Calcium ions, drug action and platelet function

Pharmac. Ther. Vol. 18, pp. 249 to 270, 1982

0163-7258/82/020249-22511.00/0 Copyright © 1982 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editor: M. A. DENBOROUGH

CALCIUM

IONS, DRUG

PLATELET

ACTION

AND

FUNCTION

NEVILLE G.

ARDLIE

The Australian National University, Department of Medicine and Clinical Science, Central Health Laboratory Buildin 9, Woden Valley Hospital, Garran, A.C.T. 2606, Australia

1. INTRODUCTION As an essential feature of their role in hemostasis, blood platelets undergo a sequence of reactions to form a plug and arrest blood flow (Mustard and Packham, 1970). Following injury to the vessel wall, platelets adhere to subendothelial connective tissue including collagen. This induces a secretory process known as the release reaction in which contents of certain cytoplasmic granules are expelled from the platelets into the surrounding medium. Released substances such as adenosine diphosphate (ADP) form a hemostatic plug by causing a change in cell shape and platelet aggregation. The platelet also promotes coagulation and influences vasomotor activity which are involved in the arrest of bleeding. These various platelet reactions, which are also considered to be important in thrombosis and atherosclerosis, have been shown to depend on calcium. A variety of biochemical events which support these reactions are also dependent on calcium. Calcium is believed to have a function in many, and perhaps all, cells by mechanisms involving either an influx of extracellular calcium or an intracellular translocation of calcium (Rubin 1974). A large number of studies in platelets have established beyond doubt that calcium ions and cyclic nucleotides are interrelated factors in the coupling of extracellular messages to eventual intracellular responses such as platelet aggregation and the release reaction (Rasmussen and Goodman, 1977; Feinstein, 1978; Feinstein et al., 1981; Gerrard et al., 1981). Indeed, the involvement of calcium in platelet responses to stimulation encompasses nearly every aspect of the cellular response. This review considers the evidence for the involvement of calcium in platelet activation, the regulation of calcium disposition in platelets, the various roles of calcium in platelet activation, and the effects of drugs which interact with calcium dependent functions of platelets. These drugs include local anesthetics, some antihypertensive drugs and some agents used in the treatment of heart disease. 2. CALCIUM MOVEMENTS IN PLATELET ACTIVATION There is evidence for several kinds of movements of calcium ions in the process of platelet activation (Massini et al., 1978) (Fig. 1). 2.1. INTRACELLULAR TRANSLOCATION OF CALCIUM

The first manifestations of platelet activation are a change in cell shape and reversible platelet aggregation. Extracellular calcium is not required since the shape change occurs in the presence of high concentrations of EDTA (Born, 1970). However, prolonged exposure to EDTA prevents the shape change and this may be due to depletion of membrane-bound calcium (Born, 1972). Indirect evidence has been obtained for an increase in the cytoplasmic calcium concentration during the platelet shape change. Chlortetracyc249

250

NEVILLE G. ARDLII

EXTRACELLULAR

Ca 2+

(iv) Surface Binding

Ca 2+ (iii) Influx Ca 2+

\\

:Co,+

//

FIG. 1. Movement of calcium ions in the activation of platelets. (i) Liberation of calcium into the platelet cytoplasm from intracellular stores (superficial membrane binding sites and dense tubular system). This type of calcium movement is considered to play a key role in the activation of platelets. (ii) Eflux of calcium through the plasma membrane due to an increase in membrane permeability. The calcium is derived from dense bodies and the response represents part of the release reaction. (iii) Infux of calcium from the exterior medium. This is not necessary for the platelet release reaction to occur and therefore platelets differ in this respect from many other secretory cells in which secretion is dependent on calcium ions entering the cell from the outside (Carafoli et al., 1975). However, there is evidence that epinephrine, unlike other platelet stimuli, initiates platelet activation by inducing calcium uptake in platelets (Owen et al., 1980). (iv) Binding of calcium to the plasma membrane. This is essential for platelet aggregation. The calcium is involved in the bond linking adherent platelets (Gerrard et al., 1981) and also the interaction of clotting factors with the platelet membrane which occurs in blood coagulation. This calcium readily exchanges with extracellular calcium.

line forms a complex with calcium which is fluorescent in a lipid environment, and Le Breton et al. (1976a) showed that fluorescence with low concentrations of chlortetracycline decreased by about 10~o when the platelet shape change was induced by ADP or A23187. This observation suggests that calcium ions are made available from a membrane. The shape change is also caused by the calcium ionophore A23187 (Gerrard et al., 1974; Le Breton et al., 1976a) indicating that the alteration is induced by an intracellular translocation of calcium. When higher concentrations of agonists are used, platelet activation is not restricted to the shape change and reversible aggregation. The release reaction occurs and this is believed to be, like the shape change, a manifestation of platelet contractile activity dependent on calcium. The release reaction is considered to be a consequence of an increase in the cytoplasmic calcium concentration, the calcium being mobilized from special calcium storing organelles. The intracellular release of membrane-bound calcium measured by chlortetracycline fluorescence has been shown to precede platelet secretion consistent with its proposed role in stimulus-response coupling (Feinstein, 1980). Further evidence for an intracellular calcium flux is obtained by the use of divalent cation ionophores which are presumed to act by making membranes more permeable to specific ions. The divalent cation ionophore A23187 causes platelets to undergo aggregation and secretion in a manner nearly identical to that caused by thrombin (Feinman and Detwiler, 1974; Massini and Luscher, 1974; Kinlough-Rathbone et al., 1977). This response was not dependent on extracellular calcium (Feinman and Detwiler, 1974). Although there are alternative explanations for the effect of A23187 on platelets, the most attractive explanation is that the ionophore causes an intracellular flux of calcium ions, and evidence of this has been obtained (Owen and Le Breton, 1981).

Calcium ions, drug action and platelet function

251

Several compounds which interfere with the movement or action of calcium in muscle cells have been shown to have inhibitory effects on platelets. The drug 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate HC1 (TMB-8), which has been reported to act as a calcium antagonist in muscle (Malagodi and Chiou, 1974), inhibits the platelet release reaction (Charo et al., 1976). Another compound which has been shown to interfere with calcium mobilization in muscle is deuterium oxide (D20) (Kaminer and Kimura, 1972). Le Breton et al. (1976b) found that D20 inhibits the platelet shape change. They also showed that a high concentration of chlortetracycline, which chelates intracellular calcium (Taljedal, 1974), inhibited the shape change and aggregation caused by ADP. In summary, there is general agreement that calcium, from an intracellular source, plays a key role in the regulation of platelet function. Holmsen (1974) proposed that the various platelet responses (i.e. shape change, aggregation, release reaction) significantly influence each other but are essentially independent of each other. Furthermore, each of these responses is proposed to be due to cellular contraction, the intensity of which depends on the strength of the stimulus. This concept obtains support from the observation that the platelet response to the calcium ionophore A23187 increases from a shape change and aggregation to the release reaction as a function of the concentration of ionophore. This implies that the magnitude and hence the nature of the platelet response is determined by the amount of internal calcium that is mobilized. Several studies suggest that the calcium ions are released from the membranes of special calcium storing organelles within the platelet. Nevertheless, the source of the calcium released within the platelet remains controversial. A vesicular fraction of platelet homogenate, similar in many respects to the sarcoplasmic reticulum vesicles of muscle cells which store calcium, has been shown to accumulate calcium ions actively (Statland et al., 1969; Robblee et al., 1973; Kaser-Glanzmann et al., 1978). White (1972a) has provided evidence that the subcellular component of the platelet analogous to the sarcoplasmic reticulum of muscle is the dense tubular system. On the other hand Sato et al. (1975) have described a pool of calcium, localized in certain platelet c~-granules, which is mobilized when platelets are stimulated by thrombin. However, these e-granules which contain nucleoids may represent an early stage in the formation of dense bodies (Gerrard et al., 1981). Thus, the mobilization of calcium from these granules may represent part of the release reaction (see below) rather than the involvement of calcium in the process of platelet activation, although the calcium from this e-granule pool is discharged more quickly than calcium from dense bodies. Yet another possible calcium storage site, analogous to other cells (Lehninger et al., 1967), are the mitochondria. However, agents such as antimycin A which readily release calcium from mitochondria in other cells do not induce platelet aggregation or secretion or the mobilization of intracellular calcium (Feinstein, 1980). This suggests that mitochondrial calcium is not involved in platelet activation. It is quite conceivable that there are several calcium storage pools in the platelet, each serving a different function. It is not known with certainty whether the calcium movements within the cytoplasm which cause the shape change and reversible aggregation on the one hand, or secretion on the other, represent two (or more) distinct fluxes from different pools, or whether the different fluxes occur from the same pool and differ only in magnitude. Indeed there is evidence that activation of different intracellular processes involved in platelet secretion alone may require calcium from different pools (Shaw and Lyons, 1982a, b). Using chlortetracycline fluorescence as a measure of intraplatelet calcium mobilization, it was found that TMB-6, a calcium antagonist, inhibited secretion, but did not inhibit calcium mobilization associated with the shape change (Le Breton and Dinerstein, 1977). Similar results were obtained with local anesthetics which have calcium antagonist properties (Feinstein et al., 1976). These observations are consistent with the concept that calcium is released from a membrane (possibly the surface membrane) to cause the shape change, and that a larger internal pool of calcium (possibly from the dense tubular system) is mobilized to cause secretion.

