Mechanisms of Platelet Activation: Thromboxane 4 as an Amplifying Signal for Other Agonists Garret A. FitzGerald,

Thromboxane (TX) 42 is a product of cyclooxygenase catalyzed metabolism of arachiinic acid. lt is formed via prostaglandin (PG) endoperoxide intermediates (PGG, and PGHJ by a specific synthase. PGH, appears to exert the same biologic efFects as T%. The cDNA for a IkA, receptor has been cloned from a human placental library. Although pharmacologic and biochemial studies suggest the presence of multiple isoforms, thii remains to be confirmed at the molecular level. A hydropathy plot of the deduced amino acid sequence of the available clone suggests that it has 7 transmembrane spannlng domains, typical of a G protein linked receptor. Pharmacologic studies imply that 7x receptors in platelets are linked to phospholipase C actiiation via pertussis toxin Insensitive G protelns. Candidates include the 42 kD Gq and the 60 kD 6,. TXA, acts as an ampiiing signal for platelet agonists and the response to thii eicosanoid is tiily regulated. Mechanisms include rapid hydrolysis of the agonist to the inactive lx&, autolnactiiation of 7x synthase, rapid homologous TXA, receptor desen= sitlzation due to receptor-G protein uncoupling, coincidental sensitization to counterregulatory Gs linked receptor systems and stimulation of prostacyclin formation by T*. Due to its role as an amplifimion signal in platelet activation, inhibition of 7x synthesis and actlon is an effectiie mechanism for preventing platelet-dependent vascular occlusion. Aspirin ls of proven efficacy in thii regard. 7x synthase inhlbttors and antago= nists are under clinlcal investigation. (Am J Cardiil166~66zllLl6B)

From the Division of Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee. This study was supported by grants from the National Institutes of Health (HL 30400 and GM 15431) and from Daiichi Seiyaku. Dr. FitzGerald is the William Stokes Professor of Experimental Therapeutics. Address for reprints: Garret A. FitzGerald, MD, Division of Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee 37232.

MD

hromboxane (TX) A2 is the predominant cyclooxygenaseproduct formed from arachidonic acid (AA) in human platelets1 AA is mobilized from platelet membranes by phospholipases; it is thought that direct phospholipase AZcatalyzedreleaserepresents the predominant mechanism by which this occurs during the process of platelet activation.’ TxA, is neither stored in platelets, nor formed in the absence of activation. However, it is synthesized in response to platelet activation by a wide variety of receptor-dependent and -independent platelet agonists.3 In this respect, it resembles serotonin, which like TxA,, itself activates platelets and causes vasoconstriction. Thus, both compounds act as amplifying signals for more potent platelet activators, such as thrombin, collagen, adenosine diphosphate and platelet activating factor in vitro (Figure 1). These agonists possessthe capability of activating platelets directly. The time course of TXA, formation at the site of vascular injury in humans corresponds to the phase of platelet-platelet interaction, which occurs subsequent to deposition of the platelet monolayer on the exposed subendothelium.4 The most convincing evidence for the functional importance of TxAz in precipitating plateletdependent coronary vascular occlusion in unstable vascular disease is provided by the efficacy of aspirin5-9in reducing the incidence of myocardial infarction and death in such patients. Aspirin inhibits platelet cyclooxygenase activity, first by binding reversibly to the active site of the enzyme prostaglandin (PG) G/H synthase” and then by rendering its interaction irreversible by acetylating Ser530.11 Deletion mutant analysis of the ram seminal vesicle PGG/H synthase suggeststhat enzyme inhibition results from stearic hinderance by the presence of the bulky acetyl group close to, rather than within, the active site.” We have recently cloned the enzyme from human platelets13and confirmed these latter results. Although other mechanisms of action have been proposed to explain the efficacy of aspirin in the treatment of cardiovascu-

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iiB

AGGREGATING

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I VESSEL WALL I AA I PGHz

IO

3

PGl2 RECEPTORS AGGREGATION

RGURE L A wide variety of aggregating stimuli, InciudiryS thrombin, adenosine diphosphate and platelet activating factor stimulate the reiease of platelet arachidonic acid (AA) and Rs mataboiism to thromboxane (TX) 4 via the endoperoxide intermediate, prostagkndin (PG) II,. Both PGH, andact at piateiet receptors to ampiify further the aggregation response (I). Both PGH, and TXA, activate vasokstriction. During pharmacologic blockade of platelet TXsynthase, accumulated PGR, may increase prostacyciin (PGIJ synthesis by mobilizing vascular AA (2) and by providing substrate directly for vascular PGI, synthase (3). Both TXsynthase Inhibitors and receptor antagonists, alone and in combination, are undergoing clinical evaluation.

