The Vascular Effects of Clopidogrel Metabolites

项目摘要： 使用低剂量阿司匹林和嘌呤能受体2Y12（P2Y12）拮抗剂的双重抗血小板治疗是最常见的 预防动脉血栓的常用方法。抗血小板药物，如氯吡格雷， 增加脑微出血和脑内出血的风险。在服用氯吡格雷的患者中， 至少一年后，31%的患者出现脑微出血，其中47%的患者还出现脑内出血 出血氯吡格雷抑制ADP诱导的血小板聚集，但这种作用与血小板聚集率无关。 不良出血增加。这表明氯吡格雷的不良反应不是由活性成分介导的。 代谢物，母体化合物或许多非活性代谢物之一可能是负责的。一个更 需要完全了解氯吡格雷及其代谢产物影响的细胞通路， 来降低这种风险。氯吡格雷通过细胞色素P450酶转化为活性代谢物 （CYP450）。只有大约5%的氯吡格雷被激活，CYP 450的功能多态性丧失 进一步减少这个。DT 678是氯吡格雷活性代谢物的结合物，其转化为活性代谢物。 通过与谷胱甘肽的非酶促巯基交换反应形成。我们之前报道过，DT678 在兔模型中，与氯吡格雷相比，我的初步数据显示， 自发性肌张力（MT）和ADP诱导的血管舒张在氯吡格雷治疗的受试者中受到抑制， DT678处理的大脑中动脉（MCA）。我的中心假设是氯吡格雷调节P2Y1， P2Y2、P2Y4、P2Y6、P2Y11和/或P2Y14信号传导，并且这损害血管功能 其导致出血副作用增加而不引起血小板的相关减少 聚合来为了检验我的中心假设，我将追求以下三个具体目标：1。识别 氯吡格雷和DT 678调节的受体信号通路。我的假设是 在稳定转染的细胞中，氯吡格雷抑制P2Y1、P2Y2、P2Y4、P2Y6、P2Y11和/或P2Y14以及P2Y12。 此外，我假设DT-678仅抑制P2Y12受体，而对其他嘌呤能受体没有影响。 受体。2.确定特异性嘌呤能激动剂在大脑中动脉中的作用， 氯吡格雷和DT 678处理的家兔。我的工作假设是氯吡格雷代谢物与 P2Y1、P2Y2、P2Y4、P2Y6、P2Y11和/或P2Y14在MCA中损害MT生成和扩张。3.评价 氯吡格雷和DT 678在P2Y12-/-小鼠实质小动脉中的作用。我的假设是 氯吡格雷调节血管系统中的P2Y1、P2Y2、P2Y4、P2Y6、P2Y11和/或P2Y14以抑制肌原性 P2Y12-/-小鼠的张力、收缩和扩张。从该项目获得的信息将允许 开发新的抗血小板疗法，提高疗效，降低脑内出血风险 出血

Project Summary: Dual antiplatelet therapy using low-dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. Antiplatelet agents, such as clopidogrel, cause an increased risk of cerebral microbleeds and intracerebral hemorrhage. Of patients who have taken clopidogrel for at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral hemorrhage. Clopidogrel inhibits ADP-induced platelet aggregation, but this effect does not correlate with the increase in adverse bleeding. This suggests adverse effects of clopidogrel are not mediated by the active metabolite and that the parent compound or one of the many inactive metabolites might be responsible. A more complete understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order to decrease this risk. Clopidogrel is converted into the active metabolite by cytochrome P450 enzymes (CYP450's). Only approximately 5% of clopidogrel is activated and loss of function polymorphisms in CYP450's reduce this further. DT678 is a conjugate of the clopidogrel active metabolite that is converted into the active form through a non-enzymatic thiol exchange reaction with glutathione. We have previously reported that DT678 has a reduced bleeding risk compared to clopidogrel in a rabbit model. My preliminary data revealed that spontaneous myogenic tone (MT) and ADP-induced vasodilation are inhibited in clopidogrel-treated, but not DT678-treated middle cerebral arteries (MCA's). My central hypothesis is that clopidogrel modulates P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 signaling in the vasculature, and this impairs vascular function which results in the increased bleeding side effects without causing a correlated decrease in platelet aggregation. In order to test my central hypothesis, I will pursue the following three specific aims: 1. Identify the receptor signaling pathways modulated by clopidogrel and DT678. My working hypothesis is that clopidogrel inhibits P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 as well as P2Y12 in stably transfected cells. Additionally, I hypothesize that DT-678 only inhibits the P2Y12 receptor without having effects on other purinergic receptors. 2. Determine the effect of specific purinergic agonists in the middle cerebral artery from clopidogrel- and DT678-treated rabbits. My working hypothesis is that clopidogrel metabolites interact with P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 in the MCA to impair MT generation and dilation. 3. Evaluate the effects of clopidogrel and DT678 in the parenchymal arterioles of P2Y12-/- mice. My working hypothesis that clopidogrel modulates P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 in the vasculature to inhibit myogenic tone, constriction and dilation in P2Y12-/- mice. The information obtained from this project will allow for development of new antiplatelet therapies that have improved efficacy with a decreased risk of intracerebral hemorrhage.