A B S T R A C T Platelets secrete lysosomal enzymes during the "platelet release reaction" early in clot formation. This study was undertaken to identify primary lysosomes of platelets and to determine their origin in megakaryocytes. Using electron microscopy and cytochemistry, we localized two lysosomal enzymes, arylsulfatase and acid phosphatase, in megakaryocytes and platelets of normal and thrombocytopenic rats. In platelets and mature megakaryocytes, reaction product for both enzymes is confined to vesicles measuring 175-250 nm. These vesicles, which are primary lysosomes, first appear in the earliest recognizable megakaryocytes and increase in number during cellular maturation. In immature and maturing megakaryocytes, arylsulfatase and acid phosphatase can also be demonstrated in an organelle similar to GERL (Golgi-endoplasmic reticulumlysosome), i.e., a single smooth-surfaced cisterna with associated vesicles near the stacked Golgi cisternae. Scant reaction product for acid phosphatase is also sometimes seen in Golgi cisternae and endoplasmic reticulum. No reaction product was found in a-granules at any stage of megakaryocyte maturation, nor in a-or serotonin granules of platelets. Thus, our findings indicate that the primary lysosomes of megakaryocytes and platelets are small vesicles derived from GERL early in megakaryocyte differentiation. They can be identified only after cytochemical staining and are distinct from both a-and serotonin granules.

The differentiation of leukocytes in the bone marrow and blood of normal adult male rats was studied by electron microscopy and peroxidase cytochemistry. Tissue samples were fixed in glutaraldehyde, or paraformaldehyde-glutaraldehyde, and incubated in a peroxidase medium containing 3,3'-diaminobenzidine and H2O2 ad pH 7.6. Mature cells of blood were identified, and then the earlier stages of maturation in bone marrow were analyzed. In immature cells of four cell lines, neutrophils, monocytes, basophils, and eosinophils, peroxidase is synthesized and could be demonstrated in the rough endoplasmic reticulum (RER), Golgi complex, and in cytoplasmic granules. Later in maturation, reaction product for peroxidase could not be found in RER or Golgi complex, indicating that peroxidase synthesis had ceased. In two cell lines, neutrophils and monocytes, peroxidase-negative granules were formed, and the mature cells contained two populations of cytochemically distinct granules. All granules of mature eosinophils were peroxidase-positive. In mature basophils, some granules were clearly peroxidase-positive; others displayed variable density, making interpretation uncertain. Mast cells were never seen in blood, but were abundant in bone marrow; peroxidase was never found in their granules by either electron microscopic cytochemistry or a variety of light microscopic methods. Hence, these cells differ from basophils, not only in morphology but also in the enzyme content of their granules.

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