title: 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.
abstract: 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 Ca2+ or in the presence of EDTA. To test the possibility that the anionic Gla domain of FXa, when not neutralized by Ca2+ ions, prevents heparin binding to FXa, the heparin and pentasaccharide dependence of FXa inactivation by AT in both the absence (100 microM EDTA) and presence of Ca2+ (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 Ca2+ (k2 = 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 Ca2+ (k2 = 5.7-8.0 x 10(5) M-1 s-1). However, in the presence of an optimum concentration of heparin ( approximately 50 nM) the inactivation rate constant of FXa in the presence of Ca2+ (k2 = 4.4 x 10(7) M-1 s-1) was 13-fold higher than the rate constant in the absence of Ca2+ (k2 = 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 Ca2+ (k2 = 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 approximately 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 Ca2+.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA. alireza-rezaie@omrf.ouhsc.edu
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000990:Antithrombin III / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D065427:Factor Xa Inhibitors; D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin; D015951:Factor Xa; D002118:Calcium