A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma.
Human plasma contains an inhibitor of activated protein C (APC) which is termed according to its function protein C inhibitor (PCI). High purification of functionally active PCI with a yield of 18% is achieved by an improved procedure consisting of 4 steps: precipitation by rivanol, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE Sephacel and chromatography on dextran sulfate Sepharose. This purification results in the isolation of a homogeneous PCI which migrates in immunoelectrophoresis with the beta-globulins of human plasma and in SDS PAGE as one single band at Mr = 57,000 both under reducing and nonreducing conditions. The specific activity of the highly purified PCI was determined to be 226 units/mg, 1 unit being equivalent to the activity of 1 ml fresh human citrated plasma. PCI forms complexes with 1:1 stoichiometry (Ki: 1.4 x 10(-8) M) resulting in a loss of the amidolytic activity of APC as measured on Tos-Glu-Pro-Arg-pNA (S 2366). The inhibition rate of APC by PCI (k: 7.5 x 10(5) M-1 min-1) is significantly increased in the presence of 5 i.u./ml heparin (kH: 2.2 x 10(7) M-1 min-1). PCI also blocks the amidolytic activities of urokinase plasminogen activator (u-PA), thrombin and factor Xa on their chromogenic substrates in a heparin-dependent manner. According to the Ki-values measured for these reactions PCI is a noncompetitive inhibitor of these proteases. The Ki-values calculated do not differ significantly from those obtained for the inhibition of APC by PCI. Immunodepleted PCI-deficient plasma still contains an inhibitory activity against APC which, however, only slowly inactivates the amidolytic activity of APC and in a time and concentration-dependent manner. Addition of heparin has no influence on the inhibition rate. This finding suggests the existence of a second, heparin-independent PCI present in human plasma.
Radtke
K P
KP
Forschungslaboratorien der Behringwerke AG, Marburg/Lahn.
Stief
T W
TW
Heimburger
N
N
D001798:Blood Proteins / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011480:Protease Inhibitors / Q000302:isolation & purification*; D011486:Protein C / Q000037:antagonists & inhibitors*; D018046:Protein C Inhibitor
D001798:Blood Proteins; D011480:Protease Inhibitors; D011486:Protein C; D018046:Protein C Inhibitor; D006493:Heparin