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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3791-3799
Activated Platelets Release Two Types of Membrane Vesicles:
Microvesicles by Surface Shedding and Exosomes Derived From
Exocytosis of Multivesicular Bodies and  -Granules
By
Harry F.G. Heijnen,
Anja E. Schiel,
Rob Fijnheer,
Hans J. Geuze, and
Jan J. Sixma
From the Division on Thrombosis and Haemostasis, the Department of
Hematology, University Hospital Utrecht; and the Department of Cell
Biology, Medical Faculty, Institute of Biomembranes, Utrecht
University, Utrecht, The Netherlands.
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ABSTRACT |
Platelet activation leads to secretion of granule contents and to
the formation of microvesicles by shedding of membranes from the cell
surface. Recently, we have described small internal vesicles in
multivesicular bodies (MVBs) and  -granules, and suggested that these
vesicles are secreted during platelet activation, analogous to the
secretion of vesicles termed exosomes by other cell types. In the
present study we report that two different types of membrane vesicles
are released after stimulation of platelets with thrombin receptor
agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm
to 1 µm, and exosomes measuring 40 to 100 nm in diameter, similar in
size as the internal vesicles in MVBs and -granules. Microvesicles
could be detected by flow cytometry but not the exosomes, probably
because of the small size of the latter. Western blot analysis showed
that isolated exosomes were selectively enriched in the tetraspan
protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed
this observation. Membrane proteins such as the integrin chains
IIb- 3 and 1, GPIb, and
P-selectin were predominantly present on the microvesicles. IEM of
platelet aggregates showed CD63+ internal vesicles in
fusion profiles of MVBs, and in the extracellular space between
platelet extensions. Annexin-V binding was mainly restricted to the
microvesicles and to a low extent to exosomes. Binding of factor X and
prothrombin was observed to the microvesicles but not to exosomes.
These observations and the selective presence of CD63 suggest that
released platelet exosomes may have an extracellular function other
than the procoagulant activity, attributed to platelet microvesicles.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELET ACTIVATION encompasses a variety
of cellular responses, including shape change, translocation of
membrane glycoproteins, exocytosis of granule contents, and the
formation of microvesicles. Formation of microvesicles has been
demonstrated in vitro using flow cytometry after stimulation with the
agonists calcium ionophore A23187, thrombin, collagen, and the thrombin receptor agonist peptide SFLLRN.1,2 Microvesicles have also been shown in the absence of agonists, under conditions of high shear
stress.3,4 In vivo they have been observed in a
standardized bleeding time wound,5 and in clinical
situations associated with platelet activation, including idiopathic
thrombocytopenic purpura6 and cardiopulmonary
bypass.7 Although the physiological role of platelet
microvesicles remains to be established, both procoagulant8,9 and anticoagulant10 activity
have been attributed to them. Formation of microvesicles is closely
associated with the exposure of phosphatidylserine at the outer
membrane leaflet of the platelet cell surface. Exposure of
phosphatidylserine has also been described on the outer membrane
leaflet of released microvesicles,11,12 and it provides an
anionic surface for the binding of coagulation factors
VIII,13 Va,14 and Xa.15 Thus,
microvesicles have the potential to exert a procoagulant function at a
distance from the site of platelet activation. Platelet microvesicles
contain several platelet surface glycoproteins (GP) such as GPIb, the
integrin chains  IIb 3 and
15,7,16 and also P-selectin,5
which is translocated from intracellular compartments to the cell
surface after activation. The presence of these glycoproteins on
released microvesicles may be of importance for their interaction with
other cells17 or with fibrin.18
Extracellular vesicles can also originate from exocytosis of endocytic
multivesicular bodies (MVBs), by which the internal vesicles are
released from the cell as so-called exosomes.19 This
phenomenon has been shown in several cell types, in particular in cells
of the hematopoietic lineage (B cells, T cells, dendritic cells,
monocytes, and reticulocytes). For example, fusion of the multivesicular MHC class II compartment (MIIC) with the plasma membrane
in B-cell lines generates exosomes in the medium,20 analogous to the release of exosomes during exocytosis of
multivesicular cytolytic granules in cytotoxic T
lymphocytes.21 Exosomes may be involved in specific
extracellular functions like antigen presentation20,22 and
target cell killing.23
We have recently identified MVBs and subclasses of -granules as
multivesicular compartments,24 and have suggested that these internal vesicles are released during platelet activation as
exosomes. In the present article we report that during platelet activation two distinct vesicle populations are released: a population of vesicles derived from the plasma membrane (microvesicles), and a
separate population of exosomes. Because of their different origin and
specific protein composition, platelet exosomes may have an
extracellular function beside the procoagulant activity attributed to
platelet microvesicles.
