Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) ∼1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [³H]NaBH₄. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [³H]2,5-anhydro-D-talitol (ATal_R), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which ∼6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing ∼2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO₄)→ATal_R(4-SO₄) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO₄)→GalNAc(4-SO₄)→IdoA(2-SO ₄)→GalNAc(4-SO₄)→IdoA(2-SO₄)→ ATal_R(4-SO₄). Since IdoA(2-SO₄)→GalNAc(4-SO₄) comprises only ∼5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.