Thrombin-activable Fibrinolysis Inhibitor Zymogen Does Not Play a Significant Role in the Attenuation of Fibrinolysis

Abstract

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...

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A

“…Only a relatively small percentage (<1%) of available TAFI is activated in plasma when clotting is initiated with low levels of thrombin [31]. As recently reported, the percentage of FVIII in plasma greatly influences TAFI activation over the interval between clot formation and tPA induced clot lysis [14] especially in the range of 0–10% FVIII.…”

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

“…Because protein MCP inhibitors eliminate the activity of both TAFI and TAFIa, it is difficult to design experiments that distinguish between the activities of the two forms. Consequently, studies with MCP inhibitors against TAFIa activity to argue that TAFI activity is irrelevant will have to be reconsidered (69,70).…”

Identification of a novel, nanobody‐induced, mechanism of TAFI inactivation and its in vivo application

“…Foley et al . demonstrated that TAFI is effective in cleaving a small substrate, but due to its inability to cleave plasmin‐modified fibrin, the authors concluded that it had no effect on the attenuation of fibrinolysis. In addition to the intrinsic zymogen activity, Mishra et al .…”

“…However, Willemse et al [13] suggested that the clot lysis experiments reported by Valnickova were compromised by in vitro activation of TAFI. Foley et al [14] demonstrated that TAFI is effective in cleaving a small substrate, but due to its inability to cleave plasmin-modified fibrin, the authors concluded that it had no effect on the attenuation of fibrinolysis. In addition to the intrinsic zymogen activity, Mishra et al [15] demonstrated that it is possible to enhance the zymogen activity of TAFI by nanobodies.…”