Generation of affinity for antithrombin III by supplemental sulfation of heparin species with low affinity for the protein.

The tributylammonium salt of a porcine heparin subfraction with low affinity for antithrombin III (Mr 7,500-18,000; anti-clotting activity, 7 USP units/mg), having degrees of sulfate substitution at D-glucosamine and L-iduronic acid residues of GlcNS 0.786, GlcN6s 0.628, and IdoA2s 0.682 mol, was reacted with 10 or 20 mol of pyridine-sulfur trioxide per mol equiv. of available hydroxyl groups in N,N-dimethylformamide at -10 degrees C for 1 h. Both chemical and NMR spectroscopic analyses revealed that sulfation proceeded exclusively at HO-6 in D-glucosamine and HO-2 in L-iduronic acid residues, according to the amount of the sulfating reagent used (GlcNS: 0.825, 0.830; GlcN6s: 0.872, 0.928; IdoA2s: 0.687, 0.749 mol, respectively). Affinity chromatography of the sulfated products on antithrombin III-Sepharose gel indicated that the polysaccharide acquired some affinity for the protein following the sulfation, as shown by the increase in the proportion of the high-affinity heparin fraction (%) from 1.1 to 6.7. Biological examination of these products indicated that sulfation at natural positions along with the polysaccharide chain resulted in significant increases in all the activities (blood anti-clotting, anti-Factor IIa, and anti-Factor Xa), and in the strength of intrinsic fluorescence of antithrombin III.