The Oligosaccharide Side Chain on Asn-135 of α-Antithrombin, Absent in β-Antithrombin, Decreases the Heparin Affinity of the Inhibitor by Affecting the Heparin-Induced Conformational Change

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Abstract
The β-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated α-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of α- and β-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to α-antithrombin was only slightly higher than that for the binding to the β-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to α-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for α-antithrombin than for β-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the α-form than for the β-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of α-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of α-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in α-antithrombin, leading to a decrease in heparin binding affinity.