Thrombin Receptor-G Protein Signaling Mechanisms

描述(由申请人提供)：G蛋白偶联的蛋白酶激活的受体的蛋白水解性切割激活了一系列非常不同的生理反应。这些包括血小板的激活和聚集、细胞的增殖/凋亡、细胞的迁移和蛋白水解酶的归巢、血管的生成以及血管损伤后的止血和炎症反应。目前已鉴定出四种蛋白水解酶激活受体：PAR1、PAR2、PARS和PAR4。基质金属蛋白水解酶也是血管生物学中重要的蛋白水解酶。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，由于它们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及其相关的金属蛋白酶去整合素成为新的焦点。最近，我们发现基质金属蛋白酶MMP1也可以作为PAR1的蛋白酶激动剂，裂解受体产生依赖PAR1的钙信号，迁移，激活血小板和细胞形状改变。基质金属蛋白酶-1在血小板和血管内皮细胞高水平表达。急性心肌梗死患者血浆中循环中的基质金属蛋白酶-1水平显著升高，提示活化的血小板和内皮细胞是基质金属蛋白酶-1的潜在来源。然而，目前还不清楚(S)MMP1-PAR1信号在止血、血栓形成和控制血管完整性中起什么作用，也不清楚MMP1是否激活了与凝血酶相比不同的G蛋白途径。在各种体外条件下，PAR1与Gq、Gi、GI2/I3和(3y)偶联。PAR1如何与G蛋白相互作用，以及在活体条件下差异G蛋白激活的相对重要性和时间顺序仍不清楚。人们对非G蛋白效应器的身份知之甚少，这些效应器可能也与PAR的细胞内环相互作用。这些研究的主要目标是：1)研究活体条件下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MMP1-PAR1信号的机制和生理意义；3)研究脓毒症时PAR1内皮细胞MMP1激活的机制以及MMP1-PAR1信号对内皮功能的功能影响；4)确定新发现的PAR1效应器BicD1如何调节PAR1信号、信号终止和细胞运动。在血管成形术和支架置入术中，血小板的活化被证明是高水平的，这可能会导致包括急性心脏病发作和死亡在内的临床并发症。长期以来，PAR1受体一直被认为是心脏疾病患者治疗干预的明显候选者，这里提出的工作可能会导致开发新的治疗方法，用于治疗动脉血栓患者和那些患有危及生命的脓毒症的患者。

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.