From INSERM E9907, Faculté Xavier Bichat, 75870 Paris Cedex 18, Paris, France, § INSERM U362,
Institut Gustave Roussy, Villejuif 94805, France, and ¶ Millenium
Pharmaceuticals Inc., Cambridge, Massachusetts 02139
Received for publication, October 5, 2000, and in revised form, December 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, the expression and
function of the platelet collagen receptor glycoprotein VI
(GPVI) were studied in human megakaryocytes during differentiation and
maturation of mobilized blood and cord blood derived
CD34+ cells. By flow cytometry, using an anti-GPVI
monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was
detected only on CD41+ cells including some
CD41+/CD34+ cells, suggesting expression at a
stage of differentiation similar to CD41. These results were confirmed
at the mRNA level using reverse transcription-polymerase chain
reaction. GPVI expression was low during megakaryocytic
differentiation but increased in the more mature megakaryocytes
(CD41high). As in platelets, megakaryocyte GPVI associates
with the Fc receptor At sites of vascular injury, platelets adhere and are activated by
contact with collagen fibers exposed with the subendothelium matrix
leading to thrombus formation. Several collagen receptors are present
on platelets: integrin Expression of platelet receptors during megakaryocyte differentiation
discriminates different stages of megakaryocyte maturation (13, 14).
GPIIb/GPIIIa (CD41/CD61) are platelet proteins that are the first
expressed during megakaryocyte differentiation, although their
expression is not entirely specific of this cell lineage (15, 16). A
fraction of megakaryocyte progenitors, colony-forming unit
megauaryocytes, express GPIIb and GPIIIa (17). Later in
differentiation, promegakaryoblasts and megakaryoblasts also express
the GPIb·GPIX complex (CD42) and CD36. More recently, it has
been shown that GPV, in which the promoter has been cloned, is
expressed during megakaryocyte differentiation even later than the
GPIb·GPIX complex (18).
Our study was designed to characterize GPVI expression during
megakaryocytopoiesis and to determine more precisely the kinetics of
GPVI expression and its functionality in megakaryocytes. Cell surface expression of GPVI was studied by flow cytometry in comparison with cell surface expression of CD49b and by blotting experiments using
specific antibodies or one specific ligand, convulxin. Expression of
GPVI and FcR Materials--
Convulxin was purified from the venom of
Crotalus durissus terrificus (from
Fundação Ezequiel Dias (FUNED), Minas Gerais, Brazil or
Latoxan, Valence, France) as previously described (19). Convulxin was
labeled with 125I using the Iodogen procedure (Pierce) and
Na125I (Amersham Pharmacia Biotech) or was coupled
to fluorescein (FITC) as previously described (7).
The anti-GPVI monoclonal antibody (mAb), 3J24·2, was produced
by immunizing Balb/c mice with the DNA encoding a fusion protein corresponding to the extracellular domain of GPVI (residues 1-269) fused at its C terminus via a 3 Ala linker to the human
IgG1 Fc sequence (shGPVI-Fc)7 using the Rapid
Immunization Gene Gun delivery as described (20). Mice were
boosted with 100 µg of intravenous shGPVI-Fc 4 days prior to fusion.
Spleen cells of one mouse were fused with SP2/0 myeloma cells using PEG
1500, and hybridoma lines were screened for secretion of GPVI-specific
antibodies by enzyme-linked immunoassay on plate-bound shGPVI-Fc.
Antibodies were isotyped, and 3J24·2 was shown to be
IgG1. Selected cell lines were cloned using
ClonalCellTM-HY medium D (Stem Cell Technology, Vancouver,
British Columbia, Canada). Ascitic fluids were produced, and antibodies
were purified by chromatography on protein A-Sepharose (Amersham
Pharmacia Biotech). Purified 3J24·2 does not inhibit collagen- or
convulxin-induced platelet activation but behaves as a mild platelet
agonist inducing a low level of aggregation (~10%) after prolonged
incubation (15 min) at 37 °C with stirring. 3J24·2 recognizes
nonreduced GPVI in immunoblotting experiments. 3J24·2 was conjugated
to FITC by the same method as used for convulxin (7). Polyclonal
anti-GPVI IgGs were purified from a patient plasma (21), kindly
provided by Dr. M. Okuma, and Fab fragments were from the same
batch as in previous studies (4). The anti-integrin
Collagen type I (from equine tendon) or collagen from fetal calf skin
was purchased from Chrono-Log Corp. (Haverton, PA) or Horm
(Hormon-Chemie, Munich, Germany) and Bio/Data Corporation (Horsham,
PA), respectively. The following mAbs were purchased from
Beckman-Coulter (Marseille, France): FITC anti-CD49b mAb (anti-integrin
Purification of CD34+ Cells--
After obtaining
informed consent, an aliquot of leukapheresis from adult patients,
after mobilization by chemotherapy and granulocyte/colony-stimulating
factor, was obtained for research purposes. Human umbilical
blood cells were obtained from full term deliveries under guidelines
established by the Ethical Committee. Cells were separated over
a Ficoll-metrizoate gradient (Lymphoprep, Nycomed Pharma, Oslo,
Norway). CD34+ cells were then isolated by a positive
selection using the Miltenyi immunomagnetic bead technique according to
the manufacturer's protocol. The purity was about 90% after two
passages through the column as estimated by flow cytometry.
