FACTOR-VII CENTRAL - A NOVEL MUTATION IN THE CATALYTIC DOMAIN THAT REDUCES TISSUE FACTOR-BINDING, IMPAIRS ACTIVATION BY FACTOR XA AND ABOLISHES AMIDOLYTIC AND COAGULANT ACTIVITY

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII m olecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII ant igen was 38% of normal, but expressed <1% factor VII procoagulant acti vity. DNA sequence analysis of the patients factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII tha t results in a novel amino acid substitution of phe(328) to Ser. The p atient was homozygous for this mutation, whereas each parent of the pa tient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S fact or VII exhibited < 1% factor VII procoagulant activity and a 2-fold de creased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic act ivity in the presence of tissue factor. The rate of F328S factor VII a ctivation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning o f SDS gels. Temporal analysis, of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S fac tor VII degradation products (40 and 9 kDa) resulting from factor Xa p roteolysis of the Arg(315)-Lys(316) peptide bond in intact F3285 facto r VII. Computer modeling and molecular dynamics simulations of the ser ine protease domain of factor VIIa suggested that the inability of F32 85 factor VIIa to cleave substrates may result hom the apparent format ion of a hydrogen bond between Tyr(377) and Asp(338), a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings sugg est that Phe(328), which is conserved in prothrombin, factor IX, facto r X, factor VII, and trypsin, is important for factor VIIa catalysis.