The aim of the study was to determine the effect of clopidogrel on adenosine diphosphate (ADP)- induced platelet activation in human volunteers. Platelets from human volunteers before and after a 7-day treatment with clopidogrel (75 mg/kg), were tested for their sensi tivity to ADP by measuring ADP-induced aggregation, adenylyl cyclase downregulation, and [3H]-2-MeS-ADP binding. Platelet membrane glycoprotein (GP IIb-IIIa; GP Ib, GMP-140) expression was measured by flow cy tometry using fluorescent-labeled antibodies or fibrino gen. After oral administration to human volunteers (75 mg/day for 7 days), clopidogrel, a novel ADP-selective antiplatelet agent, inhibited ADP-induced aggregation of platelets ex vivo. This effect was irreversible in nature, and no activity could be detected in the plasma of treated subjects. Although clopidogrel did not modify ADP- induced shape change, it prevented the inhibitory effect of ADP (but not that of epinephrine) on the prostoglandin- E 1 (PGE1)-induced increase in platelet cAMP. The num ber of binding sites for [ 3H]-2-MeS-ADP, a stable ana logue of ADP that labels ADP binding sites linked to the inhibition of stimulated adenylyl cyclase, was reduced from 525 ± 62 sites/cell in the controls to 32 ± 5 sites/cell after treatment with clopidogrel (p < 0.001). This effect occurred with no consistent change in the binding affinity of [3H]-2-MeS-ADP, indicating that inhibition of platelet functions by clopidogrel was mainly due to a selective and irreversible reduction of ADP binding sites on plate lets. Flow cytometry experiments showed that clopi dogrel selectively inhibited ADP-inducing binding of fi brinogen to platelets. This effect occurred through a ma jor reduction of the ADP-induced activation of the GP IIb-IIIa complex. These findings therefore indicate that clopidogrel downregulates platelet responses via a selec tive and direct interaction with the ADP receptors, me diating the inhibition of stimulated adenylyl cyclase ac tivity in human platelets.
Mehta J., Mehta P.Comparison of platelet function during exercise in normal subjects and coronary artery disease patients: potential role of platelets in myocardial ischemia . Am Heart J1982;103:49-53.
2.
Fitzgerald DJ , Roy L., Catella F., FitzGerald GAPlatelet activation in unstable coronary disease . N Engl J Med1986; 315:983-9.
3.
Antiplatelet Trialists' Collaboration.Secondary prevention of vascular disease by prolonged antiplatelet treatment . Br Med J1988;296:320-31.
4.
Fuster V., Badimon L., Cohen M., Ambrose JA, Badimon JJ, Chesebro J.Insights into the pathogenesis of acute ischemic syndromes. Circulation1988;77:1213-20.
5.
Mustard JF, Packham MA, Kinlough-Rathbone RL. Platelets, blood flow and the vessel wall. Circulation1990;81: 24-7.
6.
Tschoepe D., Schultheiss HP, Kolarov P., et al. Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA. Circulation1993 ;88:37-42.
7.
Scharf RE, Tomer A., Merzec UM, Teirstein PS, Ruggieri ZM, Harker LAActivation of platelets in blood perfusing angioplasty-damaged coronary arteries, flow cytometry detection. Arterioscler Thromb1992;12:1475-87.
8.
Topol E., Califf RM, Ellis SG, et al. Randomized trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet1994;343:881-6.
9.
TheIPIC investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med1994;330:956-61.
10.
DeWood MA, Spores J., Notsk ERPrevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med1980;303:897.
11.
Davies MJ, Thomas AC, Knapman PA, Hangartner JRIntramyocardial platelet aggregation in patients with unstable angina suffering sudden ischemic cardiac death. Circulation1986;73:418-27.
12.
Herbert JMClopidogrel and antiplatelet therapy. Exp Op Invest Drugs1994;3:449-55.
13.
Féliste R., Delebassée D., Simon MF, et al. Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the suppression of the effects of released ADP. Thromb Res1987;48:403-15.
14.
Maffrand JP , Bernat A., Delebassée E., Defreyn G., Cazenave JP, Gordon JLADP plays a key role in thrombogenesis in rats. Thromb Haemost1988;59:225-30.
15.
Cairns JA, Gent M., Singer J.Aspirin, sulfinpyrazone, or both in unstable angina. N Engl J Med1985 ;313:1369-75.
16.
Janzon L., Bergqvist D., Boberg J., et al. Prevention of myocardial infarction and stroke in patients with intermittent claudication; effects of ticlopidine. Results from STIMS, the Swedish Ticlopidine Multicentre Study. ScandJ Intern Med1990;227:301-8.
17.
Gent M., Easton JD, Hachinski VC, et al. The CanadianAmerican Ticlopidine Study (CATS) in thromboembolic stroke. Lancet1989;8649(1):1215-20.
18.
Hass WK, Easton JD, Adams HP, et al. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. N Engl J Med1989; 321:501-7.
19.
Defreyn G., Gachet C., Savi P., Driot F., Cazenave JP, Maffrand JPTiclopidine and clopidogrel (SR 25990) selectively neutralize ADP inhibition of PGE,-activated platelet adenylate cyclase in rats and rabbits . Thromb Haemost1991 ;65: 186-90.
