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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2313-2325
Multivesicular Bodies Are an Intermediate Stage in the Formation
of Platelet  -Granules
By
Harry F.G. Heijnen,
Najet Debili,
William Vainchencker,
Janine Breton-Gorius,
Hans J. Geuze, and
Jan J. Sixma
From the Department of Hematology, University Hospital Utrecht, and
Department of Cell Biology, Medical Faculty, and Institute of
Biomembranes, Utrecht University, Utrecht, The Netherlands; INSERM U
362, Institut Gustave Roussy, Villejuif; and INSERM U 91, Hopital Henri
Mondor, Creteil, France.
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ABSTRACT |
We have used ultrathin cryosectioning and immunogold cytochemistry
to study the position of  -granules in the endocytic and biosynthetic
pathways in megakaryocytes and platelets. Morphologically, we
distinguished three types of granules; so-called multivesicular bodies
type I (MVB I) with internal vesicles only, granules with internal
vesicles and an electron dense matrix (MVB II), and the -granules
with mainly a dense content and often internal membrane vesicles at
their periphery. The MVBs were prominent in cultured megakaryocytes and
the megakaryoblastic cell line CHRF-288, but were less numerous in bone
marrow megakaryocytes and platelets, whereas -granules were most
prominent in mature bone marrow megakaryocytes and in platelets. The
internalization kinetics of bovine serum albumin-gold particles and of
fibrinogen positioned the MVB subtypes and -granules sequentially in
the endocytic pathway. MVBs contained the secretory proteins von
Willebrand factor (vWF) and  -thromboglobulin (-TG), the platelet-specific membrane protein P-selectin,
and the lysosomal membrane protein CD63. Within the MVBs, endocytosed fibrinogen and endogenous -TG were restricted to the matrix, while vWF was predominantly associated with internal vesicles. CD63 was
also observed in association with internal membrane vesicles in the
-granules. These observations, and the gradual morphologic transition from granules containing vesicles to granules containing predominantly dense material, suggest that MVBs represent a
developmental stage in -granule maturation.
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INTRODUCTION |
AT SITES OF VASCULAR injury,
circulating platelets adhere rapidly to the subendothelium
and become activated. They subsequently release their granule content
and the released proteins in turn contribute to the formation of a
platelet aggregate. Most functional proteins are delivered to the major
storage compartment in platelets, the -granules. These proteins may
derive from different origins. Von Willebrand factor (vWF) and
-tromboglobulin (-TG) are synthesized in the
platelet precursor cell, the megakaryocyte, while fibrinogen, also a
major component of -granules, is not synthesized by the megakaryocytes,1-3 but is exclusively recruited from
plasma.4-6 The platelet -granule therefore represents a
unique storage compartment that stores both endogenously synthesized
proteins, as well as proteins derived from endocytic origin. It has
been well documented that uptake and the delivery of fibrinogen to
-granules is mediated by glycoprotein IIb-IIIa
(GPIIb-IIIa).7-9 Receptor-mediated endocytosis is a process
by which cells internalize surface-bound molecules efficiently via
specialized domains on the plasma membrane. In many cell types,
clathrin-coated pits are considered to play a key role in the selective
uptake of ligands by their appropriate receptor,10 but
clathrin-independent mechanisms of internalization have been described
as well.11 Endocytosis and incorporation of fibrinogen into
the -granules require surface binding, internalization, and proper
intracellular targeting. Little is known about the intracellular
trafficking of proteins in megakaryocytes and platelets, especially
with regard to the intracellular routing of fibrinogen following
internalization and delivery to -granules. The purpose of the
present study was to examine the position of -granules in the
intracellular pathways of endocytosis and biosynthesis. We used
ultrathin cryosectioning and immunoelectron microscopy on platelets,
bone marrow megakaryocytes (ex vivo and cultured), and on the
megakaryoblastic cell line CHRF-288. The formation of -granules with
respect to the endocytic and biosynthetic pathways is discussed and a
multivesicular precursor organelle of -granules is described.
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MATERIALS AND METHODS |
Tracers and antibodies.
Bovine serum albumin (BSA) was conjugated to 5 nm colloidal gold
particles according to standard procedures.12 Before use, the gold probe was dialyzed against culture medium without albumin. Rabbit polyclonal GPIIb-IIIa and rabbit polyclonal P-selectin antibodies were a gift from Dr M.C. Berndt (Melbourne, Australia) and
have been previously described.13 Rabbit polyclonal human fibrinogen and human von Willebrand factor (vWF) antibodies were purchased from Dakopatts (Glostrup, Denmark). The rabbit polyclonal -tromboglobulin (-TG) antiserum was exclusively
directed to human platelet -TG.14 Endosomes were
distinguished by using a rabbit polyclonal antibody directed against
the 300-kD cation-independent mannose-6-phosphate receptor
(CI-MPR), which is present in endosomes and absent from
lysosomes.15-17 To distinguish lysosomes, a monoclonal antibody RUU-SP 28, that recognizes the lysosomal integral membrane protein CD63,18,19 was used, and rabbit polyclonal
antibodies directed to the lysosomal enzymes
-hexosaminidase, cathepsin-D,17,20 and lysosomal
acid phosphatase.21 Secondary antibodies swine antirabbit
IgG and rabbit antimouse IgG were purchased from Sigma (St Louis, MO).
