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 activi...