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Surface chemical studies of human platelets Chiu, Basil 1983

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SURFACE CHEMICAL STUDIES OF HUMAN PLATELETS by BASIL CHIU , S c , University of Wisconsin-Stevens Point, 1974 M.Sc, Medical College of Wisconsin, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF PATHOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER, 1983 0  B a s i l Chiu, 1983  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  understood t h a t copying or p u b l i c a t i o n of t h i s f o r f i n a n c i a l gain  Basil  PATHOLOGY  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  14 O c t . , 1983  Chiu  my  It i s thesis  s h a l l not be allowed without my  permission.  Department of  thesis  written  ABSTRACT The purpose of this project i s to investigate the surface properties of p l a t e l e t discocytes, echinocytes  and spherocytes.  Normal "non-sticky"  discoid shaped p l a t e l e t s (discocytes) can be transformed by ADP  into  i r r e g u l a r l y shaped echinocytes which are " s t i c k y " and aggregate easily i n media containing Ca  and fibrinogen.  A model i s examined here i n which  an echinocyte attains i t s " s t i c k y " properties by evagination of a surfaceconnected canalicular system. system upon hypotonic spherocytes.  P l a t e l e t s also evaginate  this canalicular  shock, i n which case the p l a t e l e t s swell up to form  By comparing the properties of the d i f f e r e n t geometric forms  of p l a t e l e t s insight into the nature of " s t i c k i n e s s " was  sought.  The  sur-  face areas of the discocyte and spherocyte measured microscopically were found to be 16.4 was  estimated  microscopic  —8 2 and 36.7x10 cm respectively while that of the —8 2  to be 23.7x10  cm  using surface chemical analysis.  examination showed that the c a n a l i c u l a r system may  t o t a l l y evaginated  i n the echinocyte.  Although i t was  echinocyte Electron  not be  found that the  spherocyte could s t i l l be agglutinated passively by r i s t o c e t i n i t had completely  lost i t s a b i l i t y to aggregate.  Microelectrophoretic studies I |  revealed 8 and 6 f o l d increases i n the density of Ca  j |  and Mg  binding  sites respectively on the echinocyte surface r e l a t i v e to the discocyte. The spherocyte on the other hand had lost most of i t s Ca  binding s i t e s .  E l e c t r o k i n e t i c analysis of l i v e , fixed and neuraminidase or a l k a l i n e phosphatase treated p l a t e l e t s showed major differences i n charge as well as amino, s i a l i c acid and phosphate group densities among the discocyte, echinocyte and spherocyte.  The evaginated  the l a t t e r two were also d i f f e r e n t .  canalicular membrane surfaces of  SDS-PAGE of p l a t e l e t s r a d i o l a b e l l e d  - iii -  v i a lactoperoxidase iodination, periodate-borohydride neuraminidase/galactose  oxidase-borohydride  t r i t i a t i o n or  t r i t i a t i o n f a i l e d to show any  difference i n the g e l patterns between the three p l a t e l e t forms. glycoprotein species appeared during the transformations.  No new  The presence of  fibrinogen interferes i n a concentration related manner with lactoperoxidase iodination of GP-III on the echinocyte surface.  An o v e r a l l picture i s  presented here showing differences between the surface properties of p l a t e l e t discocytes, echinocytes and spherocytes.  The accumulated evidence sug-  gests that changes i n the whole p l a t e l e t surface occur during a c t i v a t i o n and the model of a c l o i s t e r e d " s t i c k y " membrane may be an o v e r s i m p l i f i c a t i o n .  - iv TABLE OF CONTENTS Page Chapter 1  GENERAL INTRODUCTION  1  Chapter 2  PLATELET AGGREGATION AND MORPHOLOGY  8  2.1  INTRODUCTION  9  2.2  MATERIALS AND METHODS  11  Collection of Blood Preparation of Platelets Preparation of Echinocytes and Spherocytes . . Cocaine Induced Spheres Enumeration of Platelets Fixation of Platelets Measurement of P l a t e l e t Sizes Electron Microscopy Tests f o r the Integrity of P l a t e l e t Forms P l a t e l e t Aggregation Lectin Studies S t a t i s t i c a l Methods Materials  11 12 12 12 12 12 12 13 14 15 16 16 16  RESULTS  20  P l a t e l e t Dimensions and Morphology P l a t e l e t Integrity and Release of Contents P l a t e l e t Aggregation  20 24 41  DISCUSSION  67  P l a t e l e t Dimensions and Morphology . P l a t e l e t Integrity and Release of Contents P l a t e l e t Aggregations Summary of Chapter 2  67 68 70 77  MICROELECTROPHORESIS  78  3.1  INTRODUCTION  79  3.2  MATERIALS AND METHODS  86  Microelectrophoresis Fixation of Platelets P l a t e l e t Surface S i a l i c Acid P l a t e l e t Surface Phosphate Groups S t a t i s t i c a l Methods Materials  86 87 87 88 89 89  2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11 2.2.12 2.2.13 2.3 2.3.1 2.3.2 2.3.3 2.4 2.4.1 2.4.2 2.4.3 2.4.4 Chapter 3  3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6  -  V  -  Page 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5  RESULTS  90  P l a t e l e t Electrophoretic M o b i l i t i e s P l a t e l e t Surface S i a l i c Acid P l a t e l e t Surface Phosphate Groups Cocaine Spheres Calcium and Magnesium Ion Binding  90 98 102 110 110  DISCUSSION  121  P l a t e l e t Electrophoretic M o b i l i t i e s P l a t e l e t Surface S i a l i c Acid P l a t e l e t Surface Phosphate Groups Cocaine Spheres Calcium and Magnesium Ion Binding A Hypothesis of the Exposed Membranes  121 125 128 129 131 135  RADIOCHEMICAL LABELLING  141  4.1  INTRODUCTION  142  4.2  MATERIALS AND METHODS  148  Surface Labelling of P l a t e l e t s Isolation of Membrane Gel Electrophoresis Surface Labelling i n the Presence of Fibrinogen Materials  148 151 151 152 153  RESULTS  154  Iodination Experiments T r i t i a t i o n Experiments Surface Labelling i n the Presence of Fibrinogen  154 172 177  DISCUSSION  195  Iodination Experiments T r i t i a t i o n Experiments Surface Labelling i n the Presence of Fibrinogen Summary of Chapter 4  195 199 201 202  SUMMARY AND CONCLUSION  203  3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 Chapter 4  4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2 4.3.3 4.4 4.4.1 4.4.2 4.4.3 4.4.4 Chapter 5 REFERENCES  209  APPENDIX,  225  - vi -  LIST OF TABLES Table  Page  1  Physical Dimensions of Washed P l a t e l e t s  23  2  Electrophoretic M o b i l i t i e s of P l a t e l e t s  91  3  Apparent P l a t e l e t Surface Charge  92  4  P l a t e l e t Surface Amino Groups  93  5  P l a t e l e t Surface S i a l i c Acid  101  6  P l a t e l e t Surface S i a l i c Acid  7  P l a t e l e t Surface S i a l i c Acid  104  8  P l a t e l e t Surface Phosphate Groups  107  9  P l a t e l e t Surface Phosphate Groups  108  10  P l a t e l e t Surface Phosphate Groups  109  11  S i a l i c Acid and Phosphate Groups of Cocaine Spheres  12  Calcium Ion Binding Sites  118  13  Magnesium Ion Binding Sites  119  ....  I |  103  Ill  | |  14  S t a t i s t i c a l Comparison of Ca  15  Comparison of Calcium Ion Bindings  133  16  P l a t e l e t Surface Properties (Table 16 continued) (Table 16 continued) Highlights of Differences Between Discocytes, Echinocytes and Spherocytes  136 137 138  17  /Mg  Binding Sites  120  208  - v i i-  LIST OF FIGURES Figure  Page  1  Example of a t y p i c a l aggregation tracing  17  2  Phase photomicrographs  22  3  Electron micrographs of p l a t e l e t discocytes (Fig. 3 continued)  26 28  4  Electron micrographs of p l a t e l e t echinocytes (Fig. 4 continued)  30 32  5  Electron micrographs of p l a t e l e t spherocytes (Fig. 5 continued)  34 36  6  Electron micrographs of cocaine spheres  38  (Fig. 6 continued)  40  7  LDH leakage from spherocytes  43  8 9  Effect of ADP concentration on aggregation v e l o c i t y .... Example of aggregation tracings showing effect of ADP concentration  46 48  10  Effect of fibrinogen concentration on aggregation v e l o c i t y .  50  11  Effect of divalent cation concentration on aggregation velocity  52  Examples of aggregation tracings showing the effects of divalent cations  54  12  of different forms of p l a t e l e t s . . .  I |  13  Inhibitory effect of Mn  14  Agglutination of p l a t e l e t spherocytes and discocytes by ristocetin  59  Aggregation tracings from normal and neuraminidase platelets  61  15  on p l a t e l e t aggregation  57  treated  16  Aggregation responses to l e c t i n s  64  17  Examples of aggregation responses to l e c t i n s  66  18  The pH-electrophoretic mobility p r o f i l e s of fixed p l a t e l e t s  95  19  The pH-electrophoretic mobility p r o f i l e s of fresh p l a t e l e t s  97  20  The time-release curve of s i a l i c acid  100  - viii -  Figure  Page  21  The time-release curve of phosphate groups  22  Ca  I [  j j  and Mg  concentration on electrophoretic mobility I[  23  106  Double reciprocal plots to find Ca densities (Fig. 23 continued)  24  SDS-PAGE of  25  SDS-PAGE of  1 2 5  . . .  113  | |  and Mg  binding 115 117  I - l a b e l l e d platelets  156  1?5  26 27  I-labelled membranes 125 131 SDS-PAGE of a mixture of I-labelled discocyte and Il a b e l l e d discocyte  161  SDS-PAGE of a mixture of labelled echinocyte  163  1 2 5  I - l a b e l l e d discocyte and  1  125 28  SDS-PAGE of a mixture of l a b e l l e d spherocyte 17 5  29  SDS-PAGE of  30  SDS-PAGE of  1 2 5  1  I-  131 I165  131  If  I/  I-labelled discocyte and  3  158  I double labelled discocyte 1 3 1  1?5  I  167  double l a b e l l e d echinocyte  169  1 31  31  SDS-PAGE of  1/  I double labelled spherocyte  171  32  SDS-PAGE of t r i t i a t e d p l a t e l e t s (periodate)  174*  33  SDS-PAGE of t r i t i a t e d p l a t e l e t s oxidase  176"  (neuraminidase/galactose  34  SDS-PAGE of pseudo-double labelled p l a t e l e t s (periodate) . . .  17:9  35  SDS-PAGE of pseudo-double labelled p l a t e l e t s galactose oxidase)  181  (neuraminidase/  36  SDS-PAGE of discocytes iodinated i n the presence of fibrinogen  183  37  SDS-PAGE of echinocytes iodinated i n the presence of fibrinogen  185  38  SDS-PAGE of spherocytes  187  39  SDS-PAGE of echinocyte iodinated i n the presence of albumin  .  189  40  SDS-PAGE of echinocyte iodinated i n the presence of fibronectin  191  41  Effect of fibrinogen concentration on the iodination of echinocytes  194  iodinated i n the presence of fibrinogen  - ix -  LIST OF ABBREVIATIONS ADP  Adenosine diphosphate  Beta-Tg  Beta-thromboglobulin  CPM  Counts per minute  DTT  Dithiothreitol  EPM  Electrophoretic  GP  Glycoprotein  JBA  J e q u i r i t y bean agglutinin  LDH  Lactate  MW  Molecular weight  NAD  Nicotinamide adenine dinucleotide  PAGE  Polyacrylamide gel electrophoresis  PRP  P l a t e l e t r i c h plasma  RBC  Red blood c e l l  R C A  60  RCA  12()  mobility  dehydrogenase  Castor bean agglutinin, MW=60,000 Castor bean agglutinin, MW=120,000  SDS  Sodium dodecyl sulfate  TEMED  Tetramethylethylenediamine  WGA  Wheat germ agglutinin  -  X  -  ACKNOWLEDGEMENTS I w i s h to express my  deep and s i n c e r e g r a t i t u d e to Dr. D.E.  h i s c o n s t a n t guidance and support throughout t h i s p r o j e c t . thank Drs. J.G. F o u l k s , R.H.  Pearce and P.E.  Brooks f o r  I a l s o w i s h to  R e i d , members of my  Super-  v i s o r y Committee, f o r t h e i r a d v i c e and encouragement. I would Cavanagh, Mr. ance.  l i k e to thank the l a t e Dr. E. Anderson C. Ramey and Ms.  as w e l l as Mr. J .  R. Rupps f o r t h e i r expert t e c h n i c a l  assist-  I am a l s o g r a t e f u l to f e l l o w graduate s t u d e n t s , e s p e c i a l l y J . Janzen  and R. Snoek, f o r t h e i r h e l p f u l  advice.  The author i s f i n a n c i a l l y s u p p o r t e d by M e d i c a l Research C o u n c i l g r a n t  MT-5759.  SURFACE CHEMICAL STUDIES OF HUMAN PLATELETS  - 1 -  CHAPTER 1  GENERAL INTRODUCTION  - 2 P l a t e l e t s are c i r c u l a t i n g formed elements of mammalian blood that are essential for normal hemostasis. into two groups.  Their functions can generally be divided  The f i r s t i s hemostatic, i . e . the physical occlusion of  damaged blood vessels by masses of aggregated p l a t e l e t s . is thromboplastic,  The  second function  i . e . the p a r t i c i p a t i o n of the chemical constituents of the  p l a t e l e t s in the blood coagulation mechanism. When the blood vessel wall i s transected, p l a t e l e t s come into contact with the basement membrane as well as collagen i n the subendothelial the cut margin.  They then rapidly undergo shape change and  tissues around  degranulation.  P l a t e l e t s are normally discoid i n shape (a form referred to as a discocyte) but during the shape change reaction they become i r r e g u l a r i n shape with pseudopods extending out from a more or less spherical central body (echinocyte).  Adhesion of the p l a t e l e t to the injured vessel wall i s f i r s t  i n i t i a t e d with the pseudopods and then the p l a t e l e t s spread out over the exposed subendothelial surfaces (Mohammad and Mason, 1981;  Frojmovic and  Milton, 1982).  occurs and  During this process p l a t e l e t degranulation  substances released such as adenosine diphosphate (ADP)  and serotonin help  stimulate other p l a t e l e t s to undergo shape change and adhere to the layer of p l a t e l e t s .  first  This "second wave" of adherent p l a t e l e t s also release  their granules and causes more p l a t e l e t s to adhere and accumulate around the injured s i t e u n t i l a hemostatic p l a t e l e t plug i s formed, stopping of blood through the injured vessel (Zucker, 1972).  further loss  By 30 sec after injury  the hemostatic plug w i l l be well formed and can grow to several times the diameter of the blood vessel.  In the central part of the plug, activated  p l a t e l e t s that have lost most of their granules form a cytoplasmic  mass i n  which individual p l a t e l e t s are no longer distinguishable (Wester ^ t al., 1977).  - 3 -  During p l a t e l e t activation factor 3 becomes available for i t s thrombop l a s t i c function.  P l a t e l e t factor 3 i s a phospholipid associated with the  p l a t e l e t membrane which i s not detected u n t i l activation occurs.  Platelet  factor 3 forms a complex with factor IXa, factor VIII and calcium to activate factor X, following which i t forms a complex with factor Xa, factor V and calcium to convert prothrombin  into thrombin (Zwaal, 1978).  Other  substances  released from p l a t e l e t granules during activation such as p l a t e l e t factor 4 (anti-heparin a c t i v i t y ) , p l a t e l e t fibrinogen and factor VIII may contribute to the coagulation cascade.  also  The time course of platelet-induced  c l o t t i n g i s such that f i b r i n i s not observed in the hemostatic plug u n t i l about 3 min after the i n i t i a l activating event.  However by 30 min most of the  p l a t e l e t s i n the plug have disintegrated and have been replaced by a massive accumulation of f i b r i n (Wester et a l . , 1977). The introduction of the aggregometer allowed p l a t e l e t aggregation to be studied i n v i t r o i n d e t a i l (Born 1962; Michal and Born, 1971).  An aggrego-  meter i s a simple photometric device that measures the t u r b i d i t y of a s t i r r e d suspension of p l a t e l e t s .  A beam of l i g h t shines through the p l a t e l e t sus-  pension and the amount of light passing through i s measured.  Normal discoid  p l a t e l e t s have the maximum t u r b i d i t y and the least amount of light passes through.  During aggregation the p l a t e l e t s clump together into aggregates and  the suspension clears o p t i c a l l y , allowing more light to pass through.  A  measure of the rate of aggregation i s therefore provided by the rate of increase in transmission of l i g h t through the p l a t e l e t suspension. Among the agents which can cause aggregation, the most well studied one i s ADP.  Upon exposure to ADP,  discocyte to echinocyte.  the p l a t e l e t s undergo a shape change from  Providing the right conditions exist, the p l a t e l e t s  - 4 w i l l aggregate.  Right conditions include the presence of the divalent cations  calcium or magnesium, and fibrinogen (Zucker, 1972).  A certain amount of  agitation i s also required to bring s u f f i c i e n t numbers of p l a t e l e t s into contact with each other. this purpose.  The aggregometer has a b u i l t - i n magnetic s t i r r e r for  Shape change alone does not require any of these conditions.  The f i r s t event i n the stimulation of p l a t e l e t s by ADP  i s the binding of  this molecule onto the surface of the p l a t e l e t s (Born and Feinberg, Legrand and Caen, 1978).  A yet-to-be understood  gers the discocyte-echinocyte transformation. of the p l a t e l e t becomes " s t i c k y . "  1975;  sequence of events then t r i g -  At the same time, the surface  The development of p l a t e l e t " s t i c k i n e s s " i s  a process which p a r a l l e l s shape change but not d i r e c t l y caused by the discocyte to echinocyte transformation i t s e l f (Zucker, 1972; Barnhart, 1978). The biochemical nature of " s t i c k i n e s s " i s s t i l l uncertain and remains to be defined.  It may  involve the increased, saturable binding of fibrinogen onto  the ADP-stimulated  p l a t e l e t surface i n the presence of C a  + +  or Mg  (Mustard et a l . , 1978; Marguerie et a l . , 1979; Bennett and V i l a i r e , Peerschke et a l . , 1980).  1979;  Surface properties associated with changes i n  electrophoretic mobility (Hampton and M i t c h e l l , 1974;  Seaman and Vassar,  1977;  Stoltz, 1979) or i n the amount of neuraminidase-susceptible s i a l i c acid on the p l a t e l e t surface (Motemed et a_l., 1976; Ku and Wu, suggested as being involved.  1977) have also been  Throughout the following sections, these and  other properties that may be involved i n p l a t e l e t " s t i c k i n e s s " w i l l be explored. After echinocyte formation and the appearance of " s t i c k i n e s s " , the p l a t e l e t s begin a two stage aggregation process.  F i r s t i s primary or reversible aggre-  gation i n which the p l a t e l e t s adhere to each other to form aggregates.  Some  - 5 i n t e r n a l r e o r g a n i z a t i o n i n the p l a t e l e t s o c c u r s :  o r g a n e l l e s move towards  the  c e n t r a l part of the e c h i n o c y t e and the c i r c u m f e r e n t i a l bundle of m i c r o t u b u l e s also shifts internally.  The degree of change v a r i e s  e c h i n o c y t e but no granules  are r e l e a s e d at t h i s  c o n c e n t r a t i o n of the a g g r e g a t i o n  point  from e c h i n o c y t e t o (White, 1972).  When t h e  agent i s not high enough or the t e m p e r a t u r e  i s below 30°C , the p l a t e l e t s w i l l  eventually disaggregate  and r e v e r t t o the  d i s c o i d shape ( Z u c k e r , 1 9 7 2 ) . On the o t h e r hand, i f  the c o n c e n t r a t i o n of the a g g r e g a t i n g agent i s  high  enough and t h e t e m p e r a t u r e i s i d e a l , t h e n secondary or i r r e v e r s i b l e a g g r e g a t i o n proceeds  a f t e r primary aggregation  (White, 1972).  At l o c a t i o n s  in  the aggregates where the p l a t e l e t s are t i g h t l y squeezed t o g e t h e r , t h e i r organelles  and m i c r o t u b u l e bundles  of the p l a t e l e t granules  beome more c e n t r a l i z e d and f i n a l l y c o n t e n t s  are r e l e a s e d .  The p l a t e l e t s i n the aggregates  s u b s e q u e n t l y b e g i n t o d i s i n t e g r a t e (Rodman, 1971; W h i t e , 1 9 7 2 ) . r e a c t i o n induced by agents  the r e l e a s e  such as t h r o m b i n , a r a c h i d o n i c a c i d and A23187 can  be s t u d i e d w i t h o u t a g g r e g a t i o n by not s t i r r i n g the p l a t e l e t s u s p e n s i o n t o promote a g g r e g a t i o n  (Holmsen, 1977).  A g g r e g a t i o n induced by ADP i s  not f o l l o w e d by r e l e a s e a c t i o n (Holmsen, P l a t e l e t s have t h r e e kinds lysoscmes  1977).  of g r a n u l e s ,  alpha g r a n u l e s ,  ( S k a e r , 1981; Nurden et a l . , 1982).  and e l e c t r o n dense.  They c o n t a i n l a r g e  Dense bodies  dense bodies  and  are few i n number  q u a n t i t i e s of s e r o t o n i n ,  c a l c i u m and phosphorus as w e l l as ADP and ATP. e l e c t r o n opaque and c o n t a i n numerous  usually  inorganic  The a l p h a granules  are l e s s  proteins including fibrinogen,  f i b r o n e c t i n , thrombospondin, f a c t o r V H I - r e l a t e d a n t i g e n , f a c t o r V, low a f f i n i t y and h i g h a f f i n i t y ( a f f i n i t y f o r h e p a r i n ) p l a t e l e t f a c t o r 4, b e t a t h r o m b o g l o b u l i n , p l a t e l e t growth f a c t o r , c h e m o t a c t i c f a c t o r and b a c t e r i c i d a l  - 6 factor.  Platelet lysosomes which are also not electron dense contain various  proteinases, glycosidases , esterases and phosphatases.  Serotonin i s usually  used as a monitor f o r dense granule release while bet a-thrombo globulin and p l a t e l e t factor 4 levels r e f l e c t alpha granule release. Transformation  from a discocyte t o an echinocyte requires the platelets to  increase their apparent surface areas to provide for the formation of the pseudopods .  The shape change occurs almost instantaneously after exposure to  an aggregating agent and i s completed within seconds.  The discocyte must  therefore have a mechanism to provide f o r the increase during t h i s short time period.  The most l i k e l y mechanism seems to be the provision of excess surface  area within the discocyte.  The surf ace-connected canalicular system (open  canalicular system) i s a l i k e l y source.  This open canalicular system i s a  labyrinth of membrane channels that are connected t o the platelet surface with openings t o the outside (White and Clawson, 1980).  The evagination of this  membrane system could therefore provide the additional surface membrane required f o r transformation 1982).  (Morgenstern andKho, 1977 ; Frojmovic  and M i l t o n ,  A comparison of the surface properties of platelet discocytes and  echinocytes may therefore provide some information on the properties of " s t i c k y " membranes and/or the open canalicular membrane system.  The degree to  which the two are r e l a t e d remains to be seen. Another way to study the invaginated open canalicular system i s to evaginate i t by means of hypotopnic  stress.  By lowering the osmotic  pressure  of the suspending medium, large swollen spherical platelets (spherocytes) are creatred with t h e i r surface-connected (Milton and Frojmovic, 197 9) .  canalicular system apparently  evaginated  Treatment of the discocyte with cocaine w i l l  also produce a spherical form of platelet (Behnke, 1979) but one which i s much  - 7 s m a l l e r i n s i z e than the s p h e r o c y t e . c o c a i n e spheres stress.  Hereafter these w i l l  t o d i s t i n g u i s h them from t h e s p h e r o c y t e s  The g e n e r a l term spheres  will  be r e f e r r e d t o as  produced by h y p o t o n i c  include both spherocytes  and c o c a i n e  s pher es . The p r o j e c t d e s c r i b e d h e r e i n was aimed at comparing the s u r f a c e f e a t u r e s of the d i s c o c y t e , e c h i n o c y t e , s p h e r o c y t e and c o c a i n e s p h e r e .  Properties  s t u d i e d i n c l u d e d e l c t r o k i n e t i c b e h a v i o u r , s u r f a c e s i a l i c a c i d , phosphate amino groups, c a l c i u m and magnesium b i n d i n g and s u r f a c e l a b e l l i n g . Aggregation w i t h various  agents was a l s o s t u d i e d .  and  CHAPTER 2  PLATELET AGGREGATION AND MORPHOLOGY  - 92.1  INTRODUCTION Mild hypotonic shock to p l a t e l e t s has been used i n the blood banking com-  munity to test for p l a t e l e t v i a b i l i t y during storage. resistant to hypotonic stress.  P l a t e l e t s are very  When introduced into a hypotonic environment  with osmolarity as low as 150 mOsm, the p l a t e l e t s w i l l change into an echinoid shape but can slowly reverse back into the discoid form (Fantl, 1966; Handin eit a l . , 1970; Kim and B a l d i n i , 1974; Odink, 1976; Milton and Frojmovic, 1977a). Under more severe hypotonic stress p l a t e l e t s w i l l turn into swollen spheres or spherocytes.  Zucker-Franklin (1969) suspended p l a t e l e t s i n d i s t i l l e d water to  obtain spherocytes for m i c r o f i b r i l studies. Milton and Frojmovic (1977b) found that by lowering the osmolarity to as much as 60 mOsm they could produce spherocytes stable for at least an hour.  They also suggested that the osmotic  spherocyte formation provides an opportunity for the study of the surfaceconnected canalicular system. Treatment of p l a t e l e t s with cocaine results i n a t o t a l loss of aggrega b i l i t y (Aledort and Niemetz, 1968; O'Brien, 1976) and produces a spherical form of p l a t e l e t (Mannucci and Sharp, 1967; Behnke, 1970).  Other properties  of this cocaine-induced sphere have yet to be explored. In this chapter, the morphology and dimensions  of the normal p l a t e l e t  discocyte, the ADP transformed echinocyte, the hypotonically induced spherocyte and the cocaine-induced sphere w i l l be described using the results from phase and electron microscopy. ++  As discussed e a r l i e r , aggregation of p l a t e l e t s by ADP requires Ca Mg  ++  ions as well as fibrinogen.  ADP, C a , M g ++  ++  or  Aggregation studies to find the optimum  and fibrinogen concentrations required are reported i n  this chapter as preparation for later  experiments.  - 10 Aggregation induced by a variety of other agents was to test the aggregability of the spherocyte.  also examined, mainly  Of these, r i s t o c e t i n i s of  p a r t i c u l a r interest because i t passively agglutinates rather than pharmacol o g i c a l l y inducing p l a t e l e t aggregation, and a plasma co-factor Willebrand's factor) i s also required  ( P h i l l i p s , 1980).  agents, the l e c t i n s , were also of special i n t e r e s t .  (von  Another group of  It w i l l be shown here  that removal of surface s i a l i c acid with neuraminidase can modify the platelet's response to d i f f e r e n t l e c t i n s .  - 11 -  2.2 MATERIALS AND METHODS 2.2.1  C o l l e c t i o n of Blood  Blood was drawn from healthy human volunteers using p l a s t i c syringes by venipuncture and anticoagulated with 0.38% sodium c i t r a t e .  