Singlet Oxygen Potentiates Thrombolysis

Abstract

Activated polymorphonuclear neutrophils (PMN) participate in physiologic thrombolysis. PMN produce large amounts of urokinase (u-PA) and oxidants of the hypochlorite/chloramine-type that generate nonradical excited singlet oxygen (1O2). The u-PA/1O2—mediated thrombolysis was imitated in vitro. One hundred microliters microclots of normal human plasma were oxidized with 25 μL 0 to 5.0 μmoles of chloramine-T in physiol. NaCl in the absence or presence of 100 μL 6% bovine serum albumin or 100 μL normal plasma. Twenty-five microliters 0 to 167 IU/mL (related to 150 μL added supernatant) u-PA or 0 to 2.08 μg/mL t-PA were added. The absorbance at 405 nm was determined after 0 to 27 hours (37°C). The specific clot turbidity was calculated, subtracting the 100% lysis absorbance from the respective measured absorbance. The chloramine-effective dose 50% (ED50) after 27 hours was determined in the presence of 2.6 IU/mL u-PA. The plasminogen activator-ED25 was determined after 2 hours (37°C), and the ET25; i.e., the time needed to lyse a microclot by 25%, was determined for each respective clot-oxidation. The ED25 of u-PA depends on the oxidation of the microclots: 1.25 μmoles chloramine/100 μL clot enhances thrombolysis approximately 20-fold; here, 25% of clot lysis is achieved within 50 minutes (using approximately 20 IU/mL u-PA), whereas approximately 5 hours are needed to lyse an unoxidized microclot by 25%. The present global assay technique imitates the u-PA/1O2 aspects of physiologic thrombolysis by PMN.