Functional G Protein pathways in platelet activation

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， 纤维蛋白原受体活化、致密颗粒释放和血栓素A2（TXA 2）生成，导致 激活其他血小板。调节这些血小板生理事件的机制尚未被阐明。 完全理解所有的血小板激动剂，无论是直接还是间接，都依赖于G蛋白途径， 引起血小板活化。我们建议进一步了解G蛋白中的下游事件 途径和它们的第二信使在血小板活化中的作用， 药理学和基因敲除方法。ADP和凝血酶的激活能力不同 血小板ADP不能引起阿司匹林处理的血小板释放致密颗粒，而凝血酶可以。ADP 依赖于整联蛋白信号传导来激活磷脂酶A2，而凝血酶则不依赖。而两 这些激动剂激活Gq-磷脂酶C途径，只有凝血酶刺激G12/13途径。这 资助申请是建立在我们最近的研究证明了一个重要的作用）G12/13途径， 血小板纤维蛋白原受体活化，B）血栓烷生成中的蛋白激酶C δ同种型，和c）Gi 血小板Akt磷酸化途径。我们将检验G12/13通路有助于 致密颗粒释放、TXA 2生成以及Akt磷酸化和激活，使用药理学方法 方法补充了来自Galpha 12和Galpha 13基因敲除小鼠的血小板， 组成型活性Galpha 12和Galpha 13转基因小鼠。我们将评估以下方面的相对贡献： Gq/PLC途径和G12/13途径对激动剂诱导的致密颗粒释放、TXA 2产生和 Akt磷酸化。我们还假设HAX-1、HS-1和Src家族激酶被激活， G12/13通路的下游，其在血小板活化中起重要作用。我们将描绘一些 这些信号分子下游的G12/13途径，因为这一途径是最不了解， 血小板最后，我们将评估HS 1在血小板中的功能作用，使用体外HS 1缺陷的小鼠 血小板功能研究和体内血栓形成模型。我们有强有力的初步数据支持每一个 上述具体目标。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，并可能确定治疗血栓形成的潜在新靶点。

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.