(American Journal of Pathology. 2000;157:69-74.)
© 2000 American Society for Investigative Pathology
Platelet Production in the Pulmonary Capillary Bed
New Ultrastructural Evidence for an Old Concept
Dorothea Zucker-Franklin* and
Claire S. Philipp
From the Department of Medicine,*
New York University
Medical Center, New York, New York; and the Division of
Hematology,
Department of Medicine, Robert
Wood Johnson Medical School, University of Medicine and Dentistry of
New Jersey, New Brunswick, New Jersey
 |
Abstract
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Although there is substantial evidence indicating that platelets
are released from megakaryocytes in the capillary bed of the
lung, this concept has not been universally accepted because
much of the evidence has been indirect. To more definitively
substantiate that platelet production takes place in the lungs,
megakaryocyte and platelet production was accelerated in mice by
phlebotomy or by administration of thrombopoietin, and
ultrastructural analysis was performed on lung specimens. Intact
megakaryocytes, megakaryocyte fragments with numerous
demarcated platelet fields, dissociating intact
platelets, and denuded megakaryocyte nuclei were seen in the
pulmonary capillaries of mice. In addition, some megakaryocyte
nuclei exhibited the morphological counterpart of apoptosis.
These observations provide evidence for platelet release in the
capillary bed of the lungs during stimulated as well as reactive
thrombocytosis without precluding observations that some
"proplatelets" form in the sinusoids of the bone marrow before
transmigration of intact megakaryocytes into the
circulation.
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Introduction
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The origin of platelets is no longer controversial. No one
would question that platelets are derived from megakaryocytes, whether
this occurs by fragmentation of long megakaryocyte processes forming on
a solid substrate in vitro,1
by fragmentation
of long megakaryocyte pseudopods, so-called proplatelets, protruding
into the sinusoidal lumen in vivo,2,3
or by
more global fragmentation of megakaryocyte cytoplasm into individual
platelets as observed when the cells are kept in
suspension.4
Rather, the controversy concerns the site
where the bulk of platelet release takes place in
vivo. Evidence supporting the concept that megakaryocytes are
migratory and able to exit intact via marrow sinusoids has been
illustrated by numerous investigators.5,6
It is also
generally known that platelets are not motile and that few, if
any, are found in normal bone marrow. The latter statement pertains
equally to denuded or bare megakaryocyte nuclei.7
Therefore, it has become increasingly
clear that platelets are released from megakaryocytes into the
bloodstream when long megakaryocyte processes protrude through the
marrow sinusoids or that this process takes place in the lungs, the
first capillary bed encountered by any cell leaving the bone marrow.
Scattered reports of the latter observation have appeared in the
literature for at least 30 years.8-12
A more recent study
by Levine et al13
addressing this issue specifically has
established that in humans, 10 times as many intact megakaryocytes are
found in pulmonary artery blood than in blood obtained from the aorta.
Moreover, it was observed that 98% of megakaryocytes leaving the lung
are devoid of cytoplasm. These observations corroborated other studies
quantitating megakaryocytes in the central and arterial circulations
before, during, and after cardiopulmonary bypass in patients with
normal platelet counts who underwent cardiac surgery.14
Before cardiopulmonary bypass (CPB), the number of megakaryocytes
in arterial blood was 0.25/ml, whereas it was 4.21/ml in venous blood.
During CPB, the number of megakaryocytes in arterial blood rose to
19.49/ml. Whereas the number of megakaryocytes was always higher in
central venous than in arterial blood before and after CPB, this
difference was essentially lost during CPB. Last, on autopsy
pathologists routinely find megakaryocytes in the lungs but not in any
other organs.15
Together, these reports have markedly
strengthened the conclusion based on numerous smaller historical
studies that platelets are released from megakaryocytes in the lung.