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MATERIALS AND METHODS |
Reagents.
The D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) peptide was
generously provided by Dr J. Bouchaudon (Rhone-Poulenc-Rorer, Chemistry
Department, Centre de Recherche de Vitry, Vitry sur Seine, France). The
thrombin-receptor activating peptide (TRAP) ser-phen-leu-leu-arg-asn
(SFLLRN) was from Bachem Feinchemikalien AG (Bubendorf, Switzerland).
Phosphatidylserine (PS), phosphatidylcholine (PC), and
phosphatidylethanolamine (PE) were from Sigma (St Louis, MO).
Prothrombin was isolated from freshly frozen human citrated plasma
using a barium citrate precipitation as previously
described.25 Human factor X was purified from plasma as
described by Hackeng et al.26 Human -thrombin was kindly
provided by Dr W. Kisiel (Department of Pathology, University of New
Mexico, Albuquerque, NM).
Antibodies.
The following monoclonal antibodies (MoAbs) were purchased:
CLB-Gran1/2, 435 (IgG1, CD63). Monoclonal AK-3 (IgG1, GPIb) and polyclonal anti-Peta-3 were kindly provided by Dr M.C. Berndt (Prahan,
Australia). Biotin-conjugated monoclonal anti-RUU-SP 1.18 (P-selectin)
and biotinylated anti-CD63 were from our own department (Hematology,
University Hospital, Utrecht, The Netherlands). In flow cytometry,
RUU-SP 1.18 detects P-selectin exposed on the cell surface after
activation. AK-6 MoAb directed to P-selectin was from Serotec (Serotec
Ltd, Oxford, UK). Monoclonal ALB-6 (CD9) was a kind gift of Dr E. Rubinstein (Inserm U268, Villejuif, France). Rabbit polyclonal
anti-1 was kindly provided by Dr K.M. Yamada (Bethesda,
MD). Monoclonal anti-annexin-V (WAC 27b) and fluorescein isothiocyanate (FITC)-conjugated annexin-V were a gift from Dr C. Reutelingsperger (University of Maastricht, Maastricht, The Netherlands). Affinity-purified rabbit polyclonal anti-prothrombin antibody and polyclonal anti-FX were from our own department
(Hematology, University Hospital, Utrecht, The Netherlands).
FITC-conjugated MoAb anti-GPIb, FITC-conjugated streptavidin,
polyclonal anti-vWF, polyclonal anti-FITC, and rabbit anti-mouse IgG
were from DAKO (Glostrup, Denmark).
Platelet isolation.
Whole blood, obtained from healthy donors who had not used aspirin in
the preceding week, was anticoagulated with 1/10 vol of 0.13 mol/L
sodium citrate. Platelet-rich plasma (PRP) was obtained by
centrifugation at 180g for 10 minutes at 22°C. The PRP was mixed in a 1:1 ratio with Krebs Ringer buffer, pH 5.0 (4 mmol/L KCl,
100 mmol/L NaCl, 20 mmol/L NaHCO3, 2 mmol/L
Na2SO4, 4.7 mmol/L citric acid, 14.2 mmol/L
tri-sodium citrate), and the platelet pellet was washed twice with the
same buffer at pH 6.0. Finally, samples of the washed platelet
suspension were resuspended in Krebs-Ringer buffer (pH 7.4), containing
100 µmol/L of the IIb-3 inhibitor dRGDW
to prevent platelet aggregation. EDTA (5 mmol/L final concentration)
was added to the suspension just before activation to prevent
aggregation of released vesicles, and 5 mmol/L (final concentration) of
the protease inhibitor 4-(2-aminoethyl)-benzene sulfonyl fluoride
(AEBSF; Boehringer Mannheim, Mannheim, Germany) was also added to the suspension.
