The role of protease activated receptors on platelets.

蛋白酶激活受体对血小板的作用。

• 批准号: 8274738
• 负责人: Marvin Thomas Nieman
• 金额: $ 27.2万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8274738
• 关键词: ADP Receptors; Amino Acids; Antibodies; Binding; Biochemistry; Biological Assay; Bioluminescence; Bleeding time procedure; Blocking Antibodies; Blood Platelets; Blood Vessels; Cell surface; Chimeric Proteins; Cleaved cell; Co-Immunoprecipitations; Complex; Cysteine; Data; Development; Drug Design; Energy Transfer; Ensure; Event; F2R gene; Fluorescence; Fluorescence Resonance Energy Transfer; Future; G Protein-Coupled Receptor Genes; Goals; Hemorrhage; Human; Individual; Maps; Mediating; Modeling; Molecular; Morphologic artifacts; Mus; Mutation; Myocardial Infarction; PAR-1 Receptor; PAWR gene; Physiological; Platelet Activation; Platelet aggregation; Proteinase-Activated Receptors; Publications; Publishing; Risk; Role; Serotonin; Signal Transduction; Site; Stroke; System; Tail; Techniques; Testing; Thrombin; Thrombosis; Thromboxanes; Thrombus; Transgenic Animals; Transgenic Mice; base; crosslink; in vivo; insight; mouse model; novel; overexpression; promoter; public health relevance; receptor

DESCRIPTION (provided by applicant): The purpose of the proposed studies is to determine how PAR1 and PAR4 interact to mediate thrombin signaling in platelets. Specifically, the project will identify the PAR1-PAR4 interaction interface and determine how the anionic region on PAR4 exodomain contributes to platelet activation in vivo in the presence of PAR1. The long-term goal is to identify potential targets for anti-platelet therapies that do not pose a risk for bleeding by understanding how PAR1 and PAR4 interact with one another to mediate thrombin signaling for platelet activation. Specific Aim 1 will characterize a blocking antibody to PAR4's anionic region and determine the role of this region in platelet activation in vivo. The proposed studies will first test the hypothesis that the anionic region on PAR4 is a potential target for anti-platelet therapy. An antibody to PAR4s anionic region, CAN12, blocks human and mouse platelet activation. Since CAN12 blocks 1-thrombin-induced human platelet aggregation, studies will determine its mechanism by examining influence of CAN12 on PAR1 activation when PAR1 is co- expressed with PAR4. In addition, studies will test the hypothesis that the anionic region of PAR4 is critical for PAR4 activation in vivo using transgenic animals expressing mouse PAR4 with mutations in the anionic cluster (mPAR4-AAA), mouse PAR1 or both under the control of the GPIb1 promoter. The transgenic animals will be compared in ex vivo platelet function assays, thrombosis assays and tail bleeding times. Specific Aim 2 will determine the importance of PAR1 and PAR4 interaction on the surface of cells for efficient activation by thrombin. We will determine the regions on PAR1 and PAR4 required for homodimer and heterodimer formation using Bioluminescence Resonance Energy Transfer (BRET) and cysteine crosslinking. The cysteine crosslinking studies will use an inducible system to examine homodimers and heterodimers over a wide range of expression levels to ensure that interactions are occurring in a physiological range and are not an artifact of overexpression. The BRET and crosslinking studies will be verified with bimolecular fluorescence complementation (BiFC) studies. Finally, we will test the hypothesis that PAR1 enhances PAR4 activation by physically interacting. The proposed studies will examine the interactions between PAR1 and PAR4 on the molecular level to determine how these receptors communicate with one another to mediate thrombin signaling in platelets. These studies have the potential to identify general mechanisms of GPCR interactions to mediate a range of signals that may be transferred to other GPCRs found on the platelet. PUBLIC HEALTH RELEVANCE: A major cause of heart attacks and strokes is the development of a platelet rich thrombus in a blood vessel. Thrombin is the most potent platelet activator by binding and cleaving protease activated receptors. The overall goal of this proposal is to identify the contact sites between the thrombin-protease activated receptor complexes which will provide insights for drug design for antiplatelet therapies that do not increase bleeding.

描述（由申请人提供）：拟议研究的目的是确定 PAR1 和 PAR4 如何相互作用以介导血小板中的凝血酶信号传导。具体来说，该项目将识别 PAR1-PAR4 相互作用界面，并确定 PAR4 外结构域上的阴离子区域如何在 PAR1 存在的情况下促进体内血小板活化。长期目标是通过了解 PAR1 和 PAR4 如何相互作用来介导血小板激活的凝血酶信号传导，从而确定不会造成出血风险的抗血小板治疗的潜在靶点。具体目标 1 将表征 PAR4 阴离子区域的封闭抗体，并确定该区域在体内血小板激活中的作用。拟议的研究将首先检验 PAR4 上的阴离子区域是抗血小板治疗的潜在靶点的假设。 PAR4 阴离子区抗体 CAN12 可阻断人和小鼠血小板活化。由于CAN12阻断1-凝血酶诱导的人血小板聚集，因此研究将通过检查当PAR1与PAR4共表达时CAN12对PAR1激活的影响来确定其机制。此外，研究将使用在 GPIb1 启动子控制下表达阴离子簇 (mPAR4-AAA) 突变的小鼠 PAR4、小鼠 PAR1 或两者都表达突变的转基因动物，验证 PAR4 的阴离子区域对于体内 PAR4 激活至关重要的假设。将在离体血小板功能测定、血栓形成测定和尾部出血时间中对转基因动物进行比较。具体目标 2 将确定细胞表面 PAR1 和 PAR4 相互作用对于凝血酶有效激活的重要性。我们将使用生物发光共振能量转移 (BRET) 和半胱氨酸交联来确定同二聚体和异二聚体形成所需的 PAR1 和 PAR4 上的区域。半胱氨酸交联研究将使用诱导系统来检查各种表达水平的同二聚体和异二聚体，以确保相互作用发生在生理范围内，并且不是过度表达的产物。 BRET 和交联研究将通过双分子荧光互补 (BiFC) 研究进行验证。最后，我们将检验 PAR1 通过物理相互作用增强 PAR4 激活的假设。拟议的研究将在分子水平上检查 PAR1 和 PAR4 之间的相互作用，以确定这些受体如何相互通信以介导血小板中的凝血酶信号传导。这些研究有可能确定 GPCR 相互作用的一般机制，以介导一系列信号，这些信号可能会转移到血小板上发现的其他 GPCR。 公众健康相关性：心脏病发作和中风的主要原因是血管中形成富含血小板的血栓。凝血酶是通过结合和裂解蛋白酶激活受体而最有效的血小板激活剂。该提案的总体目标是确定凝血酶-蛋白酶激活受体复合物之间的接触位点，这将为不增加出血的抗血小板疗法的药物设计提供见解。