Heparin binding of protein-C inhibitor--analysis of the effect of heparin on the interaction of protein-C inhibitor with tissue kallikrein.
The non-specific serine-protease inhibitor protein-C inhibitor (PCI) inactivates its target enzymes by forming stable 1:1 complexes. Heparin stimulates most PCI/protease reactions, but interferes with the inhibition of tissue kallikrein by PCI by a hitherto unknown mechanism. In this study we analyzed the inhibitory effect of heparin on the tissue-kallikrein-PCI interaction. Free PCI and tissue-kallikrein x PCI complexes but not free tissue kallikrein bound to heparin-Sepharose, implying that the inhibitory effect of heparin cannot be caused by a tissue-kallikrein-heparin interaction. Heparin did not dissociate tissue-kallikrein x PCI complexes, making it unlikely that in the presence of heparin PCI becomes a substrate for, rather than an inhibitor of, tissue kallikrein. However, heparin-bound PCI, which was able to form complexes with 125I-urokinase, did not form complexes with 125I-tissue-kallikrein. This suggests that the inhibitory effect of heparin is either based on the neutralization of positive charges in the PCI molecule, which might be required for the interaction of PCI with the acidic protease tissue kallikrein, or on a change in reactivity of PCI upon heparin binding, making heparin-bound PCI no longer a tissue-kallikrein inhibitor. Neutralization of basic amino acids in the PCI molecule by glutamic acid, which prevented in a dose-dependent way the inhibitory effect of heparin, did not have any effect on the tissue-kallikrein-PCI interaction. Therefore, direct involvement of basic amino acid residues present in the heparin-binding site of PCI in the tissue-kallikrein-PCI interaction can be excluded. Heparin binding might rather cause a change in reactivity of PCI (e.g. by inducing a conformational change or by steric interference), thereby preventing its interaction with tissue kallikrein.
Ecke
S
S
Department of Vascular Biology and Thrombosis Research, University of Vienna, Austria.
Geiger
M
M
Binder
B R
BR
D007611:Aprotinin / Q000378:metabolism; D001345:Autoradiography; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007610:Kallikreins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D018046:Protein C Inhibitor / Q000378:metabolism*; D020840:Tissue Kallikreins; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D007457:Iodine Radioisotopes; D018046:Protein C Inhibitor; D018698:Glutamic Acid; D006493:Heparin; D007611:Aprotinin; D007610:Kallikreins; D020840:Tissue Kallikreins; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D005998:Glycine