lar disease,14biochemical and pharmacologic studies of aspirin in humans have been entirely explicable in terms of TxA, inhibition.‘5,‘6 The most recent overview of secondary prevention trials performed by the Antiplatelet Trialist’s Group (Oxford, 1990) suggests that aspirin reduces the combined incidence of stroke, myocardial infarction and vascular death by about 25% in such patients. The doses of aspirin that have been used in clinical trials are not biochemically selective for TXA,. Thus, long-term administration of even the lowest dose, 75 mg daily in the RISK study,g is associated with significant impairment of prostacyclin biosynthesis.1s17 Although it is surprising that such a theoretically imperfect intervention against just one of the recognized pathways of platelet activation should yield results detectable by so crude a measure as a clinical trial, this may reflect the amplifying role that TxA plays in the process of platelet activation. The place of aspirin in the primary prevention of cardiovascular disease is much more ambiguous, given the surprisingly low incidence of end points in the placebo-treated group of the only controlled study” and doubts about the magnitude of the benefit, relative to risk, in such a healthy population.‘g

PGG/H synthase and its gene is localized on chromosome 9.13s20 Recently, a second PGG/H synthase-like gene has been cloned. Mitogenic stimulation of cells by transfection with the oncogene src is associated with the splicing of an intron that separates the signal peptide from the rest of the protein and a marked increase in mRNA levels for the splice variant21 mRNAs for both forms of PGG/H synthase have recently been demonstrated in vascular endothelial and smooth muscle cells (Hia T: unpublished 1991). The function of this second enzyme and its potential role in atherogenesis remains to be clarified. Cellular specificity for TXA, formation is lent by the more restricted distribution of TxA, synthase.22 The predominant cellular sources are platelets, macrophages and glomerular mesangial cells. Pharmacologic studies raise the possibility of tissuespecific isozymes,23 although none of these enzymes has been cloned. Platelets are a particular case, because, lacking a nucleus, their ability to form TXA, is irreversibly inhibited after aspirin exposure. I2 Advantage was taken of this particular property to demonstrate that roughly 80% of the excretion of the major urinary TXA, metabolites, 2,3-dinor-TxB, and 11-dehydro-TxB,, derive from platelets under physiologic conditions in humans.” If TXA, formation is increased at another site, its relative contribution would be expected to increase.25 Nevertheless, striking, transient elevations in excretion of these metabolites have been observed during ischemic episodes in patients with unstable coronary artery disease26 and following therapeutic thrombolysis in animal models27,28 and in humans.2g,30

MECHANISM OF ACTION OF THROMBOXANE 4 TXA, causes platelet activation via specific binding sites that have been identified using multiple agonist and antagonist ligands.31”4 Receptor activation of phospholipase C, resulting in inositol phosphate turnover, an increase in intracellular calcium and activation of protein kinase C accompanies agonist-induced platelet aggregation.35Short of information at the molecular level, studies of hierarchies of receptor ligands suggest the similarity of the TXA, receptor in human, canine and rat platelets, but remain ambiguous.36,37For example, the agonist ligand, U46619, which induces platelet shape change and aggregation in human platelets, often requires priming with subthreshold concentrations of other agonists to cause these responses BIOSYNTHESIS OF THROMBOXANE A, AA is a ubiquitous constituent of the phospho- in canine platelets and evokes only the shape lipid domain of cell membranes. Most cells possess change in rat platelets. The PG endoperoxide 126

