Calcium Regulation by Calcium Antagonists - American Chemical

9 Relationship of Cyclic Nucleotides and Calcium

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in Platelet Function G. C. LE BRETON, Ν. E. OWEN, and H. FEINBERG University of Illinois College of Medicine, Department of Pharmacology, Chicago, IL 60612 Platelets are intimately involved in the prevention of blood loss upon vascular damage as well as in the genesis of certain forms of cardiovascular disease. Although the precise mechanisms involved in the control of platelet function are unknown, Ca and adenosine 3'5'-cyclic monophosphate (cAMP) appear to modulate the levels of platelet reactivity. In this regard, platelet activation is generally associated with an increase in the concentration of cytosolic Ca . Thus, an elevation in the levels of intraplatelet Ca , whether as a consequence of increased platelet membrane permeability to Ca or release of internal Ca , is linked to the processes of shape change, aggregation and secretion. On the other hand, agents which stimulate adenylate cyclase activity, e.g., prostacyclin or prostaglandin E , are potent inhibi tors of platelet function. It has been suggested that cAMP inhibits platelet activation by reducing the availability of cytosolic Ca . In this connection, it has been shown that cAMP promotes the uptake of Ca into an isolated platelet membrane fraction resembling the sarcoplasmic reticulum of muscle. Furthermore, in intact platelets, increases in cAMP are directly related to enhanced intraplatelet Ca sequestration and reduced Ca mobilization in response to various agonists. This ability of cAMP to modulate platelet Ca levels may be related to the phosphorylation of specific platelet proteins involved in a cAMP-dependent Ca pump. 2+

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1

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Blood platelets circulate in the vascular system as discrete discoid-shaped cells approximately 2-3 ym in diameter. Although anucleated, platelets contain mitochondria, glycogen particles 0097-6156/82/0201-0153$06.50/0 © 1982 American Chemical Society

Rahwan and Witiak; Calcium Regulation by Calcium Antagonists ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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CALCIUM REGULATION BY CALCIUM ANTAGONISTS

and various types of storage o r g a n e l l e s . Osmophilic granules c a l l e d dense bodies are the storage s i t e s for adenosine d i p h o s phate (ADP), adenosine t r i p h o s p h a t e (ATP), s e r o t o n i n (5-HT) and C a (I). Various p r o t e i n s and p l a t e l e t f a c t o r (4) (2) are stored i n another p o p u l a t i o n of granules designated alpFa granules. In a d d i t i o n , p l a t e l e t s possess a bundle of m i c r o t u bules which course around the circumference of the c e l l just beneath the plasma membrane (3). I t has been suggested that these microtubules f u n c t i o n to maintain the d i s c o i d shape of the unstimulated p l a t e l e t (4_) as well as to f a c i l i t a t e the s e c r e t i o n of the platelet granules during c e l l u l a r a c t i v a t i o n ( 5 J . P l a t e l e t s also possess two i n t e r n a l membranous s t r u c t u r e s ; one i s termed the external c a n a l i c u l a r system which has been shown to be an i n v a g i n a t i o n of the outer plasma membrane ( 6 ) . The other i s termed the dense t u b u l a r system which i s belTeved to f u n c t i o n i n a manner analogous to the sarcoplasmic r e t i c u l u m of muscle i n r e g u l a t i n g c y t o s o l i c C a l e v e l s (7_). When i s o l a t e d p l a t e l e t s are exposed to v a r i o u s p h y s i o l o g i c a l a g o n i s t s , e . g . ADP, they are t r i g g e r e d to undergo a sequence of morphological and f u n c t i o n a l a l t e r a t i o n s . One of the first events i n v o l v e s a t r a n s i t i o n from smooth discoid-shaped c e l l s to c e l l s which are more s p h e r i c a l i n shape with blebs or pseudopodia projecting from their surface. The p l a t e l e t shape change response i s followed by a change i n the adhesive p r o p e r t i e s of the external p l a t e l e t membrane which f a c i l i t a t e s p l a t e l e t p l a t e l e t adhesion. Under these c o n d i t i o n s the p l a t e l e t s begin to clump together or aggregate. A separate phenomenon, described by G r e t t e (8) as the r e l e a s e r e a c t i o n , i s the s e c r e t i o n of granular c o n s t i t u e n t s from the alpha granules and the p l a t e l e t dense bodies. This process appears to be an a m p l i f i c a t i o n mechanism whereby the r e l e a s e d substances, e . g . ADP, s e r o t o n i n , C a , e t c . , promote surround ing p l a t e l e t s to undergo f u r t h e r aggregation. This i s presumably the mechanism of p l a t e l e t plug formation which acts to prevent the loss of blood from a damaged vessel i n v i v o (9). Under i n v i t r o c o n d i t i o n s the p l a t e l e t response of shape change followed by aggregation followed by s e c r e t i o n appears to be a coordinated sequence of a s i n g l e a c t i v a t i n g event (10). On the other hand, aggregation need not be preceded by shape change ( e . g . as a f t e r a d d i t i o n of epinephrine) and s e c r e t i o n can occur as the i n i t i a l event. Thus shape change, aggregation and s e c r e t i o n are independent processes; however, they appear to be c o n t r o l l e d by a common i n t r a c e l l u l a r messenger, i.e. ionic calcium. 2 +

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Calcium a c t i v a t i o n of p l a t e l e t

function 2 +

There i s compelling evidence to suggest that C a serves as a common mediator of p l a t e l e t f u n c t i o n a l change. Thus, a d d i t i o n of the d i v a l e n t c a t i o n ionophore, A23187, to a p l a t e l e t


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ET AL.

