Alterations in coagulation and fibrinolytic pathways, associated with increases in circulating and pulmonary concentrations of urokinase (uPA), plasminogen activator inhibitor 1 (PAI-1), and vitronectin, are present in almost all patients with acute lung injury (ALI). Our recent findings suggest two novel mechanisms through which uPA, PAI-1, and vitronectin can contribute to the development of ALI independently of their effects on coagulation and fibrinolytic pathways: 1) by enhancing neutrophil activation and 2) by decreasing phagocytosis and clearance of neutrophils in the lungs. Our hypothesis is that: uPA, PAI-1, and vitronectin, through actions that directly affect neutrophil activation, accumulation, and clearance in the lungs, are centrally involved in determining the development, perpetuation, and severity of ALI. Our specific aims are: 1) To define the mechanisms through which uPA and PAI-1 potentiate neutrophil activation by identifying the receptors and ligands involved, examining how interactions between uPA, PAI-1, and vitronectin modulate the proinflammatory properties of uPA and PAI-1, and determining the intracellular signaling pathways that are affected by combinations of uPA, PAI-1, and vitronectin in neutrophils stimulated through TLR4 and by whole bacteria;2) To determine the mechanisms through which PAI-1 and vitronectin modulate phagocytosis and clearance of neutrophils by delineating the roles of neutrophil and macrophage associated PAI-1 and vitronectin, identifying the receptors engaged by PAI-1 and vitronectin on neutrophils and macrophages that participate in modulating phagocytosis of viable and apoptotic neutrophils, and delineating the effects of PAI-1 and vitronectin in modifying the expression of receptors and ligands involved in phagocytosis of neutrophils, including calreticulin (CRT), CD47, CD31, integrins, mer, axl, and LRP, as well as in affecting the binding of opsonins to PtdSer and of collectins to CRT and CD91;and 3) To determine the mechanisms through which PAI-1 and vitronectin contribute to the development and severity of ALI by delineating the in vivo roles of PAI-1 and vitronectin in modulating phagocytosis of apoptotic neutrophils in the lungs during ALI, and examining the importance of interactions between PAI-1 and vitronectin in contributing to the severity of ALI. The proposed studies should not only improve understanding of cellular mechanisms leading to ALI, but also are likely to suggest novel therapeutic interventions aimed at decreasing the incidence and/or severity of ALI. PUBLIC HEALTH RELEVANCE: Urokinase, plasminogen activator inhibitor 1 (PAI-1), and vitronectin have well described roles in modulating coagulation and fibrinolysis. However, our recent results suggest two novel mechanisms through which urokinase, PAI-1, and vitronectin can contribute to the development of acute lung injury independently of their effects on coagulation and fibrinolytic pathways: 1) by enhancing neutrophil activation and 2) by decreasing phagocytosis and clearance of neutrophils in the lungs. The studies proposed in this application should not only improve understanding of cellular mechanisms leading to acute lung injury, but also are likely to suggest novel therapeutic interventions aimed at decreasing the incidence and/or severity of acute lung injury.