Prostaglandin Endoperoxides and Thromboxane A₂ Activate the same Receptor Isoforms in Human Platelets

Summary

Arachidonic acid (AA) is a potent inducer of platelet aggregation in vitro; this activity is due to its conversion to biologically active metabolites, prostaglandin (PG) endoperoxides and thromboxane A₂ (TxA₂). PG endoperoxides and TxA₂ are thought to act on the same receptor; however, at least two isoforms of this receptor have been identified. The aim of our work was to clarify whether endoperoxides and TxA₂ activate the same or different receptor subtypes to induce aggregation and calcium movements in human platelets.

AA-induced aggregation and calcium rises were still detectable in platelets preincubated with thromboxane synthase inhibitors, which suppress TxA₂ formation and induce PGH₂ accumulation, suggesting that PG endoperoxides can activate platelets. Exogenously added PGH₂ was able to induce aggregation and calcium rises. Pretreatment of platelets with GR32191B or platelet activating factor, which desensitize one of the two receptor subtypes identified in platelets, did not prevent calcium rises induced by endogenously generated or by exogenouly added PGH₂, indicating that TxA₂ and PG endoperoxides share the same receptor subtype(s) to activate platelets. HEK-293 cells overexpressing either of the two thromboxane receptor isoforms cloned to date (TPα and TPβ) and identified in human platelets, stimulated with PGH₂, or with the stable endoperoxide analog U46619, formed inositol phosphates. These data show that endoperoxides and TXA₂ mediate their effects on platelets acting on both, and the same, receptor isoform(s).

Abbreviations: AA: arachidonic acid; PAF: platelet activating factor; PG: prostaglandin; TP: thromboxane receptor; Tx: thromboxane.

Keywords

Platelet aggregation - calcium - thromboxane receptor isoforms

Present address: Maxia Pharmaceuticals Inc., San Diego, CA, USA.

