lACC Vol 8. No. bDecember 1986 21 B-32B

Pharmacology of Platelet Inhibitors

LAURENCE A. HARKER, MD,* VALENTIN FUSTER, MD, FACCt

La Jolla. California and New York, New York

21B

Although many drugs have inhibitory effects on plateletfunction, none of them inhibits all of the mechanismsthat may be involved in the various forms of thrombosis.Choice of suitable drugs is hampered by lack of fullknowledge concerning the reactions that make the majorcontributions to the formation ofarterial thrombi at sitesof repeated vessel wall injury or on atherosclerotic le­sions. Drugs such as aspirin that inhibit the arachidonatepathway in platelets can only be expected to be effectiveagainst thromboembolic events in which the generationof thromboxane A2 plays a major part. If thrombin andfibrin formation are dominant, oral anticoagulant agentsor heparin should be beneficial; thus, experimental evi­dence indicates that with repeated vessel wall injury, theformation of platelet fibrin thrombi on the vessel wall isprobably influenced more by inhibitors of thrombin gen­eration than by the subendothelial constituents such ascollagen.

Phannacologic modification of platelet function reduces thethromboembolic complications in a number of arterial vas­cular disorders and in patients with prosthetic cardiovasculardevices (1-3). However, the indications for the clinical useof antiplatelet agents are not entirely clear despite extensivebasic experimental animal and clinical studies. This uncer­tainty is explained, at least in part. by our lack of knowledgeof the precise pathogenesis of many clinical vascular syn­dromes, by the lack of suitable phannacologic agents fordefinitive clinical testing and by the inconclusive resultsobtained in some of the clinical studies. In this study, thecurrent pharmacologic status of antiplatelet drugs used inclinical practice is reviewed.

From the *Roon Center for Artenosclerosis and ThrombosIs. Depart­ment of Basic and Climcal Research, Scnpps Climc and Research Foun­dation. La Jolla. California and tMount Sinai Medical Center, New York,New York. ThiS work was supported m part by U.S. Public Health ServiceGrants HL 31950 and RR 0833 from the National Institutes of Health.Bethesda, Maryland. ThiS study is publication no. 4355 BCR from theResearch InstItute of Scripps Climc. Scripps Climc and Research Foun­datIon, La Jolla, California.

Address for reprint>. Laurence A. Harker, MD, Roon Center for Ar­teriosclerosis and Thrombosis. Department of BaSIC and ClImcal Research.Scripps Climc and Research FoundatIOn. La Jolla. California 92037

©1986 by the Amencan College of CardIology

Agents like prostacyclin that raise platelet cyclicadenosine monophosphate (AMP) levels in platelets bystimulating adenylate cyclase are potent inhibitors of thereaction of platelets to all aggregating and release­inducing stimuli, but these agents are not suitable forlong-term administration. The effect of dipyridamole onplatelet cyclic AMP levels is weak, and this drug mayact through other effects on platelets or on other cells.Indeed, several of the drugs that have been tested inclinical trials may exert their effects through unrecog­nized mechanisms. Many combinations of drugs havebeen used to affect platelets or platelets and coagulation.This practice has been based on the theory that becauseseveral mechanisms may be involved in thrombus for­mation, combinations of drugs that inhibit differentmechanisms may be beneficial.

(J Am Coil CardioI1986;8:21B-32B)

Ideally, a clinically useful, platelet-modifying drug shouldbe nontoxic, orally effective, have sustained action and havegood antithrombotic potency without excessive risk of ab­normal bleeding. None of the currently available clinicalagents satisfies all these requirements. Aspirin, sulfinpyr­azone, dipyridamole, suloctidil, ticlopidine and prostacyclinhave been the agents evaluated in clinical trials to date.Many agents with more defined pharmacologic actions havebeen synthesized for evaluation and are presently at variousstages of development.

The disorders for which antiplatelet therapy has beenstudied may have quite different pathogenetic mechanisms.Because the effects of a given drug may be different foreach pathogenetic process, it is probably inappropriate toextrapolate the results for a given drug from one clinicalsetting to another. In general, however, kinetic studies (4)in patients using radioactively labeled platelets and fibrin­ogen indicate that arterial thrombosis is characterized byselective platelet consumption that can be interrupted bysome inhibitors of platelet function but not by heparin, whereasvenous thrombosis produces combined and equivalent con­sumption of both platelets and fibrinogen that is blocked byanticoagulant therapy but not by drugs that modify plateletfunction.

0735-1097/86/$3.50

228 HARKER AND FUSTERPHARMACOLOGY OF PLATELET INHIBITORS

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Pathways of Platelet Activation andTheir Modulation

Process of platelet adhesion and aggregation (Fig. 1).Vascular injury with endothelial denudation exposes sub­endothelial structures to circulating blood and induces plate­lets to adhere to subendothelial collagen (5). The processof platelet adhesion involves interaction of the platelet mem­brane glycoprotein receptor GPlb, subendothelial collagenand plasma von Willebrand factor and fibronectin (6). Plate­let interaction with collagen leads to the release of adenosinediphosphate (ADP) and activation of the prostaglandinarachidonate pathway (7,8) to release thromboxane A2 • BothADP and thromboxane A2 recruit circulating platelets (9,10)to change shape and aggregate with the layer of alreadyadherent platelets on the subendothelial surface (Fig. I). Athird pathway of platelet recruitment involves thrombin gen­eration. Thrombin formation is produced through both ex­trinsic and intrinsic coagulation processes at the injury siteand around the aggregating platelet mass. Although throm­bin induces the release of both ADP and thromboxane A2 ,

more importantly, thrombin induces platelet aggregation andrelease through a direct mechanism that is independent ofADP release or the formation of thromboxane A2 (11-13).In addition, the formation of polymerizing fibrin in closeproximity to the platelet aggregate by the action of thrombinon fibrinogen stabilizes the aggregate through the adherenceof polymerizing fibrin to the platelet surface (14). Plateletaggregates that are not stabilized by fibrin deaggregate read­ily and are broken up under the force of blood flow.