20.
Gachet C., Stierlé A., Cazenave JP, Ohlmann P., Lanza F., Bouloux C., Maffrand JPThe 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 Pharmacol1990;40:229-38.
21.
Herbert JM, Frehel D., Vallée E., et al. Clopidogrel, a novel antiplatelet and antithrombotic agent. Cardiovasc Drug Rev1993;11:180-98.
22.
Savi P., Herbert JM, Pflieger AM, et al. Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. Biochem Pharmacol1992;44: 527-32.
23.
Savi P., Laplace MC, Maffrand JP, Herbert JMBinding of [3H]-2-methylthio-ADP to rat platelets: effect of clopidogrel and ticlopidine. J Pharm Exp Ther1994;269:772-7.
24.
Mills D., Puri R., Hu C-J., et al. Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase. Arterioscler Thromb1992;12: 430-6.
25.
Hourdillé P. , Heilmann E., Combrie R., Winckler J., Clemetson KJ, Nurden ATThrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets. Blood1990;76:1503-13.
26.
Munson PJ, Rodbard D.Ligand: a versatile computerized approach for the characterization of ligand binding systems. Anal Biochem1980 ;107:220-39.
27.
Scatchard G.The attractions of proteins for small molecules and ions. An NY Acad Sci1949;51:660-72.
28.
Meurs H., Kauffman HF, Koeter G., Devries K.Extraction of cyclic AMP for the determination in the competitive protein binding assay. Clin Chim Acta1980 ;106:91-7.
29.
Pidard D., Montgomery RR, Bennett JS, Kunicki TJInteraction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets. J Biol Chem1983;258:12582-6.
30.
Shattil SJ, Cunningham M., Hoxie JADetection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood1987;70:307-15.
31.
Abrams C., Shattil SJImmunological detection of activated platelets in clinical disorders . Thromb Haemost1991 ;65: 467-73.
32.
Kieffer N., Phillips DRPlatelet membrane glycoproteins: functions in cellular interactions . Annu Rev Cell Biol1990; 6:329-57.
33.
Maffrand JP , Defreyn G., Bernat A., Delebassée D., Tissinier AMReviewed pharmacology of ticlopidine. Act Med Int Angiologie1988 ;5(supl 77):6-13.
34.
David JL, Monfort F., Herion F., Raskinet R.Compared effects of three dose-levels of ticlopidine on platelet function in normal subjects. Thromb Res1979;14:35-49.
35.
Cattaneo M. , Akkawat B., Kinlough-Rathbone RL, Packham MA, Cimminiello C., Mannucci PMTiclopidine facilitates the deaggregation of human platelet aggregated by thrombin. Thromb Haemost1994;71:91-4.
36.
Chignard M. , Lalau Keraly C, Delautier D., Sebag C., Bouloux C., Motte G., Benveniste J.Reduced sensitivity of human platelets to PAF-acether following ticlopidine intake. Haemostasis1989;19:213-18.
37.
Cimminiello C., Milani M., Uberti T., Arpaia G., Bonfardeci G.Effects of ticlopidine and indobufen on platelet aggregation induced by A23187 and adrenaline in the presence of different anticoagulants. J Int Med Res1989;17:514-20.
38.
Féliste R., Delebassée D., Simon MF, et al. Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the suppression of the effects of released ADP. Thromb Res1987;48:403-15.
39.
Di Perri T, Pasini FL, Frigerio C., et al. Pharmacodynamics of ticlopidine in man in relation to plasma and blood cell concentration. Eur J Clin Pharmacol1991;41:429-34.
40.
Hardisty R. , Pidard D., Cox A., et al. A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder . Blood1992;80:696-798.
41.
Janes SL, Wilson DJ, Chronos N., Goodall AHEvaluation of whole blood flow cytometric detection of platelet bound fibrinogen on normal subjects and patients with activated platelets. Thromb Haemost1993;70:659-66.
42.
Dunn FW, Soria J., Soria C., Thomaidis A., Lee H., Caen JPIn vivo effect of ticlopidine on fibrinogen-platelet cofactor activity and binding of fibrinogen to platelets. Agent Action1984;15:97-104.
43.
Di Minno G, Cerbone AM, Mattioli PL, Turco S., Iovine C., Mancini M.Functionally thombasthenic state in normal platelets following the administration of ticlopidine . J Clin Invest1985;75:328-38.
44.
Hardisty RM , Powling MJ, Nokes Tjc.The action of ticlopidine on human platelets. Studies of aggregation, secretion, calcium mobilization and membrane glycoproteins . Thromb Haemost1990;64:150-5.
45.
Stenberg PE , McEver RP, Shuman MA, Jaques YV, Bainton DFA platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation . J Cell Biol1985;101:880-6.
46.
Roald HE, Barstad RM, Kierulf P., Skjorten F., Dickinson JP, Kieffer G., Sakariassen KSClopidogrel—a platelet inhibitor which inhibits thrombogenesis in non-anticoagulated human blood independently of the blood flow conditions . Thromb Haemost1994 ;71:655-62.
