Modulation of Antithrombin-Protease Interactions by Semisynthetic Low-Molecular-Weight Heparins with Different Sulfation Patterns

Heparin, a natural glycosaminoglycan (GAG), is widely used for the treatment of thrombotic diseases. Most of its side effects are related to its ability to bind to different proteins, thus interfering with their target biological activity. To gain insight into structure-activity relationships, we investigated the interaction of an homogeneous series of sulfated polysaccarides, deriving from controlled desulfation of a supersulfated low molecular weight heparin (LMWH) with the target-enzymes human Antithrombin III (AT) and thrombin (T). In addition, we analyzed the activation process of the serpin AT against T and Factor Xa (Xa). A non-linear correlation between the strength of the AT-heparin complex and the polysaccharide sulfation degree was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the LMWH derivatives in activating AT towards the proteases was generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated heparin showed a serpin activation ability higher than expected from the affinity studies. Examination of the sulfation pattern in the light of the above results suggests a key role of the substitution of the iduronic acid residue in the heparin-mediated serpin binding and activation processes. Indeed, sulfation at position 2 of the uronic acid is beneficial, whereas 2,3-disubstitution generates unfavorable contacts between the GAG and AT. On turn, glucosamine sulfation at position 6 appears to grant increased catalytic efficiency. These results indicate that chemical modification of heparin sulfation pattern can be used to modulate binding specificity and activity towards its biological targets.