Further characterization of the antithrombin-binding sequence in heparin

An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin and previously found to have the following predominant structure (see formula in text) has been studied further. High-voltage, paper electrophoresis of the 3H-labelled disaccharides obtained by deamination with HNO2 (pH 1.5) followed by reduction with Na[3H]BH4 showed approximately 25% of mono-O-sulfated components, in addition to L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol (6-O-SO3). The monosulfated disaccharides were identified by high pressure, ion-exchange chromatography as L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol, L-Iduronic acid-2,5-anhydro-D-[3H]mannitol(6-O-SO3). and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol L, iduronic acid 2,5-anhydro-D-[3H]mannitol(6-O-SO3), and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol. These components originated from the reducing, terminal disaccharide residue (units 7 and 8), as indicated by selective labelling with Na[3H]-BH4. The structural variability within this region suggests that it is not part of the antithrombin-binding sequence. Neither enzymic removal of the non-sulfated L-iduronic acid unit 1 nor N-deacetylation (by hydrazinolysis) at unit 2 had any significant effect on the affinity of the octasaccharide for antithrombin. However, removal of the disaccharide corresponding to units 1 and 2, by selective deamination of the N-deacetylated octasaccharide, yielded a low-affinity hexasaccharide. In addition, a high-affinity deamination product was formed, presumably an octasaccharide containing a 6-sulfated 2-deoxy-2-C-formyl-D-pentofuranosyl unit due to ring contraction in unit 2. These results suggest that the 6-sulfate group in unit 2 may be involved in antithrombin binding. It is concluded that the antithrombin binding site in heparin is represented by the pentasaccharide sequence extending from unit 2 to unit 6 of the octasaccharide studied.