Thrombin activatable fibrinolysis inhibitor (TAFI) does not inhibit in vitro thrombolysis by pharmacological concentrations of t-PA

TAFI (thrombin activatable :fibrinolysis inhibitor) is a plasma procarboxyp eptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of a ctivated TAFI (TAFIa) has been suggested to represent a mechanism of thromb us resistance to thrombolytic therapy. However, the ability of TAFI to inhi bit fibrinolysis by pharmacological concentrations of t-PA has not been pro perly investigated. We used an in vitro model consisting of I-125-fibrin bl ood clots submerged in autologous defibrinated plasma.,Upon addition of t-P A (125-5000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the ge nerated TAFIa with the specific inhibitor PTI (50 mug/ml) or by enhancing T AFI activation through the addition of recombinant soluble thrombomodulin ( solulin, 1 mug/ml). In our clot lysis model, activation of TAFI amounted to , about 20% of inducible carboxypeptidase activity. Addition of PTI, howeve r, produced a significant increase in the extent of lysis only at concentra tions of t-PA equal to or lower than 250 ng/ml. When solulin was added to t he plasma surrounding the clot, about 70% of TAFI: was activated within 15 min, Under these conditions, inhibition of clot lysis was very marked in sa mples containing 125 or 250 ng/ml of t-PA, but negligible in those containi ng pharmacological concentrations of the activator (1000 and 5000 ng/ml). A dditional experiments suggest that loss of fibrin-dependence by elevated co ncentrations of t-PA may be one of the mechanisms explaining the lack of ef fect of TAFIa. Our data indicate that, under our experimental conditions, c lot lysis by pharmacological concentrations of t-PA is not influenced by TA FIa even after maximal activation of this procarboxypeptidase.