Binding Study of Heparin from Different Sources to Antithrombin by Affinity Capillary Electrophoresis.

Heparin, a highly sulfated polydispersed glycosaminoglycan (GAG), is the most widespread clinical anticoagulant; it binds antithrombin III (AT), a member of serine proteinases superfamily, accelerating its antagonist effect on blood coagulation. The binding interaction with AT is an important aspect of the characterization of physicochemical properties of GAGs. With the aim at profiling several clinical and experimental heparin batches from different sources (porcine, bovine and ovine mucosa), a quantitative ATheparin binding investigation was undertaken by means of Affinity Capillary Electrophoresis (ACE). In dynamic-equilibrium ACE, the electrophoretic mobility of the receptor (AT), analysed in a BGE containing the ligand (the considered GAG), is correlated to ligand concentration and binding constant. In particular, a 20 mM sodium phosphate, pH 7.4 buffer (the BGE) was chosen as the neat medium and the experiments were carried out in a highly hydrophilic poly(vinyl alcohol) coated capillary (effective length 8.5 cm). The applied sample consisted of the receptor AT (0.50 μM) and phenylacetic acid (PAA; 10.0 μM) used as a reference compound. The samples were run in triplicate at each of the studied concentration levels of the ligand (heparin, 1.0 – 10.0 x 10-7 M) supplemented to the BGE. The migration time ratio of PAA to AT was assumed as the chemical response to be correlated to the ligand concentration and the binding constant estimation was based on the application of a nonlinear regression method (rectangular hyperbola). The average analysis time was in the order of 4 min. Under these conditions, 18 heparin samples were analysed and their binding constants (Kd) were found between 13 and 54 nM (SD ≤ ± 1.2; n = 3; coefficient of determination r2 ≥ 0.98) with significant differences depending on the origin. Correlation of the Kd values to in vitro anti-factor Xa and anti-factor IIa potencies was evaluated. Both heparin activities demonstrated to be closely related to Kd, independently from the kind and the source of the sample. Commercial samples from the same manufacturer showed a certain dispersion around the fitting, but the use of the mean point of the cloud significantly improved the coefficient of determination