Contribution of Basic Residues of the D and H Helices in Heparin Binding to Protein C Inhibitor

Protein C inhibitor (PCI) is a heparin-binding serine protease inhibitor (serpin) that regulates hemostatic proteases such as activated protein C (APC) and thrombin. The work described here provides further evidence that the PCI H helix, but not the D helix, has a major role in heparin-accelerated inhibition of APC and thrombin. We previously identified Arg-269 and Lys-270 of the H helix [R269A/K270A “H1” recombinant PCI (rPCI)] as important residues both for heparin-accelerated inhibition of thrombin and APC and for heparin–Sepharose binding (Shirk, R. A., Elisen, M. G. L. M., Meijers, J. C. M., and Church, F. C. (1994)J. Biol. Chem.269, 28690–28695). H1 rPCI was used as a template for Ala-scanning mutagenesis of other H helix basic residues (H1-K266A, H1-K273A, and H1-K266A/K273A) and of the D helix basic residues (H1-K82A, H1-K86A, H1-R90A, and H1-K82A/K86A/R90A). Compared to wild-type rPCI/heparin (k2= 2.2 × 107M−1min−1for thrombin), heparin-accelerated thrombin inhibition was decreased 2.4-fold by H1 rPCI, 4.4-fold by H1-K266A rPCI, and 8-fold by H1-K273A rPCI. H1-K266A/K273A rPCI thrombin inhibition was essentially not accelerated by heparin. A similar trend was found for APC–heparin inhibition using these H helix rPCI mutants. In contrast, the D helix rPCI mutants did not have further reduced heparin-stimulated thrombin or APC inhibition compared to H1 rPCI. Interestingly, all of the H and D helix rPCI mutants had reduced heparin–Sepharose binding activity (ranging from 180 to 360 mM NaCl) compared to wild-type rPCI and H1 rPCI, which eluted at 650 and 430 mM NaCl, respectively. These data suggest that all four basic residues (Lys-266, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin binding site. Our results also imply that while the D helix basic residues (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude that the primary heparin binding site of PCI is the H helix and not the D helix as found in other homologous heparin-binding serpins such as antithrombin III, heparin cofactor II, and protease nexin 1.