The additive antiplatelet action of clopidogrel in patients with coronary artery disease treated with aspirin

Summary

We searched for additional anti-platelet effects of clopidogrel in coronary artery disease (CAD) patients treated with aspirin. Response to clopidogrel was also stratified according to aspirin resistance. Out of 76 screened aspirin-treated CAD male patients, five were aspirin-resistant based on arachidonic acid (AA) and ADP aggregometry.These five patients and 15 aspirin-sensitive patients entered the proper study. Platelet function was assessed at baseline and after one week of additional clopidogrel treatment using aggregometry, flow cytometry (ADP, TRAP-6) and platelet reactivity index (PRI) based on VASP (vasodilatorstimulated phosphoprotein) expression.We evaluated the same markers in 15 healthy men after aspirin treatment. In healthy subjects aspirin did not affect resting or ADP-induced activated GPIIb/IIIa and P-selectin expression. The P-selectin expression on ADP-activated platelets was increased (p<0.01) in aspirin treated ASA-resistant CAD patients as compared to ASA-sensitive group or aspirin-treated healthy subjects. Clopidogrel significantly decreased ADP and AA-induced platelet aggregation and overcame aspirin resistance in four of five patients. Expression of ADP-induced activation markers was significantly lowered after clopidogrel in all patients.Out of 20 patients,five did not respond to clopidogrel (<10% inhibition of ADP aggregation), and this group showed no change in expression of ADPinduced activation markers after clopidogrel. Clopidogrel treatment significantly reduced PRI only in the clopidogrel-sensitive group. In conclusion, the addition of clopidogrel to aspirin provides greater inhibition of platelets and can overcome aspirin resistance. Flow cytometric analysis of platelets is useful for monitoring of clopidogrel therapy.