Protein A-gold particles of 10 and 15 nm were prepared according to
Slot and Geuze.12
Platelets and bone marrow megakaryocytes.
Blood was taken from healthy donors and was anticoagulated with 1/10
vol of 0.13 mol/L sodium citrate. Platelet-rich-plasma (PRP) was
prepared by centrifugation at 180g for 10 minutes at 22°C.
The PRP was mixed in a 1:1 ratio with Krebs Ringer Glucose, pH 5.0 (4 mmol/L KCl, 100 mmol/L NaCl, 20 mmol/L NaHC03, 2 mmol/L Na2SO4, 4.7 mmol/L citric acid, 14.2 mmol/L
tri-sodium citrate, and 5 mmol/L glucose) and washed again with the
same solution at pH 6.0. Normal bone marrow samples were obtained after
informed consent from a patient undergoing heart surgery. The
megakaryocyte-rich cell suspension was obtained after centrifugation on
Ficoll-Paque (Research Grade, Pharmacia, Uppsala, Sweden).
Megakaryocyte culture.
Megakaryocytes were cultured as previously described.22
Briefly, bone marrow cells were obtained after informed consent from
donors undergoing bone marrow transplantation. The cells were separated
by density centrifugation on Ficoll-Paque (density < 1.077 g/mL) and
were cultured in a liquid Iscove's medium (GIBCO, BRL Life
Technologies, Paisly, UK) containing 10% aplastic plasma (from
thrombocytopenic patients after bone marrow transplantation) and 1%
deionized BSA. Nonadherent cells were transferred into culture flasks
to enrich the megakaryocyte population. Cells were cultured at 37°C
for 13 ± 2 days in a 5% CO2 fully humidified atmosphere.
Megakaryoblastic cell line CHRF-288.
The CHRF-288 cell line, originally derived from a patient with an acute
megakaryoblastic leukemia, was a kind gift from Drs M. Liebermann and
D. Witte (Cincinnati, OH).23 The cell line was cultured in
Fisher's medium (GIBCO, BRL Life Technologies), in the presence of 2 mmol/L L-glutamin, 100 U/mL penicillin, and 100 U/mL streptomycin and
20% heat inactivated horse serum at 37°C in a humidified
atmosphere with 5% CO2. An enzyme-linked immunosorbent
assay (ELISA) was performed to check the vWF and fibrinogen content in
the medium and the immunoreactivity of the antibodies. The rabbit
antihuman vWF antibody did not cross-react with vWF in horse serum.
Internalization studies.
Megakaryocytes were used for internalization studies after 11 to 13 days of culture in medium depleted of fibrinogen. The megakaryocytes
were incubated for 30 minutes, 60 minutes, and overnight in liquid
Iscove's medium, containing 20% normal citrated plasma, to which 150 U/mL desulphatohirudin was added (a kind gift of Dr R.B. Wallis,
Ciba-Geigy Pharmaceuticals, Horsham, UK). Control megakaryocytes were
directly fixed. Incubation was at 37°C in a 5% CO2,
fully humidified atmosphere. For BSA-gold uptake, the cells were rinsed
once with albumin-free medium and incubated for 5, 15, 30, 60, and 120 minutes in medium containing BSA-gold (final optical density
[OD] 2.0). Immediately after internalization, the cells
were put on ice, rinsed once with ice-cold phosphate-buffered saline
(PBS) (0.15 mol/L NaCl, 0.1 mol/L phosphate buffer, pH 7.4), and fixed
for 1 hour in a mixture of 2% paraformaldehyde and 0.2%
glutaraldehyde (final concentration) in 0.1 mol/L phosphate buffer (pH
7.4) at 4°C. Internalization studies in the megakaryoblastic cell
line CHRF-288 were performed as described for cultured megakaryocytes.
Electron microscopy.
For standard electron microscopy, cells were fixed at room temperature
for 1 hour in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4. They were then washed three
times in 0.1 mol/L sodium cacodylate buffer and postfixed for 1 hour in
1% aqueous OsO4. Cells were rinsed three times in distilled water, dehydrated in a graded series of ethanol, and embedded
in Epon.
Cryosectioning and immunolabeling.
