Antithrombin deficiency, congenital

ABSTRACT Antithrombin is a key inhibitor of blood coagulation proteases and a prototype metastable protein. Heparin binding to antithrombin induces conformational transitions distal to the binding site. We applied osmotic stress techniques and rate measurements in the stopped flow fluorometer to investigate the possibility that hydration changes are associated with these transitions. Water transfer was identified from changes in the free energy of activation, (Delta)G^^, with osmotic pressure r. The (Delta)G^^ was determined from the rate of fluorescence enhancement/decrease associated with heparin binding/release. The volume of water transferred, (Delta)V, was determined from the relationship, (Delta)G/pi = AV. With an osmotic probe of 4 Angstrom radius, the volumes transferred correspond to 158 +/- 11 water molecules from reactants to bulk during association and 162 +/- 22 from bulk to reactants during dissociation. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures were correlated with the volumes measured in solution. Volume changes in water permeable pockets were identified at the loop-insertion and heparin-binding regions. Analyses of the pockets' atomic composition indicate that residues Ser-79, Ala-86, Val-214, Leu-215, Asn-217, Ile-219, and Thr-218 contribute atoms to both the heparin-binding pockets and to the loop-insertion region. These results demonstrate that the increases and decreases in the intrinsic fluorescence of antithrombin during heparin binding and release are linked to dehydration and hydration reactions, respectively. Together with the structural analyses, results also suggest a direct mechanism linking heparin binding/release to loop expulsion/insertion.

INTRODUCTION

Antithrombin is a circulating serine proteinase inhibitor essential for the control of blood coagulation reactions (Jordan et al., 1980; Olson et al., 1993). Congenital or acquired antithrombin deficiencies are associated with thrombosis (Lane et al., 1992; Beauchamp et al., 1998). Antithrombin's inhibitory activity is potentiated by the sulfated glycosaminoglycan heparin, which induces conformational changes that increase antithrombin's binding affinity for its target proteinases by several orders of magnitude. These conformational changes are associated with spectral changes including an enhancement of -40% in intrinsic protein fluorescence (Olson and Shore, 1981; Huntington et al., 1996). Subsequent interaction of the heparin/antithrombin complex with the proteinase is linked to both reversal of the fluorescence enhancement and release of the heparin (Craig et al., 1989).

The activating conformational changes propagate throughout the antithrombin structure in ways that are not completely understood but that appear to involve allosteric mechanisms (Lawrence, 1997; Wilczynska et al., 1997; Gils and Declerk, 1998). The transition from the circulating native conformation to the activated, heparin-bound, antithrombin conformation includes the release of the reactive center loop from an inserted position between two strands of a beta-sheet structure in the protein's core. In x-ray-derived models of unbound native antithrombin, the reactive center loop is partially inserted into the P-sheet (Schereuder et al., 1994; Skinner et al., 1997; Carrell et al., 1991). In the only one available x-ray-derived model of bound, activated antithrombin, the reactive loop is free and completely exposed to solvent. In this structure, the heparin-binding groove (Ersdal-Badju et al., 1998) is partially occupied by an oversulfated heparin analog, pentasaccharide (Jin et al., 1997). It has been proposed that antithrombin circulates in a metastable state that is destabilized by heparin during initial binding. The alteration in the rate and/or sequence of conformational changes at the loop insertion-region observed in certain antithrombin mutants is considered a prototype mechanism of "conformational disease" (Beauchamp et al., 1998; Zou et al., 1999). Kinetically, considerable evidence indicates that the heparin-antithrombin interaction is a twostep reaction with a strong electrostatic component. An initial, low-affinity step equilibrates very rapidly and induces conformational transitions leading to high-affinity interactions (Olson et al., 1993; Desai et al., 1998). The subsequent inhibition of the proteinase is also a two-step process with initial fast equilibration and readily reversible formation of a ternary complex). Interactions in the ternary complex lead via an acyl-enzyme intermediate to heparin release and formation of an irreversible antithrombin-proteinase complex (Kvassman et al., 1998). Recent structure/ function analyses also indicate that proteinase inhibition and heparin release are linked to reinsertion of the reactive loop into the beta-sheet and translocation of the proteinase from its initial low-affinity interaction site in the exposed reactive loop to the other end of the antithrombin molecule (Stratikos and Gettins, 1999).

This work was supported by NSF grant MCB-9601411 and National Institutes of Health-HL 57936. The computational geometry component was supported in part by NSF grant DBI-0078270 to J. Liang.

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Maria P. McGee,* Jie Liang,t and James Luba*

*Wake Forest University Medical School, Medicine and Biochemistry Departments, Medical Center Boulevard, Winston-Salem, North Carolina 27157 USA; and tUniversity of Illinois at Chicago, Bioengineering Department, Chicago, Illinois 60607 USA

Submitted May 10, 2001, and accepted for publication November 15, 2001.

Address reprint requests to: Maria P. McGee, Wake Forest University Medical School, Medicine and Biochemistry Departments, Medical Center Boulevard, Winston-Salem, NC 27157. Tel.: 336-716-6716; Fax: 336-7169821; E-mail: mmcgee@wfubmc.edu.

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