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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.

SUBMITTER: Mertens K

PROVIDER: S-EPMC1144343 | biostudies-other | 1984 Nov

REPOSITORIES: biostudies-other

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Project description:The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.

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

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