The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

Abstract

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.