Analysis of the functions of the first epidermal growth factor-like domain of factor X.

Abstract

Upon activation, factor X participates in the prothrombin activation complex. Similar to 4-carboxyglutamic acid (Gla)-domainless protein C, the Gla-domainless factor X (GDFX) contains a high affinity Ca(2+)-binding site critical for the function of these molecules. In the case of protein C, we recently demonstrated that the high affinity Ca(2+)-binding site critical for activation is outside the first epidermal growth factor (EGF) homology domain. To examine if this is also true for factor X, we have expressed in human 293 cells a deletion mutant of factor X (E2FX) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of factor X. Direct binding studies by equilibrium dialysis indicate that E2FX contains a single Ca(2+)-binding site with a dissociation constant (Kd) of 154 +/- 48 microM. The functional properties of E2FX are equivalent or improved over those of GDFX. For instance, the factor X coagulant protein of Russell's viper venom activates E2FX three times faster than recombinant GDFX. Kinetic analysis of prothrombin activation in the absence of membranes indicates that activated GDFX and E2FX bind to factor Va with equal affinity (Kd = 4.1 microM). The Ca2+ concentration required for half-maximal prothrombin activation rates in the above activation system shifted from 721 +/- 113 microM for activated GDFX to 193 +/- 64 microM for activated E2FX. GDFX and E2FX activation rates with the soluble tissue factor-factor VIIa complex were identical as was the Ca2+ dependence of the reaction. We conclude that E2FX retains a high affinity Ca(2+)-binding site and that the first EGF homology domain does not appear to have a positive functional role in the GDFX molecule. However, Ca2+ occupancy of the Ca(2+)-binding site in the first EGF domain of intact factor X may be essential for optimal prothrombin activation.