Volunteers were  mostly from within the Pathology Department but i n a few cases arrangements were made through the l o c a l Red Cross.  Red Cross blood was collected i n  citrate-phosphate-dextrose (Masouredis, 1972). the handling of blood and p l a t e l e t s .  P l a s t i c ware was used i n a l l  Samples remained at room temperature at  a l l times .  2.2.2  Preparation of P l a t e l e t s  Within one hour after the c o l l e c t i o n of blood, p l a t e l e t - r i c h plasma (PRP) was prepared by centrifugation at 120xg f o r 15 min (Day et a l . ,  1976).  P l a t e l e t s were then i s o l a t e d from the PRP by centrifugation at 1200xg f o r 15 min.  They were washed twice i n a calcium and magnesium free Tyrode's solution  made up of 136.75 mM sodium chloride, 2.68 mM potassium chloride, 11.90 mM sodium bicarbonate, 0.36 mM sodium dihydrogen phosphate The pH was 7.4 unless otherwise indicated.  and 5.55 mM glucose.  To prevent platelet loss during  the i s o l a t i o n and washing procedures, the PRP and Tyrode's solution were f i r s t a c i d i f i e d with c i t r i c a c i d to pH 6.5 according t o the method of Zucker and Grant (1978). solution.  A f t e r wshing, the platelets were resuspended i n pH 7.4 Tyrode's  I t was found that washing with the a c i d i f i e d Tyrode's solution was  essential.  I f platelets are centrifuged i n a pH 7.4 solution they w i l l  aggregate.  Fibrinogen as well as calcium and magnesium ions were added  separately as required on an individual experiment  basis.  - 12 2.2.3  Preparation of Echinoctyes and Spherocytes  Echinocytes were prepared by the addition of 2x10 of ADP to suspensions of p l a t e l e t s at room temperature. then gently mixed by inverting the test tube twice.  f i n a l concentration The suspensions were  Vigorous mixing or  shaking was avoided. Spherocytes were prepared by the addition of water to isotonic suspensions of p l a t e l e t s ( i n PRP or Tyrode's solution) i n a r a t i o of three parts water to one part suspension (Milton and Frojmovic, 1979).  Water was added slowly  through the period of about a minute and vigorous mixing was also avoided. The osmolality of the f i n a l suspension was therefore lowered to about 75 mOsmol. 2.2.4  Cocaine Induced  Spheres  P l a t e l e t suspensions were incubated for half an hour with cocaine at 10 mM f i n a l concentration (Behnke, 1970) to produce the cocaine spheres.  Sphering  occurred at both room temperature and 37°C.  2.2.5  Enumeration of P l a t e l e t s  P l a t e l e t s were counted using a hemocytometer under phase contrast microscopy. 2.2.6  Fixation of P l a t e l e t s  P l a t e l e t s i n various forms were fixed at room temperature i n 0.35% glutaraldehyde i n Tyrode's solution.  After one hour the p l a t e l e t s were spun down and  resuspended with fresh glutaraldehyde solution for overnight f i x a t i o n at 4°C. Fixation with glutaraldehyde was found to be very rapid (Vassar e_t ajL., 1972). 2.2.7  Measurement of P l a t e l e t Sizes  The morphology of fresh and fixed p l a t e l e t s was examined under phase contrast microscopy.  Photomicrographs were taken with a Carl Zeiss Photo-  - 13 -  microscope II using a 40x objective and overall magnification of 800x. of O.l^jLwas also photographed i n the same manner.  A grid  To measure the sizes of the  p l a t e l e t s , the negatives from the photomicrographs were projected onto a piece of paper using a photographic  enlarger.  Diameters measured from the outside  edges and the inside edges of the d i f f r a c t i o n ring of each p l a t e l e t were averaged as described by -Frojmovic and Panjwani (1976). that of the grid traced out i n the same way.  They were compared to  Dimensions for the spherical  forms of p l a t e l e t s were calculated from their measured r a d i i (r) using simple geometric  formulae for spheres: Area = 4t<r  2  3 and  Volume = 4/3"rr -  Dimensions for the discocytes were calculated according to Frojmovic  and  Panjwani (1976) using a model of an oblate spheroid: Area = (^/2)d + (n/4)t {(l+r)/(l-r)} log 2  2  R  _1  2 and  Volume = (fl/6)d t  where  d = diameter t = thickness  and  R = t/d  2.2.8  E l e c t r o n microscopy  Glutaraldehyde 0.1M  fixed p l a t e l e t s were postfixed with 2% osmium tetroxide i n  cacodylate buffer (pH 7.35)  for 1 hour.  After dehydration in a graded  series of ethanol solutions the p l a t e l e t s were embedded i n Epon 812. sections were cut with an ultramicrotome  Thin  equipped with a diamond knife and  contrast of the sections enhanced by staining with uranyl acetate and lead citrate.  P l a t e l e t samples from three healthy volunteers were viewed with a  P h i l l i p s EM 300 electron microscope and photographs taken.  - 14 -  2.2.9  Tests f o r the Integrity of P l a t e l e t Forms  2.2.9.1  Lactate Dehydrogenase (LDH) Leakage  LDH assays were performed according to the k i n e t i c method of Kachmar (1970). This method i s based on the reverse reaction of pyruvate (substrate) to lactate and monitored as a decrease i n absorbance at 340 nm when NADH i s oxidized to NAD. LDH Pyruvate + NADH  >NAD + lactate  The unit of enzyme a c t i v i t y i s the Wroblewski Unit which i s the drop of absorbance per minute per volume of sample.  Supernatants from discocyte,  echinocyte and spherocyte suspensions were assayed for the leakage of the enzyme.  Total LDH i n p l a t e l e t s was assayed using supernates from frozen and  thawed samples.  More detailed time studies for spherocytes were also  performed. 2.2.9.2  Beta-Thromboglobulin (beta-Tg) Release  Beta-thromboglobulin was assayed using an Amersham radioimmunoassay k i t . Release of beta-Tg from echinocytes and spherocytes was monitored by comparing supernatant and p l a t e l e t contents released by freeze-thawing.  Some discocytes  were also stimulated to release their granules by the addition of thrombin at 0.4 NIH units/ml.  2.2.9.3  14 C-Serotonin Release  P l a t e l e t (discocyte) dense bodies were f i r s t loaded with 14 the  method of Clark and Harms (1978).  incubated with each m i l l i l i t e r  About 25jj.Ci of  14 C-serotonin by  C-serotonin were  of PRP for one hour at room temperature.  The  p l a t e l e t s were then washed as before and resuspended i n Tyrode's solution. 14 . . After transformation to echinocytes or spherocytes C-serotonin i n the  - 15 -  supernatants counting.  and p l a t e l e t s were m o n i t o r e d u s i n g l i q u i d s c i n t i l l a t i o n  Samples were mixed w i t h A t o m l i g h t s c i n t i l l a n t f l u i d (New E n g l a n d  N u c l e a r , Boston) i n a r a t i o of 1 ml sample t o 5 ml A t o m l i g h t and then counted w i t h a Beckman LS-233 s c i n t i l l a t i o n c o u n t e r .  2.2.10  Platelet  Platelet England).  Aggregation  a g g r e g a t i o n was s t u d i e d w i t h a B o r n - M i c h a l Aggregometer  (London,  The v e l o c i t y of a g g r e g a t i o n was measured as t h e r a t e of decrease i n  the o p t i c a l d e n s i t y o f the p l a t e l e t s u s p e n s i o n (Born and C r o s s , 1963). was done by drawing a l i n e tangent t o t h e s t e e p e s t  part of t h e s l o p e of t h e  a g g r e g a t i o n t r a c i n g r e c o r d e d by the c h a r t r e c o r d e r ( F i g . 1.). t h i s l i n e was measured as the v e l o c i t y of a g g r e g a t i o n u n i t of inches  (of c h a r t paper) per m i n u t e .  This  The s l o p e  of  and had an a r b i t r a r y  A l l a g g r e g a t i o n experiments  were  o 8 done at 37 C and at a c o n c e n t r a t i o n of about 2.5x10 p l a t e l e t s / m l of Tyrode's  solution.  A series  of experiments was undertaken t o determine the e f f e c t s of  d i f f e r e n t concentrations magnesium ions comparisons  of ADP and f i b r i n o g e n as w e l l as c a l c i u m and  on p l a t e l e t a g g r e g a t i o n .  In o r d e r t o make  inter-sample  p o s s i b l e a n o r m a l i z a t i o n procedure s i m i l a r t o t h a t used by  F r o j m o v i c (1973) was adopted.  A c o n c e n t r a t i o n at which maximum  aggregation  v e l o c i t y always o c c u r r e d was r e f e r r e d to as p r o d u c i n g a 100% v e l o c i t y . Velocities  at other c o n c e n t r a t i o n s s t u d i e d were t h e n e x p r e s s e d as  of t h i s maximum v e l o c i t y . from d i f f e r e n t samples  I n t h i s way, percentage vs c o n c e n t r a t i o n  curves  c o u l d be combined.  ADP c o n c e n t r a t i o n s t u d i e s were performed w i t h p l a t e l e t s i n s o l u t i o n c o n t a i n i n g 0.5 mg/ml of f i b r i n o g e n and 4 mM CaCl„ <* 2. 0  percentages  0  Tyrode's  _., . Fibrinogen  - 16 -  concentration studies were done using platelets i n Tyrode's solution containing 4 mM C a  + +  and then 2x10 ^M ( f i n a l concentration) of ADP added  as aggregating agent.  Ca  +  and M g  ++  concentration studies were performed  using platelets i n Tyrode's solution containing 0.5 mg/ml of fibrinogen. added was also 2x10 "*M. M n  ++  and S r  + +  ADP  were also investigated f o r t h e i r  a b i l i t i e s t o support aggregation induced by ADP. A panel of aggregating agents including ADP, thrombin, epinephrine, A23187, arachidonic acid and r i s t o c e t i n was used t o survey the aggregability of the spherocyte and cocaine sphere. 2.2.11  L e c t i n Studies  Lectins and ADP were used t o study the effect of neuraminidase treatment g on the aggregation of p l a t e l e t s .  Platelets (2.5x10 /ml) were incubated with  0.04 IU/ml of neuraminidase (Vibrio cholerae) for 90 min at 37° i n Tyrode's solution (see Chapter 3 f o r more information).  The l i s t  of l e c t i n s used i n  these experiments includes wheat germ agglutinin (WGA) , j e g u i r i t y bean agglutinin (JBA) and the two castor bean agglutinins  (RCA^Q a  n  d  RCA^Q) -  Sugars N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and D-galactose were used f o r blocking experiments. 2.2.12 S t a t i s t i c a l Methods A l l s t a t i s t i c a l methods were according to K a l b f l e i s c h (1974). 2.2.13  Materials  Fibrinogen was prepared and kindly supplied by Mr. Johan Janzen of our laboratory by p r e c i p i t a t i o n with polyethylene glycol 6000 (Janzen, 1983).  The  fibrinogen was further p u r i f i e d by a f f i n i t y chromatography using l y s i n e sepharose 4B and collagen-sepaharose 4B to remove plasminogen and f i b r o n e c t i n  - 17 -  Fig. 1.  Example of a t y p i c a l aggregation tracing.  v e l o c i t y was  Aggregation  found by drawing a l i n e tangent to the steepest  part of the tracing. aggregation v e l o c i t y .  The slope of t h i s l i n e was ADP  taken as the  i s introduced into the p l a t e l e t  suspension as indicated by arrow. occurred before aggregation.  A rapid shape change phase  - 18 -  - 19 respectively. preparation was  SDS-PAGE demonstrated no detectable contaminant and the considered to be 99% pure.  ADP, arachidonic a c i d , epinephrine, r i s t o c e t i n s u l f a t e , WGA,  JBA,  D-galactose, N-acetyl-D-glucosamine, N-acetylgalactosamine, NADH and sodium phruvate were a l l from Sigma (St. Louis, Missouri). 14 The beta-throm bo globulin RIA k i t and (Arlington Heights, I l l i n o i s ) .  C-serotonin were from Amersham  The neuraminidase, both castor bean  agglutinins and A23187 were from Calbiochem (La J o l l a , C a l i f o r n i a ) . was from Park, Davis and Co. of D e t r o i t , Michigan.  Thrombin  Cocaine hydrochloride was  purchased through the UBC Department of Family Practice pharmacy from A l l e n and Hanburys of Toronto, Ontario. Arachidonic acid and A23187 were dissolved i n ethanol. dissolved i n i s o t o n i c saline (pH 7.4) solution.  A l l others were  Appropriate ethanol controls  were included i n a l l relevant experiments. Calcium, magnesium, strontium and manganese were added as the chloride.  - 20 2.3  -  RESULTS  2.3.1  Platelet Dimensions  and Morphology  Under phase microscopy, platelet discocytes could be seen to tumble through the f i e l d of view demonstrating t h e i r discoid shape.  No differences  were observed i n the morphology of discocytes i n pi at el e t - r i ch plasma or i n Tyrode's solution.  Therefore the washing procedure proved to be quite  s a t i sfactory. Addition of 2x10 "*M of ADP echinocytes ( F i g . 2).  to the platelet suspension produced the  They appeared more "stationary" than the discocytes.  Careful observations by focusing the microscope up and down showed long slender pseudopods which were extremely d i f f i c u l t to photograph. appeared as single platelets i n suspension.  Small aggregates would form i f  the suspension was shaken or s t i r r e d even i n the absence of C a fibrinogen.  Echinocytes  and  + +  Hypotonic shock at 75 mOsm produced the spherocytes which were  much larger than the spheres induced by treatment with 10 mM  of cocaine ( F i g .  2). Table 1 shows the measured diameters for the discocyte, spherocyte and -4  cocain sphere.  The discocyte has a mean diameter of 3.18x10 cm and a -4 thickness of 0.89x10 cm. The thickness to diameter r a t i o i s therefore —8  0.28.  Its surface area and volume were calculated to be 16.48x10  and 4.71x10  cm  respectively.  Since the discocyte tumbles  2  cm  through the  f i e l d of view, only p l a t e l e t images considered as presenting the edge-on view were measured for thickness.  Discocytes i n PRP had similar dimensions.  Table  1 also shows the spherocyte has a 123% and a 345% increase i n surface area and volume respectively over the discocyte.  The cocaine sphere i s much smaller  than the spherocyte and i t s increase i n volume and area over the discocyte i s not  as large.  No differences were found between f i x e d and non-fixed  - 21 -  F i g . 2.  Phase photomicrographs of d i f f e r e n t forms of  platelets. (a) Discocytes (xl200 magnification*) (b) Echinocytes (xl400 magnification) (c) Spherocytes  (xlOOO magnification)  (d) Cocaine spheres  (xl200 magnification)  *Magnification here and i n a l l following figures depicts magnification of the p l a t e l e t s as they appear on the photographic p r i n t s .  - 23 -  Table 1.  Physical Dimensions  of Washed P l a t e l e t s *  Diameter" " (cm)  Surface Area (cm )  Volume (cm )  16.48xl0(+0. 35xl0" )  4.71xl0 (+0.14xl0 )  3. 42x10 (+0. 017X10 )  36. 7 x l 0 (+0.36xl0 )  20. 9 4 x l 0 (+0.31xl0 )  2. 90X10 (+0.025X10 )  26. 3 8 x l 0 (+0.45xl0 )  12. 7 7 x l 0 (+0. 33x10" )  1  Discocyte**  2  3.18X10 (+0.03xl0 ) -4  _4  Spherocyte  8  ++  8  -4  - 8  -4  Cocaine spheres  -8  -4  -8  -4  -8  3  -12  -2  - 1 2  -12  12  12  *Measured diameter of discoycte i n PRP=3. 17xl0"4cm (+0 . 035X10 ) +1000 p l a t e l e t s measured i n each category **Thickness f o r di scocyte=0. 89X10 cm (+0.06X10 cm; n=130) Standard deviation -4  -4  -4  - 24 platelets.  Addition of ADP  (2x10 ^M)  to the spherocytes and cocaine spheres  produced no further change. Transmission electronmicrographs of p l a t e l e t discocytes, echinocytes, spherocytes and the cocaine spheres are shown i n Figs. 3 to 6.  At least 20  f i e l d s of view for each type of p l a t e l e t were examined.  A l l the p l a t e l e t s  appeared to have retained their complement of granules.  The discocytes ( F i g .  3) ranged i n appearance from discs (top view) to cigar shapes (edge-on view). The echinocytes ( F i g . 4) were irregular in shape with long pseudopods.  The  spherocytes ( F i g . 5) were large and swollen but the plasma membranes were intact. ance.  The spherocyte cytoplasm was  less dense and had an "empty" appear-  There were large vacuoles apparent besides the storage granules.  Because of the "empty" appearance of the cytoplasm the plasma membrane as well as the granules and vacuoles were highlighted better than i n the discocytes and echinocytes.  The cocaine spheres ( F i g . 6) were round but smaller than the  spherocyte. The surface-connected canalicular system was obvious i n the discocyte. Numerous openings to the outside could be seen. echinocytes was d i l a t e d .  The canalicular system i n the  In the cocaine spheres d i l a t i o n of the canalicular  channels was even more obvious.  No opening connecting the dilated channels of  the echinocyte and cocaine spheres to the outside could be seen.  2.3.2  P l a t e l e t Integrity and Release of Contents  The supernatants of washed discocytes showed no sign (zero) of lactate dehydrogenase (LDH) a c t i v i t y .  After the p l a t e l e t suspension had been frozen  and thawed once to damage the p l a t e l e t s , LDH a c t i v i t y was detected i n the supernatant at 1.047 Wroblewski unit per ml per 10 n=3).  p l a t e l e t s (+0.350 s.d.;  This i s considered as the t o t a l p l a t e l e t LDH a c t i v i t y .  After  - 25 -  F i g . 3.  E l e c t r o n micrographs of p l a t e l e t (a) x33600 m a g n i f i c a t i o n (b) x22000 m a g n i f i c a t i o n  discocytes.  - 27  Fig. 3  -  (continued) (c) schematic representation of (a) (d) schematic representation of (b)  Two prominent features are the channels of the surfaceconnected canalicular system (CS) with openings on the p l a t e l e t surface (arrows) and the alpha granules Microtubules seen.  (MT)  (AG).  from the circumplatelet bundles can be  Also v i s i b l e are glycogen deposits (GY) as well as  an occasional mitrochondrion  (MC).  - 29 -  F i g . 4.  E l e c t r o n micrographs of p l a t e l e t (a) xl6900  magnification  (b) x27600 m a g n i f i c a t i o n  echinocytes.  - 31 -  Fig. 4  (continued) (c)  schematic  r e p r e s e n t a t i o n of  (a)  (d) schematic  r e p r e s e n t a t i o n of  (d)  The  e c h i n o c y t e s are i r r e g u l a r  The  surface-connected  The  granules  s t i l l be  (AG)  seen.  i n shape with  canalicular  long pseudopods.  system (CS) i s d i l a t e d .  as w e l l as the glycogen d e p o s i t s (GY)  can  - 33 -  F i g . 5.  E l e c t r o n micrographs of p l a t e l e t spherocytes. (a) x29500 magnification (b) x20800 magnification  - 35 -  F i g . 5 (continued) (c) schematic representation of (a) (d) schematic representation of (b) The p l a t e l e t s are swollen but the plasma membranes appear intact.  There are swollen vacuoles (VO) as well as storage  granules (AG).  - 37 -  F i g . 6.  Electron micrographs of the cocaine (a) xl8200 magnification (b) x20800 magnification  spheres.  -  38 -  - 39  -  F i g . 6 (continued) (c) schematic representation of (a) (d) schematic representation of (d) The p l a t e l e t s here are spherical but much smaller than the spherocytes.  Storage granules (AG) are present and channels  of the canalicular system (CS) can be seen.  - 41 transformation to echinocytes with ADP and to spheres with cocaine the supernatant remained negative.  A more detailed timed study of LDH leakage from the  spherocytes was done and shown i n F i g . 7.  After 90 min of hypotonic shock  about 3.87% (+0.80%) of the t o t a l p l a t e l e t LDH a c t i v i t y was found i n the supernatant.  The percentage increased to 10.19% (+^1.70%) a f t e r 2-^hours and 25.51%  (+1.23%) after 4 hours. The p l a t e l e t release experiments consisted of two parts, the  14 C-  serotonin released from dense bodies and beta-thromboglobulin (beta-Tg) released from alpha-granules.  It was found that p r a c t i c a l l y no release of  14 C-serotonin had occurred after echinocyte and spherocyte transformations. Nearly a l l of the r a d i o a c t i v i t y (97.8% i n echinocyte and 97.5% i n spherocyte) could be recovered from the p e l l e t s after centrifugation. The mean beta-Tg content of washed human discocytes from two determinations was 22.21 jug/10  9  p l a t e l e t s (22.23 and 22.19^ug/10 p l a t e l e t s ) . 9  About 13%  of this was found i n the supernatant of the washed p l a t e l e t s as free beta-Tg. It was possible that some release of beta-Tg had occurred during the washing procedure.  Additional releases of 4% during echinocyte transformation and 27%  during spherocyte transformation were also found.  If the discocytes were  stimulated with 0.4 NIH units/ml of thrombin, 94% of their beta-Tg contents was released. 2.3.3  P l a t e l e t Aggregations  F i g . 8 demonstrates how aggregation v e l o c i t y increased with ADP concentration.  The curve was constructed from aggregation measurements of five  d i f f e r e n t samples of washed p l a t e l e t s suspended 0.5 mg/ml of fibrinogen and 4 mM Ca . +  maximum at 2x10 "*M ADP.  i n Tyrode's solution containing  Aggregation v e l o c i t y was about  Aggregations at this concentration were therefore  - 42  F i g . 7.  -  LDH leakage from the spherocytes (as percentage of  t o t a l p l a t e l e t LDH a c t i v i t y ) as a function of time of suspension in hypotonic Tyrode's solution.  Values are means of three  series of experiments; error bars indicate +1 standard deviation.  Vo total LDH release  1  - 44 considered as 100% i n a l l samples for the normalization procedure mentioned before.  This concentration was also used throughout  cyte transformation.  this project for echino-  An example of the actual aggregation tracings showing  the effect of d i f f e r e n t ADP concentration i s shown i n F i g . 9. The effect of fibrinogen concentration on aggregation v e l o c i t y i s shown i n F i g . 10. In this case fibrinogen and 4 mM of C a  + +  ions were added to the  p l a t e l e t suspensions before ADP (2x10 "JM). Maximum v e l o c i t y achieved at 0.5 mg/ml of fibrinogen was designated as 100% according to the normalization procedure above. tration  F i g . 10 i s a composite  of five d i f f e r e n t series of concen-  experiments.  The curves showing the effect of divalent cation concentration on the aggregation v e l o c i t y are shown i n F i g . 11. Fibrinogen at 0.5 mg/ml together with either C a ADP  + +  or M g  ++  ions were added to the p l a t e l e t suspensions before  (2x10 M). A biphasic effect was observed i n both the Ca  curves with maximum aggregation at 4 mM of C a  + +  ion.  and Mg  Velocity of this  optimum concentration therefore serves as the 100% aggregation v e l o c i t y for both the C a  + +  and M g  the a b i l i t y of Mg Ca  + +  ++  ion throughout  ++  series.  In this way, i t can be demonstrated that  ion to support aggregation i s only about half that of the concentrations tested. Examples of individual  aggregation tracings are shown i n F i g . 12. Two more divalent cations, strontium and manganese were tested for their a b i l i t i e s to support ADP induced p l a t e l e t aggregation. mg/ml of fibrinogen, Sr  In the presence of 0.5  ions at concentrations between 0-20 mM f a i l e d to  support ADP (2x10 "*M) induced aggregation ( F i g . 12). ++ .  already containing 4 mM of Ca  If added to a system  ++  ions, Sr  would neither augment nor  ++ .  reduce the supportive role of Ca  ions i n ADP induced aggregation.  - 45 -  F i g . 8.  The effect of ADP concentration on the aggregation  v e l o c i t y of washed p l a t e l e t s suspended i n Tyrode's  solution  containing 0.5 mg/ml of fibrinogen  Aggregation  v e l o c i t y at 2x10 "*M ADP  and 4 mM C a . + +  i s taken as 100% and v e l o c i t i e s at  lower concentrations are expressed as percentages of i t . Values are means of f i v e series of experiments; error bars indicate +1 standard  deviation.  - 47  F i g . 9. of ADP at  Examples  arrow.  of a g g r e g a t i o n t r a c i n g s showing the e f f e c t  c o n c e n t r a t i o n on p l a t e l e t  (a) 2x10  M;  -  (b) 2x10  aggregation v e l o c i t y .  M or (c) 1x10  M was  added at  ADP  1 MIN.  time  - 49 -  F i g . 10.  The e f f e c t of fibrinogen concentration on the  aggregation v e l o c i t y of p l a t e l e t s . Washed p l a t e l e t s were suspended i n Tyrode's solution containing fibrinogen and 4 mM Ca  +  ions.  Aggregation was induced by 2x10 ^M of ADP.  Aggregation v e l o c i t y at 0.5 mg/ml of fibrinogen was taken as 100% and v e l o c i t i e s at lower concentrations were expressed as percentages of i t . Values are from means of f i v e series of experiments; error bars indicate +1 standard deviation.  Fibrinogen  cone, (mg/ml)  -  F i g . 11.  51 -  The effect of divalent cation concentration on the ++  aggregation v e l o c i t y of p l a t e l e t s : and S r  + +  (-A-).  Ca  ++  (-•-), Mg ( - O - )  Washed p l a t e l e t s were suspended i n Tyrode's  solution containing 0.5 mg/ml fibrinogen and one of the cations. Aggregation was induced by 2x10 "*M of ADP. v e l o c i t y at 4 mM C a  + +  Aggregation  ions i s taken as 100%.  Values are  means from f i v e series of experiments; error bars indicate +1 standard deviation.  °/ol  CATION  CONC. (mM)  - 53 -  F i g . 12. Examples of aggregation tracings showing the e f f e c t s of d i f f e r e n t divalent cations.  P l a t e l e t s were suspended i n  Tyrode's solution containing 0.5 mg/ml of fibrinogen and 4 mM (a) C a  + +  ions; (b) M g  ++  ions or (c) S r  + +  2x10 ~*M was added as indicated by arrows.  ions.  ADP at  v—  1 MIN.  <  >~  time  - 55 It was found that Mn  ion was an i n h i b i t o r of p l a t e l e t aggregation.  ++  Aggregation induced by 2x10 "*M of ADP i n the presence of 0.5 mg/ml of fibrinogen and 4 mM Ca inhibited by 10 mM Mn  ++  ions was greatly reduced by 2.5 mM and completely ions ( F i g . 13).  Aggregation induced by 0.1 NIH  -4 unit/ml of thrombin or by 1x10 M of arachidonic acid was also f u l l y .  ++ .  .  inhibited by 10 mM of Mn  ions.  A panel of aggregating agents including ADP (2x10 ~*M with 0.5 mg/ml of fibrinogen and 4 mM of Ca  ions), epinephrine (5x10 ^M with 0.5 mg/ml of  fibrinogen and 4 mM of C a ions), thrombin (0.1 NIH unit/ml), A23187 -5 -3 (1x10 M) and arachidonic acid (1x10 M) were tested for their a b i l i t i e s + +  to aggregate  spherocytes.  They a l l f a i l e d to do so.  These aggregating agents  at the concentrations indicated, would strongly aggregate normal discoid platelets.  The cocaine spheres also f a i l e d to aggregate upon stimulations  with ADP, thrombin and arachidonic acid (same concentrations as above). Although spherocytes have lost their a b i l i t y to aggregate to r i s t o c e t i n , an agglutinating agent, i s quite d i f f e r e n t .  their reaction  When 1.5 mg/ml of  r i s t o c e t i n alone was added to either discocyte or spherocyte there was no response.  I f plasma was added to provide for the von Willebrand's factor then  agglutination occurred i n both spherocyte and discocyte samples.  In fact, the  agglutination of the spherocytes was twice the rate (velocity) of the discocytes ( F i g . 14).  