Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Abstract: Fibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-β or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-β, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment. [Copyright &y& Elsevier]