BINDING OF HEPARIN TO H-KININOGEN

Abstract

The binding of heparin to kininogen was analyzed by competition of kininogen with anti thrombin for high-affinity heparin. Residual heparin binding to anti thrombin was quantified by the accelerating effect on the anti thrombin-thrombin reaction. The rate of the latter reaction was monitored by displacement of the fluorescent probe, p-aminobenzamidine, from the enzyme. A linear dependence of the observed pseudo-first-order rate constant (kobs) for the heparin-accelerated anti thrombin-thrombin reaction on heparin concentration was achieved by use of catalytic amounts (≤30 nM) of heparin, a 20-fold ratio of anti thrombin to thrombin and thrombin concentrations (0.25 μM) much below the apparent of heparin for thrombin at the high (1 mM) p-aminobenzamidine concentration used. The two-chain form of H-kininogen minimally affected the heparin-accelerated rate of the anti-thrombin-thrombin reaction at pH 7.4 in the absence of metal ions. However, at saturating concentrations of Zn2+ (10 μM), kobs was reduced to 50% at ˜15 nM kininogen and to that of the uncatalyzed reaction at ≥˜0.25 μM. Conversely, at saturating kininogen, a 50% decrease of kobs was observed at ˜0.6 μM Zn2+, i.e. in the plasma concentration range. Other metal ions were effective in the order: Zn2+˜Ni2+>Cu2+>Co2+˜Cd2+. Single-chain H-kininogen and H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent as the two-chain form, whereas L-kininogen had no effect. In the absence of metal ions, the binding of heparin to two-chain H-kini-nogen increased with decreasing pH below 7.4 in a manner consistent with involvement of protonated histidine residues. Thus, heparin presumably binds to the histidine-rich region of the light chain portion of H-kininogen. The elution of two-chain H-kininogen from immobilized dextran sulfate at pH 7.4 was shifted to higher salt concentrations in the presence of 10 μM Zn2+, indicating that metal ions may also enhance H-kininogen binding to surfaces relevant to contact activation reactions. The sensitivity of H-kininogen-surface interactions to divalent metal ions and pH suggest regulation of the interactions by these factors. Like histidine-rich glycoprotein, H-kininogen may also compete with anti thrombin for heparin during heparin therapy.