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Nucleotides,

Ca,

and

Platelets

155

suspension r e s u l t s i n shape change, aggregation and s e c r e t i o n (11-17). Since A23187 can induce a net uptake of C a i n many c e l l types (11), i t was suggested that p l a t e l e t a c t i v a t i o n i s t r i g g e r e d by Tue C a brought i n by the ionophore. However, A23187-stimulation of both p l a t e l e t shape change and s e c r e t i o n also occurs in the absence of external C a (11,12,13,17). Therefore, the C a involved i n t h i s process oT" a c t i v a t i o n must be derived from an i n t r a p l a t e l e t source. On t h i s basis i t was proposed that p l a t e l e t s maintain an i n t e r n a l l y r e l e a s e a b l e pool of C a , which when m o b i l i z e d , serves to i n i t i a t e p l a t e l e t f u n c t i o n a l change. One p o s s i b l e source f o r t h i s releaseable Ca i s the p l a t e l e t dense t u b u l a r system (DTS) ( 7 ) . Thus, a stimulus which causes the release of C a from t u i s membrane system would u l t i m a t e l y lead to an e l e v a t i o n of c y t o s o l i c C a levels. Although t h i s model was c o n s i s t e n t with r e s u l t s obtained using A23187, which i s known to a l t e r i n t r a c e l l u l a r c a t i o n gradients (18,19>), a s i m i l a r mechanism of a c t i o n by p h y s i o l o gical platelet agonists, e.g. ADP, epinephrine, thrombin, thromboxane A2, e t c . had not been demonstrated. 4 5

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2

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2

4

ADP-stimulated Ca " " m o b i l i z a t i o n . On t h i ^ b a s i s , we i n v e s tigated! ân increase Tn i n t r a c e l l u i a r C a as a p o s s i b l e mechanism of p l a t e l e t a c t i v a t i o n induced by ADP. In these studies i t was f i r s t demonstrated that ADP-stimulated shape change and aggregation i s not associated with a transmembrane Ca influx (20). This observation was subsequently confirmed by Massini and Luscher {21). Based on these f i n d i n g s i t was concluded that an e l e v a t i o n i n c y t o s o l i c C a in response to ADP must be derived from i n t r a - p l a t e l e t storage s i t e s . In order to pursue t h i s question we developed a technique which permitted the measurement of intracellular Ca movements i n i n t a c t p l a t e l e t s (22). This was accomplished through the use of a C a ^ - s e n s i t i v e fluorescent probe, c h l o r t e t r a c y c l i n e (CTC), and a photon counting m i c r o s p e c t r o fluorometer. CTC had been p r e v i o u s l y used as a means of monitoring changes in i n t r a c e l l u l a r C a b i n d i n g i n many c e l l systems i n c l u d i n g sarcoplasmic r e t i c u l u m (23), red blood c e l l s (24) and mitochon d r i a ( 2 5 ) . CTC i s known to form a h i g h l y f l u o r e s c e n t pH i n s e n sitive~~[pH 6 . 0 - 8 . 5 ) adduct when chelated with d i v a l e n t c a t i o n s bound to b i o l o g i c a l membranes (26). Even though CTC c h e l a t e s both M g and Ca , i t was p r e v i o u s l y reported using red blood c e l l s (24) and confirmed by our measurements i n p l a t e l e t s (22) that the observed fluorescence stems almost e n t i r e l y from the Ca-CTC adduct. The c h a r a c t e r i s t i c which makes CTC a v a l u a b l e i n t r a c e l l u l a r C a ? probe i s that the fluorescence i n t e n s i t y of the Ca-CTC complex markedly decreases when the i s released from the membrane to a more p o l a r environment, e . g . the c y t o s o l (27_). Consequently, r e l a t i v e changes i n c e l l u l a r fluorescence 2 +

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156


can be used as an index of t r a n s i e n t a l t e r a t i o n s i n the a v a i l a b i l i t y of i n t r a p l a t e l e t i o n i c c a l c i u m . Using t h i s procedure we demonstrated that the a d d i t i o n of ADP (1 μΜ) to p l a t e l e t r i c h plasma r e s u l t e d i n a m o b i l i z a t i o n of platelet C a (as evidenced by a decrease i n fluorescence) which occurred c o i n c i d e n t with a change in platelet shape (Figure 1). In order to e l i m i n a t e the p o s s i b i l i t y that the observed change in fluorescence represented alterations in C a * binding to the p l a t e l e t external membrane, the external Ca pool was e l i m i n a t e d by p r i o r a d d i t i o n of EGTA (3 mM). Furthermore, when the p l a t e l e t s were p r e t r e a t e d with ATP, which acts as a s p e c i f i c ADP antagonist (28), p l a t e l e t shape change and the change i n Ca-CTC fluorescence were both i n h i bited. It can also be seen that s t i m u l a t i o n of p l a t e l e t shape change by the d i v a l e n t c a t i o n ionophore, A23187, was a s s o c i a t e d with a s i m i l a r r e d i s t r i b u t i o n of i n t r a p l a t e l e t C a . These f i n d i n g s provided evidence that p l a t e l e t a c t i v a t i o n induced by ADP i s mediated through an i n t e r n a l r e l e a s e of membrane bound Ca . T h i s notion was supported by a d d i t i o n a l studies i n which platelet water was totally exchanged with deuterium oxide (D2O) (29). D2O had been p r e v i o u s l y shown to block the r e l e a s e of C a from the sarcoplasmic r e t i c u l u m of barnacle muscle and inhibit muscular contraction (30). Since the p l a t e l e t DTS i s thought to f u n c t i o n i n a manner analogous to the sarcoplasmic reticulum i n muscle, we examined the p o s s i b i l i t y that D2O would have a s i m i l a r i n h i b i t o r y e f f e c t i n p l a t e l e t s . T h i s was indeed found to be the case, i . e . , D2O i n h i b i t e d both shape change and aggregation stimulated by ADP (29). Other studies have suggested that D?0 treatment (2^60%) can r e s u l t i n enhanced aggregation (31). In these experiments, however, aggregation was induced with the d i v a l e n t cation ionophore A23187, r a t h e r than ADP. Since i t i s known that D2O will also facilitate Ca-stimulated muscle contraction (32,33) the effects of D2O on ionophore-induced platelet a c t i v a t i o n would be c o n s i d e r a b l y d i f f e r e n t from i t s e f f e c t s on ADP-induced p l a t e l e t a c t i v a t i o n . Thus^ with A23187, D 0 would augment the e f f e c t s of a v a i l a b l e C a whereas with ADP, D2O would i n h i b i t C a release. 2 +

2

2

2 +

2 +

2

2

2

2

Epinephrine-stimulated C a * i n f l u x . Our r e s u l t s with D2O a l s o provided evidence that a d i f f e r e n t p l a t e l e t a g o n i s t ^ i . e . epinephrine, does not act through the same mechanism of C a m o b i l i z a t i o n as does ADP. In t h i s regard, D2O was not found to be effective in blocking epinephrine-induced platelet a c t i v a t i o n (29). T h e r e f o r e , we i n v e s t i g a t e d the possibility that epinephrine, in contrast to ADP, increases platelet membrane p e r m e a b i l i t y to C a . Using t r a c e amounts of Ca i t was demonstrated that epinephrine does i n f a c t induce a transmembrane C a i n f l u x which i s concomitant with the 2

2

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4 5

9.

