Characterization of anticoagulant heparinoids by immunoprofiling

Abstract

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and-when additional saccharides are present-inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.

Fig. 1

Agarose gel electrophoresis of heparinoids. The different classes of GAGs in the drugs were visualized by a combined azure A–silver staining procedure. The standard contains 40 ng HS, 20 ng DS, and 20 ng CS

Fig. 2

Thirty-three percent polyacrylamide gel electrophoresis of undigested (a) and enzymatically digested (b) heparinoids. Heparinoids were visualized by a combined alcian blue-silver staining procedure. Treatment with heparinase I resulted in cleavage of dalteparin and enoxaparin, whereas treatment with chondroitinase ABC did not. Sulodexide was cleaved by heparinase I as well as chondroitinase ABC

Fig. 3

Immunofluorescence staining of human renal cryosections with anti-heparin antibodies HS4C3 and HS4E4 in the absence and presence of heparinoids. Staining was abolished when renal cryosections were incubated with HS4C3 or HS4E4 in the presence of heparin or sulodexide. HS4C3 staining was decreased in the presence of nadroparin, whereas HS4E4 staining was unaffected. Bar represents 50 μm; magnification is identical for each photograph. G Glomerulus, BC Bowman’s capsule, T renal tubule