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precursor of TxA,, PGH,, shares its biologic prop- platelet activation, in much the sameway as hemoerties and is thought to act at a shared receptor.38 globin A,, is used to reflect control of blood Recent evidence, obtained with the ligands glucose.49 A second mechanism by which the response to I-BOP and GR 32191, suggeststhat there is more than one form of the PGHJTxA, receptor in receptor-dependent stimuli is regulated is by homolIndeed, GR 32191 binds irre- ogous desensitization.SoThe kinetics of such desenhuman platelets.39’40 versibly to the site (GR,) that mediates phospholi- sitization are rapid (t 1,2approximately 2 minutes) pase C activation and aggregation, but reversibly and involve initial uncoupling of PGH,/TxA, recep(GR,) to the site that mediates agonist-evoked tors from G proteins that couple them to phosphoplatelet shape change and the majority of the lipase C.35Prolonged exposure to agonist results in increase in intracellular calcium. It is unknown progressive loss of binding sites from the cell whether these sites represent distinct receptor surface. A similar process is observed in vascular isoforms rather than varied degrees of posttransla- smooth muscle cells.46Both PGH,/TxA,-induced tional modification and whether they differ in their platelet shape change and aggregation are heterolaffinity for TxA, and PGH,. The recent cloning of a ogously desensitized by thrombin, whereas only the PGH/TxA, receptor from a human placental li- aggregation response is desensitized by plateletbrary should initiate the clarification of eicosanoid activating factor.47Protein kinase C appears to play only a marginal role in the process of homologous receptor classification.41 There is reason to presume that tissue-specific desensitization. A third mechanism by which the response to isoforms of the PGHJTxA, receptor exist. Although to some extent disputedy3 most studies of PGHJTxA, receptor stimulation is regulated is by ligand hierarchies comparing vascular smooth mus- sensitization of platelet adenylate cyclase stimulacle cells and platelets suggestsuch a distinction.44,45 tion. Thus, in platelets desensitized with U46619, Although some of these studies were across spe- stimulation of adenylate cyclase, either directly by cies, more recent studies37,46 confirm such a distinc- forskolin or indirectly through receptors coupled tion within species. Thus, GR32191 dissociates to the stimulatory G protein, G,, is enhanced.47 rapidly from vascular smooth muscle cells and This might have particular biologic relevance given competes reversibly with agonist-induced contrac- the stimulation by PGH,/TxA, analogues of endotion of both rat and human vascular smooth muscle thelial release of prostacyclir? and the coincidencells, in contrast to its irreversible inhibition of tal increase in biosynthesis of both eicosanoids in agonist-induced human platelet aggregation. Simi- syndromesof platelet activation and vascular occlularly, platelet-activating factor, which heterolo- sion.23,52 Interestingly, these observations were not gously regulates the GRi,,, but not the GR platelet made when platelets were desensitized with either receptor? does not desensitizePHG,/TxA,-evoked platelet-activating factor or thrombin. These agoincreasesin intracellular calcium in vascular smooth nists differ from PGH,/TxA, in the nature of the G muscle cells.46Clearly, any definitive nomenclature proteins that couple them to phospholipase C and for these receptor subtypes, or indeed prostaglan- in exerting negative regulatory control on adenydin receptors in general,@ awaits provision of late cyclase via Gi.53,54 Platelet PGHJTxA, recepfurther information on their primary sequence or tors have been shown recently to couple to the sequences. pertussis toxin insensitive 42 kD protein GT as well as to a novel 60 kD G protein Ge.56Such cross talk between the phospholipase C and adenylate REGULATION OF THE RESPONSE OF HUMAN cyclase signaling systems may reflect coordinate PlATEW TO PROSTAGLANDIN regulation of their respective G protein message HJTHROMBOXANE 4 levels57or G protein subunit rearrangement.58’59 If TXA, plays such an amplifying role in the process of platelet activation, one would anticipate that the response to this agonist would be tightly THERAPEUTIC DEVELOPMENTS regulated. One such mechanism, recently deThe successof aspirin in preventing cardiovascuscribed, is suicide inactivation of the TxA, syn- lar fatalities has both solved a problem and created thase. Thus, as substrate is consumed to generate a new one: the disincentive that a cheap and product, the enzyme is progressively inhibited. It effective remedy presents for the clinical develophas been proposed that such covalently modified ment of theoretically more promising intervenenzyme might serve as a marker of prior TxA, tions.60 Biochemically, more selective modes of generation and provide an integrated index of inhibiting either TxA, synthesis or action have A SYMPOSIUM: MANAGEMENT OF UNSTABLE ANGINA

