Akt-regulated pathways in platelet function

Akt 调节血小板功能的途径

• 批准号: 7340485
• 负责人: DONNA S WOULFE
• 金额: $ 31万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-12 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340485
• 关键词: Actins; Agonist; Arrestin; Arrestins; Binding; Biological Assay; Blood Platelets; Cell Survival; Cell physiology; Cells; Clot retraction; Complex; Cytoplasmic Granules; Data; Defect; Enzymes; Excision; Family; Family member; Fibrinogen; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Human; Immunoprecipitation; In Vitro; Integrins; Knockout Mice; Mammals; Metabolism; Mouse Strains; Mus; Pathway interactions; Phosphorylation; Phosphotransferases; Platelet Activation; Platelet aggregation; Play; Protein-Serine-Threonine Kinases; Rate; Receptor Signaling; Recruitment Activity; Regulation; Research Personnel; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Tail; Testing; Thrombosis; actin 2; arrestin 2; glucose metabolism; in vivo; knock-down; receptor; receptor coupling; rho; scaffold; tau Proteins

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.

该提案的目标是更好地定义Akt在血小板信号传导和血栓形成中的作用。述Akt 激酶是丝氨酸-苏氨酸激酶，其在细胞存活、增殖和分化中具有已充分描述的作用。 新陈代谢.我们和其他人已经表明Akt激酶在血小板功能中也起重要作用。 具体来说，我们以前的研究表明，Akt 2在促进纤维蛋白原结合和致密化中起作用。 颗粒分泌，Akt 2敲除小鼠对动脉血栓形成有抗性。但 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GSKS β和整合素由外向内的信号通路， 动脉血栓形成我们将在以下3个具体目标中检验这一假设，重点是 阐明血小板中Akt活化的上游和下游信号传导途径。目的1是阐明 Akt被G蛋白偶联受体激活的机制，重点是抑制蛋白-2的能力， 作为PIS激酶亚基和GPCR的支架。我们的初步研究表明， 人血小板中p85-PI 3 K亚单位的激动剂依赖性复合物。通过抑制arrestin表达 在巨核细胞和研究arrestin-2-/-小鼠，我们建议确定arrestin的成分， 复合物，以确定血小板中Akt的活化是否依赖于抑制蛋白，并确定血小板中Akt的表达。 抑制蛋白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。GSKS β是一种丝氨酸/苏氨酸激酶，经常抑制细胞功能 由Akt正向调节的细胞我们的假设是GSKS β抑制血小板功能， GSKS β的去除或抑制会增强血小板聚集或血栓形成。我们 初步数据表明情况确实如此。目的3是通过以下方式来定义Akt对由外向内信号传导的影响： 整联蛋白β 1-β 5。我们的初步数据表明，血栓收缩和扩散的速度 纤维蛋白原依赖于血小板中的Akt 2。由于这些功能依赖于β-淀粉样蛋白，我们 将通过研究β-淀粉样蛋白的磷酸化来确定Akt如何调节β-淀粉样蛋白信号传导， 肌动蛋白装配，以及缺乏Akt或表达活化Akt的血小板中rho家族成员的活化。