The role of protease activated receptors on platelets.

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，该项目将确定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如何相互作用介导凝血酶信号以激活血小板，从而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Aim 1将表征PAR4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。拟议的研究将首先验证PAR4上的阴离子区域是抗血小板治疗的潜在靶点的假设。PAR4s阴离子区的抗体CAN12可阻断人和小鼠的血小板活化。由于CAN12阻断1-凝血酶诱导的人血小板聚集，研究将通过检测PAR1与PAR4共表达时CAN12对PAR1激活的影响来确定其机制。此外，研究将在GPIb1启动子控制下，通过转基因动物表达阴离子簇突变的小鼠PAR4 （mPAR4-AAA）、小鼠PAR1或两者同时表达的小鼠PAR4，来验证PAR4阴离子区对体内PAR4激活至关重要的假设。转基因动物将在离体血小板功能测定、血栓形成测定和尾出血次数等方面进行比较。特异性Aim 2将确定PAR1和PAR4在细胞表面相互作用对凝血酶有效激活的重要性。我们将利用生物发光共振能量转移（BRET）和半胱氨酸交联来确定PAR1和PAR4上形成同型二聚体和异型二聚体所需的区域。半胱氨酸交联研究将使用诱导系统在广泛的表达水平范围内检查同型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，我们将验证PAR1通过物理相互作用增强PAR4激活的假设。这些研究将在分子水平上研究PAR1和PAR4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传导。这些研究有可能确定GPCR相互作用的一般机制，介导一系列可能转移到血小板上发现的其他GPCR的信号。

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.