IHepa rlnas~

All platelet recruitment requires the rapid expression ofthe platelet-membrane complex GPllb/GPIlIa as the fibrin­ogen receptor (6,9,15). That is, platelet-platelet aggregatesare formed through calcium-dependent fibrinogen bindingwith the expressed receptor. This reaction may also be mod­ulated by platelet alpha-granule proteins, fibronectin, vonWillebrand factor or thrombospondin (16).

Limitation of thrombus formation. Intact endotheliumactively limits thrombus formation and its extension by mul­tiple mechanisms including I) inactivation of released ADP(17), 2) active clearance of vasoactive amines (18), 3)thrombin-mediated release of prostacyclin (19), 4) directinactivation of circulating thrombin by plasma protease in­hibitors (20), 5) inactivation of thrombin by enhancementof the endothelial surface thrombin-antithrombin III com­plex formation (21), 6) thrombin complexing with throm­bomodulin on the endothelial cell surface to activate proteinC (which reduces thrombin formation by destroying factorsVa and Villa both in plasma and bound to the plateletsurface) (22), and 7) thrombin-mediated release of tissueplasminogen activator from intact vascular wall (23) (Fig.2). Thus, thrombin initiates important negative feedbackmechanisms to control its own generation and extent ofeffect.

Calcium-dependent platelet activation. There is in­creasing evidence that some platelet responses occur throughcalcium-regulated contractile processes (24), which are intum modulated by cyclic adenosine monophosphate (AMP)(25) and prostaglandin or arachidonic acid products (26).In general, contractile and secretory processes are thought

Figure 1. Pathways of platelet recruitment. Endo­thelial disruption initiates the attachment of plateletsto the subendothelium through the interaction of aplatelet receptor (GPIb), subendothelial collagen anda plasma cofactor VIII/von Willebrand factor (vWF).Fibronectin and thrombospondin derived from plasma,platelets and endothelium may also play roles in thisprocess. An unstable platelet mass forms through sev­eral interactive but independent recruitment mecha­nisms, including the generation of thromboxane (TX)A2 release of dense granule, adenosine diphosphate(AOP) and thrombin-mediated platelet activation.Aggregation requires the rapid expression of plateletmembrane fibrinogen receptor complex (GPIIb andGPIIIa) and calcium-dependent interplatelet bridgingby fibrinogen. Thrombin is generated locally on theplatelet surface through both extrinsic and intrinsicpathways. Thrombin-modified factor V (factor Vm)binds to platelets. Platelet-bound factor Vm serves asa high affinity platelet receptor for factor Xa in thefinal convetsion of prothrombin to thrombm. Thus,the generation of thrombin initiates potent positivefeedback mechanisms on the platelet surface for ex­plosive activation of the coagulation cascade and fi­brin formation. f3TG = f3-thromboglobulin; FOP =fibrin degradation products; POGF = platelet-de-rived growth factor; PF4 = platelet factor 4.

lACC Vol 8. No.6December 1986'2IB-32B

Figure 2. Mechanisms limiting throm-bus extension. Intact endothelium ac­tively resists thrombus formation. Plate­let aggregate formation is prevented bythe endothelium despite the presence ofADP, epinephrine or thrombin throughthe inactivation of ADP, active clearanceof vasoactive amines, facilitated com­plexing of thrombin with antithrombin IIIand the explosive thrombin-mediatedsynthesis and release of inhibitory POb.These mechanisms markedly decrease thepossibility of thrombus forming in thepresence of intact endothelium. The po­tent effects of thrombin are also activelylimited to the site of vascular injury byplasma protease inhibitors, enhancementof the endothelial surface of thrombin­antithrombin III complex formation,binding of circulating thrombin to throm­bomodulin (a receptor of luminal surfaceof the endothelial cells) thereby activatingprotein C to destroy factors V and VIIIand induced release of tissue plasminogenactivator from vascular endothelium. Thus,thrombin initiates negative feedbackmechanisms controlling its own genera­tion. Abbreviations as in Figure I.

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to be mediated by an increase in the concentration of cy­toplasmic calcium derived from intracellular storage sitesor from the extracellular environment. Calcium-dependentplatelet activation occurs through actomyosin-mediated con­tractile mechanisms including pseudopod formation, aggre­gation, release and clot retraction. It appears that the releasereaction is triggered by release of calcium from an intra­cellular storage site, possibly the dense tubular system, intothe cytoplasm. Calcium regulation of contractile proteins inplatelets is similar to the processes of contraction in muscle.Calcium and the calcium-binding protein calmodulin areinvolved in activation of the specific enzyme myosin lightchain kinase and, hence, promotion of the interaction be­tween platelet actin and myosin (27).