Isolation of vesicles.
Microvesicles and exosomes were isolated from 5 mL releasates of 4 to
12 × 106/µL washed platelets. The platelet
suspension described above was stimulated with 15 µmol/L TRAP (fc)
for 10 minutes at 37°C and centrifuged at 750g for 20 minutes
(platelet fraction). The supernatant platelet releasate was placed on
ice and centrifuged at 10,000g for 30 minutes at
4°C to obtain the microvesicle fraction (104-g fraction).
The supernatant was resuspended in 2.0 mol/L sucrose in 20 mmol/L
HEPES/1 mmol/L EDTA, pH 7.2 and a sucrose gradient (0.8 mol/L to 0.25 mol/L sucrose in 20 mmol/L HEPES/1 mmol/L EDTA, pH 7.2) was layered on
top of this fraction. The gradient was centrifuged at
65,000g for 16 hours at 4°C using an SW50.1
rotor (Beckman Instruments, Inc, Fullerton, CA), and 500-µL fractions were collected from the top of the gradient. The fractions were diluted
in 4.5 mL phosphate-buffered saline (PBS; 0.1 mol/L sodium phosphate,
0.15 mol/L NaCl, pH 7.4), with 5 mmol/L EDTA added, and
ultracentrifuged for 60 minutes at 200,000g using an SW50.1 rotor (Beckman Instruments, Inc). Samples of the pellets were solubilized in reducing or nonreducing sodium dodecyl sulfate (SDS)
loading buffer, incubated for 5 minutes at 95°C, and analyzed by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
using 125I-protein G. Protein concentrations were
determined using the BCA assay from Pierce Chemical Co (Rockford, IL).
For electron microscopy, the fractions were fixed in 1%
paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4 (see below).
Flow cytometry.
Washed platelets were activated with 15 µmol/L TRAP or 1 U/mL of
-thrombin. The samples were diluted to a concentration of 3 × 108 platelets/mL, and 40-µL aliquots
were incubated for 30 minutes at room temperature with specific
antibodies. To detect platelets and microvesicles, FITC-conjugated
monoclonal anti-GPIb or biotinylated anti-P-selectin antibody (RUU-SP
1.18) were used. For detection of exosomes a biotinylated anti-CD63
antibody (5 µg/mL) was used. Binding of the biotinylated
antibodies was monitored with FITC-conjugated streptavidin. An
irrelevant nonplatelet antibody and FITC-conjugated streptavidin alone
were used as controls. From each sample 5,000 positive events in the
platelet gate were analyzed in a Facs Calibur flow cytometer (Becton
Dickinson, Mountain View, CA) at a wavelength of 488 nm. Fluorescence
and light scatter data were obtained at logarithmic settings. The
samples were analyzed on the basis of their forward scatter and 90°
light scatter profile, using special gate thresholds to detect
platelets and microvesicles.4 Samples of isolated exosome
fractions were analyzed using biotinylated anti-CD63 antibody.
SDS-PAGE and Western blotting.
Samples of each pellet were run on 7.5%, 10%, or 12.5%
polyacrylamide gels and transferred to Immobilon-P membrane (Millipore, Bradford, MA). The membranes were blocked for 90 minutes in PBS containing 5% (wt/vol) nonfatty dry milk Protifar (Nutricia,
Zoetermeer, The Netherlands), and 0.1% (wt/vol) Tween 20, then
incubated for 90 minutes with primary antibodies followed by incubation
with 0.1 µg/mL 125I-labeled protein G (Zymed Lab, San
Francisco, CA). For detection of MoAbs, a polyclonal anti-mouse IgG was
used as intermediate. The radioactivity of all fractions was quantified
with a Phospho-Imager (Molecular Dynamics, Sunnyvale, CA).
