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Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling

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Abstract

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

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Acknowledgements

This study was supported by SENSHIN Medical Research Foundation (T.O.), Ishizu Shun Memorial Scholarship (H.S.), and Jichi Medical University Graduate Student Start-up Award (H.S.). Additional funding support was provided by Bayer (T.O.). The authors are grateful to Professor N. Komatsu (Juntendo University) for providing UT-7/TPO cells. The authors also thank Yaeko Suto, Mika Kishimoto, Tamaki Aoki, Hiromi Ozaki, and Hiroko Hayakawa (Jichi Medical University) for their excellent technical assistance. BD LSRFortessa (BD Biosciences) used in this study was subsidized by JKA through its promotion funds from KEIRIN RACE. The authors would like to thank Enago (www.enago.jp) for the English language review.

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Authors and Affiliations

  1. Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi, 329-0498, Japan

    Hiroshi Saito, Morisada Hayakawa, Nobuhiko Kamoshita & Tsukasa Ohmori

  2. Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan

    Atsushi Yasumoto & Yutaka Yatomi

  3. Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Yamanashi, 409-3898, Japan

    Katsue Suzuki-Inoue

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  1. Hiroshi Saito
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  2. Morisada Hayakawa
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  3. Nobuhiko Kamoshita
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  4. Atsushi Yasumoto
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  5. Katsue Suzuki-Inoue
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  6. Yutaka Yatomi
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  7. Tsukasa Ohmori
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Correspondence to Tsukasa Ohmori.

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Conflicts of interest

T.O. received research supports from Mitsubishi-Tanabe, Novo Nordisk, Bayer, Japan Blood Products Organization, Dai-ichi Sankyo, Otsuka, Chugai, and CSL Behring, outside of this study. All other authors declare that they have no competing financial interests.

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Saito, H., Hayakawa, M., Kamoshita, N. et al. Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling. Int J Hematol 111, 786–794 (2020). https://doi.org/10.1007/s12185-020-02853-6

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  • DOI: https://doi.org/10.1007/s12185-020-02853-6

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