AN INDIRECT KINETIC METHOD FOR ESTIMATING THE AFFINITY BETWEEN HEPARIN AND HEPARIN COFACTOR II

The heparin-dependent inactivation of alpha-thrombin by heparin cofactor II was studied in buffer media with a pH ranging from 6 to 9 and an ionic strength from 0.05 M to 0.80 M. We used a heparin fraction with a mean M_r of 16,000 .The log dose response curves (logarithm of the 2nd order inactivation constant vs. the logarithm of heparin concentration) display a sigmoidal behavior. The lower and upper limiting plateau and the steepness of the ascending limb are characteristic for every pH and ionic strength. Similar log dose response curves can be observed for the heparin-mediated inactivation of factor Xa and plasmin by ATIII,indicating that enhancement of the inhibition only depends on heparin-inhibitor binding despite the presence of heparin-enzyme complexes.This type of inactivation mechanism is clearly different from the one observed for the thrombin- and factor IXa - ATIII interactions which are characterized by a maximum in the log dose response curves.Therefore we can make the assumption that heparin-inhibitor binding is of major importance in the heparin-mediated thrombin-HCII interaction.Our experimental data were fit to a model which describes the dependence of the observed inactivation constant upon the concentration of heparin-HCII complex.This complex concentration is a function of the initial heparin and HCII concentrations and the dissociation constant K₀ of the heparin-HCII complex.The model allows the estimation of K₀ in various buffer media.A decrease of pK₀ with increasing buffer concentration can be observed.The upper limiting plateau value for k_obs which is often referred to as the intrinsic activity of heparin also decreases with increasing ionic strength.At pH 7 and 8 and an ionic strength of 0.4 M we-found K₀ values of 1.00E-07 M.At pH 6 and an ionic strength of 0.8 M Kq eguals 4.00E-04 M indicating a markedly decreased affinity of heparin for HCII.Through a detailed analysis of the pH profile for K₀ we might gain insight in the nature of the binding sites for heparin on the inhibitor.