Measurement of different forms of tissue plasminogen activator in plasma

Abstract

Background: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor. Methods: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1- inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1- inhibitor, and anti-tPA antibodies conjugated to peroxidase. Results: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% ± 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% ± 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% ± 20% of the expected tPA/PAI-1 vs recovery of only 38% ± 16% when monoclonal anti- PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r2 = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% ± 7% of measured total tPA. Conclusion: Optimized assays based on a single antitPA capture antibody can be used to accurately measure the major forms of tPA in plasma.