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J Biol Chem, Vol. 273, Issue 13, 7478-7487, March 27, 1998

Mechanism of Heparin Activation of Antithrombin
ROLE OF INDIVIDUAL RESIDUES OF THE PENTASACCHARIDE ACTIVATING SEQUENCE IN THE RECOGNITION OF NATIVE AND ACTIVATED STATES OF ANTITHROMBIN

Umesh R. Desai, Maurice Petitou^§, Ingemar Björk^¶, and Steven T. Olson

From the  Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612, ^§ Sanofi Recherche, Ligne Hémobiologie, 195 route d'Espagne, 31036 Toulouse Cedex, France, and the ^¶ Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Box 575, S-75123, Uppsala, Sweden

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of ~15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated 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 conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

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