Project Summary Coronavirus disease 2019 (COVID-19) is a potentially life threatening disease caused by the novel viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Approximately 20% of COVID-19 patients experience severe disease, typically presenting with bilateral pneumonia, and about 5% progress to acute respiratory distress syndrome (ARDS). ARDS results from a combination of virally induced lung injury and the rapid influx of immune cells that release inflammatory mediators leading to a hyper-activated state known as cytokine storm. COVID-19 ARDS is further exacerbated by a unique diffuse coagulopathy leading to thrombus formation in the venous and arterial circulations and microthrombi in capillaries of the lungs. Predisposing factors for this coagulopathy include increased fibrinogen, activated coagulation cascade, platelet activation, hyper- inflammation, neutrophil extracellular trap (NET) formation, and endothelial cell damage. Understanding the pathophysiology of COVID-19 coagulopathy and ARDS is critical to finding effective therapeutic interventions. Accumulating evidence indicates critical roles of neutrophils in both ARDS and immunothrombosis in COVID-19. Our preliminary studies identified a novel population of low-density neutrophils (LDN) which expresses intermediate levels of CD16 (CD16Int LDN) in COVID-19 patients. The number of CD16Int LDN correlated with disease severity, levels of inflammatory cytokines IL-6/TNF-a, D-dimer levels, and clinical outcomes. In addition, CD16Int LDN showed spontaneous NET formation and evidence of in vivo platelet activation and granule exocytosis. Based on these findings, we postulate that CD16Int LDN play a critical role in the induction of coagulopathy and pulmonary inflammation in severe and critical COVID-19 patients. Three specific Aims are proposed to further dissect the underlying mechanisms. Aim 1 will comprehensively characterize LDN subsets using proteomics and transcriptomics approaches. The information gained from those studies will be used to refine our CyTOF antibody panel. We will use this panel to track differential neutrophil clusters in longitudinal patient samples. Aim 2 will determine LDN subsets functional changes during disease progression and their contributions to dysregulated inflammatory response and coagulopathy in severe and critical COVID- 19 patients. Neutrophil degranulation, NET formation, phagocytosis, chemotaxis, apoptosis, and cytokine release will be examined. We will also determine if LDN promote coagulopathy in COVID-19 patients. Aim 3 will determine whether inhibition of neutrophil granule exocytosis using our novel TAT-fusion protein inhibitors prevents activated neutrophil-mediated functional changes and hypercoagulation. We will also use a hACE2 Tg mouse model to determine the in vivo efficacy of TAT-fusion proteins on lung inflammation and impaired function. Successful completion of this proposal will provide novel insights in...