Singlet Oxygen ( 1O 2) Disrupts Platelet Aggregates

Abstract

Introduction: Important mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which generate the nonradical photon-emitting oxidant singlet oxygen ( 1O 2). Since 1O 2 inhibits platelet aggregation, we became interested in a possible oxidant mediated reversibility of platelet aggregation. Methods: Chloramine T (CT) is a stable 1O 2 generator that mimics the natural chloramine N-chloro-taurine. Platelet-rich plasma (PRP) was incubated with CT 0–8 min after addition of the aggregation agonist (10 μM adenosine-5′-diphosphate, ADP, or 5 μg/ml collagen) and the aggregation was monitored. Platelet function was also analyzed by the platelet function analyzer, PFA-100. Fifty microliters of 200 μmol/l ADP was added to 400 μl PRP. After 1 min at 37 °C, 50 μl of 0 or 30 mmol/l CT was added, and after an incubation for 3 min at 37 °C, 50 μl of 25% glutaraldehyde was added. The samples were analyzed in a transmission microscope at ×3000 and ×7000 magnification. Results: Chloramines inhibit platelet function in PRP: about 1 mM CT suppresses 50% of the aggregatory capacity of thrombocytes in normal PRP (effective dose 50%, ED 50=1 mM chloramine), which is identical to the ED 50 for CT in whole blood. The ADP- or collagen-induced platelet aggregation can be reversed by addition of CT: up to 2 min after the addition of ADP as the aggregation inducer, the aggregation is reversible to more than 70% by addition of a 1O 2 release-inducer (3 mM CT). In contrast, addition of CT 8 min after the addition of ADP results only in about 50% reversal of platelet aggregation. The electron microscopic images of platelets before ADP, after incubation for 4 min at 20 μmol/l ADP, after incubation for 1 min at 20 μmol/l ADP, and a further incubation for 3 min at 3 mmol/l CT demonstrate an ADP-dependent formation of platelet aggregates, which are disrupted by 1O 2 into the single platelets; a phenomenon comparable to the decomposition of a puzzle or the continental drift of the major earth plates. The morphology of oxidized and unoxidized platelets is similar. Conclusion: This study demonstrates that 1O 2 inhibits and reverses platelet aggregation. The physiologic signal action and the direct anticoagulant action of 1O 2 might be a new principle for pharmacologic intervention in atherothrombosis.