Relative Contributions Of Thrombin And Antithrombin III Affinities Of Heparin Fractions To The Rate Of Inactivation Of Thrombin By Antithrombin III

Abstract

Heparin enhances the rate of inactivation of thrombin (IIa) and other coagulant proteases by antithrombin III (AT III). The anticoagulant activity of heparin is associated with heparin moieties which have high affinity to AT III. However, the contribution of the IIa affinity to the anticoagulant activity has not been as clear. Standard porcine mucosal heparin was fractionated on affinity columns consisting of purified human AT III and IIa immobilized on agarose to determine the effect of heparins of various affinities on the second order rate constant (K") for the inactivation of IIa by AT III. Results are expressed as the rate enhancement factor (REF) which is defined as the ratio of the K" in the presence of 50 ng/ml heparin to the K" in the absence of heparin. The REF for unfractionated heparin was 9.5. Chromatography on either AT III-agarose or IIa-agarose resulted in elution of three heparin fractions corresponding to a void volume fraction, a low affinity fraction and a high affinity fraction. The REF’s of the fractions eluted from AT III-agarose were 1.3, 1.4 and 18.8, respectively while the REF’s for the corresponding IIa fractions were 5.6, 14.9 and 18.1, respectively. Rechromatography of the high affinity fraction from AT III-agarose on the IIa-agarose column resulted in the elution of three fractions with REF’s of 17.9, 21.1 and 27.0, respectively. Conclusions: 1. Heparin affinity to AT III is critical for anticoagulant activity as all of the activity is found in the high affinity fraction. 2. Heparin affinity to IIa also contributes to anticoagulant activity but is not critical as significant activity is observed in low affinity fractions. 3. High affinity to both IIa and AT III results in greater activity than that seen with fractions of high affinity to either protein alone.