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been developed (Figure 1) and shown to be effica- Prostaglandins,Leukotrienes and Lipoxins. New York: Plenum Press,1985:205211. cious in animal models of coronary occlusion, both 3. SiessW, Weber PC, Lqtina EG. Different functions of phosphoinositide alone61d3and in combination.28,6467 However, large turnover and arachidonatemetabolismfor platelet activation. In: Prostaglandins, trials with either synthase inhibitors or receptor Leukotrienes and Lipoxins. Bailey,J.M. (Ed). PlenumPress,NY, 1985~387-403. 4. Nowak J, FitzGerald GA. Redirection of prostaglandinendoperoxidemetaboantagonists have not been published at the time of lism at the platelet-vascularinterface in man.J Clin Invest1989;83:38&385. writing, although promising results have been ob- 5. Lewis HD, Davis JW, Archibald DG, Steinke WE, SmithermanTC, Doherty tained in humans in small studies of patients with JE, Schnaper HW, LeWinter MM, Lianares E, Pouget JM, Sabhanval SC, Chesler E, DeMots H. Protective effects of aspirin against acute myocardial renal disease.68 infarction and death in menwith unstableangina.NEnglJMed 1983;309:39&403. Another approach to the issue of biochemical 6. Cairns JA, Gent M, SingerJ, Finnie KJ, Froggatt GM, Holder DA, Jabolonsky selectivity has been to determine the rate of aspirin G, Kosti WJ, Melendez LI, Myers MG, Sackett DL, SealeyBJ, Tauser PH. Aspirin, sultinpyrazone, or both in unstable angina: Results of a Canadian delivery from a matrix formulation that will confine multicentre trial. NEnglJMed 1985;313:1369-1374. its action to the presystemic circulation,6g thereby 7. Theroux P, Quimet H, McCans J, I&our JG, Levy JP, Pelletier E, JuneauM, StasiakJ, de Guise P, Pelletier GB, Rinzler D, Water DD. Aspirin, heparin or limiting exposure of endothelial cell cyclooxygen- both to treat acuteunstableangina.NEnglJMed 1988;319:1105-1111. ase to aspirin?’ Such a controlled release prepara- 8. SecondInternational Study Group & Infarct Survival (ISIS-2) Collaborative tion has been developed and shown to cause a Group. Randomized trial & intravenous streptokinase, oral aspirin, both, or in 17,187casesof acutemyocardialinfarction. Lancet 1988;2:349. minor decrement in prostacyclin synthesis when 9.neither The RISC Group: Risk of myocardial infarction and death during treatment administered chronically.‘j’ However, it appears with low dose aspirin and intravenous heparin in men with unstable coronary that this results from inhibition of the cyclooxygen- artery disease.Land 1990,336:827-830. 10. Humes JL, Winter CA, Sadowski SJ, Kuehl FA Jr. Multiple sites on ase in the presystemic vasculature, since the in- prostagkmdii cyclooxygenaseare determinants in the action of nonsteroidal crease in prostacyclin synthesis evoked by systemic antiinilammatory agents. Pm NatlAcad Sci USA. 1984,81:2052-2056. infusion of bradykinin is unimpaired by the con- 1L Dewitt DL, Smith WL. Primary structure of prostaglandin G/H synthase from sheepvesiculargland determinedfrom the complementmyDNA sequence. trolled release preparation, but depressed signifi- ProcNatlAcad Sci USA. 1988;85:1412-1416. cantly by the same dosage provided in a regular l2. Smith WL, Mamett LT. Prostaglandinendoperoxide synthase:structure and &o&m BiophysActa 1991;1083:1-17. formulation.71 This compound will likely provide a catalysis. 13. Funk CD, Funk LB, Kennedy M, PangA, FitzGerald GA. Human platelet/ cheap and convenient tool with which to address erythroleukemia cell PGG/H synthase:cDNA cloning, expression,mutagenesis and genechromosomalassignment.FASEBJ. 