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 molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patients factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of phe(328) to Ser. The patient was homozygous for this mutation, whereas each parent of the patient 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 factor VII exhibited < 1% factor VII procoagulant activity and a 2-fold decreased 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 activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis, of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg(315)-Lys(316) peptide bond in intact F3285 factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F3285 factor VIIa to cleave substrates may result hom the apparent formation 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 suggest that Phe(328), which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.