Platelet cyclic AMP. This compound inhibits both plate­let secretion and aggregation (28). The amount of cyclicAMP in the platelet is determined both by the activity ofmembrane adenyl cyclase and by the specific phosphodi­esterase that hydrolyzes cyclic AMP to adenosine triphos­phate (ATP); that is, platelet cyclic AMP is increased byagents that either increase the activity of adenyl cyclase ordecrease the activity of phosphodiesterase. The basal levelof cyclic AMP appears to modulate release and aggregationby controlling the storage and release of calcium from plate­let intracellular storage sites.

Role of prostaglandins. As discussed in more detail inthe first section of the symposium by Vermylen et al. (29)(also see Fig. I and 2 in reference 29), prostaglandins (PG)

are important in the regulation of platelet activation for tworeasons (26,30). First, as mentioned, stimulated plateletsproduce biologically active prostaglandins and derived sub­stances that regulate platelet function. Second, other pros­taglandins produced by other cells (such as PGh by vascularcells) may either inhibit or activate platelets. Platelet stim­ulation results in the hydrolysis of arachidonic acid frommembrane phospholipid, possibly through a calmodulin­mediated process. Both phosphatidylinositol (through hy­drolysis by phospholipase C) and phosphatidylcholine (throughhydrolysis by phospholipase Az) may serve as the sourceof arachidonic acid. The arachidonic acid thus liberatedbecomes converted by cyclooxygenase to labile prostaglan­din endoperoxide intermediates (PGGz and PGHz), whichare potent platelet-aggregating agents, or through the Ii­poxygenase pathway to other prostaglandin derivates. Bymeans of thromboxane synthetase, the most important con­version of PGHz is to the highly labile nonprostaglandinsubstance thromboxane Az, which is the most potent plate­let-aggregating agent derived from arachidonic acid yet dis­covered. In addition, thromboxane Az is a potent vasocon­strictor. PGHz is also converted to several other (stable)prostaglandins, namely, PGEz and PGFza , which do notdirectly affect platelets but may potentiate aggregation byother substances, and PGDz, which inhibits aggregation andcould play the role of a negative feedback mechanism inregulating aggregation.

On the other hand, vascular tissue, cultured endothelial

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cells and postinjury neomtIma synthesize POlz from thearachidonic acid pathways (26,31,32). Moreover, endo­thelial cells can utilize POOz(Hz) derived from platelets tosynthesize POIz (33). POIz is about 30 to 40 times morepotent than POE) as an inhibitor of platelet aggregationinduced by ADP, epinephrine, collagen, thrombin and ar­achidonic acid. Its inhibitory effects are mediated throughelevation of platelet cyclic AMP through activation of ad­enylcyclase (34).

Finally, prostaglandins, cyclic AMP and Ca2+ interactin a number of complex ways to enhance platelet function(7,8,35). For example, both phospholipase Az and phos­pholipase C are activated by Caz + , and the inhibitory effectof cyclic AMP on platelet aggregation may act by main­taining calcium in a bound form, thereby preventing acti­vation of phospholipases. Thromboxane A2 also has prop­erties resembling those of a calcium ionophore, and someof the platelet-stimulatory effects of thromboxane Az maybe related to its ability to mobilize intracellular calcium fromstorage sites.

Drug TestingThe first assessment of a new compound to test for plate­

let-inhibitory activity usually involves adding it to citratedplatelet-rich plasma in vitro and examining the effect onaggregation and the release of granule contents in responseto such substances as ADP, epinephrine, collagen and ar­achidonic acid. Suspensions of washed or gel-filtered plate­lets are more suitable for tests with thrombin and also permitexperiments in the presence of physiologic concentrationsof ionized calcium and magnesium. Unfortunately, theseresults have little predictability regarding antiplatelet effi­cacy in vivo, and it is for this reason that the effects of theseagents on in vitro testing will not be addressed in detail inthis review. Thus, although many agents that strongly inhibitaggregation and release in vitro have been identified, fewhave proved useful in human patients, particularly for long­term administration. Most of the drugs that have been usedin clinical trials were in use long before their effects onplatelets were recognized, and their value as antithromboticagents would not have been recognized by the type of routinescreening systems now being used. There have been noreports of major trials of new drugs that have been designedspecifically as inhibitors of platelet function. Other in vitrotests of platelet function of uncertain interpretation includestudies of platelet adherence to glass beads, artificial sur­faces, collagen or subendothelium and examination of thecontribution of platelets to the acceleration of coagulation.In vivo testing in experimental animals has a number ofadvantages: 1) the drug is tested at concentrations that canbe achieved clinically, 2) active metabolites of the drug arealso assessed, 3) the time during which effective concen­trations of the drug or its metabolites remain in the circu-

lation can be determined, and 4) side effects of the drugmay become apparent.

The most common tests of in vivo platelet function inhuman beings after drug administration involve bleedingtime (36), platelet survival and turnover (37), examinationof plasma for materials released from platelets (platelet fac­tor 4 and beta-thromboglobulin) (38) and determination ofthe number of circulating platelet aggregates (39,40). Inaddition, there are suggestions of platelet hypersensitivity,circulating platelet aggregates or circulating materials re­leased from platelets associated with clinical complicationsof atherosclerosis (41). In almost all of these conditions,administration of drugs that inhibit the arachidonate pathwayin platelets decreases their sensitivity to aggregating agentsand reduces the number of circulating platelet aggregates.These variables, however, may be a result of the clinicalcomplications rather than the cause. If so, drugs that depressthe sensitivity of the platelets may have little effect on theclinical complications.

