Dual antiplatelet therapy using aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. The favored P2Y12 antagonist clopidogrel is a prodrug that is absorbed in the intestine and then converted into the active metabolite by cytochrome P450 enzymes (CYP450s). However, only about 5% of clopidogrel is converted into this active metabolite. The remainder is converted into more than 15 additional metabolites to which the effects have been previously thought inactive. Antiplatelet agents, such as clopidogrel, cause an increased risk of developing cerebral microbleeds and intracerebral hemorrhage. Cerebral blood flow regulation is essential in maintaining blood-brain barrier homeostasis. Larger arteries, like the middle cerebral artery and posterior cerebral artery, generate myogenic tone to regulate cerebral blood flow. Myogenic tone is the contraction of vascular smooth muscle cells in response to increasing pressure. Tone generation protects the capillary bed from changes in pressure that could lead to cerebral microbleeds and intracerebral hemorrhage. When pressure increases in cerebral arteries, these vessels constrict to decrease blood flow. Arteries also undergo active contraction when damaged to reduce blood flow. Both acute vasoconstriction (myogenic tone) and chronic vasoconstriction (in response to injury) are crucial in the prevention and protection of arteries from major bleeds. P2Y1, P2Y2, P2Y4, and P2Y6 are expressed in the endothelium of cerebral arteries, while P2Y2, P2Y4, and P2Y6 are expressed in the smooth muscle. P2Y4 and P2Y6 in the smooth muscle are key regulators of myogenic tone. Due to the structural similarity between purinergic receptors, additional clopidogrel metabolites might modulate P2Y1, P2Y2, P2Y4, or P2Y6 on the vasculature. A major focus of the work described in this dissertation was to identify which receptor(s) clopidogrel or its metabolites inhibit. My studies tested the hypothesis that clopidogrel metabolites modulate P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and/or P2Y14 signaling in the vasculature which results in increased bleeding side effects. I addressed this hypothesis using pharmacological and genetic approaches. My studies show that clopidogrel inhibits ADP-induced platelet aggregation, but this effect does not correlate with the increase in adverse bleeding. Additionally, my studies demonstrate endothelial P2Y2-mediated vasoconstriction is inhibited in clopidogrel-treated rabbits. Furthermore, the prodrug clopidogrel, the M1 metabolite, and the M2 metabolite are not responsible for inhibiting P2Y2 signaling. Lastly, I discovered both P2Y12-/- mice and clopidogrel-treated mice have decreased distensibility and increased arterial stiffness. My studies could have wide implications for the development of new antiplatelet agents. The finding of P2Y2 inhibition by clopidogrel allowed us to better define the mechanism to which clopidogrel causes adverse bleeding. This provides a basis for what to avoid in the development of improved antiplatelet therapies.
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