Leukocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions.

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Abstract

Because of the many potent biological capabilities of the blood granulocytes, and their contact with platelets in various physiologic and pathologic states, a possible interaction between granulocytes and platelets was investigated. Platelets were purified by gel filtration and via a dialysis membrane were separated from suspensions of autologous granulocytes prepared by dextran sedimentation and resuspended in modified Tyrode's buffer. After 20 min at 37 degrees C platelet aggregation was shown to be diminished by such exposure, as compared to the aggregation of platelets incubated with dialysates of buffer only. When granulocytes were stimulated by the addition of 1.1-muM latex spheres as target particles for phagocytes, the dialysate of these cells exhibited greatly enhanced platelet-inhibitory properties. The addition of catalase to the platelets abolished the effect of exposing these cells to the dialysate of resting granulocytes and markedly inhibited the effect of exposing the platelets to the dialysate of phagocytosing granulocytes. Catalase treated with 3-amino-1,2,4-triazole had no platelet-protective capacity. Purified suspensions of lymphocytes released no platelet-inhibitory principle under these experimental conditions. Hydrogen peroxide in the dialysate of granulocytes was measured directly with an assay involving an H2O2-induced decrease in the fluorescence of scopoletin catalyzed by horseradish peroxidase. The dialysate of phagocytosing granulocytes contained 0.86 +/- 0.55 nmol H2O2/2.5 X 10(7) granulocytes when sampled at 20 min. By an alternate measurement technique in which scopoletin and horseradish peroxidase were present in the dialysate from time zero, the mean amount of H2O2 in the dialysate reached 4.0 +/- 1.3 nmol/2.5 x 10(7) granulocytes at 20 min. This discrepancy suggested the consumption of H2O2, possibly mediated by the granulocytes themselves. This possibility was investigated by the addition of exogenous H2O2 to the test system. Both granulocytes and platelets enhanced the disappearance of H2O2 from the dialysate, and the amount consumed was proportional to the amount of H2O2 added to the system. Glucose oxidase at 12 M U/ml plus glucose in excess resulted in the production of H2O2 at a rate and final amount comparable to that produced by phagocytosing granulocytes. This mixture, when substituted for phagocytosing granulocytes in the standard dialysis membrane experiment, induced an inhibition of platelet aggregation similar to that caused by the granulocytes. The observation that the release of H2O2 by the blood granulocyte influences platelet function suggests a potential role for the granulocyte in the regulation of hemostasis or thrombosis.

Authors

P H Levine, R S Weinger, J Simon, K L Scoon, N I Krinsky