Abstract

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring beta-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Björk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-A long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central beta-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

Highlights

  • Pharmaceutical heparin is ubiquitously employed in modern medicine

  • This is in general agreement with an investigation of salt effects on the binding constant between high affinity heparin and antithrombin III (ATIII) which showed that five to six charged groups directly mediate the interaction between antithrombin and its cofactor [24]

  • Heparin Binding Site Identification—Recognizing that further advances in understanding of the antithrombin heparin cofactor activation mechanism would be aided by a more precise understanding of the ATIII heparin binding site, we undertook to define it at high resolution by alanine scanning mutagenesis of basic residues in the helix D region

Read more

Summary

Introduction

Pharmaceutical heparin is ubiquitously employed in modern medicine. Preparations of this sulfated glycosaminoglycan are given to increase anticoagulant activity in patients who have, or who are at risk for, venous and arterial thrombosis. As first described by Stein and Chothia [14], the conformational change associated with reactive loop insertion and inhibition is due to movement of a well defined fragment of serpin structure, consisting of helix F and strands 1, 2, and 3 of sheet A on joints formed by helices D and E This s123AhDEF fragment moves, as a unit, away from the remainder of the serpin to open a gap between strands 3A and 5A of the native structure. Structure/function studies with synthetic pentasaccharides have shown that six of the negatively charged groups are essential for heparin activation of ATIII and that two others play significant roles in the process (reviewed in Ref. 23) This is in general agreement with an investigation of salt effects on the binding constant between high affinity heparin and ATIII which showed that five to six charged groups directly mediate the interaction between antithrombin and its cofactor [24]

Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call