It is known that hepatic metabolism limits the anti-aggregatory activity of clopidogrel and by consequence, its clinical benefits. In this study, we investigated whether other factors exist, that could account for clopidogrel's sub-optimal antithrombotic activity. Using an in vivo murine FeCl3 thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of ADP-induced platelet aggregation, clopidogrel (50mg/kg oral) failed at reproducing the phenotype associated with P2Y12-deficiency. On the other hand, elinogrel (60mg/kg oral), a direct-acting reversible P2Y12 antagonist, achieved maximal levels of inhibition in vivo and its administration (1 mg/kg IV) abolished the residual thrombosis associated with clopidogrel dosing. Since elinogrel is constantly present in plasma, while the active metabolite of clopidogrel exists for ~ 2 hours, we evaluated whether an intracellular pool of P2Y12 exists, that would be inaccessible to clopidogrel and contribute to its limited antithrombotic activity. Using saturation [3H]-2MeSADP binding studies, we first demonstrated that platelet stimulation with thrombin and convulxin (mouse) and TRAP (human) significantly increased surface expression of P2Y12 relative to resting platelets. We next found that clopidogrel dose-dependently inhibited ADP-induced aggregation, signaling (cAMP) and surface P2Y12 on resting mouse platelets, achieving complete inhibition at the highest dose (50mg/kg), but failed at blocking this inducible pool. Thus, an inducible pool of P2Y12 exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not completely blocked by clopidogrel, contributes to thrombosis in vivo, and can be blocked by elinogrel.
- Received May 16, 2011.
- Revision received June 30, 2011.
- Accepted July 1, 2011.
- The American Society for Pharmacology and Experimental Therapeutics