Quantitative analysis of antithrombin III binding site in low molecular weight heparins by exhausetive heparinases digestion and capillary electrophoresis.
The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method.
['Zhang|Mingyu|M|', 'Li|Gong|G|', 'Zhang|Yi|Y|', 'Kang|Jingwu|J|']
D000990:Antithrombin III D001665:Binding Sites D019075:Electrophoresis, Capillary D017984:Enoxaparin D019763:Heparin Lyase D006801:Humans D016014:Linear Models
Quantitative analysis Antithrombin III binding site Low molecular weight heparin 3-O-sulfated tetrasaccharides Enoxaparin