Summary
Inhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated
by heparin, whereas inhibition by α1-antitrypsin (AAT) is heparin-independent. Three
lysine residues located in a positively charged cluster in the serine protease domain
of protein C (PC) were mutated to probe their involvement in the heparin stimulation
of inhibition by PCI. These mutations were selected after analysis of the three-dimensional
structure of APC and of molecular models for PCI and the APC-PCI complex. A double
mutant, K62[217]N/K63[218]D, a single mutant, K86[241]S, and wild-type PC were expressed
in embryonic human kidney 293 cells. Heparin stimulated the rate of inhibition of
wt-APC by PCI approximately 400-fold, with second order rate constants (k
2
) in the absence and presence of heparin of 0.72 × 103 M–1s–1 and 2.87 × 105 M–1s–1, respectively. In contrast, heparin only yielded a 52-fold stimulation of the rate
of inhibition of the double mutant APC by PCI as the rate constants in the absence
and presence of heparin were k
2
= 2.44 × 103 M–1s–1 and k
2
= 1.26 × 105 M–1s–1, respectively. The double mutant K62N/K63D eluted at approximately 10% lower NaCl
concentration from a heparin Sepharose column than the K86S mutant or wt-APC. These
data suggest K62 and K63 in APC to be part of a heparin binding site which is important
for heparin-mediated stimulation of inhibition of APC by PCI.
Abbreviations: APC, activated protein C; PC, protein C; PCI, protein C inhibitor, AAT, α1-antitrypsin
also called α1-proteinase inhibitor, AT, antithrombin; TM, thrombomodulin. The chymotrypsinogen
nomenclature for APC (1) is used in the text while the PC numbering is indicated between
brackets whenever appropriate. P1, P2... and P1’, P2’.. designate inhibitor residues
amino- and carboxy-terminal to the scissile peptide bond, respectively, and S1, S2..
and S1’, S2’.. the corresponding subsites of the protease (2). The antitrypsin numbering
(3) for PCI is used along this article while the PCI numbering is mentioned between
brackets whenever appropriate.
