Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.