Electron microscopy and immunogold cytochemistry.
Electron microscopic analysis was performed on isolated vesicle
fractions as follows. Immediately after the 200,000g
centrifugation step, the pellets were resuspended in 30 µL PBS (pH
7.4) containing 5 mmol/L EDTA and fixed by adding an equal volume of
2% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). All
fractions from the gradient were adsorbed for 10 minutes to
formvar-carbon coated grids by floating the grids on 5 µL drops on
parafilm. Grids with adhered vesicles were rinsed in PBS and examined
in the electron microscope after uranyl staining and embedding (as
described below).
Exocytosis was examined in platelet aggregates formed after 5 minutes
of stimulation with 1 U/mL -thrombin, in the absence of dRGDW and
EDTA. After fixation with a mixture of 2% paraformaldehyde and 0.2%
glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4), the samples were
infiltrated in 2.3 mol/L sucrose and frozen in liquid nitrogen. The
subcellular localization of CD63 and GPIb was studied on ultrathin
cryosections of platelet aggregates.
Immunogold labeling of vesicle fractions and of ultrathin cryosections
was performed at room temperature by floating the grids on drops
containing the diluted antibodies, essentially as described previously.27 After immunolabeling, the samples were washed in distilled water, 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.28 Double immunogold labeling
was performed essentially as described previously.29 Briefly, single-labeled vesicle fractions were put successively on
drops containing PBS, 1% glutaraldehyde in PBS (5 minutes), PBS/0.02%
glycine, and PBS/1% bovine serum albumin (BSA) before starting the
next immuno incubation. We used protein-A gold sizes of 5 nm and 10 nm
for the double labeling experiments. The specificity of immunolabeling
was verified using an irrelevant control antibody and protein-A gold alone.
Binding of annexin-V, prothrombin, and factor X.
For annexin-V binding studies, isolated exosome fractions were
resuspended in 100 mmol/L HEPES containing 5 mmol/L CaCl2
(HEPES/CaCl2). The fractions were adsorbed for 10 minutes
to formvar-carbon coated grids by floating them on 5-µL drops on
parafilm. The grids were washed on successive drops of
HEPES/CaCl2, blocked with 0.1% BSA in
HEPES/CaCl2, and incubated for 30 minutes at room
temperature with 10 µg/mL FITC-conjugated annexin-V in the presence
of 5 mmol/L CaCl2. After 3 rinses with
HEPES/CaCl2, the vesicle fractions were fixed in 0.2%
glutaraldehyde in HEPES/CaCl2 and immediately used for
immunogold labeling as described above, using a rabbit polyclonal
anti-FITC antibody and 10 nm protein A gold.
In a similar way, the binding of prothrombin and factor X was monitored
to isolated exosomes, microvesicles, and phospholipid vesicles.
Phospholipid vesicles containing 20% PS, 40% PC and 40% PE were
prepared as described by van Wijnen et al.30
All vesicle samples were adsorbed for 10 minutes to formvar-carbon coated grids, rinsed on successive drops of 10 mmol/L TBS
(Tris-buffered saline, 10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4)
containing 5 mmol/L CaCl2 (Tris/CaCl2), and
blocked with 0.1% BSA in Tris/CaCl2. The samples were
incubated for 30 minutes at room temperature with 10 µg/mL
prothrombin or 10 µg/mL factor X, thoroughly rinsed with
Tris/CaCl2, and fixed with 0.1% glutaraldehyde in
Tris/CaCl2. After rinsing on successive drops of TBS,
PBS/0.02% glycine and PBS/1% BSA, immunogold labeling was performed
as described above using rabbit polyclonal anti-prothrombin and rabbit
polyclonal anti-factor X antibodies, followed by 10 nm protein-A gold.
| |
RESULTS |
Platelet releasates contain microvesicles and exosomes.
