title: Generation and characterization of a highly stable form of activated thrombin-activable fibrinolysis inhibitor.
abstract: Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is approximately 40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.
authors.affiliation: Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, The Netherlands. P.F.Marx@amc.uva.nl
authors.forename: Pauline F
authors.initials: PF
authors.lastname: Marx
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Havik
authors.forename: J Arnoud
authors.initials: JA
authors.lastname: Marquart
authors.forename: Bonno N
authors.initials: BN
authors.lastname: Bouma
authors.forename: Joost C M
authors.initials: JC
authors.lastname: Meijers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D043424:Carboxypeptidase B / Q000737:chemistry; D025901:Carboxypeptidase B2 / Q000737:chemistry*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000737:chemistry; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D018180:Thrombomodulin / Q000737:chemistry; D013997:Time Factors
chemical_list: D011993:Recombinant Fusion Proteins; D018180:Thrombomodulin; D005340:Fibrinogen; D010958:Plasminogen; D043424:Carboxypeptidase B; D025901:Carboxypeptidase B2; D005341:Fibrinolysin