Binding of a glycosaminoglycan to HCII appears to displace the inhibitor's N-terminal acidic domain, which normally occupies the glycosaminoglycan-binding site. The acidic domain of HCII then binds to exosite I of thrombin, thereby facilitating the inhibitory reaction.
Previous studies have shown that the binding sites for heparin and dermatan sulphate in HCII overlap but are not identical. Mutations of Lys-173 decrease the affinity for heparin, mutations of Arg-189 decrease the affinity for dermatan sulphate, and mutations of Lys-185 decrease the affinity for both glycosaminoglycans.
To investigate the mechanism by which these polyanions stimulate HCII,
we have determined their effects on the rate of inhibition of thrombin
by native recombinant HCII in comparison with HCII variants having glycosaminoglycan-binding
site mutations or deletion of the N-terminal acidic domain. These
experiments revealed the extent to which each polyanion resembles heparin
or dermatan sulphate and whether it stimulates HCII predominantly by the
allosteric mechanism described above.
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