Flow cytometric analysis of washed platelets showed an increase in the
mean fluorescence intensity after stimulation with 15 µmol/L TRAP,
using anti-P-selectin and anti-CD63 antibodies (not shown). Thus, both
markers were translocated from intracellular compartments to the cell
surface. The flow cytometric scatter patterns show the formation of
microvesicles in the lower lefthand gate of the scatter plot (M in Fig
1B). To further investigate the release of
membrane vesicles, the supernatant of platelets stimulated with 15 µmol/L TRAP was subjected to centrifugation at 10,000g. This
104-g fraction contained predominantly large membrane-bound
vesicles (100 nm to 1 µm) with often an electron-dense cytoplasmic
content (Fig 2a). By virtue of their size
and morphology, these probably represent the population of vesicles
that is formed by shedding from the cell surface, and which have been
shown repeatedly by others using flow cytometry
(microvesicles).3,4 The much smaller exosomes, analogous to
those released by reticulocytes,19,30 and recently isolated
from B cells,31 ie, the internal membrane vesicles of MVBs
and -granules in platelets, were rare in this fraction. To separate
exosomes from soluble granule proteins, the supernatant of the
104-g fraction was subjected to centrifugation to
equilibrium in a sucrose gradient, leaving the soluble proteins at the
bottom of the gradient and membranes in the floating fractions.
Whole-mount electron microscopy (EM) analysis showed that
the fractions equilibrating at densities 1.14 to 1.18 g/mL were highly
enriched in membrane-bound vesicles, ranging in size from 40 to 100 nm
(Fig 2b), similar in size to those observed in MVBs and -granules. A
limited number of microvesicles was still present in this fraction (Fig
2c and d). When the isolated exosome fraction was analyzed by flow
cytometry, using biotinylated anti-CD63 antibody and
streptavidine-FITC, the mean fluorescence intensity remained within
background levels, indicating that the exosomes were too small to be
detected by flow cytometry.
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| Fig 1.
Flow cytometric detection of platelets and microvesicles.
Platelets were stimulated with 15 µmol/L TRAP. P and M indicate the
gates for platelets and microvesicles, respectively. (A) Nonstimulated
platelets; (B) TRAP-stimulated platelets. Data are shown of a
representative single experiment.
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| Fig 2.
Electron micrographs of 104-g fraction, and
fractions from the sucrose gradient. (a) Plasma membrane-derived
microvesicles; (b) exosomes; (c and d) microvesicles and exosomes. (c)
Immunolabeling for CD63; note the difference in size between the CD63
positive exosomes and the microvesicle; (d) immuno-double labeling for
GPIb and CD63; exosomes are enriched in CD63, while microvesicles
(upper left in d) contain predominantly GPIb. Bars: (a)
500 nm; (b through d) 100 nm.
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Platelet exosomes are enriched in the tetraspan protein CD63.
The tetraspan protein CD63, a marker for late endocytic compartments in
many cell types and present in platelet lysosomes and dense granules,
has recently been identified in multivesicular endocytic compartments
and -granules in platelets.24 GPIb, a plasma membrane
marker, is absent from these organelles (Heijnen et al, unpublished
observations, 1998). To analyze the presence of both
markers on microvesicles and exosomes, equal aliquots of platelet
lysate (PLlys), the 104-g fraction, and all
fractions from the sucrose gradient were analyzed by SDS-PAGE and
Western blotting using MoAbs AK-3 (GPIb) and CLB-Gran 1/2, 435 (CD63).
As illustrated in Fig 3A,
CD63 and GPIb were clearly detected in the PLlys and
104-g fraction (first two lanes), and were also present in
fractions equilibrating at a density of ~1.16 g/mL on the sucrose
gradient (lanes 5 and 6). The amounts of GPIb and CD63 in these
fractions were quite different. A relative enrichment for
GPIb was found in the 104-g fraction, while CD63 was
enriched in the fractions containing the exosomes (Fig
3a).
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| Fig 3.