Pharmacology of Drugs That ModifyPlatelet Function

The pharmacology of the drugs that inhibit platelet func­tion and thrombus formation will be considered in four broadcategories: I) drugs that inhibit the arachidonate pathway,2) drugs that affect platelet cyclic AMP levels, 3) drugs thatinhibit thrombin formation and action, and 4) drugs that actthrough less well defined mechanisms.

Drugs that inhibit the arachidonate pathway. Drugsthat affect this prostaglandin pathway include agents thatinhibit platelet cyclooxygenase, drugs that block the actionof phospholipases and drugs that inhibit thromboxane syn­thetase. Because these drugs affect only thrombus formationthat is dependent on thromboxane Az, thrombus formationthat is predominately mediated by the other pathways andstimuli (that is, thrombin) will be largely unaffected by thisclass of drug.

Drugs that inhibit cyclooxygenase include the nonsteroidanti-inflammatory drugs such as aspirin, indomethacin, ibu­profen and many others (1-3,42). (Although the uricosuricagent sulfinpyrazone has effects on cyclooxygenase, its an­tithrombotic mechanism is unclear and is, therefore, dis­cussed later.)

Aspirin and platelet function. Aspirin inhibits the secondbut not the first wave of ADP-induced platelet aggregation.Aspirin affects, in part, collagen-induced platelet aggre­gation caused by its inhibitory effect on platelet secretion.In addition to inhibiting the release of ADP, aspirin in vitroand in vivo inhibits the collagen-induced release of ATP,serotonin and platelet antiheparin activity (platelet factor 4).Similar results have been obtained with epinephrine-inducedaggregation. The effects of aspirin on thrombin-induced

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aggregation and secretion are dose-related. Inhibition occursat low but not high concentrations of thrombin (43). Finally,the inhibitory effects of aspirin on platelet aggregation todifferent agents are detected for 4 to 7 days after a singleoral dose when salicylate is no longer detectable in theblood. This period of inhibition reflects an irreversible effectof aspirin on the platelet.

Aspirin irreversibly acetylates the cyclooxygenase inplatelets (44). Because platelets are unable to synthesizenew enzyme to replace that which has been inactivated, itis not until new platelets enter the circulation that plateletfunction is restored. However, aspirin also acts on mega­karyocyte cyclooxygenase. This is shown by the finding thathuman platelets harvested at times up to 48 hours afteradministration of aspirin cannot be acetylated in vitro withradiolabeled aspirin (45), and also by direct experimentswith megakaryocytes that indicate that aspirin administra­tion abolishes their ability to synthesize prostaglandins (46).The long-term administration of I mg/kg of aspirin once aday effectively inhibits platelet cyclooxygenase and throm­boxane A~ formation in humans.

In contrast, aspirin inhibition of the cyclooxygenase ofthe cells of the vessel wall is not irreversible because thesecells synthesize new cyclooxygenase. Regeneration of theability of cultured human endothelial cells from umbilicalveins to synthesize PGI~ requires many hours (47). Indeed,long-term administration of I mg/kg of aspirin once a daywill also effectively inhibit vascular cyclooxygenase andprostaglandin production in humans (48). Thus, the notionthat low dose, infrequently administered aspirin exerts itsantithrombotic effects by preferentially sparing vascularprostacyclin production may not be correct. Furthermore,high doses of aspirin over prolonged periods to patients withrheumatoid arthritis may decrease thrombosis despite in­hibition of PGI~ production (49). Earlier data concerningthe low incidence of myocardial infarction as a cause ofdeath of patients with rheumatoid arthritis who consumedlarge amounts of aspirin also support this view (50).

Under physiologic conditions of flow and protein con­centration and hematocrit, aspirin does not inhibit adherenceof the initial layer of platelets to the subendothelium (51 ,52).Aspirin and indomethacin also do not inhibit the release ofamine storage granule contents from platelets that adheredirectly to collagen or the subendothelium (53), and releasedADP and serotonin from these platelets contribute to throm­bus formation in the presence of the cyclooxygenase inhib­itors. Because aspirin does not inhibit release of platelet­derived growth factor from the alpha granules of the ad­herent platelets, it is not surprising that these drugs do notprevent smooth muscle cell proliferation in response to ves­sel damage (54). However, these drugs may inhibit plateletaggregate formation on the layer of adherent platelets (52),presumably by blocking thromboxane A~ release.

In some experimental animal studies, aspirin has par-

tially inhibited thrombus formation. In other studies no sig­nificant effects have been observed and, in a few, aspirinhas been reported to potentiate thrombus formation (55).The last effect has been attributed to aspirin inhibiting PGI~

production by the vessel wall.Aspirin in unstable angina and ischemia. In the myo­

cardial and cerebral circulations, platelet aggregate forma­tion and, perhaps, associated vasospasm (related to the re­lease of thromboxane A2 or platelet-derived growth factor[56,57]) may lead to the acute coronary syndromes (that is,unstable angina, myocardial infarction, arrhythmia-relatedsudden death) or transient ischemic attacks, respectively.In dogs with experimentally stenosed coronary arteries, as­pirin inhibits the formation of platelet plugs and the occur­rence of fatal arrhythmias (58). Studies (59,60) with isch­emic dog and cat myocardium have shown that aspirin canprevent ventricular fibrillation caused by ischemia. The ben­eficial effects of aspirin in patients with unstable angina ortransient ischemic attacks and in the experimental prepa­rations just referred to, may be related not only to its abilityto inhibit platelet aggregation, but also to the inhibition ofrelease of thromboxane A~ and platelet-derived growth fac­tor, preventing vasoconstriction so that thromboemboli donot readily lodge and persist in the microcirculation.