1991;5:23&2312. the importance of biochemical selectivity. l4. Patron0 C. Aspirin and human platelets: from clinical trials to acetylation of In conclusion, TXA, is an evanescent lipid cyclooxygenase and back. TrendsPhamacol Sci 1989;10:45%458. mediator released from activated platelets that IZPatrignani P, Filabozzi P, Patron0 C. Selective cumulative inhibition of amplifies the response to more potent platelet platelet thromboxane production by low-dose aspirin in healthy subjects..l Clin 1982;69:136&1372. agonists. Such a role probably facilitates the detec- Invest 16. FitzGerald GA, Oates JA, Hawiger J, Maas RL, Roberts LJ, Brash AR. tion of an effect of TxA, blockade in clinical trials Endogenoussynthesisof prostacyclin and thromboxane and platelet function and implies that the process of TxA,-dependent during chronic aspirin administrationin man..I C&nInvest1983;71:676688. 17. Braden G, Knapp HR, Fitzgerald GA: Suppressionof eicosanoidformation platelet activation would be both rapidly and tightly during coronary angioplastyby fish oil and aspirin.Circulation 1991;68:679-685. regulated. Suicide inactivation of TXA, synthase, LB. SteeringCommittee of the Physicians’Health Study ResearchGroup. Final report on the aspirin componentof the ongoingphysicians’health study.NE&J homologous desensitization due to receptor-G Med 1989;321:129-135. protein uncoupling and postreceptor sensitization 19. Patron0 C, FibGerald GA. Physicians’ Health Study. Aspirin and the of platelet adenylate cyclase provide three mecha- primary prevention of coronary heart disease(letter). NE& JMed 1989;321:26. Yokoyama C, Tanabe T. Cloning of human gene encoding prostagkmdin nisms by which such regulation occurs in human 20. endoperoxidesynthaseand primary structureof the enzyme.Biochem Biophys Res platelets. It is probable that tissue-specificisoforms Commun1989;165:88&894. of PGHJTXA, receptors exist and that at least 2 2L Xie W, Chipman JG, Robertson DL, E&son RL, SimmonsDL. Expression of mitogen-responsivegene encoding prostaglandin synthase is regulated by functionally distinct forms exist in human platelets. mRNA splicing.pioc NatlAcad Sci USA 1991;88:2692-2696. The clinical efficacy of aspirin has slowed the 22. FitzGerald GA. Prostaglandinsand related compounds.In: WyngaardenJB, development of theoretically superior drugs. Stud- Smith LH, eds. Cecil Loeb Textbook of Medicine, 18th ed. Philadelphia: WB Saunders,1988;1271-1277. ies in animal models66,72 suggestthat the therapy of 23. Patrignani P, Filabozi F, Catella F, Pugliese F, Patron0 C. Differential occlusive coronary vascular disease may involve a effects of dazoxiben,a selectivethromboxane synthaseinhibitor on platelet and renal prostaglandin-endoperoxidemetabolism.JPhmn E.q lkr 1984;228:472combination of approaches, directed at distinct 477. mechanisms of activation of platelets and the 24. Catella F, FitzGerald GA. Paired analysis of urinary thromboxane B, metabolitesin humans.ThrombRes 1987;47:647-656. soluble coagulation cascade.73 REFERENCES L Hamberg M, SvenssonJ, SamuelssonB. 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reocclusionfollowing coronary thrombolysiswith tissue type plasminogenactivator. Proc NatlAcad Sci USA 1989;86:7585-7589. 29. Fitzgerald DJ, Catella F, Roy L, FitzGerald GA Marked platelet activation in viva after intravenous streptokinasein patients with acute myocardial infarction. Circulation 1988;77:142-150. 30. Kerins DM, Roy L, FitzGerald GA Fitzgerald DJ. Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator. Circulation 1989;80:1718-1725 3L Halushka PV, MacDermot J, Knapp D, Eller T, SaussyD, Mais D, Blair I, Dollery C. A novel approachfor the study of thromboxaneAZand prostaglandii Hz receptorsusingan ‘“I-labelled ligand.Biuckm Pharmacol1985;34:1165-1170. 