Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3

Biol Chem Hoppe Seyler. 1987 Oct;368(10):1427-33. doi: 10.1515/bchm3.1987.368.2.1427.

Abstract

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.

MeSH terms

  • Amino Acids / analysis
  • Electrophoresis, Polyacrylamide Gel
  • Glycoproteins / analysis*
  • Heparin / analysis
  • Humans
  • Plasminogen Inactivators
  • Protein C / antagonists & inhibitors*
  • Urokinase-Type Plasminogen Activator / antagonists & inhibitors*

Substances

  • Amino Acids
  • Glycoproteins
  • Plasminogen Inactivators
  • Protein C
  • Heparin
  • Urokinase-Type Plasminogen Activator