The effect of a reducing-end extension on pentasaccharide binding by antithrombin.
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.
Belzar
K J
KJ
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.
Dafforn
T R
TR
Petitou
M
M
Carrell
R W
RW
Huntington
J A
JA
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
D000991:Antithrombins; D009844:Oligosaccharides; C406807:SR 80356A; D007067:Iduronic Acid; C049714:IC 831423; D006493:Heparin