L E BRETON ET AL.

Cyclic

Nucleotides,

Τ 1

Ca, and Platelets

157

' % c h o n g e m light scatter (90°)

Δ

Δ23Ι87 I ' M F (sec) Figure 1. Relationship between light scattering ( ) and fluorescence ( ) of platelets containing chlortetracycline. (Reproduced, with permission, from Ref. 22. Copyright 1976, Biochemical and Biophysical Research Communications.) Human platelets were incubated at 25°C with 50 μΜ CTC for 40 min. The plasma was supplemented with 3 mM EGTA to prevent aggregation. Platelets from a portion of the plasma were then pelleted through silicone oil, andfluorescencewas determined in a microspectrofluorometer (400 nm excitation: 530 nm emission). A: ADP (1 μΜ) was added to the plasma, and samples were pelleted and analyzed forfluorescenceat 60, 130, and 220 s; each time point represents the mean ± SEM of 25 samples from three different blood donors. Platelet shape change in response to ADP (1 μΜ) was separately determined by measuring the intensity of scattered light at 90°C. The change in platelet shape represents a typical platelet response in the presence of 50 μΜ CTC. B: Inhibition of ADP induced shape change and plateletfluorescencechange by treatment with ATP (10 Μ). ADP (1 Μ) was added 60 s after ATP. Thefluorescencevalues at each time point represent the mean ± SEM of 16 samples from two different blood donors. C: Relationship between platelet shape change induced by A23187 (1 μΜ) and fluorescence change. The fluorescence values at each point represent the mean ± SEM of eight samples from two blood donors. μ

μ


CALCIUM

158

R E G U L A T I O N BY C A L C I U M

ANTAGONISTS

4 b

aggregation response (34_) (Figure 2 ) . This C a uptake was dependent upon the dose of epinephrine employed (10-0.1 μΜ) and was not stimulated by d-epinephrine which is biologically i n a c t i v e ( 3 A ) . Evidence that a change in p l a t e l e t membrane permeability to Ca was specifically mediated through a receptor i n t e r a c t i o n was provided by the f i n d i n g that the α - r e c e p t o r a n t a g o n i s t , phentolami ne, blocked C a uptake as well as p l a t e l e t aggregation (34). F i n a l l y , pretreatment of the platelets with v e r a p a m i l , "which selectively blocks Ca channels in c a r d i a c c e l l s , i n h i b i t e d both e p i n e p h r i n e - s t i m u l a t e d ^ C a uptake ( F i g u r e 3) and p l a t e l e t aggregation but d i d not i n h i b i t aggregation s t i m u l a t e d by ADP (34). The i n h i b i t i o n b^ verapamil could in turn be reduced by external Ca supplementation. These f i n d i n g s provided evidence that the mechanism by which epinephrine stimulates p l a t e l e t f u n c t i o n a l change i s through enhanced membrane p e r m e a b i l i t y to c a l c i u m . This e f f e c t of epinephrine appears to be in marked c o n t r a s t to the mechanism of A D P - s t i m u l a t i o n which i s presumably mediated through i n t e r n a l Ca release. 2 +

4 5

2 +

2

2 +

ADP-stimulated Na* i n f l u x . Although the nature of the ADP i n t e r a c t i o n with p l a t e l e t receptors to induce calcium r e d i s t r i bution i s unknown, i t i s c l e a r t h a t ADP does not penetrate the membrane (35). I t i s p o s s i b l e that the A D P - p l a t e l e t i n t e r a c t i o n involves the f l u x of other ions as part of the s t i m u l u s - t r a n s f e r pathway. In t h i s r e s p e c t , p l a t e l e t s , l i k e other c e l l s , maintain low i n t r a c e l l u l a r Na l e v e l s and high K l e v e l s (36J; presum a b l y , as a consequence of an Na e f f l u x pump. We therefore investigated the possibility that ADP-induced platelet a c t i v a t i o n i s associated with an a l t e r e d f l u x of N a . ADP added to r e s t i n g p l a t e l e t s induced a sudden r i s e i n N a uptake, an increase i n p l a t e l e t N a , and a decrease i n K (Figure 4) (37). E a r l i e r s t u d i e s had shown that p l a t e l e t water volume does not increase a f t e r ADP-induced aggregation (38), and using C 1 , we found no increase i n C 1 uptake (37). An uptake of Na was not seen after GDP a d d i t i o n . Furthermore, the uptake of N a occurred s i m u l t a n e o u s l y w i t h the onset of shape change and aggregation (37^,39^,40). On the other hand, epinephrine-induced aggregation, shown e a r l i e r to induce Ca uptake, had no e f f e c t to induce Na uptake (Figure 5) (39^,40). Consequently i t appears that c a t i o n i n f l u x i s agonist s p e c i f i c , at l e a s t with respect to a c t i v a t i o n by ADP or by epinephrine. Whether the uptake of Na associated with ADP-induced shape change and aggregation i s the primary s t i m u l u s - t r a n s f e r pathway of a c t i v a t i o n has not been e s t a b l i s h e d . Platelets maintain a membrane p o t e n t i a l (negative inside) (41), and a c t i v a t i o n may ensue i n a s s o c i a t i o n with a chancje i n membrane p o t e n t i a l and an increase i n p e r m e a b i l i t y to Na . A sudden +

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2 2

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3 6

3 6

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2 2

4 5

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2 2

+


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L E BRETON E T AL.