Keywords

Aggregation - aspirin - clopidogrel - flow cytometry - platelets

• References

• 1 Lopez AD, Murray CC. Theglobal burden of disease, 1990-2020. Nat Med 1998; 4: 1241-1243.
• 2 Fuster V, Badimon L, Badimon JJ. et al. The pathogenesis of coronary artery disease and the acute coronary syndromes(2). N Engl J Med 1992; 326: 310-318.
• 3 Gum PA, Kottke-Marchant K, Poggio ED. et al. Profile and prevalence of aspirin resistance in patients with cardiovascular disease. Am J Cardiol 2001; 88: 230-235.
• 4 Gum PA, Kottke-Marchant K, Welsh PA. et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol 2003; 41: 961-965.
• 5 Szczeklik A, Musial J, Undas A. et al. Aspirin resistance. J Thromb Haemost 2005; 3: 1655-1662.
• 6 Yusuf S, Zhao F, Mehta SR. et al. Effects of clopidogrel in addition to aspirinin patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001; 345: 494-502.
• 7 Chen ZM, Jiang LX, Chen YP. et al. Addition of clopidogrel to aspirin in 45,852 patients with acute myocardial infarction: randomised placebo-controlled trial. Lancet 2005; 366: 1607-1621.
• 8 Steinhubl SR, Berger PB, Mann 3rd JT. et al. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. J Am Med Assoc 2002; 288: 2411-2420.
• 9 Schmitz G, Rothe G, Ruf A. et al. European Working Group on Clinical CellAnalysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998; 79: 885-896.
• 10 Geiger J, Brich J, Honig-Liedl P. et al. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel. Arterioscler Thromb Vasc Biol 1999; 19: 2007-2011.
• 11 Leytin V, Mody M, Semple JW. et al. Flow cytometric parameters for characterizing platelet activation by measuring P-selectin (CD62) expression: theoretical considerationand evaluationinthrombin-treated platelet populations. Biochem Biophys Res Commun 2000; 269: 85-90.
• 12 Wong S, Morel-Kopp MC, Chen Q, Appleberg M, Ward CM, Lewis DR. Overcoming aspirin resistance: increased platelet inhibition with combination aspirin and clopidogrel and high dose aspirin therapy in aspirin resistantpatientswith peripheral vascular disease. Thromb Haemost 2006; 95: 1042-1043.
• 13 Eikelboom JW, Hankey GJ, Thom J. et al. Enhanced antiplatelet effect of clopidogrel in patients whose plateletsare least inhibited by aspirin: a randomized crossover trial. J Thromb Haemost 2005; 3: 2649-2655.
• 14 Klinkhardt U, Bauersachs R, Adams J. et al. Clopidogrel but not aspirinreduces P-selectin expression and formation of platelet-leukocyte aggregates in patients with atherosclerotic vascular disease. Clin Pharmacol Ther 2003; 73: 232-241.
• 15 Seyfarth HJ, Koksch M, Roethig G. et al. Effect of 300- and 450-mg clopidogrel loading doses on membrane and soluble P-selectin in patients undergoing coronary stent implantation. Am Heart J 2002; 143: 118-123.
• 16 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for P2T(AC) and alpha(2A) receptors. J Biol Chem 1999; 274: 29108-29114.
• 17 Li Z, Zhang G, Le Breton GC. et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. J Biol Chem 2003; 278: 30725-30731.
• 18 Frelinger 3rd AL, Furman MI, Linden MD. et al. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: a 700-patient study of aspirin resistance. Circulation 2006; 113: 2888-2896.
• 19 Macchi L, Christiaens L, Brabant S. et al. Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate. Thromb Res 2002; 107: 45-49.
• 20 Kawasaki T, Ozeki Y, Igawa T. et al. Increased platelet sensitivity to collagen in individuals resistant to low-dose aspirin. Stroke 2000; 31: 591-595.
• 21 Sciulli MG, Renda G, Capone ML. et al. Heterogeneity in the suppression of platelet cyclooxygenase-1 activity by aspirin in coronary heart disease. Clin Pharmacol Ther 2006; 80: 115-125.
• 22 Mills DC. ADP receptors on platelets. Thromb Haemost 1996; 76: 835-856.
• 23 Maclouf J, Folco G, Patrono C. Eicosanoids and iso-eicosanoids: constitutive, inducible and transcellular biosynthesis in vascular disease. Thromb Haemost 1998; 79: 691-705.
• 24 Santos MT, Moscardo A, Valles J. et al. Participation of tyrosine phosphorylation in cytoskeletal reorganization, alpha(IIb)beta(3)integrin receptor activation, and aspirin-insensitive mechanism of thrombin-stimulated human platelets. Circulation 2000; 102: 1924-1930.
• 25 Yusuf S, Zhao F, Mehta SR. et al. Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001; 345: 494-502.
• 26 Valles J, Santos T, Aynar J. et al. Erythrocyte promotion of platelet reactivity decreases the effectiveness of aspirin as an anthithrombotic therapeutic modality. The effect of low-dose aspirin is less than optimal in patients with vascular disease due to pro-thrombotic effects of erythrocytes on platelet reactivity. Circulation 1998; 97: 350-355.
• 27 Ikeda Y, Handa M, Kamata T. et al. Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation. Thromb Haemost 1993; 69: 496-502.
• 28 Moake JL, Turner NA, Stathopoulos NA. et al. Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin. Blood 1988; 71: 1366-1374.
• 29 Cadroy Y, Bossavy JP, Thalamas C. et al. Early potent antithrombotic effect with combined aspirin and a loading dose of clopidogrel on experimental arterial thrombogenesis in humans. Circulation 2000; 101: 2823-2828.
• 30 Nguyen TA, Diodati JG, Pharand C. Resistance to clopidogrel: a review of the evidence. J Am Coll Cardiol 2005; 45: 1157-1164.
• 31 Cattaneo M. Aspirin and clopidogrel resistance. Efficacy, safety, and the issue of drug resistance. Arterioscler Thromb Vasc Biol 2004; 109: 3171-3175.
• 32 Wenaweser P, Dorffler-Melly J, Imboden K. et al. Stent thrombosis is associated with an impaired response to antiplatelet therapy. J Am Coll Cardiol 2005; 45: 1748-1752.
• 33 Muller I, Besta F, Schulz C. et al. Prevalance of clopidogrel non-responders among patients with stable angina pectoris scheduled for coronary stent placement. Thromb Haemost 2003; 89: 783-787.
• 34 Gurbel PA, Bliden KP, Hiatt BL. et al. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003; 107: 2908-2913.
• 35 Bhatt DL, Fox KA, Hacke W. et al. CHARISMA Investigators. Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events. N Engl J Med 2006; 354: 1706-1717.
• 36 Kunapuli SP, Dorsam RT, Kim S. et al. Platelet purinergic receptors. Curr Opin Pharmacol 2003; 3: 175-180.
• 37 Schwarz UR, Geiger J, Walter U. et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets--definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-1152.
• 38 Aleil B, Ravanat C, Cazenave JP. et al. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases. J Thromb Haemost 2005; 3: 85-92.
• 39 Grossmann R, Sokolova O, Schnurr A. et al. Variable extent of clopidogrel responsiveness in patients after coronary stenting. Thromb Haemost 2004; 92: 1201-1206.
• 40 Pampuch A, Cerletti C, de Gaetano G. Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor. Thromb Haemost 2006; 96: 767-773.
• 41 Michelson AD, Barnard MR, Krueger LA. et al. Evaluation of platelet function by flow cytometry. Methods 2000; 21: 259-270.
• 42 Trip MD, Cats VM, van Capelle FJ. et al. Platelet hyperreactivity and prognosis in survivors of myocardial infarction. N Engl J Med 1990; 322: 1549-1554.