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Ultrastructure of platelet formation by human megakaryocytes cultured with
the Mpl ligand
EM Cramer, F Norol, J Guichard, J Breton-Gorius, W Vainchenker, JM Masse and N Debili
INSERM U.91-Hopital Henri Mondor, Creteil, France.
The site and mechanism of platelet production by bone marrow megakaryocytes
(MKs) has been the subject of extensive studies, but is still a matter of
controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also
called megakaryocyte growth and development factor (MGDF) or
thrombopoietin, has resulted in considerable progress in the understanding
of the maturation of the MK lineage. To better understand the mechanism of
platelet production, we examined the late stage of MK maturation by
electron microscopy in cells cultured in the presence of Mpl-l. Human bone
marrow CD34+ CD38+ cells, which contain late MK progenitors, were purified
by flow cytometry and cultured in a serum- free liquid medium containing
recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the
majority of cultured cells were large MKs with lobulated polyploid nuclei.
The MKs displayed a smooth surface with harmonious cytoplasmic maturation
and abundant, regularly distributed demarcation membranes and
alpha-granules, and even some dense granules. Interestingly, approximately
30% of the MKs observed displayed morphologic evidence of platelet
production: at optical microscopy, MKs formed long filamentous cytoplasmic
extensions (proplatelets) that fragmented into platelet-sized particles.
Moreover, flow cytometric analysis of this cultured cell population showed
GPIIb- positive particles of the size of platelets. Electron microscopic
observation showed that MKs producing platelets displayed thin pseudopods
on the surface, and that the channels of the demarcation membrane system
were dilated, allowing long strands of cytoplasm to extend from the cell
periphery. These cytoplasmic strands displayed beading with constrictions
separating platelet-sized segments; the more distal to the cell core, the
smaller the fragments were. They eventually detached from the cell core
into the culture medium either occasionally still elongated or, more often,
separated into individual platelets. Parallel longitudinal and
perpendicular microtubules were visualized in the constricted regions of
these cytoplasmic strips; immunogold study of tubulin localization
confirmed this subcellular distribution. On both sides of the constricted
areas, vacuoles were noted, the fusion of which might have led to the
detachment of individual platelets. Finally, in close proximity to the
platelet- forming MKs, numerous microparticles were shed. Although some of
these particles might correspond to transverse sections of pseudopods, this
did not seem to be the case, since they were rarely seen around
thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow
cytometry showed that apart from shed cytoplasmic fragments of platelet
size, numerous smaller particles strongly labeled for CD41 were also
released by mature MKs. In conclusion, this study describes the
ultrastructure of human platelet production in cultured MKs, involving the
formation of proplatelets and the shedding of microparticles.
Volume 89,
Issue 7,
pp. 2336-2346,
04/01/1997
Copyright © 1997 by The American Society of Hematology
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