Detection of CD63 and GPIb in platelet lysate,
microvesicles, and exosomes. (A) Platelet releasates were centrifuged
at 750g (PLlys) and 10,000g
(104-g) after stimulation with 15 µmol/L TRAP. The
exosome-enriched fraction was obtained after centrifugation of the
supernatant at 65,000g. The membrane pellet was dissolved and
then floated into a linear sucrose gradient. Top-bottom gradient
fractions were diluted in SDS-sample buffer and analyzed by Western
blotting for the presence of GPIb and CD63. (B) Relative distribution
of CD63 and GPIb quantified from (A). CD63 is enriched in the fractions
5 and 6 from the sucrose gradient, equilibrating at density ~1.16
g/mL. The majority of GPIb is recovered in PLlys, and the
104-g fraction and somewhat overlapping in the exosome
fraction. PLlys, platelet pellet; 104-g,
microvesicle fraction.
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| Fig 4.
Binding of annexin-V, prothrombin, and factor X to
isolated vesicle fractions. Microvesicles from the 10,000g
fraction, exosomes from the sucrose gradient, and PS/PE/PC phopholipids
were adsorbed to carbon-coated EM grids. The vesicles were incubated
either with FITC-conjugated annexin-V, factor X, or prothrombin in the
presence of 5 mmol/L CaCl2. After fixation, the membranes
were immunolabeled with their respective antibodies and protein-A gold.
(a) Annexin-V binding monitored with a polyclonal anti-FITC antibody
and protein-A gold; (b) immunogold double labeling as indicated on the
figure. Annexin-V binding is most prominent on the larger
microvesicles. (c and d) Binding of prothrombin (c) and factor X (e) to
microvesicles, but not to the exosomes (d and f); (g) binding of
prothrombin to phospholipid vesicles. Bars: 100 nm.
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To further investigate the protein composition, whole-mount
preparations of exosomes were immunolabeled with antibodies against CD63, GPIb, 1-integrin, Peta-3, P-selectin, and Pecam-1.
These antibodies recognized the extracellular domains of these
molecules, and were therefore suitable for whole-mount immunolabeling.
CD63 appeared abundantly associated with the exosomes (Fig 2c and d). GPIb was only detected on the microvesicles (Fig 2d), together with
Pecam-1, the platelet integrins IIb-3 and
1, and the two tetraspan proteins Peta-3 and CD9 (not
shown). P-selectin was detected on a limited number of the exosomes as
well (not shown). When the number of labeled and nonlabeled exosomes
was determined, it appeared that about 50% or more of the exosomes
were not labeled, depending on the antibody used. CD63 gave the highest
percentage of labeled exosomes (~50%), while Pecam-1, Peta-3, GPIb,
and 1-integrin were hardly present on exosomes (Table
1). Thus, while the composition of
microvesicles reflects that of the platelet surface after activation, including that of P-selectin, exosomes are selectively enriched in
CD63.
Exosomes derive from exocytosis of MVBs and
-granules.
To investigate exosome release morphologically, washed platelets were
stimulated at 37°C with 1 U/mL of -thrombin, in the absence of
dRGDW and EDTA. Under such conditions, a majority of the -granules
had fused with the platelet cell surface and open canalicular system
(OCS). Figure 5a shows an
exocytotic profile of an MVB with externalized CD63+
vesicles (ie, exosomes). Exosomes were also found adjacent to the
pseudopodal extensions formed during platelet aggregation (arrowheads
in Fig 5b). The exosomes were similar in size to those previously
described in multivesicular bodies and -granules in nonstimulated
platelets, and to those found in platelet releasates. Immunogold
labeling for GPIb resulted in the specific labeling at the platelet
cell surface, particularly on pseudopodal extensions, but not in
labeling of exosomes (not shown). We conclude that the exosomes found
in the platelet releasate after stimulation derive from exocytosis of
multivesicular compartments and -granules.
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| Fig 5.