After fixation, the cells were rinsed three times in 0.1 mol/L
phosphate buffer (pH 7.4), and resuspended in 10% gelatin in PBS. The
gelatin was solidified at 4°C and cut into small blocks. After
infiltration in 2.3 mol/L sucrose at 4°C, the cells were frozen in
liquid nitrogen. Ultrathin cryosectioning was performed on a Reichert
Ultracut S microtome (Leica Aktiengesellschaft, Reichert Division,
Wien, Austria), using a cryo-diamond knife (Diatome, Biel,
Switzerland). Sections were picked up with a 2.3 mol/L sucrose drop, or
using a recently developed method,24 with a mixture of 2.3 mol/L sucrose and 2% nonbuffered methyl cellulose (25 centipoise;
Fluka AG, Buchs, Switzerland), followed by immunolabeling.
Alternatively, sections were picked up using a mixture of 1.8% methyl
cellulose and 2% uranyl acetate, transferred to formvar carbon-coated
copper grids, and immediately embedded in a mixture of 1.8% methyl
cellulose and 0.3% uranyl acetate at 4°C ("direct view"
method). Immunolabeling was performed at room temperature by floating
on drops containing the diluted antibodies, as described
previously.25 After incubation, the sections were washed in
distilled water and stained for 5 minutes with uranyl-oxalate, pH 7.0, washed again, and embedded in a mixture of 1.8% methyl cellulose and
0.3% uranyl acetate at 4°C.26
Immunogold double labeling was performed as described by Slot et
al.27 Briefly, the single-labeled sections were put
successively on drops containing PBS, 1% glutaraldehyde in PBS (5 minutes), PBS, PBS/0.02% glycine, and PBS/1% BSA before starting the
next incubation. In the internalization experiments where 5 nm BSA-gold was used, we used protein-A gold sizes of 10 nm and 15 nm for the
detection of the marker proteins. In studies of fibrinogen uptake, we
usually combined the 5-nm gold probe for fibrinogen detection
with subsequent 10-nm and 15-nm gold probes for other marker proteins.
The specificity of immunolabeling was verified using an irrelevant
control antibody. The sections were examined in a Philips CM 10 (Philips, Eindhoven, the Netherlands) or JEOL 1200 EX (JEOL, Tokyo,
Japan) electron microscope.
In an attempt to study the kinetics of endocytosis, the number of gold
particles associated with each compartment was scored at selected time
points. Similarly, kinetic data of endocytosis were obtained in
CHRF-288 cells by comparing BSA-gold uptake in MPR- and CD63-positive
structures after 10-minute and 60-minute incubations, respectively. The
intracellular localization of internalized fibrinogen was determined
after overnight incubation with medium containing 20% normal plasma.
The subcellular distribution of GPIIb-IIIa, P-selectin, and CD63
antigen, and the biosynthetic proteins vWF and -TG was
determined in the cultured megakaryocytes, as well. For all
quantitative data, the megakaryocytes were selected at low
magnification and the number of gold particles associated with the
various intracellular compartments were counted on micrographs at a
nominal magnification of 12,000×.
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RESULTS |
Structural characteristics of MVBs and -granules.
The cultured megakaryocytes showed all stages of maturation. Immature
megakaryocytes contained only a few -granules, and generally no
demarcating membrane system (DMS), while the large mature
megakaryocytes, like those from bone marrow, had a well developed DMS
and numerous -granules. Megakaryoblasts and immature megakaryocytes
could also be identified by their differential immunoreactivity for
platelet-specific marker proteins such as GPIIb-IIIa, P-selectin, vWF,
and -TG (see below). All maturation stages showed
multivesicular bodies (MVBs), and -granules
(Figs 1-3). MVBs were most numerous in
the immature megakaryocytes in culture, but were found in bone marrow
megakaryocytes (see Fig 6B) and in platelets as well
(Fig 4A and
C). In the megakaryocytes, MVBs were often located in the trans-Golgi area. The internal membrane
vesicles measured 30 to 70 nm and were best visualized using recently
developed modifications for the processing of ultrathin cryosections.24 Based on their internal morphology, two
classes of MVBs could be distinguished: those showing abundant internal membrane vesicles only (MVB I) (Fig 4A and C), and those containing electron-dense material, together with internal membrane vesicles (MVB
II) (Fig 1B). In many type II MVBs, the internal vesicles were situated
at the periphery, leaving an electron-dense matrix either in the center
or somewhat eccentric (Fig 3B). Internal vesicles were also observed in
the periphery of -granules (Fig 4B and D), but we have not found the
tubular elements in the -granules.28
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| Fig 1.
Demonstration of -TG and CD63 in cultured
megakaryocytes. Immunogold double-labeling as indicated on the figure.
(A) Specific labeling of -TG is demonstrated in the Golgi
complex (g), -granules, and MVB II (arrows). Colocalization with
CD63 (arrowheads) is restricted to the MVBs. (B) Detailed demonstration
of colocalization of -TG with CD63 in a MVB II. Note that
the labeling of CD63 is associated with membranes of the internal
vesicles and that of -TG with the matrix. Bars A, 500 nm; B,
200 nm.
