Abstract
A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.
Highlights
Materials other small heparin oligosaccharides, which are spe- The sodium salt of heparin from porcine intestinal mucosa (167 cific for coagulation factor Xa, it inhibits both factors USP units/mg) was obtained from Hepar Industries
IIa and Xa through antithrombin111. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor I1 to inhibit thrombin and shows over half of heparin’s heparin cofactor 11-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin 111, and heparin superfine, Sephadex G-10, and cyanogen bromide-activated Sepharose 4B were from Pharmacia P-L Biochemicals
HPLC gel-permeation chromatography was done on Toyo Soda TSK-Gel G3000SW 0.75 X 50-cm and G2000SW 0.75X 50-cm columns with a 0.75 X 10-cm guard column
Summary
The abbreviations used are: ATIII, antithrombin III; HCII, hep- frozen, freeze-dried, and stored at -70 “C. arin cofactor 11; p, pyranose; DSS, 3-(trimethylsilyl)-l-propanesul- Low-pressure GPC of Heparin-derived Oligosaccharides-The fonic acid sodium salt; GPC, gel-permeation chromatography; SAX, freeze-dried oligosaccharide mixture was reconstituted with distilled strong-anion exchange; HPLC, high-pressure liquid chromatography; water to a concentration of 83 mg/ml One ml of this solution was aPTT, activated partial thromboplastin time. Oligosaccharide binding to ATIII was demonstrated by loading 200 pg of sample on a 4-ml ATIII-Sepharose column equilibrated with 100 mM TrisHC1 buffer at pH 7 and eluting with a 50-ml linear sodium chloride gradient (0-2 M)in the same buffer [24]. The Kd was determined by subtracting nonspecific binding (not displaceable by heparin) from total binding and assuming a binding stoichiometry of 1:l
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