Evaluation of two 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 (España et al, Thromb Res 64, 309, 1991) (Method A). After incubation of samples with heparin and an excess 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 residual amidolytic activity of the APC added to the sample. In all cases a good correlation was obtained with both methods. For 20 healthy subjects and 20 patients before surgery, there were no significant between-assay differences in the levels of functional PCI activity. On the other hand, there were significant between-assays differences in the levels of functional PCI of the 20 patients measured 1 and 3 days after surgery. 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% at day 3) (mean +/- SD). The level of alpha 1-antitrypsin (alpha 1 AT), the second major plasma APC inhibitor, was significantly increased in patients at day 1 and day 3 (133% +/- 30% and 190% +/- 57%, respectively). Furthermore, there was a positive correlation between the level of alpha 1AT 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 purified alpha 1AT, the level of PCI activity measured by method B increased parallel to the increase in alpha 1AT added, but that measured by method A did not. Determination of APC:inhibitor complexes in aliquots of the final mixtures utilized in the PCI assays revealed that the level of APC:alpha 1AT complex increased parallel to the increase in the amount of alpha 1AT added, whereas the APC:PCI complex level remained unchanged. We conclude that current functional PCI assays based on the determination of APC residual activity in plasma are not specific for PCI, are influenced by the level of plasma alpha 1AT and are not reliable for the determination of functional PCI levels in patients with altered levels of alpha 1AT.(ABSTRACT TRUNCATED AT 400 WORDS)
Research Center, Hospital Universitario La Fe, Valencia, Spain.
F
F
España
S
S
Hendl
J
J
Gilabert
A
A
Estellés
J
J
Aznar
D000595:Amino Acid Sequence; D002863:Chromogenic Compounds; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005833:Genital Neoplasms, Female / Q000097:blood; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides; D015849:Plasminogen Inactivators / Q000032:analysis*; D011486:Protein C / Q000037:antagonists & inhibitors*; D018046:Protein C Inhibitor; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D012307:Risk Factors; D012680:Sensitivity and Specificity; D013923:Thromboembolism; D000515:alpha 1-Antitrypsin / Q000032:analysis
D002863:Chromogenic Compounds; D009842:Oligopeptides; D015849:Plasminogen Inactivators; D011486:Protein C; D018046:Protein C Inhibitor; D000515:alpha 1-Antitrypsin; C040190:S 2366; D006493:Heparin; D011761:Pyrrolidonecarboxylic Acid