within  This  f o r the  series  presence  platelet  of and  preparations  CCD.  o f 35-S-Labeled P l a t e l e t s , Double L a b e l e d w i t h 3 - H - S e r o t o n i n  I n t h i s s e r i e s , p l a t e l e t c o h o r t s were d o u b l e - l a b e l e d , i n v i v o w i t h 35 a 3  high  level  of  S-sulphate,  and  in  vitro  H - s e r o t o n i n , and s u b j e c t e d t o c o u n t e r c u r r e n t To  improve  analyses  the  sensitivity  o f the c o h o r t  of  the  with  a  low  level  of  distribution.  method,  in  addition  l a b e l r e l a t i v e t o the p l a t e l e t  to  CCD  p a r t i c l e number,  35 the  in_ v i v o  p.i.)  were  analyses.  S-sulphate also  labeled  Because  distribution  age-labeled  (i.e.,  with  3  serotonin  platelets  H-serotonin  labeling  platelets  in  was  (Days 1,3,5 vitro, level  polymer t h a t CCD  the  problems  s o l u t i o n s . The  of  counting  distribution  (without p r i o r p l a t e l e t  o f d i f f e r e n t i a t i n g p l a t e l e t age 3.11.1  to  CCD the  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  b e t t e r i n d i c a t o r o f p l a t e l e t s than was  considering  prior  2,4,6  throughout  d i s t r i b u t i o n t a k e 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 a  and  electronic particle  coincidence 35 of  considered  this  and 3  5/ H  to  counting,  aggregation ratio  be  in  indicated  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  characteristics.  Experimental 35  Sulphate  Injection:  (Appendix T a b l e animals  A.45), and  on days 5 and  Rabbits blood  were  injected  samples were t a k e n  6 o f t h e experiment.  with  S-sulphate  from each o f  the  Samples were a l s o t a k e n  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 ( h a r v e s t e d on two s u c c e s s i v e days)  from  three  rabbits  - 128 were  labeled  by  incubation  3 H-serotonin  with  tryptamine,  creatinine sulphate,  .223,  66, Amersham), washed w i t h Tyrode's, and s u b j e c t e d  Batch  Platelets  and  radioactivity  500 mCi/mmole, 1 m l , 0.1  (Philips  4700  3 G- H  (5-Hydroxy  counter,  3  H  mCi, TRA t o CCD.  and  ^C  program) were d e t e r m i n e d . 3.11.2 R e s u l t s 35 CCD in  vitro  A.12; 35  distributions with  of the i n vivo  "^H-serotonin  and F i g u r e s  staggered  S-cohorts,  were p l o t t e d (Appendix F i g u r e s  3.11.2.1 and 3.11.2.2),  labeled  A.9 through  and t h e d i s t r i b u t i o n s  of  3 S/ H  ratios  which  exhibited  less  than  either  3%  o r 10% combined  e r r o r (CE) compared ( T a b l e s 3.11.2.1 and 3.11.2.2). T a b l e 3.11.2.1 A n a l y s i s o f S / H R a t i o S l o p e , CCD Enrichment 3 5  10% CE Mean + S.Dev. n 3% CE Mean + S.Dev. n Corr Slope  3  Day One R a t i o 2.33 x I O " 3.65 x 10-4 9  Day Three R a t i o 5.93 x IO"8.57 x I O " 11  Day F i v e R a t i o 6.42 x 1 0 ~ 6.91 x I O " 11  2.24 x I O " 1.54 x I O " 4 -0.815 -9.70 x 1 0 ~  5.50 x I O " 2.70 x I O 7 -0.929 -1.16 x I O  6.10 x I O " 2.23 x 1 0 ~ 7 0.176 -1.82 x 1 0 ~  3  3  4  3  4  3  4  - 4  T a b l e 3. 11.2.2 A n a l y s i s o f S / H R a t i o S l o p e , 3 5  10% CE Mean +_ S.Dev. n 3% CE Mean + S.Dev. n Corr Slope  CCD  3  5  Enrichment  Day Two R a t i o 3.66 x 1 0 ~ 2.51 x I O " 11  Day Four R a t i o 6.66 x 10"^ 3.52 x I O " 15  Day S i x R a t i o 4.81 x 1 0 ~ 2.45 x I O " 11  3.74 x I O " 1.63 x I O " 6 -0.810 -7.06 x I O "  4.56 x I O " 1.34 x I O " 7 -0.844 -5.25 x I O "  4.75 x I O " 1.33 x 1 0 ~ 8 0.542 2.94 x I O "  3  •  4  3  - 4  5  3  4  3  3  3  4  4  3  4  4  5  3  5  5  - 129 F i g u r e 3.11.2.1 CCD o f Day One, Three, and F i v e , S/ H-Labeled 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 p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; S / H a t 3% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3 5  3 5  3  3  Figure 3.11.2.2 CCD o f Day Two, Four, and S i x , S / 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 p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; S / H a t 3% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3 3  3 5  3  3  - 130 3.11.3 D i s c u s s i o n 35 In  vivo  1,3,5, and  S-sulphate  age-labeled  platelets  and 2,4,6, p . i . were a l s o l a b e l e d w i t h  subjected  platelet  t o simultaneous  in_ v i v o c o h o r t  CCD  label  l a b e l was more s e n s i t i v e  analyses.  t o another  harvested  on  ^H-serotonin  Comparing  days  in vitro,  the r a t i o  i n vitro platelet  than a comparison t o p l a t e l e t  of a  population  particle  number,  as p r e v i o u s l y d i s c u s s e d . 35 3 On day one t h e s l o p e o f S/ H r a t i o through t h e CCD p l a t e l e t d i s t r i b u t i o n was n e g a t i v e , i n d i c a t i n g t h a t t h e f i r s t p l a t e l e t s , l a b e l e d 35 in  t h e bone marrow  and  newly  relative which  released  into  injection  up  especially  cohort  labeled  (P  less  platelet  platelets  and on day slightly  than with  four  less  evenly.  low  the slope  than  on  day  cohort  were o f low  On  day two, and 35 3  this  S/ H  partition, was  still  three  and  a l l of on  day  enrichment  of  became  increasingly  numerically was  label,  partition  population d i s t r i b u t i o n ,  0.03),  a  S-sulphate  circulation  serotonin, r e l a t i v e l y  three  apparent,  of the  the blood  t o t h e normal a g e i n g  take  however  after  not  negative,  statistically  significant. On  day  five,  the ^S/^H  slope  day s i x t h i s s l o p e became p o s i t i v e , of the cohort l a b e l e d p l a t e l e t s .  was  zero,  indicating  or h o r i z o n t a l  and  on  a s h i f t i n the p a r t i t i o n  At t h i s t i m e , t h e c i r c u l a t i n g  platelet  35 S-sulphate  radioactivity  3.11.2.2), i n d i c a t i n g ageing,  neared  that cohort  i t s peak  (see F i g u r e s  labeled platelets  3.11.2.1  were both  and b e i n g removed from t h e c i r c u l a t i o n , w h i l e s t i l l  and  maturing  b e i n g newly  r e l e a s e d from t h e bone marrow. 35 The platelet  presence,  and  distribution  shift  of  the  were i n d i c a t i o n s  3 S/ H  slope  of the fact  through  the  CCD  t h a t the enrichment  - 131 of  35  S-sulphate  cohort  d i s t r i b u t i o n was  labeled  an enrichment  -  platelet,  of  the  o f young p l a t e l e t s , newly r e l e a s e d  into  t h e 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  aggregates.  shifts  That  these  in  the  3 5  to  the  left  t o the presence  S/ H  slopes  3  of p l a t e l e t  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 b e i n g compared s i m u l t a n e o u s l y w i t h i n a s i n g l e CCD,  and were r e p e a t e d the day a f t e r , a l s o l e n d g r e a t v a l i d i t y  t o t h i s approach and t h e s e  3.12  CCD To  o f 35-S-Labeled retest  young  the  interpretations.  P l a t e l e t s , Double L a b e l e d w i t h  f i n d i n g s of  " S-labeled  platelet  5 5  the  previous  cohort  was  of  Section lower  3-H-Serotonin (3.11;  i . e . , the  partition  than  the  35 platelet and  p o p u l a t i o n ) , another  blood  was  taken 35  on  of  enrichment  of  harvested,  labeled i n CCD  day  three  was  injected  p . i . the  with  time  S-sulphate,  during  which  the  therefore  the  3  magnitude  transfer  rabbit  S/ H  slope  young  was  platelets vitro  providing  was  with  the  the  greatest,  most  pronounced.  ^H-serotonin,  greatest  and  and  possible  Platelets subjected  resolution  were  to  120  without  introducing population overlap. 3.12.1 E x p e r i m e n t a l Approximately three  days  1.49  x  greater (Section  ml o f PRP, 10  ml  p . i . of  98.75% p l a t e l e t s , from 30  60  1 0  size,  3 3  and  of blood  S  1.25%  particles  labeled i n v i t r o ,  counted  were t a k e n  iri vivo. Platelets  platelets  3.11.1).  ( i n ACD)  in  (90.7% the  (2.38  of greater and  x  lO"^  CCDLx) were  and  9.27%  distributed,  were p r e p a r e d  rabbit,  platelets;  s i z e ) were  s u b j e c t e d t o CCD.  platelets,  Cytocentrifuge slides  from one  harvested A total  particles and  of  analyzed  f o r each  and t h e 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 c o n t a m i n a t i o n was  of  CCDF,  determined.  - 132  -  3.12.2 R e s u l t s From data CCDF, t h e full  obtained  total  recovery  (a l e v e l  by  leukocytes  i n the  CCD  determined  CCD  and  The  of  assuming  5  1.49  x 10"*"° p l a t e l e t s  distribution  distributions  S/ H  a f t e r 120 t r a n s f e r CCD,  ratio  of  leukocytes  was  were p l o t t e d ( F i g u r e s 3.12.2.3  slope  magnitude  was  (Table  highly  significant  3.12.2.1;  Figures  (P  =  3.12.2.4)  t h a n a f t e r 40 t r a n s f e r s ( T a b l e 3.11.2.1).  T a b l e 3.12.2.1 A n a l y s i s o f 3 5 / 3 S  10% CE Mean + S.Dev. n Corr Slope P  x 10 ,  3  greater  Day  6.17  w i t h i n each  3.12.2.2).  35  0.002);  The  with  content  ( r e g i o n I I ) o f lower p a r t i t i o n than p l a t e l e t s  radioactivity  3.12.2.4).  t o be  loaded  o f 0.004% c o n t a m i n a t i o n ) .  ( F i g u r e s 3.12.2.1 and  and  was  which was  found t o be p r e d o m i n a t e l y  The  enumeration o f c e l l u l a r  R a t i o S l o p e , CCD  H  Enrichment  Three R a t i o  7.39 x I O 1.22 x I O 19 -0.909 -1.97 x I O " 0.002 - 3  - 3  4  3.12.3 D i s c u s s i o n 35 Platelets, greatest 3  CCD  H-serotonin.  different based  on  three  days  enrichment, CCD  was  p.i. by  S-sulphate,  comparison  capable  ages, c r e a t i n g a t l e a s t  of  to  a  demonstrated population  subfractionating  a partial  surface p r o p e r t i e s . Since these  platelet  the label,  platelets age  into  distribution  two-polymer phase systems  are  s e n s i t i v e t o membrane s u r f a c e c h a r g e , i t was c o n c l u d e d t h a t changes i n s u r f a c e p r o p e r t i e s were a s s o c i a t e d w i t h p l a t e l e t s o f d i f f e r e n t age.  - 133 - Figure 3.12.2.1 CCD Leukocyte Contamination S/ H-Labeled Platelets: leukocytes (solid line 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 5  3  of Day Three histogram) p e r  3.40E+04  r  T"  100  •  110  3. 45E+04  I 0. 00E+00  120  Contamination and Day Three Figure 3.12.2.2 CCD Leukocyte H-serotonin (solid l i n e ) dpm S/ H-Labeled Platelets: platelet p e r CCDC, l e f t o r d i n a t e ; l e u k o c y t e s (dashed l i n e h i s t o g r a m ) p e r t o t a l CCDF, r i g h t o r d i n a t e ; CCDC a b s c i s s a .  3 5  3  3  - 134 Figure 3.12.2.3 CCD of Day Three S/ H-Labeled Platelets: H-serotonin ( s o l i d l i n e ) dpm p e r CCDC, l e f t o r d i n a t e ; c o h o r t S (dashed l i n e ) , dpm p e r CCDC, r i g h t o r d i n a t e ; S/ H ratio (dotted l i n e ) , mean r a t i o a t h a l f graph h e i g h t ; CCDC a b s c i s s a . 3 5  3  3  3 5  3 5  0  10  20  30  40  50  60  70  80  3  90  100  110  120  Figure 3.12.2.4 CCD of Day Three S/ H-Labeled Platelets: H-serotonin ( s o l i d l i n e ) dpm p e r CCDC, l e f t o r d i n a t e ; c o h o r t S (dashed l i n e ) , dpm p e r CCDC, r i g h t o r d i n a t e ; S / H r a t i o a t 10% CE ( d o t t e d l i n e ) , peak r a t i o a t h a l f graph h e i g h t ; CCDC a b s c i s s a . 3 5  3  3  3 5  3 5  3  - 135 3.13  -  S i n g l e Step P a r t i t i o n o f P l a t e l e t s During Cancer Chemotherapy Patients  model  being  system  t r e a t e d f o r bone marrow c a n c e r  to  analyze  the.  membrane  were  s t u d i e d as  charge-associated  a  surface  p r o p e r t i e s o f 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  peripheral  chemotherapy  and/or  varies  to  due  regeneration  blood  platelet  radiation  successive  brought  on  therapy bouts  by  of  treatment  subpopulations peripheral  and  can  blood  be cell  thrombocytopenia.  bone  therapy  and  undergoing  myelogenous  marrow  recovery,  relatively  separately  selected.  to  patients  leukemia  suppression  recovery  is  common  one  of  young Upon  and  old  By  of  the  platelet  presentation,  the  l e u k o c y t o s i s , anemia,  and  precursors,  w h i t e b l o o d c e l l compartment c a u s e s "crowding  and  process.  together with previous experience  picture  Due  of  for active  of  the  f o l l o w i n g the p l a t e l e t c o u n t , course  count  proliferation  from  the  o u t " or d e p r e s s i o n o f 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. Initiation  of  treatment,  inhibits  rapidly  platelet  production  decreases  even f u r t h e r .  are  being  dividing  produced  to  and  which  cells,  decline, For  strategically  the and  megakaryocyte the  remaining  i n the  effectively  included,  circulating  a p e r i o d of time,  those  and  causes  platelet  s i n c e no  new  circulation  count  platelets are  being  removed by t h e r e t i c u l o e n d o t h e l i a l system, presumably as they age, ageing  platelets  platelet  are  survival,  period  of  obtain  a  platelets  time  present.  about  (e.g.,  subpopulation and  deficient  ten  Thus, w i t h days  in  seven d a y s ) post significantly i n newly  knowledge  humans,  of  after  the an  length  in  produced p l a t e l e t s .  