Unfortunately, even the report by Levine et al was not able to lay the
controversy to rest. A subsequent study carried out in mice, after
perturbation of platelet production by several means, concluded that
the fraction of thrombopoiesis occurring in murine lung, even during
periods of greatly increased platelet production, is
insignificant.16
However, the agents used to increase
thrombopoiesis in this study, such as platelet antiserum,
5-fluorouracil, and radioactive strontium, are known to affect
megakaryocyte and platelet physiology in ways not commonly operative
during physiological, reactive thrombopoiesis. For instance, platelet
antiserum cross-reacts with megakaryocytes, which could affect their
motility. The detrimental effect of 5-fluorouracil on megakaryocytes is
well known to hematologists.
Thus, although the evidence that platelet release occurs in the lung is
substantial, most of the data are indirect and are still questioned by
some investigators. Therefore, we decided to reexamine the issue
by increasing thrombopoiesis in mice with more physiological stimuli,
namely by phlebotomy and by the administration of thrombopoietin, and
subjecting lung specimens to ultrastructural analysis. The large number
of intact megakaryocytes, megakaryocyte fragments, and denuded
megakaryocyte nuclei seen in the pulmonary capillary bed of these
animals was remarkable, leaving little room for further doubt that
platelets are released at this site.
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Materials and Methods
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Six 8–12-week-old CB6Fl/J mice (Jackson Laboratories, Bar Harbor,
ME) were given recombinant human megakaryocyte and development factor
(polyethylene glycol conjugated), a truncated Mpl ligand containing the
receptor-binding N-terminal domain of thrombopoietin (50 µg/kg/day)
(a gift from Amgen, Thousand Oaks, CA) for 4 days subcutaneously as
previously described.17
Three mice were bled (200 µl)
daily for 4 days via tail vein incision, and three mice were injected
with saline for 4 days. Mice were sacrificed on day 5. After 5 days,
platelet counts had risen from 1 x 106/µl
to 1.23 x 106/µl in bled mice and
3.6 x 106/µl in thrombopoietin-treated
animals. At this time, pegylated recombinant human megakaryocyte and
development factor is active in stimulating megakaryocyte maturation
and platelet production in mice.18
Lung tissue was harvested for electron microscopy on day 5 from
sacrificed mice and placed in 3% phosphate-buffered glutaraldehyde.
Tissue was postfixed in osmium tetroxide, dehydrated, and embedded in
PolyBed 812 (Polysciences, Warrentown, PA) as previously
described.19
Thin sections were stained with uranyl
acetate and lead citrate, and sections from untreated mice,
thromboietin-treated mice, and phlebotomized mice were examined with a
Siemens Elmskop I electron microscope.
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Results
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Megakaryocyte fragments exhibiting many demarcated platelet fields
were found within pulmonary capillaries (Figure 1)
in phlebotomized mice, when the
peripheral blood platelet count had risen from 1 x
106/ul to 1.23 x
106/µl. These findings were even more evident
in thrombopoietin-treated mice, the peripheral blood platelet count of
which had risen to 3.6 x l06/µl.
Megakaryocyte nuclei and cytoplasmic fragments were also seen in the
lungs of untreated mice, confirming reports by
others.11,14,15
However, finding megakaryocyte fragments
required more extensive search and numerous ultrathin sections in
specimens from control mice with normal platelet counts.
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Figure 1. Representative sections of lung from bled mice, showing large fragments
of megakaryocyte cytoplasm
(arrows)
within capillary lumen. The demarcated platelet fields are clearly
seen. Also note that the megakaryocyte fragment in A is
considerably larger than the lymphocyte
(L) or neutrophil
(N) within the same
capillary lumen. In B an erythrocyte
(RBC) is seen within the
same lumen as the megakaryocyte fragment. Original magnifications:
A, x8000; B, x7500.
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Figure 2
illustrates an intact
megakaryocyte, which fills the entire capillary lumen. Its peripheral
zone appears to be uninterrupted. A large intraluminal megakaryocyte,
which appears to have been fixed in the process of release of numerous
platelets, is shown in Figure 3
. It
should be noted that the platelets exhibit no evidence of activation, a
conclusion that may be safely drawn from their uniformly lenticular
shape and the even distribution of their granules. Fragmenting
megakaryocytes were not seen in the bone marrow.
