Functional Determination of Plasminogen Activatorin Arginine-stabilized Plasma

Abstract

Reliable data on plasminogen activator (PA) activities in blood of patients receiving fibrinolytic treatment are lacking. This is due to the continuing in vitro action of PA after blood withdrawal. We have elaborated a new simple stabilization technique for plasma involving the addition of arginine in final concentrations greater than 500 m M. In this study, new assays for PA in stabilized plasma are developed. The assay was performed with substrate plasma, that is, pooled normal plasma, preoxidized with chloramine-T; oxidant amount and oxidation time were optimized. The chloramine consumption by plasma was assayed with a KJ-assay (absorbance increase at 405 nm by addition of 200 μL 4 M KJ to 25 μL oxidized plasma). The substrate plasma concentration in the PA assay and the PA acting time was optimized. The inhibition of PA by the cations Na+, K+, Mg2+, and Ca2+ was evaluated. The optimized PA 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 M CT in PBS. After 30 minutes (37°C), 175 μL 15 m M CT oxidized EDTA plasma are added. After 40 minutes (37°C), 75 μL Stop-CS Reagent is added and ΔA at 405 nm was determined, giving PA + plasmin activity in plasma. A control value (basal plasmin activity) consists of the addition of Stop-CS Reagent before 175 μL oxidized EDTA plasma. To obtain plasmatic PA activity, the control value has to be subtracted from the PA main value. The assay is matrix-independent and linear up to 1250 IU/mL t-PA, 790 U/mL reteplase, or 199 IU/mL u-PA (37 n M). With arginine stabilization of plasma and the described determination of plasminogen activator activity in arginine-stabilized plasma, it is feasible to determine the activity of plasminogen activators in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes of the samples.