of  appropriate  agent a d m i n i s t r a t i o n , one enriched  only  isologously  can aged  Eventually there  i s a p e r i o d o f t i m e , t r a n s i e n t however, d u r i n g which t h e r e a r e very  few  - 136 to  negligible  transfusion necessary  is to  required prevent  approximately Again,  circulating  one  day  period  platelets,  to  maintain  hemorrhage.  and  basal  homologous  platelet  p e r i p h e r a l blood  However  there  is  a  levels lag  of  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 .  i f antineoplastic  appropriate  -  of  therapy time  i s continued  post  and  transfusion,  i f one  one  waits  can  the  harvest  a  s u b p o p u l a t i o n o f homologously aged p l a t e l e t s s i m i l i a r t o t h o s e above. Upon c e s s a t i o n o f t r e a t m e n t cells  begin  to  regenerate.  have p r e f e r e n t i a l young  platelet  peripheral  blood  in  platelet  over again  numbers. The  i n c r e a s e and by s a m p l i n g enriched  Usually  capability  production  and r e c o v e r y from l e u k e m i a , bone marrow  newly  the  normal  precursors  t h e i r leukemic ensues,  platelet  cell  restoring  count  young  platelets  their  i s observed  count approaches normal w i t h i n a few  can  cells  counterparts  p e r i p h e r a l blood at t h i s time, a  produced  stem  be  circulating to  steadily  subpopulation obtained.  days and  and  even  The  surpasses  t h i s normal l e v e l s i n c e f o r some reason t h e bone marrow p r e c u r s o r c e l l s tend t o overcompensate. Young  and  peripheral recovery been  old  blood cycle.  shown t o  abnormality  taken These  be  and  b,  1983a and  charge-associated differential  at  subpopulations  various  sized,  indication  of  platelet  J a m i e s o n , 1974;  b). Platelets membrane  were  through  were  and b, 1982;  partition  stages  platelets  ( O ' B r i e n and  Bessman e t a l . , 1981a and  platelet  harvested this  therapy  since platelet age,  Goldschmidt  has  heterogeneity,  and  and S o r l a n d ,  Thompson e t a l . , 1981a  i n charged  p r o p e r t i e s as two-polymer  and  size  and  were a l s o examined f o r t h e i r  surface  from  reflected  aqueous phase  b,  1976; 1982a  relative by  their  systems,  s i n c e p l a t e l e t age has been shown t o 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  since  platelets  from  malignant  d i s e a s e s have  been  shown  in  s t u d y t o be reduced by 24% i n membrane n - a c e t y l n e u r a m i n i c a c i d et a l . , be  one  (Martin  1982). P l a t e l e t membrane p h o s p h o l i p i d s have a l s o been shown t o  abnormal  in  myeloproliferative  disorders  (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 d e t e c t e d b o t h i n p l a t e l e t  by  scattering,  laser  light  reflected  by  a  variety  and  of  in  membrane  two-phase  surface  systems  size  properties  with  varying  as salt  c o m p o s i t i o n s and p o t e n t i a l d i f f e r e n c e s . 3.13.1 E x p e r i m e n t a l Specimens: myelogenous  Blood  from p a t i e n t s  leukemia  (AML),  Hospital,  Department  of  w i t h 3.8%  sodium c i t r a t e  undergoing chemotherapy  was  obtained  Hematology,  taken  from by  for active  Vancouver  venepuncture  (1 p a r t / 9 p a r t s b l o o d ) . B l o o d was  General and  mixed  also  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 p e r s o n n e l . Platelet  Preparation:  B l o o d i n 10 ml volumes t e m p e r a t u r e . PRP,  after  with  of  0.5-1.0  resuspended  ml  i n 0.5  Only  was  equipment  was  used  throughout.  c e n t r i f u g e d a t 150 g f o r 20 minutes a t room  b e i n g s e p a r a t e d from t h e c e l l s ,  ACD, ml  plastic  centrifuged  Tyrode's  at  solution.  3000  g  was  f o r 20  acidified  minutes  These resuspended  and  platelets  were used i m m e d i a t e l y f o r a n a l y s e s o f s u r f a c e 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 : stock  solutions  of  20%  Phase w/w  t w i c e - i s o t o n i c s o l u t i o n s o f pH chloride.  A set of f i v e  phase  systems  D  T500  7.2  were composed by  ( l o t 7830),  sodium phosphate  systems  (5/4:A-l  30%  w/w  buffer,  weight  from  PEG,  and  and  sodium  t h r o u g h 5/4:E-l)  with  v a r y i n g s a l t c o m p o s i t i o n s was made by f i r s t p r e p a r i n g a s o l u t i o n o f 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  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).  r a t i o s of twice  - 138 A s m a l l sample o f p l a t e l e t s , 50 u l , o f known p l a t e l e t was  added  phase D  t o each  tube  (Falcon p l a s t i c disposable  systems, each composed o f one ml PEG  rich  lower  inversion,  phase,  of  various  and s e t t l i n g was  concentration  tubes) of a set of  r i c h upper phase and one  salt  compostions.  Mixing  was  ml by  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 t h e upper phase by s a m p l i n g (200 u l i n t o 10 ml Diluid)  and c o u n t i n g  w i t h the E l e c t r o z o n e C e l l o s c o p e Model 112 CLTH/RWP  (48 um d i a m e t e r 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). Platelet disorders  (MPD)  scattering printout.  Sizing: were  (LS)  Platelets  from  sized  Diluid  cell  in  counter  and  saline,  (Ortho  Histogram s i z i n g p l o t s  o f each s i z e accumulated ( i n mm  normals  myeloproliferative  using  a  Diagnostics)  laser  with  light  histogram  were t a b u l a t e d , e x p r e s s e d as  platetets  from the x a x i s ) v e r s u s % window.  3.13.2 R e s u l t s S t a t i s t i c a l Comparison  o f P a r t i t i o n s : The p a r t i t i o n s were e x p r e s s e d  as t h e q u a n t i t y o f p l a t e l e t s i n the upper phase total  platelets  partitioned from  a  group  partitioned MPD  Platelets  of  age/sex  matched  A.49).  from  Student  C)  subjects,  were  platelets  (to  t tests used  f o r which  (HP  partitions  platelet  i n phase systems A - l t h r o u g h D-1  normals  normals  also  organized:  w i t h p l a t e l e t count r i s i n g  concurrently  partitioned  were  Platelets are  were then  separately  S t a t i s t i c s Volume 1 P a r t  t o compare 22  and  patients)  (PCD), MPD  partitioned  matched normals  patients  (Appendix T a b l e A.47).  (Appendix Table A.48). The  normal  platelets  from  compared c o n c u r r e n t l y  w i t h p l a t e l e t count d e c l i n i n g  (PCR),  Rev.  and  added.  as a p e r c e n t a g e o f t h e  the p a r t i t i o n s  with  MPD,  and  (Appendix  Table  No.  of p l a t e l e t  09815-15000, from  p a r t i t i o n measurements were  normal  collected  (*only 18 i n phase system E-1) and the  - 139 platelet  partitions  addition, platelet  MPD  p a t i e n t s were d i v i d e d i n t o  survival  declining rising  MPD  of patients with  dynamics;  either  with  ( 1 0 ) , (Table  3.13.2.1).  two c a t e g o r i e s based circulating  on t h e  platelet  (PCD; o l d ) a g e i n g a f t e r chemotherapy, o r w i t h p l a t e l e t  (PCR; young) d u r i n g  significance  platelet  regeneration.  At  95%  count count  level  of  (p = 0.05), t h e r e 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  o f t h e phase systems f o r n o r m a l s compared t o a l l o f t h e p l a t e l e t s MPD  In  i f platelet  survival  dynamics  were  disregarded  (PCD  from  and PCR).  However, when p l a t e l e t s from MPD were s u b d i v i d e d i n t o PCD o r PCR, t h e r e were d i f f e r e n c e s , compared t o n o r m a l s , system 5 / 4 : A - l ;  and d i f f e r e n c e s found  found  f o r MPD  PCD u s i n g  phase  f o r MPD PCR u s i n g phase systems  5/4:B-1 t h r o u g h 5 / 4 : E - l .  T a b l e 3.13.2.1 Comparison o f P l a t e l e t P a r t i t i o n s , Normal and MPD  5741  A^i  E^T  BZJ  (>1  f>l  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 t 1.21 30 df 0.1179 P 88.2%  42.8 + 18.2 0.706 30 0.2428 75.7%  36.9 + 17.4 0.9377 30 0.1779 82.2%  24.8 + 12.5 1.0142 30 0.1593 84.1%  16.4 + 9.28 0.8545 26 0.2003 80.0%  PCD (7) t df P  72.0 + 10.9 2.66 27 0.0065 99.4%  52.6 + 10.1 0.943 27 0.1771 88.3%  45.1 + 13.4 0.254 27 0.4009 59.9%  30.6 + 10.2 0.111 27 0.4562 54.4%  20.9 + 7.2 0.251 23 0.402 59.8%  PCR (3) t df P  41.7 + .6.3 1.5784 23 0.0641 93.6%  19.9 + 7.6 3.1372 23 0.0023 99.8%  17.6 + 6.41 2.4093 23 0.0122 98.8%  11.4 + 2.9 2.322 23 0.0147 98.5%  6.0 + 0.26 2.2448 19 0.0184 98.2%  - 140  -  S t a t i s t i c a l Comparison o f S i z e s : The  mean modal s i z e o f 6.40  (5) % window found f o r normal p l a t e l e t s , and % window f o r MPD  p l a t e l e t s , was  a s i z e o f 5.38  a s i g n i f i c a n t (p = 0.03)  _+  1.14  + 0.58  (8)  difference.  3.13.3 S p e c i f i c D i s c u s s i o n Partition  studies  diagnoses  (acute  leukemia,  leukemia,  drastically surface taken  and  properties  by  and  daunorubicin  bone  marrow  although  for  therapy,  no  become  decreasing  the  post-therapy,  platelet  shown normal  with  various  leukemia-acute  counts  that  had  been  differences  in  controls, platelets those  taken  when  the  production.  a combination of  circulation  the  have  d e c l i n i n g , and  t o renewed  varying  leukemia,  platelet  between  and  (TAD/TRAMCO),  acute  patients  thioguanine,  eradicates  lengths  of  cytosine  platelet  time  arabinoside  synthesis  (several  days  in  to  the  weeks),  the drugs are c l e a r e d much more r a p i d l y (Young et a l . , 1981).  Following  If  evident  cancer  whose  chemotherapy  count i s low  treatment,  leukemia,  lymphoma)  were  count i s i n c r e a s i n g due The  p l a t e l e t s from  myelogenous  reduced  when t h e  on  p l a t e l e t s are  progressively  circulating  older  platelet  formed; the with  count  production  i s observed  has  Samples t a k e n  resumed, early  to  rise,  platelets in and  are  About  the  removed,  seven  days  are s u b s t a n t i a l l y aged.  i t i s an  releasing in this  time  count.  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  platelet  circulation.  new  new  i n d i c a t i o n that  platelets into  process w i l l  be  enriched  the in  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 systems with  containing  varying  varying  potental  were performed i n a s e r i e s o f 5% isotonic ratios  of  d i f f e r e n c e s between the  High phosphate c o n t a i n i n g  phosphate upper and  systems have been shown t o  be  D,  4%  PEG  to c h l o r i d e , lower  phases.  s e n s i t i v e to  - 141 cell  s u r f a c e c h a r g e , whereas t h e p a r t i t i o n i n c h l o r i d e - r i c h  been c o r r e l a t e d  w i t h membrane l i p i d  (hydrophobicity, l i p i d Platelets recovery  associated properties i n red c e l l s  packing).  were t a k e n a t v a r i o u s s t a g e s t h r o u g h a chemotherapy  cycle  in  charge-associated patients  whose  order  surface  platelet  to  characterize  properties. count  was  their  declining  platelets  phase  respectively. marrow  recovery  system  The few "young" regeneration  phase,  exhibited  ( 7 2 % compared  platelet  and  membrane  a  from  result  of  h i g h e r t h a n normal  t o 63%, i n 5/4:A-l,  subpopulations obtained during  the r i s i n g  platelet  l o w e r than normal  f o u r o f t h e f i v e phase systems t e s t e d  and  obtained  (PCD) as  a partition coefficient slightly  i n one  relative  "Old" p l a t e l e t s ,  therapy, exhibited  bone  systems has  count  (PCR), t h e  partition coefficients i n  ( 2 0 % compared t o 47%, i n 5 / 4 : B - l ;  18% compared t o 43%, i n 5/4-.C-1; 1 1 % compared t o 30%, i n 5/4:D-l; and 6% compared t o 20%, i n 5 / 4 : E - l ) . When t h e p a r t i t i o n c o e f f i c i e n t s all  MPD,  partitions t h e s e phase  PCD  and PCR  were  pooled  together against the  o f n o r m a l s , 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 o f systems.  That p l a t e l e t s  from p a t i e n t s w i t h MPD, t a k e n from t h e r i s i n g  d u r i n g r e c o v e r y from chemotherapy, associated p a r t i t i o n coefficient lower  and t e s t e d  from  membrane  surface  charge  demonstrated than normal  a lower surface  platelets,  i s associated  with  phase charge  suggests that  young  platelets.  However t h e r e a r e o t h e r e x p l a n a t i o n s : p l a t e l e t s and megakaryocytes  from  c a n c e r p a t i e n t s have been shown t o be a t y p i c a l  (Rodman e t a l . , 1978;  Cooper  Platelets  e t a l . , 1978; G e r w i t z  e t a l . , 1983).  produced i n  r e s p o n s e t o t h i s extreme c o n d i t i o n o f t h r o m b o c y t o p e n i a a r e analogous t o s.tress-induced  reticulocytes  i n hemopoiesis  which  have  been  shown t o  - 142 differ  i n their  surface  p r o p e r t i e s , as d e t e c t e d  r e t i c u l o c y t e s produced n o r m a l l y . (Young e_t a l . , responsible are  also  f o r t h e observed known  aggregation The  1981), through  to  affect  The chemotherapeutic  behavior. platelet  surface  new p l a t e l e t s can be d e t e c t e d  a t an e a r l y  approach  (platelets  sizing  agents  from  themselves  Tumor c e l l s and leukemic  ( G a s i c e t a l . , 1977; Z a w i l s k a e t a l . ,  platelet  partition,  m e t a b o l i t e s o r i n d i r e c t e f f e c t s , might be  r e s u l t s are o f considerable i n t e r e s t  or  by  properties,  blasts  affecting  1981).  