Contribution of chemical 6-O-sulfation of the aminodeoxyhexose residues in whale heparin with high affinity for antithrombin III to its anticoagulant properties.
The tributylammonium salt of whale (Balaenoptera borealis L.) intestinal heparin with high affinity for antithrombin III, whose degrees of sulfate-substitution in D-glucosamine and L-iduronic acid residues are GlcNS 0.738, GlcN6S 0.384, and IdoA2S 0.510 mol, was reacted with 2.5, 5.0, or 10.0 mol of pyridine-sulfur trioxide/mol of available hydroxyl groups in N,N-dimethylformamide at -10 degrees C for 1 h. Both chemical and NMR spectroscopic analyses revealed that an exclusive 6-O-sulfation of the D-glucosamine residues proceeded, according to the amount of the sulfating reagent used (GlcN6S: 0.476, 0.585, and 0.641 mol, respectively), the degree of sulfation at other natural substitution positions in the polysaccharide being unchanged, without any detectable unnatural sulfate-substitution. Biological examination of these products indicated that the 6-O-sulfation in the original whale heparin resulted in significant increases in blood clotting and anti-Factor IIa activities (maximal 43 and 82% increases, respectively), and in a moderate increase in the ability to bind antithrombin III, that is, in anti-Factor Xa activity and in intrinsic fluorescence enhancement of the protein (maximal 28 and 30% increases, respectively), together with a maximal 10% increase in the proportion of heparin species with higher affinity for antithrombin III, released with 1.0-3.0 M NaCl from antithrombin III-Sepharose.
Uchiyama
H
H
School of Pharmaceutical Sciences, Kitasato University, Tokyo.
Metori
A
A
Ogamo
A
A
Nagasawa
K
K
D000818:Animals; D000925:Anticoagulants; D000990:Antithrombin III / Q000032:analysis*; D002417:Cattle; D002591:Cetacea / Q000378:metabolism*; D005453:Fluorescence; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000032:analysis; D006493:Heparin / Q000032:analysis* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013431:Sulfates / Q000032:analysis; D013552:Swine; D013696:Temperature; D014907:Whales / Q000378:metabolism*
D000925:Anticoagulants; D013431:Sulfates; C047200:1-deoxyglucosamine; D000990:Antithrombin III; D006493:Heparin; D005944:Glucosamine