Plasma was added at a r a t i o of 0.1 ml per ml of p l a t e l e t  suspension. Under phase microscopy discocytes treated with neuraminidase retained their discoid shape. fibrinogen and 4 mM of C a  + +  still  Tested at 2x10 "*M of ADP (with 0.5 mg/ml of  added) the aggregation rates were the same for  the treated and non-treated discocytes ( F i g . 15).  - 56 -  F i g . 13. gation.  The inhibitory effect of Mn Washed p l a t e l e t s were suspended  on p l a t e l e t aggrei n Tyrode's  solution  ++ containing 4 mM Ca and 0.5 mg/ml of fibrinogen.  Mn  (a) 0 mM;  Aggregation  (b) 2.5 mM or (c) 10 mM was then added.  was induced by adding 2x10 "*M of ADP as indicated  ++  at  by arrows.  1  1  1 MIN.  y>  time  - 58  F i g . 14.  -  Agglutination of p l a t e l e t (a) spherocytes and  discocytes by r i s t o c e t i n .  (b)  Discocytes were suspended i n i s o -  tonic and spherocytes in hypotonic Tyrode's solution.  Plasma  was added to each sample at 0.1 ml of plasma per ml of p l a t e l e t suspension, to provide von Willebrand's factor. means from two series of experiments.  Values are  Ristocetin Cone, (mg/ml)  - 60 -  Fig. 15.  Aggregation tracings from normal ( l e f t ) and  neuraminidase treated (right) p l a t e l e t s .  Both were suspended  in Tyrode's solution containing 0.5 mg/ml of fibrinogen and 4 mM C a  + +  ions.  Aggregation was induced by 2x10 "*M of ADP  as indicated by arrows.  ;  .  1 MIN.  i  time  -  6 2  -  Wheat germ agglutinin (WGA) aggregated normal discocytes strongly.  Afte  neuraminidase treatment, the aggregation v e l o c i t i e s dropped substantially at a l l the concentrations tested ( F i g . 1 6 ) . shown i n F i g . 1 7 .  Typical aggregation tracings are  Aggregation was inhibited by EDTA or EGTA (both at 2 . 2 5  mg/ml) as well as by N-acety1-glucosamine ( 5 0 mM) but galactose and N-acetyl galactosamine at this concentration were without e f f e c t . Both the castor bean agglutinins  ( R C A ^ Q  and  R C A ^ Q )  aggregated normal  p l a t e l e t s only to a small extent but after neuraminidase treatment aggregati was greatly enhanced (Figs. 1 6 and 1 7 ) . 2 . 2 5 mg/ml of EDTA or EGTA. acetyl-galactosamine  ( 5 0  Aggregation was also inhibited by  Aggregation by RCA  mM) and D-galactose  ( 5 0  &0  was inhibited by both NmM).  Aggregation by  R C A ^ Q  was inhibited only by D-galactose while N-acetyl-galactosamine had no effect Neither were affected by N-acety1-glucosamine. J e g u i r i t y bean agglutinin (JBA) did not aggregate non-treated p l a t e l e t s and after neuraminidase treatment the p l a t e l e t s were aggregated moderately (Fig. 1 6 ) .  The aggregation was i n h i b i t e d by 2 . 2 5 mg/ml of EDTA or EGTA as  well as 5 0 mM D-galactose.  N-acetyl-galactosamine and N-acety1-glucosamine  were without e f f e c t . Aggregation of glutaraldehyde fixed discocytes by WGA could not be demon strated with the aggregometer but under the microscope numerous small aggregates of two or three p l a t e l e t s were seen.  - 63 -  F i g . 16.  Aggregation responses of normal (-•-)  neuraminidase treated (-O-)  and  p l a t e l e t s to the l e c t i n s .  - 64 -  Lectin Cone, (jjg/ml)  - 65 -  F i g . 17. Examples of p l a t e l e t aggregation responses to lectins. ( i ) Normal (a) and neuraminidase-treated (b) p l a t e l e t s aggregated by WGA ( i i ) Normal (c) and neuraminidase-treated (d) p l a t e l e t s aggregated by  R C A ^ Q  ( i i i ) Normal (e) and neuraminidase-treated ( f ) p l a t e l e t s aggregated by R C A  12Q  A l l l e c t i n s were added at 100 u,g/ml as indicated by arrows.  - 66 -  i  t  1 MIN. time  - 67 -  2.4  DISCUSSION  2.4.1  Platelet Dimension and Morphology -A The mean diameter of the discocyte measured here 3.18x10 cm i s similar -L  to 3.2x10  cm reported by Milton and Frojmovic (1979).  The R value of 0.28  for human discocyte measured at room temperature was close to that of 0.26 measured at 37°C by Frojmovic and Panjwani  (1976) .  Although there was a  small difference between the R values, the r e s u l t i n g difference i n surface area 8 —8 2 (16.48x10 vs 16.42x10 cm ) was minimal. Born et jil (1978) reported R=0.25 for rabbit p l a t e l e t s . Using electronic p a r t i c l e counting Gear (1981) —  -12 3 found the human platelet volume t o be 4.94x10 cm and Yamazaki and -12 3 Motcmiya (1980) found a similar value 4.66x10 cm . These two values are -12 3 quite compatible with 4.71x10 cm reported i n t h i s chapter. Hypotonic stress at 75 mOsm produced spherocytes with a mean diameter of -4 3.42x10  cm. An i d e n t i c a l value was found by M i l t o n and Frojmovic (197 9).  Recently Boneu et a l . (1982) found hypotonic treatment at 120 mOsm increased platelet diameter to 3.07x10"^ cm. The surface area and volume of the echinocyte cannot be found from the geometrical means used here because of i t s i r r e g u l a r shape.  However i t w i l l be  shown i n Chapter 3, from indirect calculations, that the surface area of the echinocyte i s probably close to that of the cocaine sphere.  Born et al (1978),  using a high speed centrifugation radiolabel d i l u t i o n technique found that -12 3 rabbit platelets had a mean volume of about 5x10  cm . They found no  change i n platelet volume after echinocytic transformation. The morphologies of the discocyte, echinocyte and the cocaine sphere under electron microscopy were not unlike those reported elsewhere (White, 1972;  - 68  Behnke, 1970).  The surface-connected  -  canalicular system was  discocyte, echinocyte and the cocaine spheres.  present i n the  The channels of the system  were d i l a t e d i n the echinocyte and severely d i l a t e d i n the cocaine  spheres.  Presence of the canalicular system i n the echinocyte suggested that evagination of the system was not t o t a l .  This could be supported  surface area of the spherocyte the echinocyte  (Chapter 3).  by the fact that the  appears to be considerably larger than that of  Our o r i g i n a l hypothesis, that the increase i n  membrane surface area originates from the c a n a l i c u l a r system does not d i c t a t e the degree of evagination of the canals.  In fact White and Clawson (1980)  suggested that the canalicular system within the cytoplasm  i s so tortuous  and  interwoven that complete evagination would result i n the t o t a l destruction of the p l a t e l e t . —8 70x10  P l a t e l e t s spreading on a surface can a t t a i n a surface area of  2 cm  (Frojmovic and Milton, 1982), double that of the  This implies that even i n the spherocyte  evagination may  spherocyte.  not have been t o t a l .  The large empty vacuoles i n the spherocytes might be swollen canalicular channels.  P l a t e l e t morphology and surface area related to surface  chemistry  w i l l be discussed further i n Chapter 3. 2.4.2  P l a t e l e t Integrity and Release of Contents  The enzyme LDH  i s a cytoplasmic enzyme (Gogstad ejt al_., 1981)  used extensively as a marker for cytoplasmic and Frojmovic,  1979;  Sturk et a l . , 1982).  and has been  leakage from p l a t e l e t s (Milton  Fratantoni and Poindexter,  1981;  Ostermann et a l . , 1982;  The p o s s i b i l i t y that the transformation of p l a t e l e t s ,  especially during hypotonic  shock could result i n leakage or breaks i n the  plasma membrane i s of considerable concern here.  If even a small number of  p l a t e l e t s burst, released material could coat the surfaces of the  remaining  intact ones and the resulting properties could be misinterpreted as p l a t e l e t  - 69 -  surface changes during transformation. At 60 mOsm Milton and Frojmovic (1979) found that considerable enzyme leakage had occurred after only 10 min of hypotonic shock.  In the present work i t was found that LDH leakage was not measur-  able after 1 hour of stress at 75 mOsm (Fig. 7) and that the amount remained 3 below 4% for 90 min, the time required to .perform the H -borohydride l a b e l l i n g procedure i n Chapter 4. Much shorter time periods (20-30 min) were needed to do c e l l electrophoresis (Chapter 3).  Therefore within the time  periods of the experiments the p l a t e l e t spherocytes appeared to be intact by this c r i t e r i o n . Beta-Tg i s present i n the alpha-granules and the sole source of plasma beta-Tg i s from the p l a t e l e t s .  Increased amounts of beta-Tg i n the plasma  indicate vascular events involving in vivo p l a t e l e t activation Owen, 1981).  The radioimmunoassay  (Kaplan and  k i t developed by Amersham i s intended for  use with plasma but can e a s i l y be adopted to assay p l a t e l e t beta-Tg with l i t t l e change i n the assay protocol.  This k i t has already been used by a  number of groups for i n v i t r o p l a t e l e t experiments as an indicator for alpha-granule release (Ludlam and Cash, 1976; Bolton et^ a l . , 1980; Gogstad et^ a l . , 1981).  Kaplan and Owen (1981) have reported that the mean p l a t e l e t 9 9 beta-Tg content i s 17.7^ig/10 p l a t e l e t s , a l i t t l e lower than 22.2jxg/10 p l a t e l e t s found here. release.  It seems that i t i s extremely easy to induce beta-Tg  It was recommended by the manufacturer and others (Ludlam and Cash,  1976) that the blood should be collected i n EDTA and handled at 4°C.  This  was not possible i n our circumstances and a considerable amount of beta-Tg was released during washing and hypotonic shock as a r e s u l t .  Examination of the  p l a t e l e t s under electron microscopy, on the other hand, did not show the p l a t e l e t s to be grossly depleted of t h e i r granules. bodies was evident as monitored using 14  C  -serotonin.  No release from the dense  - 70 -  2.4.3  P l a t e l e t Aggregations  The effect of ADP studied.  concentration on p l a t e l e t aggregation has been well  The results shown i n F i g . 8 were not s i g n i f i c a n t l y d i f f e r e n t from  those found by others ( M i l l s et a l . , 1968; 1973;  Stibbe and Holmsen, 1977;  2x10 ^M of ADP was  Tangen et a_l., 1971;  Marguerie et a l . , 1979).  gives the maximum aggregation v e l o c i t y .  therefore used throughout this project.  Frojmovic,  It i s seen that This concentration  In most of the experiments i n  Chapters 3 and 4 shape change (to echinocyte), and not aggregation, desired.  was  Fibrinogen and divalent cations were withheld from the p l a t e l e t s to  avoid aggregation.  Born (1970) has shown that the speed and extent of shape  change also depend on ADP  concentration.  Michal and Born (1971) found that  the v e l o c i t y of aggregation was d i r e c t l y related to the v e l o c i t y of shape change.  In the present work i t was  also found, based on the magnitude of the  i n i t i a l decrease i n light transmission preceding the increase due to aggregation,  that higher ADP  concentrations brought about larger shape change  responses. The effect of fibrinogen concentration on p l a t e l e t aggregation ADP  induced  has also been well studied and F i g . 10 i s similar to concentration  reported by others (McLean e_t al., 1964; a l . , 1977; Marguerie e_t al.,  1979;  Deykin et al. , 1965;  Harfenist et al., 1980).  by  curves  Niewiarowski e_t The fibrinogen  concentration study here forms the basis for the fibrinogen adsorption experiments to be described i n Chapter 4. F i g . 11 shows the biphasic p l a t e l e t aggregation response to increasing concentrations of C a  and M g  + +  ++  ions.  Such biphasic responses were also  observed by Born and Cross (1964) and H e p t i n s t a l l (1976). .  .  .  .  that aggregation v e l o c i t i e s increased with Ca  ++  or Mg  ++  .  It was  found here  ion concentration  - 71 u n t i l the optimum occurred at 4 mM. a decrease i n aggregation  Further increases i n concentration caused  v e l o c i t y , the reason for which is not clear although  i t i s l i k e l y associated with the high i o n i c strength of the suspending medium at the higher C a optimum C a  + +  + +  or M g  ++  concentrations.  concentrations.  Born and Cross  Born and Cross  (1964) found the  (1964) found the optimum C a  concentrations to be 1.7 mM while H e p t i n s t a l l (1976) found i t at 2.5 mM. also found, as shown here, that M g ADP  induced  platelet  ++  + +  They  ions were less effective i n supporting  aggregation.  The requirement of C a  + +  or M g  ++  ions for the binding of fibrinogen t o  ADP activated platelets has been discussed e a r l i e r . found that t h i s requirement i s also biphasic.  Marguerie ^ t a l . (1980)  The calcium or magnesium ion  concentration optimal f o r fibrinogen binding i s about 1 mM and binding decreases below or above t h i s concentration. found that M g  ++  was less effective i n f u l f i l l i n g the requirement for  fibrinogen binding than was C a . + +  about 3x10^  Marguerie ^ t _al. (1980) also  At the optimum calcium ion concentration,  fibrinogen molecules bind t o each platelet i f magnesium i s  substituted. aggregation  The calcium and magnesium ion concentration curves for p l a t e l e t (Born and Cross, 1964; H e p t i n s t a l l , 1976 and reported here) and  fibrinogen binding (Marguerie,  1980)  are s t r i k i n g l y similar, suggesting the  presence of a close r e l a t i o n s h i p between platelet aggregation  and fibrinogen  binding. The contribution of strontium ions ( S r ) t o platelet aggregation i s not + +  as clear.  It was found here that between 0 and 20 mM, S r  ADP-induced aggregation. of S r  + +  + +  support  Lages et a l . (1975) found that the presence of 0.2 mM  had l i t t l e or no effect on ADP and adrenalin induced  aggregation.  d i d not  platelet  On the other hand, Best et a l . (1981) reported that addition of  Sr  ions alone could result i n p l a t e l e t activation with thromboxane  production and serotonin release. Unlike magnesium, calcium and strontium, manganese i s not a type IIA element.  Its contribution to p l a t e l e t aggregation  i s also uncertain.  It has  been proposed by Bosmann (1972) and Wu and Ku (1978) that s i a l y l t r a n s f e r a s e on the surface of p l a t e l e t s may be involved i n the p l a t e l e t aggregation The hypothesis  i s that the enzyme on one p l a t e l e t interacts with the substrate  s i a l i c acid on the surface of another p l a t e l e t forming a "bridge." never been substantiated. factor.  This enzyme has a requirement for Mn  However, present results suggest that M n  p l a t e l e t aggregation.  ++  ++  +  This has as co-  i s an i n h i b i t o r of  It i s therefore unlikely that s i a l y l t r a n s f e r a s e  any essential part i n p l a t e l e t aggregation. Mn  process.  plays  The optimum concentration of  ions required by the enzyme i s between 0.8 and 2 mM as reported by Wu  and Ku (1978) and 20 to 60 mM according to Bosmann (1972).  Manganese ions at  a few millimolar concentration have an inhibitory effect on the movement of calcium ions across the sacroplasmic reticulum membranes i n muscle fibres (Saida and Suzuki, 1981).  Platelet activation by various aggregating agents  involves the massive movement of compartmentalized calcium into the (Detwiler et: a l . , 1978;  Massini £t aj.., 1978).  cytoplasm  Whether manganese i n h i b i t s  p l a t e l e t aggregation by i n t e r f e r r i n g with calcium ion movements or not remains to be determined.  ++ ++ ++ ++ The fact that Ca , Mg , Sr and Mn a l l have  very d i f f e r e n t effects on ADP-induced aggregation at the same concentration strongly implies that s p e c i f i c binding of the divalent ions to, presumably, membrane sites i s involved i n their a c t i v i t y , since generalized e l e c t r o s t a t i c effects would depend only on the ionic strength, not on the chemical nature of the ions (Diamond and Wright, 1969).  - 73 -  After spherocyte transformation, the p l a t e l e t s were found to have l o s t their a b i l i t y to aggregate.  The most l i k e l y explanation i s that hypotonic  shock has caused p l a t e l e t damage to the point that aggregation no longer i s possible.  The loss of aggregability i n cocaine spheres i s also expected  (O'Brien, 1962; Aledort and Niemetz, 1968).  In both types of spheres the loss  of aggregability may be attributed to the t o t a l disruption of c o n t r a c t i l e and cytoskeletal elements (Zucker-Franklin, 1969; Nachmias et al_., 1977; Nachmias et a l . , 1979).  Membrane changes to be described i n Chapter 3 may  also be  involved. R i s t o c e t i n i s not a pharmacological aggregating agent but i s a d i r e c t agglutinating agent.  That i s , no physiological response from the p l a t e l e t i s  required to induce agglutination.  In fact, r i s t o c e t i n has been shown to  agglutinate formaldehyde fixed p l a t e l e t s ( A l l a i n e_t a l . , 1975). Agglutination requires the co-binding of r i s t o c e t i n and a plasma factor (von Willebrand's factor) to the surface of p l a t e l e t s .  The exact nature of the interaction i s  s t i l l uncertain and a variety of possible models have been reviewed (Kirby, 1977;  elsewhere  Solum and Peterka, 1977; Coller, 1978; P h i l l i p s , 1980).  c e t i n binds to the surface of p l a t e l e t s (Hashimoto and Suzuki, 1979)  Ristoand  produces a condition that favours the binding of von Willebrand's factor to the p l a t e l e t surface (Hahsimoto and Suzuki, 1979; Kao e_t a l . , 1979; et al., 1979).  Schneider  The von Willebrand's factor and/or r i s t o c e t i n then cross-bridge  receptors on the surfaces of adjacent p l a t e l e t s , causing agglutination.  The  receptor for von Willebrand's factor was found to be associated with the surface glycoprotein designated lb ( P h i l l i p s , 1980).  Patients with Bernard-  Soulier syndrome lack this glycoprotein and their p l a t e l e t s f a i l to agglutinate upon exposure to r i s t o c e t i n and the plasma factor.  On the other hand persons  - 74 with von Willebrand s disease lack the factor i n their plasma and their p l a t e 1  lets w i l l agglutinate only after external von Willebrand's factor i s added. Although spherocytes have lost their a b i l i t y to aggregate they are s t i l l able to agglutinate i n response to r i s t o c e t i n and added plasma.  Hypotonic  shock has therefore not destroyed the receptors for either r i s t o c e t i n or von Willebrand's factor.  The reason why  the rate of agglutination i s greater for  the spherocyte than the discocyte i s uncertain.  It may be related to the  increase i n surface area or p l a t e l e t volume i n the spherocyte, perhaps increasing the number of receptor s i t e s per p l a t e l e t for either type of molecule.  The increase i n volume might also lead to an increase in the number  of p l a t e l e t - p l a t e l e t c o l l i s i o n s i n the spherocyte suspension, since a larger f r a c t i o n of the suspension volume would be occupied by the spherocytes.  The  decrease i n negative charge density on the surface of the spherocyte (Chapter 3) might also contribute to the increased aggregation rate.  Ristocetin i s a  p o s i t i v e l y charged molecule and i t has been proposed that i t s function i n the agglutination i s to neutralize some of the p l a t e l e t surface negative charge ( P h i l l i p s , 1980).  If the surface charge density has already been lowered  during spherocyte transformation then the action of r i s t o c e t i n might be much enhanced.  F i n a l l y , since the spherocytes were suspended  i n hypotonic Tyrode's  solution while the discocytes were i n isotonic Tyrode's solution, the low ionic strength might also play a role i n f a c i l i t a t i n g agglutination of the spherocytes. As expected, neuraminidase treatment of p l a t e l e t s did not a l t e r their shape or their aggregability.  Hovig (1965) as well as Bowles and Brunton  (1982) found no a l t e r a t i o n i n p l a t e l e t morphology under transmission and scanning electron microscopy after neuraminidase treatment.  Greenberg e_t a l .  - 75 -  (1975) found only a s l i g h t enhancement of ADP platelets.  induced aggregation  The enhancement was most obvious at a low (4.5x10 ^M)  i n treated ADP  concentration with p r a t i c a l l y no difference being observed at a higher (9x10 ^M)  concentration.  At 2x10  "'M no differences were found here.  Greenberg and Jamieson (1974) reported that human p l a t e l e t s can be aggregated by WGA  and to a lesser extent by RCA.  Ganguly and Fossett (1979) found  that neuraminidase treatment of p l a t e l e t s reduced the extent of aggregation WGA  as well as the number of WGA  by  binding s i t e s on the p l a t e l e t surface.  Patscheke and Worner (1977) and Nairn et aj.. (1982) observed that removal of terminal s i a l i c acid residues from the p l a t e l e t surface by neuraminidase actually enhanced the aggregation observed by us.  Similar phenomena were  The effects of l e c t i n concentration on the aggregation  treated and control p l a t e l e t s was WGA  response to RCA.  of  also studied here ( F i g . 16).  binding can be s p e c i f i c a l l y inhibited by N-acetyl-glucosamine but i n  the absence of this sugar i t can interact strongly with the terminal s i a l i c acid residues of glycoproteins (Goldstein and Hayes, 1978; K a t l i c , 1979;  Peters et a l . , 1979;  Wright, 1980).  demonstrated by a number of workers that WGA  In p l a t e l e t s i t has been  binds to glycoprotein-Ib (GP-Ib)  on the surface of the c e l l (Ganguly and Fossett, 1979; 1979;  McGregor _et a l . , 1979;  Nairn et a l . , 1982).  either the binding of r a d i o l a b e l l e d WGA acrylamide  Nairn et a l . (1982).  Marchesi and Chasis,  These experiments involved  by separated glycoproteins in poly-  gels or the i s o l a t i o n of s o l u b i l i z e d GP-I  a f f i n i t y chromatography.  Bhavanandan and  using WGA-Sepharose  Caution i n interpreting these results was  urged by  Moreover Rock et a l . (1980) found that Bernard-Soulier  syndrome p l a t e l e t s which lack GP-Ib could s t i l l be aggregated by WGA. therefore seems u n l i k e l y that GP-Ib i s the only receptor on p l a t e l e t s .  It The  - 76 hypothesis  binds to terminal s i a l i c acid residues of the glyco-  i s that WGA  p r o t e i n s ) on the p l a t e l e t surface and causes p l a t e l e t aggregation v i a the usual cross-linking of adjacent surfaces by the multivalent l e c t i n .  Removal  of s i a l i c acid by neuraminidase therefore reduces the p l a t e l e t ' s response to WGA  (Ganguly and Fossett, 1979). The removal of terminal s i a l i c acid residues apparently exposes galactose  or N-acety1-D-galactosamine residues. of  RCA^Q,  RCAj^o  an<  * ^ ' B A  a  ^  (Goldstein and Hayes, 1978)  °^  w  n  i  c  n  This suggestion i s based on the effect have a high a f f i n i t y for galactose  and a l l of which show enhanced a c t i v i t y with  neuraminidase treated p l a t e l e t s (Patscheke et. al., 1977).  These galactose-  binding l e c t i n s probably induce aggregation by the same mechanism(s) as does WGA.  That galactose or N-acety1-galactosamine residues can be exposed after  neuraminidase removal of terminal s i a l i c acid has been found i n many systems (Steck and Dawson, 1974; Wright, 1980).  The combined use of neuraminidase and  galactose oxidase followed by reduction with t r i t i a t e d NaBH^ to label c e l l surface glycoproteins has become a standard procedure i n recent years ( P h i l l i p s , 1979).  This l a b e l l i n g procedure as applied to p l a t e l e t membrane  studies w i l l be discussed i n Chapter 4. F i n a l l y , a small degree of agglutination was were exposed to WGA.  observed when fixed platelets  Similar to our study Ganguly and Fossett (1980) observed  agglutination of fixed p l a t e l e t s by WGA demonstrate this with aggregometry.  microscopically but were unable to  They therefore suggested that  two  mechanisms, passive agglutination and active aggregation, might be at work i n this situation.  -  2.4.4  77 -  Summary of Chapter 2  The dimensions and morphology of the d i f f e r e n t forms of p l a t e l e t s were established.  The most important point found was that most spherocytes formed  v i a hypotonic shock remained  intact for a reasonable period of time, although  some release of beta-Tg occurred.  The second half of Chapter 2 dealt with  p l a t e l e t aggregation i n general, and i t was found that the spherocytes had lost a l l a b i l i t y to aggregate.  They could only be agglutinated passively by  r i s t o c e t i n and von Willebrand s factor. 1  - 78 -  CHAPTER 3  MICROE IE CTROPHORE SIS  - 79 -  3.1  INTRODUCTION Microelectrophoresis or c e l l electrophoresis i s a technique used to probe  the e l e c t r o k i n e t i c properties of c e l l surfaces.  An i n depth t h e o r e t i c a l  discussion of microelectrophoresis and the e l e c t r o k i n e t i c behaviour of c e l l s can be found i n a review by Seaman, 1975. B r i e f l y , the electrophoretic mobility (EPM) of a c e l l i s measured by observing  the v e l o c i t y of i t s motion  under the influence of the p a r t i c u l a r e l e c t r i c f i e l d applied.  I t i s defined  as the v e l o c i t y per unit f i e l d strength and i s usually expressed i n terms of 2  -1  -1  cm .sec .V  .  The EPM (ja.) of a c e l l , assuming i t behaves as a smooth  p a r t i c l e bearing a uniform charge density, i s d i r e c t l y related to i t s zeta potential (IS),  i . e . the e l e c t r o s t a t i c potential at the shear plane.  potential i s i n turn related to the charge density (c?) apparently the plane of shear:  where 1/K = double layer thickness ~ 8A at 1=0.15 = v i s c o s i t y of suspending medium N = Avogadro's number I = ionic strength of suspending medium =1^ .. .th . c^ = molar concentration of l ionic . .th . . z. = valence of I ionic n  I  £ = dielectric  . species  species  constant  k = Boltzman's constant T = absolute temperature e = electronic charge unit (4.8x10""^ esu)  C^Z^  The zeta  located at  - 80 Equation  [1] holds providing the smallest radius of curvature of the p a r t i c l e  i s much larger than the double layer thickness 1/K. Equation providing z^e^^kT.  [2] i s v a l i d  Both conditions are f u l f i l l e d by p l a t e l e t s suspended i n  physiological buffers.  Combining [1] and [2] gives:  CT  [3]. 2  The charge density,cr, i s expressed as esu/cm 2 . . number of charges per cm  but can be converted  into the  by dividing by e.  The surface of most c e l l s contains both p o s i t i v e and negative charge groups but i n general the net surface charge i s a negative one at physiological pH. The charge groups on the surface of a c e l l attract ions of opposite charge (counter-ions) and repel charges of l i k e sign (co-ions), the net effect being the formation of the diffuse double layer adjacent to the c e l l surface. In some instances i t i s found that a fraction of the counterions adsorb to the p a r t i c l e surface reducing the net surface charge density and forming what i s known as a Stern layer.  