Clinical studies with clopidogrel or prasugrel show that although increased inhibition of P2Y 12 and platelet function improves efficacy, bleeding is also increased. Other preclinical and clinical studies have suggested a greater therapeutic index (TI) with reversible inhibitors and disproportionate effects of thienopyridines on bleeding at high doses. We used multiple in vivo (FeCl 3 -induced arterial thrombosis in mesenteric arteries, blood loss after tail transsection, and platelet deposition and wound closure time in a micropuncture model in mesenteric veins) and ex vivo (light transmittance aggregometry, prothrombin time, and activated partial thromboplastin time) mouse models to 1) compare the TI of clopidogrel, prasugrel, and elinogrel, a reversible, competitive antagonist, with that in P2Y 12 (Ϫ/Ϫ) mice and 2) determine whether the bleeding consequences of the thienopyridines are attributed only to the inhibition of P2Y 12 . Data indicated greater (elinogrel) and decreased (thienopyridines) TI compared with that in P2Y 12 (Ϫ/Ϫ) mice. The impaired TI associated with the thienopyridines was not attributed to non-P2Y 12 activities on platelet function or coagulation but was related to a direct effect at the vessel wall (inhibition of vascular tone). Further analysis showed that the prasugrel off-target effect was dose-and time-dependent and of a reversible nature. In conclusion, the TI of thienopyridines in the mouse may be decreased by P2Y 12 -independent off-target effects at the vessel wall, whereas that of elinogrel may be enhanced by the reversible, competitive nature of the antiplatelet agent.

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 ϳ2 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) 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.

Mercuric chloride was injected intracranially into the rat brain in the region of the fourth ventricle via the foramen magnum. Responses to a single treatment of 0.3 mg/kg HgCl2, 0.03 mg/kg HgCl2, or carrier solution were followed for 10 days. Changes in the rats' behavior and motor coordination were observed 24-48 hr after treatment with HgCl2. Underwater swimming and decreased grooming and feeding were characteristic of mercury-treated rats. Decreased balancing ability and crossing of hind limbs suggested altered motor coordination. Rats treated with mercury often exhibited decreased body temperature, matting of fur about the eyes and perineal region, apparent visual impairment, decreased body weight, and diarrhea.

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