252

NEVILLE G. ARDLIE

2.2. RELEASE OF CALCIUM FROM PLATELET DENSE BODIES Another movement of calcium during platelet activation represents part of the release reaction in which the contents of dense bodies are discharged to the external medium of the platelet (Murer and Holme, 1970). The platelet dense bodies contain most of the platelet's calcium as well as other substances including adenine nucleotides and amines (Skaer et al., 1976).

2.3. INFLUX OF CALCIUM DUE TO MEMBRANE PERMEABILITY INCREASE No calcium is taken up by the platelet in the early stages of activation (i.e. the shape change and reversible aggregation) caused by ADP, thrombin and collagen (Massini and Luscher, 1976). However, the plasma membrane acquires an increased permeability with a resultant influx of calcium in the later phase of activation, usually after the earlier release of calcium from the cytoplasm (Massini and Luscher, 1976). The permeability of the membrane is therefore increased in both directions. Further evidence that the influx of external calcium is not necessary for activation by ADP, thrombin and collagen is provided by the observation that these agents can activate platelets in the presence of EDTA (Mueller-Eekhardt and Luscher, 1968). Furthermore, the uptake of calcium is inhibited when the release reaction is prevented by various inhibitors (Massini and Luscher, 1976). These findings suggest that the uptake of calcium might be a consequence rather than the cause of platelet activation. Whereas ADP, thrombin and collagen cause a shape change before aggregation and initially release calcium from a membrane, epinephrine initiates platelet aggregation without a shape change or secretion (O'Brien, 1966). Epinephrine is known to induce two phases of aggregativn of human platelets, the second phase involving secretion. During the first phase of epinephrine-induced aggregation there is an associated calcium uptake by platelets (Owen et al., 1980). ADP does not cause calcium uptake, and the calcium antagonist, verapamil, blocks calcium uptake and epinephrine-induced aggregation, but not ADP-induced aggregation (Owen et al., 1980; Owen and Le Breton, 1981). These results suggest that epinephrine initiates aggregation by causing calcium uptake. Movement of calcium into the plasma membrane may cause changes in the membrane that enable platelets to stick to each other. Epinephrine also causes calcium mobilization within platelets, and this is inhibited when calcium uptake is blocked by verapamil (Owen and Le Breton, 1981). This finding suggests that epinephrine cannot directly initiate the release of calcium within the platelet but rather requires an initial influx of calcium to cause internal mobilization. This calcium-induced calcium release may occur through a mechanism comparable, at least in some respects, to that in muscle cells (Fabiato and Fabiato, 1977). However, part of the proposed calcium-induced calcium release in platelets may be due to stimulation by calcium of phospholipase A 2 (see below), since calcium mobilization in response to epinephrine is partially blocked by indomethacin (Owen and Le Breton, 1981) which inhibits production of reactive metabolites of arachidonic acid.

2.4. BINDING OF CALCIUM TO THE PLASMA MEMBRANE Platelet aggregation requires the presence of calcium in the external medium (Heptinstall, 1976; Taylor and Heptinstall, 1980; Gerrard et al., 1981). After internal changes take place in platelets, calcium must be present at the surface for the adhesion of platelets to each other. Surface binding of calcium ions (Massini and Luscher, 1976) may be due to a rearrangement of the plasma membrane caused by activation, which results in an increase in the number of membrane calcium binding sites. Other evidence, however, suggests that additional calcium is not bound during platelet stimulation and it is presumed that the calcium necessary for aggregation is already associated with the platelet

Calcium ions, drug action and platelet function

253

membrane (Peerschke et al., 1980). Loosely bound surface calcium is readily removed by EGTA and easily repleted by exchange with extracellular calcium (Robblee and Shepro, 1976; Taylor and Heptinstall, 1980).

3. EFFECTS OF INTRACELLULAR CALCIUM IN PLATELETS The mobilization of calcium within platelets has several effects which are involved in platelet activation. 3.1. DISRUPTIONOF MICROTUBULES Normal circulating platelets contain in the equatorial plane a circular bundle of microtubules, thought to act as a cytoskeleton which is responsible for the typical disc shape (Behnke, 1965). The existence of this bundle close to the inner side of the plasma membrane prevents the transformation of the platelet shape from a disc to a sphere. During platelet activation, transient disassembly and rearrangement of these microtubules has been shown to occur. The appearance of calcium in the cytoplasm leads to the disappearance of this submembranous bundle since calcium depolymerizes microtubules (Borisy et al., 1974). The ionophore A23187 also causes the disassembly of platelet microtubules (Kenney and Chao, 1980; Ikeda et al., 1981). 3.2. ACTIVATION OF THE CONTRACTILE SYSTEM The shape change and other manifestations of platelet activation are considered to be the result of activation of the platelet contractile system. The platelet contains actomyosin, a material similar to smooth muscle actomyosin (Bettex-Galland and Luscher, 1959), and its contraction is calcium dependent (Cohen and Cohen, 1972). Thus, in many respects platelet activation is a process similar to smooth muscle cell contraction. Platelet secretion and inhibition of platelet activation by cyclic AMP (see below) both appear to depend on protein phosphorylation (Lyons et al., 1975; Haslam and Lynham, 1977; Bennett et al., 1979; Lyons and Shaw 1980; Gerrard and Carroll, 1981). Collagen, thrombin, A23187, arachidonic acid and platelet-activating factor all cause secretion of 5-hydroxytryptamine and phosphorylation of peptides of 20,000 daltons, 40,000 daltons and 260,000 daltons. The 20,000 dalton protein has been tentatively identified as the light chain of myosin, and the 260,000 dalton protein as actin, the 40,000 dalton protein being of unknown function (Daniel et al., 1977; Gerrard and Carroll, 1981). Phosphorylation of the proteins precedes secretion (Lyons and Shaw, 1980; Wallace and Bensusan, 1980) and is inhibited by TMB-8 which acts as an intracellular calcium antagonist (Charo et al., 1976). These observations suggest a role for protein phosphorylation in the release reaction. Feinstein (1980) has shown that platelet secretion is preceded by the release of intracellular calcium, and it is therefore possible that the release of calcium stimulates protein phosphorylation by activating calcium-dependent protein kinases. Calmodulindependent myosin light chain kinase has been isolated from human platelets (Hathaway and Adelstein, 1979) and there is evidence which suggests that calcium interacts with calmodulin and a protein kinase to initiate phosphorylation of myosin light chain (Dabrowska and Hartshorne, 1978; Hathaway and Adelstein, 1979). Phosphorylation of myosin light chain is necessary for the interaction of myosin with actin and development of tension (Lebowitz and Cooke, 1978). Thus contraction in platelets, as in muscle cells, is calcium dependent through regulation of myosin phosphorylation by the calmodulin calcium complex. 3.3. MEMBRANEFUSION IN THE RELEASEREACTION In the course of the release reaction, the membranes of the platelet storage organelles (e.g. dense bodies) fuse with the plasma membrane or with the membrane of the surface-

254

NEVILLEG. ARDLIE

connected canalicular system of the platelet (White, 1972b). In this way a gap is formed through which the contents of the storage organelles are expelled to the exterior of the ~latelet. Calcium ions have been shown to play a crucial role in the fusion of membranes Zakai et al., 1976). 3.4. PROSTAGLANDINMETABOLISM H u m a n platelets synthesize prostaglandins in response to a number of physiologically important agents (Bills et al., 1978). Arachidonic acid is the major precursor of these prostaglandins, and the metabolism of this fatty acid by human platelets and the endothelial cells lining the vessel wall is summarized in Fig. 2. In response to agonists, arachidonic acid is cleaved from platelet m e m b r a n e phospholipids by the activity of phospholipases C and A2 (see below). Free arachidonic acid is utilized by the enzyme cyclooxygenase to produce the cyclic endoperoxides, prostaglandin G2(PGG2) and prostaglandin H2 (PGH2) (Hamberg and Samuelsson, 1973). These compounds are then converted to the unstable c o m p o u n d thromboxane A2 (TxAz) which is capable of inducing platelet aggregation and the release reaction (Hamberg et al., 1975). Since the liberation of arachidonic acid from phospholipids is a prerequisite for prostaglandin synthesis, the activation of phospholipases is important and this may be the limiting step in the generation of this fatty acid (Bergstrom et al., 1964). Both phospholipase C (Mauco et al., 1979; Rittenhouse-Simmons, 1979) and phospholipase As (Derksen and Cohen, 1975; Rittenhouse-Simmons and Deykin, 1978: Jesse and Franson, 1979) require calcium for activity. This supports the concept that the availability of free intracellular calcium is a major regulatory step in the generation of arachidonic acid for prostaglandin biosynthesis in platelets. At high concentrations extracellular calcium may inhibit the release of arachidonic acid (Best et al., 1979: Stuart et al., 1980). This could explain previous observations that high concentrations of calcium inhibit aggregation (Born and Cross, 1964). ARACHIDONIC ACID

clooxygenase (PLATELETAND ENDOTHELIAL CELL)

ENDOPEROXIDE PGG2

HPETE

/\ HETE

1

ENDOPEROXIDE POH2

THETE

/ / d ~Prostaglandin (PLATELET) (ENDOTHELI " ~~Synthetase CELL)AL / / / i ! A 2 MDA HHT

PGE2 PGF2,~ TxB2 PGD~

PG~ 6KETO PGFI~

FIG. 2. Metabolism of arachidonic acid by platelets and endothelial cells. Arachidonic acid is transformed in a step-wise fashion, and this plays an important role in platelet and endothelial cell function. Platelet stimulation causes the release of arachidonic acid from membrane phospholipids by the action of phospholipases. The enzyme cyclooxygenase,which is inhibited by aspirin, converts free arachidonic acid to endoperoxides and then thromboxanes and other end products. Thromboxane A2 (TxA2) causes platelet aggregation and secretion, and vasoconstriction. On the other hand, endothelial cells transform endoperoxides to prostacyclin (PGI2) which opposes the action of TxA2. Endothelial cells can utilize endoperoxides released from stimulated platelets, or endogenous arachidonic acid for the synthesis of PGI 2. Thus, metabolites of arachidonic acid are revolved not only in platelet activation but also its inhibition. PGI2 inhibits platelet aggregation and release by increasing intracellular cyclic AMP (Gorman et al., 1977a: Tateson et al., 1977). At present, no role for the products of the lipoxygenase pathway of arachidonic acid metabolism in platelets has been determined.

Calcium ions, drug action and platelet function

255

A model for the regulation of platelet activation by TxA 2, cyclic AMP and calcium has been proposed by Gerrard et al. (1978b). In this model, an agent which aggregates platelets acts on the plasma membrane. The close association of the surface-connected canalicular system with the dense tubular system of the platelet allows transmission of the signal from the aggregating agents to the site where arachidonic acid is released. The platelet dense tubular system is considered to be equivalent to the sarcoplasmic reticulum of muscle cells, and the available evidence suggests that it is an internal calcium store in platelets and also the site of prostaglandin synthesis. It has been proposed that TxA2 is a physiologic ionophore which transports calcium from the dense [ubular system into the cytoplasm where the calcium is released to initiate the release reaction. Cyclic AMP promotes reuptake of calcium into the dense tubular system or extrusion from the cell, resulting in inhibition or reversal of platelet activation. Stimulation of platelets by arachidonate or the calcium ionophore A23187 leads to a rapid phosphorylation of three proteins (Gerrard and Carroll, 1981). These three proteins include actin (260,000 daltons), a protein of unknown function (40,000 daltons), and the light chain of myosin (20,000 daltons). Aspirin completely inhibited both platelet aggregation and protein phosphorylation caused by arachidonic acid. Since the light chain of myosin is phosphorylated by a protein kinase which is calcium- and calmodulin-dependent (Hathaway and Adelstein, 1979), these results are consistent with the mobilization of calcium as the principal role of the arachidonate-thromboxane pathway. It is apparent that there are several mechanisms for the mobilization of cellular calcium and that TxA2 can be regarded as an intermediary amplifier of the cellular reactions involving calcium which lead to platelet aggregation and the release reaction.

3.5. RELEASE OF PLATELET-AcTIVATING FACTOR Platelet-activating factor is a phospholipid, first described in rabbit leukocytes which mediates allergic and inflammatory reactions and causes platelet aggregation and the release reaction (Benveniste et al., 1972). Its molecular structure has recently been identified as 1-O-alkyl-2-acetyl-glyceryl-3-phosphorylcholine (Demopoulos et al., 1979). The calcium ionophore A23187 causes the release of this lipid from human alveolar macrophages (Arnoux et al., 1980), and it acts on platelets independently of prostaglandin synthesis and the release reaction (Cazenave et al., 1979). Since platelet-activating factor is synthesized by rabbit platelets stimulated by the ionophore A23187 (Chignard et al., 1979; Chap et al., 1981), it may, like TxA2, mediate platelet responses to stimuli. Rabbit platelets stimulated with thrombin or collagen also produced platelet-activating factor. In contrast, ADP and arachidonic acid did not cause formation of platelet-activating factor (Chignard et al., 1980). Conflicting results, however, have been published by Marcus et al. (1981). They obtained evidence that washed platelets from humans did not generate significant quantities of platelet-activating factor upon stimulation with thrombin or ionophore A23187. Further investigation is necessary to determine whether platelet-activating factor is another mediator of platelet aggregation as proposed by Chignard et al. (1979). The formation of platelet-activating factor appears to be calcium dependent (Chignard et al., 1979), and nonspecific inhibitors of phospholipase A2 suppress the formation of platelet-activating factor (Vargaftig et al., 1981). Platelet-activating factor has been shown to induce an influx of calcium into rabbit platelets (Lee et al., 1981). Thus, this factor, like other lipids, may function as an endogenous ionophore in mediating platelet activation.

3.6. ACTIVATIONOF PHOSPHOLIPASES There is evidence that in addition to prostaglandin biosynthesis, there is another consequence of phospholipase activation, independent of a metabolic product of arachi-

256

NEVILLEG. ARDLIE

donate, which may play a central role in mediating platelet aggregation. In recent years the idea has evolved that there is some ubiquitous calcium mobilizing mechanism which may be involved in cellular events after receptor interaction with the plasma membrane. It has been proposed that this mechanism involves phosphatidylinositol breakdown (Michell, 1979). Breakdown of phosphatidylinositol and rapid formation of phosphatidic acid are observed when platelets are stimulated by ADP, collagen and thrombin (Lloyd et al., 1972; Lloyd and Mustard, 1974; Lapetina et al., 1978, 1981a: Lapetina and Cuatrecasas, 1979; Broekman et al., 1980). Platelets possess a phospholipase C which specifically initiates the degradation of phosphatidylinositol (Rittenhouse-Simmons, 1979; Billah et al., 1980). The resultant 1,2-diacylglycerol is rapidly and completely phosphorylated to phosphatidic acid (Lapetina et al., 1978, 1979, 1981a; Broekman et al., 1980; Walenga et al; 1980). The formation of phosphatidic acid precedes the release of arachidonic acid from the phospholipids of stimulated platelets and the rate of formation parallels the rate of serotonin release (Lapetina et al., 1979). Recently the existence of a particulate phospholipase A2 enzyme that appears to act selectively on phosphatidic acid to produce arachidonic acid and lysophosphatidic acid has been reported (Billah et al., 1981). Both phosphatidic acid and lysophosphatidic acid have calcium ionophoretic properties (Tyson et al., 1976; Ikeda et al., 1979; Putney et al., 1980; Serhan et al., 1981), and lysophosphatidic acids have been shown to cause the release of calcium from a platelet membrane fraction, possibly the dense tubular system (Gerrard et al., 1979). These phospholipids could therefore act as endogenous ionophores in platelets, and it has been proposed that ADP, thrombin and collagen initiate platelet activation by stimulating the breakdown of phosphatidylinositol to 1,2-diacylglycerol, which is then converted to phosphatidic and lysophosphatidic acids which promote an intracellular flux of calcium to trigger activation independent of thromboxane synthesis (Gerrard et al., 1978a; Lapetina et al., 1978). It has been postulated that phosphatidylinositol turnover is involved in ~1 adrenergic, and adenylate cyclase inhibition is involved in ~2 adrenergic effects of catecholamines in various tissues (Fain and Garcia-Sainz, 1980) but these relationships have not been extensively studied in platelets. The interaction of epinephrine with human platelets appears to be mediated by ~2-adrenoreceptors (Grant and Scrutton, 1979), and only 20 30}~,of donors show a platelet response to ~l-agonists (Scrutton and Wallis, 1981). It is conceivable that particular receptors may be coupled to both adenylate cyclase activity and phosphatidylinositol turnover in platelets. Furthermore, in contrast to ADP and other agents which stimulate platelets, breakdown of phosphatidylinositol caused by epinephrine might occur as a result of the influx of calcium. Regulation of the enzymatic release of phosphatidic and lysophosphatidic acids and of arachidonic acid for prostaglandin formation may involve changes in the calcium concentration at the sites of the phospholipases. In support of this postulate are the observations that the various phospholipases are calcium dependent. Both phospholipase A 2 and C require calcium for activity, but phospholipase C requires a much lower concentration of the cation (Billah et al., 1980). The preferred substrates for phospholipase A 2 are the phospholipids, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, whereas phospholipase C specifically degrades phosphatidylinositol (Billah et al., 1980). The phospholipase A 2 which is specific for phosphatidic acid, is also calcium dependent and is inhibited by mepacrine in a calcium-dependent manner (Billah et al., 1981). The phospholipase A 2 that degrades phosphatidylethanolamine and phosphatidylcholine requires a higher concentration of calcium than the phosphatidate-specific phospholipase A2 (Billah et al., 1981). Although the exposure of platelets to ionophore A23187 causes some activation of phospholipase C, thrombin causes the formation of up to six times more diglyceride (Rittenhouse-Simmons, 1981). This observation suggests that optimal activation of phospholipase C in platelets requires more than a flux in intracellular calcium. In contrast, the ionophore A23187 induces phospholipase A2 activity which is greater in magnitude than that induced by thrombin (Pickett et al., 1977). Furthermore, thrombin induces phospholipase A 2 activity in intact human platelets washed in a cal-

Calcium ions, drug action and platelet function

257

cium-free buffer and resuspended in a fluid containing 5 mM EGTA (Pickett and Cohen, 1976). This is consistent with the concept that mobilization of calcium from an intracellular depot is involved in activation of phospholipase A2. Three alternative pathways have been postulated for the mobilization of arachidonic acid, and all of the enzymes involved have been shown to either require calcium or are stimulated by calcium when assayed in vitro. The three pathways are: (1) Hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase A2, yielding free arachidonic acid and lysophospholipids (McKean et al., 1981). (2) Conversion of phosphatidylinositol to diglyceride by phospholipase C, followed by hydrolysis of the diglyceride to arachidonic acid by a diglyceride lipase (Bell et al., 1979). (3) Conversion of phosphatidylinositol to diglyceride by phospholipase C, which is then converted to phosphatidic acid by diglyceride kinase. A phosphatidate-specific phospholipase Az then leads to release of arachidonic acid (Billah et al., 1981). Since the initial action of thrombin is to cause the conversion of phosphatidylinositol to phosphatidic acid preceding the production of arachidonic acid (Lapetina and Cuatrecasas, 1979; Lapetina et al., 1981a), it is evident that phospholipase C is activated before phospholipase A2. The metabolic route to arachidonate production may therefore involve the sequential activation of phospholipase C followed by phospholipase Az. Although both phospholipases require calcium, phospholipase A z requires a much higher concentration than phospholipase C (Billah et al., 1980) and therefore the available calcium may determine the sequence of activation of phospholipases. Phosphatidic acid produced by the action of phospholipase C may, through its ionophoretic properties, activate phospholipase A2 and phosphatidate-specific phospholipase Az to produce arachidonic acid from various phospholipids. It is pertinent that inhibition of phospholipase C by serine esterase inhibitors prevents the formation of arachidonic acid (Walenga et al., 1980). Furthermore, phospholipase C is not affected even when the production of arachidonic acid is completely inhibited by mepacrine which is a calmodulin antagonist (Billah et al., 1981; Lapetina et al., 1981b). The ionophore A23187 bypasses the formation of phosphatidic acid during the release of arachidonic acid, possibly by directly stimulating phospholipase Az (Lapetina et al., 1981a). This observation is consistent with the proposal that the first step in the formation of arachidonic acid is the activation of phospholipase C which degrades phosphatidylinositol to produce phosphatidic acid. The production of phosphatidic acid then results in activation of phospholipase A2 through its calcium ionophoretic action. The ability of mepacrine to inhibit phospholipase A 2 but not phospholipase C, and the demonstration that A23187 appears to activate phospholipase A2 directly, suggests that mobilization of calcium is more important in phospholipase A2 activation than phospholipase C activation. Phosphatidic acid has been shown to stimulate phospholipase A2 (Apitz-Castro et al., 1981), and phospholipase A2 may require calmodulin for stimulus activation (Wong and Cheung, 1979; Walenga et al., 1981), while phospholipase C may not (Walenga et al., 1981). The calmodulin antagonist trifluoperazine (and other phenylthiazine and non-phenylthiazine calmodulin antagonists) inhibits arachidonic acid production, but not phosphatidic acid formation (Walenga et al., 1981). These results suggest that the activity of phospholipase A2 accounts for the bulk of arachidonic acid released and metabolized. Broekman et al. (1980) have shown that whereas the amount of phosphatidic acid formed and phosphatidylinositol degraded are nearly equivalent at later times after thrombin stimulation, there is a discrepancy between the two lipids at 15 sec after stimulation. This might be accounted for by the conversion of the diglyceride formed initially to free arachidonic acid. Thus, while the enzyme diglyceride lipase may play a minor role with regard to the total amount of arachidonic acid liberated, it may be involved in the early release of a small amount of the fatty acid. Phosphatidic acid is a key intermediate in the phosphatidylinositol cycle. In this cycle, four consecutive enzyme activities including phospholipase C are involved in the degradation and resynthesis of phosphatidylinositol. Calcium has been shown to inhibit the

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resynthesis of phosphatidylinositol from phosphatidic acid after thrombin stimulation, thereby increasing the concentration of phosphatidic acid (Lapetina et al., 1981b). On the other hand, cyclic AMP, which has effects opposite to those of calcium (see below), seems to increase the rate of conversion of phosphatidic acid to phosphatidylinositol, thus decreasing the concentration of phosphatidate (Lapetina et al., 1981b). If the proposal is correct that the production of arachidonic acid is dependent on the formation of phosphatidate, the action of cyclic AMP on the phosphatidylinositol cycle may be the explanation for cyclic AMP-induced inhibition of arachidonate production. Lysophosphatidic acid augments the effects of thrombin on the formation of phosphatidic acid (Lapetina et al., 1981c). Since calcium prevents the conversion of phosphatidic acid to phosphatidylinositol, the calcium ionophoretic properties of lysophosphatidic acid might explain the accumulation of phosphatidic acid. 4. CALMODULIN AND PLATELET ACTIVATION Recent studies have suggested that many of the effects of ionic calcium in cells are mediated by the ubiquitous intracellular calcium-binding protein, calmodulin (Cheung, 1970, 1980; Means and Dedman, 1980; Scharff, 1981). Calmodulin serves as an intracellular calcium-receptor and regulates a number of fundamental cellular activities such as cyclic nucleotide and glycogen metabolism, secretion, microtubule assembly and disassembly, calcium transport, protein phosphorylation and activation of other enzymes such as phospholipase A2 (Cheung, 1980; Means and Dedman, 1980; Scharff, 1981). The mode of action of calmodulin was first established with the enzyme phosphodiesterase (Cheung, 1980). Calmodulin itself is not active, the active form being the calmodulin-calcium complex. Calmodulin assumes a more helical conformation once it is bound to calcium, and this active form binds to phosphodiesterase resulting in the formation of an active enzyme. An ordered binding of calcium by calmodulin has been demonstrated and this property may allow calmodulin to translate quantitative calcium signals into qualitatively different cellular responses (Haiech et al., 1981). The identification of calmodulin in platelets (Smoake et al., 1974; White et al., 1981 ; Young et al, 1981) raises the distinct possibility that this protein participates in calciumdependent reactions involved in platelet aggregation and the release reaction. Indeed, inhibitors of calmodulin have been shown to inhibit platelet aggregation and the release reaction (Kindness et al., 1980; Rao et al., 1980: White and Raynor, 1980: Kambayashi et al., 1981; Suda and Aoki, 1981). These agents achieve inhibition by binding to calmodulin. In addition to inhibiting platelet aggregation and secretion by exogenous arachidonate, the release of arachidonic acid from platelet membrane phospholipids is also inhibited by phenylthiazine and non-phenylthiazine calmodulin antagonists (Kambayashi et al. 1981; Walenga et al., 1981) and the sulfonamide derivative, N-(6-aminohexyl)-5chloro-l-naphthalene sulfonamide (Suda and Aoki, 1981). These observations suggest that in the platelet response to stimulation, there are at least two steps in which calmodulin is involved, namely the activation of phospholipases and secondly the contraction of proteins after the formation of TxA2. Calmodulin has been reported to stimulate phospholipase A2 activity in isolated platelet membranes (Wong and Cheung, 1979), but no such stimulation has been reported for phospholipase C. Walenga et al. (1981) demonstrated that trifluoperazine and other phenylthiazine and non-phenylthiazine calmodulin antagonists inhibit the release of arachidonic acid from phosphatidylcholine but the release of phosphatidic acid from phosphatidylinositol was not inhibited. These results indicate that in platelets, phospholipase A2 may require calmodulin for activation, whereas phospholipase C may not. Calmodulin, isolated from platelets, also enhances the activity of cyclic nucleotide phosphodiesterase and phosphorylase kinase (Gergely et al., 1980; Young et al., 1981). Calmodulin-dependent myosin light chain kinase has also been isolated from human platelets (Drabowska and Hartshorne, 1978; Hathaway and Adelstein, 1979). These enzymes are all involved in platelet activation.

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It has been reported that phenylthiazine (viz. trifluoperazine) and non-phenylthiazine antagonists of calmodulin also inhibit calcium uptake by a platelet microsomal fraction, and it was postulated that calmodulin might be involved in lowering the cytoplasmic calcium level by promoting calcium uptake into this microsomal fraction (White and Raynor, 1982). However, if the role of calmodulin is to be assessed from the effects of trifluoperazine on intact platelets, the ability of high concentrations of trifluoperazine to directly antagonize enzymes without the involvement of calmodulin, or the ability of these levels of trifluoperazine to inhibit intracellular mobilization of calcium (Walenga et al., 1981), has to be kept in mind. 5. INTERRELATIONSHIPS BETWEEN CALCIUM AND CYCLIC AMP IN INHIBITION OF PLATELET ACTIVATION The regulation of calcium mediated events in platelets by intracellular cyclic AMP appears to be important in inhibition of platelet activation by various drugs and therefore needs to be discussed briefly here. A more detailed review has recently been published (Feinstein et al., 1981). The relationship between calcium and cyclic AMP can be viewed as a bidirectional control system in platelets. While calcium links reactions at the cell membrane to ensuing intracellular events in platelet activation, cyclic AMP mediates the recovery from or opposes the action of calcium. Cyclic AMP can regulate the level of free calcium in platelets or control calcium dependent reactions which are involved in platelet activation. Therefore, an improved understanding of the interrelationship between cyclic AMP and calcium may be fundamental to the achievement of altered platelet function in the management of thrombotic disorders and atherosclerosis. 5.1. ADENYLATE CYCLASE AND PLATELET ACTIVATION

Whether aggregating agents must inhibit the cyclic AMP synthesizing enzyme adenylate cyclase or lower cyclic AMP levels by other means to activate platelets remains uncertain. From studies in vitro it has been concluded that aggregating agents do not have to reduce basal cyclic AMP levels to induce aggregation (Haslam et al., 1978). However, if PGI2 is a circulating hormone (Gryglewski et al., 1978; Moncada et al., 1978), then the basal activity of adenylate cyclase and hence the cyclic AMP level may well be much. higher in circulating platelets than in vitro, in which case reduction of basal cyclic AMP levels may be essential for platelet activation in hemostasis. Cytochemical studies in platelets have shown adenylate cyclase localized in the dense tubular system (Cutler et al., 1978). This adenylate cyclase activity was only apparent when stimulated by PGE1, PGD2 or PGI2. In broken cell preparations platelet adenylate cyclase activity has been found (Rodan and Feinstein, 1976) in a particulate fraction which accumulates calcium (Robblee et al., 1973). 5.2. REGULATION OF ADENYLATE CYCLASE BY CATIONS Both adenylate cyclase activation and inhibition depend on the cations, magnesium and calcium. Magnesium is required for activation of the enzyme (Johnson et al., 1979). Calcium probably competes with magnesium for the cation regulatory site in the enzyme, which accounts for the inhibition of adenylate cyclase in membrane fractions of platelets by calcium (Rodan and Feinstein, 1976). The effect of calmodulin on platelet adenylate cyclase has not yet been reported. 5.3. REGULATION OF ADENYLATE CYCLASE BY ARACHIDONIC ACID METABOLITES Metabolites of arachidonic acid, notably PGI 2, PGE1 and PGD2 are the most potent stimulators of adenylate cyclase and inhibition of platelet aggregation by these prosta-

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glandins is believed to be due to their ability to stimulate the enzyme. PGD2 is synthesized by platelets, and PGI2 is synthesized by endothelial cells during hemostasis and may act as feedback inhibitors of aggregation and release (Oelz et al., 1977), thus determining the size of the hemostatic plug. A decrease in platelet adenylate cyclase responsiveness to PGD 2 has been reported in some patients with deep vein thrombosis (Cooper, 1979). PGD 2 was also less effective in inhibiting collagen-induced sccretion of 5-hydroxytryptamine from platelets in these patients. The reason for the loss of sensitivity is not known. Platelets from hypercholesterolemic subjects also show an impaired ability to accumulate cyclic AMP when challenged with PGI2, PGD2 and PGE1 (Jakubowski et al., 1980). Since platelets have such an important role in atherogenesis, this observation may, in part, explain the increased risk of developing vascular disease in hyperlipidemic subjects. The activity of adenylate cyclase is influenced by its lipid environment (Gazzotti and Peterson, 1977), and this may be the reason for the impaired ability of platelets from hypercholesterolemic subjects to accumulate cyclic AMP. The prostaglandin endoperoxides PGG2 and PGH 2 and TxA2 inhibit cyclic AMP production by PGI2 and PGE1 (Gorman et al., 1977a, 1978: Salzman, 1977). The effect of the endoperoxides appears to be due to their conversion to TxA 2 (Gorman et al., 1977b). The suggestion that TxA 2 may act as an ionophore for calcium raises the possibility that inhibition of cyclic AMP production is mediated through calcium. The possibility that PGI2 is a circulating hormone (Gryglewski et al., 1978; Moncada et al., 1978) suggests that cyclic AMP levels of platelets in rivo are higher than under in vitro conditions. If this is so, then the ability of TxA2 to reduce cyclic AMP becomes more significant in determining the outcome of stimulus platelet interaction. 5.4. CALCIUM SEQUESTRATION AND CYCLIC A M P

A membrane fraction has been isolated from platelets which can accumulate calcium at the expense of energy derived from ATP (Statland et al., 1969; Robblee et al., 1973; George et al., 1976; Kaser-Glanzmann et al., 1977, 1978, 1979). The calcium accumulating membrane fractions of platelets have many properties similar to those of sarcoplasmic reticulum of muscle. They possess calcium-stimulated ATPase activity and the enzymes for prostaglandin synthesis (Kaser-Glanzmann et al., 1978). In addition, a protein with a molecular weight of 22,000 in these membrane fractions is phosphorylated by a cyclic AMP-dependent protein kinase and stimulates the uptake of calcium (Fox et al., 1979; Haslam et al., 1979; Kaser-Glanzmann et al., 1979). The protein phosphorylated in the platelet membrane has the same molecular weight as a protein in the sarcoplasmic reticulum of heart muscle which is phosphorylated by a cyclic AMP-dependent protein kinase (Tada et al., 1975). It is suggested that phosphorylation of a membrane-bound protein by a cyclic AMP-dependent protein kinase may promote the active transport of calcium out of the platelet cytosol. This appears to be important in inhibition of platelet activation by various agents, and may also be involved in the recovery of platelets from the activated state. 5.5. EFFECT OF CYCLIC AMP ON THE PHOSPHATIDYLINOSITOL CYCLE AND ARACHIDONIC ACID METABOLISM

Dibutyryl cyclic AMP and other agents that increase cyclic AMP levels such as PGE1, PGD2 and theophylline inhibit the formation of TxB2 elicited by collagen in platelet-rich plasma (Malmsten et al., 1976). Subsequently, it was shown that mobilization of arachidonic acid was the main site of inhibition by cyclic AMP (Feinstein et al., 1977; Gerrard et al., 1977; Minkes et al., 1977). The enzymes involved in the cleavage of arachidonic acid from membrane phospholipids are phospholipases C and A2, and both are calciumdependent enzymes (see above). Cyclic AMP mediated reactions have been shown to influence the phospholipase C pathway (Lapetina et al., 1977; Lapetina and Cuatrecasas, 1979; Rittenhouse-Simmons, 1979) but not phospholipase A2 activity in platelet hom-

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ogenates (Rittenhouse-Simmons and Deykin, 1978). Dibutyryl cyclic AMP inhibited diglyceride production from phosphatidylinositol by phospholipase C only in intact platelets, and this effect persisted after solubilization of the platelets (Billah et al., 1979). This suggests that cyclic AMP affects the phospholipase C pathway by a mechanism other than, or in addition to, its ability to restrict availability of calcium in intact platelets. Inhibition of formation of arachidonic acid metabolites by cyclic AMP has been variously ascribed to inhibition of conversion of arachidonic acid to its active products (Malmsten et al., 1976), inhibition of the release of arachidonic acid from phospholipids (Feinstein et al., 1977; Gerrard et al., 1977; Lapetina et al., 1977, 1978; Minkes et al., 1977), and inhibition of formation of phosphatidic acid (Lapetina et al., 1978; Lapetina and Cuatrecasas, 1979). Initially, the ability of cyclic AMP to inhibit phosphatidic acid production was attributed to inhibition of phospholipase C (Billah et al., 1979), but more recently evidence has been obtained that the phosphatidylinositol cycle is not inhibited by cyclic AMP despite a reduction in formation of phosphatidic acid (Lapetina et al., 1981b). The findings indicate that calcium interrupts the phosphatidylinositol cycle and causes accumulation of phosphatidic acid (Lapetina et al., 1981b), whereas cyclic AMP, which has effects opposite to those of calcium, enhances the phosphatidylinositol cycle, thereby decreasing the amount of phosphatidic acid and inhibiting the release of arachidonic acid from phospholipids. 6. DRUGS, CALCIUM IONS AND PLATELET ACTIVATION The redistribution of calcium in platelets is obviously important in platelet activation, and this is confirmed by the inhibitory effects on platelets of various drugs which prevent calcium movement in different ways or which antagonize the action of calcium. Drugs which interfere with calcium distribution or calcium action may prove to be important therapeutically by preventing platelet activation which contributes to the development of vascular disease. Such drugs have already been shown to have a major impact on the therapy of cardiovascular disorders such as cardiac arrythmias, myocardial ischemia and hypertension. Drugs or other agents which interfere with the actions of calcium in platelets may be divided into the following categories. 6.1. CALCIUM-ENTRY BLOCKING AGENTS

The calcium-channel or calcium-entry blocking agents are a group of drugs which share a common effect of reducing the transmembrane transport of extracellular calcium ions (Cohn, 1982). Marked differences among the many agents suggest either that the blocking action of each of these drugs is relatively specific for different tissues, or that additional actions of these agents account for their varied effects. The increase in the concentration of calcium in the cytoplasm, which triggers the contractile process in vascular smooth muscle cells, can be due either to an increased permeability of the cell membrane for extracellular calcium (i.e. calcium entry), or to mobilization of calcium from cellular stores such as sarcoplasmic reticulum (Vanhoutte, 1982). The entry of calcium into activated vascular smooth muscle cells can be antagonized by low to moderate concentrations of calcium-entry blockers such as nifedipine and verapamil (Vanhoutte, 1982). At similar or higher concentrations, some of these calcium-entry blocking agents also affect the cellular mobilization of calcium (Thorens and Haeusler, 1979; Church and Zsoter, 1980). In platelets, low concentrations of verapamil (25-50 #M) have been shown to block transmembrane calcium flux in response to epinephrine (Owen et al., 1980; Owen and Le Breton, 1981). When calcium entry was blocked by verapamil, intracellular calcium mobilization in response to epinephrine and epinephrine-induced aggregation were inhibited (Owen et al., 1980; Owen and Le Breton, 1981). These results are consistent with the concept that epinephrine causes entry of calcium into platelets to induce calcium J.P.T. 182 t

