title: The effect of a reducing-end extension on pentasaccharide binding by antithrombin.
abstract: 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.
authors.affiliation: University of Cambridge, Department of Haematology, Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom.
authors.forename: K J
authors.initials: KJ
authors.lastname: Belzar
authors.forename: T R
authors.initials: TR
authors.lastname: Dafforn
authors.forename: M
authors.initials: M
authors.lastname: Petitou
authors.forename: R W
authors.initials: RW
authors.lastname: Carrell
authors.forename: J A
authors.initials: JA
authors.lastname: Huntington
mesh_terms: D000991:Antithrombins / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D006493:Heparin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007067:Iduronic Acid; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation
chemical_list: D000991:Antithrombins; D009844:Oligosaccharides; C406807:SR 80356A; D007067:Iduronic Acid; C049714:IC 831423; D006493:Heparin