The antithrombin-binding sequence of heparin. Location of essential N-sulfate groups. Academic Article uri icon

abstract

  • Products obtained by partial depolymerization of pig mucosal heparin with nitrous acid were fractionated by affinity chromatography on immobilized antithrombin. A high affinity octasaccharide fraction was recovered and reduced with sodium [3H]borohydride, yielding terminal 2,5-anhydro-D-[1-3H]mannitol residues. Partial N-desulfation of this material by treatment with aqueous dimethylsulfoxide resulted in the formation of two distinct species, with low and medium affinity for antithrombin, respectively, in addition to components that remained a high affinity for the protein. The low and medium affinity fragments were converted into high affinity components by sulfation of free amino groups; N-acetylation was without effect. The N-substituents on the 2 glucosamine units at positions 4 and 6 (the 3H-labeled end group being at position 8) were defined by gel chromatography of the 3H-labeled oligosaccharides released on selective deamination of Nor N-unsubstituted D-glucosamine residues. In the high affinity fraction, both glucosamine residues, at positions 4 and 6, respectively, were N-sulfated. The medium affinity fraction contained an N-sulfated glucosamine unit in position 4 and an N-unsubstituted unit in position 6. The low affinity components were either N-unsubstituted in both positions or N-unsubstituted in position 4 and N-sulfated in position 6. It is concluded that high affinity binding of heparin to antithrombin requires the presence of two consecutive N-sulfated glucosamine residues in specific positions of the antithrombin-binding sequence; loss of either one of these N-sulfate groups (with or without subsequent N-acetylation) results in a distinct, and appreciable, decrease in binding affinity (and in anticoagulant activity).

published proceedings

  • J Biol Chem

altmetric score

  • 6

author list (cited authors)

  • Riesenfeld, J., Thunberg, L., Hk, M., & Lindahl, U.

citation count

  • 95

complete list of authors

  • Riesenfeld, J||Thunberg, L||Höök, M||Lindahl, U

publication date

  • March 1981