Role of calcium ions and the heavy chain of factor XIa in the activation of human coagulation factor IX

Since optimal rates of factor IX activation by factor XIa require the presence of calcium ions and the heavy chain of the enzyme as well as the active-site-containing light chain, we have studied the effects of calcium ions and the heavy chain on the reaction kinetics. Whereas the amidolytic activities of factor XIa and of its active-site-containing light chain were almost indistinguishable, the two enzymes behaved quite differently when factor IX was the substrate. Factor XIa was 100-fold more potent in the presence of Ca2+ than in its absence. On the contrary, the presence or absence of Ca2+ made very little difference in the case of the isolated light chain of factor XIa. Moreover, the enzymatic activity of the light chain was almost identical with that of intact factor XIa when Ca2+ was absent. Using an optimal concentration of Ca2+, we studied the activation in the presence of various concentrations of two monoclonal antibodies, one (5F4) directed against the light chain of factor XIa and the other (3C1) against its heavy chain. Analysis of 1/V vs. 1/S plots showed that whereas inhibition by 5F4 was noncompetitive, 3C1 neutralized the enzyme in a classical competitive fashion. We conclude that in the calcium-dependent activation of factor IX by factor XIa the heavy chain of the enzyme is involved in the binding of the substrate and this is essential for optimal reaction rates.