There i s no evidence  for the formation of a Stern  layer on b i o l o g i c a l c e l l s i f only monovalent ions are present i n the suspending medium, as no dependence of mobility on the chemical nature of the ions has been found (Heard and Seaman, 1960). or M g  ++  When multivalent cations such as C a  +  are present, however, binding does occur and the mobility decreases  associated with these events can be used to estimate the binding parameters, as w i l l be discussed Equations  subsequently.  [1] to [3] assume the surface of the p a r t i c l e i s smooth and well  defined, although i t may be irregular i n shape.  However, the surfaces of  b i o l o g i c a l c e l l s are not smooth on the scale of double layer dimensions. The l i p i d bilayer anchors a diffuse layer of charged and neutral glycoproteins and g l y c o l i p i d s known as the glycocalyx which extends some distance out from the  - 81 plane of the l i p i d head groups.  Recently,  theories have been developed which  e x p l i c i t l y model the effects of this layer on the electrophoretic mobility (Donath and Pastushenko, 1979;  Wunderlich, 1982;  results provide a modification of [3]  o-F=^nK  Levine et. _al., 1983).  The  i n the form of:  [4].  The function F includes terms involving the thickness of the glycocalyx  and  the average size and volume concentration of the polymer segments in this region. [3]  None of these parameters are known for p l a t e l e t s , however.  Equation  i s therefore used to interpret electrophoretic m o b i l i t i e s i n this work,  the assumption being made throughout that the function F remains constant  or  changes i n a consistent manner among the p l a t e l e t forms examined as conditions are varied. Like most other types of c e l l s p l a t e l e t s have a negative charge.  net  surface  The e l e c t r o k i n e t i c behaviour of p l a t e l e t s has been reviewed by Seaman  and Brooks (1970), Mason and Shermer (1971) and Seaman ( 1 9 7 6 ) . phoretic mobility of p l a t e l e t s was  The e l e c t r o -  f i r s t investigated by Abramson i n 1928.  He  suspected that the e l e c t r i c a l charge on p l a t e l e t s could have something to do with p l a t e l e t aggregation and thrombosis.  However the lack of  information  about p l a t e l e t s at that time prevented him from further examining this point. More recently Seaman and Vassar (1966) found that addition of lyag/ml of  ADP  to p l a t e l e t s i n PRP  caused an 18% decrease i n mobility.  the decrease i n EPM  might somehow be associated with aggregation induced by  ADP.  They suggested that  Hampton and M i t c h e l l (1966) reported a biphasic change i n the EPM  p l a t e l e t s (in PRP)  induced by ADP.  At extremely low concentrations  i l i t y increased, possibly due to the binding of ADP  of  the mob-  onto the p l a t e l e t  surface.  - 82 -  / i (about i , _ 1x10 , , „ 7M) the e l e c t r o p h o r e t i c m o b i l i t y g/ml  TTU »tm was above v c i n~2 When ADP 5x10  _  v  ddecreased w i t h i n c r e a s i n g c o n c e n t r a t i o n .  This  biphasic  J  phenomenon was  o b s e r v e d by S t o l t z (1971) as w e l l as K o s z t o l a n y i et a l . (1980) but  also  several  groups i n c l u d i n g Grottum (1968), B e t t s et a l . (1968) and Seaman and V a s s a r (1966) c o u l d not c o n f i r m t h i s e f f e c t .  A drop i n the s u r f a c e n e g a t i v e  w o u l d mean a drop i n t h e mutual r e p u l s i v e f o r c e between p l a t e l e t s .  charge  This  might  b r i n g about a more f a v o u r i t e c o n i d t i o n f o r p i at e l e t - p i at e l et i n t e r a c t i o n and aggregation  (Grotum, 1968 and Seaman, 1976).  undertaken by t h e s e authors decrease i n m o b i l i t y .  No m o r p h o l o g i c a l s t u d i e s  t o c o r r e l a t e between p l a t e l e t shape  were  change  and the  A French group, on the o t h e r hand, u s i n g 2x10  of  ADP have r e p o r t e d t h a t d i s c o c y t e t o e c h i n o c y t e t r a n s f o r m a t i o n i n PRP r e s u l t s a m o b i l i t y increase S i a l i c acid is  in  ( B o i s s e a u e t ^ 1 . , 1977). a major c o n t r i b u t o r t o p l a t e l e t s u r f a c e n e g a t i v e  charge.  Jung et _al. (1982) found a l i n e a r c o r r e l a t i o n between p l a t e l e t s u r f a c e  sialic  a c i d content and EPM.  negative  charge  It  has been e s t i m a t e d t h a t about 41% of t h e net  d e t e c t e d e l e c t r o k i n e t i c a l l y comes from t e r m i n a l s i a l i c a c i d r e s i d u e s  (Seaman, 1976).  Another 28% d e r i v e s from phosphate  groups.  Neuraminidase  removal of the t e r m i n a l s i a l i c a c i d groups has been found t o r e s u l t i n a 40 t o 60% drop i n human p l a t e l e t e l e c t r o p h o r e t i c m o b i l i t y (Madoff _et a l . , 1964; Seaman and V a s s a r , 1966; B r a y and A l e x a n d e r , 1969; S t o l t z and N i c o l a s , 1 9 7 9 ) . F r a n the drop i n m o b i l i t y one can c a l c u l a t e the decrease i n charge d e n s i t y and p r o v i d i n g the s u r f a c e a r e a i s known, c o n v e r t t h a t i n t o the a c t u a l number s i a l i c a c i d m o l e c u l e s r e l e a s e d (Seaman and V a s s a r , 1966).  of  One must be aware  that t h i s number r e f l e c t s o n l y s i a l i c a c i d r e l e a s e d from near the e f f e c t i v e p l a n e of s h e a r .  O t h e r s i a l i c a c i d r e s i d u e s l o c a t e d at d i f f e r e n t d i s t a n c e s  t h i s p l a n e c o n t r i b u t e somewhat l e s s  t o the e l e c t r o k i n e t i c p r o p e r t i e s  of  from  - 83 the  p l a t e l e t s and their release can be expected to have l i t t l e e f f e c t on the  mobility.  The t o t a l number of s i a l i c acid molecules liberated by the enzyme  from the p l a t e l e t membrane into the supernatant can be assayed chemically. The e f f e c t i v e number of s i a l i c acid molecules at the plane of shear, determined using equation [3], can be expressed as a f r a c t i o n of the t o t a l p l a t e l e t surface s i a l i c acid removable by neuraminidase.  This f r a c t i o n i s approximately  46% for human red blood c e l l s for example (Cook et al., 1961).  It w i l l be  shown here that this r a t i o can be very h e l p f u l i n the determination of the echinocyte surface area. The enzyme alkaline phosphatase has also been used to remove negatively charged phosphate groups from the surface of p l a t e l e t s (Mehrishi, 1979 and Stoltz e_t al_., 1975).  Decreases i n mobility of 15 to 30% were reported.  Similarly, the number of phosphate groups eliminated at the plane of shear can be calculated based on equation [3] and expressed as a f r a c t i o n of the t o t a l amount of phosphate liberated from the p l a t e l e t surface by the enzyme. Alkaline phosphatase has a s p e c i f i c i t y for monester orthophosphates,  C-O-P  (Fernley, 1971). Fixation of p l a t e l e t s with acetaldehyde has been reported to bring about a 20% increase i n negative mobility (Seaman and Vassar, 1966).  Fixation of red  blood c e l l s with glutaraldehyde also results i n an increase i n RBC mobility (Vassar e_t a_l., 1972).  The aldehydes block the p o s i t i v e l y charged amino  groups eliminating their positive charge (Jentoft and Dearborn, 1979). result net negative charge increases.  The number of negative charges  increased, calculated from the r i s e i n EPM, positive amino groups neutralized.  gives the apparent number of  As a  - 84 -  The electrophoretic m o b i l i t i e s of p l a t e l e t discocytes compared to those of the p l a t e l e t spheres as well as the electrokinetic properties of the d i f f e r e n t forms of p l a t e l e t s modified with neuraminidase, alkaline phosphatase and aldehydes are discussed i n this chapter. Electrophoretic mobility measurements have also been used to estimate the number of calcium ion binding sites on c e l l s (Seaman et a l . , 1969).  If red  c e l l s are suspended i n solutions of constant ionic strength containing d i f f e r e n t concentrations of calcium ions, their m o b i l i t i e s w i l l drop with increasing concentrations of calcium. the decrease  By p l o t t i n g the relationship between  i n apparent charge density ( A c ) and calcium concentration  according to the equation:  L_ _J  J  +  Ao- ~ 2en  where  +  1 2enK ' tCo*]-exp(2eX/kT)  '  K = exp(AG/kT)/55.6 2 n = number of binding sites per cm AG = chemical free energy of adsorption [ C a ] = calcium concentration ++  t  =  zeta potential of p l a t e l e t at that calcium concentration  the number of calcium ion binding s i t e s as well as the chemical free energy of binding can be evaluated.  If the binding obeys the above model, a plot of  I/ACT versus the reciprocal of the concentration-zeta potential function w i l l be a straight l i n e .  The intercept with the ordinate w i l l give l/(2en) and  slope w i l l represent l/(2enK).  the  This operation can also be used to find the  number of magnesium ion binding s i t e s .  Attempts had been made previously to  determine the number of calcium ion binding sites on p l a t e l e t surfaces by 45 ++ measuring the amount of Ca adsorbed after an equilibrium incubation  - 85 (Peerschke et a l . , 1980; 1982).  Taylor and H e p t i n s t a l l , 1980;  Brass and  Shattil,  However this technique suffers from the serious drawback that p l a t e l e t s  also have an active process of calcium ion i n t e r n a l i z a t i o n or uptake and i t becomes d i f f i c u l t to d i s t i n g u i s h between surface associated and i n t e r n a l i z e d ^~*Ca  ++  (Peerschke et a l . , 1980).  isotope for this kind of study.  Moreover, there i s no suitable magnesium The microelectrophoresis technique described  here therefore offers a unique opportunity to investigate calcium and magnesium ion binding to p l a t e l e t surfaces.  - 86  -  3.2 MATERIALS AND METHODS 3.2.1 Mi cro ele ctro phor esi s The electrophorecti c mobil i t i e s of platelets were measured i n a c y l i n d r i c a l chamber e s s e n t i a l l y as described by Seaman and Heard (1961).  The chamber was  immersed i n a water bath at 25°C and measurements were made at 40V. were of s i l v e r / s i l v e r chloride.  Electrodes  M o b i l i t i e s of between 10 to 20 p l a t e l e t s from  each sample were usually measured with t y p i c a l standard deviations of -4 +0.07x10  2 - 1 - 1 cm .sec  .V  . Unless indicated a l l  in pH 7.4 i s o t o n i c Tyrode's  the samples were measured  solution except f o r the non-fixed spherocytes which  were measured i n the hypotonic Tyrode's described e a r l i e r samples were usually examined under phase microscopy  (pH 7.4).  Platelet  before electrophoresis.  The m o b i l i t i e s of fresh human red blood c e l l s were measured as a control each day to ensure proper working conditions f o r the apparatus. RBC  at pH 7.4 i n 0.15MNaCl i s -1.08x1 O^cm .sec" .V -4 2 -1-1 2  deviation of +0.05x10  cm .sec  .V  1  _1  The EPM  of the human  with a standard  . Measurements from the d a i l y  controls f e l l within one standard deviation of the quoted mean. The pH-mobility profiles were constructed from measurements of platelets suspended i n Tyrode's solutions having different pH values. Adjustments of the pH's were made with HC1 or NaOH shortly before the platelets were introduced into the electrophoresis chamber. ++ The Ca  ++ and Mg  ion binding experiments  were done with platelets  suspended i n Tyrode's solutions containing different concentrations of c a l c i um or magnesium chloride. maintained.  However constant i o n i c strength and osmolality had to be  With increasing C a  of the solution increases. solution has to decrease.  + +  or M g  ++  concentration, the i o n i c strength  Therefore the amount of sodium chloride In the At the same time, osmolality decreases  as a r e s u l t  - 87 -  because the ionic strength, I, varies as the square of ionic valence so to maintain I constant as Ca necessity drops. compensate.  concentration i s increased the t o n i c i t y of  The addition of an appropriate amount of glucose i s used to  The resulting changes i n v i s c o s i t y have to be accounted for when  calculating surface charge (Seaman et a l . , 1969). by a Cannon viscometer.  V i s c o s i t i e s were measured  Spherocytes were suspended i n a solution with 1/4  i s o t o n i c i t y f o r a l l measurements. 3.2.2  Fixation of P l a t e l e t s  Fixation of p l a t e l e t s with glutaraldehyde was the same as i n Chapter 2. Formaldehyde  f i x a t i o n of p l a t e l e t s was done with 3.7% formaldehyde  concentration) overnight at 4°C.  (final  Some samples of the formaldehyde fixed  p l a t e l e t s were further treated with 5 mM temperature (Jentoft and Dearborn, 1979).  sodium borohydride for 30 min at room They were then washed and resus-  pended i n Tyrode's solution.  3.2.3  P l a t e l e t Surface S i a l i c  Acid  Surface s i a l i c acid from glutaraldehyde fixed p l a t e l e t s was removed with neuraminidase (Vibrio cholerae).  Digestion time was 90 min at 37°C.  incubation period was determined by a time-release study. t r a t i o n was 2.5x10 /ml and neuraminidase 0.04  IU/ml.  This  P l a t e l e t concen-  For optimum conditions  (Ada et a_l., 1961) the pH of the Tyrode's solution was lowered to 6.5 and 0.1 mM of Ca  was added.  After digestion the p l a t e l e t s were spun down.  The  supernatants were used for the chemical assays of released s i a l i c acid and the p l a t e l e t p e l l e t s were resuspended i n pH 7.4 Tyrode's solution for microelectrophoresis.  - 88  -  The charge density of control and enzyme treated p l a t e l e t s can be calculated from equation [3],  The number of s i a l i c acid molecules removed per unit  area at the plane of shear can be calculated by d i v i d i n g the drop in charge density with the electron charge unit (e). The procedure of Culling e_t a_l. (1977) was used to monitor the amount of s i a l i c acid released  into the supernatant.  Sialic  acid was f i r s t  oxidized  with sodium metaperiodate and then estimated c o l o r i m e t r i c a l l y with thiob a r b i t u r i c acid reagent.  N-acetyl-neuraminic acid was used as standard.  To find the t o t a l s i a l i c acid content, fixed whole p l a t e l e t s were hydrolysed with 0.1N  sulphuric  acid at 80°C for 1 hour.  Sialic  this way was assayed by the Warren (1959) procedure.  acid  released  To test for 0-acetyl  substitution at the C-4 position of s i a l i c acid, fixed p l a t e l e t s were saponified with 0.1N potassium hydroxide at room temperature (Reid et a l . , 1975) before neuraminidase digest.  Neuraminidase i s not able to attack s i a l i c  acid residues bearing such a substitution, therefore more s i a l i c acid w i l l be released  3.2.4  after saponification i f C-4 i s acetylated.  P l a t e l e t Surface Phosphate Groups  Alkaline phosphatase (calf intestine) was used to liberate terminal phosphate groups from the surface of glutaraldehyde fixed p l a t e l e t s . was at 37°C for 45 min using 0.1 mg/ml of the enzyme.  Incubation  Due to the presence  of phosphate i n Tyrode's solution, p l a t e l e t s were resuspended i n pH 10 (optimum pH) Tris-buffered  saline before enzyme treatment.  After  digestion  the p l a t e l e t s were spun down, the supernatant was used for chemical assay of phosphate and the p l a t e l e t p e l l e t was resuspended i n pH 7.4 Tyrode's solution for microelectrophoresis  - 89 The method of Chen jet _al. (1956) was used to assay phosphate ions liberated by the enzyme into the supernatant.  Phosphate forms a complex with  ammonium molybdate and color was developed by reduction of the complex with as corbie a c i d .  3.2.5  S t a t i s t i c a l Methods Linear regressioin and other s t a t i s t i c a l comparisons were according t o  K a l b f l e i s c h (1974).  3. 2. 6  Further discussion can be found i n the appendix.  Materials  Neuraminidase was from Calbiochem (La J o l l a , C a l i f o r n i a ) and c a l f intestine alkaline phosphatase from Boehringer-Mannheim  (Dorval, Quebec).  Both  enzymes were tested for the presence of proteolytic a c t i v i t y with azocoll (Rinderknecht et a l . , 1968).  The enzymes were incubated with 3 mg/ml of  azocoll (Calbiochem) overnight at 37°C under the same conditions, such as pH, medium and concentration, as used i n the platelet experiments. used as standard.  Trypsin was  No proteolytic a c t i v i t y was detected i n either enzyme.  N-acetyl neuraminic a c i d and trypsin were from Sigma (St. Louis, Missouri).  A l l other reagents were from Fisher (FairLawn, N. J.) .  - 90 -  3.3  RESULTS  3.3.1  P l a t e l e t Electrophoretic M o b i l i t i e s  Electrophoretic m o b i l i t i e s of the d i f f e r e n t forms of p l a t e l e t s are shown i n Table 2.  Table 3 shows the mobility values converted into number of charges  per p l a t e l e t and per unit area. induced by 2x10 "*M of ADP  Shape change from discocyte to echinocyte  resulted in a 13% drop in mean mobility and mean  e l e c t r o k i n e t i c charge density.  Since the surface area of the echinocyte i s  not known at this point, one cannot calculate the actual number of charges echinocyte from the data.  We were not able to detect any change i n the  mobility of p l a t e l e t s stimulated with 1x10 ^M ADP.  The spherocyte had the  lowest charge density although the mobility for the non-fixed spherocyte r e l a t i v e l y high.  per  was  This i s because non-fixed spherocytes were measured i n low  ionic strength hypotonic Tyrode's solution.  Adding ADP  to spherocytes did not  change the mobility. Formaldehyde f i x a t i o n raised platelet m o b i l i t i e s by about 17%.  Table 4  shows how  these changes are translated into increases i n net negative charge  density.  The apparent number of amino groups on the p l a t e l e t surface can then  be calculated. The surface densities of amino groups on the echinocyte and spherocyte were both lower than on the discocyte.  Because of the increase i n  surface area the number of amino groups per spherocyte actually increased. Not knowing the surface area, the number of amino groups per echinocyte cannot be calculated. The m o b i l i t i e s of fixed platelets further treated with borohydride were the same as the untreated ones.  Therefore there was no evidence  for reversal of the f i x a t i o n process. The pH-mobility curves for fixed and non-fixed p l a t e l e t s are shown i n F i g . 18 and F i g . 19 respectively.  The plateau values for glutaraldehyde fixed  T a b l e 2.  The E l e c t r o p h o r e t i c  Mobilities  Non-fixed Platelets  Discocyte  Echinocyte  Spherocyte  -1.08 (+0.016)  sphere  i n xlcAcm .sec .V 2  Glutaraldehyde Fixed P l a t e l e t s  - 1  -1.26 (+0.012)  -0.93 (+0.035)  -1.21 (+0.008)  -1.10 (+0.007)  -1.55*  -1.00 (+0.010)  -0.90 (+0.007)  +  - 1  Formaldehyde Fixed P l a t e l e t s  -1.41 (+0.020)  (+0.016) Cocaine  of P l a t e l e t s  -1.40 (+0.017)  *Non-fixed spherocytes measured i n h y p o t o n i c Tyrode's measured i n r e g u l a r i s o t o n i c Tyrode's.  solution;  a l l others  "•"Values i n b r a c k e t s r e p r e s e n t standard e r r o r o f the mean d e r i v e d from m o b i l i t y d e t e r m i n a t i o n s o f a t l e a s t 10 d i f f e r e n t p l a t e l e t samples i n each category.  - 92 -  Table 3.  Apparent P l a t e l e t Surface Charge  Discocyte Electrophoretic Mobility (xl0 cm .sec- .V- ) 4  5  1  Spherocyte  Echinocyte  -1.552 (+0.016)  -0.933 (+0.035)  -1.082 (+0.016)  Cocaine sphere -1.405 (+0.017)  1  Negative Charge Density (esu/cm )  3.74xl0 (+0.056xl0 )  3.23xl0 (+0.121xl0 )  2.60xl0 (+0.026xl0 )  4.87xl0 (+0.058xl0 )  Number of Charge Groups per cm  7.80xl0 (+0.12xl0 )  6.73xl0 (+0.25xl0 )  5.43xl0 (+0.05xl0 )  10.14xl0 (+0.12xl0 )  2  2  Surface Area (cm )  3  3  12  12  16.4xl0"  3  3  12  12  -  8  3  3  12  12  36.7xl0  3  3  12  12  26.4xl0  -8  -8  2  Number of Charge Groups per Platelet  1.28xl0 (+0.019xl0 ) 6  1.99xl0 (+0.020xl0 ) 6  6  -  6  2.67xl0 (+0.032xl0 ) 6  6  Table 4.  P l a t e l e t Surface Amino Groups Calculated from Charge Density Increases After Formaldehyde Fixation  Discocyte  Echinocyte  Spherocyte  Charge density of non-fixed p l a t e l e t s (esu/cm )  3.74xl0 (+0.056xl0 )  3.23xl0 (+0.121xl0 )  2.60xl0 (+0.026xl0 )  Charge density of formaldehyde-fixed p l a t e l e t s (esu/cm )  4.38xl0 (+0.041xl0 )  3.80xl0 (+0.024xl0 )  3.12xl0 (+0.023xl0 )  Increase i n charge density (esu/cm )  6.40xl0 (+0.526xl0 )*  5.70xl0 (+0.271xl0 )*  5.20xl0 (+0.226xl0 )*  1.33xl0 (+0.109xl0 )  1.19xl0 (+0.047xl0 )  1.08xl0 (+0.056xl0 )  3  3  3  3  3  3  2  3  3  3  3  3  3  2  2  2  2  9  12  12  Ammo groups per cur-  Surface area  2  2  12  12  16.4xl0  (cm ) 2  -  -8  -  2.18xl0 (+0.179xl0 ) 5  Amino groups per p l a t e l e t  2  2  12  12  36.7xl0-  3.97xl0 (+0.172xl0 )  *Combined standard deviations from fixed and non-fixed p l a t e l e t charge densities using the formula (Kalbfleisch, 1974): -s  2 +  V  1  5  5  5  S=(  8  .c2VS  where S i s the combined standard deviation, s and s^ are standard deviations of the two individual components, and n and nj, are number of samples from the two individual components. a  a  - 94  Fig.  18.  platelet (-X-).  The  -  p H - e l e c t r o p h o r e t i c m o b i l i t y p r o f i l e s of  d i s c o c y t e s (-•-), e c h i n o c y t e s Solid  and  l i n e s represent glutaraldehyde fixed  spherocytes platelets  and dash l i n e s r e p r e s e n t formaldehyde f i x e d p l a t e l e t s . are from means of f o u r s e r i e s of experiments. r e p r e s e n t one  standard d e v i a t i o n .  fixed  Values  E r r o r bars  discocyte 4I echinocyte  1  spherocyte  - 96  Fig. 19.  -  The pH-electrophoretic mobility p r o f i l e s of fresh  p l a t e l e t discocytes (-•-), echinocytes (-A-) (-T-).  Values are means from three series of  E r r o r bars represent one standard deviation.  and  spherocytes  experiments.  1 1  J  ^spherocyte  -discocyte -ir echinocyte  i  7  i  8  - 98 p l a t e l e t s are about 10% higher than those of the formaldehyde fixed ones ( F i g . 18 and Table 2).  At low pH's two problems were encountered.  was p l a t e l e t agglutination which occurred at pH 3 or below. was i r r e v e r s i b l e changes i n the mobility at low pH.  The f i r s t problem The second problem  For example, when the pH  of the discocyte suspension was brought down to 4.0 from 7.4 the mobility went _4  from -1.08  to -0.75x10  2 -1 -1 cm .sec .V .  Upon returning to pH 7.4, the disco_4 2 -1-1  cyte mobility went back only to -0.98x10  cm .sec  .V  .  Therefore mobilities  below pH 4.5 were not explored further. 3.3.2  P l a t e l e t Surface S i a l i c Acid  The neuraminidase digest-time curve i s shown in F i g . 20.  After 30 min.  most of the neuraminidase susceptible s i a l i c acid was released from the glutaraldehyde fixed discocytes.  An arbitrary time of 90 min (3x30 min) was  therefore used for further enzyme digestions. The amount of s i a l i c acid liberated from the p l a t e l e t surfaces was determined i n two ways:  f i r s t , by chemically assaying the p l a t e l e t suspending  media and second, by c a l c u l a t i o n from the drop i n p l a t e l e t electrophoretic mobility.  The chemical analysis data i s shown i n Table 5.  No major  differences were found i n t o t a l s i a l i c acid content between the discocyte, echinocyte and spherocyte, t o t a l s i a l i c acid being s i a l i c acid liberated by acid hydrolysis.  It represents s i a l i c acid from within as well as on the  surface of the p l a t e l e t s .  Neuraminidase, on the other hand, removed more  s i a l i c acid (17-18%) from the echinocyte and spherocyte than from the discocyte surface (Table 5).  Neuraminidase digestion of saponified p l a t e l e t s  f a i l e d to release any more s i a l i c acid than from the non-saponified ones. 0-acetyl substitution was therefore evident.  No  - 99 -  F i g . 20.  The time-release curve for the digestion of s i a l i c  acid from the surface of fixed platelet discocytes by g neuraminidase.  2.5x10 /ml of p l a t e l e t s were incubated with  0.04 IU/ml of enzyme. of experiments.  Values are means from three series  E r r o r bars represent one standard deviation.  - OOL -  -  Table 5.  101  -  P l a t e l e t Surface S i a l i c Acid Determined Chemically  Discocyte  Echinocyte  Spherocyte  205 (+6.0)  203 (+3.8)  204 (+4.9)  4.00xl0 (+0.117xl0 )  3.96xl0 (+0.074xl0 )  3.98xl0 (+0.095xl0 )  116 (+1.9)  137 (+3.3)  136 (+3.2)  2.26xl0 (+0.037xl0 )  2.67xl0 (+0.064xl0 )  2.65xl0 (+0.062xl0 )  56.6%  67.5%  66.7%  Total s i a l i c acid released by acid hydrolysis: (n=10) jLAg/10  1 0  platelet  molecules/platelet  7  7  7  7  7  7  Neuraminidase removable s i a l i c acid: (n=18) jxg/10  10  platelet  molecules/platelet  7  7  Percent of t o t a l s i a l i c acid removed by neuraminidase  7  7  7  7  - 102 -  Table 6 shows how neuraminidase  the decrease in p l a t e l e t electrophoretic mobility after  treatment translates into molecules of s i a l i c acid removed.  By  comparing the electrophoresis data to the chemical analysis data (Table 7), i t i s obvious that only 2.9% of the neuraminidase  susceptible s i a l i c acid on the  surfaces of the discocyte and spherocyte contributed to the p l a t e l e t e l e c t r o kinetics.  An opportunity i s therefore presented to estimate the surface area  of the echinocyte.  Assuming the 2.9% r a t i o to be true for the echinocyte as  well, then the number of s i a l i c acid molecules removed from the plane of shear per echinocyte can be calculated ( l i n e (a) i n Table 7). 7.74x10 cm  2  (2.67x10  x 0.029).  This i s found to be  Since the number of molecules removed per  . 12 at the plane of shear i s known (3.52x10 ), the echinocyte surface  area i s calculated to be 22.0xl0~ cm 8  3.3.3  2  (7.74xl0 ± 3 . 5 x l 0 ) . 5  12  P l a t e l e t Surface Phosphate Groups  The time-digest curve for alkaline phosphatase i s shown i n F i g . 21.  It  seems only 15 min i s required to liberate the phosphate groups from the fixed discocytes.  Therefore the phosphate experiments  (3x15 min) of incubation with the enzyme.  