because i f p r o d u c t i o n o f  stage, using t h i s from  t h e MPD  partition  patients are  s m a l l e r than n o r m a l ) , i t would p r o v i d e 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 t h e management o f t h e s e p a t i e n t s .  - 143  -  GENERAL DISCUSSION AND  The of  potential  platelets  platelets  o f p a r t i t i o n i n g and  in  two-polymer  a c c o r d i n g t o age  p r e s e n t e d which i n d i c a t e s based on in  was  systems  case  to  technique  for  separate  l a b e l i n g and Since  platelet  of  age-related  behavior  was  platelets  distribute evidence  on  The  the  method,  basis  selected  of  membrane  been shown t o d i s t r i b u t e o t h e r  (Section  1.6).  In o r d e r t o  distribution  analysis,  varying  3.2)  specimens  in  density  characteristics  investigated  using  by  two-polymer  have  several  rabbit,  phase  been  (Section  comparative  from  density platelet fractions  partition platelets partition.  1.3)  CCD  man,  systems  shown  of  and  of  than of  intermediate  mean age),  The  (containing  CCD  density  partition  density  separated  be  The  results  sensitive  while high density  distributions  of  fractions  platelets  a l l three  variable  subpopulations  the  subpopulations,  individual  Examination  volumes,  of  of  especially  platelet  platelets  from  size both  evident (Section  rabbits  in 3.3)  and  lower  (containing  were o f  density  to  rabbit,  o l d p l a t e l e t s ) were o f  platelet  have  their  monkey.  known t o  to  were i n d i c a t i v e o f s u r f a c e p r o p e r t y h e t e r o g e n e i t y  modal  CCD  means  o v e r l a p p e d and  platelets.  cells  assess t h i s  c h a r g e - a s s o c i a t e d membrane p r o p e r t i e s showed t h a t f o r human and low  was  o b t a i n i n g p l a t e l e t s o f v a r i o u s ages were examined. platelets  different  has  (CCD)  p l a t e l e t p o p u l a t i o n i s heterogeneous  platelets  age  to  demonstrated. In a d d i t i o n ,  t h a t the  b a s i s o f i n v i v o age  (Section  phase  a n a l y s e s o f membrane s u r f a c e p r o p e r t i e s .  this  the  countercurrent d i s t r i b u t i o n  aqueous  charge-associated properties, on  CONCLUSION  the  high  within density  showed t h a t humans,  the  increased  - 144  -  w i t h 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 Rabbit function  platelets,  labeled  of  survival  time  used i n the  standard  clinical  platelet  survival  labeling  at a s p e c i f i c in  the  from  the  or  marrow  with  after  survival  (Section  in  vivo  should  and  the  this  entire  be  studied  to  compare  the  CCD  3.6).  Immediately  distribution  platelets  of  a  revealed  after  specific  of  i n vitro ^Cr  In  vitro,  a  5  is  *Cr  population  5 1  Cr  labeled  rabbit  of  injection,  aged,  platelet  than  of  released  intravenous  i n uptake o f lower  function  newly  platelet  5  partition,  experimental  as  properties  heterogeneity  for  platelet  p l a t e l e t s , w i t h t h o s e o f the t o t a l normal u n l a b e l e d (Section  a  nuclide  platelets  CCD  surface  as  p o s s i b l e t o f o l l o w changes  since  unlabeled.  studied  This  1.7).  p l a t e l e t s , at various i n v i v o ageing times a f t e r were  and  population  ageing,  be  were  injection.  1.3  i t should of  ^Cr,  time t e s t  i d e n t i f i c a t i o n of  properties  time,  vitro  vivo,  t i m e p o i n t , and  surface  circulation  in  studies  p e r m i t s the  in  exception.  labeled  population  labeling,  radioactivity  the  mean  for  the by the  51 population. of  This  platelets  of  human p l a t e l e t s , and  in_  vivo  exhibiting labeled  heterogeneity specific and  platelet  in  uptake  such  was  c o n f i r m e d by  for  population  lower,  uptake  partition  circulation  enhanced  progressively  was  in  the 51  by  a  demonstrable  rabbit Cr  the  Cr  double-labeling. this  rapidly  aged,  that  of  the  details  for  of  rabbit  After  platelet  decreased.  total  subpopulation  CCD  injection  population 51 As the Cr  radioactivity the  and  became  radioactivity  d i s t r i b u t i o n c o u l d not be d i s t i n g u i s h e d . I t has  been shown t h a t the l a r g e dense p l a t e l e t s  terms o f a g g r e g a t i o n low  concentrations  ability, of  ^Cr  and  a r e most a c t i v e i n  can be p r e f e r e n t i a l l y (i.e.,  greater  uptake  l a b e l e d at and  very  specific  - 145 activity  than  the  majority  of  the  platelet  population)  successively higher concentrations  ( S e c t i o n 1.3).  subpopulation  uptake  exhibits  active  c o n c e n t r a t i o n s . The  CCD  rabbit  double  platelets,  p a t t e r n s and  for  with  5  "^Cr  ^Cr  not  I t appeared t h a t at  specific activity  labeled  but  and  at this  these  low  distributions  "'""'""'"In  at  of  various 51  concentrations had  label  been r e p o r t e d  with  distinct  partition  in  "*"Cr  not  but  5  activity. of  of  relative  enrichment was  reduce nor  the  system, to  the  was  by  a  a  phenomenon  with  "^In  "^C-serotonin  CCD),  nor f o r H - a d e n i n e (not shown).  (used  which  as  Cr  subpopulation  relatively  strongly  population  for  low  enriched  average  in  specific  s a t u r a b l e , as p r o g r e s s i v e l y h i g h e r  enrichment,  with  showed t h a t a  characterized  two-phase  '^"''In  where a c t i v e uptake  ( S e c t i o n 3.7),  properties  charged in  The  ^Cr  to occur  surface a  i n c l u d i n g those  levels  was  not  observed  standards  for  platelet  3  The  characteristics  exhibited  " a c t i v e " uptake  associated) platelets active  of  partition were  uptake  t h i s dense 51 for  Cr  coefficient 51  labeled occurs,  with and  this  phenomenon had  not  into  subpopulations  according  and was  Cr,  with  platelet  demonstrated further  at  low  "''"'""''In and  been r e p o r t e d .  subpopulation low  (charge-  investigated.  concentrations "^C  serotonin  These p l a t e l e t s  to density  a  and  then  two-polymer aqueous phase systems. In t h e s e c a s e s ,  which  for  which  were  analyzed a direct  exhibited  was  found. enhanced  "^C-serotonin,  was  However, uptake again  the  dense  for  found  to  platelet  *^Cr, have  but a  a c c o r d i n g t o the young dense p l a t e l e t model.  low  Rabbit  at  separated by  CCD  platelet  subpopulation not  for  partition,  in  correlation  o f i n c r e a s i n g modal ( 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 w i t h density  which  "^In  which nor  unexpected  - 146 Using cohort is  S-sulphate  3 5  of platelets  intravenous  injection  into  rabbits,  was l a b e l e d ( S e c t i o n s 3.8 t h r o u g h  a  3.12).  young  Sulphate  i n c o r p o r a t e d i n t o t h e megakaryocyte w i t h i n t h e bone marrow and o n l y  p l a t e l e t s which a r e newly r e l e a s e d i n t o t h e c i r c u l a t i o n appear l a b e l e d , while  ageing  3.8).  Both  p l a t e l e t s remain u n l a b e l e d and a r e b e i n g removed ( S e c t i o n  o f these  processes  occur  simultaneously  and e v e n t u a l l y t h e 35  labeled  cohort  ages. V a r i o u s  l a b e l i n g technique "^H-serotonin  combinations  of t h i s  i n vivo  S  w i t h 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  as a s t a n d a r d  f o r comparison),  density  p l a t e l e t membrane s u r f a c e p r o p e r t y a n a l y s i s p r o v i d e d  cohort  (i.e.,  with  s e p a r a t i o n , and  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 o f 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  labeled  young p l a t e l e t  density  platelet  centrifugation; (LDP)  injection cohort  fraction.  On  day  the  was found  (HDP) f r a c t i o n the opposite  (p.i.),  obtained  was  found  in  vivo  "^S-sulphate  t o be e n r i c h e d  i n the high  by S t r a c t a n d e n s i t y g r a d i e n t i n t h e low d e n s i t y  s i x p . i . , this  difference  platelet  i n density  of  35 S-sulphate  labeled  platelets  was  less  dramatic  (Section  3.10). These f i n d i n g s were i n t e r p r e t e d t o mean t h a t p l a t e l e t s  3.9  and  decreased  their  mean d e n s i t y as a f u n c t i o n o f age i n t h e c i r c u l a t i o n , t h e young  being  enriched  Platelet  i n HDP  membrane  separated  and  that  young  the  and t h e o l d c o n s i s t i n g  surface  unseparated platelet  property  analyses  "^S-sulphate cohort  not  predominately  by  CCD  labeled only  had  o f both  platelets different  o f LDP. density indicated density  c h a r a c t e r i s t i c s b u t a l s o had d i s t i n c t i v e s u r f a c e p r o p e r t i e s which were more  pronounced  activity  on day two i n comparison  was c o n s i s t e n t l y  distribution  curves  of  t o day s i x . The  e n r i c h e d i n t h e low p a r t i t i o n platelets  (even  without  specific  r e g i o n o f CCD prior  density  - 147 f r a c t i o n a t i o n ) when drawn two o r t h r e e days p . i . , an enrichment decreased  on subsequent days ( S e c t i o n s 3.11 and 3.12).  Platelets  from  model o f p l a t e l e t of  which  patients  cancer ageing  patients  ( S e c t i o n 3.13) were  i n v i v o . The p e r i p h e r a l b l o o d  undergoing  chemotherapy  f o r leukemia  studied  as a  platelet  count  varies  due  to  s u c c e s s i v e b o u t s o f bone marrow s u p p r e s s i o n and r e g e n e r a t i o n brought on by t h e therapy platelet present  and r e c o v e r y  I n i t i a t i o n o f treatment  s y n t h e s i s and f o r a p e r i o d o f t i m e and a r e g r a d u a l l y removed  treatment  permits  peripheral  blood  various single  process.  stages  the  from  production  only ageing  p l a t e l e t s are  the c i r c u l a t i o n .  Cessation of  of  numbers a r e once a g a i n i n the therapy  tube p a r t i t i o n  i n order  eradicates  young  platelets  and  restored. Platelets  and r e c o v e r y  cycle  were  to characterize their  their  taken  at  examined  by  relative  charge-  a s s o c i a t e d s u r f a c e p r o p e r t i e s o f human p l a t e l e t s o f d i f f e r e n t ages. Knowledge o f an a l t e r a t i o n i n p l a t e l e t membrane s u r f a c e charge w i t h platelet  ageing,  as examined  analyses  of platelet  i n t h e work  membrane s i a l i c  acid  of others, cleaved  i s limited  to  by enzymes and t o  e l e c t r o p h o r e t i c m o b i l i t y measurements. S t u d i e s have been c a r r i e d out on platelets  separated  according  d e n s i t y and s i z e , c o m b i n a t i o n s density least do  t o other  such  as  o f d e n s i t y and s i z e , and c o m b i n a t i o n s  of  and e l e c t r o p h o r e s i s , a l l o f which  partial  conflict  experimental  platelet and  not  systems;  platelets.  platelet  Also  properties  a r e thought  t o provide at  age based s e p a r a t i o n s . C e r t a i n o f t h e f i n d i n g s only there  because  most  age and p l a t e l e t  of  are admittedly  i n c o n s i s t e n c i e s and p o s s i b l e t e c h n i q u e in  physical  the  differences  technique  in  (e.g.,  ( e . g . , g r a d i e n t ) induced  o f t h e c o r r e l a t i o n s which denstiy are incomplete,  do e x i s t  total  gradient) changes between  i n that there  is a  - 148 young p l a t e l e t the  direction  density d i s t r i b u t i o n o f higher  which i s o n l y  density, i . e . , there  slightly  shifted i n  are p l a t e l e t s  which a r e  young and o f low d e n s i t y , b u t w i t h t h e mean d e n s i t y f o r young p l a t e l e t s being  higher.  interesting overlaps  The megakaryocyte-produced p l a t e l e t h e t e r o g e n e i t y  and worth of  considering  platelet  density,  as a  partial  size  and  i s also  explaination of the  surface  properties  as  demonstrated i n t h i s t h e s i s . The al.,  e v i d e n c e t h u s f a r , based  1975; Isobe,  (Rand,  1976; Isobe  Ph.D. 1983),  surface  charge  density,  age.  Therefore,  negative,  area.  total  assayed  of s i a l i c  by  i n abstracts  1976) and i n a  platelet  platelet  a c i d , should  (Carty e t  surface  thesis  charge and  electrophoresis  and  correlate directly  with  d e n s i t y which i s t h o u g h t t o be i n v e r s e l y r e l a t e d t o p l a t e l e t providing  young p l a t e l e t s , net  found  and Yamanaka,  i n d i c a t e s that  neuraminidase cleavage platelet  on d a t a  HDP  higher  The o p p o s i t e  t h e p r o p e r t i e s o f HDP  ( c o n t a i n i n g young p l a t e l e t s ) s h o u l d total  should  s u r f a c e charge and s u r f a c e be t r u e  upon  stimulation  those  of  be o f h i g h e r  charge p e r u n i t  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 t o t h e mean f o r t h e p l a t e l e t Platelets  do r e f l e c t  population.  