A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography.

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.