ADP Receptor Induced Activation of Guanine Nucleotide Binding Proteins in Rat Platelet Membranes– An Effect Selectively Blocked by the Thienopyridine Clopidogrel

Summary

The thienopyridine clopidogrel, a potent analog of ticlopidine, is a powerful inhibitor of ADP induced platelet aggregation and ADP induced inhibition of cyclic AMP accumulation in intact platelets but not of ADP induced shape change. We have recently demonstrated that ADP stimulates the binding of GTPγS to GTP binding proteins (G proteins) in human platelet membranes. We now studied the effects of clopidogrel, a specific inhibitor of ADP induced platelet aggregation on the stimulation of GTPγS binding to rat platelet membranes by ADP. Using the non hydrolyzable stable analog of ADP, 2MeSADP, we demonstrate that 2MeSADP stimulates the binding of [³⁵S]GTPγS to rat platelet membranes in a concentration dependent manner, that this effect is inhibited by the specific ADP receptor antagonist Sp-ATPαS and that clopidogrel completely and selectively blocks the stimulation by 2MeSADP of [³⁵S]GTPγS binding to platelet membranes of treated rats. We conclude that: i) rat platelet membranes possess an ADP receptor coupled to unidentified G protein(s) and ii) the thienopyridine clopidogrel impairs the interaction of the ADP receptor with its G protein by an irreversible modification the ADP receptor itself or its putative G protein.

• References

• 1 Panak E, Maffrand JP, Picard-Fraire C, Vallée E, Blanchard J, Roncucci R. Ticlopidine: a promise for the prevention and treatment of thrombosis and its complications. Haemostasis 1983; 13 (Suppl 1) 1-52
• 2 Saltiel E, Ward A. Ticlopidine: a review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficiency in platelet-dependent disease states. Drugs 1987; 34: 222-262
• 3 Maffrand JP, Defreyn G, Bernat A, Delebassée D, Tissinier AM. Reviewed pharmacology of ticlopidine. Angiologie 1988; 77: 6-13
• 4 Delebassée D, Bernat A, Frehel D, Defreyn G, Maffrand JP. Antiaggregating and antithrombotic effects in the rat of two thienopyridine enantiomers structurally related to ticlopidine. Xth International Congress on Thrombosis, Athens 1988 Abst. 197
• 5 Féliste R, Delebassée D, Simon MF, Chap H, Defreyn G, Vallée E, Douste-Blazy L, Maffrand JP. Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the supression of the effects of released ADP. Thromb Res 1987; 48: 403-415
• 6 Dunn FW, Soria J, Soria C, Thomaidis A, Lee H, Caen JP. In vivo effect of ticlopidine on fibrinogen-platelet cofactor activity and binding of fibrinogen to platelets. Agents Actions Suppl 1984; 15: 97-103
• 7 Di Minno G, Cerbone AM, Mattioli PL, Turco S, Iovine C, Mancini M. Functionally thrombasthenic state in normal platelets following the administration of ticlopidine. J Clin Invest 1985; 75: 328-338
• 8 Gachet C, Stierlé A, Cazenave JP, Ohlmann P, Lanza F, Maffrand JP. The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man. Biochem Pharmacol 1990; 40: 229-238
• 9 Hardisty RM, Powling MJ, Nokes TJC. The action of ticlopidine on human platelets: studies on aggregation, secretion, calcium mobilization and membrane glycoproteins. Thromb Haemostas 1990; 64: 150-155
• 10 Gachet C, Cazenave JP, Ohlmann P, Bouloux C, Defreyn G, Driot F, Maffrand JP. The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE₁ . Biochem Pharmacol 1990; 40: 2683-2687
• 11 Defreyn G, Gachet C, Savi P, Driot F, Cazenave JP, Maffrand JP. Ticlopidine and clopidogrel (SR 25990 C) selectively neutralize ADP inhibition of PGE₁-activated platelet adenylate cyclase in rats and rabbits. Thromb Haemostas 1991; 65: 186-190
• 12 Gachet C, Cazenave JP, Ohlmann P, Hilf G, Wieland T, Jakobs KH. ADP-receptor-induced activation of guanine nucleotide binding proteins in human platelet membranes. Blood 1991; 78: 140 (abstract)
• 13 MacFarlane DE, Srivastava PC, Mills DCB. 2-methylthio-adenosine [β-³²P] diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets. J Clin Invest 1983; 71: 420-428
• 14 Cusack NJ, Hourani SMO. Adenosine 5’-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors. Br J Pharmacol 1982; 76: 221-227
• 15 Meurs H, Kauffman HF, Koeter G, Drevries K. Extraction of cyclic AMP for the determination in the competitive protein binding assay. Clin Chim Acta 1980; 106: 91-97
• 16 Hilf G, Gierschick P, Jakobs KH. Muscarinic acetylcholine receptor-stimulated binding of guanosine 5’-0-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes. Eur J Biochem 1989; 186: 725-732
• 17 Féliste R, Simon MF, Chap H, Douste-Blazy L, Defreyn G, Maffrand JP. Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets. Biochem Pharmacol 1988; 37: 2559-2564
• 18 Vickers JD, Kinlough-Rathbone RL, Packham MA, Mustard JF. Inositol phospholipid metabolism in human platelets stimulated by ADP. Eur J Biochem 1990; 193: 521-528
• 19 Vickers JD, Kinlough-Rathbone RL, Mustard JF. The decrease in phosphatidylinositol 4,5-bisphosphate in ADP stimulated washed rabbit platelets is not primarily due to phospholipase C activation. Biochem J 1986; 237: 327-332
• 20 Brass LF, Woolkalis MJ, Manning DR. Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase. J Biol Chem 1988; 263: 5348-5355
• 21 Crouch MF, Winegar DA, Lapetina EG. Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein G_iα-2 and a 38-kDa phosphorylated protein in the human platelet. Proc Natl Acad Sci 1989; 86: 1776-1780
• 22 Simonds WF, Goldsmith PK, Codina J, Unson CG. G_i2 mediates α₂-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with GαC-terminal antibodies. Proc Natl Acad Sci 1989; 86: 7809-7813
• 23 Derian CK, Friedman PA. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization. Thromb Res 1988; 50: 65-76
• 24 Siess W. Molecular mechanisms of platelet activation. Pharmacol Rev 1989; 69: 58-178