title: Determination of plasminogen activator inhibitor (PAI) capacity of human plasma in presence of oxidants: a novel principle.
abstract: 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 afford the 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 microliter 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 mumol/l final), CT (2.5 mmol/l final), tranexamic acid (0.9 mmol/l final) for 5 min. at 37 degrees 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 delta A405/t in the range of 0.2/min for normal plasma was recorded. In an endpoint version of the test the chromogenic substrate can be added together with plasminogen resulting an A/t2-kinetic. 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) (x = 26 years, sigma = 4.2) were determined. They ranged from 0.4 - 6.9 (x = 1.3, sigma = 0.9) AU/ml plasma. Plasma samples containing more than 14 AU/ml were prediluted with PAI-deficient plasma. Intra- and inter-assay 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 those 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 A2-PI mimics the leukocyte attack phase, suggesting that activated leukocytes create a microenvironment of uncontrolled plasmin activity.
mesh_terms: D000328:Adult; D002700:Chloramines / Q000494:pharmacology*; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors*; D015849:Plasminogen Inactivators; D014105:Tosyl Compounds; D014148:Tranexamic Acid / Q000494:pharmacology*
chemical_list: D002700:Chloramines; D003509:Cyclohexanecarboxylic Acids; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D014105:Tosyl Compounds; C016300:chloramine-T; D014148:Tranexamic Acid; D010960:Plasminogen Activators