THE N-TERMINAL SEGMENT OF ANTITHROMBIN ACTS AS A STERIC GATE FOR THE BINDING OF HEPARIN

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibit ion of thrombin and factor Xa. The areas of antithrombin involved in b inding heparin and stabilizing the interaction in the high-affinity fo rm have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding wa s investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved an tithrombin had 5.5-fold lowered affinity for heparin pentasaccharide a nd 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the c leavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the se gment constituting residues 22-46 in the N terminus of antithrombin hi nders access to the binding site for heparin, hence the increased init ial binding for the cleaved form, whereas, when heparin is bound, segm ent 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with the rmolysin, it protects the N-terminal cleavage site, implying that segm ent 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.