32. Mais DE, Burch RM, SaussyDL, Kochel PJ, Halushka PV. Binding of a tbromboxane A,/prostaglandin H, antagonist to washed human platelets. J Phannacol E+ Ther 1985;235:72%734. 33. Lie1N, Mais DE, Halushka PV. Binding of a thromboxaneA,/prostaglandm Hz agonist [‘HjU46619 to washed human platelets. Pmstaglandins 1987;33:789797. 34. SaussyDL Jr, Mais DE, Butch RM, Halushka PV. Identification of a putative thromboxaneA,/prostaglandin H, receptor in human platelet membranes.JBiol Chem1986;261:3025-3029. 35. Murray R, FitzGerald GA Regulation of thromboxanereceptor activation in humanplatelets.ProcNatIAcad Sci USA 1989;86:124-128. 36. Mais DE, SaussyDL Jr, Chaikhouni A, Kochel PJ,Knapp DR HamanakaN, Halusbka PV. Pharmacologiccharacterization of human and canine thromboxane A,/prostaglandin H, receptors in platelets and blood vessels:Evidence for different receptors.JPharmacolw Ther 1985;233:418424. 37. Masuda A, Mais DE, Oatis JE Jr, Halushka PV. Platelet and vascular thromboxaneA,/prostaglandin Hz receptors.Evidencefor diierent subclassesin the rat. Biachem Phamucol199O;(m press). 36. Mayeux P,Morton HE, Gillard J, Lord A Morinelli TA BoehmA, Mais DE, Halushka P. The affinities of prostaglandin Hz and thromboxane AZ for their receptor are similar in washed human platelets. BiochemBiophys Res Common 1988;157:733-739. 39. Darn GW, II. Distinct platelet thromboxane A,/prostaglandin Hz receptor subtypes.A radioligand binding study of human platelets. J Clin Invest3989;84: 188%1891. 40. Takahara K Murray R, FitzGerald GA, Fitzgerald DJ. The response to tbromboxaneAZin human platelets: discrimination of two binding siteslied to distinct effector systems.JBiol Chem 1990;265:683&6844. 4L Hirata M, Hayashi Y, Ushiibi F, Yokota Y, Kageyama R, Nakanishi S, Narumiya S. Cloning and expression of cDNA for a human thromboxane A2 receptor.Natnre 1991;349:617+520. 42. Swayne GTG, Dolan JMJ, Ravel P, Dane G, Greener M, Gwen DAA. Evidence for homogeneity of thromboxane AZ receptors using structurally different antagonists.EurJPharaco2 1988;152:311-319. 43. Jones RI+ Peesapata V, Wilson NH. Antagonism of the thromboxanesensitive contractile systems of the rabbit aorta, dog saphenous vein and guinea-pigtrachea.BrJPhannacol1982;67:423-429. 44. Morhtelli TA, Okwu AK, Mais DE, Halushka PV, John V, Chen C-K, Fried J. Diiluorothromboxane A2 and stereoisomers:Stablederivativesof thromboxane AZwith differential effects on platelets and blood vessels.Prcx NatlAcad Sci USA 1989;86:560%5604. 45. Hanasaki K, Nakano K Kassai H, Kurihara H, Arita H. Identification of thromboxane AZ receptors in cultured vascular endothelial cells of rat aorta. Bioch.em Biophys Res Commun 1988;151:1352-1357. 46. Furci L, Fitzgerald DJ, FitzGerald GA. Heterogeneity of prostaglandin H,/thromboxane AZreceptors:Distinct subtypesmediatevascularsmoothmuscle contraction and platelet aggregation,JPharmacol Exp Ther 1991;(inpress). 47. Murray R, ShippL, F&Gerald GA Prostaglandinendoperoxideithromboxane AZ receptor desensitization. Cross-talk with adenylate cyclase in human platelets.J&l Chem1990;265:21670-21675. 48. ColemanJR, Humphrey P, Kennedy I, Lumley P. Prostanoidreceptorsthe developmentof a working classification.Trends Phamucol Sci 1984;5:303-306. 49. Jones DA, Fitzpatrick FA. “Suicide” inactivation of thromboxane A2 synthase.JBiul Chem1990;265:20166-20171. 50. Hausdorlf WP, Caron MG, Lefkowitz RJ. Turning off the signal desensitization of B-adrenergicreceptor function. FASEEJ 1990;4:2881-2889. 51 Jeremy JY, Mikhaihdis DP, Dandona P. Thromboxane AZ analogue (U46619)stimulatesvascularPGI, synthesis. EwJPhamucol1985;107:25%262, 52. Reilly LAG, Roy L, FitzGerald GA Thromboxanebiosynthesisis increasedin systemicsclerosiswith Raynaud’sphenomenon.BrMed J 1986;292:1087-1089.

