Thrombopoietin can influence mature megakaryocytes to undergo further nuclear and cytoplasmic maturation

Objective We studied the effects of TPO on the nuclear and cytoplasmic maturation of mature megakaryocytes. Materials and Methods We prepared mature megakaryocytes by velocity sedimentation method and investigated the maturational effect of TPO on such mature megakaryocytes from the aspects of DNA ploidy and cytoplasmic features using a flow cytometry and Br-dU incorporation. We also studied the effects of TPO on expression of Gpllb and NF-E2 transcripts by RT-PCR and on proplatelet formation. Results DNA content increased when megakaryocytes were cultured for 2 days with TPO, resulting in generation of megakaryocytes with a DNA content of 32N or 64N. SCF had a similar, but weaker, effect, while IL-6 did not influence mature megakaryocytes. The proportion of megakaryocytes incorporating Br-dU into their nuclei was 44.3% ± 6.9% after culture with TPO, 19.5% ± 4.6% with SCF, 7.5% ± 3.5% with IL-6, and 5.2% ± 2.7% in control culture. Flow cytometry showed that TPO did not generate larger megakaryocytes with more complex intracellular structures when compared with cells of the same DNA content class (16N) cultured without TPO. In addition, TPO also did not enhance the expression of NF-E2 transcripts, but it delayed proplatelet formation by cultured megakaryocytes. Conclusion TPO first affected endomitosis by mature megakaryocytes and then altered their cytoplasmic maturation, with both maturation processes remaining proportionate to each other.