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

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 (IC 50) 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 IC 50 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 IC 50 values for fucosylated chondroitin sulphate, chondroitin sulphate E, dextran sulphate, and fucoidan were minimally affected. Only in the case of heparin was the IC 50 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.