The effect of reducing end extension on pentasaccharide binding by antithrombin

KJ Belzar, Timothy Dafforn, M Petitou, RW Carrell, RA Huntington

Research output: Contribution to journalArticle

22 Citations (Scopus)


Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >/=0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.
Original languageEnglish
Pages (from-to)8733-41
Number of pages9
JournalJournal of Biological Chemistry
Issue number12
Publication statusPublished - 1 Jan 2000


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