Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor.
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.
Bajzar
L
L
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Manuel
R
R
Nesheim
M E
ME
D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D015951:Factor Xa / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D006868:Hydrolysis; D007700:Kinetics; D011486:Protein C / Q000378:metabolism; D013386:Succinates / Q000494:pharmacology; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*
D004792:Enzyme Precursors; D011486:Protein C; D013386:Succinates; C022014:guanidinoethylmercaptosuccinic acid; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D013917:Thrombin; D015951:Factor Xa