Amino acid residues of heparin cofactor II required for stimulation of thrombin inhibition by sulphated polyanions

Niall S. Colwell, Michael J. Grupe, Douglas M. Tollefsen

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Abstract

A variety of sulphated polyanions in addition to heparin and dermatan sulphate stimulate the inhibition of thrombin by heparin cofactor II (HCII). Previous investigations indicated that the binding sites on HCII for heparin and dermatan sulphate overlap but are not identical. In this study we determined the concentrations (IC50) of various polyanions required to stimulate thrombin inhibition by native recombinant HCII in comparison with three recombinant HCII variants having decreased affinity for heparin (Lys-173→Gln), dermatan sulphate (Arg-189→His), or both heparin and dermatan sulphate (Lys-185→Asn). Pentosan polysulphate, sulphated bis-lactobionic acid amide, and sulphated bis-maltobionic acid amide resembled dermatan sulphate, since their IC50 values were increased to a much greater degree (≥8-fold) by the mutations Arg-189→His and Lys-185→Asn than by Lys-173→Gln (≤1.5-fold). By contrast, the IC50 values for fucosylated chondroitin sulphate, chondroitin sulphate E, dextran sulphate, and fucoidan were minimally affected. Only in the case of heparin was the IC50 increased to a greater degree by both Lys-173→Gln and Lys-185→Asn (≥6-fold) than by Arg-189→His (≤1.5-fold). None of the polyanions significantly stimulated inhibition of thrombin by an N-terminal deletion mutant of HCII (Δ1-74). These results suggest that, like dermatan sulphate and heparin, other polyanions stimulate HCII primarily by an allosteric mechanism requiring the N-terminal acidic domain. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)148-156
Number of pages9
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1431
Issue number1
DOIs
StatePublished - Apr 12 1999

Keywords

  • Dermatan sulphate
  • Heparin
  • Heparin cofactor II
  • Polyanion
  • Thrombin

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