Calcium enhances factor Xa activity independent of gamma-carboxyglutamic acid residues

We investigated the gamma-carboxyglutamic acid (Gla) independent effect of calcium on the activity of human factor Xa. The effect of calcium on the reaction rate of factor Xa was compared using native and Gla-modified forms of human factor Xa [chemically decarboxylated (Gla-modified, 10 Gla residues modified/mol) and Gla-domainless (chymotrypsin-treated)]. Factor Xa activity was assessed by hydrolysis of a synthetic tripeptide nitroanilide substrate, by p-aminobenzamidine binding to the active site and by inhibition with antithrombin III. Calcium (1 mM) increased, by 25-35%, the amidolytic hydrolysis rates of all three factor Xa derivatives. Calcium had an apparent Kd of approximately 200 uM with both native and modified forms of factor Xa. However, there was no change in binding of p-aminobenzamidine, a small fluorescent probe, to factor Xa in the presence of calcium. Calcium (1 mM) increased the inhibition reaction rates of native and modified forms of factor Xa with antithrombin III by 20-30%. Magnesium (1 mM) showed greatly reduced effects on factor Xa activity relative to activities with calcium. We conclude that Gla-independent calcium interactions with factor Xa are important for some catalytic activities of this blood coagulation protease.