Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products. Thrombin-activable fibrinolysis inhibitor (TAFI)3 is a 60-kDa carboxypeptidase-like protein that circulates in plasma at a concentration of ϳ75 nM (1). TAFI (also known as procarboxypeptidase U, plasma carboxypeptidase B, and carboxypeptidase R) was discovered independently by several groups (2-6), and its role in fibrinolysis was subsequently characterized (6). Thrombin in complex with thrombomodulin activates TAFI with a catalytic efficiency of 1.2 M Ϫ1 s Ϫ1, which is ϳ1250-fold greater than that with thrombin alone (7). Plasmin has also been shown to activate TAFI (0.008 M Ϫ1 s Ϫ1 ), and the second-order rate constant of this reaction increases by 16-fold in the presence of unfractionated heparin (8). Activated TAFI (TAFIa) plays a significant role in attenuating fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which can then solubilize the fibrin clot by cleaving fibrin after specific arginine and lysine residues (9). TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues on fibrin (6, 10), thereby decreasing the tissue-type plasminogen activator (tPA) cofactor activity of plasmin-modified fibrin (11). Removal of these C-terminal lysine residues suppresses plasminogen activation (12, 13) and down-regulates the conversion of Glu-plasminogen to Lysplasminogen, which is a 20-fold better substrate for tPA than . TAFIa is intrinsically unstable, with its inactivation being highly temperature-dependent (15). The inactivation of TAFIa is thought to be accompanied by a large structural change (16). The existence of a TAFIa concentration threshold has been demonstrated, such that TAFIa at a concentration above the threshold inhibits fibrinolysis. On...