Affinity of Antithrombin II/III to Insolubilized Heparin

Five heparin fractions of different molecular weight range were obtained from commercial sodium heparin (Sigma) by gel chromatography on Sephadex G-200. Each fraction was bound separately to Biogel A-15 m by the cyanogen bromide reaction. Microcolumns containing equal amounts of the individual heparin agarose batches were prepared. A protein fraction from human plasma containing antithrombin II/III but no fibrinogen, which was obtained by polyethylene glycol precipitation, was passed through the columns. After extensive washing with 0.15 and 0.3 M buffered sodium chloride antithrombin was eluted with 1.0 M buffered sodium chloride. The uptake of antithrombin did not differ greatly in all five heparin agarose preparations. However, the amount of antithrombin eluted with 1.0 M NaCl varied considerably depending on the insolubilized heparin fraction. Almost no antithrombin activity was eluted from the agarose grafted with the highest molecular weight heparin, while all of the antithrombin was recovered from the agarose with the intermediate molecular weight heparin.Our findings of different affinities of antithrombin to insolubilized heparin in dependance of its molecular weight suggest similar implications for the practical use of heparin in anticoagulant therapy.