GRANULOPHYSIN IS LOCATED IN THE MEMBRANE OF AZUROPHILIC GRANULES IN HUMAN NEUTROPHILS AND MOBILIZES TO THE PLASMA-MEMBRANE FOLLOWING CELL STIMULATION

Granulophysin, a protein described in platelet dense granule membranes , has been shown to be similar or identical to CD63, a lysosomal membr ane protein. We have previously shown granulophysin to be present ill neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochem istry. Granulophysin expression on the surface membrane of the neutrop hil is increased following stimulation of the cells, demonstrated by f low cytometry and fine structural immunocytochemistry. A similar patte rn is shown for an anti-CD63 antibody. Incubation of activated neutrop hils with D545, a monoclonal antibody to granulophysin, blocks subsequ ent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cyt ometry and immunoblotting. Our results support the homology of CD63 an d granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granu le membrane marker of neutrophils.