title: Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation.
abstract: The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165-->Ala, Lys166-->Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158-->Arg, Ser160-->Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TF(R158G160). In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TF(A165A166) as the cofactor, but only 3.5-fold with TF(R165G160). The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TF(R158G160) and 7-8-fold with TF(A165A166). This suggests that the molecular recognition of VII in complex with TF by the enzymes TF-VIIa and factor Xa are similar. Generation of factor IXa by TF(R158G160)-VIIa was unaltered, but reduced 2-fold with TF(A165A166). In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TF-VIIa complex.
authors.affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: S
authors.initials: S
authors.lastname: Dittmar
authors.forename: W
authors.initials: W
authors.lastname: Ruf
authors.forename: T S
authors.initials: TS
authors.lastname: Edgington
mesh_terms: D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D010743:Phospholipids; D011994:Recombinant Proteins; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa