title: Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage.
abstract: Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).
authors.affiliation: Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. P.F.Marx@lab.azu.nl
authors.forename: P F
authors.initials: PF
authors.lastname: Marx
authors.forename: T M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: P E
authors.initials: PE
authors.lastname: Dawson
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
authors.forename: J C
authors.initials: JC
authors.lastname: Meijers
authors.forename: B N
authors.initials: BN
authors.lastname: Bouma
mesh_terms: D025901:Carboxypeptidase B2; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D018180:Thrombomodulin; D005337:Fibrin; D002268:Carboxypeptidases; D025901:Carboxypeptidase B2; D013917:Thrombin; D008239:Lysine