Involvement of lys 62(217) and lys 63(218) of human anticoagulant protein C in heparin stimulation of inhibition by the protein C inhibitor

Inhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated by heparin, whereas inhibition by alpha 1-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 x 10(3) M(-1)s(-1) and 2.87 x 10(5) M(-1)s(-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 x 10(3) M(-1)s(-1) and k(2) = 1.26 x 10(5) M(-1)s(-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 PCT.