Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II

Inhibition of thrombin by heparin cofactor (HCII) is accelerated ∼1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg¹⁸⁹ → His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg⁴⁷ and Lys¹²⁵ of anti-thrombin (homologous to Arg¹⁰³ and Lys¹⁸⁵ of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg¹⁰³ → Leu, Gln, or Trp; Lys¹⁸⁵ → Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg¹⁰³ variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg¹⁰³ → Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg¹⁰³ → Gln and Arg¹⁰³ → Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu⁴⁴⁴-Ser⁴⁴⁵) of HCII. All of the Lys¹⁸⁵ variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required >10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys¹⁸⁵ of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys¹⁸⁵ but not Arg¹⁰³, both of which were predicted to be involved by homology to anti-thrombin.