Arginine 200 of heparin cofactor II promotes intramolecular interactions of the acidic domain - Implication for thrombin inhibition

Heparin cofactor II (HCII) is presumed to be a physiological inhibitor of the serine proteinase thrombin, The reaction between HCII and thrombin is quite unique, because it involves an unusual HCII-reactive site loop sequence of Leu(444)-Ser(445), requires the presence of glycosaminoglycans for optimal activity and involves a protein-protein interaction besides the reactive site loop-active site interaction characteristic of serine proteinase inhibitor-serine proteinase pairs, Two mutations at a unique HCII residue, Arg(200) --> Als or Glu, were generated by site-directed mutagenesis, The mutations did not alter either HCII binding to heparin-Sepharose or HCII inhibition of thrombin in the presence of heparin or dermatan sulfate, suggesting that Arg(200) is not part of the glycosaminoglycan binding site of HCII. In the absence of glycosaminoglycan, there was a significant increase in alpha-thrombin inhibition by the Arg(200) mutants as compared with wild type recombinant HCII (wt-rHCII), whereas inhibition rates with chymotrypsin were identical, Inhibition of gamma(T)-thrombin, which lacks anion-binding exosite 1 ((ABE-1), the region of alpha-thrombin that interacts with the acidic domain of HCII), was significantly reduced compared with alpha-thrombin, but the reduction was more dramatic for the Arg(200)-rHCII mutants. Hirugen, which binds to ABE-1 of alpha-thrombin, also diminished inhibition of alpha-thrombin by the Arg(200)-rHCII mutants to nearly wt-rHCII levels. Both Arg(200)-rHCII mutants had significantly increased k(d) values as compared with wt-rHCII, whereas the k(d) rates were unchanged. Collectively, these results suggest that the improved inhibitory activity of the Arg(200)-rHCII mutants is mediated by enhanced interactions between the acidic domain and ABE-1, resulting in an increased HCII-thrombin association rate.