Safety and efficacy of targeting platelet proteinase-activated receptors in combination with existing anti-platelet drugs as antithrombotics in mice.

BACKGROUND AND PURPOSE: Developing novel anti-platelet strategies is fundamental to reducing the impact of thrombotic diseases. Thrombin activates platelets via proteinase-activated receptors (PARs), and PAR antagonists are being evaluated in clinical trials for prevention of arterial thrombosis. However, one such trial was recently suspended due to increased bleeding in patients receiving a PAR1 antagonist in addition to anti-platelet drugs that most often included both aspirin and clopidogrel. Therefore, it remains unclear how to best manipulate PARs for safe antithrombotic activity. To address this, we have examined potential interactions between existing anti-platelet drugs and strategies that target PARs. EXPERIMENTAL APPROACH: We used in vivo mouse models in which interactions between various anti-platelet strategies could be evaluated. We examined the effects on thrombosis and haemostasis in PAR4 -/- mice (platelets unresponsive to thrombin) treated with therapeutic doses of either aspirin or clopidogrel. KEY RESULTS: Using a model in which occlusive thrombosis occurred in PAR4 -/- mice or wild-type mice treated with aspirin or clopidogrel, PAR4 -/- mice treated with either anti-platelet agent showed marked protection against thrombosis. This antithrombotic effect occurred without any effect on haemostasis with aspirin, but not clopidogrel. Furthermore, specifically targeting thrombin-induced platelet activation (via PARs) improved the therapeutic window of non-specifically inhibiting thrombin functions (via anticoagulants). CONCLUSIONS AND IMPLICATIONS: Our results indicate that PAR antagonists used in combination with aspirin provide a potent yet safe antithrombotic strategy in mice and provide insights into the safety and efficacy of using PAR antagonists for the prevention of acute coronary syndromes in humans.

Atherosclerosis proceeds independently of thrombin-induced platelet activation in ApoE-/- mice.

Platelet activation has long been postulated to contribute to the development of atherosclerotic plaques, although the mechanism by which this might occur remains unknown. Thrombin is a potent platelet activator and transfusion of thrombin-activated platelets into mice increases plaque formation, suggesting that thrombin-induced platelet activation might contribute to platelet-dependent atherosclerosis. Platelets from protease-activated receptor 4-deficient (Par4-/-) mice fail to respond to thrombin. To determine whether thrombin-activated platelets play a necessary role in a model of atherogenesis, we compared plaque formation and progression in Par4+/+ and Par4-/- mice in the atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) background. Littermate Par4+/+ and Par4-/- mice, all ApoE-/-, were placed on a Western diet (21% fat, 0.15% cholesterol) for 5 or 10 weeks. The percent of aortic lumenal surface covered by plaques in Par4+/+ and Par4-/- mice was not different at either time point (2.2+/-0.3% vs. 2.5+/-0.2% and 5.1+/-0.4% vs. 5.6+/-0.4% after 5 and 10 weeks, respectively). Further, no differences were detected in the cross-sectional area of plaques measured at the aortic root (1.53+/-0.17 vs. 1.66+/-0.16x10(5)microm(2) and 12.56+/-1.23 vs. 13.03+/-0.55x10(5)microm(2) after 5 and 10 weeks, respectively). These findings indicate that thrombin-mediated platelet activation is not required for the early development of atherosclerotic plaques in the ApoE-/- mouse model and suggest that, if platelet activation is required for plaque formation under these experimental conditions, platelet activators other than thrombin suffice.