Binding of heparin to human high molecular weight kininogen

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Abstract
The binding of heparin to high molecular weight kininogen (H-kininogen) was analyzed by the effect of kininogen in decreasing the heparin-induced enhancement of the rate of inactivation of thrombin by antithrombin. The conditions were arranged so that the heparin-catalyzed antithrombin-thrombin reaction, monitored in the presence of the reversible thrombin inhibitor p-aminobenzamidine, followed pseudo-first-order kinetics and the observed rate constant (kobsd) varied linearly with the heparin concentration. In the absence of metal ions, H-kininogen minimally affected kobsd, measured at a constant concentration of heparin with high affinity for antithrombin (30 nM), at I = 0.15, pH 7.4 and 25.degree. C. However, at a saturating concentration of Zn2+ (10 .mu.M), kobsd was reduced to 50% at .apprx. 20 nM H-kininogen and to that of the uncatalyzed reaction at .gtoreq. .apprx. 0.2 .mu.M H-kininogen. Conversely, at a saturating concentration of H-kininogen (0.5 .mu.M), kobsd was decreased to 50% at .apprx. 0.6 .mu.M Zn2+ and to the Kobsd of the uncatalyzed reaction at .gtoreq. 10 .mu.M Zn2+. Other metal ions were effective in the order Zn2+ .apprx. Ni2+ > Cu2+ .apprx. Co2+ .apprx. Cd2+. The single-chain and two-chain forms of H-kininogen and the H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent, whereas low molecular weight kininogen had no influence. Heparin with low affinity for antithrombin reversed the effect of H-kininogen, together with Zn2+, in decreasing the rate enhancement caused by high-affinity heparin at concentrations consistent with the two heparin species binding similarly to H-kininogen. In the absence of metal ions, the effect of H-kininogen on the rate of the heparin-catalyzed antithrombin-thrombin reaction increased with decreasing pH below 7.4 in a manner indicating involvement of protonated histidine residues. A lower metal-dependent heparin-neutralizing ability was observed in H-kininogen-deficient than in normal plasma. These findings suggest that heparin with both high and low affinity for antithrombin can bind with appreciable affinity to the histidine-rich region of the light-chain portion of H-kininogen. At physiological pH, such binding must be mediated by divalent metal ion binding to unprotonated histidine residues, while at lower pH the polysaccharide binds directly to protonated histidines. Like histidine-rich glycoprotein, H-kininogen may compete with antithrombin for heparin during heparin therapy.