We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (

Wakabayashi et al., J. Biol. Chem. 279:12677–12684, 2004
). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

Topics:
factor viii, binding sites, point mutation, factor xa, factor ixa, factor x, thrombin, amino acid substitution, science of chemistry, time factors

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2005, The American Society of Hematology
2004
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