SITE-DIRECTED MUTAGENESIS BUT NOT GAMMA-CARBOXYLATION OF GLU-35 IN FACTOR VIIA AFFECTS THE ASSOCIATION WITH TISSUE FACTOR

Factor VIIa is a vitamin K-dependent enzyme whose gamma-carboxyglutami c acid (Gla)-containing domain is important for calcium ion-dependent binding to the cofactor tissue factor and membrane surfaces, This doma in contains 10 Gla residues, the individual roles and importance of,wh ich are not known, Comparisons with the homologous protein C, factor I X and prothrombin may provide functional information on the first nine cia residues, whereas no data can be extrapolated to Gla-35 in factor VIIa. Therefore, the effects of posttranslational gamma-carboxylation and site-directed mutagenesis of Glu-35 were investigated. Mutations to Asp, Gin or Val all lead to a lower affinity for tissue factor by d ecreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Glu or Gla side chains at position 35 appear to fulfil the functional roles equally well.