with  thrombin  have  been  shown  to  d e c r e a s e i n d e n s i t y ( C i e s l a r e t a l . , 1979). A f t e r s t i m u l a t i o n w i t h ADP, they  demonstrate an i n c r e a s e  indicating possible  an i n c r e a s e  that  such  endothelial-platelet  i n neuraminidase-susceptible  in total  i n vitro  surface  charge  s t i m u l a t i o n does  sialic  (Chiu,  1983).  not r e f l e c t  encounters w i t h i n the c i r c u l a t i o n ,  All  analyses  of  platelets  and  density  Iti s  platelet-  but i f i t does  t h e n 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 c o u l d demonstrate lower and h i g h e r r e l a t i v e s u r f a c e c h a r g e than u n s t i m u l a t e d  acid,  mean d e n s i t y  platelets. separated  platelet  - 149 subpopulations were  rely  on t h e a s s u m p t i o n s t h a t  f r e e o f a g g r e g a t e s and c e l l  often  invalid  as g r e a t  preparations.  care  the p l a t e l e t  contamination.  must  be  The d i f f e r e n c e s observed  taken  preparations  These assumptions a r e  to obtain  pure  platelet  i n the properties 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 ) a r e s m a l l and c o u l d o f t e n be e x p l a i n e d o r altered  by  the presence  of contamination  (e.g.,  platelet  aggregates  and/or l e u k o c y t e s ) o f h i g h e r d e n s i t y than p l a t e l e t s . Separations centrifugation  of  blood  cells  i s seldom  according  sufficient  to  for a  size  by  complete  differential separation  of  p l a t e l e t s from c o n t a m i n a n t s and v i s u a l e s t i m a t i o n o f p u r i t y a t b e s t i s o n l y an e s t i m a t e . However, a c e r t a i n degree o f a c c u r a c y  can be  obtained  u s i n g t h e 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 w i s e t o monitor  f o r t h e presence o f c e l l u l a r c o n t a m i n a t i o n  separation relative  procedures, to  erythrocyte partition  the  although  phenomenon  contamination  i t s presence  sought.  This  i n the present  was work,  throughout a l l c e l l  may  be  insignificant  the  case  i t being  for rabbit o f such  as not t o i n t e r f e r e w i t h any CCD r e s u l t r e g a r d i n g  However,  this  was  not t h e c a s e  f o r l e u k o c y t e s , which  carefully  by m i c r o s c o p y . I n a d d i t i o n t o t h e s e ,  low  platelets.  were  monitored  o t h e r methods were used  h e r e i n t o d e f i n e t h e "pure p l a t e l e t " p o p u l a t i o n f o r comparison w i t h t h e age-related  subpopulation,  particularly  double  labeling  techniques  u s i n g 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 g e n e r a l  label.  That p a r t i t i o n i s s e n s i t i v e t o 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 t h r o u g h 1.7). However, t h e p r e c i s e c h a r a c t e r o f the  surface  detected  charge  detected  by  partition  may  by e l e c t r o p h o r e s i s o r s u r f a c e s i a l i c  be d i f f e r e n t a c i d analyses  from  that  (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  distribution  o f charge  on membrane g l y c o p r o t e i n s  and  glycolipids  (Seaman and Brooks 1977; L e v i n e , e t a l . , 1983) and no d e t a i l e d a n a l y s i s of  the relation  Membrane s i a l i c of  terminal  from  acid analyses sialic  glycoproteins. derive  between t h e two has been c a r r i e d  groups  by e n z y m a t i c c l e a v a g e a s s a y s t h e r e l e a s e  acid,  A large  predominately  fraction  other  out f o r p l a t e l e t s .  from  integral  membrane  o f membrane s u r f a c e charge appears t o  than  sialic  acid,  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 t o t h e t o t a l s u r f a c e at  a l l depths  other  i n t h e membrane, and hence d i f f e r s  two methods mentioned  certain  findings  sensitive (shifts)  i n the l i p i d that  Also  that  are  environment  newly  released  s e n s i t i v e systems  e i t h e r of the  (D.E. B r o o k s , p e r s o n a l communication). charge-sensitive  t o a l t e r a t i o n s i n membrane  observation charge  indicate  from  charge  charge  phase  systems  resulting  from  o f t h e membrane ( W a l t e r , platelets  have  i s not n e c e s s a r i l y  a  changes  1982). The  low p a r t i t i o n i n  i n conflict  with  the  l i t e r a t u r e , therefore. The  evidence presented  here  i n d i c a t e s that  platelets  i n t h e HDP  f r a c t i o n a r e no h i g h e r i n p a r t i t i o n than t h e b u l k o f t h e p o p u l a t i o n and a r e v e r y heterogeneous i n systems which r e f l e c t membrane s u r f a c e charge associated  properties.  regard  the t o t a l  to  This  cannot  surface  be  charge  interpreted specifically or  surface  charge  density  platelets  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 n o t been e s t a b l i s h e d  this cell  type.  However, a r e a s o n a b l e  with  i n t e r p r e t a t i o n o f the data  of with  would  be t h a t , a l l o t h e r membrane p a r a m e t e r s b e i n g t h e same f o r a l l p l a t e l e t s within  the population  difference, (containing  regardless young  studied of  (i.e.,  spacial  i f surface expression  p l a t e l e t s ) a r e not o f h i g h e r  charge of  i s the only  such),  average  then  total  HDP  surface  - 151 -. charge  than  the majority  distribution certain  o f HDP  fraction  partition  equal  with the rabbit That  of the population.  particularly  fact  i s heterogeneous  of the p l a t e l e t s t o or higher  The  contained  LDP i s o f l o w e r  HDP  t h a n IDP, as was found  partition  c o r r e l a t e s with  t h e CCD  indicates  within  and i n t h e c a s e o f human d e n s i t y  that  that  are also of  i n certain  separated other  a  cases  platelets.  assays  of c e l l  surface charge. The  present  situation ranging  on p l a t e l e t s  found when t h e m a t u r a t i o n  from a c o r r e l a t i o n  correlation sensitive two  results  a r e i n a sense analogous t o t h e  of erythrocytes  o f decreasing  s u r f a c e c h a r g e w i t h age, t o no  a t a l l have been r e p o r t e d )  i s analyzed  phase systems. Here t h e r e t i c u l o c y t e s  day "mature" e r y t h r o c y t e s  erythrocytes are again  ( f o r which e v i d e n c e  are o f high  o f low p a r t i t i o n  by CCD  i n charge  a r e o f low p a r t i t i o n ,  partition,  similar  and 48 day o l d  t o t h e youngest  cells,  reticulocytes. There  i s some  precedent  "reticulocyte-like" the  the  circulation  1970;  the p l a t e l e t  erythrocytes platelets to  of large  functionally  and  and  maturation-ageing as  has  demonstrate  been  Jamieson,  1974;  population.  The  system i n platelets  and Charmatz,  Corash,  1982).  sequence has n o t been documented  f o r the erythrocyte.  i n that  low s u r f a c e  be a  then change i n  Based  can be drawn between t h e p a r t i t i o n  platelets,  might  active  from t h e megakaryocyte which  1979; O b r i e n  analogies  the platelet  there  ( K a r p a t k i n , 1974, 1977, 1978a; K a r p a t k i n  a similar  experiments  that  i n the megakaryocyte-platelet  consisting  released  Djaldetti,  However, for  counterpart  megathrombocyte  or p o l y p l a t e l e t s  t o t h e thought  the data  indicate  charge-sensitive fact  that  this  on  CCD  behavior that  of  young  partition  relative  age-related  platelet  - 152 subpopulation platelets  was  of  maturation  heterogeneous. partition,  e n r i c h e d . i n a c o n f i n e d a r e a o f the CCD  other  or  -  ages  present  ageing,  i.e.  However,  LDP  different  their  partition  (containing  i n agreement w i t h  other  membrane  old  indicates that properties  behavior platelets)  methods o f  surface  upon  becomes  more  are  lower  charge  of  analyses  and a n a l o g o u s t o t h e e r y t h r o c y t e model as w e l l .  Conclusion The  purpose o f t h i s t h e s i s  heterogeneity  of  distribution  platelet  possibility  age  distributions  by  countercurrent  of  process  eventually  a t the membrane l e v e l ; and  developing  a  relatively  information  to assess  rapid,  the  technically  i n e x p e n s i v e , 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 o f  clinical  specimens. T h i s would p r o v i d e  distribution  in  newly-released, Whether  a  single  regulated occurring antigen,  processes  associated  the  immune  ageing  cell  cells  are  enabling  not  yet  been  with  related,  system,  immunoglobulins due have  the  to  and  platelet  detection  perhaps  the  proven.  by  removed has  from been  the  general,  circulation  proposed  binding  appearance o f In  that  removal  whether t h e s e  ( p l a t e l e t ) s u r f a c e are o f i n t e r e s t  it  of the p l a t e l e t  age of  young p l a t e l e t s i n t h e c i r c u l a t i o n .  the  by  an e s t i m a t e  experiment,  c i r c u l a t i o n a r e random or age  system,  e s s e n t i a l l y t w o - f o l d : t o examine the  i n two-polymer aqueous phase systems, g a i n i n g  on the p l a t e l e t a g e i n g  simple,  was  by  cell  a  the  from  the  processes  are  of  new  age  aspects  related of  the  i n themselves. Since o l d the  reticuloendothelial  senescence  involves  t r a n s f o r m a t i o n which t a k e s p l a c e a t the c e l l membrane s u r f a c e , the m i c r o e n v i r o n m e n t w i t h i n t h e  naturally  system t o r e c o g n i z e  the  cell  a  enabling as  being  - 153 old,  and  such  eliminate  membrane  i t from t h e  age  related  advance our u n d e r s t a n d i n g environment  in  associated removal. physically  of  the  harvesting  present  distribution platelet  no of  drawbacks  release,  a  patient's  provide  partial  (platelet)  might  since  the  information  distribution and  indeed their  communications  ageing,  provide  differing  arrest  some  and  means  s u r v i v a l capacity  population.  l a b e l i n g with procedure  patients' platelet  age  elucidate  would  surface  maturation,  platelet  age  platelet  to  method i s a v a i l a b l e t o d e t e r m i n e an  the  the  examine and  of  and/or  platelet longevity.  complex, and for  surface  p l a t e l e t s of  s u r v i v a l t i m e by  several  To  transformations  information  biochemically manipulating At  molecular  the  platelet  Moreover,  circulation.  o f the i n t e r a c t i o n s o f p l a t e l e t s w i t h i n  terms  with  -  gained  i s hazardous,  incomplete,  population.  after  density  sometimes  separation/analysis  I n d i u m - I l l or  Determination  of  Chromium-51  the c a l c u l a t i o n a mean  attempts to  analyze  based  separations  correlations,  is  has  only  size  conflicting  procedure  age  providing  Since  and  unequivocal  needed  which  a  cell  is  more  s e n s i t i v e t o c h a r a c t e r i s t i c s which change d u r i n g c e l l u l a r a g e i n g ,  e.g.,  membrane s u r f a c e p r o p e r t i e s . One  such c e l l  distribution separates and on  has the  in  s u r f a c e membrane a n a l y s i s t e c h n i q u e two-polymer  aqueous  phase  countercurrent This  technique  on the b a s i s o f a c o n t r o l l a b l e v a r i e t y o f s u r f a c e p r o p e r t i e s , been shown t o be  capable of separating  b a s i s of i n vivo maturation  and  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 which  systems.  is  are  shown t o be  distribution.  r e l a t e d , at  These b l o o d  elements,  ageing.  mammalian I t was  erythrocytes  applied i n  t o membrane s u r f a c e  l e a s t i n p a r t , t o the while  being  this  properties  platelet  heterogeneous  due  age to  - 154 various  age  surface  p r o p e r t i e s which  different  and  ageing  combination  with  c o n s t i t u e n t s ) can  non-age  related are  different  characteristics. in vitro  sources,  provide a l t e r n a t i v e  time r e l e v a n t i n a c l i n i c a l  setting.  distributed  for platelet  Thus,  labeling  are  subpopulations  i t i s possible  procedures  (or assays  information  based  that  CCD  on of in  of  platelet  to p l a t e l e t  survival  - 155 -  REFERENCES A l b e r t s s o n , P.A. 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(1967) K i n e t i c s t u d i e s o f p l a t e l e t a g g r e g a t i o n i n d u c e d 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 a g e n t s , g u a n i d i n o compounds, and adenosine. Thrombos D i a t h . Haemorrh. 18:713-725. Z u c k e r , M.B., and P e t e r s o n , J. (1968) I n h i b i t i o n o f adenosine diphosphate-induced secondary aggregation and other platelet 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 . P r o c . Soc. E x p t l . B i o l . Med. 127:547-551.  - 176 -  APPENDIX  - 177 Table A . l Freezing Point Depression A n a l y s i s of Deionized  Stractan  StRSB  Tonicity  (mOsm, mean + S.Dev.)  0.00 8.06 10.75 16.13 32.26 + 0.16 (5) S t d 100 S t d 500  3.50 27.67 33.60 50.67 160.67 100.0 502.5  2.29 0.57 1.15 2.08 8.0  (3) (3) (3) (3) (3)  0.00 6.18 7.42 9.28 12.37 18.55 37.09 + 0.38 (4) S t d 100 S t d 500  5.66 28.33 33.00 36.00 45.00 73.00 219.67 130.33 524.67  + + + + + + + + +  1.1 1.2 1.0 1.0 1.0 3.6 5.5 5.1 3.1  (3) (3) (3) (3) (3) (3) (3) (3) (3)  2.25 4.55 9.00 18.0 36.0 + 0.73 (4) S t d 100 S t d 500  2.8 5.5 14.3 46.8  + + + +  0.4 0.8 1.2 1.4  (3) . (3) (3) (3)  89.3 457.3  + 1.2 + 2.1  1.2 2.4 4.8 9.6 19.10 + 0.14 (2) 40 S t d 100 S t d 500  1.50 2.50 6.75 13.70 27.75 80.50 101 512  + + + + + +  Refer t o Section  (3) (3)  2.12 0.71 2.47 0.42 1.77 0.71  (2) (2) (2) (2) (2) (2)  2.8  T a b l e A.2 D e n s i t y o f S t r a c t a n S t o c k 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  + + + + +  2.8  0.0025 0.0042 0.0019 0.0019 0.0017  (3T (4) (3) (2) (2)  - 178 T a b l e A.3 T o n i c i t y by E r y t h r o c y t e Volume, O s m o l a r i t y v . s . H e m a t o c r i t Salt mOsM  Hmct (%)  Hmct (%) @ 10 min  Salt mOsM  Hmct (%)  Hmct (%) @ 10 min  235 240 245 250 255 260 265 270 275 280 285 290  46.5 46.0 47.5 47.0 44.5 45.5 43.5 45.0 45.5 45.0 44.5 44.0  42 41 38 43 44 43 42.5 42 43.5 43 43 43.5  295 300 305 310 315 320 325 330 335 340 345 350  44.0 44.0 43.5 43.0 42.5 42.5 42.0 41.5 41.5 40.5 40.0  43.5 43 43 43 42.5 42 42 41.5 41.5 38.5 40.0  2.8  Table A.4 D e n s i t y D i s t r i b u t i o n o f Human P l a t e l e t s on S t r a c t a n StR (%)  HuPlt 11/23 (% T o t )  HuPlt 11/27 (% T o t )  HuPlt 11/29 (% T o t )  12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0  0.00 3.26 7.87 4.34 11.58 13.73 22.91 31.42 52.45 65.10 77.68 87.93 92.99 97.95 95.90 100  0.00 1.47 5.88 6.67 22.79 38.97 24.51 44.61 51.72 60.05 71.81 • 99.26 100 91.42 95.1 82.35  6.28 4.20 0.00 5.94 4.77 4.77 5.89 9.33 13.31 27.65 40.25 55.95 69.65 84.68 98.16 100  2.8  - 179 T a b l e A.5 B l o o d C e l l Recovery StR%  StR  13.5  1.055 1.061 1.062 1.065 1.066 1.070 1.071 1.074 1.075 1.080 1.084  15.0 16.0 17.0 18.0 19.0 20.0  (g/ml)  from S t r a c t a n D e n s i t y G r a d i e n t  R a P l t (%)  A p e P l t (%)  H u P l t (%)  14.7+2.3  17.42+6.68  43.3+4.2  38.61+7.05  29.3+3.1  29.95+6.67  Hu MNC (%)  6.0 11.7 32.0  18.5 33.8 27.1 62.0 12.7+3.1  14.03+3.58  9.0  2.8  T a b l e A.6 S t r a c t a n and D e x t r a n , C o n c e n t r a t i o n and O s m o l a r i t y Stractan% (g/ml)  Tonicity mOsm  % Tonic % 290 mOsm  Dextran% (g/ml)  Tonicity mOsm  % Tonic % 290 mOsm  14.0 16.0 18.0 32.0  44 50 64 160  15.2 17.2 22.1 55.2  14.0 16.0 18.0 32.0  17.0 22.5 29.5 200  5.9 7.8 10.2 69.0  2.9 T a b l e A.7 Two-Polymer Phase System 5/4: D e x t r a n (g)  PEG (g) NaCl (g) NaPB (g) H 0 (g) 2  2.9  A 1X3  , 6.67 25.0 6.83  B  Composition C  D  E  F  G  IT75  1775  LT75  LT75  I D  6.67 2.08 20.88 8.87  6.67 4.15 16.75 10.93  6.67 6.25 12.63 12.95  6.67 8.33 8.48 15.02  6.67 6.67 10.43 12.5 4.83 2.18 16.57 17.15  iTTT"  - 180 T a b l e A.8 A l t e r n a t e S a l t C o n c e n t r a t i o n and R e c i p e 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 HP04 (M) N a H P 0 (M) NaCl (M) NaPB/NaCl  °~^9 0.0345 0.0 10/0  0.0872 0.0276 0.03 8/2  0.0654 0.0207 0.06 6/4  0.0436 0.0138 0.09 4/6  0.0218 0.0069 0.12 2/8  0.0 0.0 0.15 0/10  2  2  4  2.9  T a b l e A.9 L i n e a r i t y o f C e l l o s c o p e Counts U s i n g 76 M i c r o n O r i f i c e Stock Counts V o l ( u l ) /255 u l 1.25 2.50 3.75 5.0 7.5 10.0 15.0 20.0 40.0 60.0 80.0 100.0  Counts /255 u l  4149 8658 11981 16369 22343 28729 40769 52697 88616 115794 132834 146273  Counts /255ul  8254  9062  15964  15526 22343 28542 39873  28916  Counts /255 u l  Count Less Rate/255 u l C o u n t ( % )  10000  2.24  20000  4.48  40000 60000 80000 100000  8.96 13.43 17.90 22.38  140000 160000 180000  31.33 35.81 40.28  17616 41665 52697  2.12 T a b l e A.10 Volume and Diameter v . s . T h r e s h o l d , w i t h 76 um O r i f i c e Window Setting  Volume (um )  Diameter (um)  5 10 20 30 40 50 60 70 80 90 100  1.55 3.09 6.19 9.28 12.37 15.47 18.56 21.66 24.75 27.84 30.94  1.44 1.81 2.28 2.61 2.87 3.09 3.28 3.46 3.62 3.76 3.89  2.12  3  Standards (um d i a m e t e r )  Fixed C e l l s  FxRaPlt 2.02  3.05  FxHuRBC  - 181 T a b l e A.11 N o r m a l i z e d Data Summary, R a b b i t LDP, IDP, and HDP CCD Tr n  Peak CCDC/n  Mod CCDC/n  Mid CCDC/n  Spd CCDC/n  Spd CCDC /(n)  28.2 35.5 36.4  1--3 40 41--43 81--83  0.5789 0.5789 0.5789  0.5921 0.5921 0.5921  0.5721 0.5797 0.5829  0.297 0.317 0.317  1.875 2.006 2.006  LDP IDP HDP  62.1 26.6 11.2  1--3 40 41--43 81--83  0.6316 0.7368 0.6316  0.6447 0.6447 0.6447  0.6105 0.6074 0.6105  0.305 0.317 0.288  1.929 2.003 1.820  1/ 14 28/ 17.5 80 20  LDP IDP HDP  4.1 81.8 14.1  1--3 40 41--43 81--83  0.3816 0.5921 0.6974  0.4342 0.5921 0.5921  0.3889 0.6082 0.4945  0.501 0.359 0.697  3.170 2.269 4.405  10/ 14. 8 7/ 17. 8 80 20  LDP IDP HDP  16.3 73.7 10.0  81--83 40 41--43 1--3  0.6184 0.5658 0.5658  0.6447 0.5921 0.5395  0.6000 0.5650 0.5471  0.309 0.335 0.280  1.951 2.119 1.796  1/ 16 12/ 18 82 20  LDP IDP HDP  5.2 45.6 32.4  1 31 61  29  0.3214 0.7500 0.7143  0.3393 0.7500 0.7500  0.3214 0.6775 0.6804  0.268 0.323 0.268  1.445 1.740 1.445  1/ 16 28/ 82 18  LDP 26.5 IHHD 78.7 LIDP 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 21/ 18 83 20  LDP IDP HDP  14.1 68.6 17.3  1--3 37 41--43 81--83  0.5143 0.5714 0.6000  0.5429 0.5571 0.5429  0.5351 0.5443 0.5443  0.246 0.240 0.246  1.496 1.462 1.496  8/ 16 27/ 18 83 20  LDP IDP HDP  9.0 64.0 27.0  1--3 37 41--43 81--83  0.4286 0.4571 0.5143  0.4571 0.4571 0.4571  0.4171 0.4294 0.4323  0.309 0.343 0.360  1.879 2.087 2.191  11/ 16 17/ 18 83 20  LDP IDP HDP  14.9 74.2 10.8  1--3 37 41--43 81--83  0.4571 0.5714 0.4857  0.4571 0.5286 0.4857  0.4571 0.5200 0.4837  0.200 0.200 0.212  1.218 1.218 1.287  11/ 16 23/ 18 83 20  LDP IDP HDP  24.5 67.6 7.9  1--3 37 41--43 81--83  0.5143 0.6571 0.4286  0.5000 0.6429 0.4429  0.4957 0.6454 0.4263  0.177 0.166 0.189  1.078 1.009 1.148  4/ 16 23/ 18 83 20  LDP IDP HDP  15.4 67.7 16.9  1--3 37 41--43 81--83  0.4000 0.4571 0.5429  0.4000 0.4286 0.4571  0.3763 0.4263 0.4443  0.215 0.240 0.275  1.305 1.462 1.670  4/ 16 29/ 18 83 20  LDP IDP HDP  29.5 50.2 20.3  1--3 37 41--43 81--83  0.4429 0.4429 0.3857  0.4286 0.4571 0.4000  0.4306 0.4580 0.3797  0.283 0.300 0.275  1.723 1.826 1.670  Exp Grad MDY % S t R  Pit Pit Pop %  11/ 16 15/ 17. 5 79 20  LDP IDP HDP  1/ 5/ 80  3.2  16 18 20  Load CCDC  1 / 2  - 182 T a b l e A.12 N o r m a l i z e d Data Summary, Human LDP, IDP, and HDP CCD Exp Grad MDY % S t R  Pit Pop  Pit  %  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 40 41-43 40 81-83 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 5/ 18 79 20  LDP IDP HDP  34.1 54.0 11.9  1--3 40 41--43 40 81--83 40  0 .3125 0 .4125 0 .6125  0.300 0.425 0.5375  0.3298 0.401 0.5329  0.3376 0.1372 0.3112  2.135 0.867 1.968  11/ 16 8/ 18 79 20  LDP IDP HDP  29.9 38.3 31.8  1--3 40 41--43 40 81--83 40  0.6125 0.525 0.525  0.525 0.525 0.5625  0.5408 0.5118 0.5448  0.4220 0.3482 0.2559  2.669 2.202 1.618  11/ 16 9/ 18 79 20  LDP IDP HDP  42.9 33.0 24.1  1--3 40 41--43 40 81--83 40  0.4125 0.4125 0.5125  0.450 0.4375 0.575  0.431 0.441 0.509  0.449 0.285 0.485  2.837 1.801 3.069  LDP IDP HDP  16.3 60.8 22.9  1--3 40 41--43 40 81--93 40  0.50 0.50 0.60  0.50 0.50 0.675  0.498 0.508 0.603  0.375 0.180 0.334  2.372 1.140 2.114  1/ 15 10/ 18 80 20  LDP IDP HDP  5.5 82.5 12.0  1--3 40 31--33 40 81--83 40  0.21 0.51 0.61  0.225 0.463 0.60  0.210 0.462 0.623  0.361 0.264 0.390  2.285 1.668 2.469  1/ 15 24/ 18 80 20  LDP IDP HDP  16.5 69.8 13.7  1--3 40 41--43 40 81--83 40  0.51 0.61 0.6125  0.525 0.575 0.650  0.507 0.558 0.661  0.301 0.277 0.277  1.902 1.752 1.752  1/ 15 31/ 18 80 20  LDP IDP HDP  10.0 84.0 6.0  1--3 40 41--43 40 81--83 40  0.4125 0.4125 0.3125  0.450 0.45 0.325  0.492 0.450 0.426  0.441 0.240 0.557  2.787 1.518 3.520  2/ 6/ 80  LDP IDP HDP  13.0 83.6 3.4  1--3 40 41--43 40 81--83 40  0 .6125 0 .6125 0 .5125  0.5625 0.5625 0.575  0.545 0.618 0.464  0.377 0.586 0.607  2.385 3.703 3.837  11/ 15.5 12/ 18 81 20  1.5 LDP IDP 65.6 HDP 31.3 HDP* 1.6  1--3 40 41--43 40 81--83 40  0.5875 0.5875 0.5875  0.6125 0.575 0.60  0 .593 0 .551 0 .596  0.314 0.271 0.205  1.983 1.726 1.299  11/ 16 18/ 18 81 20  LDP IDP HDP  20.0 53.0 27.0  1--3 37 41--43 37 81--83 37  0.6351 0.6891 0.6351  0.6216 0.6530 0.6530  0.5389 0.6238 0.6443  0.2922 0.2221 0.2162  1.777 1.351 1.315  1/ 8/ 80  3.2  15.5 18 20  15 18 20  - 183 T a b l e A.13 N o r m a l i z e d Data Summary, Monkey LDP, IDP, and HDP CCD Tr n  Peak CCDC/n  Mod CCDC/n  Mid CCDC/n  1--3 40 41--43 40 81--83 40  0.1125 0.1125 0.1125  0.0625 0.1125 0.1125  0.08188 0.1899 0.10025 0.1424 0.11475 0.1556  1.201 0.9008 0.9842  Phase System 5/3.5:A 2/ 15 LDP 26.5 1--3 40 19/ 18 IDP 67.2 41--43 40 80 20 HDP 2.3 81--83 40 HHD 4.0  0.2125 0.2125 0.3125  0.2000 0.2250 0.3000  0.1845 0.2255 0.3035  0.2058 0.1978 0.4430  1.3013 1.2507 2.8018  Phase System 5/3.3:A 3/ 15 LDP 16.1 1--3 40 10/ 18 IDP 72.5 41--43 40 80 20 HDP 8.8 81--83 40 HHD 2.6  0.2125 0.3125 0.2125  0.2125 0.2625 0.2125  0.1953 0.2560 0.1861  0.1319 0.2532 0.2242  0.8341 1.6014 1.418  0.2125 0.4125 0.3125  0.2625 0.3750 0.3125  0.2586 0.3760 0.3074  0.2532 0.2770 0.3350  1.6014 1.7516 2.1186  Exp Grad MDY % S t R  Pit Pit Pop %  Load CCDC  Phase System 5/4:A 2/ 15 LDP 10.1 11/ 18 IDP 81.3 80 20 HDP 7.3 HHD 1.3  Phase System 4.85/3.3:A 3/ 15 LDP 6.1 1--3 40 11/ 18 IDP 85.7 41--43 40 80 20 HDP 8.2 81--83 40  Spd CCDC/n  Spd CCDC /(n) 1 / 2  3.2 T a b l e 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 F u n c t i o n o f CCDC CCDC  K = r/n  G = r/(n-r)  CCDC  K = r/n  G = r/(n-r)  0 5 10 15 20 25 30 35 40 45 50  0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50  0.052632 0.111111 0.176471 0.250000 0.333333 0.428571 0.538462 0.666667 0.818182 1.000000  55 60 65 70 75 80 85 90 95 100  0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95  1.22222 1.50000 1.85714 2.33333 3.00000 4.00000 5.66661 9.00000 19.00000  3.2  - 184 T a b l e A.15 S t u d e n t t T e s t o f R a b b i t LDP, IDP, and HDP CCD Parameters Peak LDP 0.487 paired nonpaired IDP 0.578 paired nonpaired LDP 0.487 paired nonpaired Mode LDP 0.498 paired nonpaired IDP 0.567 paired nonpaired LDP 0.498 paired nonpaired Middle LDP 0.479 paired nonpaired IDP 0.552 paired nonpaired LDP 0.479 paired nonpaired Spread LDP 0.285 paired nonpaired IDP 0.290 paired nonpaired LDP 0.285 paired nonpaired Spread* = LDP 1.727 paired nonpaired IDP 1.752 paired nonpaired LDP 1.727 paired nonpaired 3.2  + 0.098 - IDP t = 2.441 d f t = 2.257 , d f + 0.10 - HDP t = 0.495 , d f t = 0.336 d f + 0.098 - HDP t = 1.825 d f t = 1.918 d f  0 .578 = 11, = 22, 0 .564 = 11, = 22, 0 .564 = 11, = 22,  = - 0.091 + 0.129, S.E. 0.037 (12) = 0.016 98.4% = 0.017 98.3% 0.014 + 0.096, S.E. 0.028 (12) 0.10 = = 0.315 68.5% = 0.370 63.0% 0.100 = - 0.077 + 0.147, S.E. 0.042 (12) = 0.048 95.2% = 0.034, 96.6%  + 0.10  P P +  P P +  P P  + 0.096 - IDP 0 .567 + 0.092 = - 0.069 + 0.125, S.E. 0.036 (12) t = 1.914 d f — 11, P = 0.041, 95.9% t = 1.780 d f = 22, P = 0.044 95.6% + 0.092 - HDP 0 .537 + 0.098 = 0.030 + 0.059, S.E. 0.017 (12) t = 1.810 d f = 11, P = 0.049 95.1% t = 0.616 d f — 22, P = 0.272, 73.0% + 0.096 - HDP 0 .543 + 0.10 = - 0.045 + 0.133, S.E. 0.038 (12) t = 1.162 d f = 11, P = 0.135, 86.5% t = 1.109 d f = 22, P = 0.140 86.0% + 0.095 - IDP t = 2.203 d f t = 2.016 d f + 0.081 - HDP t = 1.658 d f t = 0.954, d f + 0.095 - HDP t = 1.180, d f t = 1.079 d f  0.552 = 11, = 22, 0 .518 = 11, = 22, 0 .517 = 11, — 22,  + 0.082 - IDP t = 0.364, d f t = 0.312, d f + 0.063 - HDP t = 0.776 d f t = 0.481, d f + 0.082 - HDP t = 1.659, d f t = 0.638, d f Spd x ( n ) ' ^ + 0.541 - IDP t = 0.289, d f t = 0.134, d f + 0.388 - HDP t = 0.798, d f t = 0.558, d f + 0.541 - HDP t = 1.639, d f t = 0.607, d f  0.290 = 11, 23, 0.313 = 11, = 23, 0.313 = 11, = 22,  1  + 0.081 = - 0.073 + 0.114, S.E. 0.033 (12)  P = 0.025, 97.5% P = 0.028, 97.2% + 0.089 = 0.034 + 0.071, S.E. 0.021 (12) 93.7% = 0.063, P P = 0.175, 82.5% + 0.089 = - 0.038 + 0.113, S.E. 0.033 (12) P = 0.131, 86.9% P = 0.146, 85.4% + 0.063 = - 0.005 + 0.050, S.E. 0.014 (12)  P = 0.361, 63.9% P = 0.379, 62.1% + 0.131 = - 0.023 + 0.103, S.E. 0.030 (12) P = 0.227, 77.3% P = 0.318, 68.2% + 0.131 = - 0.028 + 0.059, S.E. 0.017 (12) P = 0.063, 93.7% P = 0.265, 73.5%  1 752 + = 11, P - 22, P 1 902 + = 11, P = 22, P 1 902 + = 11, P = 22, P  0.388 = - 0.026 + 0.310, S.E. 0.090 (12) = 0.390, 61.1% = 0.447, 55.3% 0.842 = - 0.149 + 0.649, S.E. 0.187 (12) = 0.221, 77.9% = 0.291, 70.9% 0.842 = - 0.175 + 0.370, S.E. 0.107 (12) = 0.065, 93.5% = 0.275, 72.5%  - 185 T a b l e A.16 Student t Test o f Human LDP, IDP, and HDP CCD P a r a m e t e r s Peak LDP 0.475 paired nonpaired IDP 0.526 paired nonpaired LDP 0.475 paired nonpaired Mode LDP 0.471 paired nonpaired IDP 0.512 paired nonpaired LDP 0.471 paired nonpaired Middle LDP 0.463 paired nonpaired IDP 0.510 paired nonpaired LDP 0.463 paired nonpaired Spread LDP 0.347 paired nonpaired IDP 0.284 paired nonpaired LDP 0.347 paired nonpaired -Spread* = LDP 2.186 paired nonpaired IDP 1.789 paired nonpaired LDP 2.186 paired nonpaired 3.2  + 0.136 - IDP 0.526 + 0.092 = - 0.052 + 0.101, S.E. 0.030 ( I D  t = 1.696, d f t = 1.039, d f + 0.092 - HDP t = 0.793, d f t = 0.570, d f + 0.136 - HDP t = 1.526, d f t = 1.493, d f  = 10, = 20, 0.549 = 10, = 20, 0.549 = 10, = 20,  P = 0.060, 94.0% P = 0.156, 84.4% + 0.092 = - 0.022 + 0.094, S.E. 0.028 ( I D P = 0.223, 77.7% P = 0.287, 71.3% + 0.092 = - 0.074 + 0.161, S.E. 0.048 (11) P = 0.079, 92.1% P = 0.076, 92.5%  + 0.123 - IDP t = 1.704, d f t = 0.936, d f + 0.073 - HDP t = 2.383, d f t = 1.683, d f + 0.123 - HDP = 2.476, d f t = 2.153, d f  + 0.073 = - 0.040 + 0.079, S.E. 0.024 (11)  r  0.512 = 10, = 20, 0.572 = 10, = 20, 0.572 = 10, = 20,  P = 0.060, 94.0% P = 0.180, 82.0% + 0.094 = - 0.060 + 0.084, S.E. 0.025 (11) P = 0.019, 98.1% P = 0.054, 94.6% + 0.094 = - 0.101 + 0.135, S.E. 0.041 ( I D P = 0.016, 98.4% P = 0.022, 97.8%  + 0.112 - IDP t = 1.873, d f t = 1.180, d f + 0.072 - HDP t = 1.469, d f t = 1.203, d f + 0.112 - HDP t = 2.077, d f t = 2.075, d f  0.510 = 10, = 20, 0.550 = 10, = 20, 0.550 = 10, = 20,  + 0.072 = - 0.047 + 0.084, S.E. 0.025 (11) P = 0.045, 95.5% P = 0.126, 87.4% + 0.082 = - 0.040 + 0.090, S.E. 0.027 (11) P = 0.086, 91.4% P = 0.122, 87.9% + 0.082 = - 0.087 + 0.139, S.E. 0.042 ( I D P = 0.032, 96.8% P = 0.026, 97.5%  + 0.086 - IDP t = 1.501, d f t = 1.449, d f + 0.116 - HDP t = 2.410, d f t = 1.736, d f + 0.086 - HDP t = 0.686, d f t = 1.684, d f Spd x ( n ) ^ + 0.548 - IDP t = 1.497, df t = 1.431, d f + 0.736 - HDP t = 2.416, d f t = 1.731, d f + 0.548 - HDP t = 0.991, d f t = 0.688, d f  0.284 = 10, = 20, 0.382 = 10, = 20, 0.382 = 10, = 20,  + 0.116 =  1  1.789 = 10, = 20, 2.412 = 10, = 20, 2.029 = 10, = 20,  P P +  P P +  P P  0.063 + 0.139, S.E. 0.042 (11) = 0.082, 91.8% = 0.081, 91.9% 0.148 = - 0.098 + 0.135, S.E. 0.041 (11) = 0.018, 98.2% = 0.049, 95.1% 0.148 = - 0.035 + 0.168, S.E. 0.051 ( I D = 0.254, 74.6% = 0.251, 74.9%  + 0.736 =  P P +  P P +  P P  0.397 + 0.880, S.E. 0.265 (11) = 0.083, 91.7% = 0.084, 91.6% 0.940 = - 0.623 + 0.855, S.E. 0.258 ( I D = 0.018, 98.2% = 0.049, 95.1% 0.908 = 0.157 + 0.527, S.E. 0.159 (11) = 0.173, 82.8% = 0.250, 75.0%  - 186 T a b l e A.17 S t u d e n t t Test o f Monkey LDP, IDP, and HDP CCD P a r a m e t e r s Peak LDP 0.188 paired nonpaired IDP 0.263 paired nonpaired LDP 0.188 paired nonpaired Mode LDP 0.184 paired nonpaired IDP 0.244 paired nonpaired LDP 0.184 paired nonpaired Middle LDP 0.180 paired nonpaired IDP 0.239 paired nonpaired LDP 0.180 paired nonpaired Spread LDP 0.195 paired nonpaired IDP 0.218 paired nonpaired LDP 0.195 paired nonpaired Spread* = LDP 1.235 paired nonpaired IDP 1.376 paired nonpaired LDP 1.235 paired nonpaired 3.2  + 0.050 - IDP t = 1.567 , d f t = 1.083 , d f + 0.129 - HDP t = 0.522 , d f t = 0.310 , d f + 0.050 - HDP t = 1.732 , d f t = 0.926 , d f  0.263 + 0.129 = = 3, p =. 0.108, = 6, p =: 0.160, 0.238 + 0.096 = = 3, p =: 0.319, = 6, p == 0.384, 0.238 + 0.096 = = 3, p =: 0.091, = 6, p =: 0.195,  - 0.075 + 0.096, S.E. 0.048 (4) 89.2% 84.0% 0.025 + 0.096, S.E. 0.048 ( 4 ) 68.1% 61.7% - 0.050 + 0.058, S.E. 0.029 (4) 90.9% 80.5%  + 0.086 - IDP 0.244 + 0.108 = - 0.059 + 0.037, S.E. 0.019 (4)  t = 3.181 d f t = 0.860 , d f + 0.108 - HDP t = 0.301 i d f t = 0.132 d f + 0.086 - HDP t = 2.450 d f t = 0.793 d f  = 3, p = 0.025, = 6, p =: 0.211, 0.234 + 0.093 = = 3, p = 0.392, = 6, p = 0.450, 0.234 + 0.093 = = 3, p = 0.046, = 6, p = 0.229,  97.5% 78.9% 0.009 + 0.062, S.E. 0.031 (4) 60.8% 55.0% - 0.050 + 0.041, S.E. 0.020 (4) 85.4% 77.1%  + 0.073 - IDP 0.239 + 0.113 = t = 2.791 d f = 3, p = 0.034, t = 0.881 d f = 6, p = 0.206, + 0.113 - HDP 0.228 + 0.094 = t = 0.317 d f = 3, p = 0.386, t = 0.156 d f = 6, p = 0.441, + 0.073 - HDP 0.228 + 0.094 = t = 1.794 d f = 3, p = 0.085, t = 0.803 d f = 6, p = 0.226,  - 0.059 + 0.043, S.E. 0.021 (4) 96.6% 79.4% 0.011 + 0.072, S.E. 0.036 ( 4 ) 61.4% 56.0% - 0.048 + 0.053, S.E. 0.027 (4) 91.5% 77.4%  + 0.050 - IDP t = 0.621, d f t = 0.573 d f + 0.060 - HDP t = 1.189, d f t = 1.028, d f + 0.050 - HDP t = 1.695, d f t = 1.388, d f Spd x (n) ^ + 0.317 - IDP t = 0.621, d f t = 0.523, d f + 0.380 - HDP t = 1.189, d f t = 1.028, d f + 0.317 - HDP t = 1.695, d f t = 1.388, d f  - 0.022 + 0.072, S.E. 0.036 (4) 71.1% 70.6% - 0.072 + 0.121, S.E. 0.061 ( 4 ) 84.0% 82.8% - 0.094 + 0.111, S.E. 0.056 (4) 90.6% 89.3%  0.218 + 0.060 = = 3, p = 0.289, = 6, p = 0.294, 0.290 + 0.126 = = 3, p = 0.160, = 6, p = 0.172, 0.290 + 0.126 = = 3, p = 0.094, = 6, p = 0.107,  1  1.376 + 0.380 = - 0.142 + 0.456, S.E. 0.228 (4)  = 3, p = 0.289, 71.1%  = 6, p = 0.291, 1.831 + 0.799 = = 3, p = 0.160, =• 6, p = 0.172, 1.831 + 0.799 = = 3, p = 0.094, = 6, p = 0.107,  70.6% - 0.455 + 0.765, S.E. 0.382 ( 4 ) 84.0% 82.8% - 0.596 + 0.704, S.E. 0.352 (4) 90.6% 89.3%  - 187 T a b l e A.18 S i z e A n a l y s i s o f R a b b i t LDP, IDP, and HDP Exp  P i t Subpop  17 28/ 80  LDP IDP HDP PRP  Peak% W  Mode% W  26725 33.75 42.5 31.25  (Celloscope)  Mid% W  26 30 40 30  Spd% W  26792 37.47 43.54 40.90  53783 74.93 87.08 81.79  3.3 T a b l e A.19 R a b b i t and Rat LDP, IDP, and HDP, S i z e by L a s e r S c a t t e r i n g Wn  %  RaPRP 10/8/80  2 3 0.25 4 1.95 5 2.3 6 2.15 7 1.6 8 1.45 9 0.95 10 0.95 0.6 11 12 0.25 13 0.25 14 0.25 15 16 17 18 19 20 21-•26  RaPRP 10/16/80  1.75 8.95 8.1 6.75 3.15 1.75 1.45 0.95 0.6 0.6 0.45 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25  RaLDP RalDP RaHDP 10/8/80  RtLDP RtlDP RtHDP 2/25/81  0.25 1.45 6.5 4.95 3.15 1.8 1.25 0.6 0.25 0.25  0.25 1.45 1.45 0.95 0.75 0.6 0.45 0.45 0.25  4.5 2.65 3.5 3.5 2.65 2.15 1.25 1.1 0.75 0.55 0.4 0.25 0.25  1.65 2.15 2.3 1.95 1.45 0.95 0.95 0.75 0.6 0.25 0.25  1.45 1.45 0.75 0.95 0.75 0.75 0.6 0.25 0.45  0.25 1.0 0.75 2.1 1.6 1.45 0.6 0.6 0.45 0.25 0.45 0.25 0.25  0.25 0.25  3.3 P l a t e l e t s were a l s o s i z e d u s i n g 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 c o u n t e r . P l a t e l e t s i z e h i s t o g r a m s were t a b u l a t e d 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 n c e n t r a t i o n s were: (10/8/80) LDP ( 5 7 ) , IDP ( 5 2 ) , HDP ( 3 5 ) , RaPRP ( 3 3 ) , (10/16/80) RaPRP ( 3 4 3 ) , WBC ( 5 . 9 ) , RBC ( 5 . 5 6 ) ; and f o r t h e f o l l o w i n g t a b l e , (10/23/80) H u P l t (228), HuRBC (4.37) 33% W, HuWBC (8.7) 4 0 % W, (10/8/80) H u P l t ( 5 0 5 ) .  - 188 T a b l e A.20 Human LDP, IDP, and HDP, S i z e by L a s e r S c a t t e r i n g % w  HuPRP 10/23/80  HuPRP 10/8/80  3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 39 40 41 43 44  0.25 1.1 1.6 1.95 2.1 2.25 2.45 2.1 1.95 1.6 1.95 1.6 1.45 1.6 0.95 1.1 1.1 0.95 0.6 0.95 0.75 0.75 0.45 0.6 0.6 0.45 0.6 0.45 0.25 0.45 0.45 0.45 0.45 0.25 0.25  1.25 5.75 7.05 7.6 7.6 7.05 6.25 5.2 4.35 4.35 3.6 2.45 2.1 1.75 1.6 1.45 1.1 1.25 0.75 0.75 0.55 0.55 0.55 0.45 0.25 0.45 0.25 0.45 0.25 0.45 0.25 0.25 0.25 0.25 0.25 0.25  3.3  0.25 0.25 0.25 0.25  HuLDP HuIDP HuHDP 11/12/81  6.0 7.5 6.0 4.5 4.5 4.5 2.5 2.5 2.5  4.5 2.3 3.65 3.45 2.95 2.95 2.95 2.45 2.3 2.15 1.95 1.75 1.45 1.1 0.95 0.6 0.75 0.6 0.6 0.45 0.25 0.25 0.25 0.25 0.25  0.75 0.95 1.1 0.95 1.25 1.1 1.45 1.1 0.75 0.95 0.75 0.6 0.45 0.6 0.45 0.25 0.25 0.25 0.25 0.25 0.25  HuLDP HuIDP HuHDP 11/18/81 0.75 0.95 0.75 0.75 0.75 0.6 0.45 0.45 0.25 0.45 0.25 0.25  2.5 1.6 2.45 3.1 2.6 2.6 2.1 2.1 1.95 1.6 1.6 1.45 1.25 1.1 0.95 0.95 0.95 0.75 0.75 0.6 0.45 0.6 0.45 0.25 0.25 0.25 0.25 0.25  0.25 0.25 0.25 0.25  1.1 1.75 1.45 1.75 1.6 1.6 1.6 1.45 1.6 1.45 1.25 1.45 0.95 1.1 0.75 0.75 0.45 0.45 0.45 0.6 0.6 0.45 0.45 0.45 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25  - 189 T a b l e 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 o f P l a t e l e t s and RBC S t a n d a r d s  (3/10/80)  (12/19/80)  (1/22/81) (2/25/81)  Sample  Electrophoretic Mobility  Monkey P i t i n PRP Monkey P i t i n PRP Human P i t i n PRP Human RBC (HuRBC) Monkey P i t ( M o P l t ) Monkey P i t Rabbit P i t (RaPlt) Rabbit P i t Human RBC Human RBC  1.021 1.047 0.939 1.127 1.408 1.413 1.214 1.231 1.087 1.096  + + + + + + + + + +  0.301 0.113 0.058 0.108 0.082 0.078 0.062 0.061 0.025 0.037  (23) (40) (40) (19) (20) (20) (20) (20) (10) (10)  3.4  T a b l e 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 o f D e n s i t y S e p a r a t e d P l a t e l e t s Sample MoPlt MoPlt HuPlt FxRtPlt  LDP EPM 0.988 1.089 1.094 -1.078 1.323  + + + + +  0.170 0.143 0.026 0.024 0.043  (21) (40) (10) (10) (10)  IDP EPM  HDP EPM  1.043 + 0.144 (23) 1.076 + 0.065 (40) 1.081 + 0.046 (10)  1.081 + 0.120 (23) 1.083 + 0.098 (40) 1.087 + 0.037 (10)  1.320 + 0.046 (10)  1.330 + 0.033 (10)  3.4 T a b l e A.23 S e p a r a t i o n o f F i x e d R a b b i t P l a t e l e t s by S i z e Sed Fr #  Distance V o l (ml)  FxRaPlt Cone x l O  1 2 3 4 5 6 7 8 9 10 11 12  10 15 20 25 30 35 40 45 50 55 57.5 60  0.167 0.436 1.76 3.09 3.46 3.77 3.81 4.15 4.13 4.12 3.93 17.4  2.02 S t d 4.04 S t d 3.5  - 7  Sedimentation  Mode  Size % W Mid Spd  18 19 25 30 30 30 30 30 30 30 30 30  18 19 30 35 37 36 39 38 38 37 38 38  46 100  46 100  36 35 39 50 52 53 55 56 55 58 53 55 6 6(2)  - 190 T a b l e A. 24 EPM o f S i z e S e p a r a t e d , F i x e d  Platelets  Sed Fr #  FxMoPlt 1/26/81  FxRaPlt 1/27/81  1.376 + 0.0654 1.378 + 0.0389  1.275 + 0.0343  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 3.5  FxHuPlt 12/19/80  1.362+0.0744 1.370 + 0.0789 1.373+0.0890 1.409 + 0.0774 1.409+0.0754 1.403 + 0.0764 1.416 + 0.0762 1.417 + 0.0873 1.449+0.0676 1.408 + 0.0668 1.417 + 0.0710 1.395 + 0.0604 1.436 + 0.0734  FxHuPlt 1/22/81  1.388+0.0577 1.394 + 0.0480 1.406+0.0443 1.404 + 0.0411 1.432 + 0.0420 1.421 + 0.0435 1.416+0.0530 1.423 + 0.0519  1.371 + 0.0428 1.400 + 0.0626 1.410+0.0432 1.424 + 0.0400 1.425+0.0532 1.432 + 0.0457 1.435+0.0386 1.435 + 0.0386 1.436 + 0.0510  1.278 + 0.0355 1.282+0.0522  1.424+0.0315  1.290+0.0328 1.290 + 0.0436 1.303 + 0.0507 1.294 + 0.0479 1.290+0.0328  1.431 + 0.0311  1.300 + 0.0491  1.429 + 0.0363  1.306 + 0.0518  1.435 + 0.0432  1.306 + 0.0430  1.428 + 0.0463 1.425 + 0.0563  1.303 + 0.0442  - 191 F i g u r e A . l CCD o f 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 p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) w i t h t h e mean s p e c i f i c a c t i v i t y a t 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 l  8.10E+02  5. 31E+06-  1.52E-04  0.00E+00  ~i  i  i  —  —i  i  i  \—  •0. 00E+00  1.73E+08  2.20E+02  0.00E+00  0.00E+00  Figure A.2 CCD o f C r - l a b e l e d P l a t e l e t s , Two days Circulation: p l a t e l e t s p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; cpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t p e r CCDC ( d o t t e d l i n e ) , w i t h t h e mean s p e c i f i c a c t i v i t y a t 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 1  - 192 T a b l e A.25 N o r m a l i z e d CCD o f 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 5 l  Time Days S l T=0 S l T=0 S l T=2 S l T=2 S2 T=0 S2 T=3  Sample CCD Pit CPM Pit CPM Pit CPM Pit CPM Pit CPM Pit CPM  Ld CCDC  Tr n  1-3  60  Peak CCDC/n  0.620 0.522 61-63 60 0.522 0.422 1-3 60 0.422 0.517 61-63 60 0.623 0.522 1-3 120 0.463 0.463 1-3 120 0.712 0.712  Mod CCDC/n  Mid CCDC/n  Spd CCDC/n  Spd CCDC /(n)l/2  0.575 0.522 0.575 0.470 0.470 0.475 0.575 0.575 0.463 0.432 0.735 0.735  0.598 0.497 0.540 0.447 0.508 0.508 0.515 0.515 0.483 0.444 0.723 0.723  0.280 0.283 0.352 0.310 0.310 0.323 0.283 0.283 0.181 0.181 0.269 0.250  2.17 2.19 2.72 2.40 2.40 2.50 2.19 2.19 1.65 1.65 2.46 2.28  T=0  Pit CPM CPM  1-3  57  0.418 0.418 0.296  0.446 0.416 0.298  0.449 0.381  0.167 0.257  1.258 2.080  T=0  Pit CPM  1-4  36  0.528 0.422  0.494 0.458  0.450 0.414  0.406 0.353  2.43 2.12  T=0  Pit CPM CPM Pit CPM CPM Pit CPM CPM  1-4  36  0.408 0.353 0.131 0.406 0.353 0.131 0.408 0.408 0.128  0.369 0.369 0.147 0.383 0.369 0.142 0.400 0.369 0.106  0.386 0.286  0.256 0.447  1.53 2.68  0.392 0.308  0.233 0.383  1.40 2.30  0.411 0.356  0.272 0.344  1.63 2.07  T=0 T=0  3.6  41-44 36 81-84 36  - 193 T a b l e A. 26 Grouped S t a t i s t i c s , Statistic  Platelets  - L a b e l e d P l a t e l e t s Aged i n v i v o  Radioactivity  Statistic  S3T=1 (40 CCDF/120 CCDC) Ef 4.023 x 1 0 Exf 1.123 x i o i o 3.233 x i o n Ex2f XCCDF 2.792 x i o i S 2.430 x i o i  3.989 1.084 3.084 2.717 3.498  X  10 105 10  x-x  X  ioi ioi  S3T=3 (40 CCDF/120 CCDC) 4.012 x 10* Ef Exf 1.149 x i o n Ex2f 3.339 x 1012 2.864 x i o i XCCDF S2 1.213 x i o i  5.863 1.641 4.736 2.799 2.453  X  10  X-X Err t  6.519 x 1 0 " 6.469 x l O " 10.079  S4T=1 (40 CCDF/120 CCDC) 8.988 x 108 Ef Exf 2.853 x i o i o 9.186 x i o n Ex2f XCCDF 3.174 x i o i S2 1.437 x i o i  6.489 1.979 6.199 3.051 2.449  X-X Err t  1.236 1.942 63.594  S4T=3 (40 CCDF/120 CCDC) Ef 1.110 x 1 0 Exf 3.228 x 1 0 Ex2f 9.639 x 1 0 XCCDF 2.908 x 1 0 S 2.285 x 1 0  1.801 1.801 Xx 1 lO^ 0 105 4.867 x 0 X 1 1.391 1.391 x X 1 1 00 2.702 X i o i 4.193 X i o i  X-X E rrr r E t t  2.054 4.825 x 10" 42.560  8  2  9  1 0 1 1  1  2  S4T=6* (40 Ef Exf Ex2f XCCDF(-20) S 2  1  X  X X X X  X X X X X  3  lO  3  10 10 10  6  10* 10 lO? 6  ioi ioi  X  ioi  1 0  X  1 1  X  10 10 10  8  1  S4T=4* (40 CCDF/120 CCDC) Ef 2.035 x 1 0 Exf 2.694 x 1 0 Ex2f 3.664 x 1 0 XCCDF(-20) 1.332 x 1 0 S 4.739  4.330 5.585 7.530 1.328 7.530  2  9  1 0  1 1  1  X  2 3  ioi  X X  lO^ 105 10  X  ioi  X  10  X  10 10 10^ 10  X X X  1 2  X-X Err t  1.022 4.959 2.017  1  X  X  2  1  7 7  CCDF/120 CCDC) 9.506 x 1 0 5.131 1.272 x 1 0 6.342 1.758 x 1 0 8.462 1.338 x 1 0 1.237 5.795 1.237  Err t  7.481 x l O " 9.365 x l O " 7.988  1  5  2.203 7.045 2.301 3.197 2.207  3.6  X  6  4  S4T=2 (40 CCDF/120 CCDC) 1.797 x 10* Ef 5.804 x i o i o Exf 1.904 x l O * Ex2f XCCDF 3.230 x i o i S2 16.111  2  X  3  Comparison  7  X-X Err t  3.296 x 1 0 " 3.164 x l O " 10.413  X-X Err t  3.426 x i o - i 1.320 x i o - i 2.595  1 2  1  2  3  1  1  - 194 T a b l e A.27 S p e c i