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| Fig 2.
Colocalization of vWF and CD63 in cultured
megakaryocytes. Labeling as indicated on the figure. Characteristic
labeling of vWF in Golgi-stacks (g), -granules (arrows), but also in
MVBs containing CD63 (asterisks). Note that vWF is often associated with the internal vesicles. Bar, 200 nm.
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| Fig 3.
Demonstration of P-selectin and CD63 in cultured
megakaryocytes. Labeling as indicated on the figure. P-selectin is
present in the Golgi-complex (g), -granules (), and MVBs
(asterisks). Colocalization with CD63 (arrowheads) is restricted to the
MVBs. (B and C) Selected MVBs from megakaryocyte origin. Immunolabeling as indicated on the figure. Bar, 250 nm.
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| Fig 6.
Internalization of fibrinogen. (A and C)
Overnight incubation of cultured megakaryocytes with medium
containing 20% normal plasma. Labeling as indicated on the figures.
(A) Type I MVB in close aposition to the Golgi complex (g) containing
fibrinogen. (B) Demonstration of fibrinogen in a type I MVB
(arrowheads) in bone marrow megakaryocyte. (C) Typical matrix labeling
of internalized fibrinogen in type II MVBs. Inset: Detail of a type II
MVB showing fibrinogen associated with the matrix, and CD63 typically
with the internal vesicles. Bars, 100 nm.
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| Fig 4.
Multivesicular bodies and -granules in human
platelets. Modified pick up approach applied to thin frozen sections as
described in Materials and Methods. (A and B) Methyl cellulose sucrose
pick up after immuno-gold labeling with a nonspecific control antibody and protein-A gold. (C and D) "direct-view" method. (E and F) Immunolabeling as indicated on the figure. The arrow in (B) indicates an -granule containing internal vesicles at the periphery. Inset: Higher magnification of an -granule with eccentrically located internal vesicles. (D) Arrowheads indicate peripheral intragranular vesicles. (E) Immunolabeling of vWF using 10 nm protein-A gold. Gold
labeling is located at the eccentric rims of the -granules. Arrowheads indicate two internal vesicles  40 nm in diameter. (F)
Demonstration of CD63 in platelet -granules using 5 nm protein-A gold. Arrowheads indicate CD63 associated with internal vesicles, which
are often located at extended tips of the -granules. MVB, multivesicular body, , -granules. Bars, 100 nm.
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Immunocytochemical characterization of MVBs and -granules in
cultured megakaryocytes.
Immunogold labeling studies were performed on cryosections of cultured
megakaryocytes and platelets with antibodies directed against platelet
specific membrane proteins (GPIIb-IIIa and P-selectin), secretory
proteins (vWF and -TG), and lysosomal markers (CD63, cathepsin-D and lysosomal acid phosphatase), to further characterize the MVBs and -granules.
-TG and vWF.
-TG (Fig 1) and vWF (Fig 2) were present in the
Golgi-complex and trans-Golgi reticulum (TGR), in both types of MVBs
and in the -granules. A quantitative evaluation of the gold labeling patterns is presented in Table 1. Of both
vWF and -TG, the majority of the labeling occurred on the
-granules. There was threefold more labeling of -TG in
the Golgi/TGR than of vWF, while in MVB I and II, -TG
labeling was about half of that of vWF. VWF was often associated with
the internal vesicles (Fig 2), whereas -TG was predominantly
associated with the matrix of type II MVBs (Fig 1B). The pattern of
labeling for vWF within the -granules was eccentric, as has been
described before,28 and colocalized with the
electron-lucent area and occasionally with the small internal vesicles
at the eccentric rims (Fig 4E). -TG was predominantly associated with the matrix of the -granules (Fig 1A and B).
P-selectin and GPIIb-IIIa.
Although P-selectin (Fig 3), as well as GPIIb-IIIa localized to Golgi
stacks, TGR, both types of MVBs, and -granules, there were
quantitatively major differences between the two membrane receptors
(Table 1). Most of the GPIIb-IIIa was found in -granules and minor
amounts in MVBs, while P-selectin showed a more equal distribution over
MVBs I and II and -granules. In the -granules, P-selectin was
confined to the limiting membrane, and P-selectin was almost absent
from the plasma membrane.
CD63 and lysosomal enzymes.
The integral lysosomal membrane protein CD63 was predominantly present
in both types of MVBs (Figs 1-3), and specific labeling was
observed in a small number of the -granules, as well, which was best
visualized when the immunolabeling was performed with 5 nm protein-A
gold (Fig 4F). The lysosomal enzymes -hexosaminidase, cathepsin-D, and lysosomal acid phosphatase showed only weak
immunoreactivity in type I and II MVBs (not shown) and were
undetectable in -granules. The late endosomal marker CI-MPR could
not be detected in MVBs and -granules at all. The labeling of CD63
within the -granules was observed at the periphery and was
consistently associated with the internal vesicles (arrowhead in Fig
4F).
