In vitro Simulation of Therapeutic Plasmatic Fibrinolysis

Abstract

One type of therapy for thromboembolism is plasmatic thrombolysis. Several plasminogen activators (PA) are clinically available, including urokinase (u-PA), tissue plasminogen activator (t-PA), streptokinase (SK), plasminogen-streptokinase-activator-complex (PSAC), or mutants of t-PA such as reteplase (RP) or tenecteplase (TP). Therapeutic plasmatic fibrinolysis was simulated, using the PA at relevant plasma concentrations, and plasmin (Pli) and PA activities were determined. Normal citrated plasma was supplemented with 31 to 1,000 IU/mL u-PA, 0.31 to 20 μg/mL t-PA, 125 to 4,000 IU/mL SK, 12.5 to 400 U/mL PSAC, 125 to 4,000 U/mL RP, or 0.31 to 10,μg/mL TP. Ten IU/mL urokinase was also incubated with pooled plasma of stroke patients, that was previously oxidized with the singlet oxygen (¹O₂) donor chloramine T® (CT), to destroy plasmatic PAI-1 and a2-antiplasmin. After 0 to 80 minutes (37°C), 50-μL samples were withdrawn and added to 100 μL 1.5 M arginine, pH 8.7, and oxidized with 50 μL of 20 mM CT. For determination of plasmin activity, 10 μL thereof was incubated with 150 μL 1.5 M arginine, pH 8.7, and 100 μL 20 mM CT preoxidized (15 minutes 37°C) pooled normal citrate buffered EDTA-plasma for 30 minutes (37°C). For determination of [PA+Pli]-activity, arginine was added after this incubation. 25-μL 6 mM Val-Leu-Lys-pNA were added and AA/h at room temperature (RT) was monitored, using a microtiterplate reader. [PA+Pli]-Pli = PA. The PA concentration required to induce 25% [ED₂₅] of the maximally inducible Pli-activity in plasma (= 1 U/mL = 45 mg/L = 0.53 Amol/L active Pli; AA = 363 + 8 mA/h RT) after 10 minutes (37°C) were 320 IU/mL u-PA, 8 μg/mL t-PA, 140 U/mL PSAC, 6,000 IU/mL SK, 720 U/mL RP, and approximately 150 μg/mL TP. The approximate activity half-lives of the PA in plasma were 30 minutes for u-PA, 30 minutes for t-PA, greater than 80 minutes for SK, greater than 80 minutes for PSAC, 50 minutes for RP, and 80 minutes for TP. The present study shows-for the first time-a combined kinetic in vitro simulation of the plasmatic activity of six different PAs. At clinically used concentrations, RP induces the highest plasmatic Pli activity. Due to unselective generation of plasmin in plasma, all PA are of some danger in inducing severe hemorrhagias. Clinical thrombolysis might be improved by usage of more physiologic activators of thrombolysis, such as activators of polymorphonuclear neutrophils.