were carried out with 45 min  The amounts of phosphate terminal  groups released by alkaline phosphatase as determined  chemically and e l e c t r o -  p h o r e t i c a l l y are shown i n Table 8 and Table 9 respectively. phosphate groups at the plane of shear as determined about 3.9% of the t o t a l amount determined  The percentage of  electrophoretically i s  chemically (Table 10).  Again i f one  takes the percentage also to be true for the echinocyte, then i t s area can be back calculated as before.  —8 2 This calculation gave 25.3x10 cm , only  s l i g h t l y higher than the area found from the s i a l i c acid calculations. —8 2 average of the two (23.7x10 cm ) i s therefore taken.  An  - 103 -  Table 6.  P l a t e l e t Surface S i a l i c Acid Determined from the Decrease i n Electrophoretic Mobility after Neuraminidase Treatment  Discocyte  Echinocyte  Spherocyte  EPM:glutaraldehyde fixed p l a t e l e t (xi0 cm .sec" .V~ )  -1.41 (+0.020)  -1.21 (+0.008)  -1.00 (+0.100)  EPM:neuraminidase treated fixed p l a t e l e t (xl0 cm .sec .V )  -0.86 (+0.010)  -0.72 (+0.010)  -0.70 (+0.007)  EPM decrease (xl0 cm .sec .V )  0.55 (+0.014)*  0.49 (+0.005)*  0.29 (+0.004)*  4  4  4  2  1  2  2  1  _1  -1  _1  _1  Decrease i n charge density (esu/cm )  1.91xl0 (+0.047xl0 )  1.69xl0 (+0.016xl0 )  1.02xl0 (+0.015xl0 )  S i a l i c acid molecules removed per cm  3.98xl0 (+0.098xl0 )  3.52xl0 (+0.033xl0 )  2.12xl0 (+0.031xl0 )  S i a l i c acid molecules removed per p l a t e l e t  6.53xl0 (+0.161xl0 )  -  7.80xl0 (+0.114xl0 )  2  i  Electrophoretic experiments.  3  3  12  12  3  3  5  5  12  12  3  3  12  12  mobility (EPM) values are means from 10 series of paired  *Combined standard deviation (same as i n Table 4)  5  5  - 104 -  Table 7.  Ratio of P l a t e l e t Surface S i a l i c Acid Removed by Neuraminidase as Determined by Microelectrophoresis to That Determined Chemically  Discocyte 16.4xl0  -8  S i a l i c acid molecules removed per cm  3.98xl0  12  S i a l i c acid molecules removed per p l a t e l e t (a)  6.53xl0  Surface area (cm ) 2  Echinocyte  Spherocyte 36.7xl0  Electrophoresis data: 3.52xl0  12  2.12xl0  2  5  —  7.80xl0  5  2.65xl0  7  Chemistry data: S i a l i c acid molecules removed per p l a t e l e t (b) Ratio (a)/(b)  2.26xl0  0.0288  7  2.67xl0  -  7  0.0294  12  -8  - 105 -  Fig. 21.  The time-release curve for the digestion of phos-  phate groups from the surface of fixed p l a t e l e t discocytes g  by alkaline phosphatase.  2.5x10 /ml of p l a t e l e t s were  incubated with 0.01 mg/ml of the enzyme. from three series of experiments. standard deviation.  Values are means  Error bars represent one  - 106 -  £]3Pld  Jed  p9SD6]ey  sdncug  epqdsoiy  - 107 Table 8.  P l a t e l e t Surface Phosphate Groups Released After Alkaline Phosphatase Treatment Determined Chemically  Discocyte Phosphate groups/ platelet n=5  9.44xl0 (+0.199xl0 ) 6  6  Echinocyte  Spherocyte  12.50xl0 (+0.184xl0 ) 6  6  18.51xl0 (+0.130xl0 ) 6  6  - 108 Table 9.  P l a t e l e t Surface Phosphate Groups Determined from the Decrease i n Electrophoretic Mobility after Alkaline Phosphatase Treatment  Discocyte  Echinocyte  Spherocyte  EPM:glutaraldehyde fixed p l a t e l e t (xl0 cm .sec- .V )  -1.41 (+0.020)  -1.21 (+0.008)  -1.00 (+0.010)  EPM:alk. phosphatase treated fixed p l a t e l e t (xl0 cm .sec .V )  -1.10 (+0.005)  -0.94 (+0.008)  -0.73 (+0.009)  EPM decrease (xl0 cm .sec .V" )  0.31 (+0.015)*  0.27 (+0.008)*  0.27 (+0.009)*  4  4  4  2  2  2  1  -1  _1  -1  -1  1  Decrease i n charge density (esu/cm )  1.09xl0 (+0.052xl0 )  0.94xl0 (+0.027xl0 )  0.94xl0 (+0.032xl0 )  Phosphate groups removed per cm  2.28xl0 (+0.108xl0 )  1.96xl0 (+0.056xl0 )  1.96xl0 (+0.067xl0 )  Phosphate groups removed per p l a t e l e t  3.74xl0 (+0.177xl0 )  2  2  3  3  12  12  3  3  5  5  12  12  -  3  3  12  12  7.19xl0 (+0.245xl0 )  Electrophoretic mobility (EPM) values are means from 10 series of paired experiments. *Combined standard deviation (same as i n Table 4)  5  5  - 109 Table 10.  Ratio of P l a t e l e t Surface Phosphate Groups Removed by Alkaline Phosphatase as Determined by Microelectrophoresis to Those Determined Chemically  Discocyte 16.4xl0  -8  Phosphate groups removed per cm  2.28xl0  12  Phosphate groups removed per p l a t e l e t (a)  3.74xl0  5  9.44xl0  6  Surface area (cm ) 2  Echinocyte  Spherocyte 36.7xl0  Electrophoresis data: 1.96xl0  12  1.06xl0  12  2  -  7.19xl0  5  Chemistry data: Phosphate groups removed per p l a t e l e t (b) Ratio (a)/(b)  0.0396  12.50xl0  6  18.51xl0  0.0389  6  -8  - 110 3.3.4  Cocaine Spheres  Non-fixed cocaine spheres were treated with neuraminidase and alkaline phosphatase. determined  The amounts of s i a l i c acid and phosphate groups liberated as  chemically from the supernatant and determined  from the reductions  in electrophoretic mobility after enzyme treatment are shown i n Table 11. Since the e l e c t r o k i n e t i c properties of the cocaine sphere are much d i f f e r e n t from the other forms of p l a t e l e t s (Table 3), no attempt was made to compare the fixed cocaine spheres to the other forms of fixed p l a t e l e t .  3.3.5  Calcium and Magnesium Ion Binding  F i g . 22 shows how the electrophoretic mobility of p l a t e l e t s decreases with increasing divalent ion concentration.  F i g . 23 i s a plot of the double  reciprocal relationship between the drop i n negative charge density and divalent cation concentration according to the formula given i n the Introduction.  The number of binding sites calculated from the intercepts i n  Fig. 23 are shown i n Table 12 for calcium ions and Table 13 for magnesium ions.  There are twelve times more Ca  and eight times more Mg  sites on the echinocyte than on the discocyte.  binding  On the other hand, the amount  ++ of Ca binding to the spherocyte i s 61% lower than to the discocyte. ++  S t a t i s t i c a l comparisons of the binding density and free energy of Ca Mg  ++  adsorption to the discocyte, echinocyte and spherocyte are summarized  in Table 14. ++ Ca  and  It i s evident that the binding densities and free energies of  ++ and Mg  to the echinocyte are s i g n i f i c a n t l y d i f f e r e n t from those to  the discocyte.  ++  The binding of Ca  and of Mg  ++  .  .  to the discocyte i s not  s i g n i f i c a n t l y d i f f e r e n t and the same applies for the echinocyte.  - Ill T a b l e 1 1 . S i a l i c A c i d and Phosphate Groups R e l e a s e d by Enzymes from C o c a i n e Sphere* D e t e r m i n e d E l e c t r o p h o r e t i c a l l y and C h e m i c a l l y  Electrophoresis  Akl a l i n e Phos phat as e treatment  -1.40 (+0.017)  -0.67 (+0.012)  -1.00 (+0.018)  data:  EPM ( 1 0 c m . s e c . V - ) n=8 4  Control  Neuraminidase treatment  2  _ 1  1  Char ge dens i t y (esu/ cm )  4. 87x10 (+0. 0 5 8 x 1 c )  2. 34x10 (+0 . 041x10 )  3.48x10 (+0.062x10 )  Number of charges per c m  10.14x10 (+0.121xl0 )  4.88x10 (+0 . 0 8 5 x 1 0 )  7. 2 5 x 1 0 (+0.129xl0 )  Number of charges per p i at e l et  2.68x10 (+0.032x10 )  1.29x10 (+0.022x10 )  1.91x10 (+0.03xl0 )  1.39x106  0. 7 6 x 1 0  3  2  3  1 2  2  12  6  6  Drop i n number of charges per p l a t e l e t  3  3  1 2  12  6  6  (a)  Chemistry data: S i a l i c a c i d molecules r e l e a s e d per p l a t e l e t (b) (n=4)  (a)/(b)  - 8  2  1 2  12  6  6  6  1.27x107 (+0 . 02 3 x 1 0 ) 7  0. 0515  *Area=2 6. 4 x 1 0 c m  3  2. 69x107 (+0. 031x107)  Phosphate groups r e l e a s e d per p l a t e l e t (b) (n=4) Ratio  3  0.0601  - 112 -  F i g . 22.  Influence of calcium and magnesium ion concentration  on the electrophoretic mobility of the p l a t e l e t discocyte, echinocyte, and spherocyte.  Each data point represent a mean  of 100 mobility determinations. standard deviation.  E r r o r bar represent  one  -  113  i  -1-6-  -1-5-  spherocyte  -1-1-10CO  Z>  e  -0-9-  discocyte  -0-8-. -0 7 echinocyte -0-6-  ov> • X  i  i  I  I  I  J -1-1-  z: Q_  l±J  -10-0-9-  discocyte  -0-8-07^eclinccyte  -0-6i  0  l  l  2  4  I  I  6  8  I  10  Cation Cone.  i  i  i  12  14  16  (mM)  i  i  18 20  - 114 -  Fig. 23.  The double reciprocal plots to find the densities  of calcium and magnesium ion binding sites on the discocyte and echinocyte.  E r r o r bars represent one standard deviation,  [cat. ] c o n c e n t r a t i o n of either C a  + +  or Mg  ++  - 116 -  F i g . 23 (continued).  The double reciprocal plot to find the  density of calcium ion binding sites on the E r r o r bars represent one standard deviation, [cat. ] c o n c e n t r a t i o n of Ca  spherocyte.  - 117 -  - 118 -  Table 12.  Calcium Ion Binding Sites  Discocyte  Echinocyte  Spherocyte  Intercept on ordinate (esu/cm ) !  8.15xl0 (+6.8xl0 )  9.50xl0~ (+2.07xl0 )  4.69xl0 (+1.5xl0~ )  Slope  4.54xl0 (+8.45xl0 )  4.03xl0~ (+2.24xl0 )  2.43xl0 (+4.06xl0 )  0.98  0.95  2  -  -4  _4  -5  -6  Correlation c o e f f i c i e n t  5  -4  5  -6  0.88  -3  3  -4  -5  Binding s i t e s : Density  (cm ) -2  1.28xl0 (+1.07xl0 )  1.09xl0 (+2.39xl0 )  (+7.17xl0 )  2.10xl0  2.58xl0 *  8.16xl0  2.89 (+0.03)  4.13 (+0.10)  12  12  Per p l a t e l e t -AG (Kcal/mole)  4.09 (+0.11)  5  13  13  6  *Calculated using the surface area of 23.7x10 "cm .  2.22X10  11  10  4  - 119 Table 13.  Magnesium Ion Binding Sites  Discocyte  Echinocyte  Intercept on ordinate (esu/cm ) !  9.66xl0 (+3.49xl0 )  1.58xl0~ (+2.55xl0 )  Slope  4.03xl0" (+5.32xl0 )  3.57xl0 (+3.38xl0 )  2  -  -4  -4  5  -6  Correlation c o e f f i c i e n t  0.97  4  -4  -5  -6  0.98  Binding s i t e s : Density  (cm ) -2  1.08xl0 (+3.89xl0 )  6.60xl0 (+1.07xl0 )  1.77xl0  1.56xl0 *  12  n  Per p l a t e l e t -AG (Kcal/mole)  5  4.26 (+0.08)  *Calculated using the surface area of 23.7x10 °cm%  12  13  6  3.26 (+0.06)  - 120 -  Table 14.  S t a t i s t i c a l Ccmparison of C a and M g Binding t o the Discocyte, Echinocyte and Spherocyte (Sig. = significant differences; not=no s i g n i f i c a n t difference) + +  ++  Binding s i t e density  Free energy ( G)  C a , discocyte vs echinocyte  s i g . , 0.025 p 0.05  sig-,  C a , discocyte vs spherocyte  s i g . , p 0.005  s i g . , 0.01 p 0.02 5  C a , echinocyte vs spherocyte  s i g . , p 0.005  sig-,  Mg , discocyte vs echinocyte  s i g . , 0. 005 p 0. 01  sig-, p 0.0005  discocyte, C a  not, 0.50 p 0.75  not., 0. 50 p 0. 75  not, 0.50 p 0.75  not., 0.50 p 0.75  ++  + +  ++  ++  + +  echinocyte, C a  + +  vs M g  ++  vs M g  ++  0.0005 p 0.001  0.005 p 0.01  S t a t i s t i c a l analysis using Analysis of Variance Tables and F-dis t r i but i o n according to K a l b f l e i s c h (1974). See also the appendix.  - 121 3.4  DISCUSSION  3.4.1  P l a t e l e t Electrophoretic M o b i l i t i e s  The electrophoretic mobility of the human discocyte i n Tyrode's solution -4 was found to be -1.08x10  2 - 1 - 1 cm .sec  .V  (non-fixed).  to the mobility of the fresh human RBC.  A review of the l i t e r a t u r e shows a  variety of values for human p l a t e l e t mobility. Zarrabi, 1981), -0.71 1980), -1.07  (Boisseau  This i s similar  They are -0.635 (Coller and  et a l . , 1971), -0.806 (Kosztolanyi et a l . ,  (Hampton and M i t c h e l l , 1974) and -1.17xl0~ cm .sec .V 4  2  _1  -1  (Seaman and Vassar, 1966) for p l a t e l e t s measured i n p l a t e l e t - r i c h plasma and -0.84  (Mehrishi,  1970), -0.85  a l . , 1980), -1.08 (Ross and Ebert, 1959), -1.4  (Seaman and Vassar, 1966), -0.86  (Bray and Alexander, 1969), -1.09 1959), -1.14  (Bosmann, 1972),  (Madoff et a l . , 1964), -1.25 2  -1.1  (Kirschmann et a l . ,  (Yamazaki et a l . , 1980) and -1.58xl0~ cm . s e c . V 4  (Kosztolanyi et  _ 1  _ 1  (Shimizu e_t ajL., 1979) for washed p l a t e l e t s measured i n simple buffered s a l i n e _4 2 -1-1 type media.  Our result of -1.08x10  cm .sec  .V  for washed p l a t e l e t s  is similar to the ones obtained by Bray and Alexander and Bosmann.  The great  range of mobility values probably r e f l e c t s differences in the electrophoretic systems involved as well as the conditions of the p l a t e l e t samples (this point i s to be discussed  further below).  -4 2 -1 -1 An electrophoretic mobility of -1.08x10 cm .sec .V translates 3 2 6 into a charge density of 3.74x10 esu/cm or 1.28x10 net negative charge groups per discocyte (Table 3).  The number of net negative charge  groups i s the actual ( t o t a l ) number of negative charge groups minus the positive charge groups. After stimulation by 2x10 ~*M ADP, there was a 13.7% drop in mobility -4 2 - 1 - 1 from -1.08 to -0.93x10 cm .sec .V . The net negative charge density  - 122 3 3 2 correspondingly from 3.74x10 to 3.23x10 esu/cm (Table 3).  decreased  As discussed e a r l i e r , Seaman and Vassar  (1966) reported an 18% drop i n human  p l a t e l e t electrophoretic mobility after addition of lyo-g/ml (2.3x10 ^M) of ADP.  The decrease was concentration dependent and the reduction was up to 40% -4  at 100^jug/ml (2.3x10  M) of ADP.  However, they f a i l e d to confirm the  biphasic mobility behaviour reported by (1970) and Kosztolanyi et a l . (1980).  Hampton and M i t c h e l l (1966), Stoltz These workers reported that at low ADP  concentrations, the p l a t e l e t mobility increased up to a concentration of -2 5x10  -7 jxg/ml  tration.  (1x10  M) after which the mobility decreased  with concen-  However the maximum mobility increase at 1x10 ^M was only about 8%  in a l l three reports.  Confirming  Seaman (1976) we were able to observe only  the decrease i n electrophoretic mobility.  At 1x10 ^M ADP concentration we  were not able to detect any change i n p l a t e l e t mobility.  Takano and Suzuki  (1981) working on rabbit p l a t e l e t s recorded only decreases in mobility with increasing ADP concentration.  F i n a l l y , Boisseau —6  increase i n p l a t e l e t mobility of 10% at 2x10  a l . (1977) reported an  M ADP concentration.  Interestingly, p l a t e l e t electrophoretic m o b i l i t i e s were a l l measured i n p l a t e l e t - r i c h plasma i n the above papers.  This work i s the f i r s t to report a  change i n the mobility of washed p l a t e l e t s induced by ADP.  Seaman and Vassar  (1966) f a i l e d to find any change i n the mobility of washed p l a t e l e t s i n saline after exposure to ADP.  The mobility -0.85  m.sec \ v  ''.cm for their washed  p l a t e l e t s was very low, i n fact close to the mobility of our echinocyte. washed their p l a t e l e t s simply by centrifuging them down i n saline.  They  The d i f f i -  c u l t i e s encountered i n washing p l a t e l e t s have already been discussed i n Chapter 2.  By repeating of the Seaman and Vassar washing procedure by us produced  p l a t e l e t aggregates of various sizes.  Therefore i t i s possible that the above  - 123 -  authors were actually measuring the mobility of small p l a t e l e t aggregates or echinocytes.  This could well be the reason why  not respond to ADP. mentioned above may procedures.  their washed p l a t e l e t s would  The great v a r i e t y of published p l a t e l e t m o b i l i t i e s also in part be caused by d i f f e r e n t p l a t e l e t  P a r a l l e l microscopic monitoring  that done here i s therefore considered  handling  of p l a t e l e t morphology similar to  e s s e n t i a l . Handling and  transformation  (to echinocyte and spherocyte) protocols were set up and standardized with  the  help of electron microscopy and a l l p l a t e l e t samples were examined under phase microscopy before electrophoresis. _4 2 -1 -1 The spherocyte had an electrophoretic mobility of -1.55x10 cm .sec .V 3 measured in hypotonic  Tyrode's solution.  The charge density was 2.60x10  2 esu/cm  (Table 3).  Presumably due to the increase i n surface area, the  number of charge groups per spherocyte increased by The hypotonic  55%.  Tyrode's solution has an osmolality of 75 mOsmol and  ionic strength of 0.0379.  The relationship between mobility and  strength i s an inverse one  (see equation  i n Introduction).  an  ionic  Kirschmann et a l .  (1959) have examined the effect of low ionic strength on the e l e c t r o k i n e t i c properties of human p l a t e l e t s . increased.  As ionic strength was  They found that the change i n mobility was  p l a t e l e t s were suspended i n 0.015  decreased the mobility t o t a l l y reversible.  ionic strength medium and then resuspended  back i n an i s o - i o n i c strength medium, the mobility returned to normal. made similar observations.  ionic strength -4 2 isotonic Tyrode's solution had a mobility of about -2.08x10 cm . sec ^".V ^ and after resuspension i n regular Tyrode's solution the mobility -4 2 -1 -1  was  Discocytes suspended i n a 0.04  We  returned back to -1.08x10  cm .sec  .V  .  No shape change was  observed i n the discocyte due to change i n ionic  strength.  If  - 124 The pH-mobility curves are shown i n F i g . 18 and 19.  It was hoped that  information concerning p l a t e l e t surface ionogenic groups could be obtained by constructing these curves.  However, as a result of the problems  encountered  at low pH already discussed, the curves cannot be extrapolated below pH and values such as pKa for the p l a t e l e t surfaces cannot be deduced.  4.5  The  m o b i l i t i e s of glutaraldehyde fixed p l a t e l e t s were about 10% higher than the m o b i l i t i e s of formaldehyde fixed p l a t e l e t s .  This phenomenon has also been  found i n red blood c e l l s (Vassar et a l . , 1972) 1973).  and lymphocytes (Vassar et a l . ,  The reason for the apparent additional charge remains unclear.  Because of i t s fast action (Vassar et a l . , 1972)  glutaraldehyde remained the  f i x a t i v e of choice for microscopic and enzyme digestion studies.  Since the  10% increase occurred s i m i l a r l y to the discocyte, echinocyte and spherocyte correction i s required for the enzyme digestion studies.  no  Acetaldehyde  f i x a t i o n tends to agglutinate p l a t e l e t s during f i x a t i o n . To study the amino groups on p l a t e l e t surfaces formaldehyde f i x a t i o n chosen.  was  The reaction of formaldehyde with c e l l u l a r components to induce  f i x a t i o n i s complex but presumably formaldehyde interacts with amino groups i n the following way:  R-NH + HC=0 3  2  > '^0H R  *  R  -  N  =  C  H  2  +  H  2 °  The positive charge i s thus eliminated (Jentoft and Dearborn, 1979).  To test  the p o s s i b i l i t y that reversal of the f i x a t i o n process could have occurred the p l a t e l e t s were further treated with borohydride  (Jentoft and Dearborn, 1979):  R-N=CH ^^R-N-CH 2  ;  - 125  -  A stable neutral species i s then obtained.  If there were s i g n i f i c a n t reversal  of the f i x a t i o n process, the borohydride treated fixed p l a t e l e t s ought to have had a greater increase i n mobility than the non-treated ones.  We found no  differences, indicating no reversal of f i x a t i o n had occurred. The value obtained here for the number of amino groups per p l a t e l e t was 2.18x10"* (Table 4) which compares well with those of Stoltz and Nicolas (1979), 3 x l 0 , and Mehrishi (1970), 2.2 to 2.5xl0 . 5  The amino group  5  surface density on the echinocyte i s 10.5% lower and on the spherocyte 18.8% lower than on the discocyte. 3.4.2  P l a t e l e t Surface S i a l i c Acid  By acid hydrolysis one can find the t o t a l s i a l i c acid content i n the platelets.  Mild hydrolysis with l^ESO^ released s i a l i c acid from the surface as  well as from within the fixed p l a t e l e t s . assayed.  The released s i a l i c acid was then  The t o t a l s i a l i c acid content i n the discocyte was found to be 205  yug/10^^ p l a t e l e t s or 3.99x10^ molecules/platelet.  The discocyte, echino-  cyte and spherocyte a l l had similar t o t a l s i a l i c acid contents (Table 5) showing that during the transformations no s i a l i c acid i s l o s t .  Both Motamed  et a l . (1976) and Martin et a l . (1982) found 207 ^ig of s i a l i c acid per p l a t e l e t s by acid hydrolysis.  10  10  Ku and Wu (1977) found 185 and Stoltz and  Nicolas (1979) found about 1 9 7 y u g / 1 0  10  platelets.  Neuraminidase (Vibrio cholerae) removed 56.6% of the t o t a l s i a l i c acid from the d i s c o c y t e .  Higher p e r c e n t a g e s ,  67.5% and 66.7% of t o t a l were removed  from the e c h i n o c y t e and s p h e r o c y t e r e s p e c t i v e l y ( T a b l e 5 ) . neuraminidase  In o t h e r words,  s u s c e p t i b l e s i a l i c a c i d goes from 116 y x g / 1 0 ^ d i s c o c y t e s  1 3 7 y x g / 1 0 ^ e c h i n o c y t e s and 136 jig/10^  spherocytes.  Ku and Wu (1977)  to  - 126  -  found that 48% of the t o t a l p l a t e l e t s i a l i c acid i s released and  after ADP  (5x10  ^M)  et a l . (1976) found 40% stimulated ones.  stimulation  by neuraminidase  the percentage increased to 65%.  for the unstimulated p l a t e l e t s and  Motamed  about 49%  for  ADP  Peerschke et a l . (1978), on the other hand, found no  difference between the amount of s i a l i c acid removed by the enzyme from the surface of unstimulated and ADP  stimulated p l a t e l e t s .  No explanation  was  offered by them for the discrepancy. Neuraminidase (Vibrio cholerae) has a molecular weight of 90,000 (Gottschalk and  Bhargava, 1971;  d i f f u s i o n c o e f f i c i e n t s i t has 1961).  Behring, 1979).  an estimated radius of 32A  (Pye  and  Curtain,  It i s the b e l i e f of most workers that i t w i l l not gain access to the  i n t e r i o r of the p l a t e l e t and  i t s enzymatic action i s s t r i c t l y on the p l a t e l e t  surface (Stoltz and Nicholas, 1979; 1981).  From sedimentation and  Packham et^ _al., 1980;  Coller and  Zarrabi,  It i s our hypothesis that an enzyme of this size w i l l not be able to  penetrate into the surface-connected canalicular system.  Electron microscopic  studies by Behnke (1968) showed that horseradish peroxidase, which has molecular weight of 40,000 (Worthington, 1972) some of the canalicular system. the system by using two o  n i t r a t e (forming 25A  a  did stain the inside surface of  White (1970) made a more detailed study of  electron dense tracers of d i f f e r e n t sizes.  diameter p a r t i c l e s ) penetrates e a s i l y into the  Lanthanum surface-  ° connected canalicular system.  On the other hand, ruthenium red (forming  diameter p a r t i c l e s ) stains just the outside of p l a t e l e t s and " s u p e r f i c i a l l y " into the canalicular system.  After ADP  50A  only penetrates  stimulation,  ruthenium  red readily penetrates into the canalicular system demonstrating an apparent opening up process.  White (1980) further showed that the openings of  the  - 127 the channels at the discocyte surface are rather c o n s t r i c t i v e (see F i g . 3) and may  not be as simple as once thought. Our results show quantitative differences between the discocyte and  echinocyte  as well as between the discocyte and the spherocyte.  the  The  additional s i a l i c acid susceptible to neuraminidase most probably comes from evaginated  or newly exposed membrane surfaces.  Given white's results and  increase i n surface area believed to occur upon echinocyte (Frojmovic and Milton, 1982)  the  transformation  i t seems u n l i k e l y that neuraminidase penetrates  into the discocyte canalicular system.  The same argument applies to the  enzyme alkaline phosphatase whose molecular weight i s considerably larger at 140,000 (Frenette, 1980). Electrophoretic measurements of fixed p l a t e l e t discocytes showed a 39% drop i n mobility after neuraminidase treatment (Table 6).  Madoff et a l .  (1964) found a 42% decrease while Seaman and Vassar (1966) and Bosmann (1972) found 53% reductions.  The decrease i n mobility was  only 29% for the spherocyte (Table 6). acid charge density on the Only 2.9%  40% for the echinocyte  and  This r e f l e c t s the much lower s i a l i c  spherocyte.  of the neuraminidase susceptible s i a l i c acid on the p l a t e l e t  surface i s apparently responsible for the e l e c t r o k i n e t i c charge properties of the p l a t e l e t (both discocyte and spherocyte).  This figure derives from the 30  fold difference between the amount of s i a l i c acid assayed chemically i n the enzyme digest supernatant and that calculated from electrophoretic mobility measurements.  S t o l t z and Nicolas (1979) found a figure of 3.2%.  percentage i s higher i n polymorphonuclear leukocytes higher s t i l l for red blood c e l l s (46%) 1969).  (10%) and  (Cook et a l . , 1961  If we assume the percentage for the echinocyte  The considerably  and Vassar et a l . ,  to be also 2.9%,  then  - 128 -  the surface area of the echinocyte can be back calculated from the chemistry -8 2 data as described above (Table 7), giving the value of 22.0x10 cm . 3.4.3  P l a t e l e t Surface Phosphate Groups  Alkaline phosphatase removed 9.44x10 discocyte (Table 8). a l . (1982).  phosphate groups from each fixed  This value i s double that of 4.2x10  found by Bik et  The enzyme liberates 32% and 96% more phosphate from the surfaces  of the echinocyte and spherocyte respectively. The electrophoretic mobility of the discocyte decreased by 22% after enzyme digestion corresponding to a removal of 3.74x10"' phosphate groups from the discocyte plane of shear (Table 9).  Mehrishi (1970) found a drop i n  mobility of between 30 and 37%, corresponding to a removal of 5x10^ groups per p l a t e l e t .  Stoltz (1975) estimated p l a t e l e t surface phosphate groups to be  between 3 and 5x10^ per p l a t e l e t from microelectrophoresis experiments. Again, by comparing electrophoresis data with chemistry data (Table 10) i t was found that only 3.9% of the phosphate groups removed are apparently responsible for the e l e c t r o k i n e t i c charge contribution of the discocyte and spherocyte.  If i t i s assumed that the same percentage applies to the echino-  cyte i t s surface area can again be calculated.  The value based on phosphatase  —8 2 —8 2 digestion data was 25.3x10 cm , quite close to 22.0x10 cm obtained —8 from the s i a l i c acid calculations.  An average of 23.7x10  2 cm  was  therefore taken to be the estimate of the echinocyte surface area accessible to these two enzymes. —8 The established area of 23.7x10  2 cm  i s larger than that of the disco-  cyte but much smaller than that of the spherocyte (Table 1).  Presuming that  the newly-exposed membrane originates from the surface-connected canalicular  - 129 system, i t i s apparent cyte.  that i t could not be t o t a l l y evaginated in the echino-  This conclusion i s consistent with the morphological evidence for a  p a r t i a l l y intact canalicular system presented i n Chapter  2.  In a recent review, Frojmovic and Milton (1982) made an estimate of the surface area of an echinocyte based on microscopic observations.  By assuming  2 an echinocyte to have a central spherical body of 13jum  with thin  c y l i n d r i c a l pseudopods extending out of the central body, they arrived at an area of between 17.7  and 22.4x10  cm .  