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Figure 2. Intact megakaryocyte occupying the lumen of a pulmonary capillary. The
specimen was obtained from a thrombopoietin-treated mouse. Original
magnification, x9000.
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Figure 3. Megakaryocyte in the lumen of a pulmonary capillary adjacent to
numerous platelets (Plt)
likely to leave been released by the cell. RBC, erythrocyte. Original
magnification, x4000.
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In addition, the pulmonary capillaries of animals with accelerated
thrombopoiesis contained numerous megakaryocyte nuclei, which were
almost completely denuded of cytoplasm (Figure 4)
. Many of these nuclei also exhibited
morphological evidence of apoptosis (see arrow in Figure 4
and inset).
Although present, such nuclei were much more difficult to find in
control animals with baseline platelet counts.
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Figure 4. Denuded megakaryocyte nuclei seen in the capillaries of lungs obtained
from thrombopoietin-treated mice. Note erythrocytes
(RBC) within the same
lumen. The chromatin distribution in the megakaryocyte nuclei is
suggestive of apoptosis. The arrow indicates an
"apoptic" body. Original magnification, x4500; inset,
x3000.
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Discussion
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The illustrations presented in this report provide convincing
evidence that intact megakaryocytes reach the pulmonary capillaries and
that they release platelets by fragmentation of their cytoplasm at this
site. The fragments of megakaryocyte cytoplasm seen in Figure 2
are
likely to fragment further before reaching the systemic circulation,
because they are several times larger than giant platelets or even
leukocytes seen on routine blood smears. Although the uninitiated
reader may question whether the large accumulation of platelets
depicted in Figure 3
could represent a thrombus, this concern is
obviated by the lack of adherence of one platelet to another, as well
as the absence of shape changes and degranulation. Even centralization
of granules, which occurs very early after activation and before
degranulation takes place, is not evident.
These observations do not gainsay reports by several investigators
showing that long megakaryocyte processes exhibiting demarcated
platelet fields, referred to as proplatelets, can be seen to protrude
into the lumen of marrow sinusoids (reviewed in ref 3
). Presumably, the
proplatelets subsequently fragment into individual platelets. In this
case, the denuded megakaryocyte nucleus may not exit from the medullary
cavity. However, although a few denuded megakaryocyte nuclei can be
found in normal bone marrow, their number does not increase during
reactive thrombocytosis (ref 7
and unpublished observations).
Intravascular megakaryocytes in the lung were first described by
Aschoff in 1893,20
and the concept that platelet
production occurs primarily in the lung was first published in
1937.21
Since that time, numerous reports of the
occurrence of pulmonary and circulating megakaryocytes in human
subjects have been published. Interestingly, the largest number of
these reports are found in the nonhematological
literature.15
For instance, the prevailing theory
attempting to explain the etiopathology of hypertrophic pulmonary
arthropathy associated with arteriovenous shunts, either in the heart
or in the lungs, attributes the phenomenon to circulating
megakaryocytes or their fragments. It is postulated that these reach
the fingertips in the axial vascular stream, releasing growth factors
there, such as platelet-derived growth factor, which hyperstimulates
fibroblasts.22,23
Together with convincing indirect evidence, provided by an unusually
large number of reports, the illustrations presented here should leave
no further doubt that in health, a substantial number of megakaryocytes
arrest in the pulmonary capillary bed and release platelets at this
site.
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Footnotes
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Address reprint requests to Dr. Dorothea Zucker-Franklin, New York University Medical Center, UH 445, 550 First Avenue, New York, NY 10016.
Supported in part by American Heart Association grant-in-aid 9707978A (NJ Affiliate), the Foundation of UMDNJ, and the Robert Wood Johnson Cardiovascular Institute.
Accepted for publication April 13, 2000.
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References
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Top
Abstract
Introduction
