A SECOND INHIBITOR OF ACTIVATED PROTEIN C (APC)

Abstract

The inactivation of APC in plasma, as measured with the chromogenic substrate S2366, follows, in the absence of heparin, pseudo-first order kinetics. The ti of about 20 minutes is independent of the APC concentration (31-500 nM) and increases linearly with the dilution of the plasma. These observations suggest that the concentration of the APC inhibitor in plasma is much higher than 500 nM, which is much higher than the concentration of 88 nM reported by Suzuki for the protein C inhibitor (PCI).In the presence of heparin (5 IU/ml) the inactivation of APC becomes biphasic. Fast inactivation with an apparent ti of 8 minutes is followed by slower inactivation with a ti of 20 minutes.Removal of PCI from the plasma with α-PCI antibodies (kindly provided by Dr. Suzuki) has no effect on APC inactivation in the absence of heparin. However, in this plasma APC inactivation could not be stimulated by addition of heparin (absence of fast phase of APC-inactivation). These data suggest the presence of two APC inhibitors in plasma: the heparin dependent PCI (PCI-I), earlier reported by Suzuki, and a sofar unknown heparin-independent inhibitor (PCI-II).Further experiments showed that this PC I-11 has a molecular weight of 60 kD and is different from the APC-binding protein reported by Kisiel. Using Heparin-Sepharose affinity chromatography we could separate PCI -II from both PCI-I and the APC binding protein. In this PC I-11 preparation APC inactivation was accompanied by the formation of an APC-PCI -11 complex of about 105 kD as demonstrated by immunoblotting with a-PC antibodies after SDS-PAGE. The identity of PCI-II is unknown; however, it is different from antithrombin III, heparin cofactor II, α1-antitrypsin and β2-antiplasmin.The demonstration of the presence of two APC inhibitors in plasma will require a re-evaluation of the current functional assays for the APC inhibitor.