Page 327 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
Evidence for a cytoplasmic proplatelet promoting factor that triggers platelet production
Megakaryocytes are specialized cells that enlarge and be- come polyploid through repeated cycles of DNA replication without cell division. During development, megakaryocytes become full of platelet-specific granules, expand their cy- toplasmic content of cytoskeletal proteins, and develop a demarcation membrane system. Megakaryocytes then gen- erate platelets by remodeling their cytoplasm into proplate- let extensions, which serve as assembly lines for platelet production.1-3 Several laboratories have made fundamental discoveries in the mechanics of platelet biogenesis, includ- ing identifying the cytoskeletal forces that power proplatelet elongation, defining the mechanics of organelle transport and packaging, and establishing new mechanisms of the final stages of platelet production.4-7 Despite this progress, our understanding of the cellular and molecular basis of the process by which megakaryocytes trigger proplatelet production has clearly lagged behind. Identification of a master regulator that triggers platelet production from megakaryocytes has the potential to reveal novel targets for treating thrombocytopenia. Here we report a rigorous set of microinjection experiments that reveal a cytoplasmic factor, designated proplatelet-promoting factor (PPF), that triggers platelet production in megakaryocytes. PPF was first detected when cytoplasm from proplatelet-produc- ing megakaryocytes was isolated and microinjected back into round megakaryocytes lacking proplatelets, and this led to the immediate initiation of proplatelet production. Furthermore, the frequency with which proplatelet pro- duction was initiated was proportional to the volume of injected cytoplasm. Using various agents and treatments, we show that PPF was inactivated by protein modification procedures but resistant to inactivation by methods that modify nucleic acids. Specifically, PPF remained active after treatment with UV, nucleases, weak detergents, and dialysis, but was inactivated by treatment with proteases, sodium dodecylsulfate (SDS), phenol extraction, and heat. These observations provide compelling evidence that there is a protein, or multiple proteins, expressed during proplatelet formation that serve as an internal molecular trigger for proplatelet production.
We hypothesized that megakaryocytes undergoing pro- platelet production may contain a cytoplasmic agent re- sponsible for the initiation of proplatelet formation. This led us to ask if the microinjection of cytosol from pro- platelet-producing megakaryocytes is sufficient to trigger round megakaryocytes to start proplatelet production. To test this, we adapted a microinjection approach that was initially used to identify mitosis promoting factor (Figure 1A).8 We first separated proplatelet-producing megakaryo-
cytes from round megakaryocytes (Figure 1B) and generat- ed separate supernatant (S100) from both populations to use as the starting material for microinjection into round non-proplatelet-producing megakaryocytes (Figure 1C). Following injection of cytosol from proplatelet-producing megakaryocytes into round megakaryocytes, 83+6% of recipient megakaryocytes formed proplatelets within 1 hour (hr) (Figure 1D, E). Conversely, cytoplasm prepared from round, non-proplatelet-producing donor megakary- ocytes had almost no effect when injected into recipient round megakaryocytes (3.8+2.1% of megakaryocytes made proplatelets). Proplatelet initiation was also minimally observed in control non-injected and sham saline-in- jected recipient round megakaryocytes at 1 hr (Figure 1E), demonstrating the mechanical force of microinjection did not cause proplatelet initiation. To ensure that round megakaryocytes injected with PPF did not just initiate proplatelet formation, but also continued to elongate and elaborate proplatelets, we examined these cells again after 12 hr. Round megakaryocytes microinjected with cytosol containing PPF continued to progress beyond proplatelet initiation and formed highly developed proplatelets with the hallmark beads-on-a-string appearance, emphasizing the physiological significance of this assay (Figure 2A, B). Of note, recipient cells injected with cytosol from round megakaryocytes had still not initiated proplatelet forma- tion after 12 hr. Proplatelet initiation was also minimally observed in control non-injected and sham saline-injected recipient round megakaryocytes at the 12-hr time point (Figure 2B). However, cells injected with cytosol from pro- platelet-producing megakaryocytes, round megakaryocytes, non-injected and saline-injected controls had all formed proplatelets at similar rates by 24 hr, suggesting our obser- vations were not likely to be due to injection of an inhibitor in the cytosol from round donor megakaryocytes (Figure 2C). In order to confirm that the initiation of proplatelet formation was due to PPF, we performed a dose response, which revealed that the frequency of proplatelet initiation was directly proportional to the volume of injected cytosol from proplatelet-producing megakaryocytes (Figure 2D). To test the need for the cytosol being injected into cells versus megakaryocytes being incubated with the cytosol, we added S100 into the culture medium of round megakaryocytes. Cytosol at dilutions of 1:10 and 1:100 from both round and proplatelet-producing megakaryocytes was cultured with round (day 4) megakaryocytes (Figure 2E). Cytoplasm pre- pared from proplatelet-producing donor megakaryocytes had little effect when cultured with round megakaryocytes for 1 hr (1:10 dilution: 4.7+1.5% of megakaryocytes made
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