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mobilization from a cellular store within the platelet. In contrast, it is postulated that other aggregating agents such as ADP, collagen and A23187 cause activation by mobilization of calcium from cellular stores (e.g. superficial binding sites and dense tubular system) without calcium entry, and much higher concentrations of verapamil (100-500/tM) are needed to inhibit aggregation caused by these agents (Ikeda et al., 1981). These higher concentrations of verapamil may be affecting cellular mobilization of calcium as occurs in smooth muscle (Thorens and Haeusler, 1979; Church and Zsoter, 1980). When verapamil was administered to healthy volunteers, inhibition of platelet aggregation caused by epinephrine, collagen and ADP was observed two hours after ingestion (Ikeda et al., 1981). This observation suggests a potential role for verapamil in antithrombotic therapy. Nifedipine, another calcium-entry blocking agent, also inhibits platelet secretion and aggregation (Johnsson, 1981). 6.2. INHIBITORS OF CALMODULIN The calcium-binding protein, calmodulin, mediates a variety of calcium-dependent functions in cells. Calmodulin appears to be involved in at least two different steps in platelet activation, namely activation of phospholipase A2 and the contraction of platelet actomyosin (see above). Calmodulin antagonists have been shown to inhibit platelet aggregation and the release reaction induced by ADP, collagen, epinephrine, thrombin, arachidonic acid and A23187 (Kindness et al., 1980; Rao et al., 1980; White and Raynor, 1980; Kambayashi et al., 1981; Suda and Aoki, 1981). The release of arachidonic acid from platelet membrane phospholipids is also inhibited by phenylthiazine (trifluoperazine, chlorpromazine, fluphenazine) and non-phenylthiazine (mepacrine, dibucaine) calmodulin antagonists (Kambayashi et al., 1981, Walenga et al., 1981), and the sulfonamide derivative, N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide (W-7) which binds to calmodulin (Suda and Aoki, 1981). Walenga et al. (1981) demonstrated that calmodulin antagonists inhibit the release of arachidonic acid from phosphatidylcholine, but the conversion of phosphatidylinositol to phosphatidic acid was not inhibited. These results indicate that phospholipase A2 requires calmodulin for activation while phospholipase C does not. 6.3. CALCIUM ANTAGONISTS AND OTHER DRUGS The drug 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HC1 (TMB-8), which acts as an intracellular calcium antagonist in muscle (Malagodi and Chiou, 1974), inhibits platelet secretion induced by either thrombin or A23187 (Charo et al., 1976). Similar results were obtained with TMB-6 (Le Breton and Dinerstein, 1977). TMB-8 also inhibits the conversion of arachidonic acid to TxA2 and this inhibition could be overcome by increasing the calcium concentration (Shaw, 1981). Inhibition of phospholipase A2 by TMB-8 has also been reported and this could not be reversed by high concentrations of A23187 (Rittenhouse-Simmons and Deykin, 1978). TMB-6 (100/XM) inhibited secretion but did not block platelet calcium redistribution associated with the shape change (Le Breton and Dinerstein, 1977). This finding suggests that platelets may contain two pools of calcium, one being involved in the shape change and aggregation and the other in secretion. Deuterium oxide (D20) which blocks calcium mobilization in muscle (Kaminer and Kimura, 1972), and high concentrations of chlortetracycline which chelate intracellular calcium (Taljedal, 1974), have been shown to inhibit platelet shape change and aggregation (Le Breton et al., 1976b). Certain local anesthetics c#,n inhibit intracellular calcium movements. These include dibucaine, tetracaine, lidocaine, benzocaine, QX572 and procaine as well as chlorpromazine and propranolol which possess significant local anesthetic activity. All of these drugs have been shown to inhibit platelet aggregation, the release reaction and phospholipase A2 mediated mobilization of arachidonic acid following stimulation of platelets by ADP,

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thrombin, collagen, arachidonic acid and the ionophore A23187 (Feinstein et al., 1976; Vanderhoek and Feinstein, 1979; Glass et al., 1980). It is apparent that local anesthetics have many different effects and the mechanism of their inhibitory action on platelets is not fully understood. Vitamin E has been shown to inhibit the release of calcium from a platelet membrane fraction containing membranes of the dense tubular system (Butler et al., 1979). At a similar concentration, vitamin E also inhibited platelet aggregation and secretion but did not inhibit phospholipase A2 induced release of arachidonic acid. It was therefore suggested that vitamin E prevents secretion by inhibiting efflux of calcium ions from the dense tubular system (Butler et al., 1979). It is believed that the principal mechanism of action of nonsteroidal antiinflammatory agents is to prevent metabolism of arachidonic acid by inhibiting the enzyme cyclooxygenase. However, it has recently been shown that indomethacin, sodium meclofenamate and sodium flufenamate were able to inhibit a purified calcium-dependent phospholipase A2 isolated from human platelets (Franson et al., 1980). The sensitivity of the enzyme to inhibition by the three agents was found to depend on the calcium concentration (Franson et al., 1980) and thus, inhibition of phospholipase A2 by nonsteroidal antiinflammatory agents via calcium antagonism may contribute to the mechanism of action of these drugs. 6.4. DRUGS THAT INCREASE CYCLIC A M P

The role of cyclic AMP in regulating platelet function has already been discussed. This nucleotide causes the sequestration of calcium, removing ionized calcium from the cytoplasm and thus inhibiting platelet activation or mediating recovery after stimulation. Therefore, drugs which increase cyclic AMP inhibit platelet function. The pyrimidopyrimidine drugs and related compounds inhibit ADP-induced aggregation and this effect has been attributed to their ability to increase platelet cyclic AMP by inhibiting phosphodiesterase, the enzyme which degrades cyclic AMP (Vigdahl et al., 1971; McE1roy and Philp, 1975). There are several other drugs that inhibit phosphodiesterase, increase platelet cyclic AMP levels, and inhibit platelet aggregation and release in vitro. This group includes the methylxanthines such as theophylline, caffeine and aminophylline (Ardlie et al., 1967; Brinson, 1972). Cyclic AMP is synthesized by the membrane bound enzyme adenylate cyclase, and the prostaglandins PGI2, PGE1 and PGD 2 are the most potent stimulators of platelet adenylate cyclase. Inhibition of platelet aggregation by these prostaglandins is believed to be due to their ability to stimulate adenylate cyclase and increase cyclic AMP. PGI2 has been reported to be up to 30 times more potent a stimulator of adenylate cyclase than PGD2 (Best et al., 1977; Gorman et al., 1977a; Tateson et al., 1977). Indeed PGI2 is the most potent inhibitor of platelet aggregation known. It has been proposed that a balance between the formation of TxA 2 and PGI2 is important for the homeostasis of the cardiovascular system. Analogues of PGI 2 or selective inhibitors of TxA2 may prove to be valuable in the treatment of vascular disease. 7. SUMMARY Although there are considerable gaps in our knowledge, the evidence for a role for calcium in mediating platelet activation by physiological stimuli is impressive. The response of platelets to vascular injury involves adhesion to subendothelial connective tissue, the formation of platelet aggregates, the secretion of substances stored in cytoplasmic organelles, the synthesis of metabolites of arachidonic acid, increased glycogenolysis and consumption of ATP. All of these reactions depend on calcium. A tentative scheme for the regulation of platelet function by calcium is presented in Fig. 3. It is proposed that ADP, thrombin and collagen initiate platelet activation by releasing a limited amount of calcium from binding sites in the plasma membrane. This is achieved

