title: Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor.
abstract: Thrombin-activatable fibrinolysis inhibitor (TAFI) circulates as an inactive proenzyme of a carboxypeptidase B-like enzyme (TAFIa). It functions by removing C-terminal lysine residues from partially degraded fibrin that are important in tissue-type plasminogen activator mediated plasmin formation. TAFI was classified as a metallocarboxypeptidase, which contains a Zn(2+), since its amino acid sequence shows approximately 40% identity with pancreatic carboxypeptidases, the Zn(2+) pocket is conserved, and the Zn(2+) chelator o-phenanthroline inhibited TAFIa activity. In this study we showed that TAFI contained Zn(2+) in a 1:1 molar ratio. o-Phenanthroline inhibited TAFIa activity and increased the susceptibility of TAFI to trypsin digestion. TAFIa is spontaneously inactivated (TAFIai) by a temperature-dependent intrinsic mechanism. The lysine analogue epsilon-ACA, which stabilizes TAFIa, delayed the o-phenanthroline mediated inhibition of TAFIa. We investigated if inactivation of TAFIa involves the release of Zn(2+). However, the zinc ion was still incorporated in TAFIai, indicating that inactivation is not caused by Zn(2+) release. After TAFIa was converted to TAFIai, it was more susceptible to proteolytic degradation by thrombin, which cleaved TAFIai at Arg(302). Proteolysis may make the process of inactivation by a conformational change irreversible. Although epsilon-ACA stabilizes TAFIa, it was unable to reverse inactivation of TAFIa or R302Q-rTAFIa, in which Arg(302) was changed into a glutamine residue and could therefore not be inactivated by proteolysis, suggesting that conversion to TAFIai is irreversible.
authors.affiliation: Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Centre, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. P.F.Marx@azu.nl
authors.forename: Pauline F
authors.initials: PF
authors.lastname: Marx
authors.forename: Bonno N
authors.initials: BN
authors.lastname: Bouma
authors.forename: Joost C M
authors.initials: JC
authors.lastname: Meijers
mesh_terms: D025901:Carboxypeptidase B2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis; D013054:Spectrophotometry, Atomic; D013917:Thrombin / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D025901:Carboxypeptidase B2; D013917:Thrombin; D015032:Zinc