Regulation of the early events of platelet activation

血小板活化早期事件的调节

• 批准号: 8065935
• 负责人: LAWRENCE F BRASS
• 金额: $ 58.49万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-26 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8065935
• 关键词: ADP Receptors; Acute; Affect; Agonist; Arteriosclerosis; Binding; Biology; Blood Platelets; Blood Vessels; Cardiovascular system; Cells; Chinese Hamster Ovary Cell; Chronic; Collagen; Complex; Coupled; Cyclic AMP; Defect; Development; Disease; Disease Progression; Dissociation; Doctor of Philosophy; Drug usage; Endothelium; Ensure; Epoprostenol; Event; Evolution; Feedback; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Proteins; Goals; Health; Heart; Hemorrhage; Hemostatic Agents; Hemostatic function; Heterotrimeric GTP-Binding Proteins; Human; In Vitro; Knock-out; Learning; Left; Link; Long-Term Effects; Modeling; Morbidity - disease rate; Mus; Mutation; PTPN6 gene; Phosphorylation Site; Pilot Projects; Platelet Activation; Protein Dephosphorylation; Protein Tyrosine Phosphatase; RGS Proteins; Regulation; Resistance; Rest; Role; Scaffolding Protein; Signal Transduction; Stroke; Testing; Thrombin; Thrombosis; Time; Tissues; Tyrosine; United States; Vascular Diseases; Work; adverse outcome; base; brass; cerebrovascular; gain of function; in vivo; insight; interest; loss of function; member; mouse development; mouse model; mutant; novel; prevent; public health relevance; receptor coupling; response; response to injury; spinophilin

DESCRIPTION (provided by applicant): Regulation of the early events of platelet activation Lawrence F. Brass, MD PhD Platelets are essential for normal hemostasis, but can also contribute to thrombosis and to the evolution and consequences of common diseases of the vessel wall including arteriosclerosis. Although a great deal is known about platelet activation, much less is known about the events within platelets that modulate responsiveness, limiting platelet activation when it is not needed and ensuring that the response to injury halts bleeding without causing vascular occlusion. The focus of this proposal is on the events of platelet activation that occur immediately downstream of agonists such as thrombin, ADP and TxA2, all of whose receptors are coupled to heterotrimeric G proteins. Our hypothesis is that dysregulation of G protein dependent events is prothrombotic and potentially contributes to vascular disease progression. This hypothesis will be tested in studies with human platelets and selected mouse models. In our preliminary studies, we have identified a previously-undescribed regulatory complex in resting platelets in which at least two RGS (regulators of G protein signaling) proteins and the tyrosine phosphatase, SHP-1, are bound to the 130 kDa scaffold protein, spinophilin (SPL), which in resting platelets is tyrosine phosphorylated. Platelet activation causes SHP-1- dependent dephosphorylation of spinophilin and agonist-selective dissociation of the SPL/RGS/SHP-1 complex. These events can be recapitulated in transfected CHO cells. Based on these observations, we propose that spinophilin first sequesters RGS proteins, allowing signaling to begin, and then releases them in order to limit signaling magnitude and duration. Support for this model is drawn from our studies on SPL(-/-) mice and mice expressing Gi2a(G184S), a mutation that renders the a subunit of the G protein, Gi2, resistant to inactivation by RGS proteins. Those studies show that 1) loss of spinophilin impairs platelet responses to agonists, 2) this defect is limited to agonists that can cause dissociation of the SPL/RGS/SHP-1 complex, and 3) blocking RGS-dependent negative feedback on Gi2 produces, as the model would predict, a gain of platelet function in vitro and in vivo. The proposed studies are divided into three specific aims. Aim 1 will test our hypothesis that RGS proteins help to regulate platelet responsiveness by limiting the duration of G protein signaling during platelet activation. Aim 2 will test our hypothesis that the decay of the SPL/RGS/SHP-1 complex provides a timed brake on G protein signaling and identify the mechanisms involved. Finally, Aim 3 will focus on the role of RGS proteins and the SPL/RGS/SHP-1 complex in regulating the conversion of adherent platelets to a fully activated state and in avoiding the adverse consequences of a chronic increase in platelet reactivity. PUBLIC HEALTH RELEVANCE: Platelet activation occurring at the wrong place and at the wrong time remains a major cause of cardiovascular and cerebrovascular morbidity in the United States. In the proposed studies we will examine novel mechanisms that we believe helps to regulate the extent of platelet responses to injury, ensuring that the response is neither inadequate nor so robust that the health of the surrounding tissues is endangered. A better understanding of this critical, but under-explored aspect of platelet biology is key to understanding and preventing what goes wrong when people have heart and strokes.

描述（由申请人提供）：血小板活化早期事件的调控血小板对正常止血至关重要，但也可能导致血栓形成以及包括动脉硬化在内的血管壁常见疾病的演变和后果。虽然我们对血小板活化了解很多，但对血小板内调节反应性、在不需要时限制血小板活化并确保对损伤的反应在不引起血管闭塞的情况下止血的事件知之甚少。本提案的重点是发生在激动剂（如凝血酶、ADP和TxA2）下游的血小板激活事件，所有这些受体都与异源三聚体G蛋白偶联。我们的假设是G蛋白依赖事件的失调是血栓形成的前兆，并可能导致血管疾病的进展。这一假设将在人类血小板和选定小鼠模型的研究中得到验证。在我们的初步研究中，我们在静息血小板中发现了一种以前未描述的调节复合物，其中至少有两种RGS （G蛋白信号的调节因子）蛋白和酪氨酸磷酸酶SHP-1结合到130 kDa的支架蛋白-亲脊髓蛋白（SPL）上，该蛋白在静息血小板中被酪氨酸磷酸化。血小板活化导致嗜脊髓蛋白依赖于SHP-1的去磷酸化和SPL/RGS/SHP-1复合物的激动剂选择性解离。这些事件可以在转染的CHO细胞中重现。基于这些观察结果，我们提出嗜脊髓蛋白首先隔离RGS蛋白，允许信号开始，然后释放它们以限制信号的强度和持续时间。我们对SPL（-/-）小鼠和表达Gi2a（G184S）的小鼠的研究支持了这一模型，Gi2a（G184S）是一种突变，可以使G蛋白的a亚基Gi2抵抗RGS蛋白的失活。这些研究表明，1)嗜脊髓蛋白的缺失会损害血小板对激动剂的反应，2)这种缺陷仅限于可以导致SPL/RGS/SHP-1复合物解离的激动剂，以及3)阻断RGS依赖的Gi2负反馈，正如该模型所预测的那样，在体外和体内产生血小板功能的增强。拟议的研究分为三个具体目标。目的1将验证我们的假设，即RGS蛋白通过限制血小板激活过程中G蛋白信号传导的持续时间来帮助调节血小板反应性。目的2将验证我们的假设，即SPL/RGS/SHP-1复合物的衰变对G蛋白信号传导提供了定时制动，并确定了相关机制。最后，Aim 3将重点关注RGS蛋白和SPL/RGS/SHP-1复合物在调节粘附血小板向完全活化状态的转化以及避免血小板反应性慢性增加的不良后果中的作用。