Characterization of anticoagulant heparinoids by immunoprofiling.
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.
Wijnhoven
Tessa J
TJ
Department of Matrix Biochemistry, Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
van de Westerlo
Els M
EM
Smits
Nicole C
NC
Lensen
Joost F
JF
Rops
Angelique L
AL
van der Vlag
Johan
J
Berden
Jo H
JH
van den Heuvel
Lambert P
LP
van Kuppevelt
Toin H
TH
D000818:Animals; D000918:Antibody Specificity; D000925:Anticoagulants / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006496:Heparinoids / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D051379:Mice; D013552:Swine
D000925:Anticoagulants; D006496:Heparinoids; D007106:Immune Sera