PDPN/CLEC-2 axis modulates megakaryocyte subtypes in a hematopoietic stem cell-regulating megakaryocyte-dominant manner

Rikuto Nara; Hinako Notoh; Tomoyuki Sasaki; Nagaharu Tsukiji; Toshiaki Shirai; Ayuka Kamata; Nobuaki Suzuki; Atsuo Suzuki; Shuichi Okamoto; Takeshi Kanematsu; Naruko Suzuki; Akira Katsumi; Tetsuhito Kojima; Katsue Suzuki-Inoue; Tadashi Matsushita; Shogo Tamura

doi:10.1016/j.thromres.2024.109230

PDPN/CLEC-2 axis modulates megakaryocyte subtypes in a hematopoietic stem cell-regulating megakaryocyte-dominant manner

Thromb Res. 2024 Nov 26:245:109230. doi: 10.1016/j.thromres.2024.109230. Online ahead of print.

Authors

Rikuto Nara¹, Hinako Notoh¹, Tomoyuki Sasaki², Nagaharu Tsukiji², Toshiaki Shirai², Ayuka Kamata¹, Nobuaki Suzuki³, Atsuo Suzuki⁴, Shuichi Okamoto⁵, Takeshi Kanematsu⁶, Naruko Suzuki⁶, Akira Katsumi⁷, Tetsuhito Kojima⁸, Katsue Suzuki-Inoue², Tadashi Matsushita⁹, Shogo Tamura¹⁰

Affiliations

¹ Graduate School of Health Sciences, Hokkaido University, Japan.
² Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Chuo, Yamanashi, Japan.
³ Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Japan.
⁴ Department of Medical Technique, Nagoya University Hospital, Japan.
⁵ Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.
⁶ Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan.
⁷ Department of Hematology, National Center for Geriatrics and Gerontology, Obu City, Japan.
⁸ Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan; Aichi Health Promotion Foundation, Nagoya, Japan.
⁹ Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Japan; Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan.
¹⁰ Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Japan; Department of Clinical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan. Electronic address: stamura@hs.hokudai.ac.jp.

PMID: 39615442
DOI: 10.1016/j.thromres.2024.109230

Abstract

Introduction: Megakaryocytes are classified into several subtypes including LSP1-positive immune-skewed, MYLK4-positive hematopoietic stem cell (HSC)-regulating, and BMAL1-positive platelet-producing megakaryocytes. Podoplanin (PDPN)-expressing stromal cells generate a microenvironment that promotes megakaryopoiesis in the bone marrow. In this context, PDPN interacts with C-type lectin-like receptor-2 (CLEC-2) on megakaryocyte progenitors, which induces megakaryocyte proliferation. However, the megakaryocyte subtypes developed by the regulation of the PDPN/CLEC-2 axis have not yet been elucidated.

Materials and methods: We established an immortalized bone marrow PDPN-expressing stromal cell line and a PDPN-knockout line (PDPN WT and KO feeder cells, respectively). Bone marrow hematopoietic progenitors were committed to megakaryocytes in co-culture with PDPN WT or KO feeder cells. The number and ploidy of megakaryocytes, resultant platelets, and the polarization of megakaryocyte subtypes were investigated.

Results: The number of megakaryocytes was significantly increased in the co-culture with PDPN WT feeder cells compared to that with PDPN KO feeder cells. The megakaryocytes on the PDPN WT and KO feeders showed their main ploidy at 16 N∼32 N and 8 N∼16 N, respectively. The number of platelets was decreased in the co-culture with the PDPN WT feeder compared to that in the co-culture with the PDPN KO feeder. For each megakaryocyte subtype, the percentage of MYLK4-positive megakaryocytes significantly increased and the percentage of BMAL1-positive megakaryocytes significantly decreased when co-cultured with the PDPN WT feeder. These results were also confirmed in the co-culture of CLEC-2 conditional KO megakaryocytes with PDPN WT feeder cells.

Conclusion: The PDPN/CLEC-2 axis modulates megakaryocyte subtype differentiation, with a predominance of HSC-regulating megakaryocytes.

Keywords: C-type lectin-like receptor-2; Megakaryocyte; Megakaryocyte subtype; Podoplanin.