Cyclic

Nucleotides,

Ca, and Platelets

42

159

Epi ( I 0 M ) 6

a

Q-

JADP(IO" M) 6

o

Saline

σ O

60

150

240

Time (sec) Figure 2. or ADP.

Ca uptake associated with primary aggregation induced by epinephrine (Reproduced, with permission, from Ref. 34. Copyright 1980, The American Physiological Society.) 2+

Aspirin (1 mg/mL) or indomethacin (20 μΜ) treated human blood platelets were isolated by albumin density gradient centrifugation and resuspended in Tyrode buffer (pH 7.4) in which the ionic calcium concentration had been adjusted to 100 μΜ with CaCl . Thirty min after CaCl addition, the platelet suspension was supplemented with fibrino gen (0.1 mg/mL), I-human serum albumin, and Ca. Three min later, aggregation was induced by epinephrine (1 μΜ), or ADP (1 μΜ), and 1 mL aliquots of the platelet suspension" were withdrawn at 60, 150, and 240 s following the addition of aggregating agent. The platelets were then analyzed for Ca * uptake. Each point represents the mean ± SEM of 16 samples from four blood donors. 2

2

125

45

2

Figure 3. Inhibition of epinephrine-induced Ca uptake by verapamil in resus pended platelets. (Reproduced, with permission, from Ref. 34. Copyright 1980, The American Physiological Society.) 2+

Platelets were isolated and Ca uptake was determined (conditions were as stated in Figure 2). Resuspended platelets were incubated with verapamil (50 μΜ) for 1 min before addition of epinephrine (1 μΜ). Each point represents mean ± SEM of 28 samples from seven separate blood donors. 2+


CALCIUM REGULATION BY CALCIUM

160

ANTAGONISTS

Figure 4. Effect of ADP on platelet Na and K and Na 'space.' Platelet rich plasma was incubated at 37°C in the absence (panel A) or presence (panel B) of ouabain (1 μΜ). The arrow indicates the time of addition of ADP to an aliquot of the sample. Key: Φ, Na+ 'space'; Δ , Na content; and O , K content of the unstimulated platelets. The separate symbols aligned with the arrow depict the same measurements obtained 60 s after the addition of ADP. Samples were taken at slightly different times in four experiments; the time ranges are shown by the horizontal bars. Each point represents the mean ± SEM. (Reproduced, with permission, from Ref. 37. Copyright 1977, Elsevier Biomedical Press B.V.) +

22

+

+

22

+

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Nucleotides,

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Ca, and Platelets

AN OVA TABLE SS DF MS F ADP 250 3 0833 BetweenEPI , OU 3 0038 ADP-14 124 t. ADP

Within EPI

620 105

005E

.567 124

004*

Epi -0828

ω Q) Q.

σ Φ c

Q.

Control t 60" Treatment

120"

180

Time after Agonist Figure 5. Effect of epinephrine on Ν a* uptake by platelets. Uptake of Na* (neq/ JO platelets) is based on an average value of 165 meq/1 of Ν a in cit rated plasma. Aggregation initiated by ADP 10 μΜ) or by epinephrine (fâ, 10 μΜ); values represent mean ± SEM of 10 experiments. (Reproduced, with permission, from Ref. 40. Copyright 1981, Elsevier Biomedical Press B.V.) 8


162


+

r i s e i n p l a t e l e t N a l e v e l s could f u n c t i o n to a c t i v a t e p l a t e l e t s by d i s p l a c i n g C a ^ from bound s i t e s . This p o s s i b i l i t y i s c o n s i s t e n t with the f i n d i n g s of S t e i n e r and T a t e i s h i (42J that Ca^ s e q u e s t r a t i o n by p l a t e l e t membranes i s i n h i b i t e d by N a and enhanced by K . In t h i s connection we found that ouabain, which increases the Na level in p l a t e l e t s , induces an increased s e n s i t i v i t y to both ADP and to epinephrine a c t i v a t i o n (39). +

+

+

+

+

Involvement of Ca^* i n p o t e n t i a t e d aggregation. I t has been known f o r some time that Tôw doses of epinephrine wi 11 p o t e n t i a t e p l a t e l e t aggregation induced by ADP ( 4 2 , 4 4 ) . We next i n v e s t i g a t e d whether these same d i s t i n c t mechanisms of ADP and epinephrine are o p e r a t i v e during p o t e n t i a t i o n of aggregation by each agonist ( 4 5 ) . On t h i s b a s i s we examined whether the mechanism of epinephrine p o t e n t i a t i o n i s due to a l t e r a t i o n s i n membrane p e r m e a b i l i t y to C a ^ . I t was found that a requirement for epinephrine p o t e n t i a t i o n i s indeed a transmembrane Ca f l u x , since the p o t e n t i a t e d response was completely a b o l i s h e d by verapamil. This i n h i b i t i o n was i n turn p a r t i a l l y reversed by Ca supplementation. Based on these r e s u l t s , i t was oroposed that low doses of epinephrine induce an uptake of C a ^ which promotes the a b i l i t y of ADP to r e d i s t r i b u t e C a from i n t e r n a l Ca stores. This p o t e n t i a t i o n could proceed through a process of c a l c i u m - i n d u c e d - c a l c i u m r e l e a s e as has been suggested to occur i n the sarcoplasmic r e t i c u l u m of muscle c e l l s ( 4 6 ) . Support f o r t h i s notion was provided in a study which i n v e s t i g a t e d the e f f e c t s of low dose epinephrine on i n t r a - p l a t e l e t C a ^ b i n d i n g . Using i n t a c t p l a t e l e t s and the C a - s e n s i t i v e probe CTC, we found that sub-aggregatory doses of epinephrine can indeed lead to a m o b i l i z a t i o n of i n t r a p l a t e l e t C a ^ , s t o r e s (47) (Figure 6 ) . An e s s e n t i a l feature of t h i s " e p i n e p h r i n e stimulated C a ^ , r e d i s t r i b u t i o n " was a transmembrane Ca^ f l u x , since the m o b i l i z a t i o n process was blocked by v e r a p a m i l . Thus, i t appears that an i n i t i a l increase i n i n t r a p l a t e l e t C a , as a consequence of C a ^ uptake, can i n turn promote the release of C a from i n t e r n a l b i n d i n g s i t e s . The s i g n i f i c a n c e of the i n t e r n a l C a ^ r e l e a s e observed with epinephrine i s u n c l e a r . Presumably i t does not c o n s t i t u t e a major source of the increase i n c y t o s o l i c C a ^ , s i n c e D^O which would be expected to s t a b i l i z e the DTS does not block epinephrine-induced aggregation. Rather, this ability of C a to stimulate additional Ca^ release may serve as an amplification mechanism by which the e f f e c t of C a ^ i n f l u x i s enhanced. +

