血小板p66Shc/NO/VASP-P信号通路介导糖尿病患者氯吡格雷抵抗的作用及机制

Clopidogrel plays a pivotal role in the treatment of patients with cardiovascular disease. Clopidogrel resistance increases the risk of major adverse cardiovascular events (MACE) in patients. However, Most research based on pharmacokinetics can only partially explain the variability in clopidogrel pharmacodynamics. Recent research demonstrated that p66Shc expressed in platelets and our previous study found that platelet p66Shc unregulated in diabetic patients, which is associated with the VASP-P, the target molecular of clopidogrel. These findings support the suggestion that p66Shc mediates the platelet aggregation in diabetic patients. In this grant, we aim to investigate the role of p66Shc in high platelet aggregation and the mechanism by which the ROS/NO/VASP-P signal pathway is regulated under hyperglycemic conditions by in vivo using siRNA transgenic mice, and in vitro using murine and diabetic patients platelets at all levels, from molecular, cellular and patients standpoints. Furthermore， Our hypothesis is that hyperglycemic conditions stimulate p66Shc expression in platelets, decreasing the inhibition of VASP-P of clopidogrel, leading to the hyperreactivity and hyperaggregation of clopidogrel. Our study is expected to provide novel insights into platelet aggregation and clopidogrel resistance in diabetic patients, in order to provide the basis for innovative intervention strategies in clinical practice and provide a target for drug therapy.

氯吡格雷是心脑血管疾病防治的基石，氯吡格雷抵抗显著增加患者心血管事件风险，然而目前针对氯吡格雷药物代谢的研究仅能部分解释氯吡格雷抵抗的机制。最新研究发现，血小板氧化应激蛋白p66Shc参与血小板聚集的调控。申请者预实验发现：糖尿病患者血小板p66Shc表达水平上调，且与氯吡格雷靶分子VASP的磷酸化相关，提示血小板p66Shc可能通过调控VASP介导高糖诱导的血小板聚集。基于以上研究基础，本项目拟从临床、整体动物、细胞和分子水平，综合应用siRNA敲减动物模型、流式细胞术、激光共聚焦显微镜等方法，阐明血小板p66Shc通过调控ROS/NO/VASP-P信号通路介导血小板聚集的分子机制；并进一步探讨血小板p66Shc通过抑制VASP的磷酸化，削弱氯吡格雷的抗血小板作用，从而导致氯吡格雷抵抗的作用及机制。本项目有助于深入阐明糖尿病患者氯吡格雷抵抗的分子机制，为临床干预策略创新提供理论依据。

氯吡格雷抗血小板治疗是冠心病（Coronary heart disease，CHD）患者急性致死性血栓栓塞的主要治疗策略，然而约30%的患者会出现氯吡格雷抵抗（Clopidogrel resistence，CR），2型糖尿病（T2DM）合并症是CR的重要危险因素，探索CR的机制将为CHD合并T2DM患者的抗血小板治疗提供理论依据和新靶点。.本项目从临床样本和细胞水平两个方面出发，对氯吡格雷抵抗的作用和机制进行了探究。临床样本的研究结果证实合并T2DM的CHD患者，其基础血小板聚集率及服用氯吡格雷后发生氯吡格雷抵抗和低反应的发生率相比较于未合并T2DM的CHD患者更高，并显著增加了再发MACE的风险。CHD+T2DM+CR组患者与CHD-T2DM-CR组相比，其血浆和血小板内的NO、NOS水平显著偏低，血小板p66Shc的表达水平显著偏高，因此证实了CHD合并T2DM患者的p66Shc/NO信号通路水平与血小板聚集及心血管不良事件之间的相关性。细胞水平的研究发现高糖条件与p66Shc蛋白水平的相关性即高糖可显著提升p66Shc蛋白水平；在敲低实验中，发现p66Shc可显著上调NO、NOS水平，氯吡格雷在p66Shc敲低条件下，可改变细胞ROS水平；此外在高糖条件下，氯吡格雷可显著上调VASP磷酸化水平；最后，课题组发现p66Shc可在氯吡格雷存在情况下调控VASP磷酸化水平，从而证实了p66Shc/NO/VASP-P 信号通路通过调控与氯吡格雷共同的靶分子VASP磷酸化介导了糖尿病患者氯吡格雷抵抗的作用及分子机制。.本项目从全新的角度探索的氯吡格雷抵抗的新机制，将为氯吡格雷临床用药选择提供了新的科学依据。.