Their pseudopods had a mean radius  of 0.075 jdm and t o t a l length of between 10 and 20 y.m.  The closeness of this  estimation to the one we obtained v i a surface chemical means i s remarkable. 3.4.4  Cocaine Spheres Incubating the p l a t e l e t discocyte with 10 mM  of cocaine for half an hour _g  2  produces a "cocaine sphere" with a surface area of 26.4x10  cm . The -4 2 - 1 - 1 electrophoretic mobility increases dramatically from -1.08x10 cm .sec .V -4 2 - 1 - 1 for the discocyte to -1.40x10 cm .sec .V for the cocaine sphere. This 2 corresponds  to an apparent  30% increase in charge density (charge per cm )  and a 108% increase i n the number of charges per p l a t e l e t (Table 3) at the plane of shear.  Having a surface area s l i g h t l y larger than that of the echino-  cyte, i t has 68% more charge than the echinocyte.  This change cannot be  attributed to increase i n surface area alone (see below). Treatment of the cocaine spheres (unfixed) with neuraminidase resulted i n a 52.6%  reduction i n mobility representing 1.39x10^ s i a l i c acid groups per  p l a t e l e t removed from the plane of shear. fixed echinocytes.  This i s 66.4% more than i n the  The chemistry data, on the other hand, shows that the  t o t a l amount of s i a l i c acid removed from the p l a t e l e t surface i s about the same for the fixed echinocyte and unfixed cocaine sphere.  In other words, the  - 130 percentage of s i a l i c acid groups on the p l a t e l e t surfce situated at the plane of shear whose loss i s seen electrophoretically, has increased from 2.9% to 5.1%. 6.0%  Similarly the percentage for the phosphate groups went up from 3.9% to (Table 11).  Since the t o t a l enzyme susceptible s i a l i c acid and  phosphate  groups on the surfaces of the cocaine sphere and the echinocyte are similar i t i s reasonable to believe the t o t a l number of surface molecules bearing these groups i s similar i n the two forms.  The change in surface architecture  associated with cocaine binding apparently allows a larger f r a c t i o n of the s i a l i c acid and phosphate groups to contribute to the mobility, hence the increase i n surface charge. Cocaine has the molecular structure:  0  Its mechanism as a l o c a l anesthetic i s s t i l l unclear, however i t i s commonly believed to act on the membrane of nerve c e l l s i n t e r f e r i n g with their function (Papahadjopoulos,  1972; Mather and Cousins, 1979).  The l i p o p h i l i c benzene  ring buries i t s e l f into the membrane l i p i d layer and the hydrophilic end remains i n solution.  It causes expansion of the membrane and conformation  changes (Mather and Cousins, 1979).  Although cocaine has a p o s i t i v e l y charged  t e r t i a r y amine group, i t i s doubtful that this w i l l contribute to the e l e c t r o k i n e t i c properties of the p l a t e l e t at the plane of shear.  It i s more  - 131 l i k e l y to interact with the negative (Papahadjopoulos , 1972). the c e l l membrane may  charges of the phospholipid head groups  I t i s thought that t h i s molecule's unique position i n  somehow i n t e r f e r e with Na /K +  +  fluxes and the  transmission of nerve signals. Cocaine and r e l a t e d l o c a l anesthetics also transform biconcave  erythrocytes i n t o cup-shaped forms by disrupting the  inside/outside i o n i c environment of the erythrocytes displacing C a  + +  (Deuticke, 1968).  By  from anionic sites on the membrane and i n the cytoplasm,  cocaine and r e l a t e d anesthetics also cause the breakdown of microfilaments, microtubules 1976)  and cytoskeleton i n c e l l s (Pos te jat jal., 1975 ; Nicolson et a l . ,  and platelets (Nachmias et al ., 1977 ; Nachmias et al ., 1979;  Palek, 1982).  Davies and  No matter which i s the mechanism for the sphering of the  p l a t e l e t , the change i n surface electrokinetic properties i s overwhelming.  3. 4. 5 Calcium It was  and Magnesium Ion Binding  estimated that 2.10x10"' C a  + +  binding sites are present  on the  e l c t r o k i n e t i c surface of each discocyte, assuming that equation [3] and model the s i t u a t i o n adequately.  ADP  [5]  stimulation of the discocyte increases  this f i g u r e twelve-fold to 2.58x10^ s i t e s .  The density of binding sites  12 2 13 increases eight-fold from 1.28x10 per cm on the discocyte to 1.09x10 2 per cm  on the echinocyte.  time drops from -4.09  The average binding free energy ( G) at the same  to -2.89 Kcal/mole (Table 12) . Using similar procedures  Seaman et a l . (1969) found the Ca  ++  2 per cm  1 2  binding s i t e density to be 4.97x10 12  for the human erythrocyte and 3.84x10  morphonuclear leukocyte (Table 15).  2 per cm  for the poly-  Their binding free energies were -3.8  -4.0 Kcal/mole r e s p e c t i v e l y . The density of C a  + +  binding sites on the  discocyte is therefore comparatively low, although the average binding free  and  - 132 -  energy i s similar t o those of the erythrocyte and leukocyte.  After  transformation i n t o the echinocyte the density becomes much higher than on the RBC  and leukocyte but the binding i s less strong.  The estimated  between adjacent sites decreases from about 9nm to 3nm after transformation.  Data obtained for M g  different from those of C a (Table 13 and 14).  ++  binding were not  distance  echinocyte  significantly  although the number of sites i s somewhat lower  + +  A f t e r transformation of discocytes i n t o spherocytes by  hypotonic shock the density of the C a  + +  binding sites decreased  by over 80%  representing apparently a drastic change i n p l a t e l e t membrane properties. Using the ^ C a  binding techniques, Taylor and Heptins t a l l (1980)  + +  found 2.20xl0 , Brass and S h a t t i l 5  (1982) found 4.85xl0 , and Peerschke et 5  a l . (1980) found 1.66xl0 sites per discocyte. 6  45 found no increase i n Shattil  Peerschke ej: _al. (1980)  ++ Ca binding after ADP stimulation while Brass and  (1982) found a 28% increase. . .  45 ++  s u s c e p t i b i l i t y of the  Ca  The range of values shows the  experiments to calcium i n t e r n a l i z a t i o n .  However the microelectrophoresis method used here also has some drawbacks. I t only estimates C a platelets. Mg  ++  + +  and M g  ++  binding t o the e l e c t r o k i n e t i c surface of the  It i s therefore l i k e l y t o underestimate the t o t a l C a  binding t o the whole plasma membrane surface.  The estimates  from microelectrophoresis depend on an i d e a l i z e d model.  + +  and obtained  They ignore, by  depending on equation E3], the distributed nature of the surface charge throughout the glycocalyx.  Despite these uncertainties, differences i n the  binding free energy and the number of binding sites on various platelet forms are much less l i k e l y to be i n error than the individual absolute 45 ++ Given t h e i r l i m i t a t i o n s , comparisons of the results are d i f f i c u l t t o i n t e r p r e t .  Ca  values.  and the electrophoresis  - 133 -  Table 15.  Comparison of Calcium Ion Binding on P l a t e l e t s , Erythrocytes and Polymorphonuclear Leukocyte  Density of Binding Sites (cm ) -2  - G (Kcal/mole)  Discocyte  1.28xl0  12  4.09  Echinocyte  1.09xl0  13  2.89  Spherocyte  2.22X10  11  RBC*  4.97xl0  1 2  3.82  PMN*  3.84xl0  1 2  4.01  *From Seaman et a l . (1969)  4.13  134 -  -  The necessity for fibrinogen binding t o precede platelet aggregation and the requirement of C a  + +  and M g  ++  for fibrinogen binding t o the surface of  the activated platelet has already been discussed i n previous chapters and w i l l be discussed further i n the next chapter. between 40,000 (Bennett  I t has been estimated that  and V a l a r i e , 1979; Plow and Marguerie, 1980) and  50,000 (Hawiger jat ^1., 1980) fibrinogen molecules bind s p e c i f i c a l l y t o surface of each echinocyte. to 60 C a  + +  This i n effect gives a binding r a t i o of about 50  per fibrinogen molecule.  The fibrinogen molecule has been  estimated to be about 45nm long and 6. 5nm i n diameter (Weisel et _al., 1981). The maximum area per C a 9nm  2  + +  binding s i t e on the echinocyte surface i s about  . 13 2 ( i . e . the r e c i p r o c a l of the sites density of 1.09x10 /cm ).  Therefore on the average only a maximum of 30 C a each fibrinogen molecule. echinocyte  + +  could be associated with  From this estimatin about h a l f of the C a  + +  on the  could be associated with fibrinogen providing fibrinogen molecules  maximize thier area of contact with the surface and the C a  + +  binding sites  are uniformly distributed. The  above discussion i s based on the assumption that a l l the C a  binding sites are saturated.  + +  Obviously, under physiological conditions t h i s  i s not the case.  The optimum C a  4mM (Chapter  2).  One can back calculate using F i g . 23 and equation [5] that  only 5. 7x10^  Ca  ions bind t o each echinocyte at t h i s concentration.  + +  + +  concentration for platelet aggregation i s  Only about 2% of the binding sites are therefore occupied. relationship of 1 to 1 .4 C a  + +  ion per fibrinogen molecule.  This also gives a  - 135 -  3.4.6  A Hypothesis Regarding Membrane Exposed by ADP Stimulation or Hypotonic Swelling  Using the above data, a table outlining the surface properties of the p l a t e l e t discocyte, echinocyte and spherocyte as well as their "new membranes" can be constructed (Table 16). "New membrane" i s defined as the increased or evaginated surface areas of the echinocyte and spherocyte.  Total negative  charge i s the apparent net negative charge (Table 3) corrected by adding the number of p o s i t i v e l y charged amino groups (Table 4).  The s p e c i f i c properties  of the "new membrane" are calculated by assuming that the surface properties of the o r i g i n a l "old membrane" of the p l a t e l e t remain unchanged during shape transformation and that the additional charged groups which appear are distributed uniformly over the exposed area added during formation of the echinocyte or spherocyte. There seem to be only small differences between the discocyte and echinocyte membrane considering their respective s i a l i c a c i d / t o t a l charge, phosphate/total charge and s i a l i c acid/ phosphate r a t i o s , although absolute quantitative differences are obvious.  The spherocyte, and especially the  spherocytic "new membrane," on the other hand, are quite different from the others both quantitatively and q u a l i t a t i v e l y . It was proposed  at the beginning of this work that the newly exposed,  evaginated membrane of the echinocyte might be responsible for the sticky nature of the p l a t e l e t surface.  In other words, the non-sticky platelet  discocyte has a cloistered sticky surface that can be unfolded to produce a sticky p l a t e l e t . interpretation.  The data presented i n this chapter argue against this F i r s t l y , the unfolding of the evaginated canalicular membrane  system using hypotonic shock produces a spherocyte with completely  Table 16a. P l a t e l e t Surface Properties by Microelectrophoresis  Area (cm ) 2  Discocyte  Spherocyte  Spherocyte New Membrane  Echinocyte  Echinocyte New Membrane  16.4xl0~  36.7xl0"  20.3xl0"  23.7xl0"  7.3xl0~  8  0.38xl0 5 .21x10  6  8  8  8  8  Total negative charge per  platelet 2 per cm^  1.50xl0 9.13x10  6  2.39xl0 6.50x10  6  0.89xl0 4.38x10  6  1.88xl0 7.92x10  2.18xl0 1.33x10  5  3.97xl0 1.08x10  5  1.79xl0 0.88x10  5  2.82x10!? 1.19x10  0 .64xl0 0 .88x10  5  6.53x10^ 3.98x10  7.80xl0 2.12x10  5  1.27x10^ 0.62x10  8.34xl0 3.52x10  1 .18xl0 2 .48x10  5  3.74xl0 2.28x10  7.19xl0 1.96x10  5  3.45xl0 1.70x10  4.62x10^ 1.95x10  0 ,88xl0 1 .20x10  5  6  Amino groups per per  platelet cm 2  S i a l i c acid groups per per  platelet cm 2  5  Phosphate groups per per  platelet cm 2  S i a l i c acid/Total charge Phosphate/Total charge S i a l i c acid/Phosphate  43.5% 25.0% 1.75  5  32.6% 30.1% 1.08  14.3% 38.8% 0.37  5  44.4% 24.6% 1.80  47.6% 23.1% 2.07  Table 16b.  P l a t e l e t Surface Enzyme Released S i a l i c Acid and Phosphate Group (Chemistry Data)  Discocyte  Spherocyte  Spherocyte New Membrane  Echinocyte  Echinocyte New Membrane  S i a l i c acid groups per p l a t e l e t per cm 2  2.26x102.65xl0 , 1.38x10 0.72x10 ?  0.39xl0 0.19x10  ?  2.67x100.41xl0 , 1.13x10 0.56x10 ?  Phosphate groups per p l a t e l e t per cm 2  S i a l i c acid/Phosphate  9.44xl0 5.75x10 2.39  6  18.52xl0 5.04x10 1.43  6  9.08x10^ '4.47x10  12.48xl0 ~ 5.28x10  3.04xl0 4.17x10  0.43  2.14  1.34  6  6  - 138 -  Table 16c.  Platelet Surface C a  Discocyte Ca  + +  and M g  Binding Sites  ++  Spherocyte  Echinocyte  Echinocyte New Membrane  8.16xl0 2.22xlO  2.58xl0 1.09xl0  2.37xl0 3.25xl0  + +  per p l a t e l e t per cm 2  Mg  2.10xl0 1.28xl0  5  4  12  6  n  6  13  13  ++  per p l a t e l e t per cm'-  1. 77xl0 1.08xl0  5 12  1.56xl0 6.60xl0  6  1.38xl0 1.89xl0 6  12  13  - 139 d i f f e r e n t surface properties.  Secondly, i t does not seem possible that the  o r i g i n a l discocyte surface ("old membrane") remains unchanged during cyte transformation.  echino-  This becomes most evident i f the p l a t e l e t surface C a  binding data i s considered  (Table 16c).  model almost a l l of the C a  + +  + +  Under the sticky invaginated membrane  binding sites on the echinocyte surface must  originate from the "new membrane."  Yet during spherocyte  t o t a l number of sites actually decreases.  transformation the  Not only do no contributions appear  from the "new membrane", but i n this case C a  + +  binding sites are  disappearing. This contradiction implies that the spherocyte membrane cannot consist of unchanged echinocyte membrane plus "new membrane."  Rather, the average  properties of the whole surface are l i k e l y to have changed during  sphering.  F i n a l l y , i t was mentioned e a r l i e r that about 40,000 fibrinogen molecules bind s p e c i f i c a l l y onto the surface of an activated p l a t e l e t .  Considering the  fibrinogen molecule as a c y l i n d r i c a l rod of 45nm long and 6.5nm i n diameter (Weisel et al., 1981), 40,000 molecules w i l l cover about 11.7x10  cm i f  the molecules l i e f l a t , about half of the t o t a l echinocyte surface area. I f they are standing up on ends, the 40,000 fibrinogen molecules could occupy an —8 2 area as small as 1.3x10 cm . "new  Since the surface area of the echinocyte _g 2  membrane" i s about 7.3x10  cm , i t i s possible, given a favourable  orientation for the fibrinogen molecules, involved i n the binding.  However, the Ca  region would be extremely high.  that only "new" membrane could be binding s i t e density i n this  It remains to be seen whether independent  evidence, perhaps u l t r a s t r u c t u r a l , i n favour of this model w i l l appear. In conclusion, although  i t appears that there are s i g n i f i c a n t differences  between the discocyte membrane and the "new membrane" of the echinocytes and spherocytes,  i t i s not clear that the "new membrane" i s responsible for the  stickiness of the echinocyte surface.  The changes i n C a , Mg + +  ++  and  - 140 fibrinogen binding s i t e s could just as well involve the whole p l a t e l e t and just the "new  e  membrane."  not  - 141 -  CHAPTER 4  RADIOCHEMICAL LABELLING OF PLATELET SURFACE STRUCTURES  - 142 4.1  INTRODUCTION Polyacrylamide gel electrophoresis (PAGE) of r a d i o l a b e l e d p l a t e l e t  membranes dissolved i n sodium dodecyl sulphate (SDS) has been an important tool i n the understanding of the surface chemistry of p l a t e l e t s .  The gel  electrophoresis patterns and their interpretations have been discussed and reviewed extensively elsewhere (Jenkins et ^al., 1979; P h i l l i p s , 1979; McGregor et a l . ,  1979; Nurden et a l . , 1981; Connellan et a l . , 1982; Bowles and Brunton,  1982; Toor ej: a l . , 1982).  Three major platelet membrane glycoproteins were  o r i g i n a l l y found by r a d i o l a b e l l i n g techniques and SDS-PAGE ( P h i l l i p s , P h i l l i p s , 1980; Okumura and Jamieson, 1976; George et aJL., 1980).  1979;  They are  glycoprotein I (GP-I) with an apparent molecular weight of between 130,000 and 160,000; glycoprotein II (GP-II) with a molecular weight of between 110,000 and 130,000 and glycoprotein III (GP-III) with a molecular weight of between 90,000 and 110,000.  Subsequent work revealed another glycoprotein with a  molecular weight of between 150,000 and 170,000.  This glycoprotein ran very  near the GP-I peak and was resolved only through improved SDS-PAGE techniques. This l a s t glycoprotein was named GP-Ia and the o r i g i n a l GP-I was then called GP-Ib.  Careful SDS-PAGE experiments demonstrated more minor glycoprotein  peaks below the GP-III peak:  GP-IV (sometimes referred to as GP-IIIb) was  found with a molecular weight of between 85,000 and 100,000; GP-V has a molecular weight of between 68,000 and 89,000.  If one uses the nomenclature  of GP-IIIb for GP-IV, then GP-V becomes GP-IV.  A variety of nomenclature  systems have been developed i n d i f f e r e n t laboratories.  Wide ranges of  molecular weight have also been reported i n the l i t e r a t u r e s for the glycoproteins species, resulting i n some d i f f i c u l t y and confusion i n the i n t e r pretation of data.  The nomenclature system using GP-Ia, GP-Ib, GP-II, GP-III,  - 143 -  GP-IV, GP-V  and GP-VI generally referred to as the P h i l l i p s system  (Phillips,  1979) w i l l be used throughout this work. SDS-PAGE has proved useful for the separation of proteins according to their molecular weights (Reynold and Tanford, 1970; Lasky, 1978).  The useful-  ness of SDS-PAGE for molecular weight determination depends on the a b i l i t y of the anionic detergent SDS to interact with and denature a wide variety of proteins i n a similar manner.  Native proteins having a wide difference in  charge, size and shape c h a r a c t e r i s t i c s are converted, upon d i s u l f i d e bond reduction and SDS-binding, to SDS-protein complexes of their constituent polypeptide chains.  The complex i s a rod-like structure, the length of which  varies with the molecular weight of the protein.  One interesting aspect of  this complex i s that d i f f e r e n t proteins bind i d e n t i c a l amounts of SDS on a gram per gram basis.  The charge per unit mass of protein i s therefore  approximately constant.  In other words, the charge densities of different  SDS-protein complexes are about equal and the e l e c t r o k i n e t i c properties of the complex w i l l be mainly a function of the protein's molecular weight.  The PAGE  gel i s formed by the cross-linking of acrylamide and bis-acrylamide to give a three-dimensional polymer meshwork.  The degree of cross-linking depends on  the gel (acrylamide) concentration.  This three-dimensional meshwork forms a  molecular sieve.  During electrophoresis, the smaller proteins w i l l  encounter  less resistance from the gel matrix than the larger ones and hence migrate farther along the g e l .  When the distances migrated by d i f f e r e n t proteins are  plotted vs. the logarithm of their molecular weights, i t i s generally found that the proteins f a l l on a straight l i n e .  Ideally, the molecular weight of  an unknown protein can be read o f f from such a c a l i b r a t i o n plot, providing i d e n t i c a l experimental conditions are used.  However, errors i n determining  - 144 -  molecular weights can occur (Lasky, 1978).  F i r s t , the semi-logarithmic  c a l i b r a t i o n plot may not be linear, especially towards the higher molecular weight region of the plot (top of the g e l ) .  Second, the action of SDS may not  be perfect (uniform) towards a l l proteins and errors of as much as 15% i n the determination of molecular weight can occur (Lasky, 1978).  Finally, l i t t l e i s  known about how the carbohydrate moieties of glycoproteins may affect the molecular weight determination. Three methods are commonly used for the r a d i o l a b e l l i n g of membrane glycoproteins ( P h i l l i p s , 1979 and McGregor et a l . , 1979).  The f i r s t method i s  lactoperoxidase-catalyzed iodination which covalently attaches the radioactive iodine to tyrosine residues of the glycoproteins. GP-III and a moderate GP-II peak on SDS-PAGE.  This method gives a strong  The second method requires the  removal of terminal s i a l i c acid residues from the surface glycoproteins with neuraminidase.  Galactose oxidase i s then used to oxidize the hydroxyl groups  at the C-6 position of the exposed galactose residues into aldehyde groups. 3 Next,  H-borohydride i s used to reduce the aldehyde groups, resulting i n the 3  incorporation of  H into the galactose molecules.  The third method uses  periodate to cleave oxidatively the carbon-carbon bonds between C7-C8 and C8-C9 positions of the terminal s i a l i c acid residues. The aldehyde groups so 3 . . 3 formed are then reduced with  H-borohydride resulting i n  incorporated into the s i a l i c acid molecules.  H being  The second and the third  l a b e l l i n g methods give similar patterns on SDS-PAGE.  GP-Ib has the strongest  peak while peaks of GP-Ia, GP-II and GP-III are moderately strong.  A l l three  methods w i l l be used here to detect possible changes i n p l a t e l e t surface glycoproteins after transformation to echinocytes and spherocytes.  -  145  -  Bunting et_ _al. (1978) using the p e r i o d a t e - t r i t i a t i o n method found no differences between the gel patterns of normal and ADP activated p l a t e l e t s . 125 George «jt _al. (1980) using the diazotized  I-diiodosulfanilic acid  l a b e l l i n g technique found no difference.  This l a s t compound reacts with  labels amino groups on the platelet surface. 125 . . . using  and  Preliminary experiments by us  I - l act oper oxidase i o d i n a t i o n also f a i l e d to demonstrate any  difference between discocyte and echinocyte SDS-PAGE patterns.  Therefore  double label experiments were undertaken to t r y to improve the r e s o l u t i o n . Sorg and Greczy (1976) have used double r a d i o l a b e l l i n g i n conjunction with SDS-PAGE i n lymphocyte studies.  They have shown that t h i s can be a valuable  technique i n detecting small differences between test and control experiments. 125 131 Double l a b e l l i n g with I and I was used with lactoperoxidasecatalyzed iodination. This was done i n two ways. F i r s t l y , discocytes were 125 .131 l a b e l l e d with I and echinocytes or spherocytes were l a b e l l e d with I. They were then mixed, dissolved i n SDS  and run on the same gel.  This method  helps to eliminate gel to gel differences that may have masked small 125 differences i n the patterns. transformed echinocytes  I-labelled discocytes were 125 i n t o echinocytes or spherocytes. Then these I-labelled 131 131 and spherocytes were r e l a b e l l e d with I. Any I peak i n 125  the gel represents new  Secondly,  glycoprotein species exposed after  I labelling.  Controls were done by double l a b e l l i n g the discocytes without transformation step.  the  A similar double r a d i o - i o d i n a t i o n technique was  used by  P h i l l i p s and Agin (1974) t o f i n d the p l a t e l e t surface thrombin p r o t e o l y t i c s i t e . For the t r i t i a t i o n experiments, double l a b e l l i n g i s achieved by f i r s t " l a b e l l i n g " the discocytes with non-radioactive borohydride or neuraminidase-galactose  oxidase method.  using the  periodate  Then after transformation i n t o  - 146 -  echinocytes or spherocytes, the p l a t e l e t s are relabelled with  3  H-borohydride  3 using similar methods.  Any  H peak i n the gel greater than that i n the con-  t r o l represents glycoproteins exposed during the transformations.  These are  termed pseudo-double label experiments. Lactoperoxidase iodination was also used to study the surface component involved i n the binding of fibrinogen onto the echinocyte surface.  The  requirement for fibrinogen i n ADP induced aggregation has been discussed earlier.  Activation of p l a t e l e t s results i n the exposure or appearance of  receptors for fibrinogen. the activated p l a t e l e t s . binding (Chapter 2).  Fibrinogen molecules then bind onto the surface of Calcium or magnesium ions are required for the  The number of s p e c i f i c receptor s i t e s per activated  p l a t e l e t ranges from about 40,000 (Bennett and V i l a i r e , 1979; Plow and Marguerie, 1980) to 50,000 (Hawiger et a_l., 1980). found 4000 high a f f i n i t y and 9000 low a f f i n i t y  Peerschke et a l . (1980)  sites.  Recent studies suggest that the fibrinogen receptor i s associated with the GPII-GPIII complex.  Four lines of evidence point to this fact.  Firstly,  patients with Glanzmann's thrombasthenia have p l a t e l e t s that f a i l to bind fibrinogen when activated. GPII-GPIII complex.  Glanzmann's thrombasthenia p l a t e l e t s lack the  P l a t e l e t s from patients with Bernard-Soulier syndrome  bind fibrinogen normally when activated.  They have the GPII-GPIII complex but  lack GPIb (Mustard et a l . , 1979; Bennett and V i l a i r e , 1979; Peerschke et a l . , 1980; Lee et a l . , 1981).  Secondly, antibodies to the GPII-GPIII complex block  fibrinogen binding (Lee et a l . , 1981; Coller, 1981; DiMinno et a l . ,  1981).  Thirdly, isolated GPII, GPIII and GPII-GPIII complex when adsorbed to p l a s t i c microtitre plates bind to fibrinogen (Nachman and Leung, 1982).  The glyco-  proteins were isolated using l e n t i l l e c t i n a f f i n i t y chromatography and were  - 147  -  found to react with mono-specific anti-GPIIa  and I l l b antibodies.  by using fibrinogen coupled to a photoreactive  Fourthly,  agent Peerschke et a l . , (1981)  and Bennett e_t a_l. (1981) were able to demonstrate the association of fibrinogen with GPII and GPIII respectively.  This agent (methyl-4-  azidobenzoimidate) reacts with organic molecules covalently on exposure to light.  It can therefore link fibrinogen with i t s receptor, or to structures  near the receptor, when the experiment i s done i n darkness and then exposed to light.  When fibrinogen i s bound, a fibrinogen-protein supercomplex i s formed  and i d e n t i f i e d as a high molecular weight band on SDS-PAGE. weight of the putative receptor of the supercomplex and a ligand-receptor  The molecular  i s the difference between the molecular weight  that of fibrinogen.  This method does not demonstrate  relationship but only a close proximity  between the  fibrinogen molecule and GPII/III molecule. Morrison and Bayse (1970) have shown that the lactoperoxidase  enzyme  molecule must form a complex with tyrosine in a stereospecific manner before iodination can occur.  