The role of protease activated receptors on platelets.

描述（由申请方提供）：拟定研究的目的是确定PAR 1和PAR 4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，该项目将确定PAR 1-PAR 4相互作用界面，并确定PAR 4胞外结构域上的阴离子区域如何在PAR 1存在下促进体内血小板活化。长期目标是通过了解PAR 1和PAR 4如何相互作用以介导血小板活化的凝血酶信号传导，确定抗血小板治疗的潜在靶点，这些靶点不会造成出血风险。特异性目标1将表征PAR 4阴离子区域的阻断抗体，并确定该区域在体内血小板活化中的作用。拟议的研究将首先检验PAR 4上的阴离子区域是抗血小板治疗的潜在靶点的假设。PAR 4s阴离子区的抗体CAN 12阻断人和小鼠血小板活化。由于CAN 12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PAR 1与PAR 4共表达时CAN 12对PAR 1活化的影响来确定其机制。此外，研究将使用在GPIb 1启动子的控制下表达阴离子簇中具有突变的小鼠PAR 4（mPAR 4-AAA）、小鼠PAR 1或两者的转基因动物来检验PAR 4的阴离子区域对于PAR 4体内活化至关重要的假设。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比较转基因动物。特异性目的2将确定细胞表面上PAR 1和PAR 4相互作用对于凝血酶有效活化的重要性。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PAR 1和PAR 4上形成同源二聚体和异源二聚体所需的区域。半胱氨酸交联研究将使用诱导型系统在广泛的表达水平范围内检查同源二聚体和异源二聚体，以确保相互作用发生在生理范围内，而不是过表达的假象。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，我们将测试PAR 1通过物理相互作用增强PAR 4激活的假设。拟议的研究将在分子水平上研究PAR 1和PAR 4之间的相互作用，以确定这些受体如何相互沟通，以介导血小板中的凝血酶信号。这些研究有可能确定GPCR相互作用的一般机制，以介导可能转移到血小板上发现的其他GPCR的一系列信号。 公共卫生相关性：心脏病发作和中风的一个主要原因是血管中富含血小板的血栓的形成。凝血酶是最有效的血小板激活剂，通过结合和切割蛋白酶激活受体。本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部位，这将为不增加出血的抗血小板治疗药物设计提供见解。

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.