Clopidogrel treatment inhibits P2Y₂-Mediated constriction in the rabbit middle cerebral artery

Clopidogrel is an effective purinergic 2Y₁₂ receptor (P2Y₁₂) antagonist used to prevent arterial thrombosis, but its use is associated with adverse bleeding. Clinical studies have demonstrated that clopidogrel users have an increased risk of cerebral microbleeds and intracerebral hemorrhage. Our previous studies suggest that non-platelet mechanisms mediate these adverse bleeding events; we hypothesize that clopidogrel or one of its metabolites interacts with blood vessels directly to cause bleeding. New Zealand white rabbits (1.9-2.7 kg) were treated orally with vehicle or clopidogrel (3 or 10 mg/kg) for three days. On the fourth day, the rabbits were anesthetized for blood collection and then euthanized. The brain was collected, and the middle cerebral arteries were isolated. We used light transmission aggregometry and pressure myography to elucidate the mechanisms of the off-target effects associated with clopidogrel treatment. We confirmed that inhibition of P2Y₁₂ activation by clopidogrel inhibited ADP-induced platelet aggregation but had no impact on P2Y₁₂-independent arachidonic acid- or collagen-induced platelet aggregation. Analysis of middle cerebral arteries from clopidogrel treated rabbits showed that clopidogrel did not affect P2Y₄, P2Y₆, and P2Y₁₄ receptor-mediated contraction but attenuated the contractile response after P2Y₂ receptor activation. Further analysis determined P2Y₂-mediated constriction was endothelium-dependent. Vasoconstriction is a primary component of hemostasis, and impaired vasoconstriction can prolong bleeding. These results suggest clopidogrel inhibits the endothelial P2Y₂ receptor in the middle cerebral artery, which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.