Biomarker Optimization to Track the Antithrombotic and Hemostatic Effects of Clopidogrel in Rats

Abstract

We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl 2 . Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y 12 receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED 50 ) for thrombus reduction was 2.4 Ϯ 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction.The ED 50 for bleeding was 10.5 Ϯ 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED 50 s for peak platelet aggregation to 10 M ADP (11.9 Ϯ 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 Ϯ 1.3 mg/kg) approximated the higher ED 50 for bleeding. ED 50 s for ligand binding (3.0 Ϯ 0.3 mg/kg) and late aggregation (5.1 Ϯ 0.4 mg/kg) better matched the lower ED 50 for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.The thienopyridine clopidogrel is an ADP receptor antagonist that, upon conversion to an active metabolite, irreversibly blocks the platelet P2Y 12 receptor (Savi and Herbert, 2005). The structure of the active metabolite and the identity of the platelet receptor for clopidogrel were not known before phase III clinical trials had been completed (CAPRIE Steering Committee, 1996). In fact, the P2Y 12 receptor was referred to as the thienopyridine receptor in the original publication that detailed its molecular identification (Hollopeter et al., 2001). The primary activity biomarker for tracking the effects of clopidogrel has been ex vivo inhibition of ADPinduced platelet aggregation (IPA) (Nguyen et al., 2005).It is well recognized that the standard 75-mg maintenance dose of clopidogrel that reduces clinical cardiovascular events exerts a submaximal effect on ex vivo IPA (Thebault et al., 1999). The antiplatelet effect of clopidogrel in man requires oral bioavailability of parent drug, its bioactivation to the active metabolite, and productive blockade by that metabolite of platelet P2Y 12 receptors. Its effects will not prevent ADP-mediated platelet stimulation through P2Y 1 receptors. Our goal was to understand whether IPA is in fact a good predictor of clopidogrel's efficacy and bleeding ...

Modifying Clopidogrel Maintenance Doses According to Vasodilator‐Stimulated Phosphoprotein Phosphorylation Index Improves Clinical Outcome in Patients With Clopidogrel Resistance

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Multiple electrode aggregometry and vasodilator stimulated phosphoprotein-phosphorylation assay in clinical routine for prediction of postprocedural major adverse cardiovascular events

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Pretreatment with a combination of ligustrazine and berberine improves cardiac function in rats with coronary microembolization

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