The role of phospholipid and factor VIIIa in the activation of bovine factor X

Abstract

The kinetic parameters of bovine factor X activation by bovine factor IXa have been determined in the absence and presence of Ca2+, thrombin-activated bovine factor VIII (VIIIa), and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 75/25; mol/mol). Factor IXa in the absence of Ca2+, factor VIIIa, and phospholipid is able to catalyze factor X activation. The Km for factor X is 299 microM which is well above its concentration in bovine plasma, about 0.2 microM. The Vmax of factor Xa formation is 0.0022 mol of Xa . min-1 . mol of IXa-1 under these conditions. Addition of Ca2+ has little effect on the kinetic constants of factor X activation by factor IXa. In the presence of 10 mM CaCl2 the Km for factor X is 181 microM, and the Vmax is 0.0105 mol of Xa . min-1 . mol of IXa-1. The presence of 10 microM phospholipid dramatically decreases the Km for factor X to 0.058 microM, and the Vmax becomes 0.0025 mol of Xa . min-1 . mol of IXa-1. The Vmax of factor Xa formation slightly increases when more phospholipid is present in our experiments, and there is a considerable increase of the Km for factor X at higher phospholipid concentrations. Therefore, the Km measured in the presence of phospholipid has to be regarded as an apparent Km. The possible explanations for this phenomenon are discussed. For the complete factor X-activating complex (i.e. factor IXa, factor VIIIa, Ca2+, and 10 microM phospholipid) the Km for factor X is 0.0063 microM, and the Vmax is raised 200,000-fold to 500 mol of Xa . min-1 . mol of IXa-1. In order to exert its stimulating effect on factor X activation factor VIII has to be activated with thrombin. Our results show that factor IXa is an enzyme which can activate factor X at a very low rate. The stimulating effect of phospholipid in factor X activation is mainly due to an effect on the Km for factor X, bringing it within the range of the plasma concentration. The stimulatory effect of factor VIIIa is explained by its 200,000-fold increase of the Vmax of factor Xa formation.