The prothrombin gene mutation G20210A is a common risk factor for thrombosis and is associated with increased prothrombin levels. However, the mechanism whereby hyperprothrombinemia predisposes to thrombosis remains unclear. Because thrombin is the physiologic activator of TAFI (thrombin activatable fibrinolysis inhibitor), the precursor of an antifibrinolytic carboxypeptidase (TAFIa), we evaluated the influence of hyperprothrombinemia on fibrinolysis. Thirty-two heterozygous carriers of the G20210A mutation and 30 noncarriers were studied. Plasma fibrinolytic factors and TAFI levels were similar in the 2 groups. Mean lysis time of tissue factor-induced plasma clots exposed to 25 ng/mL exogenous tissue-type plasminogen activator (t-PA) was significantly longer in 20210A carriers than in control donors. This difference disappeared on addition of a specific inhibitor of TAFIa. Determination of thrombin and TAFIa activity, generated during clot lysis, revealed that G20210A mutation was associated with a significant enhancement of late thrombin formation and an increase in TAFI activation. Plasma prothrombin level was highly significantly correlated with both clot lysis time and TAFI activation. The addition of purified prothrombin, but not of factors X or VIIa, to normal plasma caused a concentration-dependent, TAFI-mediated inhibition of fibrinolysis. These findings provide a new mechanism that might contribute to the thrombotic risk in prothrombin 20210A carriers.