Effect of NaCl on the association of thrombin with heparin

The association of heparin with thrombin was investigated by fluorometric titration. A maximum of 25% of the fluorescence of fluorescein‐labeled heparin (FTC‐heparin) was quenched at thrombin saturation in the absence of NaCl. FTC‐heparin (H) associated tightly with thrombin (T) and the association constant of the ternary complex, H2T, formed in the absence of NaCl, was calculated to be 1.7 × 108M−1. However, the association was strongly influenced by the NaCl concentration, and the association constant of the equimolar complex, HT, formed in 0.15M NaCl was found to be 1 × 106M−1. The first‐order rate constant, kapp, for inactivation of thrombin by antithrombin III (AT III) and low‐affinity heparin (LA‐heparin) was comparable with that of high‐affinity heparin (HA‐heparin) in the absence of NaCl, but decreased with an increase in the concentration of NaCl. The decreased enhancement of the thrombin‐AT III reaction by LA‐heparin at high NaCl concentration appeared to result from a decreased association of thrombin with LA‐heparin, thus reducing the formation of the ternary complex, thrombin‐LA‐heparin‐AT III.