Calcium enhances heparin catalysis of the antithrombin factor Xa reaction by a template mechanism - Evidence that calcium alleviates Gla domain antagonism of heparin binding to factor Xa

It is believed that heparin accelerates factor Xa (FXa) inactivation by antithrombin (AT) by conformationally activating the inhibitor rather than by bridging AT and FXa in a ternary complex (template effect). This is derived from kinetic studies done in the absence of Ca(2+) or in the presence of EDTA. To test the possibility that the anionic Gla domain of FXa, when not neutralized by Ca(2+) ions, prevents heparin binding to FXa, the heparin and pentasaccharide dependence of FXa inactivation by AT in both the absence (100 mu M EDTA) and presence of Ca(2+) (2.5 mM) was studied using wild-type FXa and a FXa derivative that lacks the Gla domain (GDFXa). AT inactivated both FXa derivatives similarly in both the absence and presence of Ca(2+) (k(2) = 1.7-2.5 x 10(3) m(-1) s(-1)). The active AT-binding pentasaccharide also accelerated the inactivation rates of both derivatives similarly in both the absence and presence of Ca(2+) (K(2) = 5.7- 8.0 x 10(5) M(-1) s(-1)). However, in the presence of an optimum concentration of heparin (similar to 50 nM) the inactivation rate constant of FXa in the presence of Ca(2+) (k(2) = 4.4 x 10(7) M(-1) s(-1)) was 13-fold higher than the rate constant in the absence of Ca(2+) (k(2) = 3.5 x 10(6) M(-1) s(-1)). Heparin acceleration of GDFXa inactivation by AT was rapid and insensitive to the presence or absence of Ca(2+) (k(2) = 5.1-5.9 x 10(7) M(-1) s(-1)). The additional cofactor effect of heparin with all FXa derivatives was a bell-shaped curve, which disappeared if the ionic strength of the reaction was increased to similar to 0.4. These results suggest that although the major effect of heparin in acceleration of FXa inactivation is through a heparin-induced conformational change in the reactive site loop of AT, the template effect of heparin, nevertheless, contributes significantly to rapid FXa inactivation at physiological Ca(2+).