Purification and Characterization of TAFI, a Thrombin-activable Fibrinolysis Inhibitor

Abstract

Previous studies demonstrated that tissue plasminogen activator-induced fibrinolysis in vitro is retarded in the presence of prothrombin (II) activation and that the anticoagulant-activated protein C appears profibrinolytic by preventing the formation of thrombin (IIa)-like activity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assay that is sensitive to the antifibrinolytic effect of II activation was developed and was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified components, is retarded when this protein is present and when II activation occurs in situ, the protein was named TAFI (thrombin-activatable fibrinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa product correlates with both prolongation of lysis time and CPB-like activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen but does not prolong the lysis of clots formed in the presence of Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive inhibitor of CPB, completely inhibits prolongation of lysis by activated TAFI in a purified system and the prolongation induced by II activation in barium-adsorbed plasma. This suggests that TAFI accounts for the antifibrinolytic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.

Increased circulating levels of thrombin‐activatable fibrinolysis inhibitor in lung cancer patients

“…Under physiological conditions, liver cells are the main source of TAFI production, and during inflammation TAFI acts as an acute reactant protein as demonstrated in mice [18,26]. We have previously demonstrated that, in addition to hepatic cells, adipocytes, endothelial, and epithelial cells may also produce and secrete TAFI in vitro [21,27].…”

“…Recently, a new potent inhibitor of fibrinolysis, the thrombin-activatable fibrinolysis inhibitor (TAFI) or carboxypeptidase U, has been isolated and characterized from human plasma [18]. TAFI is a glycoprotein synthesized by the liver that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis.…”

Inhibition of carboxypeptidase U (TAFIa) activity improves rt-PA induced thrombolysis in a dog model of coronary artery thrombosis

“…An important regulator of the interaction between fibrin, plasminogen and t-PA is exerted by carboxypeptidase U (CPU), also known as active Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) [36,37]. The proposed mechanism of action of CPU in clotting plasma is that it removes C-terminal lysine residues from partly degraded fibrin, which results in lower plasminogen binding, and thereby lower plasmin generation and a retarded lysis rate.…”

“…The Gusto IIB trial [164] showed that hirudin and heparin in combination with rt-PA produced similar effects on the combined endpoints death and myocardial infarction in patients with acute myocardial infarction. It had been suggested that thrombin trapped in, or bound to, the fibrin in a thrombus may play an important role in resistance to thrombolysis and subsequent reocclusion [37,141]. It was therefore hypothesised that a small, potent thrombin inhibitor (MW 429) could be more effective in inhibiting fibrin-bound thrombin than the heparin: antithrombin complex (MW ≈75000) and the polypeptide hirudin (MW ≈7000).…”

The Effect of Hormone Replacement Treatment on Thrombin-Activatable Fibrinolysis Inhibitor Activity Levels in Patients with Hashimoto Thyroiditis

“…Thrombin plays a critical role in coagulation and the fibrinolytic system ( (8)(9)(10). These residues are important for binding and activating plasminogen.…”