Structure-function of cytoprotective coagulation proteases and their receptors

项目摘要 本申请旨在提高对介导细胞保护作用的分子机制的基本理解。 凝血蛋白酶对细胞的作用，并将测试潜在治疗血栓的新方法， 炎症性疾病。尽管最近取得了进展，但关于生理学和 当血管、炎症和凝血途径 在细菌或病毒感染期间的干扰，以及这些途径的不平衡调节如何引发恶性肿瘤。 循环的血管功能障碍、免疫凝血病和血栓炎症。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 其中血管、炎症和凝血途径的不平衡调节促成发病机制。 APC的这些有益作用主要是由APC的细胞保护活性介导的，所述细胞保护活性涉及 内皮蛋白C受体（EPCR）和PAR 1在Arg 46和PAR 3在Arg 41的非典型激活， 其诱导偏向的信号传导途径，有助于重新平衡组织稳态和宿主防御 系统.目标1的一个主要焦点是血栓炎症过程中EPCR功能的调节。我们 已经确定了EPCR在细胞表面上的功能调节的新机制，并且已经发现 一种EPCR稳定抗体，其极大地增强了内源性蛋白C系统的有益作用。 体外和体内研究将表征抗体增强APC的机制。 细胞保护作用及其在血栓炎症中的转化潜力。Aim 2专注于合成APC- 源自非典型PAR 1和PAR 3活化的模拟肽。这些APC模拟肽不 需要EPCR提供抗炎和内皮稳定活性，并可能提供关键的血管 血栓炎性疾病期间炎症介质使EPCR失效时的保护作用。目的 2项研究将提供关于偏向PAR 1和PAR 3信号传导的结构-功能的高度创新知识。 SARS-CoV-2感染引起的COVID-19病理生物学的新知识表明， 宿主防御免疫和血液凝固系统的失调，基于过量的 重症患者血液中循环的细胞因子/趋化因子和D-二聚体。我们假设EPCR 由循环中过量的细胞因子/趋化因子释放诱导的功能障碍有助于血管生成， APC在重症COVID-19患者中的内源性细胞保护作用的功能障碍和损害。 目的3将表征EPCR在SARS-CoV-2感染小鼠中COVID 19发病机制中的作用 表达人ACE 2。用我们的EPCR稳定抗体和我们的第二代- 将测试具有改善的APC模拟活性的第二代PAR序列衍生肽的功效， 减轻鼠COVID-19的严重影响。这些研究将提高我们对监管的认识 EPCR在血栓炎症中发挥作用，并可能促进COVID-19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.