Project Summary Tissue factor pathway inhibitor-2 (TFPI-2) consists of three Kunitz domains arranged in tandem flanked by a short acidic amino terminus and a highly basic C-terminal tail. The N-terminal Kunitz domain (KD)1 of TFPI-2 is the only inhibitory domain and it inhibits plasmin (Pm), factor (F) XIa, plasma kallikrein (pKLK), and very weakly FVIIa/tissue factor. The structural specificity of TFPI-2 KD1 for each protease is essentially unknown. The focus of this proposal is to delineate the TFPI-2 KD1 residues, which impart specificity toward Pm, FXIa and pKLK through structural and functional studies. Another goal is to design and test novel, potent and specific inhibitors of fibrinolysis, which have the potential to reduce bleeding in major surgeries without causing adverse effects attributed to broad spectrum protease inhibitors of Pm, like bovine pancreatic trypsin inhibitor (BPTI, Aprotinin). Using structure-based investigations and analyzing the serine proteases substrate specificity profiles, we have developed two TFPI-2 KD1 variants to specifically inhibit Pm. These TFPI-2 KD1 variants are dual reactive inhibitors that selectively inhibit plasmin active site as well as the activation of plasminogen by binding to the kringle domains of plasminogen/plasmin and tissue plasminogen activator. Our new data indicate that these two TFPI-2 KD1 variants are potent and selective inhibitors of Pm with no detectable inhibition of FXIa and pKLK. We will study the efficacy and toxicity of these two KD1 variants in mouse models of bleeding. Since trauma associated hemorrhage and coagulopathy are leading causes of mortality, we will also study the safety and efficacy of the potent KD1 variant in a rat trauma model, where the most potent KD1 will be selected based on the results from the mouse bleeding models. The three overlapping areas to be investigated in this proposal are: 1) Delineate the residues/segments that impart protease specificity of KD1 for Pm, FXIa and pKLK. 2) Assess the efficacy and safety of the two TFPI-2 KD1 variants in two mouse models of bleeding. 3) Evaluate safety and efficacy of the most potent KD1 variant in a rat liver hemorrhage model in acute and chronic settings. The proposed studies will significantly improve our understanding of the specificity of the proteases involved in coagulation and fibrinolysis. Further, it will lead to the development of a potent, specific inhibitor of Pm that can be used as a therapeutic agent to inhibit fibrinolysis. The applications of such therapeutic agents are broad and encompass trauma, cardiovascular surgery, as well as neurologic and orthopedic operations that are complicated by fibrinolysis.