Inhibition of Urokinase by Protein C-Inhibitor (PCI). Evidence for Identity of PCI and Plasminogen Activator Inhibitor 3

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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 unreducint 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 pentosanpoly- sulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 .times. 10-8M. 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 hightly 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.