PURIFICATION AND CHARACTERIZATION OF TAFI, A THROMBIN-ACTIVABLE FIBRINOLYSIS INHIBITOR

Previous studies demonstrated that tissue plasminogen activator-induce d fibrinolysis in vitro is retarded in the presence of prothrombin (II ) activation and that the anticoagulant-activated protein C appears pr ofibrinolytic by preventing the formation of thrombin (IIa)-like activ ity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assa y that is sensitive to the antifibrinolytic effect of II activation wa s developed tend was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified com ponents, is retarded when this protein is present and when II activati on occurs in situ, the protein was named TAFI (thrombin-activatable fi brinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 1 4-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kD a 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 pur ified system and the prolongation induced by II activation in barium-a dsorbed plasma. This suggests that TAFI accounts for the antifibrinoly tic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.