Endocytosis of BSA-gold.
Fibrinogen present in the -granules must be derived from the
extracellular environment, as megakaryocytes do not synthesize fibrinogen.1-3 Thus, -granules are connected to the
endocytic pathway of which MVBs are known to be part. To study the
relative positions of MVB types and -granules in the endocytic
pathway, we determined the arrival of internalized BSA-gold particles
in MVBs and -granules. Megakaryocytes were incubated with BSA-gold and samples were fixed after 5, 15, 30, and 120 minutes of incubation. BSA-gold was quantitated in lucent endosomes, MVBs, and -granules. As can be seen in Table 2, at 5 minutes and
15 minutes, BSA-gold could only be detected in endosomes
(Fig 5A through C) and MVB I. Starting at
about 30 minutes, MVB II and -granules became labeled.
Characteristic examples of compartments that contained internalized
BSA-gold are given in Fig 5. Thus, endosomes, MVBs type I and II, and
-granules are sequentially reached by the tracer particles. The
sequence of BSA-gold arrival and the gradual morphologic change from a
strictly multivesicular compartment toward a compartment with an
increased protein content suggest that MVBs represent developmental
stages of -granules.
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| Fig 5.
Endocytosis of BSA-gold (5 nm gold) in cultured
megakaryocytes. Immunolabeling as indicated on the figure. Compartments
that have incorporated the tracer are shown. (A) Characteristic tubulo vesicular endosome close to the plasma membrane containing also GPIIb-IIIa. (B) Endosome with adjacent small vesicles containing the
tracer (arrowhead). (C) Tubulo vesicular endosomes (asterisks) and a
type I MVB (arrow). (D) MVB type II (asterisk) and two adjacent -granules () both contain the endocytic tracer (arrowheads). Bars, 100 nm.
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Endocytosis of fibrinogen.
After incubation of cultured megakaryocytes with 20% normal plasma for
30, 60, and 120 minutes, fibrinogen was found in the DMS and
tubulo-vesicular endosomes adjacent to the plasma membrane. However,
the -granules were still negative at these times. Overnight incubation with plasma resulted in fibrinogen labeling in both types of
MVBs (Fig 6A and C), and occasionally in
-granules. In bone marrow megakaryocytes, fibrinogen was more
readily detected in the -granules, and in MVBs as well (Fig 6B). In
MVBs type II containing a matrix content, fibrinogen was predominantly
associated with this electron-dense matrix (Fig 6C and inset).
Observations in the megakaryoblastic cell line CHRF-288.
The megakaryoblastic cell line CHRF-288 has been described as a useful
model to study megakaryocyte function.23 We therefore compared immunocytochemical and endocytic characteristics of these cells with those of megakaryocytes in culture. The absence of a DMS in
CHRF-288 cells facilitated the identification of tubulo-vesicular structures as endosomes. CHRF-288 cells possessed a heterogeneous population of granules with a variable number of internal membrane vesicles with or without an electron dense matrix. These MVBs were
generally larger than those observed in platelets and in the cultured
and bone marrow megakaryocytes. They have previously been suggested to
represent -granules.23 CHRF-288 cells contained only
occasionally the classical -granules, described above.
Distribution of CI-MPR and CD63.
The distribution of GPIIb-IIIa, P-selectin, and vWF in CHRF-288 cells
was similar to that in megakaryocytes, but the reactivity was much
lower. CI-MPR was detectable in tubulo-vesicular structures close to
the plasma membrane (Fig 7A and B) and in
an occasional type I MVB with a small number of internal vesicles. Some
CI-MPR positive endosomes contained GPIIb-IIIa as well (Fig 7B). The majority of the MVBs in the CHRF-288 cells was positive for CD63 and
negative for CI-MPR (Fig 7A).
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| Fig 7.
Demonstration of endosomes in CHRF-288 cells. (A)
10-minute internalization of BSA-gold. Tracer is found in
characteristic tubulo vesicular structures that are enriched in CI-MPR
(arrowheads). MVBs positive for CD63 are negative for CI-MPR and do not
contain the endocytic tracer at these times. (B) Simultaneous
demonstration of CI-MPR (arrowheads) and GPIIb-IIIa (small arrows) in
an endosome containing tracer. (C) Overnight incubation with medium
containing 20% normal plasma. Simultaneous demonstration of fibrinogen
and GPIIb-IIIa in an endosome close to the plasma membrane. mvb,
multivesicular body; e, endosome; pm, plasma membrane. Bars, 100 nm.
