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

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.