Regulation of the early events of platelet activation

血小板活化早期事件的调节

基本信息

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
项目类别：
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

项目摘要

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. Brass，医学博士博士血小板对于正常止血至关重要，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。尽管人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以及确保对损伤的反应停止出血而不引起血管闭塞的事件知之甚少。该提案的重点是紧邻凝血酶、ADP 和 TxA2 等激动剂下游发生的血小板活化事件，所有这些受体均与异三聚体 G 蛋白偶联。我们的假设是，G 蛋白依赖性事件的失调会导致血栓形成，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的小鼠模型的研究中得到检验。在我们的初步研究中，我们在静息血小板中发现了一种先前未描述的调节复合物，其中至少有两种 RGS（G 蛋白信号传导调节剂）蛋白和酪氨酸磷酸酶 SHP-1 与 130 kDa 支架蛋白亲旋蛋白结合(SPL)，在静息血小板中被酪氨酸磷酸化。血小板激活导致 SHP-1 依赖性亲旋蛋白去磷酸化和 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，spinopilin 首先隔离 RGS 蛋白，允许信号传导开始，然后释放它们以限制信号传导的强度和持续时间。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，使 G 蛋白的亚基 Gi2 能够抵抗 RGS 蛋白的失活。这些研究表明 1) 亲旋蛋白的缺失会损害血小板对激动剂的反应，2) 这种缺陷仅限于可导致 SPL/RGS/SHP-1 复合物解离的激动剂，以及 3) 阻断 Gi2 上的 RGS 依赖性负反馈产生正如模型预测的那样，血小板在体外和体内功能的增强。拟议的研究分为三个具体目标。目标 1 将检验我们的假设，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。目标 2 将检验我们的假设，即 SPL/RGS/SHP-1 复合物的衰变对 G 蛋白信号传导提供定时制动，并确定相关机制。最后，目标 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激活状态的转化以及避免血小板反应性长期增加的不利后果方面的作用。公共健康相关性：在错误的地点和错误的时间发生的血小板激活仍然是美国心血管和脑血管发病的主要原因。在拟议的研究中，我们将研究新的机制，我们认为这些机制有助于调节血小板对损伤的反应程度，确保反应既不充分也不强烈，以致周围组织的健康受到威胁。更好地了解血小板生物学这一关键但尚未充分探索的方面，是了解和预防人们患有心脏病和中风时出现问题的关键。