Abstract This research project focuses on the fibrinolysis system and its activity in regulating innate immunity. We have shown that tissue-type plasminogen activator (tPA) functions as an antagonist of pro-inflammatory responses triggered by Toll-like Receptors (TLRs) in macrophages and in vivo in mice. Mechanistically, the activity of tPA is mediated by a receptor system that includes the N-methyl-D-aspartate receptor (NMDA-R) and LDL Receptor- related Protein-1 (LRP1). Although the function of enzymatically-active tPA in innate immunity is regulated by the Serpin, PAI1, and by plasmin generation, enzymatically-inactive tPA (EI-tPA) is resistant to these regulatory pathways. EI-tPA fails to inhibit pro-inflammatory responses mediated by Pattern Recognition Receptors (PRRs) other than TLRs; however, in mouse models of disease in which multiple PRRs function in concert, including the Dextran Sulfate Sodium (DSS) colitis model and the K/BxN serum-transfer arthritis model, EI-tPA is efficacious as a candidate therapeutic. In this application for continued support, we propose studies to elucidate the activity of tPA and its receptors in regulating inflammation and determine the potential to generate novel anti-inflam- matory drugs based on the structure of tPA. Four specific aims are proposed. In Specific Aim 1, the structural elements in tPA required for regulation of innate immunity will be determined by genetic engineering. Novel recombinant derivatives of EI-tPA will be developed and tested with the goal of optimizing tPA for use as a candidate anti-inflammatory therapeutic in vivo. Specific Aim 2 is focused on understanding the anti-inflam- matory cell-signaling pathway activated by tPA downstream of the NMDA-R. New preliminary results implicate a novel system, involving Trk receptor transactivation by Src family kinases, which is previously undescribed in macrophages and other inflammatory cells. The proposed studies in Specific Aim 2 will not only contribute to our understanding of tPA signaling in general but also may identify novel intracellular targets for anti-inflam- matory drug development based on our analysis of tPA-activated cell-signaling. In Specific Aim 3, we will breed mice available in our laboratory to generate animals in which macrophages and other cells in which the LysM promoter is active do not express the NMDA-R. This will allow us to definitively test the hypothesis that the anti- inflammatory activity of EI-tPA in vivo, for example in neutralizing LPS toxicity, requires the NMDA-R and that macrophages and/or neutrophils are EI-tPA target cells. In Specific Aim 4, we will study the activity of tPA and its anti-inflammatory receptors, the NMDA-R and LRP1, in the DSS colitis model. Single-cell transcriptome profiling studies are proposed to identify, in an unbiased manner, colon cells targeted by EI-tPA in vivo and identify novel pathways by which EI-tPA elicits a favorable response in this model system. Collectively, the s...