PAI-1 released from cultured human endothelial cells delays fibrinolysis and is incorporated into the developing fibrin clot

Abstract

The ability of plasminogen activator inhibitor type 1 (PAI-1), secreted from stimulated human umbilical vein endothelial cells (HUVEC), to prolong fibrinolysis was investigated with a model fibrin-rich thrombus. Laser light scattering kinetic analyses demonstrated that HUVEC stimulated with either TNF-α or TGF-β secreted a product that delayed rt-PA mediated fibrinolysis more than 3-fold. Furthermore, the Global Lysis Time, defined as the time required to achieve 50% clot lysis, was strongly correlated with the quantity of PAI-1 antigen present in the HUVEC releasate. Addition of a neutralizing anti-PAI-1 monoclonal antibody fully reversed this delay in lysis, whereas a control antibody had no effect. Scanning electron microscopy demonstrated that >99% of the fibrin strands in this model endothelial cell/fibrin thrombus were avidly associated with the cell surface, both early and late in the fibrinolytic process. Immunocytochemistry, coupled with intermediate voltage electron microscopy, demonstrated specific association (>80%) of the endothelial cell-released PAI-1 with these fibrin fibres. These observations indicate stimulated human umbilical vein endothelial cells secrete sufficient active PAI-1 to delay fibrinolysis. Furthermore, this HUVEC-secreted PAI-1 is incorporated into the three-dimensional fibrin clot, where it may continue to inhibit fibrinolysis by tissue-type plasminogen activator.