Akt-regulated pathways in platelet function

描述（由申请人提供）：本提案的目的是更好地定义Akt在血小板信号传导和血栓形成中的作用。Akt激酶是丝氨酸-苏氨酸激酶，在细胞存活、增殖和代谢中发挥着重要作用。我们和其他人已经证明Akt激酶在血小板功能中也起重要作用。具体而言，我们之前的研究表明Akt2在促进纤维蛋白原结合和致密颗粒分泌中发挥作用，并且Akt2敲除小鼠对动脉血栓形成具有抗性。然而，Akt调控血小板功能的机制尚不清楚。这一提议将验证G蛋白偶联受体（gpcr）激活Akt支持血小板中特定信号通路的假设，包括GSKSbeta和整合素外向内信号通路的调节，这些信号通路有助于动脉血栓形成。我们将在以下3个具体目标中验证这一假设，重点是阐明血小板中Akt激活的上游和下游信号通路。目的1是阐明G蛋白偶联受体激活Akt的机制，重点研究阻滞蛋白2作为PIS激酶亚基和gpcr支架的能力。我们的初步研究表明，在人血小板中，抑制因子与p85-PI3K亚基形成激动剂依赖复合物。通过抑制巨核细胞中阻滞蛋白的表达和对阻滞蛋白2-/-小鼠的研究，我们拟确定阻滞蛋白复合物的组分，确定Akt在血小板中的活化是否依赖于阻滞蛋白，并确定阻滞蛋白在血小板活化中的作用。目的2是确定Akt底物GSKSbeta在血小板功能和血栓形成中的作用。GSKSbeta是一种丝氨酸/苏氨酸激酶，经常抑制Akt正调控的细胞功能。我们的假设是GSKSbeta抑制血小板功能，去除或抑制GSKSbeta会增强血小板聚集或血栓形成。我们的初步数据表明情况确实如此。目的3是定义Akt通过整合素α - β - as对outside-in信号传导的影响。我们的初步数据表明，凝块在纤维蛋白原上的收缩和扩散速率依赖于血小板中的Akt2。由于这些功能依赖于α - β - as，我们将通过研究β - β - as尾部的磷酸化、肌动蛋白组装和缺乏Akt或表达活化Akt的血小板中rho家族成员的激活来确定Akt如何调节α - β - β信号。

DESCRIPTION (provided by applicant): The goal of this proposal is to better define the roles of Akt in platelet signaling and thrombosis. The Akt kinases are serine-threonine kinases that have well-described roles in cell survival, proliferation, and metabolism. We and others have shown that Akt kinases also play important roles in platelet function. Specifically, our previous studies show that Akt2 plays a role in promoting fibrinogen binding and dense granule secretion and that Akt2 knockout mice are resistant to arterial thrombosis. However, the mechanisms by which Akt regulates platelet function are not understood. This proposal will test the hypothesis that Akt activation by G protein-coupled receptors (GPCRs) supports specific signaling pathways in platelets, including the regulation of GSKSbeta and integrin outside-in signaling pathways, that contribute to arterial thrombosis. We will test this hypothesis in the following 3 Specific Aims, which focus on elucidating signaling pathways upstream and downstream of Akt activation in platelets. Aim 1 is to elucidate the mechanisms of Akt activation by G protein-coupled receptors, focusing on the ability of arrestin-2 to serve as a scaffold for PIS kinase subunits and GPCRs. Our preliminary studies show that arrestins form agonist-dependent complexes with p85-PI3K subunits in human platelets. By inhibiting arrestin expression in megakaryocytic cells and studying arrestin-2-/- mice, we propose to determine the components of arrestin complexes, to establish whether Akt activation in platelets is dependent on arrestins, and to determine the role of arrestins in platelet activation. Aim 2 is to determine the role of the Akt substrate, GSKSbeta in platelet function and thrombosis. GSKSbeta is a ser/thr kinase that frequently suppresses cellular functions that are positively regulated by Akt. Our hypothesis is that GSKSbeta acts to suppress platelet function, and that the removal or inhibition of GSKSbeta should enhance platelet aggregation or thrombosis. Our preliminary data suggest that this is the case. Aim 3 is to define the impact of Akt on outside-in signaling by integrin alphallb-betaS. Our preliminary data indicate that the rate of clot retraction and spreading on fibrinogen are dependent on Akt2 in platelets. Since these functions are dependent on alphallb-betaS, we will seek to define how Akt regulates alphallb-betaS signaling by studying phosphorylation of the betaS tail, actin assembly, and activation of rho family members in platelets lacking Akt or expressing activated Akt.