Background: Platelets are derived from megakaryocytes (MKs), although the generation process is unclear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation . The release of platelets requires cytoskeletal re-organisation involving actin. Tropomyosin TPM4 has a role in platelet formation .

Aims: To investigate the mechanisms of platelet generation from MKs in the lung using a novel ex vivo mouse model

Methods: We established an ex vivo mouse heart-lung model (Fig. 1A) for perfusion of murine MKs. MKs and their derivates were imaged by confocal microscopy. The numbers of generated platelets (GPs) in the perfusate or retained in the lung were counted by FACS and two-photon microscopy, respectively. Morphological and functional features of GPs were determined by FACS, transmission electron microscopy and in vitro thrombosis assays. A microfluidic chamber mimicking the lung vasculature system was designed to explore the mechanism of platelet generation in the lung (Fig. 1B). MKs from TPM4-/- mice were introduced to study whether TPM4 is required for platelet generation in the lung.

Results: MKs can pass multiple times through the lung vasculature, leading to the generation of physiological levels of functional platelets (approximately 1,000~4,000 per MK), through a process involving nuclear marginalization and enucleation, prior to TPM4-dependent fragmentation. GPs demonstrated morphological and functional features comparable to control platelets. Air ventilation and healthy pulmonary endothelial cells play critical roles in platelet generation.

Conclusion(s): The ex vivo mouse heart-lung model allows the detailed study of platelet generation, and has enabled us to show that MKs can repeatedly passage through the lung vasculature under air ventilation, leading to enucleation and TPM4-dependent fragmentation to generate platelets. The findings add to our understanding of why the lung is a primary site for platelet generation.

Image

Fig. 1: Mouse platelets are generated in physiological quantities from megakaryocytes passaged multiple times through mouse pulmonary vasculature ex vivo.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/platelet-generation-from-circulating-megakaryocytes-is-triggered-in-the-lung-vasculature/