The effect of a reducing-end extension on pentasaccharide binding by antithrombin

Antithrombin requires heparin for efficient inhibition of the final two pro teinases of the blood coagulation cascade, factor Xa and thrombin, Antithro mbin binds heparin via a specific pentasaccharide domain in a two-step mech anism whereby initial weak binding is followed by a conformational change a nd subsequent tight binding. The goal of this study is to investigate the r ole of a reducing-end extension in the binding of the longer oligosaccharid es that contain the cognate pentasaccharide sequence. We determined the ant ithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end dis accharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the d isaccharide addition, but at ionic strengths greater than or equal to 0.2 t he mode of heptasaccharide binding changes resulting in a a-fold increase i n affinity due to a decrease in the off-rate caused by a greater nonionic c ontribution to binding, Molecular modeling of possible binding modes for th e heptasaccharide at high ionic strength indicates a possible shift in posi tion of the pentasaccharide domain to occupy the extended heparin-binding s ite. This conclusion supports the likely presence of a range of sequences t hat can bind to and activate antithrombin in the natural heparan sulfates t hat line the vascular endothelium.