Endothelial P2Y 2 ‐mediated vasoconstriction is inhibited in middle cerebral arteries of rabbits treated with clopidogrel

Abstract

Clopidogrel is an effective P2Y12 antagonist used to prevent arterial thrombosis, however it's use is associated with adverse bleeding events. Clinical studies have demonstrated that clopidogrel users have an increased risk of both cerebral microbleeds and intracerebral hemorrhage. Recent reports from our laboratory and others suggest that the adverse bleeding events associated with clopidogrel may not be due to the inhibition of platelets alone. To elucidate the non-platelet effects of clopidogrel treatment, we used pressure myography to measure changes in vascular function. Male New Zealand white rabbits (2.0-2.7kg) were treated with vehicle or 10mg/kg clopidogrel for 3 days by oral gavage. These doses were chosen because they are clinically relevant and effective at inhibiting platelet aggregation. On the 4th day, the middle cerebral artery (MCA) was isolated and mounted in a pressure myograph. Cumulative concentrations (10-9-10-5M) of purinergic receptor agonists (MRS 2768 – P2Y2, MRS 4062 – P2Y4, MRS 2693 – P2Y6, and MRS 2905 – P2Y14) were added through the MCA lumen and the artery inner diameter was tracked. Analysis of MCAs from vehicle-treated rabbits demonstrated constriction in response to P2Y2, P2Y4, P2Y6 and P2Y14 activation. Three days of clopidogrel treatment did not affect the contraction induced by P2Y4, P2Y6, and P2Y14 activation. However, clopidogrel treatment did reduce the P2Y2 mediated contraction (vehicle: -22.486% +/- 2.107 vs. 10mg/kg clopidogrel: -14.159% +/- 3.785 percent baseline diameter change at 10-5M MRS 2768; p < 0.01). Removal of the endothelium revealed that endothelial P2Y2 receptors were responsible for the constriction observed upon receptor activation. Vasoconstriction is a key component of the hemostatic process. Attenuated vasoconstriction could result in an increase in bleeding. These results suggest clopidogrel inhibits P2Y2 receptor vasoconstriction in the MCA which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.