Singlet Oxygen Enhances Intrinsic Thrombolysis: The Intrinsic Oxidative Clot Lysis Assay (INOXCLA)

Abstract

Granulocytes are important cells of inflammation and cellular thrombolysis. They produce urokinase (u-PA) and chloramines. In this study, u-PA/chloramine—mediated fibrinolysis is imitated in a microtiter-plate. Seventy-five microliters plasma are incubated with 50 μL 50% Pathromtin SL, 6% BSA, and 38 mM CaCl2 for 30 minutes (37°C). Then, 50 μL 10 mM chloramine-T in PBS are added. After 30 minutes (37°C), 50 μL 0, 100, or 10 IU/mL u-PA in 6% BSA-PBS are added and the turbidity is determined at 405 nm after 0, 3, or 16 hours. Clot lysis was increased more than tenfold by 0.5 to 1 μmoles chloramine (ED50 after 3h = about 0.25 μmoles = 2mM final concentration). The normal range for the present intrinsic oxidative clot lysis assay (INOXCLA) is 100% ± 25% (MV ± SD; 100 relative % of norm; the normal lysis being 60 absolute %; CVs < 10%). Fifty percent lysis of adherent microclots occurred after 0.75 hours, 2 hours, 14 hours, 13 days, or 17 days when using 1000, 100, 10, 1, or 0 IU/mL u-PA reagent. If the u-PA activity is quenched by PAI-2, no clot lysis appears. Chloramines are important physiologic generators of nonradical excited singlet oxygen and enhance u-PA—mediated lysis of plasma clots. Based on the u-PA/chloramines coaction, a new global fibrinolysis assay has been derived.