2 +

2 +

+

2 +

2 +

+

2 +

+

+

+

2 +

+

2 +

+

+

2 +

+

+

d

Ca m o b i l i z a t i o n by metabolites of arachadonic a c i d . One p o s s i b l e mechanism by which t h i s a m p l i f i c a t i o n process could proceed i s through C a ^ a c t i v a t i o n of enzymes involved i n arachadonic a c i d (AA) metabolism. In t h i s regard, i t i s known that blood p l a t e l e t s metabolize endogenous AA to the potent +


9.

LE BRETON ET AL.

Cyclic

Nucleotides,

Ca, and

163

Platelets

platelet stimulant thromboxane A2 (TXA?) (48). Thus, AA which i s e s t e r f i e d i n the phospholipid pool of the p l a t e l e t i s l i b e r a t e d by the a c t i o n of phospholipase C (or A?) (49,50). The f r e e AA i s then acted upon by a e y e l o - o x y g é n a s e r e s u l t i n g i n the production of the p r o s t a g l a n d i n endoperoxide, p r o s t a g l a n d i n G2 ( P G G 2 ) . PGG2 i s i n t u r n r a p i d l y converted to p r o s t a g l a n d i n H2 ( P G H 2 ) which serves as substrate f o r the enzyme thromboxane synthetase. The rate l i m i t i n g step i n T X A 2 prod u c t i o n appears to be at the l e v e l of the phospholipase. Since phospholipase C or A 2 are C a - a c t i v a t e d enzymes (49,50J, it i s p o s s i b l e that the a b i l i t y of C a to cause a d d i t i o n a l " ( H r r e l e a s e may, at l e a s t i n p a r t , be mediated through C a - s t i m u lated TXA2 production. The T X A 2 so produced may then act to d i r e c t l y release C a from the p l a t e l e t DTS (51). 2 +

2 +

2

2 +

T h i s notion was examined by measuring"the e f f e c t s of the cyclo-oxygenase i n h i b i t o r , i ndomethacin, on e p i n e p h r i n e stimulated Ca redistribution. It was found (47), that i n h i b i t i o n of endogenous AA metabolism did i n f a c t s u b s t a n t i a l l y reduce the C a m o b i l i z a t i o n process (Figure 7). Consequently, it appears that at least a portion of the observed Ca release stimulated by epinephrine i s mediated through TXA2 production. Furthermore, d i r e c t evidence to support the notion that TXA2 is capable of causing Ca release within the p l a t e l e t was provided by the f i n d i n g that a d d i t i o n of the s t a b l e TXAg "mimetic", U46619, to intact platelets causes a m o b i l i z a t i o n of i n t e r n a l C a stores (Figure 8). In summary, i t appears that p h y s i o l o g i c a l stimulants of platelet function, e.g. ADP, epinephrine and T X A 2 all act through a Ca mediated process. Recently, Feinstein (52) demonstrated that the a b i l i t y of thrombin to cause plateTêt s e c r e t i o n i s a l s o r e l a t e d to the r e l e a s e of i n t r a p l a t e l e t membrane bound C a . On t h i s b a s i s , i t would seem that i n t r a platelet cytosolic Ca as a second messenger is directly l i n k e d to the state of p l a t e l e t a c t i v a t i o n . 2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

Inhibition of platelet monophosphate (cAffFT

function

by

3'-5'

cyclic

adenosine

One of the f i r s t i n d i c a t i o n s that cAMP might be i n v o l v e d in the regulation of platelet function was provided by experiments of Marcus and Zucker (53). In these studies i t was shown that the addition of cAMF" to platelets resulted in i n h i b i t i o n of p l a t e l e t aggregation. T h i s notion was supported by subsequent studies of A r d l i e et a l . (54) who demonstrated that phosphodiesterase inhibitors a l s o "THocked ADP-induced p l a t e l e t aggregation. It now appears that many of the agents, in particular the prostaglandins, which inhibit platelet a c t i v a t i o n , produce t h e i r e f f e c t through i n c r e a s e s i n p l a t e l e t cAMP l e v e l s . Thus, numerous i n v e s t i g a t o r s have demonstrated that p r o s t a g l a n d i n (PGE^) i s a potent stimulant of the



164

Figure 6. Effect of verapamil on Ca * mobilization in response to epinephrine. 2

Human platelets were incubated at 25°C with 50 Μ CTC for 40 min. CTC-treated platelets were incubated for 180 s with saline or verapamil (25 μ Μ ) alone, saline plus epinephrine (0.1 μΜ), or verapamil (25 μΜ) plus epinephrine (0.1 Μ). Platelets were then pelleted through silicone oil, and pellet fluorescence was determined as in Figure 1. A decrease influorescencecounts relative to control indicates mobilization of platelet Ca *. The fluorescence values are represented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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Figure 7. Effect of indomethacin on Ca * mobilization in response to epine phrine. 2

Platelets were incubated with CTC and fluorescence determined as described in Figure 6. CTC-treated platelets were incu bated with saline or indomethacin (20 Μ) alone, saline plus epinephrine (0.1 μΜ), or indomethacin (20 Μ) plus epinephrine (0.1 μΜ). A decrease influorescencecounts relative to control indicates mobilization of platelet Ca *. Fluorescence values are repre sented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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Platelets were incubated with CTC and fluorescence determined as described in Figure 6. Platelets were then treated with saline (control), ADP (0.5 Μ), or U46619 (0.5 μΜ). A decrease influorescencecounts relative to control indicates mobilization of platelet Ca \ The fluorescence change in response to the ADP is indicated for com parative purposes. Fluorescence values are represented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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9.

LE BRETON ET AL.

Cyclic

Nucleotides,

Ca, and

165

Platelets

p l a t e l e t adenylate cyclase (55-59). Furthermore, more recent studies have i n d i c a t e d that tïïe i n h i b i t o r y properties of prostaglandin D2 (PGD2) and p r o s t a c y c l i n ( P G I 2 ) are also p r i m a r i l y due to s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y (60,6^,62^. A d d i t i o n a l evidence to support t h i s concept i s provided by the f i n d i n g , that the e f f e c t s of the i n h i b i t o r y prostaglandins are markedly p o t e n t i a t e d by phosphodiesterase i n h i b i t o r s (63). F i n a l l y , the i n f l u e n c e of cAMP on p l a t e l e t r e a c t i v i t y i s c l e a r l y demonstrated by the f i n d i n g , that an increase of only 25% i n p l a t e l e t cAMP l e v e l s i s associated with a complete i n h i b i t i o n of maximal aggregation induced by e i t h e r AA or U46619 ( 6 4 ) . At present then, there i s a s u b s t a n t i a l body of evidence which suggests that increases i n p l a t e l e t cAMP l e v e l s are d i r e c t l y l i n k e d to i n h i b i t i o n of p l a t e l e t f u n c t i o n . In s p i t e of t h i s , however, the mechanism by which these i n h i b i t o r y e f f e c t s are produced remains u n c l e a r . 2

R e l a t i o n s h i p between C a * and cAMP There have been two t h e o r i e s proposed which attempt to e x p l a i n the u n d e r l y i n g mechanism by which cAMP i n h i b i t s blood platelet function. The f i r s t model suggests that cAMP i s d i r e c t l y involved i n the r e g u l a t i o n of c y t o s o l i c C a levels. Using an i s o l a t e d p l a t e l e t membrane f r a c t i o n r i c h i n the DTS, Kaser-Glanzmann et a l . (65) demonstrated that the C a accumu l a t i n g a c t i v i t y of the i s o l a t e d v e s i c l e s was markedly s t i m u l a t e d by cAMP. These r e s u l t s provided evidence that cAMP may act to promote the movement of C a from the p l a t e l e t c y t o s o l i n t o the platelet DTS, and were c o n s i s t e n t with the previous suggestion of White et à]_. ( 1 3 ) . The net e f f e c t of t h i s uptake mechanism would be a decrease i n the a v a i l a b i l i t y of free C a f o r the s t i m u l a t i o n of shape change, aggregation and s e c r e t i o n . D i r e c t support f o r t h i s concept was provided by experiments measuring C a binding in intact p l a t e l e t s . I t was found that a l t e r a t i o n s i n p l a t e l e t cAMP l e v e l s , induced by e i t h e r PGE^ or P G I 2 » were d i r e c t l y r e l a t e d to i n t r a p l a t e l e t C a seques t r a t i o n (47) (Figure 9) as i n d i c a t e d by CTC f l u o r e s c e n c e . Furthermore, the e f f e c t s of PGE^ or P G I on cAMP l e v e l s and Ca b i n d i n g were augmented by pretreatment of the p l a t e l e t s with the phosphodiesterase i n h i b i t o r RO 1724. In a d d i t i o n , i t was found that the increases i n cAMP, stimulated by e i t h e r PGEi or P G I 2 , were associated with an i n h i b i t i o n of C a m o b i l i z a t i o n induced by e p i n e p h r i n e , A23187 or U46619 ( F i g u r e 10). Thus, i t appears that one consequence of increased cAMP production i s a " s t a b i l i z a t i o n " of i n t r a p l a t e l e t C a pools. In t h i s c o n n e c t i o n , however, these experiments d i d not d i f f e r e n t i a t e between i n h i b i t e d C a r e l e a s e or enhanced Ca? r e s e questration. Consequently, i t i s p o s s i b l e that cAMP prevents the discharge of C a from the DTS i n a manner analogous to the proposed mechanism of i n h i b i t i o n by l o c a l a n e s t h e t i c s ( 6 6 ) , 2 +

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166


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centrations of A23187 (68-72). Consequently^ i f the rate of C a release J S greater than the rate of Ca^ resequestration, cytosolic C a l e v e l s w i l l r i s e . 3) In F i g u r e 10 i t can be seen that PGE^ or PGI treatment actually increased C a binding above c o n t r o l levels. If cAMP were only a c t i n g to block the release of C a i n the stimulated p l a t e l e t , such i n c r e a s e s i n C a binding would not be expected. Furthermore, in the presence of PGE^ or PGI plus a phosphodiesterase inhibitor, epinephrine (which increases platelet membrane permeability to C a ) induced a further increase in Ca binding (Figure 10). Thus, Ca influx induced by epinephrine would provide a d d i t i o n a l C a f o r cAMP promoted C a sequestion w i t h i n the p l a t e l e t . 4) Low doses of p l a t e l e t a g o n i s t s , e . g . ADP, AA, U46619, e t c . induce r e v e r s i b l e aggregation. Consequently, a mechanism f o r the r e s e q u e s t r a t i o n of C a (or C a e f f l u x ) must e x i s t i n the p l a t e l e t (13,73). z +

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In t h i s c o n n e c t i o n , i t has been shown that PGT^ not only i n h i b i t s p l a t e l e t aggregation but a l s o reverses the aggregation process (74). T h i s e f f e c t can be r e a d i l y explained on the b a s i s t h a £ cAMP-stimulated C a r e s e q u e s t r a t i o n exceeds the rate of Ca^ release. Furthermore, i t has a l s o been demonstrated that the specific TXA /PGH receptor a n t a g o n i s t , 13-azaprostanoic a c i d (13-APA) (75), causes r e v e r s a l of both p l a t e l e t shape change and aggregation i n response to AA or U46619 (76,77). Since 13-APA i s capable of b l o c k i n g the r e l e a s e of Ca^ " i n response to TXA i n isolated platelet vesicles (78), the a b i l i t y of 13-APA to reverse p l a t e l e t a c t i v a t i o n i s presumably due to a sudden inhibition of TXA -stimulated Ca release. This inhibition, i n the face of continued C a resequestration, ultimately leads to a lowering of i n t r a p l a t e l e t C a levels (Figure 11). That the mechanisms of p l a t e l e t d e a c t i v a t i o n by 13-APA and PGI are indeed separate, i s demonstrated by the f i n d i n g that the a b i l i t y of 13-APA to cause deaggregation is p o t e n t i a t e d by PGI (79). Consequently, the combined e f f e c t of each i n d i v i d u a l mechanism of i n a c t i v a t i o n , i . e . i n h i b i t e d C a * rel ease with 13-APA, and cAMP promoted Ca^ r e s e q u e s t r a t i o n with P G I , i s more e f f e c t i v e i n reducing c y t o s o l i c Ca l e v e l s than e i t h e r mechanism alone. z +

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These observations strongly support the concept that cAMP i n h i b i t i o n of p l a t e l e t f u n c t i o n i s mediated through enhanced



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Figure 10. Effect of PGE or PGI on Ca * mobilization in response to epinephrine, A23187 or Ό46619. {Reproduced, with permission, from Ref. 47. Copyright 1981, The American Physio logical Society.)

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9.

LE BRETON ET AL.

Cyclic

Nucleotides,

Ca, and

169

Platelets

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Ca sequestration. Althouah the mechanism by which cAMP may promote the b i n d i n g of C a ^ w i t h i n the p l a t e l e t i s unknown, t h i s uptake mechanism has been a s s o c i a t e d with the phosphoryla t i o n of a s p e c i f i c 22,000 dalton p r o t e i n ( 7 2 , 8 0 ) . In t h i s regard, i t has been p o s t u l a t e d (80) that p l a t e l e t s c o n t a i n an ATP-dependent C a pump, and furthermore, t h a t the e f f i c i e n c y of t h i s pump i s enhanced through phosphorylation of a 22,000 molecular weight p r o t e i n by a cAMP dependent p r o t e i n k i n a s e . An a l t e r n a t i v e mechanism by which cAMP may act to i n h i b i t p l a t e l e t f u n c t i o n was proposed by Hathaway et a l . (81). In t h i s model (Figure 12), i t i s suggested that an increase i n cAMP r e s u l t s i n i n h i b i t i o n of myosin phosphorylation and consequent i n h i b i t i o n of p l a t e l e t c o n t r a c t i l e a c t i v i t y ( s i n c e unphosphorylated myosin cannot i n t e r a c t with a c t i n ) . Thus, i t was proposed that cAMP causes the a c t i v a t i o n of a p r o t e i n kinase which i n turn phosphorylates myosin k i n a s e . In the phosphoryl a t e d form, myosin kinase i s less capable of binding calmodulin and therefore i s not as e f f e c t i v e i n phosphorylating myosin. Although t h i s model would e x p l a i n the a b i l i t y of cAMP to i n t e r f e r e with p l a t e l e t a c t i v a t i o n , the r e s u l t s to date were obtained using p u r i f i e d p l a t e l e t myosin k i n a s e . Consequently, more c o n c l u s i v e proof to support t h i s mechanism of i n h i b i t i o n would r e q u i r e the demonstration that cAMP dependent phosphoryla t i o n of myosin kinase occurs under more p h y s i o l o g i c a l c o n d i t i o n s , e . g . in i n t a c t p l a t e l e t s or p l a t e l e t membrane fragments. In a d d i t i o n to the p o s s i b l e i n t e r a c t i o n s of C a and cAMP o u t l i n e d above, c e r t a i n experimental r e s u l t s have also suggested that C a may a c t u a l l y r e g u l a t e cAMP p r o d u c t i o n . In t h i s connection, i t was demonstrated by Rodan and F e i n s t e i n (82) that Ca i s a potent i n h i b i t o r of adenylate c y c l a s e ( K i = 16 μΜ) in i s o l a t e d p l a t e l e t membranes. Support f o r t h i s concept comes from two l i n e s of evidence: 1) agents which increase c y t o s o l i c Ca l e v e l s e . g . ADP, e p i n e p h r i n e , TXA?, e t c . , reduce the e l e v a t i o n i n cAMP s t i m u l a t e d by e i t h e r PGEi or P G I (59,83); and 2) the C a antagonist TMB-8 i n h i b i t s the a b i l i t y of T X A 2 to lower cAMP ( 8 3 ) . The p h y s i o l o g i c a l s i g n i f i c a n c e of these f i n d i n g s i s u n c l e a r . However, previous r e s u l t s have suggested that C a may be involved i n other p o s i t i v e feedback systems enhancing p l a t e l e t r e a c t i v i t y , e . g . C a - i n d u c e d Ca release or C a a c t i v a t i o n of phospholipase C or A ^ . The reported a b i l i t y of C a to i n h i b i t adenylate c y c l a s e a c t i v i t y may therefore represent an a d d i t i o n a l amplification mechanism, whereby s l i g h t increases i n c y t o s o l i c C a levels lead to a d d i t i o n a l net C a mobilization. In t h i s case, Ca inhibition of cAMP production would promote platelet a c t i v a t i o n by e i t h e r decreasing the r a t e of C a resequest r a t i o n or decreasing phosphorylation of myosin k i n a s e . +

2 +

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CALCIUM REGULATION BY CALCIUM

170

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ANTAGONISTS

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Possible mechanism of calcium release and sequestration.

PROTEIN KINASE (inactive)
P 0 - MYOSIN KINASE (inactive) 4

ADP > P O 4 - MYOSIN Scheme according to Hathaway et al. (8 1 ).


171 Cyclic Nucleotides, Ca, and Platelets

9. LE BRETON ET AL.

Conclusions The above findings thereforç suggest that there is an intimate relationship between Ca and cAMP in the human blood platelet. In this respect, evidence has been presented which indicates that many, if not all, physiological platelet agonists mediate their effects through a common intracellular messenger, i.e. ionic Ca . Furthermore, it is clear that the mechanisms for this intraplatelet Ca mobilization are indeed complex. Thus, an initial increase in intraplatelet Ca , whether as a consequence of enhanced Ca membrane permeability induced by epinephrine or direct Ca release by U46619, results in additioiial Ca mobilization by activation of AA metabolism or by Ca -induced-Ca release. The ability of cAMP to modulate platelet function also appears, at least in part, to be due to regulation of cytosolic Ca levels, possibly through direct stimulation of Ca^ sequestration. 2

2+

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+

Acknowledgements This work was supported in part by a Grant-in-Aid from the American Heart Association. The expert typing services of the Word Processing Center are greatly appreciated. Literature Cited 1. Holmsen, H.; Day, H.J.; Storm, E. Biochim. Biophys. Acta 1969, 186, 254-266. 2. Moore, S.; Pepper, D.S.; Cash, J.D. Biochim. Biophys. Acta 1975, 379, 370-384. 3. White, J.G. "Platelet Aggregation"; Caen, J., Ed.; Masson et Cie: Paris, 1971; 15-52. 4. Behnke, O. J. Ultrastruct. Res. 1965, 13, 469-477. 5. White, J.G. "The Circulating Platelet"; Johnson, S.A., Ed.; Academic Press: New York, 1971; 45-121. 6. Behnke, O. Anat. Rec. 1967, 158, 121-8. 7. White, J.G.: Am. J. Pathol. 1972, 66, 295-312. 8. Grette, K. Acta Physiol. Scand. 1962, 56 Supplement 195, 1-93. 9. Gordon, J.L.; Milner, A.J. "Platelets in Biology and Pathology"; Dingle, J.T., Ed.; North-Holland: New York, 1976; 3-22. 10. Holmsen, H. "Platelets, Production, Function, Transfusion and Storage"; Baldini, M. and Ebbe, S., Eds.; Grune and Stratton: New York, 1974; 207-220. 11. Massini, P; Luscher, E.F. Biochim. Biophys. Acta 1974, 372, 109-121. 12. Feinman, R.D.; Detwiler, T.C. Nature London 1974, 249, 172-173.


172

CALCIUM REGULATION BY CALCIUM ANTAGONISTS 13. White, J.G.; Rao, G.H.R.; Gerrard, J.M. Am. J. Path. 1974, 77, 135-149. 14. Worner, P.; Brossmer, R. Thrombosis Res. 1975, 6, 295-305. 15. Feinstein, M.B.; Fraser, C. J. Gen. Physiol. 1975, 66, 561-581. 16. Murer, Ε.H.; Stewart, G.J.; Rausch, Μ.Α.; Day, H.J. Thrombos. Diathes. Haemorrh. 1975, 34, 72-82. 17. Kinlough-Rathbone, R.L.; Cahil, Α.; Packham, M.A.; Reimers, H.J.; Mustard, J.F. Thrombosis Res. 1975, 7, 435-449. 18. Pressman, B.C. Fed. Proc. 1973, 32, 1698-1703. 19. Reed, P.W.; Lardy, H.A. J. Biol. Chem. 1972, 247, 6970-7. 20. Le Breton, G.C.; Feinberg, H. Pharmacologist l974, 16, 699. 21. Massini, P.; Luscher, E.F. Biochim. Biophys. Acta 1976, 436, 652-663. 22. Le Breton, G.C.; Dinerstein, R.J.; Roth, L.J.; Feinberg, H. Biochem. Biophys. Res. Comm. 1976, 71, 362-370. 23. Caswell, A.H.; Warren, S. Biochem. Biophys. Res. Comm. 1972, 46, 1757-1763. 24. Hallet, M.; Schneider, A.S.; Carbone, Ε. J. Memb. Biol. 1972, 10, 31-44. 25. Luchra, M.; Olson, M.S. Biochim. Biophys. Acta 1976, 440, 744-758. 26. Caswell, A.H. J. Memb. Biol. 1972, 7, 345-364. 27. Caswell, A.H.; Hutchinson, J.D. Biochem. Biophys. Res. Comm. 1971, 42, 43-9. 28. Macfarlane, D.E.; Mills, D.C.B. Blood 1975, 46, 309-320. 29. Le Breton, G.C.; Sandler, W.C.; Feinberg, H. Thrombosis Res. 1976, 8, 477-485. 30. Kaminer, B.; Kimura, J. Science 1972, 176, 406-7. 31. Menche, D.; Israel, Α.; Karpatkin, S. J. Clin. Invest. 1980, 66, 284-291. 32. Eastwood, Α.Β.; Grundfest, H.; Brandt, P.W.; Reuben, J.P. J. Membrane Biol. 1975, 24, 249-263. 33. Sandow, Α.; Pagala, M.K.D.; Sphicas, E.C. Biochim. Biophys. Acta 1976, 440, 733-743. 34. Owen, N.E.; Feinberg, H.; Le Breton, G.C. Am. J. Physiol. 1980, 239, H483-8. 35. Born, G.V.R.; Feinberg, H. J. Physiol. 1975, 251, 803-816. 36. Moake, J.L.; Ahmed, K.; Sachur, N.R.; Gutfreund, D.E. Biochim. Biophys. Acta 1970, 211, 337-344. 37. Feinberg, H.; Sandler, W.C.; "Scorer, M.; Le Breton, G.C.; Grossman, B.; Born, G.V.R. Biochim. Biophys. Acta 1977, 470, 317-324. 38. Feinberg, H.; Michael, H.; Born, G.V.R. J. Lab. Clin. Med. 1974, 84, 926-934.


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Cyclic Nucleotides, Ca, and Platelets 173

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