TI:  Leukocyte-Platelet Interaction
     Release of Hydrogen Peroxide by Granulocytes as a Modulator 
     of Platelet Reactions

DT:  May 5, 1975

AU:  P.H. Levine, R.S. Weinger, J. Simon, K.L. Scoon, 
     and N.I. Krinsky

SO:  The Journal of Clinical Investigation, Vol. 57, April 1976, 
     pp 955-963

AB:  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 deg 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 uM 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 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.