EVALUATION OF 2 FUNCTIONAL ASSAYS FOR PROTEIN-C INHIBITOR PLASMINOGEN-ACTIVATOR INHIBITOR-3 ACTIVITY

Two functional assays for protein C inhibitor (PCI) were evaluated in parallel for the same plasma samples. One assay is specific for active PCI and measures its ability to form complexes with activated protein C (APC) in the presence of heparin (Espana et al, Thromb Res 64, 309, 1991) (Method A). After incubation of samples with heparin and an exc ess of APC, the amount of APC:PCI formed is measured by an ELISA. The second functional PCI assay is based on the determination of the resid ual amidolytic activity of the APC added to the sample. In all cases a good correlation was obtained with both methods. For 20 healthy subje cts and 20 patients before surgery, there were no significant between- assay differences in the levels of functional PCI activity. On the oth er hand, there were significant between-assays differences in the leve ls of functional PCI of the 20 patients measured 1 and 3 days after su rgery. In this case, method B gave higher values than method A (38% +/ - 13% versus 29% +/- 11% at day 1 and 59% +/- 16% versus 43% +/- 12% a t day 3) (mean +/- SD). The level of alpha1-antitrypsin (alpha1AT), th e second major plasma APC inhibitor, was significantly increased in pa tients at day 1 and day 3 (133% +/- 30% and 190% +/- 57%, respectively ). Furthermore, there was a positive correlation between the level of alpha1AT and the normalized difference between the level of functional PCI measured by method B and that measured by method A. Additionally, when normal plasma was supplemented with increasing amounts of purifi ed alpha1AT, the level of PCI activity measured by method B increased parallel to the increase in alpha1AT added, but that measured by metho d A did not. Determination of APC:inhibitor complexes in aliquots of t he final mixtures utilized in the PCI assays revealed that the level o f APC:alpha1AT complex increased parallel to the increase in the amoun t of alpha1AT added, whereas the APC:PCI complex level remained unchan ged. We conclude that current functional PCI assays based on the deter mination of APC residual activity in plasma are not specific for PCI, are influenced by the level of plasma alpha1AT and are not reliable fo r the determination of functional PCI levels in patients with altered levels of alpha1AT. In contrast, the method based on the determination of the APC:PCI complexes formed following incubation of plasma sample s with APC is specific for active PCI and is not influenced for the pr esence of other APC inhibitors.