f i c A c t i v i t y o f C r - L a b e l e d P l a t e l e t s R e g i o n s I I and I I I i n Comparison t o t h e E n t i r e D i s t r i b u t i o n 5 1  CCD  S1T=0 1-60 Gm m _+ S.Dev. n Corr Slope Intcp S1T=0 61-120 Gm m + S.Dev. n Corr Slope Intcp S1T=2 1-60 Gm m + S.Dev. n Corr Slope Intcp S1T=2 61-120 Gm m + S.Dev. n Corr Slope Intcp 3.6  Mean CPM/Plt  II CPM/Plt  1.90 x I O " 3.15 x 10-4 2.56 x 10-4 10 -0.952 -8.05 x I O " 6.77 x I O "  within  III CPM/Plt  4  4  5  4  1.52 x I O " 2.07 x 10-4 1.22 x I O " 8 -0.910 -4.52 x I O " 3.65 x I O "  4.78 x I O " 1.94 x 10-4 6 -0.904 -9.36 x I O " 7.12 x I O "  5  4  7.02 x 10-5 3.49 x 10-5 4 -0.857 -2.32 x I O " 1.05 x I O "  5  4  4  4  5  6  6  1.30 1.30 5.20 4 0.0 0.0 1.30  x IO" x IO" x IO"  6  x IO"  6  6 8  5  5  4  1.19 x I O " 1.98 x I O " 2 -0.999 -2.80 x I O " 1.04 x I O " 6  7  7  8  5  1.32 4.95 2 1.0 7.00 1.28  x IO" x IO" x IO" x IO"  6 8  8 6  4.86 x I O " 2.18 x I O " 2 -1.0 -3.08 x I O " 6.40 x 10-5 5  5  4  1.23 x 10-6 1.19 x I O " 1.23 x I O " 5 -0.129 -1.00 x 1 0 ~ 1.21 x I O "  2.60 x 10-4 8.49 x I O " 6 -0.774 -3.51 x I O " 3.48 x I O "  7  1.18 x 1.03 x 3 0.292 3.00 x 1.15 x  1.28 6.36 2 1.0 9.00 1.23  IO" IO"  6  IO" IO"  8  x IO" x IO" x IO" x 10~  7  6  6 8  8 6  5  CCD  - 195 T a b l e A.28 S p e c i f i c A c t i v i t y o f C r - l _ a b e l e d P l a t e l e t s R e g i o n s I I and I I I i n Comparison t o t h e E n t i r e D i s t r i b u t i o n  within  5 1  CCD  S2T=0 1-120 Gm m +_ S.Dev. n Corr Slope Intcp S2T=3 1-120 Gm m +_ S.Dev. n Corr Slope Intcp S3T=1 1-120 Gm m _+ S.Dev. n Corr Slope Intcp S3T=3 1-120 Gm m +_ S.Dev. n Corr Slope Intcp 3.6  Mean CPM/Plt  II CPM/Plt  3.93 x 10-^ 3.62 x 1 0 2.44 x 10-4 11 -0.984 -7.25 x l O " 7.25 x 10-4 A  3.94 x 1 0 ~ 3.71 x l O " 1.93 x l O " 5 -0.394 -4.82 x 1 0 " 3.81 x l O "  • III CPM/Plt  6.24 x 10-4 1.42 x l O " 4 -0.986 -1.08 x l O " 7.86 x 10-4 4  5  4  2.13 x 10-4 1.34 x l O " 7 -0.971 -6.01 x 1 0 ~ 3.93 x l O "  4  5  4  7  7  9.86 x 1 0 " 9.31 x 1 0 ~ 9.85 x l O " 10 -0.203 -6.60 x 1 0 " 9.61 x 1 0 "  3.71 x l O " 2.73 x l O " 3 -0.979 -2.67 x 1 0 " 3.97 x l O " 7  8  8  9 7  8  7  3.72 1.41 2 -1.0 -2.00 3.74  x lO" xlO"  7  9  x 10~ x lO"  9 7  6 6  7  8  6  1.45 x 1.44 x 7.23 x 8 0.0117 3.45 x 1.44 x  10" 10~ 10" 10" 10"  9.54 x 1 0 " 9.83 x l O " 5 -0.888 -5.52 x l O " 1.06 x 1 0 " 6  7  5  7  9.08 x 1.04 x 5 0.890 5.85 x 7.91 x  10~ 10~  6 6  lO" 10" 6  6 6 8  1 0 6  1.46 x 8.12 x 4 0.677 4.26 x 1.39 x  10" 10" 10" 10"  6 8  8 6  1.42 x 6.81 x 4 0.407 2.15 x 1.39 x  10~ 10" 10~ 10"  6 8  8 6  7  CCD  - 196 T a b l e A.29 S p e c i f i c A c t i v i t y o f C r - L a b e l e d P l a t e l e t s R e g i o n s I I and I I I i n Comparison t o t h e E n t i r e D i s t r i b u t i o n 5 1  CCD S4T=1 1-120 Gm m + S.Dev. n Corr Slope Intcp S4T=3 1-120 Gm m + S.Dev. n Corr Slope Intcp S4T=2 1-120 Gm m + S.Dev. n Corr Slope Intcp S4T=4 1-120 Gm m + S.Dev. n Corr Slope Intcp 3.6  Mean CPM/Plt  II CPM/Plt  7.25 x IO" 5 9.29 x IO" 5 5.79 x IO" 5 17 -0.734 -8.40 x IO" 6 1.60 x 1 0 "  1.16 x 6.69 x 10 -0.824 -1.82 x 1.98 x  1.62 x 10-5 2.11 x 10-5 1.06 x 10-5 25 -0.864 -1.24 x IO" 6 3.59 x IO" 5  A  1.23 x 1.89 x 1.20 x 28 -0.559 -8.41 x 2.96 x 2.10 x 2.07 x 1.65 x 8 0.367 2.47 x 1.99 x  IO" 5 IO" 5 IO" 5 IO" 7 IO" 5 IO" 7  10~  IO"  7  8  IO" 9 IO" 7  within  III CPM/Plt  IO" 4 IO" 5  10-5  IO" 5 IO" 4  5.98 x 4.69 x 7 0.684 1.48 x 5.54 x  2.65 x IO" 3 1.04 x 10-5 15 -0.944 -2.19 x IO" 6 4.19 x IO" 5  1.28 x 1.90 x 10 0.619 3.89 x 1.11 X  10-5  2.23 x 1.25 x 20 -0.577 -1.22 x 3.39 x  IO" 5 IO" 3 10~  IO"  6  5  2.03 x 1 0 ~ 7.12 x IO" 9 4 -0.276 -1.52 x 1 0 ~ 2.05 x 1 0 ~ 7  7  9  IO" 6  10~ 10-5 6  IO" 6 io-7  10-5  1.05 x 10-5 4.57 x IO" 6 8 -0.835 -1.56 x IO" 6 1.60 x 10-5  2.12 x 2.27 x 4 0.365 6.43 x 2.03 x  IO" 7  10-8 10-9  io-7  CCD  - 197 T a b l e A.30 S p e c i f i c A c t i v i t y o f C r - L a b e l l e d P l a t e l e t s R e g i o n s I I and I I I i n Comparison t o t h e E n t i r e D i s t r i b u t i o n 5 1  CCD 5/5/81 Gm m + S.Dev. n Corr Slope Intcp 1/21/82 Gm m + S.Dev. n Corr Slope Intcp 12/10/81A Gm m _+ S.Dev. n Corr Slope Intcp 12/10/81B Gm m + S.Dev. n Corr Slope Intcp 12/10/81C Gm m +_ S.Dev. n Corr Slope Intcp 3.6  Mean CPM/Plt 7.01 x I O " 1.77 x I O " 1.11 x I O " 8 -0.281 -1.27 x I O " 2.21 x I O "  I I CPM/Plt  within  I I I CPM/Plt  3  4  4  5  4  3.05 x 1 0 ~ A.04 x-10-5 2.13 x I O " 14 -0.252 -1.29 x 1 0 ~ 4.88 x I O "  1.90 x 10-4 1.67 x 10-4 4 -0.397 -5.15 x 1 0 ~ 2.68 x 10-4  5  1.63 x 10-4 1.79 x 10-5 4 -0.886 -1.23 x I O " 1.82 x 10-4  5  5  5  6  5  4.69 x 2.16 x 8 0.648 5.70 x 2.70 x  3.18 x 10-5 1.94 x 10-5 6 -0.932 -9.65 x I O " 5.59 x 10-5  IO" 10-5 3  IO" 10-5 6  8.53 x I O " 5.16 x I O " 1.03 x I O " 17 -0.665 -1.35 x 10-4 1.60 x I O "  1.17 x I O " 1.40 x I O " 7 -0.896 -5.81 x 10-4 2.92 x i o - 3  5.58 x 9.88 x 10 0.572 1.87 x 4.74 x  3.23 x I O " 1.33 x I O " 2.22 x I O " 19 -0.583 -2.30 x 10-4 3.41 x I O "  2.72 x i o - 3 2.98 x I O " 8 -0.567 -6.91 x I O " 5.14 x I O "  3.23 x 1.11 X 11 0.898 3.00 x 1.72 x  10-4 10-4  7.20 x I O " 4.25 x I O " 7.30 x I O " 18 -0.639 -8.73 x I O " 1.17 x I O "  8.72 x I O " 9.39 x i o - 3 8 -0.652 -2.50 x: 1 0 " 1.75 x I O "  6.71 x 1.87 x 10 0.777 4.80 x 4.55 x  IO" IO"  5  4  3  3  3 3  10-5 10~ 6  10~ 10-5 6  4  3  3  3  3  4  3  10-5 IO" 4  4  3  3  3  2  4  2  3  4 4  10-5 IO" 4  6  CCD  - 198 T a b l e A.31 S p e c i f i c A c t i v i t y o f Regions I I and I I I , N o r m a l i z e d w i t h R e s p e c t t o t h e Grand Mean, and t h e Mean a t Ten P e r c e n t Combined E r r o r SA/Gm  T=0 I I / I I I  SI  1.656/0.369 1.710/0.320 1.589/0.5A2  S2 S3 SA '  T=l  II/III  T=2 I I / I I I  T=3 I I / I I I  T=4 I I / I I I  0.967/0.962 1.012/0.981 1.003/0.946 1.003/0.977 1.636/0.791  0.968/0.921 1.599/0.825 1.816/0.855  5/5/81 1/21/82 12/10/81A 12/10/81B 12/10/81C  2.6A1/2.266 1.538/1.0A3 13.72/0.65A 8.A21/1.000 12.11/0.932  SA/m  T=0 I I / I I I  SI  1.518/0.229 1.255/0.235 1.722/0.588  S2 S3 SA  T=l  II/III  T=2 I I / I I I  0.964/1.012  T=3 I I / I I I  1.003/0.998 1.015/0.985 0.998/0.941 1.013/0.987 1.260/0.636  1.025/0.975 1.2A9/0.6AA 1.177/0.556  5/5/81 1/21/81 12/10/81A 12/10/81B 12/10/81C  T=4 I I / I I I  0.976/1.024  1.076/0.92A 1.161/0.787 2.267/0.108 2.0A5/0.2A3 2.052/0.158  3.6 T a b l e A.32 C o n c e n t r a t i o n s o f Chromium and Indium i n L a b e l i n g M i x t u r e s CCDC  51-Cr S t o c k ml  C o n c e n t r a t i o n Range nmoles ug  C o n c e n t r a t i o n Range ag amoles  1-40 41-80 81-120  0.1 0.5 1.0  0.1-0.25 0.5-1.25 1.0-2.5  25-625 125-312.5 250-625  CCDC  111-In S t o c k ml  Maximum C o n c e n t r a t i o n nmoles ng  1-40 1-80 81-120  0.1 0.2 0.5  5 10 25  3.7  2-5 10-25 20-50  45 90.1 225  0.5-1.25 1.25-3.125 5-12.5  Maximum C o n c e n t r a t i o n ag amoles 1.25 2.5 6.25  11.25 22.53 56.25  - 199 T a b l e A. 33 S p e c i f i c A c t i v i t y o f Sample  3.7  C r and in in  Labeled  Platelets  111-In CPM/Plt xlO  51-Cr CPM/Plt xlO  0.9875 1.861 12.08 0.684 1.61 9.99  1.0556 3.3523 7.5379 0.853 3.23 7.20  4  Lxl Lx2 Lx3 CCDC 1-40 CCDC 41-80 CCDC 81-120  5 1  A  - 200 F i g u r e A. 3 CCD o f 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 p e r CCDC ( s o l i d l i n e ) left o r d i n a t e ; r a d i o a c t i v i t y cpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t a t 10% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t 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 : i ; L 1  0  10  20  30  40  50  60  70  60  90  100  110  120  F i g u r e A.A CCD o f 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; c p m / p l a t e l e t a t 10% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t 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 5 1  - 201 T a b l e A. 34 S u l p h a t e I n j e c t i o n  Schedule  Date Aug  Day o f Expt  Time o f Injection  Rabbit Number  Rabbit Weight  Days O l d ( p . i .35-S) on T 17 W 18 T 19 F 20 S 21  F S S M  0 1 2 3  2:05 3:15 2:00 2:30  22 23 24 26  5.5 4.2 4.5 4.55  4 3 2 1  13 14 15 16  PM PM PM PM  5 4 3 2  6 5 4 3  7 6 5 4  8 7 6 5  3.8  Table A.35 P l a t e l e t Recovery Rabbit No.  Blood ml (g)  PRP ml (g)  Blood P i t / m l xlO"  PRP P i t / m l xl0~  P i t Recovered xlO"  R26  32.7 32.7  R23  35.0  R22  33.0  2.55 2.35 3.70 3.25 2.32 1.97 2.25 2.04  5.18  R24  16.12 14.8 18.5 16.23 14.7 12.48 14.6 13.24  8.35 7.66 12.1 10.62 8.13 6.90 7.42 6.73  3.8  8  8  6.54 5.53 5.08  9  - 202 T a b l e A. 36 Time Day  S-Sulphate Labeling Kinetics  3 3  Blood CPM/ml DPM/ml xlO" xlO-4  PRP CPM/ml DPM/ml xlO" xl0~  PPP CPM/ml DPM/ml xlO" xlO-  CCD P i t Lx ~ CPM/ml DPM/ml xl0~ xl0~  1 2 3 4  4.08 1.49 0.58 0.74  1.62 0.62 0.24 0.34  6.62 3.96 1.90 1.94  1.046 0.634 0.304 0.302  7.52 4.18 1.96 2.30  1.202 0.656 0.312 0.370  2.54 2.97 3.24 1.60*  5.45 6.54 6.95 3.67*  3 4  Activity 36.52 14.22 18.14  % 1=1 38.27 14.81 20.99  Activity 59.82 28.70 29.31  % T=l 60.61 29.06 28.87  Activity 55.59 26.06 30.59  % 1=1 54.58 26.96 30.78  Activity 116.93 127.56 62.99  % 1=1 120.00 127.52 67.34  CPM/ml xlO"  DPM/ml xlO"  CPM/ml xlO"  DPM/ml xlO"  CPM/ml xlO"  DPM/ml xlO"  CPM/ml xlO"  DPM/ml xlO"  2.93 3.01 1.06 1.06 3.70 3.90 4.00 5.00  6.40 6.63 2.70 2.75 1.45 1.51 1.40 1.60  3.42 3.52 1.48 1.46 0.74 0.68 0.70 0.70  5.94 6.14 3.18 3.18 2.14 2.06 2.08 2.10  3.28 3.36 1.54 1.62 0.76 0.74 0.92 0.60  5.76 5.92 3.28 3.40 2.16 2.16 1.94 2.42  0.39 0.40 0.47 0.59 0.57 0.55 0.35 0.36  1.14 1.15 1.25 1.44 1.39 1.38 1.23 1.24  Activity 36.18 35.22 12.63 12.96 13.65 16.61  % T=l A c t i v i t y 42.19 43.27 41.48 41.48 22.66 21.64 22.78 19.32 20.47 21.88 24.13 19.89  % T=l 53.53 51.79 36.03 33.55 35.02 34.20  A c t i v i t y % 1=1 46.95 56.94 48.21 57.43 23.17 37.50 22.02 36.49 18.29 33.68 27.38 40.88  Activity 120.51 147.50 146.15 137.50 91.28 90.50  % 1=1 109.65 125.22 121.93 120.00 107.72 107.48  A c t i v i t y ' % 1=1 100 48.19 + 10.11 23.22 + 4.49 23.22 + 5.28  Activity 100 50.25 + 23.75 + 25.42 +  A c t i v i t y % T=l 100 128.3 + 16.71 137.1 + 9.30 81.92 + 16.40  100 55.31 + 4.67 32.88 + 3.53 32.70 + 3.33  100 56.32 + 1.52 33.32 + 6.39 35.11 + 5.20  100 118.29 + 7.92 123.15 + 3.91 94.18 + 23.24  100 51.75 + 8.05 28.05 + 6.52 27.96 + 6.52  100 53.25 + 4.55 28.53 + 6.74 30.27 + 7.44  100 123.30 + 12.92 130.11 + 9.94 87.89 + 19.17  3  3  1 2 3 4  2 3 4  3  Day Hr A c t i v i t y % T=l 1 21 100 46 35.97 + 0.67 2 68 13.27 + 0.83 3 4 94 16.13 + 2.28 Based on t h r e e samples 1 21 100 2 46 40.65 + 2.08 68 20.08 + 4.57 3 94 22.33 + 1.62 4 Based on t h r e e samples 1 21 100 2 46 38.31 + 2.91 68 16.68 + 4.75 3 94 19.23 + 3.83 4 Based on s i x samples 3.8  3  3  4  3  3  3  4  3  % 1=1 4.67 2.08 6.38  3  3  3  3  - 203 T a b l e A.37 S t r a c t a n C o n c e n t r a t i o n and D e n s i t y , Rand e t a l . (1981) % StR (g/ml)  Density (g/ml)  Density (g/SBStR)  Density (g/% StR)  15 15.5 16 17 17.5 19  1.058 1.060 1.063 1.067 1.069 1.076  0.003867 0.003871 0.003938 0.003941 0.003943 0.004  0.003958 0.003922  Average  0.003929 + 0.0000441 (8)  3.9  T a b l e A.38 S t r a c t a n C o n c e n t r a t i o n and D e n s i t y , Corash and S h a f e r % StR (g/ml)  Density (g/ml)  Density (g/% StR)  Density (g/% StR)  15 16 17 20  1.059 1.063 1.067 1.082  0.003933 0.003938 0.003941 0.0041  0.003958 0.003922  Average  (1982)  0.003965 + 0.0000671 ( 6 )  3.9 T a b l e A.39 P l a t e l e t Recovery from G r a d i e n t , Day Two and Day S i x Day Two  Platelets x IO" 9  Bloody Blood2 Blood3 PRT GLx LDPT LDPLxi LDPL.X2 IDPT IDPLxj_ IDPLx HDPT HDPLxj_ HDPLx HHDT Total 2  2  8.424 8.727 9.225 11.781 11.644 1.893 1.699 2.085 7.281 4.173 8.385 2.866 2.194 3.080 0.281 12.32  Platelets % Recovered  15.4 59.1  23.3 2.3 100  Greater x IO" 9  0.6414 0.6339 0.0381 0.0633 0.0932 0.1406 0.5539 0.5572 0.0840 0.2929 0.3482 0.3426 0.5981  Greater % Recovered  5.2  23.5 14.1 57.3 100  Greater % Sample  1.60 3.59 4.28 1.89 11.72 6.23 2.85 11.78 10.16 54.91  - 204 T a b l e A.39-cont. Day S i x Bloodi Blood2 Blood3 PRT GLx LDPT LDPLxi LDPLx I DPT IDPLxi IDPLx HDPT HDPLxi HDPLx HHDT 2  P l a t e l e t Recovery from G r a d i e n t , Day Two and Day S i x  Platelets x 10-9  Platelets % Recovered  13.21 13.334 13.83 9.797 9.683 2.097 0.813  15.4  Greater x lO" 9  1.0375 1.049 0.2379 0.0834  Greater % Recovered  Greater % Sample  17.2  7.504 7.133  55.0  0.5794 0.4335  41.9  3.849 2.314 2.824 0.182 0.207 13.633  28.2  0.3661 0.1612 0.2084 0.1989 0.4658 1.3824  26.5  2  2  Total  1.3 100  14.4 100  3.9 Day Two: The b l o o d and ACD p l a t e l e t count was 1.52 x 1 0 p i t / m l , range 1.36-1.68 x 1 0 p i t / m l . Blood]_ was based on 55.5 m l ; Blood2 was based on 57.5 m l ; B l o o d 3 was based on 60.8 g. P l a t e l e t s , h a r v e s t e d from b l o o d and resuspended i n 8.6 ml o f 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% r e c o v e r y from b l o o d . The g r a d i e n t l o a d m i x t u r e (GLx) was suspended i n 8.5 ml and g r a d i e n t f r a c t i o n s LDPT, IDPT, HDPT and HHDT were suspended i n 5 ml o f Tyrode's ( T ) . Lx\ was the CCD l o a d m i x t u r e (3.1 m l ) counted w i t h t h e CCDF. L x was counted a g a i n two days a f t e r CCD a n a l y s i s . HHD was a s m a l l s u b p o p u l a t i o n o f p l a t e l e t s o f d e n s i t y even g r e a t e r t h a n t h a t o f t h e HDP, and p e l l e t s through t h e l a s t step o f 20% S t r a c t a n , along with t h e m a j o r i t y o f t h e c e l l u l a r c o n t a m i n a t i o n . The t o t a l p l a t e l e t r e c o v e r y from t h e g r a d i e n t r e p r e s e n t e d 105.81% r e c o v e r y o f t h e p l a t e l e t s , and 94.35% o f t h o s e p a r t i c l e s o f a s i z e g r e a t e r than t h e normal p l a t e l e t based on t h e d e n s i t y g r a d i e n t l o a d m i x t u r e (GLx) c o u n t s . Day S i x : The b l o o d and ACD p l a t e l e t count was 2.54 x 1 0 p i t / m l . Bloodj_ was based on 52.0 m l ; Blood2 was based on 52.5 m l ; Blood3 was based on 54.5 g. P l a t e l e t s h a r v e s t e d from b l o o d and resuspended i n 8.6 ml o f 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 73.4% r e c o v e r y from b l o o d . The t o t a l p l a t e l e t r e c o v e r y from t h e g r a d i e n t r e p r e s e n t e d 140% r e c o v e r y o f t h e p l a t e l e t s , and 133% o f t h o s e p a r t i c l e s o f a s i z e g r e a t e r t h a n t h e normal p l a t e l e t , based on t h e d e n s i t y g r a d i e n t l o a d m i x t u r e (GLx) c o u n t s ; and 102% p l a t e l e t r e c o v e r y , based on t h e b l o o d p l a t e l e t c o u n t . 