The influence of heparin, NaCl and CaCl2 on the rate of the thrombin-antithrombin III reaction

The rate of inactivation of thrombin by antithrombin III was studied in the presence of heparin using molar concentrations of the reactants in the order: antithrombin III > thrombin > heparin. Reaction rates were calculated assuming either pseudo-first-order kinetics or second-order kinetics.Heparin, up to 0.25 μg/ml (2.2 × 10−2 μM), affected the reaction in such a way that for each 0.1 μg added, the second-order constant increased by 17 × 105 M−1 min−1. Increases in the concentration of antithrombin III at a constant concentration of heparin yielded higher pseudo-first-order constants but fractional contribution of heparin to the overall increment remained approximately the same. In contrast, increases in the ratio of enzyme to heparin resulted in decreases in pseudo-first-order constants.Sodium chloride, in the range from 0.08M to 0.22M, accelerated the inactivation reaction so that an increase by 0.1M of NaCl raised the second-order constant by 12.3 × 105 M−1 min−1. Indirect evidence was obtained supporting the view that NaCl exerts its effect by promoting the recycling of non-stoichiometric amounts of heparin.Calcium chloride, in the range from 1.2 mM to 5.0 mM, also accelerated the inactivation reaction by an unknown mechanism which was distinct from that of NaCl. In the presence of CaCl2, kinetics of inactivation appear to have depended on the choice of the substrate (fibrinogen or N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide-HCl) for measuring residual thrombin activity. Possible reasons for this difference are discussed.