It is known that hepatic metabolism limits the antiaggregatory activity of
clopidogrel and, as a consequence, its clinical benefits. In this study, we investigated whether other factors exist that could account for
clopidogrel's suboptimal antithrombotic activity. Using an in vivo murine
FeCl(3)
thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of
ADP-induced platelet aggregation,
clopidogrel (50 mg/kg p.o.) failed to reproduce the phenotype associated with P2Y(12) deficiency. However,
elinogrel (60 mg/kg p.o.), a direct-acting reversible P2Y(12) antagonist, achieved maximal levels of inhibition in vivo, and its administration (1 mg/kg i.v.) abolished residual
thrombosis associated with
clopidogrel dosing. Because
elinogrel is constantly present in the plasma, whereas the active metabolite of
clopidogrel exists for ∼2 h, we evaluated whether an intracellular pool of P2Y(12) exists that would be inaccessible to
clopidogrel and contribute to its limited antithrombotic activity. Using saturation [(3)H]2-(
methylthio)ADP ([(3)H]
2MeSADP) binding studies, we first demonstrated that platelet stimulation with
thrombin and
convulxin (mouse) and
thrombin receptor activating peptide (TRAP) (human) significantly increased surface expression of P2Y(12) relative to that of resting platelets. We next found that
clopidogrel dose-dependently inhibited
ADP-induced aggregation, signaling (cAMP), and surface P2Y(12) on resting mouse platelets, achieving complete inhibition at the highest dose (50 mg/kg), but failed to block this inducible pool. Thus, an inducible pool of P2Y(12) exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not blocked completely by
clopidogrel, contributes to
thrombosis in vivo, and can be blocked by
elinogrel.