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。血小板激动剂会导致形状改变， 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。调节这些血小板生理事件的机制尚未得到证实。 完全理解。所有的血小板激动剂，无论是直接的还是间接的，都依赖于G蛋白途径 导致血小板活化。我们建议进一步了解G蛋白中的下游事件 利用互补生化激活血小板的途径及其第二信使， 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不能导致阿司匹林治疗的血小板释放致密颗粒，而凝血酶可以。ADP 依赖于整合素信号来激活磷脂酶A2，而凝血酶不能。鉴于两者 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 赠款申请是基于我们最近的研究，证明了a)G12/13途径在 血小板纤维蛋白原受体激活，b)血栓素生成中的蛋白激酶Cβ亚型，以及c)Gi 血小板Akt磷酸化的途径。我们将检验G12/13通路有助于 致密颗粒释放，TXA2产生，Akt磷酸化和激活，使用药理学 用Galpha12和Galpha13基因敲除小鼠的血小板补充的方法 结构活跃的Galpha12和Galpha13转基因小鼠。我们将评估以下各项的相对贡献 激动剂诱导致密颗粒释放、TXA2产生的GQ/PLC通路和G12/13通路 AKT磷酸化。我们还假设HAX-1、HS-1和Src家族的激酶被激活 G12/13途径下游，在血小板活化中起重要作用。我们将描述一些 在G12/13通路下游的这些信号分子中，因为这一通路是目前最不了解的 血小板。最后，我们将在体外利用HS1基因缺陷的小鼠评估HS1在血小板中的功能作用 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。这些研究将加深我们对信号通路及其相互作用的理解 在血小板活化中的作用，并可能确定潜在的治疗血栓形成的新靶点。

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.