SUBSTRATE RECOGNITION BY TISSUE FACTOR-FACTOR VIIA - EVIDENCE FOR INTERACTION OF RESIDUES LYS(165) AND LYS(166) OF TISSUE FACTOR WITH THE 4-CARBOXYGLUTAMATE-RICH DOMAIN OF FACTOR-X

Tissue factor (TF) is the protein cofactor for factor VIIa (FVIIa), th e first serine protease of the clotting cascade. Previous studies usin g alanine mutagenesis have identified TF residues Lys(165) and Lys(166 ) as important for factor X (FX) activation, hypothesizing either that these residues interact with phospholipid head groups or that they di rectly or indirectly promote macromolecular substrate binding. In the recently reported x-ray crystal structure of the isolated extracellula r domain of TF, both Lys(165) and Lys(166) are solvent-exposed and pre dicted to be near the phospholipid surface in intact TF. We hypothesiz ed that these residues may in fact be ideally positioned to interact w ith the 4-carboxy-glutamate-rich domain (Gla domain) of FX. We therefo re predicted that mutations at Lys(165) and Lys(166) should have no ef fect on the activation of Gla domainless FX. To test this hypothesis, we mutated both residues Lys(165) and Lys(166) of TF to Ala, Glu, or G ln and examined the ability of these double mutants to support FVIIa-m ediated activation of FX, Gla domainless FX, and factor IX (FM). Each TF mutant was equivalent to wild-type TF in both FVIIa binding and pro motion of FVIIa amidolytic activity, However, all three mutants were m arkedly deficient in supporting FIX and FX activation, with FX activat ion rates decreased more than FIX activation rates. In both reactions, the TF mutants exhibited different extents of activity: Gln(165)-Gln( 166) > Ala(165)-Ala(166) > Glu(165)-Glu(166). In sharp contrast, all t hree TF mutants were equivalent to wild-type TF in supporting activati on of Gla domainless FX by FVIIa. Interestingly, the deficiency of the mutants in FX activation was less pronounced when Gla domainless FVII a was used in place of native FVIIa, Together, these findings suggest that TF residues Lys(165) and Lys(166) contribute to a binding site fo r the Gla domain of FX (and perhaps other substrates) and that this in teraction may be facilitated by the presence of the Gla domain of FVII a.