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Endocytosis of BSA-gold and fibrinogen.
Internalization of BSA-gold was followed in CHRF-288 as described for
cultured megakaryocytes. Table 3 shows the
slow progression of tracer from endosomes (Fig 7A and B) to MVBs. To
further characterize the structures involved in endocytosis,
immunodouble labeling was performed for CI-MPR and CD63 after
respectively 10 minutes and 60 minutes of BSA-gold internalization.
After 10 minutes, the tracer was located for 77% over tubulo-vesicular
endosomes showing characteristic staining of CI-MPR (Fig 7A; Table 3), and for 23% over CD63 positive MVBs. Fibrinogen was not detected when
CHRF-288 cells were cultured in serum depleted of fibrinogen. After a
continuous overnight incubation with 20% normal plasma, fibrinogen was
found in endosomes situated near the cell periphery that contained also
detectable levels of GPIIb-IIIa (Fig 7C), and in many CD63 positive
MVBs (not shown).
| |
DISCUSSION |
We have studied the biogenesis of -granules in platelets, bone
marrow megakaryocytes, and in the megakaryoblastic cell line CHRF-288,
using immunoelectron microscopy of cells that had internalized exogenous tracer particles or fibrinogen. Several observations indicated the existence of a multivesicular precursor organelle of
-granules, which, like MVBs in most other cells, were endocytic compartments. First, internalized tracer transited MVBs before reaching
-granules, which showed that -granules represent a post-MVB stage
in the endocytic pathway. Next, -granules contained small membrane
vesicles similar in size to the internal membrane vesicles of MVBs. The
presence of such vesicles has not been described before and could be
detected in our study because of the improved preservation of membranes
in the cryosectioning method we used. These 30 to 70 nm vesicles were
clearly different from the nonmembranous 20 nm tubules in -granules
described by others and which are probably composed of vWF
multimers.29 The vesicles that we found most likely derive
from inward vesiculation of the limiting membrane of the MVBs, as in
other cell types.30 We have not found the tubular
multimers, probably due to the absence of osmium in our preparation
procedure.29 Also in support of a formational link between
MVBs and -granules was the identification of a MVB type sharing
characteristics of both. This MVB type II contained material similar in
density to the content of -granules and which likewise showed
labeling for fibrinogen. Fibrinogen in the -granules derives from
receptor-mediated endocytosis, which is consistent with the presence of
the fibrinogen receptor in all MVB types and -granules. The
existence of an intermediate MVB type suggests a maturation of
-granules directly from type II MVBs. However, vesicular trafficking between MVBs and -granules may also add to the biogenesis of -granules. Finally, the early MVBs were most abundant in immature megakaryocytes and in the growth-arrested CHRF-288 cells, whereas the
-granules prevailed in the mature bone marrow megakaryocytes and in
platelets. In most cell types, lysosomes represent the post-MVB stage
in the endocytic route. Dense granules were not preserved in our
cryosections, probably because of their extreme dense content.
Therefore, the precise position of these organelles in the endocytic
pathway and their relation to -granules remains to be established.
As has been shown in other cells, the endocytic pathway in
megakaryocytes probably is connected to the biosynthetic organelles, particularly the Golgi complex and trans Golgi network
(TGN) by vesicular transport. Thus, -granules contain
several proteins including P-selectin and CD63 which in resting
platelets are absent from the plasma membrane, but on stimulation are
translocated to the cell surface by exocytosis of -granules.
Although not yet established, these proteins may be transported from
the Golgi/TGN directly to the endocytic compartments, including MVBs
and -granules. Recently, we have shown that the glucose
transporter-3 (Glut-3) is mainly localized to the -granule membrane
in resting platelets.31 Glut-3 is a resident plasma
membrane protein in other cells and lacks consensus internalization
motifs in its cytoplasmic domain.32 After thrombin
activation of platelets, -granules are released and Glut-3 is
incorporated into the plasma membrane. Most likely, the Glut-3 in
-granules derives directly from the biosynthetic route in the
megakaryocyte without passing through the cell surface. The identity of
putative TGN to MVB/-granule transport intermediates has not been
established. However, small granules, emanating from the TGN during
megakaryocyte maturation, have been proposed as precursor
-granules.33 These granules did not contain
internal membrane vesicles, were smaller than the MVB that we observed, and may mediate transport from the biosynthetic apparatus to the endocytic route in megakaryocytes.
In the MVBs and -granules, we found differential protein
distributions in internal membranes, the limiting membranes, and the
electron dense material in the matrices of type II MVBs and -granules. Thus, the soluble proteins -TG and fibrinogen were predominantly localized to the matrices, vWF and CD63 were enriched in
the internal vesicles, and P-selectin and GPIIb-IIIa were associated both with the internal vesicles and limiting membrane. Differences in
intragranular protein distribution have been reported
before,14,28,34,35 and may reflect effective sorting
and/or retention mechanisms during the formation of MVBs and
-granules.
