Neue chromogene Screening-Methoden der Fibrinolyse Diagnostik

5.1. Fibrinolysis Parameters Assay (FIPA) Through FIPA a global assay of fibrinolysis diagnostics has been developed. It is a simple, rapid and economical routine test for several clinical relevant plasmatic fibrinolysis parameters like plasminogen, plasminogen activator inhibitor type 1 (PAI-1) and 2-Antiplasmin. The effect of aprotinin can, among other things, be judged by FIPA. However, the FIPA is not sensitive to physiological concentrations of prourokinase (scu-PA) or tissue-type plasminogen activator (t-PA). The following assay volumes and concentrations turned out to be optimal for the test set up: 50 µl citrate plasma and 50 µl urokinase reagent consisting of 10 IU u-PA/ml, 1,1 mmol/l tranexamic acid, 1% polygelin, 0,1% Triton X 100, PBS, pH 7,4 are incubated for 20 minutes at 37°C, the so called plasmin generation phase. Subsequently, for the plasmin determination phase 50 µl of 3 mmol/l H-D-Val-Leu-Lys-pNA, 1,05 mol/l KCl is added. The linear kinetic is determined by means of a microtiterplate reader at 405 nm through the balance of the formation of released pNA and the orginal substrate. The results are calibrated against pooled normal plasma (100% FIPA activity). The intra- and interassay coefficients of variation have been found to be less than 5%. The detection limit (sensitivity) of the functional fibrinolysis assay is 5% of the normal plasmatic FIPA activity. The normal FIPA activity amounts to 100%,  = 25%. The FIPA activity correlates negatively (r = - 0,684) with the activity of the plasminogen activator inhibitor type 1 (PAI-1) of patient samples. 5.2. Activated Fibrinolysis Parameters Assay (aFIPA) In addition to FIPA the development of aFIPA produced an additional global assay of fibrinolysis diagnostics. Due to the test principal being based on the physiological contact activation of fibrinolysis, it is sensitive also for scu-PA. aFIPA assay is carryied out as follows: 50 µl citrate plasma stabilized with 100 mM arginine pH 8,7 and 25 µl 50 mM Chloramin T in 100 mM NaHCO3, pH 8,0 is incubated at 37°C for 5 minutes. Subsequently 150 µl 0.1 g/l ellagic acid is added by a pipette and incubated for 120 minutes at 37°C. After the second incubation, 25 µl 6,0 mM chromogenic substrate H-D-Val-Leu-Lys-pNA in 3,5 M KCl is added and the linear kinetic (A405nm/min) is measured at 0 and 30 minutes at a wavelength of 405 nm through a microtiterplate reader. The intra- and interassay coefficients of variation have been found to be < 5%. A linear plasmin activity could almost be proven to recover at 90%. 5.3. Plasmatic PAI-2 Assay PAI-2 activity in the blood can be determined through the newly developed plasmatic PAI-2 assay. PAI-2 is marking placenta function and has clinical relevance differentiating normal pregnancy and placental insufficiency, for example pre-eclampsia. PAI-2 possesses a slower kinetic reaction than PAI-1. Since the PAI-1/u-PA-reaction is complete within 5 minutes at 37°C, PAI-2 activity can be calculated by determining the difference in u-PA inhibition between 35 minutes and 5 minutes incubation time. PAI-2 in plasma of pregnant women is determined as follows: 25 µl plasma is being incubated with 25 µl 40 IU/ml urokinase (u-PA) pH 8,4 in 100 mM Tris, 0,1% Triton X 100, 1% polygelin, Aqua dest. (assay buffer) for 5 minutes or incubated for 35 minutes at 37°C. Then 25 µl of 40 mM Chloramin T and 25 µl 0,4 g/l (4,34 µM) Glu-plasminogen in assay puffer, containing 60 mM tranexamic acid are added. After 5 minutes at 37°C, 50 µl 1,8 mM H-D-Val-Leu-Lys-pNA in 1,39 M KCl is added and the linear kinetic (A405nm/min) is being measured through absorbation at a wavelength of 405 nm using a microtiterplate reader. The inhibition difference between 35 minutes and 5 minutes incubation time is calculated. The assay is not sensitiv to therapeutical plasma concentractions of heparin. The intra- and interassay coefficients of variation have been found to be less than 5%. The detection limit equals 2 U/ml PAI-2 and the recovery at 95 7%. A negative linearity exists between PAI-2 and extinction. The PAI-2 activity of pregnant women at term is 30,3  19,9 U/ml. The plasmatic PAI-2 activity correlates negatively with the PAI-1 activity (r = - 0,730). In summary, three new chromogenic fibrinolysis assays are being investigated by this dissertation. In particular, the global FIPA assay, but also aFIPA and PAI-2 assay, are valuable new techniques to determine the fibrinolysis status of patients.