Affinity capillary electrophoresis for the determination of binding affinities for low molecular weight heparins and antithrombin‐III

Abstract

The anticoagulant properties of heparin stem in part from high-affinity binding to antithrombin-III (AT-III) inducing a 300-fold increase in its inhibitory activity against the coagulation protease factor Xa. The minimal structural requirements for AT-III binding are contained in the rare heparin pentasaccharide sequence containing a 3,6-O-sulfated N-sulfoglucosamine residue. ACE is used in this work to measure the relative AT-III binding affinities of the low molecular weight heparins (LWMHs) dalteparin, enoxaparin, and tinzaparin and the synthetic pentasaccharide drug fondaparinux (Arixtra). Determination of the AT-III binding affinities of the LWMHs is complicated by their inherent structural heterogeneity and polydispersity. The fractional composition of 3-O-sulfo-N-sulfoglucosamine residues was determined for each drug substance using 2D NMR and used in the interpretation of the ACE results.

Capillary Electrophoresis

“…Heparin’s high affinity for antithrombin III (AT-III) gives rise to its anticoagulant properties. Dinges et al used ACE to test the AT-III binding affinities of low-molecular weight heparins . Linear isotherm and double reciprocal plotting methods were used for determining the effective binding ligand concentration, with linear isotherm data producing values between 2 and 16 × 10 –8 M for the studied components.…”

Capillary Electrophoresis for Quantitative Studies of Biomolecular Interactions

“…Affinity interaction between the AT-III-protein and a set of ligands, including the low molecular weight heparins (LMWHs) and the synthetic pentasaccharide drug fondaparinux, was investigated by the ACE method. 37 Most of the studied ligands of AT-III were polydisperse materials containing saccharide chains of different length and structures. However, a unique bis-sulfated Nsulfoglucosamine residue is known to be the main structure responsible for specific interaction with AT-III.…”

Glycosaminoglycans: Anticoagulant and Nonanticoagulant Actions: A Short History of Symposia Held at Villa Vigoni

“…Antithrombin is required to potentiate the anticoagulant action of heparin LMWH, 12 fondaparinux, 13 and other heparin-derived oligosaccharides on coagulation proteases. 14 Structural aspects of heparins and derivatives are analyzed today using high performance size exclusion chromatography, 15 capillary electrophoresis, 16 antithrombin affinity or other matrices in connection with electrophoreses methods, 17 nuclear magnetic resonance techniques, 18 matrix-assisted laser desorption/ionization timeof-flight mass spectrometry 19 and fast atom bombardment, electrospray ionization, and tandem mass spectrometry. 20 These methods were designed to differentiate isomeric heparin disaccharides, to determine sulfation positions and uronic acid epimerization in oligosaccharides of chondroitin sulfate (another member of the GAG family), and to study the effect of the positions of sulfate groups on heparin binding.…”