Determination of plasminogen activator inhibitor (PAI) capacity of human plasma in presence of oxidants: A novel principle

A new functional assay of PAI-activity in human plasma is described.Hitherto known assays for fast acting PAI have some disadvantages:predilution and/or acidification steps of the sample to affordthe required inactivation of alpha-2-antiplasmin (A2-PI). This approach in contrast implicates test performance in presence of chloramine T(CT), an oxidant that destroys plasma antiplasmin activity without impairing significantly the activity of urokinase (u-PA) or plasmin. The following reaction conditions were found optimal: 50 μl of undiluted plasma, anticoagulated with citrate or EDTA, were first incubated with 1 IU u-PA in a Tris-buffer, pH 8.4 and then with Glu-plasminogen (0.85 μmol/l final), CT (2.5 mmol/l final), tranexamic acid (0.9 mmol/l final) for 5 min. at 37°C. After addition of 0.3 mmol/l of the chromogenic plasmin substrate H-D-Nva-CHA-Lys-pNA (pNA : para nitroanilide) and of NaCl (250 mmol/l final) a linear kinetic with e Δ 405/t in the range of 0.2/min for normal plasma was recorded. In an endpoint version of the test the chromogonic substrate can be added together with plasminogen resulting an A/t 2kinetic. Dilution studies showed a linear calibration curve from 0 to 14 arbitrary u-PA inhibiting units (AU)/ml plasma. By means of PAI-standard plasmas PAI-capacity values of 20 healthy volunteers (10 males/10 females) (× = 26 years, (σ = 4.2) were determined. They ranged from 0.4 – 6.9 × = 1.3, (F = 0.9) AU/ml plasma. Plasma samples containing more than 14 AU/ml were prediluted with PAI-deficient plasma. Intra- and interassay coefficients of variation (CV) were determined to be 1.3±0.6 and 4.3±0.5 %, respectively. The values of this assay correlate well with these obtained by acidification of the samples. However, the possibility of measuring plasma PAI (and PA) activities by means of a simple and direct approach can be considered as an important progress with regard to routine hospital practice. The presented oxidative inactivation of A 2-PI mimics the leukocyte attack phase, suggesting that activated leukocytes create a microenvironment of uncontrolled plasmin activity.