Preparation, characterization, and turnover properties of heparin-antithrombin III complexes stabilized by covalent bonds.

Abstract

High affinity heparin obtained by chromatography of clinical grade heparin on antithrombin 111-Sepharose was covalently coupled to antithrombin III using a three-step procedure: 1) introduction of free amino groups in the heparin molecule; 2) reaction of these amino groups with the bifunctional reagent tolylene-2,4-diisothiocyanate; and 3) reaction of the remaining isothiocyanate group with amino groups on antithrombin III.Amino groups were introduced in the heparin molecule by limited N-desulfation or by reaction of carboxyl groups with hexamethylenediamine with the use of a water-soluble carbodiimide.On average between l and 2 mol of N H 2 groups were introduced/ 16,000 g of heparin.The yield of coupling of the isothiocyanate-substituted heparin to antithrombin III was around 30% and coupling occurred preferentially with an apparent 1: 1 stoichiometry for both substituted heparins.The complexes were separated from free heparin by chromatography on antithrombin IJI-Sepharose or on DEAE-Sephadex and from unreacted antithrombin 111 by gel filtration on a high performance liquid chromatography column or by gradient elution from heparin-Ultrogel.