Akt-regulated pathways in platelet function

描述(由申请人提供)：本提案的目标是更好地确定Akt在血小板信号转导和血栓形成中的作用。Akt激酶是一种丝氨酸-苏氨酸激酶，在细胞存活、增殖和新陈代谢中具有广泛的作用。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颗粒分泌方面发挥作用，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。这一提议将验证G蛋白偶联受体(GPCRs)激活Akt支持血小板中特定的信号通路的假设，包括调节GSKSbeta和整合素由外向内的信号通路，从而促进动脉血栓形成。我们将在以下三个特定目标中验证这一假设，这三个目标集中在阐明血小板Akt激活的上游和下游的信号通路。目的1阐明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支架的能力。我们的初步研究表明，在人类血小板中，阻滞素与P85-PI3K亚基形成激动剂依赖的复合体。通过抑制巨核细胞中arrestin的表达和对arrestin-2-/-小鼠的研究，我们建议确定arrestin复合体的组成，确定血小板中Akt的激活是否依赖于拦阻蛋白，并确定拦阻蛋白在血小板激活中的作用。目的2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正向调节的细胞功能。我们的假设是，GSKSbeta具有抑制血小板功能的作用，移除或抑制GSKSbeta应该会增强血小板聚集或血栓形成。我们的初步数据表明，情况就是这样。目的3是通过整合素Alphallb-Betas确定Akt对外向内信号转导的影响。我们的初步数据表明，凝块回缩和在纤维蛋白原上扩散的速度取决于血小板中的Akt2。由于这些功能依赖于Alphallb-Betas，我们将通过研究Betas尾部的磷酸化、肌动蛋白组装以及缺乏Akt或表达激活Akt的血小板中Rho家族成员的激活来确定Akt是如何调节Alphallb-Betas信号的。

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.