Regulation of the early events of platelet activation

血小板活化早期事件的调节

• 批准号: 7888575
• 负责人: LAWRENCE F BRASS
• 金额: $ 59.42万
• 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/7888575
• 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; Knowledge; Learning; Left; Link; Long-Term Effects; Modeling; Morbidity - disease rate; Mus; Mutation; PTPN6 gene; Phosphorylation Site; Pilot Projects; Platelet Activation; Protein Dephosphorylation; 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; human PTPN6 protein; 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节Lawrence F.黄铜，MD PhD血小板是正常止血所必需的，但也可能导致血栓形成和血管壁常见疾病（包括动脉硬化）的演变和后果。虽然对血小板活化有很多了解，但对血小板内调节反应性的事件知之甚少，这些事件在不需要时限制血小板活化，并确保对损伤的反应停止出血而不引起血管闭塞。该建议的重点是血小板活化的事件，发生在激动剂，如凝血酶，ADP和TxA 2，所有的受体偶联到异源三聚体G蛋白的下游。我们的假设是G蛋白依赖性事件的失调是促血栓形成的，并可能导致血管疾病的进展。将在使用人血小板和选定小鼠模型的研究中检验这一假设。在我们的初步研究中，我们已经确定了一个以前未描述的调节复合物在静息血小板中，其中至少有两个RGS（G蛋白信号调节）蛋白和酪氨酸磷酸酶，SHP-1，结合到130 kDa的支架蛋白，spinophilin（SPL），这在静息血小板是酪氨酸磷酸化。血小板活化导致SHP-1依赖性的亲棘素去磷酸化和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，我们提出，亲棘素首先螯合RGS蛋白，允许信号开始，然后释放它们，以限制信号的幅度和持续时间。对该模型的支持来自我们对SPL（-/-）小鼠和表达Gi 2a（G184 S）的小鼠的研究，Gi 2a是一种突变，其使得G蛋白的a亚基Gi 2抵抗RGS蛋白的失活。这些研究表明，1）亲棘素的损失损害血小板对激动剂的反应，2）该缺陷限于可引起SPL/RGS/SHP-1复合物解离的激动剂，和3）如模型所预测的，阻断G12上的RGS依赖性负反馈在体外和体内产生血小板功能的增益。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活化过程中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将测试我们的假设，SPL/RGS/SHP-1复合物的衰减提供了一个定时制动G蛋白信号转导，并确定所涉及的机制。最后，目标3将集中于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和避免血小板反应性慢性增加的不良后果中的作用。 公共卫生相关性：在错误的时间和地点发生的血小板活化仍然是美国心血管和脑血管疾病发病的主要原因。在拟议的研究中，我们将研究我们认为有助于调节血小板对损伤的反应程度的新机制，确保反应既不不足，也不过于强烈，以至于危及周围组织的健康。更好地了解血小板生物学的这一关键但未充分探索的方面是了解和预防人们心脏病和中风时出现问题的关键。