Ticlopidine and Clopidogrel (SR 25990C) Selectively Neutralize ADP Inhibition of PGE₁-Activated Platelet Adenylate Cyclase in Rats and Rabbits

Summary

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE₁-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGEj-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGEr stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGEX-induced cAMP elevation but this effect seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGEr activated platelet adenylate cyclase in rats and rabbits.

• 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 183 (13) (Suppl. 01) 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, Bemat A, Delebassée D, Tissinier AM. Reviewed pharmacology of ticlopidine. Angiologie 1988; 77: 6-13
• 4 Delebassée D, Bemat A, Frehel D, Defreyn G, Maffrand JP. Antiaggregating and antithrombotic effects in the rat of two thienopy-ridine enantiomers structurally related to ticlopidine. Xth International Congress on Thrombosis, Athens 1988; 197
• 5 Féliste R, Delebassée D, Simon MF, Chap H, Defreyn G, Vallee 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, Stierle 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 (in press)
• 9 Sage SO, Reast R, Rink TJ. ADP evkes biphasic Ca²⁺ influx in fura-2-loaded human platelets. Biochem J 1990; 265: 675-680
• 10 Siffert W, Siffert G, Scheid P, Akkerman JWNS. Na⁺/H⁺ exchange modulates Ca²⁺ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619. J Biol Chem 1990; 264: 719-725
• 11 MacFarlane DE, Mills DCB. Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets. J Cyclic Nucleotide Res 1981; 7 (01) 7-11
• 12 Koneti RaoA, Kowalska MA. ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5’-p-fluorosulfonylbenzoyl-adenosine is a weak platelet agonist. Blood 1987; 70 (03) 751-756
• 13 Colman RW, Figures WR, Wu QX, Chung SY, Morinelli TA, Tuszynski GP, Colman RA, Niewiarowski S. Distinction between glycoprotein Ilia and the 100 kDa membrane protein (aggregin) mediating ADP-induced platelet activation. Arch Biochem Biophys 1988; 262 (01) 298-306
• 14 Lips JPM, Sixma JJ, Schiphorst ME. The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets. Thromb Res 1980; 17: 19-27
• 15 Feliste 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 (13) 2559-2564
• 16 Derian CK, Friedman PA. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization. Thromb Res 1988; 50: 65-76
• 17 Ashida SI, Abiko Y. Mode of action of ticlopidine in inhibition of platelet aggregation in the rat. Thromb Haemostas 1979; 41: 436-449
• 18 Morikawa M, Kojima T, Inoue M, Tsuboi M. Comparison of the inhibitory effects of aspirin and ticlopidine on platelet aggregation and Ca²⁺ mobilization in rat platelets. Res Commun Chem Pathol Pharmacol 1986; 54 (02) 275-278
• 19 Bonne C, Battais E. Ticlopidine and adenylate cyclase. Agents Actions Suppl 1984; 15: 88-96
• 20 Mills DCB, Fauci JB. The control of platelet responsiveness by agents that influence cyclic AMP metabolism. Ann N Y Acad Sci 1972; 201: 391-399
• 21 Aktories K, Jakobs KH. N_i-mediated inhibition of human platelet adenylate cyclase by thrombin. Eur J Biochem 1984; 145: 333-338
• 22 Lanza F, Beretz A, Kubina M, Cazenave JP. Increased aggregation and secretion responses of human platelets when loaded with the calcium fluorescent probes quin-2 and fura-2. Thromb Haemostas 1987; 58 (02) 737-743
• 23 Bom GVR, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-195
• 24 Best LC, McGuire MB, Martin TJ, Preston FE, Russel RGG. Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation, on release of 5-hydroxytryptamine and on the metabolism of 3’, 5’-cyclic AMP and cyclic GMP in human platelets. Biochim Biophys Acta 1979; 583: 344-351
• 25 Meurs H, Kauffman HF, Koeter G, Devries K. Extraction of cyclic AMP for the determination in the competitive protein binding assay. Clin Chim Acta 1980; 106: 91-97
• 26 Khym JX. An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin Chem 1975; 21 (09) 1245-1252
• 27 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
• 28 Simonds WF, Goldsmith PK, Codina J, Unson CG. G_i2 mediates _α2 ⁻adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with GaC-terminal antibodies. Proc Natl Acad Sci 1989; 86: 7809-7813
• 29 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 (11) 5348-5355
• 30 Houslay MD, Bojanic D, Gawler D, O’Hagen S, Wilson A. Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets. Biochem J 1986; 238: 109-113
• 31 Cusack NJ, Hourani SMO, Welford LA. Characterisation of ADP receptors. Adv Exp Med Biol 1985; 192: 29-39
• 32 Lanza F, Beretz A, Stierle A, Hanau D, Kubina M, Cazenave JP. Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 1988; 255: 1276-1288
• 33 Limbird LE. Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms. Faseb J 1988; 2: 2686-2695