THE OLIGOSACCHARIDE SIDE-CHAIN ON ASN-135 OF ALPHA-ANTITHROMBIN, ABSENT IN BETA-ANTITHROMBIN, DECREASES THE HEPARIN AFFINITY OF THE INHIBITOR BY AFFECTING THE HEPARIN-INDUCED CONFORMATIONAL CHANGE

The beta-form of antithrombin, lacking a carbohydrate side chain on As n-135, is known to bind heparin more tightly than the fully glycosylat ed alpha-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 recombinan t forms of alpha- and beta-antithrombin, The dissociation equilibrium constant for the first step of the two-step mechanism of binding of bo th heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharid e at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin, In contrast, the rate const ant for the conformational change induced by heparin and pentasacchari de in the second binding step was substantially lower for alpha-antith rombin than for beta-antithrombin, Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form, The carbohydrate side chain at Asn -135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosacchar ide of alpha-antithrombin is oriented away from the heparin binding si te 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 transm itted to the reactive-bond loop. The resulting decreased conformationa l flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the na tive conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrea se in heparin binding affinity.