Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y₁₂ pathway by inducing irreversible alteration of the platelet receptor P2Y₁₂, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.

In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y₁₂ pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl₃-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.

To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y₁₂. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y₁₂ double null mice, in both the tail bleeding assay and in a FeCl₃-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y₁₂ pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.