It w i l l be shown here that the presence of fibrinogen  interferes with the iodination of GPIII in the activated p l a t e l e t , demonstrating a close relationship between the adsorbed fibrinogen and GPIII.  - 148 4.2  MATERIALS AND METHODS  4.2.1  Surface L a b e l l i n g  4.2.1.1  of P l a t e l e t s  Lactoperoxidase Iodination  Lactoperoxidase i o d i n a t i o n methods o f P h i l l i p s  o f p l a t e l e t s was c a r r i e d  out a c c o r d i n g t o the  (1972).  P l a t e l e t s were i s o l a t e d  and a d j u s t e d t o 2.5x10° p e r ml w i t h Tyrode's  solution 125  as i n Chapter  2.  They were mixed w i t h 0.1 mCi/ml of c a r r i e r - f r e e Na  I or  131 Na  I and 0.1 mg/ml o f l a c t o p e r o x i d a s e .  at 0.5 mM f i n a l c o n c e n t r a t i o n . temperature label.  Hydrogen p e r o x i d e was then added  R e a c t i o n was almost  i n s t a n t a n e o u s a t room  and a f t e r 2 min the p l a t e l e t s were washed f r e e  Spherocytes were l a b e l l e d i n h y p o t o n i c Tyrode's  same c o n d i t i o n s and reagent c o n c e n t r a t i o n s .  of excess  solution  radio-  under the  Whole p l a t e l e t s were d i s s o l v e d i n  1% SDS and reduced w i t h 40 mM d i t h i o t h r e i t o l (DTT) a c c o r d i n g t o F a i r b a n k s (1971).  Double l a b e l l i n g was as d e s c r i b e d above i n the I n t r o d u c t i o n . I n  125 where I l a b e l l e d d i s c o c y t e s had t o be transformed and r e 131 125 l a b e l l e d with I , the p l a t e l e t s were f i r s t washed f r e e of excess I,  situations  transformed  and then r e i n c u b a t e d w i t h f r e s h  l a c t o p e r o x i d a s e , ^2®2  *  anC  131 Na  I.  After  l a b e l l i n g , p l a t e l e t s were counted  Compugamma c o u n t e r b e f o r e membrane i s o l a t i o n .  i n a LKB 1282 Dual-channel  - 149 -  4.2.1.2  Borohydride T r i t i a t i o n g P l a t e l e t s (2.5x10 /ml) i n Tyrode's solution were f i r s t incubated with 1  mM sodium metaperiodate for 10 min at room temperature to s e l e c t i v e l y oxidize 3 s i a l i c acid residues.  Then, 1 mCi/ml (4.4 mM)  sodium  H-borohydride was  used to reduce and label the s i a l i c acid at room temperature for 30 min (McGregor et a l . ,  1979).  Other p l a t e l e t s (2.5xl0°/ml) were treated with neuraminidase as outlined i n Chapter 3 to release s i a l i c acid and then incubated with galactose oxidase (20 U/ml)  at 37°C for 30 min at pH 7.5 to oxidize the exposed galactose residues.  Reduction using  3 H-borohydride was as above (McGregor ej: ajL. , 1979).  - 150 -  The pseudo-double  label experiments were done as outlined i n the Introduction  using cold (5 mM) and then hot borohydride.  In these cases "cold labelled"  p l a t e l e t s were washed and then transformed with ADP or hypotonic shock.  They  were treated with metaperiodate or the enzymes under the same conditions as 3 used for the discocyte before r e l a b e l l i n g with  H-borohydride.  Spherocytes  were handled and labelled i n hypotonic Tyrode's solution but otherwise under the same conditions and reagent concentrations as above.  After l a b e l l i n g , a  portion of each of the t r i t i a t e d p l a t e l e t samples was dissolved i n Amersham NCS-Tissue S o l u b i l i z e r and diluted i n an OSC-Organic Counting S c i n t i l l a n t (Amersham) f l u i d . l a t i o n counter.  The samples were then counted with a Beckman LS-233 s c i n t i l Changes i n counting e f f i c i e n c y were monitored using the  - 151 -  external standard  r a t i o output of the counter.  Other portions of the p l a t e l e t  samples were used for membrane i s o l a t i o n . 4.2.2  Isolation of Membrane  Isolation of membranes from labelled p l a t e l e t s was according  to the method  g  of Jamieson j 3 t aj.. (1979).  P l a t e l e t s (2.5x10 /ml) i n Tyrode's solution were  disrupted using a Braunsonic 1510 sonicator (A. Braun, South San Francisco, C a l i f o r n i a ) at 100W for about 30 seconds. d i f f e r e n t i a l centrifugation. 20,000xg for 20 min at 4°C. undisrupted  Membranes were then isolated using  The homogenates were i n i t i a l l y centrifuged at The p e l l e t s containing p l a t e l e t debris and  organelles were discarded.  The remaining supernatant was again  centrifuged at 150,000xg for 1 hr at 4°C.  The p e l l e t s containing membranes  were washed i n Tyrode's solution and dissolved i n 20 mM Tris-HCl pH 8 buffer containing 2% SDS and reduced with 40 mM d i t h i o t h r e i t o l (DTT) according to Fairbanks (1971).  Both centrifugation steps were done with a Beckman L5-65  Ultracentrifuge and a SW41 Swinging Bucket Rotor. 4.2.3  Gel Electrophoresis  SDS-PAGE was run on 5% c y l i n d r i c a l gels cast according Fairbanks (1971).  to the method of  The gel solution consists of 5% acrylamide, 0.18% b i s -  acrylamide, 0.2% SDS, 0.025% tetramethylethylenediamine (TEMED) and 0.15% ammonium persulfate i n a buffer system containing 40 mM T r i s , 20 mM sodium acetate and 2 mM EDTA at pH 7.4.  The gel solution was cast into 7x12.5 mm  glass tubes and allowed to harden at room temperature f o r an hour.  About 20  ug of dissolved protein material together with a small amount of bromophenol blue as tracking dye were loaded into each tube.  The electrophoresis buffer  (pH 7.4) consists of 40 mM T r i s , 20 mM sodium acetate, 2 mM EDTA and 2 gm/L of  - 152 SDS.  A Hoefer S c i e n t i f i c Instrument  (San Francisco, C a l i f o r n i a ) gel e l e c t r o -  phoresis chamber model DE102 was used.  Electrophoresis was run at 5 mA/tube  i n a 4°C r e f r i g e r a t o r u n t i l the tracking dye reached bottom.  The gels were  removed from the tubes and fixed with 3.5% perchloric acid. BDH protein molecular weight standards were run alongside the membrane samples.  They have molecular weights of 53,000; 106,000; 159,000; 212,000 and  265,000.  The molecular weight standard gels were stained with coomassie blue  according to Fairbanks  (1971).  Radioactive gels were cut into 1 mm s l i c e s using a BioRad gel s l i c e r model 195 (BioRad Laboratories, Richmond, C a l i f o r n i a ) .  Iodinated gel s l i c e s were  counted with the LKB Dual-channel Compugamma counter.  T r i t i a t e d gel s l i c e s  were incubated with 0.6 ml NCS-Tissue S o l u b i l i z e r at 50°C for 2 hr.  Radio-  a c t i v i t y was leached out of the g e l into solution which was then diluted with 6 ml of OSC-Organic Counting S c i n t i l l a n t f l u i d for counting i n a Beckman LS-233 s c i n t i l l a t i o n counter.  These procedures were recommended by the manufacturer  Amersham Corp. (Arlington Heights, I l l i n o i s ) .  Leaching was complete after 2  hrs, further incubation of the g e l with fresh NCS overnight f a i l e d to remove any more r a d i o a c t i v i t y from the g e l .  4.2.4  Surface Labelling i n the Presence of Fibrinogen  In this series of experiments iodination of the p l a t e l e t s .  fibrinogen at 0.5 mg/ml was included during  Fibronectin and albumin at the same concentration  were added to other samples as controls.  Calcium ions at 1 mM were added 125  together with the proteins.  Labelling with  I-lactoperoxidase followed.  Membrane i s o l a t i o n and SDS-PAGE were then carried out i n the same way as before.  - 153 4.2.5  Materials  Fibrinogen used was the same as mentioned i n Chapter 2.  Fibronectin was  prepared and kindly supplied by Mr. Johan Janzen using the method of Vuento and Vaheri (1978).  Fibronectin from fresh human c i t r a t e d plasma was f i r s t  bound to a gelatin-Sepharose 4B a f f i n i t y column and then eluted with 1.0M L-arginine.  Bovine serum albumin was from Miles Laboratories (Elkhart,  Indiana). Enzymes:  neuraminidase (Vibrio cholerae) was from Calbiochem (La J o l l a ,  C a l i f o r n i a ) , lactoperoxidase from Sigma (St. Louis, Missouri) and galactose oxidase (Dactylium dendroides) was from Worthington (Freehold, N.J.). 125 Carrier-free sodium i o d i d e  -  131 I and  I as well as t r i t i a t e d  sodium  borohydride were obtained from Amersham (Arlington Heights, I l l i n o i s ) .  NCS-  Tissue S o l u b i l i z e r and OCS-Organic Counting S c i n t i l l a n t were also from Amersham. SDS-PAGE reagents including SDS, acrylamide, bis-acrylamide, IEMED and DTT were a l l from BioRad (Richmond, C a l i f o r n i a ) . was from BDH (Poole, England).  The molecular weight standard  -  4.3  154  -  Results  4.3.1  Iodination Experiments  Gel electrophoresis of lactoperoxidase iodinated whole p l a t e l e t discocytes and echinocytes revealed one major glycoprotein peak (GP-III) with an apparent molecular weight of 100,000 (Fig. 24). of 120,000 corresponding spherocyte  to GP-II was  A smaller peak with molecular weight also present.  The gel pattern of the  showed two major peaks, the GP-III peak and an additional peak with  a molecular weight similar to that of GP-II ( F i g . 24). membrane from the iodinated p l a t e l e t s was  When the plasma  isolated and electrophoresed, the  patterns from the discocyte, echinocyte and spherocyte were a l l similar ( F i g . 25).  The large 120,000 molecular weight peak i n the spherocyte pattern had  apparently disappeared although the smaller underlying peak similar to that i n the discocyte and echinocyte patterns remained. 25 were t y p i c a l of seven experiments.  Gel patterns i n F i g . 24 and  Washing of the spherocytes  after  iodination had no effect on the 120,000 molecular weight protein which was apparently removed during plasma membrane i s o l a t i o n . proteins with similar molecular weights are involved:  It appears that two GP-II with a smaller  peak size, and a second protein with a larger peak size appearing only on the intact spherocyte.  The most l i k e l y explanation for the observation i s that  this second protein i s not native to the p l a t e l e t plasma membrane but an a r t i f a c t originating from inside the spherocyte.  It apparently leaks out and  attaches i t s e l f to the surface of the spherocyte during hypotonic shock. attachment i s strong enough to withstand  The  the washing procedure but the rigors  of membrane i s o l a t i o n can dislodge this protein from the spherocyte membrane. This observation w i l l be discussed further i n section 4.4.1.  In light of this  phenomenon, a l l SDS-PAGE experiments throughout the rest of this work were done using plasma membranes isolated from radiolabelled p l a t e l e t s .  - 155 -  F i g . 24.  SDS-PAGE of ""^I-labelled p l a t e l e t s .  Whole  p l a t e l e t (a) discocytes; (b) echinocytes and (c) spherocytes were dissolved  i n 1% SDS and reduced i n 40 mM DTT.  were then run on 5% gels.  They  OOO'OVn 000'09-  OOO'OS-  ooo'ootOOCto  ooo'ovi000'09l-  5 o  I o  -  F i g . 25.  157 -  SDS-PAGE of plasma membrane isolated from  labelled p l a t e l e t (a) discocyte; (c) spherocyte.  (b) echinocyte and  Isolated membranes were dissolved and  reduced as i n F i g . 24.  They were then run on 5% gels.  I-  r160,000 •140,000 -120,000 •100,000 ;80,000 "60,000 -40,000  - 159 Gel patterns of isolated p l a t e l e t membranes from the double iodination experiments are shown i n Figs. 26-31.  F i g . 27 and 28 demonstrate the f i r s t  kind of double l a b e l l i n g experiment i n which discocytes were labelled with 125 I.  Echinocytes or spherocytes derived from unlabelled discocytes were 131 125 separately labelled with I and then run on the same gels as the I— 125 discocytes.  F i g . 26 i s a control i n which  I labelled discocytes were  131 mixed with  I labelled discocytes and run on the same g e l .  Figs. 29-31 125 show the second kind of double l a b e l l i n g experiment i n which I labelled discocytes were transformed into echinocytes or spherocytes and then relabelled 131 125 with I. F i g . 29 i s the control in which I labelled discocytes were relabelled with  131  . . I without being transformed.  . The r a t i o , R, of the  125  I  131 to  1 counts i s shown under each pair of the gel patterns i n F i g . 26-31.  They w i l l be referred to as R-plots. No major differences can be observed between the patterns i n any of these figures.  They a l l show one major (GP-III) peak at molecular weight of about  100,000 and a smaller peak (GP-II) with molecular weight of about 120,000. Although some small variations are shown i n the R-plots, they are not s i g n i f i c a n t enough to demonstrate unequivocal differences between the 125  131 I-  I patterns.  The brackets  (S-S) in a l l the R-plots i n Figs.  represent +2 standard deviations of their respective means. 125  26-31  In theory, i f the  131 I and  I patterns are i d e n t i c a l the R-plot w i l l be a straight l i n e .  If there i s a s i g n i f i c a n t difference between the patterns i t w i l l show up as a large peak or valley i n the R-plot (see under the fibrinogen section below). The above patterns are t y p i c a l of three series of similar experiments. Quantitation of the counts revealed that during the a c t i v a t i o n of p l a t e l e t s from discocyte to echinocyte there was  a 21.7%  (+1.5%) increase i n the iodine  - 160 -  Fig. 26.  SDS-PAGE of membranes (reduced) from a mixture of  125  131 I-labelled discocytes and  run on the same 5% g e l .  (a)  125 and (c) r a t i o , R, of (R-plot).  Dotted  125  I-labelled discocytes 131 I-pattern; (b) I-pattern  131 I to  I a c t i v i t y i n each s l i c e  line marks the mean of the R values and the  bracket (s-s) represents +2 standard deviation.  CPM  P160JOOO  •140,000 •120,000 •100,000 •80,000 •60,000 %0,000  >  - 162 -  Fig. 27.  SDS-PAGE of membranes (reduced) from a mixture of  125  131 I-labelled discocytes and I-labelled echinocytes run 125 131 on the same 5% g e l . (2) I-pattern; (b) I-pattern and (c) R-plot, as defined for F i g . 26.  -  163 -  - 164 -  F i g . 28.  SDS-PAGE of membranes (reduced) from a mixture of  125  131 I-labelled discocytes and  on the same 5% g e l .  (a)  1 2 5  I-labelled spherocytes run  I - p a t t e r n ; (b)  (c) R-plot, as defined f o r F i g . 26.  1 3 1  I - p a t t e r n and  CPM  >  - 166 -  F i g . 29.  SDS-PAGE of membranes (reduced) from ^ " ' i - l a b e l l e d  131 discocytes which were washed and then relabelled with I. 125 131 The membranes were run on 5% g e l . (a) I-pattern; (b) Ipattern and (c) R-plot, as defined for Fig. 26.  CPM. X  [160,000 •140,000 •120,000 •100,000 •80,000 •60,000 40,000  o  - 168 -  F i g . 30.  SDS-PAGE of membranes (reduced) from *~*~ I-label led  discocytes which then transformed into echinocytes  J  and re-  131 labelled with I. The membranes were run on 5% g e l . 125 131 (a) I-pattern; (b) I-pattern and (c) R-plot, as defined f o r F i g . 26.  CPM O O o o  o  O o o o  - 170 -  F i g . 31.  SDS-PAGE of membranes (reduced) from ^^"'i-labelled  discocytes which then transformed into spherocytes and re131 labelled with (a)  1 2 5  I. The membranes were run on 5% g e l .  I - p a t t e r n ; (b)  defined f o r F i g . 26.  1 3 1  I - p a t t e r n and (c) R-plot, as  CPM cn  o o o o  -160,000  [•140,000 120,000 -100P00 -80000 •60,000 %0,000  > cn  o o o o  - 172 -  incorporation. o  39.8%  Transformation from discocytes to spherocytes resulted i n a  o  (+1.3%) increase.  single label experiments.  These were whole p l a t e l e t  125  I counts from p a r a l l e l  The percentages were means of four series of such  experiments. Morphological examinations of the p l a t e l e t s under phase microscopy were done before and after l a b e l l i n g to ensure they were i n their proper discoid, echinoid or spheroid shapes as desired. respect. 4.3.2  There were no problems i n this  No aggregation was encountered during or after  labelling.  T r i t i a t i o n Experiments  F i g . 32 shows the gel patterns of p l a t e l e t discocytes, echinocytes and spherocytes labelled with the periodate method. using the neuraminidase-galactose oxidase method. these patterns.  F i g . 33 shows them labelled More peaks are revealed i n  The four major ones are GP-Ia; GP-Ib; GP-II and GP-III with  apparent molecular weights 150,000, 140,000, 120,000 and 100,000 respectively. A few minor peaks can also be seen.  They are GP-IV (which appears as a  shoulder on the right side of GP-III); GP-V  and GP-VI with molecular weights  of 90,000, 80,000 and 65,000 respectively.  However the overall patterns for  the discocytes, echinocytes and spherocytes do not d i f f e r i n either t r i t i a t i o n l a b e l l i n g procedure.  These patterns are t y p i c a l of two series of experiments.  Quantitation of whole p l a t e l e t counts showed that the echinocyte and the spherocyte had 0.4% and 0.9% more counts respectively than the discocyte using the periodate t r i t i a t i o n method.  While using the neuraminidase-galactose  oxidase l a b e l l i n g method the echinocyte had 16.2% and the spherocyte had more counts than the discocyte. series of experiments.  These percentages were the means of two  21.1%  - 173 -  Fig. 32.  SDS-PAGE gel patterns of membranes (reduced)  from  t r i t i a t e d (a) discocyte; (b) echinocyte and (c) spherocyte. T r i t i a t i o n was by the periodate method.  i  160,000  r  -140,000 -120,000 100,000  :  -80,000 -60,000 L  40,000  10,000 i  1  CPM i  i  -  Fig. 33.  1 7 5  -  SDS-PAGE gel patterns of membranes (reduced) from  t r i t i a t e d (a) discocyte; (b) echinocyte and (c) spherocyte. T r i t i a t o n was by the neuraminidase/galactose oxidase method.  i  10,000 CPM 1  1  1  1  1  1-160,000  -140,000 -120,000 -100,000 -80,000 £0,000 •40,000  CT)  - 177  -  The gel patterns of the pseudo-double label experiments are shown i n Figs. 3 34 and 35.  Using the periodate method, l i t t l e or no l a b e l l i n g by  2nd label) has taken place ( F i g . 3 4 ) .  With the  neuraminidase-galactose  oxidase method, small peaks are present i n the gel patterns of the and spherocytes  (Fig. 35).  They correspond  H (the  echinocytes  to GP-Ia, GP-Ib, GP-II and GP-III.  In both Figs. 34 and 35,  the patterns of the discocytes served as controls. 3 Here "cold labelled" discocytes were relabelled with H-borohydride without being transformed.  Theoretically, i f the cold reaction went to completion,  no  3 H l a b e l l i n g should be observed. Morphological  examinations of the p l a t e l e t s under phase microscopy were  done as i n the iodination experiments above.  Again, no problems were  encountered. 4.3.3  Surface Labelling i n the Presence of Fibrinogen The influence of fibrinogen (0.5  mg/ml) on lactoperoxidase catalyzed  iodination of the p l a t e l e t forms i s shown in Figs. 36-38. added together with the lactoperoxidase and therefore was  The fibrinogen was present during the  whole process of iodination. P r o f i l e s of the gels of the discocyte (Fig. 36) and spherocyte  ( F i g . 38)  show that fibrinogen did not influence iodination of  the discocyte and spherocyte  surface.  On the other hand, a drop i n the peak  size of GP-III i n the patterns of the echinocytes fibrinogen can be seen ( F i g . 37).  labelled i n the presence of  This i s also reflected by a peak in the  R-plot demonstrating a difference between the control (without fibrinogen) and the test gel at that location.  Fibrinogen therefore impaired the l a b e l l i n g of  GP-III on the echinocyte but not on the spherocyte (Fig. 40)  and albumin (Fig. 39),  or discocyte.  Fibronectin  tested also at 0.5 mg/ml, had no influence on  the gel patterns of the echinocytes.  This impairment i n l a b e l l i n g was  also  - 178 -  Fig. 34.  SDS-PAGE gel patterns of membranes (reduced) from  the pseudo-double labelled (a) discocyte; (b) echinocyte and (c) spherocyte.  P l a t e l e t discocytes were f i r s t labelled with  3 cold borohydride, then transformed and relabelled with borohydride using the periodate method both times.  H-  - 179 -  Q_  C_J> 2000 0  (a)  2000 (b)  0  - 180 -  Fig. 35.  SDS-PAGE gel patterns of membranes (reduced) from  the pseudo-double labelled (a) discocyte; (b) echinocyte and (c) spherocyte.  P l a t e l e t discocytes were f i r s t labelled with  3 cold borohydride, then transformed and relabelled with  H-  borohydride using the neuraminidase/galactose oxidase method both times.  - 182 -  F i g . 36.  SDS-PAGE g e l patterns of membranes (reduced)  from  125 discocyte  I-lactoperoxidase labelled (a) without and  (b) with fibrinogen present (0.5 mg/ml).  (c) Ratio, R, of  the r a d i o a c t i v i t y i n (a) to that i n (b) for each gel s l i c e (R-plot).  Dotted l i n e marks the mean of the R values and  the bracket (s-s) represents +2 standard deviation.  CPM ~o o o  >  o o  r160,000 co  -140,000 •120P00 •100,000 •80,000 •60,000 •40,000  -  Fig. 37.  184 -  SDS-PAGE gel patterns of membranes (reduced) from 125  echinocytes  I-lactoperoxidase labelled (a) without and  (b) with fibrinogen present (0.5 mg/ml); (c) R-plot, the bracket (S-S) i s the same as that of the control i n F i g . 36.  CPM X O cn T  ro  — > •  O  &> o  1  r160,000  1-140,000  1 2 0 P 0 0 H00,000  -80,000  -60,000 -40,000  Q  - 186 -  Fig. 38.  SDS-PAGE gel patterns of membranes (reduced) from 125  spherocytes  I-lactoperoxidase labelled (a) without and  (b) with fibrinogen present (0.5 mg/ml); (c) R-plot, as defined for F i g . 36.  - 187 -  - 188 -  Fig. 39.  SDS-PAGE gel patterns of membranes (reduced) from 125  echinocytes  I-lactoperoxidase labelled (a) without and  (b) with albumin present (0.5 mg/ml); (c) R-plot, as defined for F i g . 36.  - 189 -  - 190 -  F i g . 40.  SDS-PAGE gel patterns of membranes (reduced) from 125  echinocytes  I-lactoperoxidase labelled (a) without and  (b) with fibronectin present (0.5 mg/ml); (c) R-plot as defined for F i g . 36.  </>h  ooo'oy 000'09H  000'08  :  OO0OOI-  oocfozt ooo'on000091-  5  cr  - 192 -  dependent on the concentration of fibrinogen. counts (cpm)  The decrease  in the number of  from the control (echinocyte labelled with no fibrinogen present)  increases i n magnitude with fibrinogen concentration (Fig. 41). reached at about 0.5 mg/ml. similar experiments. experiments.  A plateau was  Gel patterns are t y p i c a l of three series of  Values i n F i g . 41 are also from three sets of  - 193 -  F i g . 41.  The  effect  of f i b r i n o g e n c o n c e n t r a i o n on  l a b e l l i n g of e c h i n o c y t e s . counts  (cpm)  per e c h i n o c y t e  l a b e l l e d w i t h no  The decrease  i n the number of  from the c o n t r o l  (echinocyte  f i b r i n o g e n present) i s p l o t t e d  fibrinogen concentration.  the  against  Decrease in CPM per Echinocyte  - 195 4.4  DISCUSSION  4.4.1  Iodination Experiments The gel patterns i n Figs. 24-31  are not unlike those found by others for  lactoperoxidase iodination ( P h i l l i p s , 1972; Connellan et^ a l . , 1982). usually small.  Okumura and Jamieson,  1976;  GP-III i s most dominant while the GP-II peak i s  Those of the GP-Ia and GP-Ib are less obvious and better  demonstrated i n the t r i t i a t i o n gel patterns. L i t t l e difference can be seen between the gel patterns of single iodinated discocytes and echinocytes (Figs. 24 and 25).  However the difference between  the gel patterns of the iodinated intact spherocytes plasma membrane isolated from iodinated spherocytes  ( F i g . 24) and that of ( F i g . 25) i s obvious.  large peak with an apparent molecular weight of 120,000 had disappeared  The  during  the membrane i s o l a t i o n process leaving behind only a small peak similar to that of GP-II observed i n the discocyte and echinocyte patterns. two proteins with similar molecular weights are involved here. peak belongs to GP-II.  Apparently The  The i d e n t i t y of the other remains uncertain  i t must be a product of hypotonic  shock.  The fact that i t was  smaller although  removed during  membrane i s o l a t i o n makes i t less l i k e l y to be a native component of the membrane.  It i t believed to be a protein o r i g i n a t i n g from the inside of the  p l a t e l e t and released to the outside during hypotonic i t was  treatment.  then adsorbed onto the surface of the spherocyte  Presumably  and iodinated.  Removal from the p l a t e l e t surface occurred during membrane i s o l a t i o n .  The  adsorption would be f a c i l i t a t e d by low ionic strength condition of the hypotonic Tyrode's solution (Heard and Seaman, 1960).  After membrane  i s o l a t i o n they were washed i n isotonic Tyrode's solution and desorption might have occurred assisted by the higher s a l t concentration.  - 196 In 1974,  P h i l l i p s and Agin reported the presence of a thrombin sensitive  protein with a molecular weight similar to that of GP-II i n SDS-PAGE.  They  were not able to confirm the observation i n a l a t t e r study ( P h i l l i p s and Agin, 1977) was  and attribute i t to an a r t i f a c t .  In their i n i t i a l 1974  observation, i t  found that only 30% of the 120,000 molecular weight peak was susceptible  to thrombin indicating the presence of two overlapping components with similar molecular weights i n the g e l .  Several ideas were entertained at that point  concerning the nature of the thrombin sensitive component. suggestions was  One of the  p l a t e l e t factor XIII.  P l a t e l e t factor XIII accounts  for approximately  50% of t o t a l factor XIII  a c t i v i t y i n human whole blood and almost a l l of i t i s carried i n the p l a t e l e t cytoplasm  (Lopaciuk et a l . , 1976; Walsh, 1981).  such as beta-thromboglobulin  Leakage of p l a t e l e t  contents  and LDH to the outside during hypotonic shock has  already been discussed i n Chapter 2.  Leakage of some factor XIII out of the  spherocyte i s therefore not t o t a l l y unexpected.  Factor XIII was known to  adsorb strongly onto p l a t e l e t surfaces (Born, 1968).  P l a t e l e t factor XIII i s  a dimer of two i d e n t i c a l chains each having a molecular weight of about 110,000 (Loewy et a l . , 1961;  Ganguly, 1971;  v i t r o environment the dimer spontaneously subunits (Loewy ejt a_l., 1961).  Schwartz et a l . , 1971).  dissociates into i t s two monomer  P l a t e l e t factor XIII monomers appear to be a  l i k e l y candidate for the protein i n question here. p l a t e l e t factor XIII contains no carbohydrate  Unlike plasma factor XIII,  (Schwartz et a l . , 1971)  therefore w i l l not influence s i a l i c acid results i n Chapter 3. hand, the presence of this adsorbed protein may  and  On the other  physically interfere with the  neuraminidase molecule resulting i n underestimation the spherocyte surface.  