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.
Sequence Read Archive
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.).
Extended data figures
Extended data tables
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.
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.
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.
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.
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).
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.
Calvarium BM 2PIVM in PF4-mTmG mice. The videos show extravascular MKs (GFP+) releasing proplatelets in the BM sinusoids (arrows).
Calvarium BM 2PIVM in PF4-mTmG mice. The videos show large MKs (circled) entering the BM sinusoids and exiting the imaged BM space.
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.
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).