53. Brass LF, Shaller CC, Belmonte El. Inositol 1,4,5-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipaseC mediated by platelet thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanismdistinct from that of thrombin. J Clin Invest 198%79:1X9-1275. 54. BrassL, LaposataM, BangaH, Rittenhouse S. Regulation of the phosphoioinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitiveguaninenucleotide binding protein. J Biol C/tent 1986;261:1683816847. 55. ShenkerA, Goldsmith P, Unson CG, SpiegelAM. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel G, family. JBiol Chem 1991;266:9309-9313, 56. Moran N, FitzGerald GA. Purification of an abundant guanine nucleotide binding (G) protein liied to human platelet (HP) prostaglandm (PGH,)/ thromboxane(TX) receptors.Circulation 1990,823461A. 57. Hadcock JR, Ros M, Watkins DC, Malbon CC. Cross-regulationbetween G-protein-mediatedpathways.Stimulation of adenyl cyclaseincreasesexpression of the inhibitoty G-protein G,,. JBiol Chem 1990;265:14784-14790. 58. Ashby B. Model of prostaglandm-regulatedcyclic AMP metabolismin intact platelets: examination of time-dependent effects on adenylate cyclase and phosphodiesteraseactivities.Mel Pharmncol1989;36:866-873. 59.Hildebrandt JD, Kohnken RE. Hormone inhibition of adenyl cyclase. DifIerences in the mechanismsfor inhibition by hormones and G protein py. J Biul Chem 1990,265:982.%9830. 60. FitzGerald GA. Ticlopidme in unstable angina: a more expensiveaspirin? editorial. C&&ion 1990,82:296-298. 6L Fitzgerald DJ, Doran J, Jackson EK, FitzGerald GA Coronary vascular occlusionmediatedthrough thromboxaneA,-prostaglandmendoperoxiderecep tor activation in viva. J Clin Invest 1986;77:49&502. 62. Aiken JW, ShebuskiRJ, Miier OV, Gorman RR. Endogenousprostacyclin contributes to the efficacy of a thromboxane synthetaseinhibitor for preventing Ther1981;219:29%308. coronary artery thrombosis.JPhannacol@ 63. DeClerck F, BeetensJ, de Chaffoy de CourcellesD, Freyne E, JanssenPA? R68 070: Thromboxane AZ synthetase inhibition and thromboxane A,/ prostaglandin endoperoxide receptor blockade combined in one molecule-I. Biochemicalprolile in vitro. Thromb Haemostas 1989;61:3542. 64. Fitzgerald DJ, Fragetta J, FitzGerald GA Prostaglandin endoperoxides modulate the response to thromboxane synthase inhibition during coronary thrombosis.J Clin Invest 1988;82:1708-1713. 65. Golino P, Ashton JH, Glas-Greenwalt P, McNatt .I, Buja LM, Willerson JT. Mediation of reocclusion by thromboxane A2 and serotonin after thrombolysis with tissue-type plasminogen activator in a canine preparation of coronaty thrombosis.Circulation1988;77:678674. 66. Golino P, Ashton JH, McNatt J, Glas-Greenwalt P, Sheng-KunY, O’Brien RA, Buja LM, Willerson JT. Simultaneousadministration of thromboxane AZand serotonin S,-receptor antagonistmarkedly enhancesthrombolysis and prevents or delays reocclusion after tissue-typeplasminogenactivator in a canine model of coronary thrombosis.Circt&ion 1989;79:911-919. 67. GreseleP, Arnout J, DeckmynH, HuybrechtsE, PietersG, Vermylen .I. Role of proqgregatory and antiaggregatoryprostaglandinsin hemostasis.Studieswith combmedthromboxane synthaseinhibition and thromboxane receptor antagonism.J Clin Invest 1987;80:1435-1445. 66. Pietucci A, Siionetti BM, Pecci G, Mavrikakis G, Feriozzi S, Cinotti GA, Patrignani P, Caibattoni G, Patron0 C. Improvement of renal function with selectivethromboxaneantagonismin lupusnephritis.NEnglJMed 1989;320:421425. 69. PedersenAK, FitzGerald GA: Dose related pharmacokineticsof aspirin presystemicacetylation of platelet cyclooxygenasein man. NE+ JMed 1984;311: 1%1211. 70. FitzGerald GA, Lupinetti MD, Charman S, Charman W: Presystemic acetylation of platelets by aspirin: reduction in dose and rate of drug delivery to improve biochemical selectivity for thromboxaneA2.J. Phann Exp ti 1991. (in press). 71. Clarke RJ, Price P, Mayo G, FitzGerald GA: Simultaneouspreservation of systemicprostacyclin biosynthesisand suppressionof thromboxane AZ by controlled releaseaspirin.NEnglJMed 1991 (in press). 72. Fitzgerald DJ, FitzGerald GA: Role of thrombin and thromboxane AZ in reocclusionfollowing coronary thrombolysiswith tissuetype plasminogenactivator.PmcNatlAcad

Sci USA 1989;86:7585-7589.

73. Clarke RJ, FitzGerald GA, Fitzgerald DJ. The human pharmacology of argatroban,a specificthrombin inhibitor. Circlrlarion 1991;82:1510-1518.

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