Thrombin Receptor-G Protein Signaling Mechanisms

描述（由申请人提供）：G蛋白偶联蛋白酶激活受体的蛋白水解裂解激活了一系列非常多样化的生理反应。这些包括血小板活化和聚集、细胞增殖/凋亡、细胞迁移和蛋白酶归巢、血管生成以及血管损伤的止血和炎症反应。目前已鉴定出四种蛋白酶激活受体：PAR1、PAR2、PARS和PAR4。基质金属蛋白酶也成为血管生物学中重要的蛋白酶。MMPs最初被描述为参与组织修复和癌症侵袭的细胞外基质重塑酶，由于其在动脉粥样硬化性血栓形成疾病和血小板止血中的突出作用，MMPs和相关的金属蛋白酶崩解素重新受到关注。最近，我们发现基质金属蛋白酶MMP-1也可以作为PAR1的蛋白酶激动剂，切割受体，产生PAR1依赖性的Ca2+信号，迁移，血小板活化和细胞形状改变。MMP-1在血小板和内皮细胞中高水平表达。急性心肌梗死患者血浆中循环MMP-1水平显著升高，表明活化的血小板和内皮细胞是MMP-1的潜在来源。然而，目前尚不清楚MMP1- par1信号在止血、血栓形成和血管完整性控制中的作用，也不清楚与凝血酶相比，MMP1是否激活了不同的G蛋白途径。PAR1，已被证明在各种体外条件下与Gq， Gi， Gi2/i3和（3y）偶联。PAR1如何与G蛋白相互作用的细节以及体内条件下差异G蛋白激活的相对重要性和时间顺序仍然未知。对于可能与PARs细胞内环相互作用的非g蛋白效应物的身份知之甚少。这些研究的主要目标是：1)研究体内条件下PAR1激活G蛋白的分子基础；2)了解动脉血栓形成过程中血小板中MMP1-PAR1信号传导的机制和生理相关性；3)研究内皮细胞MMP1激活PAR1的机制以及MMP1-PAR1信号传导对脓毒症期间内皮功能的影响；4)确定新发现的PAR1效应物BicD1如何调节PAR1信号传导。信号终止和细胞运动。血小板活化已被证明在血管成形术和支架植入的情况下会升高，这可能导致包括急性心脏病发作和死亡在内的临床并发症。PAR1受体长期以来一直被认为是心脏病患者治疗干预的明显候选者，本文提出的工作可能会导致动脉血栓患者和危及生命的败血症患者的新治疗方法的发展。

DESCRIPTION (provided by applicant): Proteolytic cleavage of the G protein-coupled protease-activated receptors activates an extraordinarily diverse array of physiologic responses. These include platelet activation and aggregation, cellular proliferation/apoptosis, cell migration and protease-homing, angiogenesis, and the hemostatic and inflammatory responses to vascular injury. Four protease-activated receptors have been identified: PAR1, PAR2, PARS, and PAR4. Matrix metalloproteases have also emerged as important proteases in vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in atherothrombotic disease and platelet hemostasis. Quite recently, we found that a matrix metalloprotease, MMP-1, can also function as a protease agonist of PAR1 cleaving the receptor to generate PAR1-dependent Ca2+ signals, migration, platelet activation and cell shape changes. MMP-1 is expressed in high levels in platelets and endothelium. Circulating levels of MMP-1 have been shown to be significantly elevated in plasma from patients following acute myocardial infarction pointing to activated platelets and endothelium as potential sources for MMP-1. However, it is not clear what role(s) MMP1-PAR1 signaling plays in hemostasis, thrombosis and the control of vascular integrity or whether MMP1 activates different G protein pathways as compared to thrombin. PAR1, has been shown to couple to Gq, Gi, Gi2/i3, and (3y under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation under in vivo conditions is still unknown. Far less is known about the identity of non-G protein effectors that might also interact with the intracellular loops of the PARs. The major goals of these studies are: 1) to investigate the molecular basis of G protein activation by PAR1 under in vivo conditions, 2) to understand the mechanism and physiologic relevance of MMP1-PAR1 signaling in platelets during arterial thrombus formation 3) to investigate the mechanism of endothelial MMP1 activation of PAR1 and the functional consequences of MMP1-PAR1 signaling on endothelial function during sepsis, and 4) to determine how the newly identified PAR1 effector, BicD1, modulates PAR1 signaling, signal termination, and cell motility. Platelet activation has been shown to be heightened in the setting of angioplasty and stenting which may cause clinical complications including acute heart attacks and death. The PAR1 receptor has long been recognized as an obvious candidate for therapeutic intervention of patients with heart conditions, the work proposed here may lead to the development of novel treatments for patients with arterial blood clots and those suffering from life- threatening sepsis.