Location of the Antithrombin-binding Sequence in the Heparin Chain

The antithrombin-binding region of heparin is a pentasaccharide sequence with the predominant structure -GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-Ido A(2-OSO3)- GlcNSO3(6-OSO3)-. By using the 3-O-sulfated glucosamine residue as a marker for the anti-thrombin-binding sequence, the location of this sequence within the heparin chain was investigated. Heparin with high affinity for antithrombin (HA-heparin) contains few N-acetyl groups located outside the antithrombin-binding region, and cleavage at such groups was therefore expected to be essentially restricted to this region. HA-heparin was cleaved at N-acetylated glucosamine units by partial deacetylation followed by treatment with nitrous acid at pH 3.9, and the resulting fragments with low affinity for anti-thrombin (LA-fragments) were recovered after affinity chromatography on immobilized antithrombin. The LA-fragments were further divided into subfractions of different molecular size by gel chromatography and were then analyzed with regard to the occurrence of the nonreducing terminal GlcA-GlcNSO3(3,6-di-OS-O3)- sequence. Such units were present in small, intermediate-sized as well as large fragments, suggesting that the antithrombin-binding regions were randomly distributed along the heparin chains. In another set of experiments, HA-heparin was subjected to limited, random depolymerization by nitrous acid (pH 1.5), and the resulting reducing terminal anhydromannose residues were labeled by treatment with NaB3H4. The molecular weight distributions of such labeled LA-fragments, determined by gel chromatography, again conformed to a random distribution of the antithrombin-binding sequence within the heparin chains. These results are in apparent disagreement with previous reports (Radoff, S., and Danishefsky, I. (1984) J. Biol. Chem. 259, 166-172; Rosenfeld, L., and Danishefsky, I. (1988) J. Biol. Chem. 263, 262-266) which suggest that the antithrombin-binding region is preferentially located at the nonreducing terminus of the heparin molecule.