In an in  of s i a l i c acid density on  However, this seems unlikely because the density of  - 197 -  the phosphate groups on the spherocyte and the echinocyte surfaces are similar (Table 9) and the molecular weight of alkaline phosphatase i s much higher than neuraminidase. In  light of the a r t i f a c t involving intact spherocytes, the rest of the  SDS-PAGE experiments were done using plasma membranes isolated from radiolabelled p l a t e l e t s .  During the preliminary single iodination  experiments  (Figs. 24 and 25) no s i g n i f i c a n t difference could be observed between the gel patterns of the discocyte and the echinocyte.  The decision was therefore made  to u t i l i z e double label procedures i n hope that i t might bring out more subtle differences. The gel patterns of the double iodination experiments 26-31.  are shown i n Figs.  It seems clear that the patterns are again similar to each other,  since i n no case did the r a t i o R vary by more than two standard deviations from i t s mean value.  Apparently no new p l a t e l e t surface species are labelled  on the echinocyte or spherocyte.  F i g . 29 shows that iodination of the 125  tyrosine residues i s less than complete.  After the i n i t i a l  there was considerable tyrosine l e f t over to be labelled with second iodination.  F i g . 29 represents the control experiment  cytes were f i r s t labelled with ^ " ' i transformation.  . I-iodination, 131 I for the  i n which disco-  and then relabelled with ' ^ I without  However, f a i l u r e of the f i r s t iodination reaction to go to  completion w i l l not affect the outcome of the echinocyte and spherocyte experi125 131 ments since the experimental conditions for both  I and I iodinations 131 were kept the same. New species appearing on the echinocyte or spherocyte surface after transformation would be labelled by I together with " o l d 1  species not yet labelled by ^ " ' i .  - 198 Q u a n t i t a t i v e experiments showed t h a t the e c h i n o c y t e bound 21.7% more  125  I  l a b e l t h a n the d i s c o c y t e .  The e s t i m a t e d i n c r e a s e i n s u r f a c e a r e a , on t h e o t h e r  hand, i s 44% (Chapter 3 ) .  There a r e a number of p o s s i b l e e x p l a n a t i o n s f o r  discrepancy. high.  this  F i r s t l y , the e c h i n o c y t e area e s t i m a t e d i n Chapter 3 may be t o o  T h i s seems u n l i k e l y because the areas  o b t a i n e d by n e u r a m i n i d a s e  and  a l k a l i n e phosphatase e x p e r i m e n t s m u t u a l l y agree w i t h each other and w i t h the Frojmovic estimation.  S e c o n d l y , 1 a c t o p e r o x i d a s e may have e n t e r e d i n t o part of  the i n v a g i n a t e d s u r f a c e - c o n n e c t e d c a n a l i c u l a r system of the d i s c o c y t e t o catalyze i o d i n a t i o n there.  I n t h i s case the d i f f e r e n c e betwwen v a l u e s  o b t a i n e d f o r the d i s c o c y t e and e c h i n o c y t e w i l l  (counts)  be s m a l l e r than e x p e c t e d w i t h  r e s p e c t t o the e s t i m a t e d d i f f e r e n c e i n s u r f a c e a r e a s .  Lact oper o x i das e has  a  m o l e c u l a r weight of 93,000, l a r g e r than neuraminidase but s m a l l e r than a l k a l i n e phosphatase. phosphatase, system.  As d i s c u s s e d i n Chapter 3 f o r neuraminidase and a l k a l i n e i t is  u n l i k e l y that 1 actoperoxidase w i l l  Thirdly, i f  e n t e r the c a n a l i c u l a r  the o n l y change i n the b i o c h e m i c a l n a t u r e of the  e c h i n o c y t e s u r f a c e i s the appearance o f "new membrane" from the c a n a l i c u l a r s y s t e m , t h i s membrane may be d e f i c i e n t , r e l a t i v e t o the d i s c o c y t e s u r f a c e , iodinatable tyrosine residues.  It  in  has a l r e a d y been shown i n T a b l e 16 t h a t t h e  e c h i n o c y t e "new membrane" has lower q u a n t i t i e s of t e r m i n a l s i a l i c a c i d , phosphate and amino groups.  Fourthly, i t is  a l s o p o s s i b l e t h a t the c h e m i c a l  c h a r a c t e r of the p l a t e l e t s u r f a c e may have changed due t o ADP-induced transformation such that the o v e r a l l l a b e l l i n g i s reduced. i n v o l v i n g whole p l a t e l e t s u r f a c e s have been d i s c u s s e d i n Chapter 3.  such as  changes  c a t i o n and f i b r i n o g e n b i n d i n g s i t e s  However i t i s  f o u r t h , or both e x p l a n a t i o n s b e s t f i t s the  Membrane  uncertain i f the t h i r d , the  discrepancy.  - 199 The s p h e r o c y t e showed a 39.8% i n c r e a s e i n l a b e l l i n g w h i l e the g e o m e t r i c s u r f a c e area a c t u a l l y i n c r e a s e d by 123% (Chapter 2).  The i n c r e a s e i n l a b e l l i n g  due t o the appearance of the 120,000 m o l e c u l a r weight p r o t e i n ( f a c t o r X I I I ) , on the h y p o t h e t i c a l "new membrane" i s t h e r e f o r e m i n i m a l . reasons g i v e n above f o r e c h i n o c y t e s  A g a i n one can use  to e x p l a i n t h i s s i t u a t i o n .  4.4.2 T r i t i a t i o n Experiments G e l p a t t e r n s of t r i t i a t e d p l a t e l e t s show more peaks samples.  than the i o d i n a t e d  A g a i n , t h e r e i s no apparent d i f f e r e n c e between the g e l p r o f i l e s  the d i s c o c y t e , e c h i n o c y t e , or s p h e r o c y t e .  The p a t t e r n s of p l a t e l e t s l a b e l l e d  u s i n g t h e p e r i o d a t e method and t h o s e u s i n g t h e n e u r a m i n i d a s e - g a l a c t o s e method a r e a l s o s i m i l a r ( F i g s .  32 and 3 3 ) .  oxidase  They a r e not u n l i k e t h o s e shown  elsewhere (McGregor jet al ., 1979; Mosher e t _ a l . , 1979; M a r c h e s i 1979; P h i l l i p s and A g i n ,  of  and C h a s i s ,  1977)  The pseudo-double l a b e l experiments u s i n g the p e r i o d a t e t r i t i a t i o n method showed no  H - l a b e l l i n g at a l l ( F i g . 34).  Here t h e d i s c o c y t e s were f i r s t  l a b e l l e d with cold borohydride f o l l o w i n g periodate o x i d a t i o n . t r a n s f o r m e d i n t o e c h i n o c y t e s or s p h e r o c y t e s  They were t h e n  and r e l a b e l l e d w i t h the p e r i o d a t e  3 method u s i n g surfaces  H i m p l i e s that a l l the t e r m i n a l s i a l i c a c i d r e s i d u e s  had a l r e a d y r e a c t e d .  on t h e i r  I n o t h e r w o r d s , the p e r i o d a t e c r o s s e d t h e  membrane or e n t e r e d the c a n a l i c u l a r system and o x i d i z e d a l l the s i a l i c a c i d i n t h e membrane.  Both b o r o h y d r i d e and m eta p e r i o d a t e are v e r y s m a l l m o l e c u l e s  s h o u l d e a s i l y e n t e r the s u r f a c e - c o n n e c t e d c a n a l i c u l a r s y s t e m . membranes Andersson,  and l a b e l t h e i n t e r i o r m a t e r i a l s of i n t a c t c e l l s 1977).  They a l s o  (Gahmberg and  and  cross  - 200 The pseudo-double label experiments using the  neuraminidase-galactose  oxidase method present another picture (Fig. 35).  Small peaks were found  corresponding label gels.  to the surface glycoproteins already demonstrated in the single Since the molecular weight of neuraminidase i s 90,000 and galac-  tose oxidase i s 42,000 (Worthington, 1972), they would not be expected to enter the canalicular system s i g n i f i c a n t l y .  Therefore the glycoproteins on  the canalicular surface were probably not cold labelled i n the discocyte. Even though the cold borohydride  had access to the canalicular system, the  enzymes required to catalyze the reaction should not have been able to enter. After transformation from discocyte to echinocyte or spherocyte  the glyco3  proteins from these surfaces became available for enzymatic oxidation and labelling.  H  The fact that the peaks here are r e l a t i v e l y small compared to  those of the direct l a b e l l i n g experiments (Fig. 33), suggests that the amount of glycoproteins c l o i s t e r e d i n the discocyte c a n a l i c u l a r system i s rather small. Quantitative single label experiments using the periodate method showed that the echinocyte and the spherocyte had only 0.4% respectively than the discocyte.  Using the neuraminidase-galactose  method, the echinocyte exhibited 16.2% label than the discocyte.  and 0.9% more counts  and the spherocyte  oxidase 3  21.1% more  H  The discrepancies can be explained using the same  argument as for the pseudo-double l a b e l l i n g experiments discussed above. periodate had ready access to the invaginated canalicular system.  The  Therefore  the amount of membrane surface available for l a b e l l i n g i s the same i n the discocyte, echinocyte and spherocyte.  On the other hand, the enzymes w i l l not  be able to enter the canalicular system.  More surface w i l l therefore be made  -  201 -  a v a i l a b l e f o r l a b e l l i n g a f t e r e v a g i n a t i o n o f the c a n a l i c u l a r system i n t h e e c h i n o c y t e and s p h e r o c y t e . . . 3 The q u a n t i t a t i v e r e s u l t s from n e u r a m i n i d a s e - g a l a c t o s e o x i d a s e H l a b e l l i n g . . 125 were q u i t e s i m i l a r t o t h o s e o f the l a c t o p e r o x i d a s e I - l a b e l l i n g experiments. The p e r c e n t i n c r e a s e s i n t h e t r i t i a t i o n of t h e e c h i n o c y t e and s p h e r o c y t e were much lower than t h e percent i n c r e a s e s Chapter 2 and 3. glycoproteins  i n t h e i r s u r f a c e areas  surfaces  estimated i n  T h i s a g a i n suggests e i t h e r t h a t the d e n s i t y of t h e  on the i n v a g i n a t e d membrane system i s much lower than on t h e  e x t e r n a l plasma membrane or t h a t t h e average s u r f a c e p r o p e r t i e s of t h e t r a n s f o r m e d c e l l s have  changed.  I t has been r e p o r t e d by Cazenave j j t _al. (1976) t h a t p e r i o d a t e (1-10 mM) t r e a t m e n t of r a b b i t p l a t e l e t s i n T y r o d e ' s  s o l u t i o n caused them t o a g g r e g a t e .  However, we f o u n d no a g g r e g a t i o n d u r i n g t h e experiments w i t h human p l a t e l e t s . The c h i e f concern here i s t h a t the d i s c o c y t e s may t u r n i n t o e c h i n o c y t e s the l a b e l l i n g p r o c e d u r e s .  during  M i c r o s c o p i c e x a m i n a t i o n demonstrated t h a t t h i s d i d n o t  happen.  4. 4. 3  Surface L a b e e l i n g i n t h e Presence o f F i b r i n o g e n  I t was found here t h a t f i b r i n o g e n i m p a i r s t h e i o d i n a t i o n of e c h i n o c y t e surface G P - I I I .  I t seems that the b i n d i n g o f f i b r i n o g e n o n t o t h e e c h i n o c y t e  s u r f a c e b r i n g s i t i n t o c l o s e a s s o c i a t i o n w i t h G P - I I I , c l o s e enough f o r f i b r i n o g e n ( m o l e c u l a r weight  340,000) t o i n t e r f e r e w i t h t h e enzyme l a c t o p e r o x i d a s e w h i c h  catalyzes the i o d i n a t i o n .  While this  does n o t mean t h a t t h e f i b r i n o g e n r e c e p t o r  i s a c t u a l l y G P - I I I , i t c e r t a i n l y adds f u r t h e r evidence t o support the i d e a t h a t GP-III i s the receptor.  The phenomenon i s s p e c i f i c o n l y t o t h e e c h i n o c y t e  ( a c t i v a t e d p l a t e l e t ) and not the d i s c o c y t e o r s p h e r o c y t e .  It i s  - 202 -  s p e c i f i c to fibrinogen and does not occur with albumin or f i b r o n e c t i n . last result i s consistent with the work of Harfenist (1980) who fibronectin i s not involved i n p l a t e l e t aggregation activated p l a t e l e t s . p l a t e l e t aggregation  similar to each other. aggregation  found that  and w i l l not bind to  It has already been shown in Chapter 2 that ADP requires fibrinogen.  This  induced  F i g . 10 and Fig. 41 are extremely  They imply a close relationship between p l a t e l e t  and fibrinogen binding to or near GP-III on the p l a t e l e t surface.  This experiment f a i l e d to demonstrate any association of fibrinogen with the spherocyte surface.  It i s possible that there i s no fibrinogen receptor  on the spherocyte or that the receptors have been destroyed hypotonic  treatment.  Presence of the 120,000 molecular  spherocyte might also have p h y s i c a l l y blocked the C a Ca  + +  4.4.4  + +  binding s i t e s .  during the  weight protein on the  the fibrinogen receptors and/or  This might have caused the drastic drop i n the  binding sites on the spherocyte (section 3.3.5).  Summary of Chapter 4 Different methods have been used to label the p l a t e l e t discocyte,  echino-  cyte and spherocyte but none showed any q u a l i t a t i v e differences between the gel patterns.  Quantitative differences were demonstrated, however, but i t was  found that the l a b e l l i n g density on the evaginated system was  surfaces of the canalicular  lower than on the discocyte surface, presuming the l a t t e r  unchanged during shape transformation. and GP-III was  was  A close association between fibrinogen  also demonstrated i n the echinocyte,  the  concentration  dependence of which c l o s e l y p a r a l l e l s the fibrinogen dependence of  aggregation.  - 203 -  CHAPTER 5  SUMMARY AND CONCLUSIONS  - 204 -  The purpose of this project was to investigate the surface properties of the p l a t e l e t discocyte, echinocyte and spherocyte.  A model i s examined here  in which an echinocyte attains i t s stickiness properties by evagination of the surface-connected canalicular system.  Platelets also evaginate their  canalicular system upon hypotonic shock treatment to form spherocytes.  By  comparing the properties of d i f f e r e n t forms of p l a t e l e t s some insight into the nature of " s t i c k i n e s s " was  sought.  The following conclusions can be drawn  from the results obtained. 5.1  The surface areas of the discocyte and spherocyte were found micro-  scopically to be 16.4x  10  —8  2 —8 2 cm , and 36.7x10 cm respectively (Chapter —8  2).  The surface area of the echinocyte was estimated to be 23.7x10  2 cm  using surface chemical and c e l l electrophoresis techniques (Chapter 3).  The  surface area of the echinocyte i s therefore much smaller than that of the spherocyte. 5.2  E l e c t r o n microscopic examination  canalicular system may  showed that the surface-connected  not have t o t a l l y evaginated i n the echinocyte,  therefore supporting the surface area calculations. 5.3  Aggregometry studies demonstrated a biphasic requirement  ++ cations Ca  for the divalent  ++ or Mg  to support ADP  induced aggregation. The optimum ++ . ..... . ++ concentration i s 4 mM for both cations but Ca i s more e f f i c i e n t than Mg ++ ++ i n supporting aggregation. Sr does not support aggregation while Mn i n h i b i t s aggregation. 5.4  Microelectrophoresis studies revealed an eight fold increase i n the  density of C a  + +  binding sites on the p l a t e l e t surface during discocyte to  - 205 -  echinocyte transformation. sites was over six f o l d .  The increase i n the density of Mg  The spherocyte which has lost i t s a b i l i t y to  aggregate also lost over 60% of i t s Ca 5.5  ADP  binding  binding s i t e s .  induced aggregation requires the presence of fibrinogen (Chapter 2).  The presence of fibrinogen also interferes with the lactoperoxidase l a b e l l i n g of GP-III on the ADP activated p l a t e l e t surface (Chapter 4).  Similar concen-  t r a t i o n dependences were found between the two phenomena. 5.6  Although spherocytes have lost their a b i l i t y to aggregate they can  still  be agglutinated with r i s t o c e t i n and a plasma component, presumably von Willebrand s factor. 1  5.7  Neuraminidase  treatment has no effect on ADP  induced aggregation but the  p l a t e l e t ' s responses to d i f f e r e n t l e c t i n s are altered.  The removal of terminal  s i a l i c acid residues and the exposure of galactose by the enzyme decreases the aggregation response to WGA  5.8  and increase the responses to RCA and  JBA.  E l e c t r o k i n e t i c analysis of l i v e , fixed and neuraminidase or alkaline  phosphatase  treated p l a t e l e t s showed major differences i n charge densities as  well as amino, s i a l i c acid and phosphate group densities on the discocyte, echinocyte and spherocyte.  The evaginated "new"  membrane surfaces of the  echinocyte and spherocyte seem also considerably d i f f e r e n t .  Data are  summarized i n Table 16.  5.9  SDS-PAGE of p l a t e l e t s radiolabelled using a variety of methods including  lactoperoxidase (single and double) iodination, periodate-borohydride t r i t i a t i o n and neuraminidase/galactose oxidase-borohydride t r i t i a t i o n f a i l e d  - 206 -  to show any s i g n i f i c a n t differences i n the gel patterns of the discocyte, echinocyte and spherocyte.  No new glycoprotein species appeared after the  transformations but quantitative differences among the three forms of p l a t e l e t s were found i n the r a d i o l a b e l l i n g experiments. 5.10  Considering the amounts of s i a l i c acid removed from the p l a t e l e t forms  by neuraminidase and the quantitative differences i n r a d i o l a b e l l i n g experiments, i t appears that the "new membrane" surfaces of the echinocyte and spherocyte have lower densities of glycoproteins than the discocyte surface. In the o r i g i n a l hypothesis, i t was proposed that the discocyte surface-connected  canalicular membrane system i s inherently "sticky".  The  apparent increase i n surface area associated with the discocyte to echinocyte transformation was thought to occur v i a the evagination of this membrane system.  Since there i s no evidence that p l a t e l e t s synthesize membrane, the  "pre-formed" membrane of the canalicular system seems to be the most l i k e l y , i f not the only, choice f o r the source of the added area.  However, whether  this c l o i s t e r e d membrane system i s also inherently " s t i c k y " i s another matter.  Evagination of the canalicular membrane system by hypotonic shock  f a i l e d to create a spherocyte that was capable of fibrinogen-dependent aggregation, even though i t was found that the spherocyte had a much larger surface area than the echinocyte, indicating a greater degree of evagination.  It i s shown i n Table 16 that the echinocyte "new membrane" has lower densities of the various charge groups than the discocyte surface. Its evagination w i l l therefore cause a d i l u t i o n effect r e s u l t i n g i n the lowering of the average surface charge density on the p l a t e l e t echinocyte.  - 207 Lowering of surface charge i n p r i n c i p l e could f a c i l i t a t e contact and aggregation.  platelet-platelet  However, the spherocyte has an even lower negative  charge density than that of the echinocyte but i t s surface i s non-sticky the spherocytes do not aggregate.  and  Therefore i t seems u n l i k e l y that  " s t i c k i n e s s " results simply from the lowering of p l a t e l e t surface charge density.  Two  other properties that can be considered as a c h a r a c t e r i s t i c of  " s t i c k i n e s s " , i . e . the appearance of C a / M g ++  fibrinogen binding s i t e s were observed here.  ++  binding s i t e s and of They are unique to the  echinocyte and were not observed on the discocyte or spherocyte.  GP-III was  demonstrated i n a l l three forms of p l a t e l e t s but only those on the echinocyte surface associate with fibrinogen molecules.  This suggests that a  conformational change i n GP-III i t s e l f , or i n molecules associated with i t , i s a consequence of p l a t e l e t a c t i v a t i o n and not necessarily a r e s u l t only of shape change and area increase. In conclusion, a number of differences were demonstrated here between the surface properties of the p l a t e l e t discocyte, echinocyte and spherocyte 17).  (Table  However, the data f a i l to support the model that p l a t e l e t " s t i c k i n e s s "  i s a result only of c a n a l i c u l a r system evagination or that the c l o i s t e r e d membrane of the canalicular system i s inherently " s t i c k y " .  It appears more  l i k e l y that changes occur over the whole p l a t e l e t surface during a c t i v a t i o n and make them capable of p a r t i c i p a t i n g i n aggregation reactions.  - 208 Table 17.  Highlights of Differences* Between Platelet Discocyte, Echinocyte, and Spherocyte  Area (cm ) 2  Discocyte  Echinocyte  Spherocyte  16.4xl0~  23.7xl0~  36.7xl0~  8  8  8  Negative charge density per cm  9.13xl0  12  7.92xl0  12  6.50xl0  12  Ammo groups per cm'  1.33xl0  12  1.19xl0  12  1.08xl0  12  S i a l i c acid molecules per cm  3.98xl0  12  3.52xl0  12  2.12xl0  12  Phosphate groups per cnr-  2.28xl0  12  1.96xl0  12  1.96xl0  12  2  2  S i a l i c acid/Total charge  43.5%  44.4%  32.6%  Phosphate/Total charge  25.0%  24.6%  30.1%  1.75  1.80  1.08  1.28xl0  1.09xl0  2.22xlO  S i a l i c acid/Phosphate Ca  + +  binding sites per cm  2  12  13  Fibrinogen association with membrane  No  Yes  No  Aggregation  No  Yes  No  Membrane "stickiness"  No  Yes  No  *No differences found i n their SDS-PAGE patterns.  n  - 209 -  REFERENCES  -  210 -  Abramson, H.A. 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Acta, 515:163-205.  - 225  -  APPENDIX  STATISTICAL CONSIDERATIONS FOR THE Ca++ AND Mg** BINDING EXPERIMENTS*  The d e n s i t i e s  of the d i v a l e n t c a t i o n i c ( C )  binding sites  + +  on p l a t e l e t s  were determined a c c o r d i n g t o the f o l l o w i n g e q u a t i o n p r e s e n t e d i n Chapter  _!_ _ J _ , _L_  AC  K  "  2en * 2enK  _  exp(AG/kT)  '  55 6  tC ]exp(2eVkT) ++  '  o b t a i n a l i n e whose s l o p e r e p r e s e n t s /^enK  p a r t i c u l a r j^cy  •  Chapter  n  e  r e g r e s s i o n was used t o  i n t e r c e p t w i t h the  the mean m o b i l i t y f o r t h a t p o i n t , i . e :  are a c c o r d i n g t o K a l b f l e i s c h  of the e q u a t i o n and a f u l l 3.  t  n e a r  deviation  * A l l s t a t i s t i c a l methods + Details  l"i  ^he c o e f f i c i e n t of v a r i a t i o n ( c . v . ) f o r a  was equal to the c . v . o f  where s . d . = s t a n d a r d  +  1  was p l o t t e d a g a i n s t ^c**]exp(2e^/(<T)  o r d i n a t e g i v e s /^en  3 :  (1974).  l i s t of the symbols can be found  in  -  226  -  This i s derived by c a l c u l a t i n g the absolute value of the f i r s t derivative of l ^ a : S(ACf)  S(ka) =  Therefore  %{\<j)  (ACrf _  (VACT) since  S ( A a ) SOu) A O  cr = constant * JJ  Hence, the uncertainty i n (lAc) provided  i s given by the uncertainty i n (u),  both uncertainties are expressed as fractions of the appropriate mean.  In situations l i k e the present  one, where r e c i p r o c a l values  are plotted  against each other, linear regression i s v a l i d only i f the variances points are not s i g n i f i c a n t l y d i f f e r e n t from each other. variance of the f i r s t J&cr the variances point.  compared to  l ^ c r data points on the same l i n e point by  Pairs of variances were tested using the F - d i s t r i b u t i o n and a  i n t e r v a l of 95%.  It was  data  To t e s t t h i s , the  data point on each regression l i n e was  of a l l the other  of the  confidence  determined that the variances for the data points  each regression l i n e were not s i g n i f i c a n t l y d i f f e r e n t from each other.  along  The  regression lines i n F i g . 23 are therefore v a l i d . S t a t i s t i c a l comparisons f o r the binding site densities (N) and binding free energy (AG) between different platelet forms and cations were achieved  by  comparing the regression lines concerned using analysis of variance tables. They are shown below.  The number of degrees of freedom i s based on the number  of points on each l i n e i l l u s t r a t e d i n the f i g u r e s , each point representing the average of at l e a s t 100 individual mobility determinations.  Significance  between the two intercepts r e f l e c t s the significance between the binding site densities while that between the slopes r e f l e c t s  the s i g n i f i c a n c e between t h e A G .  - 227 Ca  binding;  Source of V a r i a n c e  -  comparing d i s c o c y t e vs e c h i n o c y t e : —  Degree of Freedom  Total  Sum of Squares  19  3.33 x 10-5  I nt er ce pt  1  1.64  Slope  1  4. 77 x 10-6  Residual  Intercept Slope  16  Source of V a r i a n c e Total  + +  binding;  x IO"  4. 27 x I O "  0.025 < p < 0.05; 0.001 < p < 0.0005; Mg  significant significant  Degree of Freedom  Sum of Squares  1.63 x  IO  Mean Square R a t i o  - 5  Slope  1  5. 18 x 10-6  16  7.44 x 10-7  - 6 1 4 -  23  55.69  0. 005 < p < 0. 0 1 ; significant difference, p < 0.0005; s i g n i f i c a n t d i f f e r e n c e . D i s c o c y t e s ; comparing C a  Source of V a r i a n c e  Degree of Freedom  + +  vs M g  binding  + +  Sum of Squares  15  2.46 x 10"5  I nt er ce pt  1  5.33 x 1 0 ~  Slope  1  6. 78 x 10-8  12  4. 19 x 10-6  Intercept Slope  -  echinocyte  1. 32 x I O  Res i dual  17. 86  difference. difference.  1  Total  6. 14  6  I nt er ce pt  Int er ce pt Slope  -  6  comparing d i s c o c y t e vs  19  R e s i dual  Mean Square R a t i o  0.50 < p < 0. 75 ; 0.50 < p < 0 . 7 5 ;  no s i g n i f i c a n t no s i g n i f i c a n t  :-  Mean Square R a t i o  8  difference. difference.  0. 1525 o.1941  - 228 Echinocyte; Source of V a r i a n c e  comparing C a  Degree of Freedom  Total  + +  vs M g  binding: —  + +  Sum of Squares  Mean Square R a t i o  15  2.20 x  10  - 5  -  I nt er ce pt  1  2.91 x  10  - 5  0.04  Slope  1  7. 36 x  10~  12  6.88 x  IO  Res i dual Intercept Slope  0.50 < p < 0. 75 ; 0.25 < p < 0. 50; Ca  + +  Source of V a r i a n c e  binding;  1.07  -  - 8  no s i g n i f i c a n t d i f f e r e n c e no s i g n i f i c a n t d i f f e r e n c e .  comparing d i s c o c y t e vs s p h e r o c y t e : —  Degree of Freedom  Total  8  Sum of Squares  Mean Square R a t i o  15  4.80 x  IO"  I nt er ce pt  1  3.71 x  10-^  345.57  Slope  1  4.48 x  10"  41.68  12  1.29 x  10-5  Res i dual Int er ce pt Slope 0.01 Ca  p < 0. 005; < p < 0.025; + +  Source of V a r i a n c e  binding;  significant significant  5  -  difference difference  comparing e c h i n o c y t e vs s p h e r o c y t e :-  Degree of Freedom  Total  -  4  Sum of Squares  Mean Square R a t i o  15  5.21 x  KT*  I nt er ce pt  1  4.15 x  IO *  510. 96  Slope  1  4.92 x  10-5  60. 58  Residual Intercept Slope  12  0.005 < p  p < 0.005; < 0.01;  9.75  X  -  IQ-6  significant difference significant difference.  —  

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