Aspirin and thromboembolism. Aspirin has been shownto be clinically effective in reducing thromboembolic com­plications of artificial heart valves in one study (61), throm­botic occlusion of arteriovenous cannulas in uremic patients(62), stroke and death in patients with transient ischemicattack (63), myocardial infarction in men with unstable an­gina (64,65), and, possibly, the occlusion of saphenous veincoronary artery bypass grafts (66). It may be important inthis context to note that an additional hemostatic abnormalitywas associated with the beneficial effect of aspirin in thestudies of artificial heart valves (oral anticoagulation) andarteriovenous cannulas (platelet dysfunction in uremic pa­tients treated with long-term hemodialysis). The reportedantithrombotic effect of aspirin in patients undergoing sa­phenous vein coronary artery bypass surgery is provisionalin that the study involved a relatively small number of pa­tients with an unusually high frequency of graft occlusionin the control group. Thus, aspirin may exert limited an­tithrombotic effects unless it is used in association withanother antithrombotic drug or perturbation of the hemo­static mechanism.

Regarding aspirin dose, the beneficial effects of low dosesof aspirin (100 to 150 mg/day) reported to prevent throm­bosis in saphenous vein aortocoronary bypass grafts andarteriovenous shunts must be considered to be provisionalbecause, as already mentioned, the former study requiresconfirmation because of questions regarding the control group,and the conclusions of the latter study may not be extrap­olated to other clinical settings because of the associatedplatelet dysfunction in those patients undergoing long-term

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hemodialysis. On the other hand, there are clinical reportsof high dose-dependent antithrombotic effects of aspirin. Inthis context, recent studies (67) in experimental throm­boembolism indicate that aspirin has antithrombotic effectsthat are independent of its effect on cyclooxygenase andthat these effects occur at high doses.

Nonsteroid anti-inflammatory drugs. Other nonsteroidanti-inflammatory drugs also inhibit platelet activation throughinhibition of platelet cyclooxygenase (67). However, theseagents differ in potency and duration of action because theyinhibit competitively. In addition, some of these drugs mayhave other effects on platelets besides inhibiting cyclo­oxygenase. For example, indomethacin may inhibit phos­pholipase activation. At equimolar concentrations, meclofe­namic acid, aspirin and indomethacin and ibuprofen werethe most active, whereas phenylbutazone was a relativelyweak inhibitor (68).

Other agents. Drugs that affect the generation of ara­chidonate from the cell membrane have been considered tobe inhibitors of phospholipase Az; they include the anti­malarial drug quinacrine hydrochloride (mepacrine hydro­chloride) and some of the steroid anti-inflammatory drugs.None of these agents has been shown to have clinicallyuseful antithrombotic effects in humans (42).

Inhibitors that block thromboxane synthetase include im­idazole and its derivatives. Although a number of thesecompounds have been synthesized and have undergone test­ing in animal models and human subjects (69), none ofthese agents has been shown to prevent thrombosis in hu­mans.

Drugs that increase platelet cyclic AMP levels. Whenthe cyclic AMP level in platelets is elevated, many plateletfunctions are inhibited (42). Platelet cyclic AMP is increasedby agents that stimulate adenylate cyclase and, to a lesserextent, by drugs that inhibit platelet phosphodiesterase (79)and, thus, prevent the breakdown of cyclic AMP in platelets(42). Adenylate cyclase is stimulated by PGlz, PGFz andPGE!. The combination of PGIz or PGE, with a phospho­diesterase inhibitor such as dipyridamole or theophyllinestrongly inhibits platelet functions (71,72).

PGE1 and PGIz. Agents that increase cyclic AMP levelsdiminish rabbit platelet adherence to damaged vessel walls(72), inhibit platelet aggregation induced by all stimuli andinhibit the release of platelet granule contents induced byall stimuli (26,42,74). When cyclic AMP levels are raised,the change in platelet shape in response to aggregating andrelease-inducing agents is also inhibited. This prevents plateletmembrane sites involved in the intrinsic coagulation path­way from becoming available to accelerate the generationof thrombin on the platelet surface. Agents such as PGE 1

or PGIz, when given in high doses, have been shown to bestrong inhibitors of platelet aggregation, thrombus formationand their consequences under a variety of situations in hu-

mans and experimental animals (75-80). PGIz is the mostpotent inhibitor of platelet aggregation thus far described,and it is 30 to 40 times more potent than PGE, in inhibitingplatelet aggregation by ADP. The duration of these effectsin vivo is very short; they disappear within 30 minutes ofdosing. PGh is a strong dilator in several vascular beds suchas those of the heart, kidney, mesenteric and skeletal muscle(81,82). Intravenous infusion of relatively small amounts(5 ng/kg per min) of PGlz in human patients produces vaso­dilation of peripheral blood vessels in the head, neck andpalms, a decrease in diastolic but not systolic blood pressureand an increase in heart rate (83). Platelet aggregation inresponse to ADP and the number of circulating aggregatesdecrease during PGIz infusion, while the bleeding time in­creases. One hour after termination of the infusion, theresponsiveness of the platelets to ADP returns to normal.