264

NEVILLE G. ARDLIE ADP THROMBIN COLLAGEN

EPINEPHRINE

PI PhospholipQse C

,Ca 2+ PA+Ca2~ PhospholipaseA 2

.L T×i2+C o 2+ i 2+

Cyclic AMP STORAGE GRANULE

PGI2

RELEASEREACTION

FIG. 3. A scheme for the regulation of platelet function by calcium. It is proposed that ADP, thrombin and collagen initiate platelet activation by mobilizing calcium (Ca 2+) from the plasma membrane. This is achieved by hydrolysis of phosphatidylinositol (PI) by phospholipase C. This leads to production of phosphatidic acid (PA) and lysophosphatidic acid which have ionophoretic properties and cause mobilization of Ca 2 +. Calcium interaction with the surface membrane and associated microtubules and contractile elements induces a shape change and aggregation (i). If sufficient Ca 2+ is mobilized by PA it will cause activation of calmodulin-dependent phospholipase A2 (ii) to release arachidonic acid (AA) and to produce TxA2 which also has ionophoretic properties and serves to amplify' the mount of Ca 2+ mobilized in platelets. The Ca 2+ is mobilized from the dense tubular system and enters the cytoplasm enabling actin and myosin to interact with the resultant release of contents of cytoplasmic storage granules. A strong stimulus may cause suflqcient Ca 2+ to be mobilized by PA to cause the platelet release reaction (iii) independent of AA release and TxA2 formation. A small amount of arachidonate might also be released from PI by phospholipase C. Whereas ADP. thrombin and collagen release intracellular Ca 2+, epinephrine initially activates platelets by causing Ca ~+ uptake, which in some way mobilizes intracellular Ca 2'. Cyclic AMP opposes the actions of Ca 2- by removing the cation from the cytoplasm, either discharging it from the platelets or returning it to the dense tubular system. PGI 2 inhibits platelet activation by elevating cyclic AMP.

by h y d r o l y s i s o f p h o s p h a t i d y l i n o s i t o l by p h o s p h o l i p a s e C. B r e a k d o w n o f p h o s p h a t i d y l i n o s i t o l l e a d s to t h e p r o d u c t i o n o f p h o s p h a t i d i c a c i d a n d / o r l y s o p h o s p h a t i d i c a c i d w h i c h c a u s e m o b i l i z a t i o n o f c a l c i u m , t h e site a n d a c t i o n of w h i c h m a y be h i g h l y l o c a l i z e d w i t h i n t h e platelet. T h e s h a p e c h a n g e , for e x a m p l e , p r o b a b l y i n v o l v e s c a l c i u m i n t e r a c t i o n with the surface membrane and associated microtubules and contractile elements. A c c o r d i n g to this h y p o t h e s i s , the s m a l l a m o u n t o f c a l c i u m t h a t is initially m o b i l i z e d m a y be insufficient by itself to a c t i v a t e c o n t r a c t i l e e l e m e n t s d e e p e r w i t h i n t h e c y t o p l a s m , a n d a d d i t i o n a l c a l c i u m is r e c r u i t e d by r e l e a s e f r o m the d e n s e t u b u l a r system. If sufficient c a l c i u m is m o b i l i z e d by p h o s p h a t i d i c a c i d (or l y s o p h o s p h a t i d i c acid) it will c a u s e a c t i v a t i o n o f c a l m o d u l i n - d e p e n d e n t p h o s p h o l i p a s e A2 to p r o d u c e T x A 2 a n d p o s s ibly p l a t e l e t - a c t i v a t i n g factor. T x A 2 , t h r o u g h its i o n o p h o r e t i c a c t i o n , serves to a m p l i f y

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the amount of calcium mobilized in stimulated platelets. The calcium is released from the dense tubular system (which is analogous to the sarcoplasmic reticulum in skeletal muscle) and enters the cytoplasm enabling actin and myosin to interact with resultant contraction and release of the contents of cytoplasmic granules. A strong stimulus may also cause sufficient calcium to be mobilized by phosphatidic acid to cause the platelet release reaction, independent of arachidonate release and TxA2 formation. A small amount of arachidonate might also be released from phosphatidylinositol by phospholipase C. Whereas ADP, thrombin and collagen release intracellular calcium and cause a change in platelet shape before aggregation, epinephrine initiates platelet aggregation without a change in platelet shape. The initial activation of platelets by epinephrine is associated with calcium uptake by platelets and does not require intraplatelet calcium mobilization. By causing a calcium flux into and through the membrane, epinephrine may cause changes in the membrane enabling platelets to adhere to each other. Thus, in platelets the initial increase in the cytoplasmic concentration of calcium which triggers platelet activation can be due either to mobilization of calcium from a cellular store (viz. plasma membrane binding sites) or to an increased permeability of the plasma membrane with entry of calcium ions from the exterior. The source of the initial trigger calcium depends on the nature of the stimulus. Accordingly, some platelet stimuli (viz, epinephrine) are more sensitive than others (viz. ADP, thrombin and collagen) to the inhibitory effects of calcium-entry blocking agents. In the past decade, the concept has been developed that interference with or blockage of the effects of calcium might be utilized therapeutically. A variety of agents capable of blocking the physiological actions of calcium have been identified, and have been found to produce beneficial effects in patients with coronary artery disease. It is possible that calcium-blocking agents may be valuable in the pharmacological therapy of patients with coronary artery disease and other disorders with platelet involvement. REFERENCES APITZ-CASTRO, R., CRUZ, M., MAS, M. and JAIN, M. K. (1981) Further studies on a phospholipase A 2 isolated from human platelet plasma membranes. Thromb. Res. 23: 347-354. ARDLIE, N. G., GLEW, G., SCHULZ, B. G. and SCHWARTZ, C. J. (1967) Inhibition and reversal of platelet aggregation by methyl xanthines. Thromb. Diath. haemorrh. 18: 67(~673. ARNOUX, B., DUVAL, D. and BENVENISTE, J. (1980) Release of platelet-activating factor (PAF-acether) from alveolar macrophages by the calcium ionophore A23187 and phagocytosis. Eur. J. clin. Invest. 10: 437-441. BEHNKE, O. (1965) Further studies on microtubules. A marginal bundle in human and rat thrombocytes. J. Ultrastruct. Res. 13: 469-477. BELL, R. L., KENNERLY, D. A., STANFORD, N. and MAJERUS, P. W. (1979) Diglyceride lipase: A pathway for arachidonate release from human platelets. Proc. hath. Acad. Sci. U.S.A. 76: 3238-3241. BENNETT, W. F., BELVILLE,J. S. and LYNCH, G. (1979) A study of protein phosphorylation in shape change and Ca + +-dependent serotonin release by blood platelets. Cell 18: 1015-1023. BENVENISTE,J., HENSON, P. M. and COCHRANE, C. B. (1972) Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils, and a platelet-activating factor. J. exp. Med. 136: 1356-1377. BERGSTROM, S., DANIELSON,H., KLEMBERG,D. and SAMUELSSON,B. (1964)The enzymatic conversion of essential fatty acids into prostaglandins. J. biol. Chem. 239: 4006-4008. BEST, L. C., JONES, P. B. B. and RUSSELL, R. G. G. (1979) Evidence that extracellular calcium ions inhibit thromboxane B E biosynthesis by human platelets. Biochem. biophys. Res. Commun. 90:1179 1185. BEST, L. C., MARTIN, T. J., RUSSELL, R. G. G. and PRESTON, F. E. (1977) Prostacyclin increases cyclic AMP levels and adenylate cyclase activity in platelets. Nature 267: 850-851. BETTEX-GALLAND, M. and LUSCHER, E. F. (1959) Extraction of an actomyosin-like protein from human thrombocytes. Nature 184: 276-277. BILLAH, M. M., LAPETINA, E. G. and CUATRECASAS,P. (1979) Phosphatidylinositol-specific phospholipase-C of platelets: association with 1,2-diacylglycerol-kinase and inhibition by cyclic-AMP. Biochem. biophys. Res. Commun. 90: 92-98. BILLAH, M. M., LAPETINA, E. G. and CUATRECASAS,P. (1980) Phospholipase A 2 and phospholipase C activities of platelets. Differential substrate specificity, Ca 2+ requirement, pH dependence, and cellular localization. J. biol. Chem. 255: 10227-10231. BILLAH, M. M., LAPETINA, E. G. and CUATRECASAS,P. (1981) Phospholipase A 2 activity specific for phosphatidic acid. A possible mechanism for the production of arachidonic acid in platelets. J. biol. Chem. 256: 5399-5403. BILLS,T. K., SMITH,J. B. and SILVER,M. J. (1978) Intracellular regulation of the metabolism of arachidonic acid in human platelets. Thromb. Haemostas. 40: 219-223.

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