Plasmin proteolysis of endothelial cell and vessel wall associated tissue factor pathway inhibitor

Plasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have sug gested that plasmin can cleave and inactivate recombinant TFPI, a major inh ibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with i mplications for thrombolysis. In a series of experiments, we examined the e ffects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cell s (SMC) in the vessel wall. Plasmin (0.2 muM) decreased cell surface and ma trix associated TFPI activity in cultured endothelial cells by 77 +/- 5 % a nd 69 +/- 6 % respectively (p < 0.01). Plasminogen, the proenzyme form of p lasmin had no such effect on cell surface TFPI or matrix TFPI, Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was als o significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a similar to 45kD TFPI on Western Blot analysis fo llowing plasmin treatment. Plasmin also proteolysed a similar to 45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincuba tion with aprotinin, a plasmin inhibitor. Incubation of similar concentrati ons of plasmin, with homogenates of normal vessel decreased a similar to 45 kD TFPI immunoreactive band on Western blot analysis. Plasmin also decrease d surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque s ections caused complete removal of TFPI immunoreactivity associated with lu minal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-ass ociated (surface and matrix) TFPI and may remove TFPI from the luminal surf ace and intima of the vessel wall. TFPI proteolysis in cultured EC was asso ciated with significant reduction in TFPI anti-coagulant activity, These da ta provide evidence that plasmin degradation Of TFPI occurs in vascular cel ls and in the vessel wall and may have implications for re-thrombosis follo wing thrombolysis in vivo.