PROJECT SUMMARY The leading cause of acute liver injury and acute liver failure in the United States is hepatotoxicity caused by overdose of the pain medication acetaminophen (APAP). Nearly 50% of patients presenting with APAP- induced liver injury require costly hospitalization, and the efficacy of available treatments (i.e., N- acetylcysteine) is very limited for patients presenting well after APAP overdose. APAP overdose patients would benefit most from treatments that maximize liver repair pathways, such as proe-pr air macrophage activation. We recently discovered a remarkable pathway of liver repair following APAP hepatotoxicity whereby the clotting protein fibrin(ogen), deposited in the injured liver of both humans and mice, engages β2 integrins and directs local pro-repair macrophage activity. The fibrin(ogen)-β2 integrin interaction is carefully regulated and acquired by modifications of fibrin(ogen) structure. Defining the mechanisms whereby fibrin(ogen) is converted to a β2 integrin ligand that elicits pro-repair macrophage activity in liver injury is critical, as this could uncover novel putative therapeutic targets to improve liver repair pathways in patients with severe hepatotoxicity or acute liver failure. Our strong preliminary studies suggest that, in contrast to the current paradigm, hepatic fibrin(ogen) accumulation caused by acute toxicity is independent of blood coagulation activity. The central hypothesis framing these studies is that liver injury stimulates coagulation-independent cross-linking of fibrin(ogen) by tissue transglutaminase-2 (TGM2) to form a novel fibrin(ogen) complex that drives macrophage- mediated liver repair. Our approach includes genetically-modified mice lacking specific fibrin(ogen) cross- linking enzymes, mice expressing fibrin(ogen) proteins with specific functional mutations, application of a coagulation-insensitive fibrin(ogen) as a putative pro-repair therapeutic, a unique cell culture system to examine primary macrophage activation by unique molecular forms of fibrin(ogen), and analysis of fibrin(ogen) deposits in livers of human patients with acute liver failure. The investigative team comprises experts in toxic liver injury and repair, coagulation and fibrin(ogen) biochemistry/function, and mouse modeling of liver disease. In our proposed studies we will: (Aim 1) Identify the mechanism whereby APAP-induced liver injury stimulates hepatic deposition of repair-promoting cross-linked fibrin(ogen); (Aim 2) Determine the role of fibrin(ogen) hemostatic function in repair of the APAP-injured liver; and (Aim 3) Determine the mechanism whereby fibrin(ogen) cross-linking drives pro-repair macrophage activity in vitro. The expected outcome of these Specific Aims is discovery of entirely novel mechanisms linking unique structural modification of the clotting protein fibrin(ogen) with macrophage-mediated liver repair. Identifying these mechanisms could pinpoint putative targets (e.g., TGM2, β2 integrin-fib...