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(申请人提供)：调节血小板激活的早期事件Lawrence F.Brass，MD PhD血小板对于正常止血是必不可少的，但也可能导致血栓形成和包括动脉硬化在内的常见血管壁疾病的演变和后果。尽管人们对血小板活化的了解很多，但对血小板内调节反应性、在不需要时限制血小板激活以及确保对损伤的反应停止出血而不会导致血管闭塞的事件知之甚少。这一建议的重点是发生在凝血酶、ADP和TXA2等激动剂下游的血小板激活事件，这些激动剂的所有受体都与异三聚体G蛋白偶联。我们的假设是，G蛋白依赖事件的失调是血栓前病变，并可能导致血管疾病的进展。这一假设将在对人类血小板和选定的小鼠模型的研究中得到验证。在我们的初步研究中，我们在静息的血小板中发现了一个以前未被描述的调控复合体，其中至少有两个RGS(G蛋白信号调节因子)蛋白和酪氨酸磷酸酶SHP-1与130 kDa的支架蛋白SPL结合，在静息的血小板中，SPL是酪氨酸磷酸化的。血小板活化可导致SHP-1依赖的Spin-phin去磷酸化和SPL/RGS/SHP-1复合体激动剂选择性解离。这些事件可以在转基因的CHO细胞中重现。在这些观察的基础上，我们认为，亲刺蛋白首先隔离RGS蛋白，允许信号开始，然后释放它们，以限制信号的大小和持续时间。对这一模型的支持来自我们对SPL(-/-)小鼠和表达Gi2a(G184S)的小鼠的研究，Gi2a(G184S)是一种突变，使G蛋白的a亚单位GI2抵抗RGS蛋白的失活。这些研究表明，1)亲刺蛋白的丢失损害了血小板对激动剂的反应，2)这一缺陷仅限于可导致SPL/RGS/SHP-1复合体解离的激动剂，以及3)阻断依赖RGS的GI2负反馈会产生如模型预测的体外和体内血小板功能的增加。建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蛋白信号的持续时间来帮助调节血小板的反应性。目的2将验证我们的假设，即SPL/RGS/SHP-1复合体的衰退对G蛋白信号提供了一个定时刹车，并确定了相关的机制。最后，目标3将侧重于RGS蛋白和SPL/RGS/SHP-1复合体在调节黏附的血小板转换到完全激活状态以及在避免血小板反应性慢性增加的不良后果方面的作用。 公共卫生相关性：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脑血管疾病的主要原因。在拟议的研究中，我们将检查新的机制，我们认为这些机制有助于调节血小板对损伤的反应程度，确保这种反应既不充分，也不太强大，以至于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未被充分探索的方面，是了解和预防当人们有心脏病和中风时出现问题的关键。