Structural Variation in the Antithrombin III Binding Site Region and Its Occurrence in Heparin from Different Sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz ¹H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as AUAp2S (l→4)-α-D-GlcNp2S6S(1→4)-β-D-GlcAp(l→4)-α-D-GlcNp2S3S6S (where ΔUA represents 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid). The ¹H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient Polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as →4)-α-D-GlcNp2S6S(1→4)-β-D-GlcAp(1→4)-α-D-GlcNp2S3S6S(l→ 4)-α-L-IdoAp2S(l→4)-α-D-GlcNp2S6S(l→ This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.