Functional Determination of Plasminin Arginine-stabilized Plasma

Abstract

Reliable data on plasmin activities in blood of patients during fibrinolytic treatment are lacking. This is due to continuing plasminogen activation by plasminogen activators after blood withdrawal. The purpose of this study was to establish a new method for stabilization of blood and to detect plasmin activity in stabilized plasma. For optimization of plasma stabilization by arginine, 50 μL pooled normal citrated plasma was incubated with 50 μL of 0 to 1500 m M arginine, pH 8.7, and 25 μL 100 IU/mL u-PA, 1250 IU/mL t-PA, 10000 U/mL reteplase, 400 U/mL plasminogen-streptokinase-activator complex, 10 μg/mL tenecteplase in 6% BSA-PBS or 25 μL 25 μg/mL plasmin in 20% glycerol. Twenty-five microliters 3 m M HDVal-Leu-Lys-pNA were added immediately (1 step) or after 90 minutes (room temperature [RT]). The same experiment was performed with pooled normal citrated plasma supplemented with 3.2 mg/mL EDTA, preoxidized with 0 m M or 20 m M chloramine-T for 10 minutes (37°C). For optimization of plasmin activity, the oxidation time of the arginine-stabilized plasma sample containing 0.5 U/mL active plasmin and the chloramine-T amount was varied. Citrated plasma is stabilized against the in vitro action of all six plasminogen activators tested if the final arginine concentration is greater than 500 mM. Neither the addition of EDTA nor the addition of chloramine-T changes this plasma-stabilizing power of arginine. The optimized functional plasmin assay consists of incubation of 10 μL arginine-stabilized plasma with 10 μL 1.5 M arginine, pH 8.7, and 10 μL 100 m MCT in PBS. After 30 minutes (37°C), 75 μL 1.2 M KCl, 1.6 M Arg, 0.75 m M Val-Leu-Lys-pNA (Stop-CS Reagent), and 175 μL 6% BSAPBS are added and the absorbance increase (ΔA) at 405 nm is determined. With the present arginine stabilization procedure of plasma and the determination of plasmin activity in arginine-stabilized plasma as described, it is feasible to determine the activity of plasmin in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes in the samples.