Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma

Current studies show a more than 50-fold variation in the estimated level of tissue-type plasminogen activator (t-PA) activity in normal resting blood samples that result from major differences in the methods used to sample, preserve, and assay t-PA activity in blood. In this study we developed optimized methods for stabilizing and measuring t-PA activity in plasma by using a coupled plasminogen-chromogenic substrate (amidolytic) assay. To maximize the recovery of t-PA activity, blood should be acidified within 60 seconds after being drawn by adding 2 parts whole blood to 1 part 0.5 mol/L sodium acetate, pH 4.2. This method prevents hemolysis and eliminates 70% of the α₂-plasmin inhibitor. Optimum conditions for measuring t-PA activity are pH 8.0 to 8.3 (37°C), ionic strength 0.02 to 0.04, 0.5 μmol/L plasminogen. 80 μg/ml CNBr-cleaved fibrinogen, and a chromogenic substrate concentration of 0.65 mmol/l D-valyl-leucyl-lysyl-p-nitroanilide, 0.25 mmol/L D-valyl-phenylalanyl-lysyl-p-nitroanalide, or 0.2 mmol/L D-norleucyl-hexahydrotyrosyl-lysyl-p-nitroanilide. The final assay is linear with respect to added one-chain t-PA, two-chain t-PA, and acidified plasma. There was no difference in t-PA activity measured with ethylenediaminetetraacetic acid anticoagulant versus that measured with citrate anticoagulant after correction for dilution effects (average resting t-PA activity in plasma from 20 healthy individuals = 1.59 IU/ml). We conclude that assay conditions can have major effects on the measurement of t-PA activity in plasma and that suboptimal conditions may result in a significant underestimation of t-PA activity.