Project Summary Sepsis is a critical clinical condition that is caused by dysregulated host immune response to infection and characterized by organ failure and high mortality. Sepsis is associated with marked inflammation and hemostatic activation that often leads to widespread microvascular thrombus formation and global clotting dysfunction, a complication termed sepsis-induced coagulopathy (SIC). Toll-like receptors (TLRs) are innate immune receptors that respond to pathogen (PAMPs) or host-derived danger molecules (DAMPs) and drive profound inflammation. We have established a murine model of SIC characterized by time-dependent thrombocytopenia, procoagulant consumption, and global clotting dysfunction, and identified a critical role for TLR7 (a receptor for single-stranded RNAs, e.g., HIV RNA or microRNA) in the pathogenesis of SIC including thrombocytopenia, procoagulant (e.g., tissue factor, TF) production, and global clotting dysfunction. Moreover, we have observed increased circulating plasma miRNAs (e.g., miR-146a-5p) and extracellular vesicles (EVs, miRNA carrier) in sepsis, and a close correlation between the plasma RNA levels and sepsis severity. Both miR-146a-5p and septic plasma EVs induce proinflammatory and procoagulant response via a TLR7-dependent manner. Based on these data and the literature, we hypothesize that miRNA→TLR7 signaling plays an important role in regulating platelet activity and contributes to platelet dysfunction in SIC. To test this hypothesis, we propose the following three Specific Aims. In Aim 1, we will test the function and mechanisms of miRNA-TLR7 signaling in platelet activation. In Aim 2, we will determine the effect of EVs loaded with miR-146a-5p and septic plasma EVs on platelet activation and function. In Aim 3, we will investigate the role of TLR7 signaling in platelet activation and dysfunction in SIC. Completion of the proposed studies will provide new insight into the role and mechanism of miRNA-TLR7 signaling in regulating platelet activity and dysfunction in SIC, and potentially offer a novel therapeutic target.