The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

Abstract

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors α 1-antitrypsin, antithrombin III and α 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca 2+, Mn 2+ and Mg 2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62·10 4 M −1-·min −1 in the absence of divalent cations to a maximum of 6.40·10 4 M −1·min −1 at 5 mM Ca 2+, 8.10·10 4 M −1·min −1 at 5 mM Mn 2+, with a slight decresae in rate at higher cation concentrations. Mg 2+ caused a gradual rise in rate constant to 5.65·10 4 M −1·min −1 at 20 mM. The rate constant for the inhibition of factor Xa by α 1-antitrypsin in the absence of divalent cations was 5.80·10 3 M −1·min −1. Ca 2+ increased the rate to 1.50·10 4 M −1·min −1 at 5 mM and Mn 2+ to 2.40·10 4 M −1·min −1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg 2+ caused a gradual rise in rate constant to 1.08·10 4 M −1·min −1 at 10 mM. The rate constant for the factor Xa- α 2-macroglobulin reaction was raised from 6.70·10 3 M −1·min −1 in the absence of divalent cations to a maximum of 4.15·10 4 M −1·min −1 at 4 mM Ca 2+, with a decreae to 3.05·10 4 M −1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca 2+ and Mn 2+ may be due to factor Xa dimerization.