知母皂苷AⅢ抑制血小板致密颗粒分泌的作用机制研究

Effective hemostasis and pathological arterial thrombosis rely upon the rapid development of a platelet aggregate at the site of blood vessel damage. Secretion of ADP from platelet dense granules forms a major positive feedback loop that is essential for thrombus growth and stability. The goal of anti-thrombotic therapies is a regimen that dampens thrombosis without inducing excessive bleeding. Inhibiting dense granule secretion in platelets has proved an effective target to accomplish this goal. In our previous study, we found Timosaponin AIII inhibits platelet aggregation and exert antithrombosis activity by diminishing TxA2 receptor-mediated, Gq/PLCβ/PKC-dependent dense granule secretion, but not the activation of G12/13 signaling pathway. So, in this project, the effect of Timosaponin AIII on the number and contents of dense granule in platelets and the vacuolar ATPase-mediated retention of quinacrine will be observed using many methods. Furthermore, we would explore the pathway involved in inhibiting dense granule secretion in platelets through observing the changes in [Ca2+]I and phosphorylation of PKC and its substrates. We will observed the effect of Timosaponin AIII on the activation of TXA2 receptor and the interaction between TXA2 receptor and its signaling proteins in the cytoplasm using many methods as well as TXA2 receptor knockout mice. Above all, we would find the binding protein of Timosaponin AIII which involved in inhibiting dense granule secretion in platelets.

致密颗粒分泌是血小板聚集正反馈调节的主要环节，参与体内止血过程；而抑制致密颗粒分泌已成为抗血小板药物研发的新靶标。前期工作发现，知母皂苷AⅢ可选择性地抑制TXA2受体激活引起的血小板致密颗粒分泌。本课题拟采用血小板静态观察与激活抑制相结合、信号转导通路追踪与靶点验证相呼应、传统药理学方法检测与模式动物研究相印证的研究策略，从血小板致密颗粒结构与功能改变、调节致密颗粒分泌的细胞内钙动员和PKC激活信号通路的抑制状况以及TXA2受体激活及其与下游信号分子相互作用的变化等多个层面揭示知母皂苷AⅢ抑制血小板致密颗粒分泌的关键信号分子或作用靶点。该研究对认识主含甾体皂苷的中成药治疗心、脑血管疾病的作用机理以及基于新靶点的创新药物研发均具有重要的理论意义和实用价值。

致密颗粒分泌是血小板聚集正反馈调节的主要环节，参与体内止血过程。抑制致密颗粒分泌已成为抗血小板药物研发的新靶标。前期工作发现，知母皂苷AⅢ可选择性地抑制TXA2受体激活引起的血小板致密颗粒分泌。为进一步研究知母皂苷AⅢ抗血小板活性是否直接与抑制TXA2受体功能相关，本课题应用TXA2受体基因敲除小鼠和野生型小鼠进行确证研究。与野生型小鼠比较，在TXA2受体基因敲除小鼠血小板，TXA2受体模拟剂U46619不能引起血小板变形、分泌和聚集，而AIII在敲除小鼠和野生型小鼠的血小板上均能引起变形。同时，具有诱导活性的甾体皂苷化合物Tg在TXA2受体基因敲除小鼠血小板上的激活性质也未发生改变。这些结果提示AⅢ不是直接抑制TXA2受体激活而抑制致密颗粒释放。在研究中本课题进一步发现了一个新的三萜类化合物viburnumtol能激活人和大鼠血小板。研究发现viburnumtol能剂量依赖地诱导大鼠血小板聚集及释放； viburnumtol诱导的血小板聚集依赖ADP释放及其介导的P2Y12受体激活，而不依赖血小板内TXA2生成；viburnumtol激活血小板与PLC-β/DAG/PKC信号通路相关。viburnumtol能与U46619、胶原、凝血酶短肽等诱导剂协同诱导血小板聚集。以上研究为揭示知母皂苷AⅢ抑制血小板致密颗粒分泌的关键信号分子或作用靶点缩小了研究范围；viburnumtol血小板活性的发现和机制研究也为研发新的止血剂奠定了实验基础。