Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.

An octasaccharide with high affinity for antithrombin was isolated after partial deaminative cleavage of heparin with nitrous acid. After conversion of the 2,5-anhydro-D-mannose end group to anhydro1-3Hmannitol, labeled pentasaccharide was released from the octasaccharide by periodate-alkali treatment. Incubation of the pentasaccharide with a recently discovered 3,O-sulfatase from human urine resulted in desulfation, suggesting the occurrence of a 3-sulfate group on the terminal glucosamine residue. The same glucosamine residue was recovered as a 2,5-anhydro1-3Hmannitol derivative by a procedure involving deamination of the octasaccharide with nitrous acid, reduction of the products with sodium boro3Hhydride, isolation of 3H-labeled tetrasaccharide by gel chromatography, and release of the labeled end-group by periodate-alkali treatment. Paper electrophoresis indicated disulfated anhydro3Hmannitol, presumably sulfated at C3 and C6, as a major component, along with smaller amounts of monosulfated (presumably 3-sulfated) anhydro3Hmannitol. Similar treatment of an analogous tetrasaccharide derived from heparin with low affinity for antithrombin failed to produce any disulfated anhydromannitol. These results suggest that 3-sulfated glucosamine is a unique component of high-affinity heparin, located at a specific position in the antithrombin-binding sequence of the molecule.