.) Despite long standing recognition of the relation between pleural fibrin deposition and organization of pleural exudates, there is only limited information concerning the molecular basis of disordered fibrin turnover in pleural injury. Exudative pleural effusions in humans were found to be characterized by increased procoagulant and depressed fibrinolytic activities and pilot studies with cells from these pleural fluids suggest that the mesothelium plays a key role in fibrin deposition. Proposed studies will address the hypothesis that mesothelial cells contribute to abnormalities of fibrin turnover and promote fibrin deposition within the pleural space. The mechanisms and regulation of procoagulant and fibrinolytic activity in mesothelial cells and pleural fluids will be determined. These experiments will involve coagulation assays, functional and immunologic assays of plasminogen activators, characterization and assay of plasminogen activator inhibitors and antiplasmins. It was found that human pleural mesothelial cells assemble procoagulant complexes on their surface. The molecular mechanisms that regulate assembly of the extrinsic activation complex (tissue factor associated with factor VII/VIIa) and the prothrombinase complex (factor Xa associated with Va) at the mesothelial cell surface will be determined. Binding studies will determine binding parameters (Kd and number of sites), cation requirements and functional activity of both the extrinsic activation complex and the prothrombinase complex. Monoclonal antibodies to factors VII and X will be used to verify the number of molecules bound and to relate binding with functional activity. In addition, binding site specificity will be probed using a series of synthetic peptides that compete with factors VII and X. Because mesothelial cells have low functional fibrinolytic activity, and because they secrete high levels of plasminogen activator inhibitor (PAI-1), we suspect that PAI-1 from the mesothelium permits fibrin deposition. The mechanisms that regulate PAI-1 in cultures of human mesothelial cells will be determined. These experiments will focus on the expression of mRNA for PAI-1 and the duration of the message under various conditions, such as cellular interaction with cytokines and the extracellular matrix.
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