• References

• 1 Gresele P, Deckmyn H, Nenci GG, Vermylen J. Thromboxane synthase inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic disorders. Trends Pharmacol Sci. 1991: 158-63.
• 2 Bhagwat SS, Hamann PR, Still WC, Bunting S, Fitzpatrick FA. Synthesis and structure of the platelet aggregation factor thromboxane A₂ . Nature 1985; 315: 511-3.
• 3 Holzgrefe HH, Buchanan LV, Bunting S. In vivo characterization of synthetic throboxane A₂ in canine myocardium. Circ Res 1987; 60: 290-6.
• 4 Thomas DW, Mannon RB, Mannon PJ, Latour A, Oliver JA, Hoffman M, Smithies O, Kaller BH, Coffman TM. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A₂ . J Clin Invest 1998; 102: 1994-2001.
• 5 Ogletree ML. Overview of physiological and pathophysiological effects of thromboxane A₂ . Fed Proc 1987; 46: 133-8.
• 6 FitzGerald GA. Mechanisms of platelet activation: thromboxane A₂ as an amplifying signal for other agonists. Am J Cardiol 1991; 68: 11B-15B.
• 7 Coleman RA, Humphrey PPA, Kennedy I, Levy GP, Lumely P. Comparison of the actions of U46619, a prostaglandin H₂ analogue, with those of prostaglandin H₂ and thromboxane A₂ on some isolated smooth muscle preparations. Brit J Pharmacol 1981; 73: 773-8.
• 8 Hornby EJ, Skidmore IF. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A₂ production. Biochem Pharmacol 1982; 31: 1158-60.
• 9 Halushka PV, Mais DE, Mayeux PR, Morinelli TA. Thromboxane, prostaglandin and leukotriene receptors. Annu Rev Pharm Tox 1989; 10: 213-39.
• 10 Dorn II GW. Distinct platelet thromboxane A₂/prostaglandin H₂ receptor subtypes. A radioligand binding study of human platelets. J Clin Invest 1989; 84: 1883-91.
• 11 Dorn II GW, DeJesus A. Human platelet aggregation and shape change are coupled to separate thromboxane A₂/prostaglandin H₂ receptors. Am J Physiol 1991; 260: H327-34.
• 12 Gallet C, Rosa JP, Habib A, Lebret M, Levy-Toledano S, Maclouf J. Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change. J Biol Chem 1999; 274: 23610-6.
• 13 Takahara K, Murray R, FitzGerald GA, Fitzgerald DJ. The response to thromboxane A₂ analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems. J Biol Chem 1990; 265: 6836-44.
• 14 Murray R, Shipp E, FitzGerald GA. Prostaglandin endoperoxide/thromboxane A₂ receptor desensitization. Cross-talk with adenylate cyclase in human platelets. J Biol Chem 1990; 265: 21670-5.
• 15 Hirata M, Hayashi Y, Ushikubi F, Yokota Y, Kageyama R, Nakanishi S, Narumiya S. Cloning and expression of cDNA for a human thromboxane A₂ receptor. Nature 1991; 349: 617-20.
• 16 D’Angelo DD, Davis MG, Ali S, Dorn II GW. Cloning and pharmacologic characterization of a thromboxane A₂ receptor from K562 (human chronic myelogenous leukemia) cells. J Pharmacol Exp Ther 1994; 271: 1034-41.
• 17 Allan CJ, Higashiura K, Martin M, Morinelli TA, Kurtz DT, Geoffroy O, Meier GP, Gettys TW, Halushka PV. Characterization of the cloned HEL cells thromboxane A₂ receptor: evidence that the affinity state can be altered by Gα13 and Gαq. J Pharmacol Exp Ther 1996; 277: 1132-9.
• 18 Raychowdhury MK, Yakawa M, Collins LJ, McGrail SH, Kent C, Ware JA. Alternative splicing produces divergent cytoplasmic tail in the human endothelial thromboxane A₂ receptor. J Biol Chem 1994; 269: 19256-61. correction: J Biol Chem 1995; 277: 7011.
• 19 Hirata T, Ushikubi F, Kakizuka A, Okuma M, Narumiya S. Two thromboxane A₂ receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation. J Clin Invest 1996; 97: 949-56.
• 20 Habib A, FitzGerald GA, Maclouf J. Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets. J Biol Chem 1999; 274: 2645-51.
• 21 Mayeux PR, Morton HE, Gillard J, Lord A, Morinelli TA, Boehm A, Mais DE, Halushka PV. The affinities of prostaglandin H₂ and thromboxane A₂ for their receptors are similar in washed platelets. Biochem Biophys Res Commun 1988; 157: 733-9.
• 22 Yukawa M, Yokota R, Eberhardt RT, von Adrian L, Ware JA. Differential desensitization of thromboxane A₂ receptor subtypes. Circ Res 1997; 80: 551-6.
• 23 Blinks JR. Use of calcium-regulated photoproteins as intracellular Ca₊₊ indicators. Methods Enzymol 1989; 172: 164-203.
• 24 Vezza R, Roberti R, Nenci GG, Gresele P. Prostaglandin E₂ potentiates platelet aggregation by priming protein kinase C. Blood 1993; 82: 2704-13.