Dipyridamole. Dipyridamole is another platelet-inhibitordrug. It has been proposed that it acts to elevate plateletcyclic AMP by both blocking platelet phosphodiesterase­dependent breakdown and increasing cyclic formation byPGIrmediated stimulation of platelet adenylate cyclase (72).It seems more likely that an increase in blood adenosinelevels may be involved because dipyridamole is a potentinhibitor of vascular and erythrocyte adenosine uptake (84,85)and produces an elevation in plasma adenosine levels (86).The higher plasma concentrations of adenosine could me­diate the inhibition of platelet reactivity by stimulating plate­let adenylate cyclase activity (87). For example, 10 fLM ofdipyridamole in vivo markedly inhibits the uptake of aden­osine by endothelial cells and produces potent antiaggre­gating activity that is not blocked by aspirin (that is, in theabsence of prostacyclin). The vasodilating effects of dipyr­idamole also appear to be related to the elevation of plasmaadenosine levels. Indeed, vasodilation may account for itsactions in some experimental situations in which it has beenshown to be beneficial (88).

Dipyridamole, when given experimentally at a very highdose, does inhibit platelet adherence to collagen (89) andthe subendothelium (90), but most importantly, when givenat a dose more comparable with that used in humans, di­pyridamole inhibits platelet activation over artificial surfaces(67,91). Indeed, the capacity of dipyridamole to decreaseexperimental thromboembolism in a dose-dependent fashioncorrelates well with its ability to normalize platelet survivalin patients with artificial heart valves (92,93) and arterio­venous cannulas (94) and its efficacy in preventing throm­boembolism in patients with prosthetic heart valves (95).Moreover, the dose (10 mg/kg per day) and resulting bloodlevels that produce complete interruption of experimentalthromboembolism at prosthetic surfaces are in reasonableaccord with the dose (5 to 7 mg/kg per day) reported tonormalize platelet survival (92,94,96) and prevent throm­boembolism in patients with artificial heart valves.

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Aspirin potentiates the efficacy of dipyridamole in ex­perimental thromboembolism on prosthetic surfaces (67).Moreover, the full potentiation depends on administering arelatively high dose of aspirin simultaneously with everydose of dipyridamole (20 mg/kg per day). These results inbaboons are in accord with the capacity of the combinationof aspirin and dipyridamole to prolong platelet survival inpatients with Dacron vascular grafts (96). On the other hand,the data regarding the beneficial effects on platelet survivaland thromboembolism of the combination of aspirin anddipyridamole in biologic surfaces such as in coronary arteryatherosclerosis (97-99), renal microvascular disease (100)and saphenous vein bypass grafting (10 I, 102) has raisedthe possibility of aspirin being the main effective compo­nent. That is, in four other different trials (103-106), thecombination of aspirin and dipyridamole was as effectiveas aspirin alone; in only one trial (107) addressing the rateof progression of peripheral vascular disease did this com­bination appear to be more effective than aspirin alone, butthis remains to be confirmed.

The mechanism whereby aspirin potentiates the anti­thrombotic activity of dipyridamole. particularly in exper­imental prosthetic surfaces. is not: I) mediated through as­pirin's conversion to salicyclic acid; 2) due to alteredpharmacokinetic results; or 3) dependent on inhibition ofthe cyclooxygenase-thromboxane Az pathway since the spe­cific blockage of thromboxane Az formation with dazoxibendoes not by itself produce detectable antithrombotic activityor potentiation of dipyridamole's antithrombotic activity (67),

Drugs that inhibit thrombin. The effects of thrombinon platelets and' fibrin formation can be prevented by in­hibiting its action with heparin. Heparin, through its effecton antithrombin III, also inhibits other serine proteasesproduced during activation of the coagulation cascade, suchas factors IXa, Xa and XIa (108).

Heparin. Reports of the effects of heparin on plateletreactions are complex and contradictory (109,110) perhaps,in part, because of the variability among different com­mercially available heparin preparations. In vitro, heparinprevents the effects of thrombin on platelets, but may po­tentiate or inhibit aggregation and release induced by otheraggregating agents (109,110). It may itself cause plateletaggregation.

It is now recognized that heparin is heterogeneous withrespect to its molecular size and its affinity for antithrombinIII (111,112). More than 60% of some commercial formsof heparin are practically inactive as an anticoagulant. Inone study (109), heparin induced platelet aggregation incitrated platelet-rich plasma, Fractions of high molecularweight (approximately 20,000 daltons) were more reactivewith platelets than were fractions of low molecular weight(approximately 7,000 daltons). These findings raise the pos­sibility that it may be feasible to select fractions of low

molecular weight and high antithrombin III activity for an­tithrombotic therapy without the undesirable effects of un­fractionated heparin on platelets.

Heparin combined with prostaglandins. Two observa­tions should be considered in the relation between the effectsof heparin and PGIz on platelet function. In the presence ofPGlz, the inhibitory effect of heparin on coagulation shouldbe enhanced (113). On the other hand, heparin has beenshown to inhibit the activation of adenylate cyclase by PGlz(114). Thus, a combination of heparin with one of theseprostaglandins might prove beneficial in preventing the for­mation of thrombi to which the coagulation pathway makesa major contribution, but might be less useful if plateletaggregation in response to collagen, ADP and thromboxaneAz has a dominant role and PGlz acts in a regulatory role.Overall, when well defined anticoagulant fractions of hep­arin become available for more extensive testing, it will beimportant to reassess the antiplatelet effects of heparin in avariety of experimental and clinical situations.

