Enzymatic Preparation of Heparin Oligosaccharides Containing Antithrombin III Binding Sites

Abstract

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One-and two-dimensional 1 H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are ⌬UAp2S (134) The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.

Viral and Cellular Determinants of the Hepatitis C Virus Envelope-Heparan SulfateInteraction

“…Highly sulfated liver-derived HS was isolated as previously described (44). Heparin oligosaccharides of 2, 4, 6, 8, 10, 14, and 20 subunits and control saccharide (decasaccharide containing the pentasaccharide antithrombin III-binding site) were synthesized or isolated as previously described (34,43). Mouse anti-E1 (11B7) and anti-E2 (16A6, 2F10, 917, and AP33) monoclonal antibodies (MAbs) have been described previously (11,49).…”

Antithrombin-binding Octasaccharides and Role of Extensions of the Active Pentasaccharide Sequence in the Specificity and Strength of Interaction

“…Earlier studies on tetrasaccharides sequences adjacent to the antithrombin-binding site have demonstrated two possible variants of AT-binding sequences, suggesting a possible role of the extensions of these sequences on binding to AT (9). Longer AT-binding sequences, such as decasaccharides, were also previously isolated (8). The influence of the position of the pentasaccharide sequence along the oligosaccharide chains together with the knowledge of the role of the residues prolonging the active sequence toward both its reducing and nonreducing side are among the major goals of current heparin research (10).…”

“…are located at different sites along the oligosaccharide chains (7,8). The increasing interest in the development of "tailored" LMWHs and very low molecular weight heparins stimulates studies aimed at a better understanding at the molecular level the mechanisms of interaction between AT and AGA*IA-containing oligosaccharides.…”

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides

“…AT-binding oligosaccharides are usually obtained after partial chemical or enzymatic depolymerization of heparin and purification using affinity chromatography on an immobilized Sepharose-AT column (12). In other studies, the AT-binding oligosaccharides were isolated from LMWHs, such as enoxaparin, using a combination of orthogonal separation techniques, including gel permeation chromatography, HPLC, and AT affinity chromatography (13).…”