8  8  2  8  - 205 T a b l e A.40 S t r a c t a n G r a d i e n t  Composition  StR% Final  22% S t o c k StR (ml)  Tyrode•s (ml)  10 16 18 20  2.2 21.82 24.55 27.27  2.64 8.18 5.45 2.73  3.10 T a b l e A.41 S t r a c t a n C o n c e n t r a t i o n and D e n s i t y StR%  Tyrode•s 10 16 18 20 22 Average  StR% (w/v) Measured*  Density (g/ml)  Density g/% S t R )  Density (g/% StR) Based on M*  0.004127 0.003189 0.004497 0.005326 0.003907 0.004209  0.003620 0.003073 0.004376 0.005072 0.003872 0.004003  -  1.45 11.4 16.6 18.5 21.0 22.2  1.04127 1.05102 1.08095 1.10652 1.08595  3TTo Table A.42 P l a t e l e t Recovery Platelet Particles x 10  Platelet Particles % Recovered  Greater Particles x 10  Greater Particles % Recovered  Greater Particles % Sample  7.675 1.624 6.834 1.791 10.251  15.85 66.67 17.48 100  6.07 0.426 4.041 1.199 5.667  7.52 71.3 21.17 100  7.33 2.54 17.91 6.70 5.24  14.6 3.744 8.259 1.716 0.193 10.391  26.91 59.37 12.33 1.38 100  5.284 4.265 9.925 2.857 0.739 17.78  23.98 55.8 16.06 4.15 100  3.49 10.23 10.73 14.27 27.27 11.34  - 9  Day Two GLx LDPLx IDPLx HDPLx Total Day S i x GLx LDPLx IDPLx HDPLx HHD Total  from G r a d i e n t , Day Two and Day S i x  - 8  3.10 On day two p . i . , t h e t o t a l p l a t e l e t s r e c o v e r e d r e p r e s e n t e d 133% o f t h e p l a t e l e t s l o a d e d on t h e g r a d i e n t (GLx) i n 8-8.73 m l ; on day s i x t h e t o t a l p l a t e l e t s r e c o v e r e d r e p r e s e n t e d 95% o f GLx.  - 206 T a b l e A. 43 P l a t e l e t Recovery from G r a d i e n t , Day Two  GLx LDPLx IDPLx HDPLx HHD Total  Platelet Particles x 10-9  Platelet Particles % Recovered  Greater Particles xlO"  Greater Particles % Recovered  Greater Particles % Sample  8.189 2.594 5.342 1.767 0.301 10.004  25.93 53.40 17.66 3.01 100  2.6997 1.717 5.765 2.735 0.246 10.463  16.41 55.1 26.14 2.35 100  3.19 6.21 9.74 13.40 7.56 9.47  8  3.10 A l l samples were resuspended i n 3.1 ml e x c e p t t h a t o f t h e GLx which was resuspended i n 9.46 g, o f which 9.2 g was a p p l i e d t o t h e 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 o f s i z e g r e a t e r than p l a t e l e t s ( G r e a t e r P a r t i c l e s ) , were e x p r e s s e d as c o u n t s , c a l c u l a t e d f o r t h e e n t i r e sample. The t o t a l p l a t e l e t s r e c o v e r e d r e p r e s e n t e d 122% o f t h e p l a t e l e t s l o a d e d on t h e g r a d i e n t i n 9.2 ml ( G L x ) ; whereas t h e t o t a l G r e a t e r P a r t i c l e s r e c o v e r e d r e p r e s e n t e d 388% o f t h a t which was l o a d e d . The number o f G r e a t e r P a r t i c l e s r e c o v e r e d was c o n s i s t a n t l y h i g h e r i n t h e 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 s u b p o p u l a t i o n .  T a b l e A.44 Recovery o f R a d i o a c t i v i t y from G r a d i e n t , Day Two 3-H DPM x 10" Glx LDPLx IDPLx HDPLx HHD Total  3  48.116 0.465 0.806 0.775 0.434 2.48  3-H % Total Recovered  35-S DPM x 10"  18.75 32.5 31.25 17.5 100  20.332 1.674 8.773 5.332 0.558 16.337  3  35-S % Total Recovered  3-H DPM/Plt x 10  35-S DPM/Plt x 10  10.24 53.70 32.64 3.42 100  5.88 0.179 0.151 0.439 1.44 0.248  2.48 0.645 1.64 3.02 1.85 1.63  6  6  3.10 H) was t h a t The radioactivity, e x p r e s s e d i n DPM ( S , or c a l c u l a t e d f o r t h e 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 r e c o v e r e d was r e p r e s e n t e d by 5.15% o f t h e H and 80.35% o f t h e S loaded onto t h e g r a d i e n t . I n c r e a s e d H - t h y m i d i n e uptake ( i n a d d i t i o n t o 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 o f h i g h e r d e n s i t y . 3  3 5  3  3  3 5  3 5  - 207 F i g u r e A.5 CCD o f Day Two S - LDP CCDC 1-40, IDP CCDC 41-80, and HDP CCDC 81-120: p l a t e l e t s p e r CCDC ( s o l i d line) left ordinate; r a d i o a c t i v i t y p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt p e r CCDC ( d o t t e d l i n e ) mean a t h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 3 5  0  10  20  30  40  50  60  70  80  90  100  110  120  F i g u r e A.6 CCD o f Day Two S - LDP, IDP, and HDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt a t 10% CE p e r CCDC ( d o t t e d l i n e ) mean a t h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 3 5  - 208 F i g u r e A.7 CCD o f Day S i x S - LDP CCDC 1-40, IDP CCDC 41-80, and HDP CCDC 81-120: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt a t 200% CE per CCDC ( d o t t e d l i n e ) mean a t h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 3 5  F i g u r e A.8 CCD o f Day S i x S - LDP, IDP, and HDP: p l a t e l e t s p e r 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 p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; dpm/plt a t 100% CE p e r CCDC ( d o t t e d l i n e ) mean a t h a l f graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.10 3 5  - 209 T a b l e A.45 S u l p h a t e I n j e c t i o n S c h e d u l e Date Jan  Day o f Expt  Time o f Injection  Rabbit Number  R a b b i t Days O l d ( p . i,. 35-S) on Wt (kg) W 12 T 13 F 14 S 15 S16  F 7 S 9 T 11  0 2 4  1:00 PM 3:00 PM 1:00 PM  40 39 37  3.65 4.15 4.16  5 3 1  6 4  7 5 3  2  8 6 4  9 7 5  3.11  T a b l e A.46 C e l l u l a r E s t i m a t i o n U s i n g t h e C y t o c e n t r i f u g e Lens Power  4x lOx 40x lOOx  Field Diameter mm  Field Area mm  Scan Area mm  Scan Cytospot % Total  4.4 1.8 0.46 0.19  15.205 2.545 0.166 0.028  30.8 12.6 3.22 1.33  80.03 32.74 8.37 3.46  2  2  3.12 The a r e a o f t h e c e l l u l a r specimen on t h e c y t o c e n t r i f u g e slide (Appendix T a b l e A.46; C y t o s p o t a r e a ) was 38.48 mm based on 7 mm d i a m e t e r . The number o f c e l l s i n t h e t o t a l specimen was c a l c u l a t e d from Scan% T o t a l . 2  - 210 F i g u r e A. 9 CCD o f Day One CCDC 1-40, Three CCDC 41-80, and F i v e CCDC 81-120, S/ H-Labeled Platelets: platelet serotonin radioactivity dpm p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed l i n e ) right ordinate; S / H p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.11 3 5  3  3 5  3  Figure A. 10 CCD o f Day One, Three, and F i v e , S/ H-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 p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; S / H a t 10% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.11 3 3  3 5  3  3  - 211 F i g u r e A. 11 CCD o f Day Two CCDC 1-40, Four CCDC 41-80, and S i x CCDC 81-120, S/ H-Labeled Platelets: platelet serotonin radioactivity dpm p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed line) right ordinate; S / H p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.11 3 5  3  3 3  3  Figure A. 12 CCD o f Day Two, Four, and S i x , S / 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 p e r CCDC ( s o l i d l i n e ) l e f t o r d i n a t e ; s u l p h a t e dpm p e r CCDC (dashed l i n e ) r i g h t o r d i n a t e ; S / H a t 10% CE p e r CCDC ( d o t t e d l i n e ) w i t h mean a t one h a l f t h e graph h e i g h t ; CCDC 1-120 a b s c i s s a . 3.11 3 3  3 5  3  3  - 212 T a b l e A.47 P l a t e l e t s o f 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 Sample  P i t 1Ct  IW F30 AER F42 WB M61  PCD PCD PCR  Normal IW F30  A-l  B-1  C-l  D-l  E-l  72.9 66.3 39.2  47.1 43.3 11.4  38.6 30.7 10.4  25.2 25.4 8.0  20.4 10.1 5.9  PCR  41.9 48.9  28.8 22.3  24.1 19.7  17.4 13.2  10.7 5.8  Normal WB M61 EVB F48  PCD PCD  66.2 61.0 71.2  66.3 53.0 65.7  60.7 52.0 62.2  41.9 39.1 48.4  22.1 26.4 31.3  Normal LHS M61 GEM M53 NKC F19  PCD PCD PCD  88.1 91.5 61.3 79.9  85.5 65.0 39.8 54.2  76.0 58.8 28.0 45.4  55.4 32.4 18.4 25.0  38.2 24.8 15.8 17.5  Normal JM M47  PCR  71.8 37.1  64.1 26.0  51.3 22.7  24.7 13.0  13.7 6.3  68.8  59.7  53.9  28.9  16.4  Normal 3.13  T a b l e A.48 P l a t e l e t s o f Age, Sex Matched Normal C o n t r o l s CRCBBNormal  A-l  B-1  C-l  D-l  NCB JAM DJS BH JBB  F20 F32 F24 F44 F58  A+ AB+ 0+ 0+  39.3 48.2 45.7 49.8 44.1  31.9 39.02 39.2 37.9 42.4  16.8 33.5 31.8 27.9 37.1  15.3 20.9 26.1 18.8 52.3  RJR ES EDO OGP TT LJ JAN DL JED PMS JC TP  M34 M52 M56 M42 M40 M58 M30 M40 M59 34 M38 M52  A+ A+ A+ AAB+ 0+ 0000+  40.9 87.2 59.2 60.9 51.4 59.4 41.2 62.0 63.6 37.3 61.2 37.3  35.0 44.5 49.4 59.9 47.1 53.4 35.5 64.2 47.9 29.5 36.7 36.2  24.2 46.0 45.3 87.2 42.0 54.4 28.4 58.8 53.2 27.1 47.7 23.5  16.7 28.7 28.0 57.4 21.2 24.4 28.6 36.1 54.1 18.3 23.1 20.3  3.13  E-l 29.6 10.9  11.8 23.0 23.0 48.0 7.9 18.9 13.0 24.1 20.7 13.2 11.5  - 213 T a b l e A.49 P l a t e l e t s  o f 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 and Normals  Sample  P i t Ct  A-l  B-1  C-1  D-1  IW AER WB EVB LHS GEM NKC JM WB IW  PCD PCD PCD PCD PCD PCD PCD PCR PCR PCR  7279 66.3 61.0 71.2 91.5 61.3 79.9 37.1 39.2 48.9  4771 43.3 53.0 65.7 65.0 39.8 54.2 26.0 11.4 22.3  3876 30.7 52.0 62.2 58.8 28.0 45.4 22.7 10.4 19.7  2572" 25.4 39.1 48.4 32.4 18.4 25.0 13.0 8.0 13.2  66.2 41.9 88.1 71.8 68.8  66.3 28.8 85.5 64.1 59.7  60.7 24.1 76.0 51.3 53.9  41.9 17.4 55.4 24.7 28.9  F30 F42M61 F48 M61 M53 F19 M47 M61 F30  Normal Normal Normal Normal Normal  E-1  2075 .  10.1 26.4 31.3 24.8 15.8 17.5 6.3 5.9 5.8 22.1 10.7 38.2 13.7 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 i n c l u d e d i n t h e p a r t i t i o n s t u d i e s , however s i z i n g a n a l y s e s were performed.  - 214 T a b l e A.50 S i z e A n a l y s e s o f P l a t e l e t s from Normals and MPD %~W  NorTW  2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 46 47 49 51  0.25 0.6 5.85 7.55 8.25 7.55 6.7 6.05 5.45 5.45 4.35 4.7 3.5 3.3 2.95 2.45 2.15 1.95 1.75 1.45 1.45 1.25 0.75 0.55 0.95 0.75 0.75 0.6 0.6 0.6 0.45 0.45 0.25 0.25 0.25 0.25 0.25  3.13  0.25 0.25  NorBb 0.25 1.45 1.95 2.15 1.95 1.75 1.45 1.45 1.25 0.75 0.95 0.75 0.6 0.45 0.45 0.25 0.25 0.25 0.45 0.25 0.25 0.25  0.25  NorBb  MPD.EEB  0.25 0.75 4.85 7.55 10.3 10.65 10.8 10.45 10.1 9.05 7.75 7.9 6.05 6.4 5.2 4.2 3.65 3.5 2.8 2.75 1.95 1.95 1.8 1.6 1.45 1.25 1.1 1.1 1.1 1.1 0.75 0.75 0.6 0.6 0.6 0.6 0.45 0.45 0.45 0.25 0.45 0.45  0.25 0.95 6.7 7.55 7.75 7.35 6.7 6.05 5.7 4.35 3.95 3.1 2.95 2.45 1.95 1.95 1.6 1.6 1.25 0.95 0.75 0.95 0.6 0.45 0.75 0.6 0.45 0.25 0.25 0.45 0.25 0.45 0.25 0.25  0.25 0.25 0.25 0.25 0.25  0.25 0.25 0.25  MPD,MLF  MPD,PMA  0.25 2.95 3.3 3.15 3.3 2.45 2.3 1.95 1.95 1.45 1.45 1.1 1.25 0.95 0.75 0.75 0.75 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.25 0.25 0.25  0.45 2.8 3.15 3.65 3.65 2.95 2.95 2.65 2.15 1.95 1.65 1.3 1.15 0.75 0.95 0.95 0.45 0.6 0.6 0.45 0.25 0.25  0.25  0.25  0.25  MPD,VSM 0.25 0.6 4.55 4.9 4.9 4.7 4.2 3.15 3.15 2.25 2.1 1.6 1.75 1.45 1.1 0.95 0.95 0.95 0.6 0.45 0.6 0.45 0.45 0.45 0.45 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25  - 215 T a b l e A.51 S i z e A n a l y s e s o f P l a t e l e t s % w  NorPS  NorBB  2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38  0.18 0.35 2.9 3.7 3.4 4.5 3.4 3.4 3.2 3.1 2.0 2.4 1.7 1.7 1.7 1.5 1.2 1.2 0.85 0.68 0.85 0.69 0.51 0.51 0.51 0.51 0.34 0.34 0.17 0.17 0.17 0.17 0.34 0.18 0.18 0.35 0.17  0.17 0.51 4.3 6.4 5.9 5.7 4.3 4.5 3.6 3.2 2.4 2.2 1.5 1.7 1.4 1.5 1.0 0.85 0.68 0.68 0.51 0.51 0.35 0.50 0.34 0.34 0.34 0.17 0.17 0.17 0.17  from Normals and MPD MPD,NKC 0.35 1.5 1.9 1.7 1.4 1.2 0.69 0.87 0.69 0.51 0.51 0.34 0.34 0.34 0.18  0.18 0.18 i  MPD,GEM  MPD,LHS  MPD, JM  0.34 0.68 5.5 6.0 5.2 5.0 4.2 3.4 2.4 2.4 1.9 2.0 1.7 1.0 1.0 1.2 0.85 0.85 0.51 0.32 0.32 0.50 0.32 0.17 0.17 0.17 0.17 0.35 0.16 0.16 0.16 0.16  0.34 1.0 6.9 8.3 7.7 7.2 5.4 4.7 4.2 3.2 2.5 2.4 1.9 1.4 1.4 1.0 1.0 0.51 0.51 0.51 0.51 0.18 0.35 0.18 0.18  0.18 1.2 1.4 1.0 1.0 0.69 0.87 0.69 0.87 0.51 0.51 0.35 0.35 0.18 0.18 0.18 0.35 0.18 0.18 0.18 0.18 0.18 0.18 0.18  0.18  0.18  0.17 0.17 0.16  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 d u r i n g s i z i n g a n a l y s e s 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 o f 649; MPD,VSM, 137; NorPS, 146; NorBb, 162; MPD,NKC, 34; MPD,GEM, 144; MPD,LHS, 182; MPD,JM, 3 1 .  

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