An alternative strategy being developed to block throm­bin-mediated platelet thrombus formation involves the syn­thesis of peptides that show specificity for the inhibition ofthrombin (115-117). A number of these agents have beenstudied in vitro and in experimental animals. No clinicalexperience is available.

Agents that inhibit platelet function by other mech­anisms. Many other agents have been shown to inhibitplatelet reactions and thrombus formation, although theirmechanisms of action remain to be defined (1-3,42).

Suljinpyrazone. Sulfinpyrazone inhibits platelet aggre­gation and secretion in vitro, but at concentrations higherthan that achieved after standard clinical doses (42). Inhi­bition of platelet aggregation and secretion after oral admin­istration of high doses of sulfinpyrazone has also been re­ported (118). However, the concentration of collagen usedin performing in vitro tests is critical in demonstrating aninhibitory effect of sulfinpyrazone after oral administrationof the drug in humans (119). Although it has been suggestedthat sulfinpyrazone, in contrast to aspirin, is a competitiveinhibitor of platelet cyclooxygenase, present data suggestthat other mechanisms are more relevant in vivo (42,67).The inhibitory effects of sulfinpyrazone on collagen-inducedaggregation in rabbits has persisted for many hours after thedrug was cleared from the blood. This prolonged inhibitoryeffect has been attributed to mediated drug metabolites inplasma (120). Sulfinpyrazone has also been found to inhibitthe formation of thrombi on subendothelium (121) and toprotect endothelium from chemical injury in vitro and pos­sibly in vivo (122). No mechanism has been identified forthis postulated endothelial protective effect. In addition, adose-dependent inhibition by sulfinpyrazone of experimen­tal thromboembolism has been reported (67) that correlateswith its capacity to normalize platelet survival in patients

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with artificial heart valves (123) and with its ability to reducethrombotic events in patients with arteriovenous cannulas(124). Experimentally on prosthetic surfaces, aspirin alsopotentiates this antithrombotic effect of sulfinpyrazone, witha maximal effect at 20 mg/kg per day, the same dose foundto optimally potentiate dipyridamole (67). These results withsulfinpyrazone on prosthetic surfaces have been more con­sistent than with biologic surfaces. Thus, despite a beneficialtrend in decreasing vascular events after myocardial infarc­tion (125) and saphenous vein coronary artery bypass (126),the results were negative in the Canadian stroke study (63)and in the Canadian unstable angina study (65).

Ticlopidine. Ticlopidine is unusual in two respects: 1)

its effects are only manifest 24 to 48 hours after adminis­tration, but they last for several days after the drug is stopped;and 2) it is an inhibitor of the primary phase of ADP-inducedaggregation (127,128). It also inhibits aggregation and re­lease induced by collagen, epinephrine, arachidonic acidand thrombin, prolongs the bleeding time, prolongs short­ened platelet survival (129) and inhibits intimal proliferation(130) in the rabbit aorta from which the endothelium hasbeen removed. As well as being an inhibitor of plateletreactions in its own right, ticlopidine has been shown toenhance the inhibitory effect of PGh on platelets (13!).Thus, of the antiplatelet drugs currently available for clinicalinvestigation, ticlopidine is one of the most potent and hasseveral important theoretical advantages over existing drugs.Ticlopidine is chemically unrelated to other antiplatelet drugsand appears to have a unique, albeit unknown, mechanismof action. It is neither a prostaglandin synthesis inhibitornor a cyclic AMP-phosphodiesterase inhibitor. There aredata that indicate that ticiopidine may act on the plateletmembrane to alter its reactivity to activating stimuli (128).Clinical evaluation of this drug is underway in a number ofEuropean and American trials.

Antibiotics. In high doses, carbenicillin and penicillinproduce a bleeding tendency in patients (132). Studies withhuman volunteers have shown that these antibiotics alsoprolong the bleeding time and inhibit platelet aggregationinduced by a variety of agents. The drugs also inhibit plateletactivation in vitro at concentrations greater than those ob­tained in vivo (133). Similar findings have been describedfor ticarcillin. In vitro studies suggest that these drugs in­terfere with the binding of substances such as ADP, epi­nephrine and factor VIII/von Willebrand factor with theirspecific receptors on the platelet membrane, perhaps as aresult of properties that permit them to bind to membraneproteins or, as shown with artificial phospholipid bilayers,to platelet membrane lipids (134). The inhibitory effects invitro are observed immediately, but the prolongation of thebleeding time occurs after 12 to 24 hours of parenteraladministration (132,133). Therefore, either a metabolite orprolonged contact of platelets with lower concentrations ofthe antibiotics may account for the inhibition of platelet

activation observed in subjects who received the drugs (134).Although these antibiotics appear to have platelet-inhibitingeffects, no studies have been performed demonstrating thatthey prevent clinical thrombosis.

Dextran infusions. Dextran of a molecular weight of65,000 to 80,000 daltons produces a prolonged bleedingtime after patients receive one or more liters (135-137).The inhibitory effects of dextran infusions on platelet func­tion are dose-related and are more pronounced with dextranof high molecular weight. The finding that platelets absorbdextran, with resultant changes in electrophoretic mobility,suggests that the inhibitory effect of dextran on plateletfunction might be due to some alteration of platelet mem­brane function. Other findings suggest that dextran maytemporarily impair platelet function by interfering with thefactor VIII/von Willebrand factor complex (138). Anti­thrombotic effects of dextran have been shown by experi­mental and clinical studies (3,42).

