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）：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至关重要，但也可以有助于血栓形成，以及包括动脉粥样硬化在内的血管壁的常见疾病的进化和后果。尽管对于血小板激活知之甚少，但对于调节反应能力的血小板中的事件的了解却少得多，在不需要时限制了血小板激活，并确保对损伤的反应在不引起血管闭塞的情况下停止出血。该建议的重点是血小板激活的事件，该事件立即发生在激动剂，例如凝血酶，ADP和TXA2的下游，所有受体都与异邻三聚体G蛋白偶联。我们的假设是，G蛋白依赖性事件的失调是血栓性的，有可能导致血管疾病进展。该假设将在对人血小板和选定小鼠模型的研究中进行检验。在我们的初步研究中，我们在静止血小板中鉴定了一种先前所描述的调节复合物，其中至少两个RG（G蛋白信号的调节剂）蛋白质和酪氨酸磷酸酶SHP-1绑定到130 kDa scaffold蛋白，微生素（Splophilin（Splophilin），这是静止的platosine platsient platosed plosphose的130 kDa支架蛋白质（Spleophilin）。血小板激活导致spl/rgs/shp-1复合物的自旋蛋白和激动剂选择解离的SHP-1-依赖性去磷酸化。这些事件可以在转染的CHO细胞中概括。基于这些观察结果，我们提出了自旋素第一隔离RGS蛋白，从而允许信号开始，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表达GI2A（G184S）的SPL（ - / - ）小鼠的研究，该突变使G蛋白的A亚基GI2，GI2的A亚基，对RGS蛋白抗活化的抗性。这些研究表明，1）旋转酚蛋白的丧失会损害血小板对激动剂的反应，2）这种缺陷仅限于可能导致SPL/RGS/SHP-1复合物解离的激动剂，以及3）阻止RGS依赖于GI2的负面反馈，因为模型可以预测，该模型可以预测，在Vivo In Vivo中获得了血小板功能。拟议的研究分为三个特定目标。 AIM 1将测试我们的假设，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有助于调节血小板的反应性。 AIM 2将检验我们的假设，即SPL/RGS/SHP-1复合物的衰减为G蛋白信号传导提供了定时制动，并识别涉及的机制。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转化为完全激活状态的转化以及避免血小板反应性慢性增加的不利后果方面的作用。 公共卫生相关性：在错误的地方发生的血小板激活仍然是美国心血管和脑血管发病率的主要原因。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程度的新型机制，以确保反应既不是不足，也不是如此强大，以至于周围组织的健康受到威胁。更好地理解这一关键但不足探索的血小板生物学方面是理解和防止人们有心脏和笔触的问题的关键。