Release of exosomes from activated platelets. Thin frozen
sections of a platelet aggregate after activation with 1 U/mL of
-thrombin in the absence of dRGDW. Immunolabeling with anti-CD63 and
protein-A gold (10 nm). (a) Exocytotic profile of
multivesicular body containing CD63+ exosomes (star). (b)
Exosomes detected in the extracellular area between platelet pseudopods
(arrowheads). Bars: 100 nm.
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Exosomes do not bind annexin-V, prothrombin, and factor X.
To examine whether released exosomes also exhibit procoagulant sites
like microvesicles, we studied first the exposure of anionic
phospholipids on the isolated vesicle fractions. In the presence of low
calcium concentrations, annexin-V binds specifically to areas in the
membrane that are enriched in phosphatidylserine.11 Isolated membrane fractions were adsorbed to carbon-coated grids and
incubated with FITC-conjugated annexin-V in the presence of 5 mmol/L
CaCl2. After fixation, the annexin-V binding to membranes was monitored by incubation with a polyclonal anti-FITC antibody and
protein-A gold. Annexin-V bound predominantly to the microvesicles (Fig
4a and b). Eighteen percent of the exosomes showed binding of annexin-V
(Table 2), while no labeling was observed
when vesicle fractions were incubated with polyclonal anti-FITC
antibody and protein-A gold alone. To investigate the possibility that
endogenous platelet annexin-V was already present on released exosomes,
we incubated isolated exosome fractions with anti-annexin-V, followed by protein-A gold. No annexin-V was detected on the isolated exosomes.
In a similar way, the binding of prothrombin and factor X to exosomes,
microvesicles, and PS/PC/PE phospholipids was tested. The vesicles were
adsorbed to carbon-coated grids and incubated with either prothrombin
or factor X in the presence of 5 mmol/L CaCl2. Binding of
both coagulation factors was monitored after fixation followed by
incubation with their corresponding antibodies and protein-A gold. Less
than 10% of the exosomes showed binding of prothrombin or factor X
(Table 2, Fig 4d and f), while under similar conditions specific
binding of prothrombin and factor X was observed to the population of
large microvesicles (Fig 4c and e). A significant amount of the
PS/PC/PE vesicles (>50%) showed also binding of prothrombin (Fig
4g). No labeling was observed when vesicles were incubated with
polyclonal anti-prothrombin or anti-factor X antibody alone.
| |
DISCUSSION |
We have studied the release of microvesicles and exosomes during
platelet activation, using Western blotting and (immuno)-electron microscopical techniques. After stimulation with 15 µmol/L TRAP, relatively large vesicles were identified (microvesicles) together with
a population of small vesicles (40 to 100 nm), showing similarities with the internal vesicles described previously in multivesicular bodies and -granules.24 Analogous to the previously
described vesicles released by reticulocytes and B
cells,19,20 these small vesicles were termed exosomes.
Western blot analysis of the membrane vesicles obtained from the
releasate of activated platelets showed that the microvesicles
contained predominantly plasma membrane glycoproteins, while the
exosomes were selectively enriched in the late endocytic marker CD63.
The selective enrichment of CD63 on exosomes was confirmed by
immunogold labeling of isolated exosome fractions and of ultrathin
cryosections of platelet aggregates. In situ CD63 was predominantly
detected on released exosomes present in exocytotic profiles, and in
between pseudopodal extensions. A limited number of the exosomes also
contained P-selectin. Flow cytometric analysis after stimulation with
15 µmol/L TRAP showed that microvesicles are readily detected with
anti-GPIb, but released exosomes were not observed using the anti-CD63 antibody.
