MECHANISM OF HEPARIN ACTIVATION OF ANTITHROMBIN - EVIDENCE FOR REACTIVE CENTER LOOP PREINSERTION WITH EXPULSION UPON HEPARIN-BINDING

A heparin-induced conformational change is required to convert antithr ombin from a slow to a fast inhibitor of factor Xa. It has been propos ed [van Boeckel et al. (1994) Nat. Struct. Biol. 1, 423-425] that the reactive center residue P14 is inserted into beta-sheet A in native an tithrombin and is displaced from the beta-sheet by heparin binding, th ereby altering the conformation of the reactive center and making it a better target for factor Xa binding. To test this hypothesis, we have characterized a P14 serine --> tryptophan antithrombin variant. From changes in tryptophan fluorescence upon heparin binding, increased aff inity for heparin, and partial activation of the variant against facto r Xa, we conclude that the proposed mechanism of heparin activation is correct with respect to loop expulsion and that it may consequently b e possible to create more highly activated antithrombin variants throu gh suitable hinge region substitutions.