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异常多样的生理反应。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、血管生成以及对血管损伤的止血和炎症反应。已经鉴定了四种蛋白酶激活受体：PAR 1、PAR 2、PARS和PAR 4。基质金属蛋白酶也已成为血管生物学中重要的蛋白酶。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，由于其在动脉粥样硬化血栓形成疾病和血小板止血中的突出作用，MMPs和相关金属蛋白酶解整合素重新成为焦点。最近，我们发现基质金属蛋白酶MMP-1也可以作为PAR 1的蛋白酶激动剂，切割受体，产生PAR 1依赖性Ca 2+信号、迁移、血小板活化和细胞形状变化。MMP-1在血小板和内皮细胞中高水平表达。已经显示在急性心肌梗死后患者的血浆中MMP-1的循环水平显著升高，这表明活化的血小板和内皮是MMP-1的潜在来源。然而，目前尚不清楚MMP 1-PAR 1信号传导在止血、血栓形成和血管完整性控制中起什么作用，或者MMP 1是否与凝血酶相比激活不同的G蛋白途径。PAR 1已显示在多种体外条件下与Gq、Gi、Gi 2/i3和β γ偶联。PAR 1如何与G蛋白相互作用以及在体内条件下差异G蛋白激活的相对重要性和时间顺序的细节仍然未知。关于可能也与PAR的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PAR 1激活G蛋白的分子基础，2）了解动脉血栓形成期间血小板中MMP 1-PAR 1信号传导的机制和生理相关性3）研究内皮MMP 1激活PAR 1的机制以及功能后果脓毒症期间MMP 1-PAR 1信号传导对内皮功能的影响，和4）确定新鉴定的PAR 1效应子BicD 1如何调节PAR 1信号传导、信号终止和细胞运动性。血小板活化在血管成形术和支架植入术中被证明是增强的，这可能导致临床并发症，包括急性心脏病发作和死亡。PAR 1受体长期以来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导致开发用于动脉血凝块患者和患有危及生命的脓毒症的患者的新治疗。

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.