We have found clathrin-coated buds on the surface of MVBs and in
agreement with previous reports on the -granules, as well (data not
shown).36,37 The presence of clathrin coats has been shown
on immature secretory granules,38,39
endosomes,40 and lysosomes.41 Clathrin-coated
buds probably represent stages in the formation of
vesicles,42,43 rather than of vesicles that
fuse,36,44 and have been implicated in protein sorting and
recycling. Specific segregation of the fibrinogen receptor from
-granules45 may be mediated by such vesicles, although we have not found an enrichment for GPIIb-IIIa in the buds.
Finally, during platelet activation, -granules undergo exocytosis by
which their content is released to the exterior. It is envisaged that
the internal vesicles of -granules and MVBs are externalized
together with the dense content. Secretion of similar internal vesicles
by exocytosis of multivesicular endocytic compartments has been shown
in many cell types, in particular in cells of the hematopoietic
lineage.46 In B and T lymphocytes, exosomes have been
suggested to be involved in antigen presentation47 and
target cell killing,48 respectively. Of interest,
extracellular microvesicles have also been observed after platelet
activation.49,50 Whether exosomes are secreted during
platelet activation and play some physiologic role is subject to
further investigations.
| |
FOOTNOTES |
Submitted September 17, 1997;
accepted November 11, 1997.
Address reprint requests to Harry F.G. Heijnen, PhD,
Department of Hematology, G03.647, University Hospital Utrecht, PO Box 85500, NL-3508 GA Utrecht, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr M.A. Liebermann for providing the CHRF-288 cells, Dr
Michael C. Berndt for providing the polyclonal anti-GPIIb-IIIa and
P-selectin antibodies, and Maurits K. Niekerk and René Scriwaneck for their excellent help in preparing the electron micrographs.
| |
REFERENCES |
1.
Lange W,
Luig A,
Dolken G,
Mertelsmann R,
Kanz L:
Fibrinogen gamma-chain mRNA is not detected in human megakaryocytes.
Blood
78:20,
1991[Abstract/Free Full Text]
2.
Louache F,
Debili N,
Cramer E,
Breton Gorius J,
Vainchenker W:
Fibrinogen is not synthesized by human megakaryocytes.
Blood
77:311,
1991[Abstract/Free Full Text]
3.
Handagama P,
Rappolee DA,
Werb Z,
Levin J,
Bainton DF:
Platelet -granules fibrinogen, albumin, and immunoglobulin G are not synthesized by rat and mouse megakaryocytes.
J Clin Invest
86:1364,
1990
4.
Handagama P,
Shuman R,
Shuman MA,
Bainton DF:
Incorporation of intraveneously injected albumin, immunoglobulin G, and fibrinogen in guinea-pig megakaryocyte granules.
J Clin Invest
84:73,
1989
5.
Harrison P,
Wilbourn B,
Debili N,
Vainchenker W,
Breton Gorius J,
Masse JM,
Savidge GF,
Cramer EM:
Uptake of plasma fibrinogen into the -granules of human megakaryocytes and platelets.
J Clin Invest
84:1320,
1989
6.
Handagama PJ,
Shuman MA,
Bainton DF:
In vivo defibrination results in markedly decreased amounts of fibrinogen in rat megakaryocytes and platelets.
Am J Pathol
137:1393,
1990[Abstract]
7.
Coller BS,
Seligsohn U,
West SM,
Scudder LE,
Norton KJ:
Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: Evidence consistent with specific roles for glycoprotein IIb/IIIa and alpha v beta 3 integrins in platelet protein trafficking.
Blood
78:2603,
1991[Abstract/Free Full Text]
8.
Handagama P,
Bainton DF,
Jacques Y,
Conn MT,
Lazarus RA,
Shuman MA:
Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea pig megakaryocyte and platelet -granules.
J Clin Invest
91:193,
1993
9.
Handagama P,
Scarborough RM,
Shuman MA,
Bainton DF:
Endocytosis of fibrinogen into megakaryocyte and platelet -granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa).
Blood
82:135,
1993[Abstract/Free Full Text]
10.
Pearse BM,
Robinson MS:
Clathrin, adaptors, and sorting.
Annu Rev Cell Biol
6:151,
1990
11.
Hansen SH,
Sandvig K,
van Deurs B:
Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors.
J Cell Biol
123:89,
1993[Abstract/Free Full Text]
12.
Slot JW,
Geuze HJ:
A new method of preparing gold probes for multiple-labeling cytochemistry.
Eur J Cell Biol
38:87,
1985[Medline]
[Order article via Infotrieve]
13.
Cramer EM,
Savidge GF,
Vainchenker W,
Berndt MC,
Pidard D,
Caen JP,
Breton Gorius J:
Alpha granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes.
Blood
75:1220,
1990[Abstract/Free Full Text]
14.
Sander HJ,
Slot JW,
Bouma BN,
Bolhuis PA,
Pepper DS,
Sixma JJ:
Immunocytochemical localization of fibrinogen, platelet factor 4, and beta-thromboglobulin in thin frozen sections of human blood platelets.
J Clin Invest
72:1277,
1983
15.
Geuze HJ,
Slot JW,
Strous GJ,
Hasilik A,
Von Figura K:
Ultra structural localization of the mannose 6-phosphate receptor in rat liver.
J Cell Biol
98:2047,
1984[Abstract/Free Full Text]
16.
Geuze HJ,
Slot JW,
Strous GJ,
Peppard J,
Von Figura K,
Hasilik A:
Intracellular receptor sorting during endocytosis: Comparative immunoelectron microscopy of multiple receptors in rat liver.
Cell
37:195,
1984[Medline]
[Order article via Infotrieve]
17.
Von Figura K,
Gieselmann V,
Hasilik A:
Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts.
EMBO J
3:1281,
1984[Medline]
[Order article via Infotrieve]
18.
Nieuwenhuis HK,
van Oosterhout JJ,
Rozemuller E,
van Iwaarden F,
Sixma JJ:
Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation.
Blood
70:838,
1987[Abstract/Free Full Text]
19.
Metzelaar MJ,
Wijngaard PL,
Peters PJ,
Sixma JJ,
Nieuwenhuis HK,
Clevers HC:
CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells.
J Biol Chem
266:3239,
1991[Abstract/Free Full Text]
20.
Hasilik A,
Pohlmann R,
von Figura K:
Inhibition by cyanate of the processing of lysosomal enzymes.
Biochem J
210:795,
1982
21.
Hille A,
Klumperman J,
Geuze HJ,
Peters C,
Brodsky CP,
von Figura K:
Lysosomal acid phophatase is internalized via clathrin-coated pits.
Eur J Cell Biol
59:106,
1992[Medline]
[Order article via Infotrieve]
22.
Debili N,
Kieffer N,
Nakazawa M,
Guichard J,
Titeux M,
Cramer E,
Vainchenker W:
Expression of platelet glycoprotein Ib by cultured human megakaryocytes: Ultra structural localization and biosynthesis.
Blood
76:368,
1990[Abstract/Free Full Text]
23.
Fugman DA,
Witte DP,
Jones CL,
Aronow BJ,
Lieberman MA:
In vitro establishment and characterization of a human megakaryoblastic cell line.
Blood
75:1252,
1990[Abstract/Free Full Text]
24.
Liou W,
Geuze HJ,
Slot JW:
Improving structural integrity of cryosections for immunogold labeling.
Histochem Cell Biol
106:41,
1996[Medline]
[Order article via Infotrieve]
25. Slot JW, Geuze HJ, Weerkamp AJ: Localization of macromolecular
compounds by application of the immunogold technique on cryosectioned
bacteria, in Mayer F (ed): Methods in Microbiology. Electron Microscopy in Microbiology (vol 20). London, UK,
Academic Press, 1988, p 211
26.
Tokuyasu KT:
A study of positive staining of ultra thin frozen sections.
J Ultrastruct Res
63:287,
1978[Medline]
[Order article via Infotrieve]
27.
Slot JW,
Geuze HJ,
Gigengack S,
Lienhard GE,
James DE:
Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
J Cell Biol
113:123,
1991[Abstract/Free Full Text]
28.
Cramer EM,
Meyer D,
le Menn R,
Breton Gorius J:
Eccentric localization of von Willebrand factor in an internal structure of platelet -granule resembling that of Weibel-Palade bodies.
Blood
66:710,
1985[Abstract/Free Full Text]
29.
Cramer EM,
Caen JP,
Drouet L,
Breton Gorius J:
Absence of tubular structures and immunolabeling for von Willebrand factor in the platelet -granules from porcine von Willebrand disease.
Blood
68:774,
1986[Abstract/Free Full Text]
30.
Van Deurs B,
Holm PK,
Kayser L,
Sandvig K,
Hansen SH:
Multivesicular bodies in Hep-2 cells are maturing endosomes.
Eur J Cell Biol
61:208,
1993[Medline]
[Order article via Infotrieve]
31.
Heijnen HFG,
Oorschot V,
Sixma,
JJ,
Slot JW,
James DE:
Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from -granules to the cell surface.
J Cell Biol
138:323,
1997[Abstract/Free Full Text]
32.
Pascoe WS,
Inukai K,
Oka Y,
Slot JW,
James DE:
Differential targeting of facilitative glucose transporters in polarized epithelial cells.
Am J Physiol
271:C547,
1996 |
Abstract
Introduction
Abstract