Clopidogrel: An antithrombotic drug acting on the ADP-dependent activation pathway of human platelets

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 sensitivity to ADP by measuring ADP-induced aggregation, adenylyl cyclase downregulation, and [H-3]-2-MeS-ADP binding. Platelet membrane glycoprotein (GP IIb-IIIa; GP Ib, GMP-140) expression was measured by flow cytometry using fluorescent-labeled antibodies or fibrinogen. 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) (PGE(1))-induced increase in platelet cAMP. The number of binding sites for [H-3]-2-MeS-ADP, a stable analogue 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 [H-3]-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 platelets. Flow cytometry experiments showed that clopidogrel selectively inhibited ADP-inducing binding of fibrinogen to platelets. This effect occurred through a major reduction of the ADP-induced activation of the GP IIb-IIIa complex. These findings therefore indicate that clopidogrel downregulates platelet responses via a selective and direct interaction with the ADP receptors, mediating the inhibition of stimulated adenylyl cyclase activity in human platelets.