title: Amino acid residues in the P6-P'3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation.
abstract: Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P'3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen Bbeta-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90-95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P'3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.
authors.affiliation: Departments of Biochemistry and Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Schneider
authors.forename: Mariko
authors.initials: M
authors.lastname: Nagashima
authors.forename: Sabine
authors.initials: S
authors.lastname: Knappe
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.forename: Michael
authors.initials: M
authors.lastname: Nesheim
mesh_terms: D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D025901:Carboxypeptidase B2 / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D001224:Aspartic Acid; D012694:Serine; D001120:Arginine; D025901:Carboxypeptidase B2; D013917:Thrombin