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: 8125073
• 负责人: Marvin Thomas Nieman
• 金额: $ 27.48万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8125073
• 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。具体而言，该项目将识别PAR1-PAR4相互作用界面，并确定PAR4外域上的阴离子区域如何在PAR1存在下在体内促进血小板激活。长期目标是通过了解PAR1和PAR4如何相互作用以介导凝血酶信号以介导血小板激活的抗血小板疗法的潜在靶标，这些抗原疗法的潜在靶标。具体目标1将表征与PAR4阴离子区域的阻断抗体，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以下假设：PAR4上的阴离子区域是抗血小板治疗的潜在靶标。 par4s阴离子区域CAN12的抗体阻断了人和小鼠血小板激活。由于CAN12块1-脑组诱导的人血小板聚集，因此研究将通过检查与PAR1共同表达PAR1时CAN12对PAR1激活的影响来确定其机制。此外，研究将检验以下假设：PAR4的阴离子区域对于使用与阴离子簇（MPAR4-AAA），小鼠PAR​​1或在GPIB1启动子控制的阴离子簇（MPAR4-AAA）中具有突变的小鼠PAR​​4的转基因动物至关重要。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比较转基因动物。特定的目标2将确定PAR1和PAR4相互作用在细胞表面上的重要性，以通过凝血酶有效激活。我们将使用生物发光共振能传递（BRET）和半胱氨酸交联所需的同二聚体和异二聚体形成所需的PAR1和PAR4区域。半胱氨酸的交联研究将使用诱导系统在广泛的表达水平上检查同二聚体和异二聚体，以确保相互作用在生理范围内发生并且不是过表达的伪像。 BRET和交联研究将通过双分子荧光互补（BIFC）研究来验证。最后，我们将检验以下假设：PAR1通过物理相互作用增强PAR4激活。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。这些研究具有鉴定GPCR相互作用的一般机制，以介导可能转移到血小板上其他GPCR的一系列信号。 公共卫生相关性：心脏病发作和中风的主要原因是血管中血小板丰富的血栓形成。凝血酶是通过结合和切割蛋白酶活化受体的最有效的血小板激活剂。该提案的总体目标是确定凝血酶 - 蛋白酶激活的受体复合物之间的接触位点，这将为抗血小板疗法的药物设计提供见解，这些抗血小板疗法不会增加出血。