Suloctidil. This compound, originally introduced to pre­vent vasospasm at high concentrations, has been reportedto have a weak effect on platelet aggregation and secretion(139). Although it has been reported to be antithromboticin some thrombotic models and to prolong shortened plateletsurvival time in patients with artificial heart valves (140,141),suloctidil is ineffective in the secondary prevention of stroke(142) and in experimental arterial thromboembolism (143).

Vitamin E. This agent inhibits collagen-induced aggre­gation and the second phase of aggregation induced by ADPor epinephrine in citrated human platelet-rich plasma (144).It has been suggested that vitamin E may prevent the internalcalcium flux that mediates internal platelet contraction andsecretion induced by aggregating and release-inducing agents(145). However. the clinical antithrombotic usefulness ofvitamin E remains unproved.

Other drugs. A variety of other drugs have been reportedto have in vitro antiplatelet effects (1-3,42): calcium chan­nel blockers, clofibrate and halofenate, furosemide and otherdiuretic drugs, serotonin antagonists, anesthetics, pheno­thiazines, tricyclic antidepressants, antihistamines, pro­pranolol and other beta-adrenergic blocking agents, glycerylguaiacolate, furadantin, monoamine oxidase inhibitors, pyr­idinal carbamate, hydroxychloroquine, alcohol, pyrizadinederivates, pyridoxal phosphate and chlortetracycline. Clin­ically useful antithrombotic effects have not been demon­strated using any of these agents.

Summary of Drug EffectsThe principal antithrombotic effects of those drugs ad­

equately evaluated are summarized as follows:Aspirin. This nonsteroid anti-inflammatory agent po­

tently and irreversibly inactivates platelet cyclooxygenaseby acetylation. Although all of aspirin's antithrombotic ef­fects have been attributed to this blockade of thromboxane

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A2 formation by platelets, there is evidence for anti throm­botic effects independent of its inactivation of cyclooxy­genase. At present, there is intense interest in aspirin therapyfor vascular disease.

Aspirin has been shown in well designed clinical trialsto be beneficial in at least five and possibly six clinicalsettings: I) reduction of thromboembolic complications as­sociated with artificial heart valves; 2) prevention of strokeand death in patients with transient ischemic attacks; 3)decrease in thrombotic occlusion of arteriovenous Silasticcannulas in uremic patients undergoing hemodialysis; 4)reduction in myocardial infarction and cardiac death in pa­tients with unstable angina; 5) probable effect in the sec­ondary prevention of myocardial infarction; and 6) possibleincrease in the patency of saphenous vein coronary bypassgrafts. These reported benefits of aspirin require some com­ment. First, the antithrombotic effects of aspirin in patientswith prosthetic mitral valves are evident in association withoral anticoagulant therapy, and this combination is associ­ated with an unacceptably high frequency of gastrointestinalbleeding. Second, the capacity of aspirin in low doses toreduce the thrombotic complications of arteriovenous can­nulas was shown in patients undergoing long-term hemo­dialysis and, thus, associated with significant platelet dys­function. It cannot be assumed that a similar dose wouldbe antithrombotic in the absence of associated platelet dys­function.

Sulfinpyrazone. This urocosuric agent has weak anti­inflammatory properties. The mechanism of antithromboticaction remains to be defined. Sulfinpyrazone reduces arte­riovenous cannula occlusion and early coronary artery sa­phenous vein graft occlusion. Since the claimed benefit inreducing mortality in the secondary prevention of myo­cardial infarction remains controversial, no recommenda­tions can be made regarding that possible indication. Thereports that it may also decrease thromboembolism in pa­tients with artificial heart valves require confirmation.

Dipyridamole. This agent, a coronary vasodilator withweak inhibitory effects on phosphodiesterase activity, ap­pears to increase inhibitory cyclic AMP levels in plateletsby elevating blood adenosine levels through the blockadeof adenosine uptake by red blood cells and vascular wallcells. Experimentally, on artificial surfaces, aspirin poten­tiates the antithrombotic effects of dipyridamole by mech­anisms independent of platelet inactivation of platelet cy­clooxygenase. Dipyridamole decreases thromboembolismin patients with artificial heart valves (in combination withanticoagulants). Dipyridamole in association with aspirinmay be more effective than aspirin alone in reducing theprogression of peripheral vascular disease, but this remainsto be confirmed. When used in combination with aspirin,it has been shown to decrease coronary mortality and non­fatal myocardial infarction in patients who have previouslysustained a myocardial infarction, to reduce both early and

late coronary artery saphenous vein graft occlusion and topreserve renal function in patients with membranoprolifer­ative glomerulonephritis. However, the benefit of addingdipyridamole to aspirin in these latter settings of biologicsurfaces remains to be established.

Ticlopidine. This agent exhibits global antiplatelet ac­tivity of prolonged duration through some apparently novelmechanism of action. The fact that ticlopidine does notinhibit prostacyclin synthesis in the arterial wall, but canstill inhibit aggregation induced by thromboxane Az andprostaglandin endoperoxide, gives it a theoretical advantageover aspirin. A number of important weB designed multi­center trials are in progress. These trials should determinethe ultimate clinical role of ticlopidine.

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