Project Summary/Abstract Activated protein C (APC) is a naturally occurring plasma serine protease that has been translated to the clinic as a recombinant wild type or mutant biologic. In a diverse collection of preclinical animal injury models, pharmacologic APC’s provides many benefits. APC and the protein C systems contribute to the regulation of thromboinflammation that is critical for host defense, including defense versus SARS-CoV-2 infection. APC can act on diverse cell types, including immune cells for suppression of graft-versus-host disease. APC not only has anticoagulant activity but also initiates cell signaling via multiple receptors, in particular, via several protease activated receptors (PAR). APC-initiated cell signaling contributes to tissue homeostasis and host defense systems. Beneficial APC-initiated biased signaling is caused by specific cleavages of PAR1 and PAR3, and for some critical biologic activities, e.g., reducing mortality in sepsis, APC requires the integrin, Mac-1. Despite recent insights, there is a major gap in knowledge about protein-protein interactions between APC and its cellular receptors. Aim 1 studies will use a very extensive library of > five dozen recombinant APC mutants to provide a database regarding APC’s receptor specificities which will then enable engineering of APC mutants with receptor-specific selectivity, e.g., an APC mutant with highly selective PAR1-specific or PAR3-specific signaling capabilities or selectively specific for the I-domain of Mac-1. Such receptor-selective APC mutants will be useful reagents for deciphering which receptors play critical roles on cells in vitro or in animals in vivo, and they may lead to translation for novel APC mutants. To address the major problem of COVID-19, Aim 2 studies will characterize how murine COVID-19-like symptoms in SARS-CoV-2 infected hACE2 mice are influenced by endogenous protein C systems, e.g., by anticoagulant systems, by cytoprotective systems including anti- inflammation, anti-apoptosis, and endothelial barrier stabilization, and by fibrinolysis systems. Studies will define how these systems are modulated to be either hyperactive or hypoactive. This will provide new insights into how the protein C systems may be intertwined with defense versus SARS-CoV-2. The most promising cytoprotective- selective APC mutant (3K3A-APC) will be tested for COVID-19 therapy to advance potential translation. Aim 3 will characterize the multifaceted roles of APC in the pathobiology of acute traumatic coagulopathy. Studies will assess mechanisms by which disproportional APC generation contributes either to the development of acute traumatic coagulopathy involving Factor V inactivation and hyperfibrinolysis or, conversely, to protection of the integrity of organ function during and after acute traumatic coagulopathy by way of APC’s cytoprotective activities. The proposed studies will provide novel mechanistic insights and new APC variants which may aid transl...