In the present study we have also shown that exocytosis of
multivesicular compartments and -granules is accompanied by the release of exosomes, the former internal vesicles of MVBs and -granules. These platelet exosomes contained CD63, in agreement with
the enrichment of CD63 on the internal vesicle membranes. At present we
do not know whether exocytosis of dense granules contributes as well to
the release of CD63-enriched exosomes. Exocytosis of multivesicular
endocytic compartments has been described in several cell types,
particularly those of the hematopoietic lineage.19-21 The
enrichment of certain molecules on exosomes suggests that such
molecules are selectively sorted from other glycoproteins via the
formation of small internal vesicles. Such a sorting in MVBs has been
described for the transferrin receptor during reticulocyte maturation31 and in B cells for major histocompatibility
complex (MHC) class II molecules.32 Release of exosomes
from these cells may serve a specialized function, respectively, in
antigen presentation,20,22 and in the clearance of
transferrin receptor during the maturation of reticulocytes into
erythrocytes.33 In platelets, release of membrane vesicles
has the potential to exert procoagulant activity at a distance from the
site of platelet activation. However, we found that in the presence of
5 mmol/L CaCl2, binding sites for FITC-conjugated annexin-V
were mainly presented on the large cell-surface-derived microvesicles,
and not on exosomes. These observations suggest that microvesicles are
enriched in aminophospholipids and, in addition to the platelet surface
itself, are most likely the sites where procoagulant activity
predominates. The relatively low number of exosomes that bound
annexin-V suggests that the outer leaflet of platelet exosomes is
probably not enriched in phosphatidylserine. This is in accord with
previous findings in reticulocytes where exosomes have relatively high
cholesterol content and only trace amounts of
phosphatidylserine.34 Endogenous annexin-V was detected in
the cytosol of resting and stimulated platelets (not shown), but was
absent from small internal vesicles and isolated exosomes, suggesting
that endogenous annexin-V is not associated with exosomes. Moreover,
the finding that factor X and prothrombin readily bound to
microvesicles but not to isolated exosomes under similar experimental conditions supports the concept that platelet exosomes are probably not
the site where the prothrombinase complex is formed.
The selective enrichment of CD63 in platelet exosomes may provide a
clue for their possible extracellular function. CD63 belongs to the
family of tetraspan proteins, together with CD37, CD81, CD82, Peta-3,
and CD9.35 The latter two are readily detected on the
platelet cell surface after activation, including released microvesicles, but they were not enriched on exosomes. Several tetraspan proteins (CD37, CD81, CD82, and CD63) have recently been
shown on the exosomes of human B lymphocytes.31 Besides CD63, none of them was detected on platelet exosomes. Tetraspan proteins may function cooperatively with each other36 in
complex formation with cell surface proteins and can interact with
integrins.37-39 Recent work has shown that CD9 is
associated with 3-integrins in both resting and
stimulated platelets.40,41 Formation of tetraspan-integrin
complexes on the cell surface may also modulate integrins,42 and contribute to the signaling and adhesive
functions of the integrin. For example, CD63 molecules have been
implicated in the transmission of activation signals in neutrophils
leading to increased adhesion of neutrophils to endothelial
cells.43 The nature of the interactions between integrins
and tetraspans is still unknown, but all these studies indicate that
the extracellular domains of tetraspan proteins are involved. Platelet
exosomes therefore may have the potential to excite signaling at a
distance from the site of platelet activation. Such a signaling role
may occur through specific association of CD63 with integrins on the cell surface of platelets (homotypic) or other cells (heterotypic) in
the circulation.
In conclusion, we have found two populations of membrane vesicles that
are released during platelet activation: platelet microvesicles and
exosomes. The absence of factor X and prothrombin binding and the low
capacity of annexin-V binding to exosomes suggest that the membranes of
exosomes have little procoagulant activity. Whether CD63 on released
exosomes serves another specific extracellular function, for example in
transmission of activation signals to neutrophils44 or
endothelial cells,45 remains to be determined.
| |
ACKNOWLEDGMENT |
We thank Margot van Gastelen and Martijn van Andel for their excellent
technical assistance, and Rene Scriwaneck for his help in preparing the
electron micrographs. We are gratefully indebted to Drs J.M. Escola and
W. Stoorvogel for their helpful suggestions during the course of this work.
| |
FOOTNOTES |
Submitted October 6, 1998; accepted June 29, 1999.
Supported in part by the Netherlands Foundation of Thrombosis, Grant
No. 97.003.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
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; e-mail:
H.F.G.Heijnen{at}lab.azu.nl.
| |
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TOP
ABSTRACT
INTRODUCTION