Abstract

Platelets are critical for haemostasis, thrombosis, and inflammatory responses1,2, but the events that lead to mature platelet production remain incompletely understood3. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis4,5,6,7. Here, by directly imaging the lung microcirculation in mice8, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow9, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

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Acknowledgements

We thank the UCSF BIDC for assistance with 2PIVM and 3D printing; A. Hérault, E. Verovskaya and S. Y. Zhang from the Passegué laboratory for assistance with hematopoietic progenitor isolation and transplantation; and D. Erle and the UCSF SABRE Functional Genomics Facility for assistance with the RNA-sequencing experiments. This work was supported in part by NIH grants HL092471 to E.P., HL107386 and HL130324 to M.R.L., the UCSF Nina Ireland Program in Lung Health (M.R.L.), and the UCSF Program for Breakthrough Biomedical Research (M.R.L.).

Author information

Author notes

    • Emma Lefrançais
    •  & Guadalupe Ortiz-Muñoz

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, University of California, San Francisco (UCSF), San Francisco, California 94143, USA

    • Emma Lefrançais
    • , Guadalupe Ortiz-Muñoz
    • , Axelle Caudrillier
    • , Beñat Mallavia
    • , Fengchun Liu
    • , Andrew D. Leavitt
    • , Emmanuelle Passegué
    •  & Mark R. Looney
  2. Department of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California 90095, USA

    • David M. Sayah
  3. Department of Pathology, University of California, San Francisco (UCSF), San Francisco, California 94143, USA

    • Emily E. Thornton
    • , Mark B. Headley
    •  & Matthew F. Krummel
  4. Cardiovascular Research Institute, University of California, San Francisco (UCSF), San Francisco, California 94143, USA

    • Tovo David
    •  & Shaun R. Coughlin
  5. Department of Laboratory Medicine, University of California, San Francisco (UCSF), San Francisco, California 94143, USA

    • Mark R. Looney

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Contributions

E.L. designed and conducted most of the experiments, analysed the data, and wrote the manuscript. G.O.-M. designed and conducted experiments and analysed the data. A.C. and B.M. conducted experiments and analysed data. F.L. performed the lung transplantation experiments. D.M.S., E.E.T., M.B.H. and T.D. assisted in designing and conducting experiments. S.R.C, M.F.K. and A.D.L. assisted in designing experiments and provided editorial support on the manuscript. E.P. assisted in designing experiments, provided technical expertise with haematopoietic progenitor analyses, and provided editorial support on the manuscript. M.R.L. designed the experiments, conducted experiments, analysed data, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark R. Looney.

Reviewer Information Nature thanks F. Ginhoux, S. Morrison, G. Zimmerman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This file contains Supplementary Tables 1a-g.

Videos

  1. 1.

    Dynamic release of platelets in the lung vasculature

    Lung 2PIVM in PF4-mTmG mice where PF4-Cre drives membrane GFP expression in MKs and platelets while all other cells are labelled with membrane tomato. Several examples are shown of large GFP+ MKs that enter the lung circulation and undergo proplatelet formation and extension. Scale bars and acquisition time are indicated.

  2. 2.

    MKs with nuclei circulating in the lung vasculature

    Lung 2PIVM in PF4-mTmG mice where PF4-Cre drives membrane GFP expression in MKs and platelets while all other cells are labelled with membrane tomato. Several examples are shown of large GFP+ MKs that enter the lung circulation and undergo proplatelet formation and extension. The evidence of a nucleus in the GFP+ cells is supported by the presence of a dark centre that appears inside the GFP+ cells. At the end of the process, when all the cytoplasm has been released, a naked nucleus is observed. Scale bars and acquisition time are indicated.

  3. 3.

    MK nuclei circulating in the lung vasculature

    Lung 2PIVM in PF4-nTnG mice where PF4-Cre drives nuclear GFP expression in MKs while all other cells are labelled with nuclear tomato. Depicted are several examples of mobile GFP+ nuclei circulating in the lung. Scale bars and acquisition time are indicated.

  4. 4.

    Surface analysis of MKs and platelets in the lung

    Surface-rendered 3D reconstruction of GFP+ MKs and platelets recorded during lung 2PIVM in a PF4-mTmG mouse. Note in example 2 that the nuclear volume is not included in the rendered GFP+ surface volume.

  5. 5.

    Quantification of MKs releasing platelets

    Lung 2PIVM in a PF4-mTmG mouse that includes 0.7 μm3 of lung volume. The number of MKs releasing platelets during this 2 hour video were counted (white circles).

  6. 6.

    MKs releasing platelets in the lung are of extrapulmonary origin

    a) Lung 2PIVM of mTmG lung (no Cre expression) transplanted into a PF4-mTmG recipient. Intravascular GFP+ MKs from the PF4-mTmG recipient (extrapulmonary) were observed releasing proplatelets. b) Reverse transplant (PF4-mTmG lung transplanted into mTmG recipient) shows extravascular MKs (GFP+), but no intravascular MKs or proplatelet formation. Scale bars and acquisition time are indicated.

  7. 7.

    Proplatelet release by MKs in the BM

    Calvarium BM 2PIVM in PF4-mTmG mice. The videos show extravascular MKs (GFP+) releasing proplatelets in the BM sinusoids (arrows).

  8. 8.

    MK migration in the BM sinusoids

    Calvarium BM 2PIVM in PF4-mTmG mice. The videos show large MKs (circled) entering the BM sinusoids and exiting the imaged BM space.

  9. 9.

    MKs releasing proplatelets in the spleen

    Spleen 2PIVM in PF4-mTmG mice. The videos show large extravascular MKs in the spleen (green circles = inactive MKs; white circles = MKs releasing proplatelets). The higher-power views show examples of extravascular MKs releasing proplatelets into the splenic sinusoids.

  10. 10.

    Sessile MKs are observed in the lung interstitium

    Lung 2PIVM in PF4-mTmG mice (examples 1 and 2) or PF4-tomato mice (examples 3 and 4) where PF4-Cre drives membrane GFP or cytoplasmic tomato expression in MKs and platelets. Shown are examples of large MKs observed in the lung interstitium that remain sessile during several hours of imaging (up to 4 hours).

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https://doi.org/10.1038/nature21706

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