• 25 Nucciarelli F, Gresele P, Nardicchi V, Porcellati S, Macchioni L, Nenci GG, Goracci G. Evidence that cytosolic phospholipase A₂ is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate. FEBS Lett 1999; 450: 39-43.
• 26 Habib A, Vezza R, Creminon C, Maclouf J, FitzGerald GA. Rapid, agonistdependent phopshorylation in vivo of human thromboxane receptor isoforms. Minimal involvement of protein kinase C. J Biol Chem 1997; 272: 7191-200.
• 27 Gresele P, Arnout J, Deckmyn H, Huybrechts E, Pieters G, Vermylen J. Role of proaggregatory and antiaggregatory prostaglandins in hemostasis. Studies with combined thromboxane synthase inhibition and thromboxane receptor antagonism. J Clin Invest 1987; 80: 1435-45.
• 28 Barry OP, Pratico D, Lawson JA, FitzGerald GA. Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 1997; 99: 2118-27.
• 29 Bundy GL. The synthesis of prostaglandin endoperoxide analogues. Tetrahedron Letters 1975; 1957-60.
• 30 Jones RL, Wilson NH, Lawrence RA. EP171: a high affinity thromboxane A₂ mimetic, the actions of which are slowly reversed by receptor blockade. Br J Pharmacol 1989; 96: 875-87.
• 31 Harris DN, Phillips MB, Michel IM, Goldenberg HJ, Heikes JE, Sprague PW, Antonaccio MJ. 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: specific stable agonist (SQ26,538) and antagonist (SQ26,536) of the human platelet thromboxane receptor. Prostaglandins 1981; 22: 295-307.
• 32 Gresele P, Deckmyn H, Huybrechts E, Vermylen J. Serum albumin enhances the impairment of platelet aggregation with thromboxane synthase inhibition by increasing the formation of prostaglandin D₂ . Biochem Pharmacol 1984; 33: 2083-8.
• 33 Gresele P, Van Houtte E, Arnout J, Deckmyn H, Vermylen J. Thromboxane synthase inhibition combined with thromboxane receptor blockade: a step forward in antithrombotic strategy?. Thromb Haemost 1984; 52: 364.
• 34 Fitzgerald DJ, Fragetta J, FitzGerald GA. Prostaglandin endoperoxides modulate the response to thromboxane synthase inhibition during coronary thrombosis. J Clin Invest 1988; 82: 1708-13.
• 35 Bosia A, Losche W, Pannocchia A, Treves S, Ghigo D, Till U, Pescarmona G. Regulation of arachidonic acid-dependent Ca⁺⁺ influx in human platelets. Thromb Haemost 1988; 59: 86-92.
• 36 Mais DE, Saussy DL, Chaikouni A, Kochel PJ, Knapp DR, Hamanaka N, Halushka PV. Pharmacologic characterization of human and canine thromboxane A₂/prostaglandin H₂ receptors in platelets and blood vessels: evidence for different receptors. J Pharmacol Exp Ther 1985; 233: 418-24.
• 37 Morinelli TA, Okwu AK, Mais DE, Halushka PV, John V, Chez CK, Fried J. Difluorothromboxane A₂ and stereoisomers: stable derivatives of thromboxane A₂ with differential effects on platelets and blood vessels. Proc Natl Acad Sci USA 1989; 86: 5600-4.
• 38 Miggin SM, Kinsella BT. Expression and tissue distribution of the mRNAs encoding the human thromboxane A₂ receptor (TP) α and β isoforms. Biochim Biophys Acta 1998; 1425: 543-59.
• 39 Audoly LP, Rocca B, Fabre JE, Koller BH, Thomas D, Loeb AL, Coffman TM, FitzGerald GA. Cardiovascular responses to the isoprostanes iPF_2α-III and iPE₂-III are mediated via the thromboxane A₂ receptor in vivo. Circulation 2000; 101: 2833-40.
• 40 Maclouf J, Kindahl H, Granstrom E, Samuelsson B. Interactions of prostaglandin H₂ and thromboxane A₂ with human serum albumin. Eur J Biochem 1980; 109: 561-6.
• 41 Maclouf J, Folco G, Patrono C. Eicosanoids and iso-eicosanoids: constitutive, inducible, and transcellular biosynthesis in vascular disease. Thromb Haemost 1998; 79: 691-705.
• 42 Neri GGSerneri, Gensini GF, Poggesi L, Modesti PA, Rostagno C, Boddi M, Gori AM, Martini F, Ieri A, Margheri M. et al. The role of extraplatelet thromboxane A₂ in unstable angina investigated with a dual thromboxane A₂ inhibitor: importance of activated monocytes. Coron Artery Dis 1994; 05: 137-45.
• 43 Cipollone F, Patrignani P, Greco A, Panara MR, Padovano R, Cuccurullo F, Patrono C, Rebuzzi AG, Liuzzo G, Quaranta G, Maseri A. Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina. Circulation 1997; 96: 1109-16.
• 44 Weber AA, Zimmermann KC, Meyer-Kirchrat J, Schror K. Cyclooxygenase-2 in human platelets as a possible factor in aspirin resistance. Lancet 1999; 353: 900.