MECHANISM OF HEPARIN ACTIVATION OF ANTITHROMBIN - ROLE OF INDIVIDUAL RESIDUES OF THE PENTASACCHARIDE ACTIVATING SEQUENCE IN THE RECOGNITIONOF NATIVE AND ACTIVATED STATES OF ANTITHROMBIN

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation o f the serpin, antithrombin, we studied the effect of deleting pentasac charide residues on this activation, Binding, spectroscopic, and kinet ic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccha ride binding energy of similar to 15-75% but did not affect the oligos accharide's ability to conformationally activate the serpin or to enha nce the rate at which the serpin inhibited factor Xa, Rapid kinetic st udies revealed that elimination of the reducing end disaccharide margi nally affected binding to the native low-heparin-affinity conformation al state of antithrombin but greatly affected the conversion of the se rpin to the activated high-heparin-affinity state, although the activa ted conformation was still favored. In contrast, removal of the nonred ucing-end residue D drastically affected the initial low-heparin-affin ity interaction so as to favor an alternative activation pathway where in the oligosaccharide shifted a preexisiting equilibrium between nati ve and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pe ntasaccharide function both to recognize the native low-heparin-affini ty conformation of antithrombin and to induce and stabilize the activa ted high-heparin-affinity conformation. Residues at the reducing end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformati on, Together, these findings establish an important role of the hepari n pentasaccharide sequence in preferential binding and stabilization o f the activated conformational state of the serpin.