Extension and structural variability of the antithrombin-binding sequence in heparin

Area of Science:

• Biochemistry
• Glycobiology
• Pharmacology

Background:

• Heparin is a complex polysaccharide known for its anticoagulant properties.
• Its biological activity is mediated through binding to antithrombin.
• Understanding the structural basis of this interaction is crucial for drug development.

Purpose of the Study:

• To investigate the structural requirements of heparin oligosaccharides for high-affinity binding to antithrombin.
• To identify the minimal sequence and specific functional groups essential for antithrombin interaction.

Main Methods:

• Partial deaminative cleavage of pig mucosal heparin using nitrous acid to generate oligosaccharides.
• Spectroscopic techniques (near-ultraviolet difference spectra, circular dichroism, fluorescence) to study oligosaccharide-antithrombin interactions.
• Determination of binding constants (Kd) for various oligosaccharide fragments and intact heparin.

Main Results:

• High-affinity binding to antithrombin was observed for oligosaccharides containing an octasaccharide sequence, with binding constants similar to intact heparin.
• Hexasaccharide fragments showed significantly lower affinity (approximately 1000-fold less), indicating the importance of specific structural elements.
• The N-sulfate group at position 6 and the pentasaccharide sequence (2-6) were identified as critical for high-affinity binding.

Conclusions:

• The N-sulfate group at position 6 of the heparin oligosaccharide is essential for high-affinity binding to antithrombin.
• A specific pentasaccharide sequence (2-6) represents the core antithrombin-binding region within the heparin molecule.
• Variations in N-acetylation/sulfation and the D-gluco configuration at position 3 influence binding affinity and may be related to heparin biosynthesis.