Singlet oxygen enhances intrinsic thrombolysis: the intrinsic oxidative clot lysis assay (INOXCLA).
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 microL 50% Pathromtin SL, 6% BSA, and 38 mM CaCl2 for 30 minutes (37 degrees C). Then, 50 microL 10 mM chloramine-T in PBS are added. After 30 minutes (37 degrees C), 50 microL 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 micromoles chloramine (ED50 after 3h = about 0.25 micromoles = 2 mM 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.
Stief
Thomas W
TW
Department of Clinical Chemistry, University Hospital Giesson and Morburg, Germany. thstief@med.uni-marburg.de
D002700:Chloramines / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D010958:Plasminogen / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000494:pharmacology; D026082:Singlet Oxygen / Q000097:blood / Q000494:pharmacology*; D014105:Tosyl Compounds / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology
D002700:Chloramines; D017396:Plasminogen Activator Inhibitor 2; D014105:Tosyl Compounds; C016300:chloramine-T; D026082:Singlet Oxygen; D005340:Fibrinogen; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator