Functional G Protein pathways in platelet activation

描述（由申请人提供）：血小板活化在止血和血栓形成中起主要作用。血小板激动剂引起形状改变、纤维蛋白原受体激活、致密颗粒释放和血栓素A2 （TXA2）的产生，导致其他血小板的激活。调控这些血小板生理事件的机制尚不完全清楚。所有的血小板激动剂，直接或间接地依赖于G蛋白途径引起血小板活化。我们建议使用互补的生化、药理学和基因敲除方法进一步了解G蛋白途径及其第二信使在血小板活化中的下游事件。ADP和凝血酶活化血小板的能力不同。ADP不能在阿司匹林处理过的血小板中引起致密颗粒释放，而凝血酶可以。ADP依赖于整合素信号来激活磷脂酶A2，而凝血酶则不是。虽然这两种激动剂都激活gq -磷脂酶C途径，但只有凝血酶刺激G12/13途径。这项资助申请是基于我们最近的研究，证明了a) G12/13通路在血小板纤维蛋白原受体激活中的重要作用，b)蛋白激酶C δ异构体在血栓素生成中的重要作用，以及C) Gi通路在血小板中Akt磷酸化中的重要作用。我们将采用药理学方法，结合来自Galpha12和Galpha13基因敲除小鼠以及组成型活性Galpha12和Galpha13转基因小鼠的血小板，验证G12/13通路有助于致密颗粒释放、TXA2生成和Akt磷酸化和激活的假设。我们将评估Gq/PLC通路和G12/13通路对激动剂诱导的致密颗粒释放、TXA2生成和Akt磷酸化的相对贡献。我们还假设HAX-1、HS-1和Src家族激酶在G12/13通路下游被激活，而G12/13通路在血小板活化中起重要作用。我们将描述G12/13通路下游的一些信号分子，因为这一通路在血小板中是最不为人所知的。最后，我们将在体外血小板功能研究和体内血栓形成模型中使用HS1缺陷小鼠来评估HS1在血小板中的功能作用。我们有强有力的初步数据支持上述每一个具体目标。这些研究将增强我们对信号通路及其在血小板活化中的作用的理解，并可能确定治疗血栓形成的潜在新靶点。

DESCRIPTION (provided by applicant): Platelet activation plays a major role in hemostasis and thrombosis. Platelet agonists cause shape change, fibrinogen receptor activation, dense granule release, and thromboxane A2 (TXA2) generation, leading to the activation of other platelets. The mechanisms regulating these platelet physiological events have not been completely understood. All the platelet agonists, either directly or indirectly, depend on G protein pathways to cause platelet activation. We propose to further understand the downstream events in the G protein pathways and their second messengers in platelet activation using complementary biochemical, pharmacological, and gene knockout approaches. ADP and thrombin differ in their ability to activate platelets. ADP fails to cause dense granule release in aspirin-treated platelets, whereas thrombin can. ADP depends on integrin signaling to activate phospholipase A2, whereas thrombin does not. Whereas both these agonists activate Gq-phospholipase C pathways, only thrombin stimulates G12/13 pathways. This grant application is built upon our recent studies demonstrating important roles for a) G12/13 pathways in platelet fibrinogen receptor activation, b) protein kinase C delta isoform in thromboxane generation, and c) Gi pathways in Akt phosphorylation in platelets. We will test the hypothesis that G12/13 pathways contribute to dense granule release, TXA2 generation, and Akt phosphorylation and activation, using pharmacological approaches complemented with platelets from Galpha12 and Galpha13 gene knockout mice and constitutively active Galpha12 and Galpha13 transgenic mice. We will evaluate the relative contributions of Gq/PLC pathways and G12/13 pathways to agonist-induced dense granule release, TXA2 generation, and Akt phosphorylation. We also hypothesize that HAX-1, HS-1, and Src family kinases are activated downstream of G12/13 pathways, which play an important role in platelet activation. We will delineate some of these signaling molecules downstream of the G12/13 pathways, as this pathway is the least understood in platelets. Finally, we will evaluate the functional role of HS1 in platelets using mice-deficient in HS1 in ex vivo platelet functional studies and in vivo thrombosis models. We have strong preliminary data supporting each of the above specific aims. These studies will enhance our understanding of the signaling pathways and their role in platelet activation, and might identify potential newer targets for the treatment of thrombosis.