Akt-regulated pathways in platelet function

这项建议的目的是更好地确定Akt在血小板信号转导和血栓形成中的作用。阿克特人 激酶是丝氨酸-苏氨酸激酶，在细胞存活、增殖和 新陈代谢。我们和其他人已经证明Akt激酶在血小板功能中也扮演着重要的角色。 具体地说，我们之前的研究表明Akt2在促进纤维蛋白原结合和致密方面发挥作用 颗粒分泌物和Akt2基因敲除小鼠对动脉血栓具有抵抗力。然而， Akt调节血小板功能的机制尚不清楚。这项提案将考验 G蛋白偶联受体(GPCRs)激活Akt支持特定信号通路的假说 在血小板中，包括调节GSKSβ和整合素由外向内的信号通路，这对 动脉血栓形成。我们将在以下三个具体目标中测试这一假设，这三个目标集中在 阐明血小板Akt激活的上下游信号通路。目标1是澄清 G蛋白偶联受体激活Akt的机制，重点是Arrestin-2 作为PIS激酶亚基和GPCRs的支架。我们的初步研究表明，逮捕形成了 人血小板中P85-PI3K亚单位的激动剂依赖复合体。通过抑制arrestin的表达 在巨核细胞和研究arrestin-2-/-小鼠中，我们建议确定arrestin的成分 以确定血小板中Akt的激活是否依赖于拦阻蛋白，并确定 抑制素在血小板活化中的作用。目的2是确定Akt底物GSKSbetain的作用 血小板功能和血栓形成。GSKSbeta是一种丝氨酸/苏氨酸激酶，经常抑制细胞功能 受到Akt的积极监管。我们的假设是GSKSbeta作用于抑制血小板功能，并且 GSKSbeta的去除或抑制应增强血小板聚集或血栓形成。我们的 初步数据显示情况的确如此。目标3是通过以下方式定义Akt对自外向内信号的影响 整合素字母-Betas。我们的初步数据显示，血栓回缩和扩散的速度 纤维蛋白原依赖于血小板中的Akt2。由于这些函数依赖于字母-Beta，因此我们 将通过研究BetaSail的磷酸化来确定Akt是如何调节Alphallb-Betas信号的， 在缺乏Akt或表达激活Akt的血小板中，肌动蛋白组装和Rho家族成员的激活。

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 GSKSbetaand 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 scaffoldfor 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, GSKSbetain platelet function and thrombosis. GSKSbetais 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 betaStail, actin assembly, and activation of rho family members in platelets lacking Akt or expressing activated Akt.