Structure-function of cytoprotective coagulation proteases and their receptors

项目摘要 这项应用旨在提高对介导细胞保护的分子机制的基本理解 凝血酶对细胞的作用，并将测试潜在的血栓治疗的新方法。 炎症性疾病。尽管最近取得了进展，但在生理和心理方面的知识差距仍然很大。 血管、炎症和凝血途径影响的病理生理机制 在细菌或病毒感染期间相互缠绕，以及这些途径的不平衡调控如何助长恶性 血管功能障碍、免疫凝血和血栓炎症的循环。内源性和药理学 活化蛋白C(APC)在多种临床前动物损伤模型中具有多种有益作用 血管、炎症和凝血通路的不平衡调节导致发病。 APC的这些有益作用主要是由APC的细胞保护活性介导的，该活性涉及 内皮蛋白C受体(EPCR)和Arg46的PAR1和Arg41的PAR3的非规范激活， 它们诱导有偏见的信号通路，有助于重新平衡组织动态平衡和宿主防御 系统。目标1的一个主要焦点将是血栓性炎症过程中EPCR功能的调节。我们 已经确定了一种新的细胞表面EPCR功能调节机制，并发现 一种EPCR稳定抗体，可大大增强内源性蛋白C系统的有益效果。 体外和体内研究将表征抗体增强APC的机制 血栓性炎症中的细胞保护作用及其翻译潜能。目标2专注于合成APC- 来自非典型PAR1和PAR3激活的模拟多肽。这些模拟APC的多肽不会 需要EPCR提供抗炎和稳定内皮的活性，并可能提供关键的血管 在血栓性炎症性疾病中，当EPCR被炎症介质禁用时的保护作用。目标 2研究将为偏向PAR1和PAR3信号的结构和功能提供高度创新的知识。 关于感染SARS-CoV-2引起的新冠肺炎的病理生物学的新兴知识表明了一个引人注目的事实 宿主防御免疫和凝血系统的失调，基于过量的 危重病患者血液中细胞因子/趋化因子和D-二聚体的变化我们假设外源基因扩增 由循环中过度释放的细胞因子/趋化因子引起的功能障碍导致血管 新冠肺炎危重病患者活化蛋白C内源性细胞保护作用的功能障碍。 目的3研究EPCR在SARS-CoV-2感染小鼠柯萨奇病毒19致病机制中的作用 表达人血管紧张素转换酶2。用我们的EPCR稳定抗体和我们的第二个- 将测试具有改进的APC模拟活性的PAR序列衍生多肽的有效性 减轻新冠肺炎对小鼠的严重影响。这些研究将提高我们对监管的认识。 电子聚合酶链式反应在血栓性炎症中发挥作用，并可能推进新冠肺炎患者的治疗。

Project Summary This application seeks to improve basic understanding of the molecular mechanisms that mediate cytoprotective actions of coagulation proteases on cells and will test novel approaches for the potential treatment of thrombo- inflammatory disease. Despite recent advances, major gaps in knowledge remain about the physiological and pathophysiological mechanisms that are affected when vascular, inflammatory, and coagulation pathways intertwine during bacterial or viral infection, and how unbalanced regulation of these pathways can fuel a vicious cycle of vascular dysfunction, immunocoagulopathy, and thromboinflammation. Endogenous and pharmacologic activated protein C (APC) has multiple beneficial effects in a diverse collection of preclinical animal injury models where unbalanced regulation of vascular, inflammatory, and coagulation pathways contribute to pathogenesis. These beneficial effects of APC are primarily mediated by the cytoprotective activities of APC that involve the endothelial protein C receptor (EPCR) and non-canonical activation of PAR1 at Arg46 and of PAR3 at Arg41, which induce biased signaling pathways contributing to rebalancing tissue homeostasis and host defense systems. A major focus of Aim 1 will be on the regulation of EPCR function during thromboinflammation. We have identified a novel mechanism for the functional regulation of EPCR on cell surfaces and have discovered an EPCR stabilizing antibody that greatly enhances the beneficial effects of the endogenous protein C system. In vitro and in vivo studies will characterize the mechanism for the antibody’s augmentation of APC’s cytoprotective effects and its translational potential in thromboinflammation. Aim 2 focusses on synthetic APC- mimetic peptides derived from non-canonical PAR1 and PAR3 activation. These APC-mimetic peptides do not require EPCR to provide anti-inflammatory and endothelial stabilizing activities and may provide critical vascular protective effects when EPCR is disabled by inflammatory mediators during thromboinflammatory disease. Aim 2 studies will provide highly innovative knowledge on the structure-function of biased PAR1 and PAR3 signaling. The emerging knowledge of the pathobiology of COVID-19 caused by SARS-CoV-2 infection suggests a striking dysregulation of host defense immune and blood coagulation systems, based on excessive levels of cytokines/chemokines and D-dimer circulating in blood of severely ill patients. We hypothesize that EPCR dysfunction, induced by the excessive cytokines/chemokines release in the circulation, contributes to vascular dysfunction and impairments of the endogenous cytoprotective effects of APC in severely ill COVID-19 patients. Aim 3 will characterize the contribution of EPCR in the pathogenesis of COVID 19 in SARS-CoV-2-infected mice expressing human ACE2. Experimental treatments with our EPCR stabilizing antibody and our second- generation PAR-sequence-derived peptide with improved APC-mimetic activities will be tested for efficacy to mitigate the serious effects of murine COVID-19. These studies will improve our knowledge on the regulation of EPCR function in thromboinflammation and may advance therapies for COVID-19 patients.