INFLUENCE OF MUTATIONS IN TISSUE FACTOR ON THE FINE SPECIFICITY OF MACROMOLECULAR SUBSTRATE ACTIVATION

The C-terminal fibronectin-type-III-like module of the tissue factor ( TF) extracellular domain plays a requisite role in the activation of m acromolecular substrates by factor VIIa (VIIa) in complex with TF. Unl ike the mutations Lys(165) --> Ala, Lys(166) --> Ala in TF, which prev ent efficient proteolysis of factor X, we found that the coagulant def ect of a site-specific Trp(158) --> Arg, Ser(160) --> Gly replacement mutant of TF is largely attributable to the inability of TF to efficie ntly support the activation of the bound zymogen VII to the active pro tease VIIa. Binding studies demonstrated comparable affinity of bindin g of VIIa or VII by wild-type TF and TFRI58G160. In comparison with wi ld-type TF, the catalytic efficiency of factor X activation was reduce d 56-fold with TFA165A166 as the cofactor, but only 3.5-fold with TFR1 58G160. The activation of VII bound to TF by factor Xa or VIIa was red uced 2-fold in the presence of TFR157G160 and 7-8-fold with TFA165A166 . This suggests that the molecular recognition of VII in complex with TF by the enzymes TF-VIIa and factor Xa are similar. Generation of fac tor IXa by TFR158G160-VIIa was unaltered, but reduced 2-fold with TFA1 65A166. In addition, the mutations affected the cleavage of the two sc issile bonds of factor IX differently, providing further support for t he idea that the cofactor, TF, influences the fine specificity of acti vation of macromolecular substrates by the TF-VIIa complex.