In Vitro Liquid Cultures of Megakaryocytes from CD34+
Cells--
CD34+ cells were grown for 5-14 days in
"serum-free" Iscove's modified Dulbecco's medium (Life
Technologies, Inc.) prepared as previously reported (23). The medium
was supplemented with a combination of pegylated recombinant human
megakaryocyte growth and development factor (PEG-rHuMGDF 10 ng/ml, a
generous gift from Kirin, Tokyo, Japan), a truncated form of
thrombopoietin (TPO), and 50 ng/ml recombinant human stem cell factor
(a generous gift from Amgen, Thousand Oaks, CA) for flow cytometry or
with PEG-rHuMGDF alone for biochemical and adhesion experiments.
Cell Sorting of Different CD41
Subsets--
Megakaryocytes at different stages of differentiation
were obtained after 6 days of culture. Cells were incubated with a
mixture of a FITC anti-CD42a, R-PE anti-CD41a, and R-PE-Cy5 anti-CD34 mAbs for 30 min at 4 °C in their culture medium. Cells were washed in culture medium and sorted into the following five populations using a FACS Vantage flow cytometer (Becton Dickinson) equipped with an
argon laser (Coherent Radiation, Palo Alto, CA) and a 100-µm nozzle:
CD34+CD41a RT-PCR--
RNA was extracted according to the technique of
Chomczynsky and Sacchi (24). Total RNA (500 ng-1
µg) was denatured at 70 °C for 10 min and then reverse-transcribed
with avian myeloblastosis leukemia virus reverse transcriptase (20 units, Promega, Madison, WI) at 42 °C for 1 h with random
hexanucleotides (200 ng, Amersham Pharmacia Biotech) as primers in a
final volume of 50 µl.
After reverse transcription, each sample was subjected to
amplification of GPVI, FcR
PCR was performed as previously reported in a 25-µl reaction
mixture (containing 30 pmol of primers for GPVI and FcR Flow Cytometry--
Cells were studied from day 5 to day 14 of
culture. To study GPVI during megakaryocyte maturation, cells were
simultaneously labeled for 30 min with R-PE-Cy5 anti-CD34 mAb, R-PE
anti-CD41 mAb, and a FITC anti-CD42 or FITC anti-GPVI mAb (3J24-2) or
FITC anti-CD49b mAb and subsequently washed. Isotype IgGs were used as
controls. In some experiments, cells were labeled by the R-PE anti-CD41
mAb or R-PE anti-CD34 mAb and FITC-convulxin (20 nM). Nonspecific labeling was measured using FITC-coupled bothrojaracin (20 nM), another protein from the same C-type lectin snake
venom protein family as convulxin but which does not bind to platelets (7). For studying the expression of the FcR Adhesion to Immobilized Collagen or Convulxin--
Adhesion
experiments were performed using cells cultured in the presence of TPO
alone, a culture condition that allows the growth of megakaryocytes
with a high level of purity (about 90%). Adhesion was measured
in static conditions on microtitration plates as previously described
(4, 7). Briefly, collagen (2 µg) and convulxin (1.5 µg) were
immobilized on Immulon II plates. After saturation with BSA,
megakaryocytes were added for 60 min at 37 °C in the presence or the
absence of 10 mM EDTA. Megakaryocyte adherence was
quantified by measuring alkaline phosphatase activity as follows: 100 µl of 0.1 M sodium citrate, pH 5.4, containing 0.3% v/v
Triton and 7 mM of paranitrophenylphosphate were added to
emptied wells. Reaction was stopped with 1 M NaOH, and
optical density was measured at 405 nm. Nonspecific adhesion was
measured using wells coated with BSA. The percentage of adherent cells was calculated from a calibration curve performed using cells in suspension.
Immunoblotting and Ligand Blotting--
Cells
(2.106/ml) were lysed in a radioimmune precipitation buffer
composed of 1% v/v Nonidet P-40 in 12 mM Tris, 300 mM NaCl, 12 mM EDTA containing 0.2 mM PMSF, 2 µM leupeptin, and 5 Kallikrein inhibitory units (KIU) of aprotinin. Proteins (40 µg) were
separated by SDS-polyacrylamide gel electrophoresis and transferred to
PVDF membranes (6, 19). Membranes were incubated with polyclonal anti-GPVI IgG or with 3J24-2 or the anti-FcR
Tyrosyl phosphorylations were studied on cells cultured in the presence
of TPO alone. Megakaryocytes (1.106 cells/ml) in Hanks
buffer containing 2 mg/ml BSA were incubated in an aggregometer at
37 °C under stirring conditions (1100 rpm). Convulxin (10 nM) or collagen (100 µg/ml) was added to the suspension for 6 min. Incubation was stopped by the addition of 100 µl from a
mixture containing 65 mM EDTA, 33 mM vanadate,
and 2 µM phenylarsin oxide (PAO) (25). Samples
were centrifuged for 1 min at 13,000 × g. Pellets were
then lysed in a buffer containing 1% v/v Triton, 0.1% w/v SDS, 5%
Immunoprecipitation--
Megakaryocytes (0.5 × 106) or platelets (2.5 × 108) were lysed
by the addition of a radioimmune precipitation buffer containing 1%
(v/v) Nonidet P-40, 5 mM vanadate, 2 mM PMSF,
0.25 mg/ml pefabloc, 500 KIU/ml aprotinin, and 5 µg/ml
leupeptin. Samples were precleared by incubation with protein
A-Sepharose for 15 min at 4 °C and centrifuged to reduce nonspecific
signal (25). Lysates were incubated with
anti-PLC Study of GPVI Expression during Megakaryocyte
Differentiation--
To determine expression of GPVI during
megakaryocyte differentiation, we used a new mAb called 3J24·2
directed against human GPVI and the binding of a specific ligand,
convulxin. Megakaryocyte cultures were performed from CD34+
cells isolated from cord blood or leukapheresis and were studied at
different days of culture from days 5 to 14. To analyze the precise
stage at which GPVI is expressed, a triple staining technique was
performed using the CD34 and CD41 antigens as markers of
differentiation; CD41+ cells corresponded to immature
megakaryocytic cells, whereas CD412+ cells to mature
megakaryocytes. In addition, expression of GPVI was compared with that
of another collagen receptor, CD49b
(
Using megakaryocytes derived in vitro by culture in TPO,
GPVI was present only on the CD41+ cell subset. GPVI was
expressed on CD41+ cells regardless of time in culture (5, 10 (Figs.
1 and 2), or 14 days (data not shown)). Expression was also not dependent on the source of CD34+
cells, blood-mobilized cells (Fig. 1), or cord blood (Fig.
2). GPVI was also present on both
CD34+ cells (Figs. 1 and 3)
but only in the CD34+CD41+ cell subset.
However, expression of GPVI was low during megakaryocytic differentiation and was just over the background in the
CD41+ cells (see histograms in Figs. 1-3). Expression of
GPVI increased with maturation in parallel with the augmentation of
CD41 antigen on the cell surface (see dot plots in Figs. 1-3). CD49b
had a similar pattern of expression to GPVI in these cultures with a
weak expression in the CD41+ cells and increased expression
in the CD412+ cells (Figs. 1-3). However, there was a
great variation in the expression of CD49b. In some experiments, the
level of CD49b was at the threshold of detection (Figs. 1 and 2),
whereas in other experiments it was at an intermediate point between
the level of CD42 and GPVI (Fig. 3). This heterogeneity in the
expression of CD49b might be the consequence of CD49b
polymorphisms(26). Expression of CD42 was slightly different from that
of GPVI. Indeed, its level was much higher and increased linearly with
the CD41 antigen (Figs. 1-3). However, it appears, especially in
experiments with cord blood cells, that a fraction of CD41+
cells did not express CD42 (see Fig. 2, Day 5).
Together, these results indicate a very weak expression of GPVI and
CD49b on immature megakaryocytes. The two molecules increase at the end
of the maturation concurrent with CD41 expression, but their expression
remains much weaker than that of CD42. To confirm that GPVI was
synthesized early in differentiation, RT-PCR analysis was performed on
different cell fractions sorted on the expression of CD34, CD41, and
CD42. GPVI mRNA was detected in all CD41+ cell
fractions including those that also express CD34, whereas it was not
detected in the CD41
To determine whether functional binding of megakaryocyte GPVI to a
specific ligand takes place, flow cytometry was performed using
FITC-convulxin and R-PE anti-CD41 mAb or R-PE anti-CD34 mAb. The
results confirmed those obtained with the anti-GPVI mAb; FITC-convulxin
labeled CD41+ cells just over the threshold when compared
with the control snake venom protein, FITC-coupled bothrojaracin (Fig.
5, Day 8). This level of
binding was low but much more pronounced in mature megakaryocytes
(CD412+) (Fig. 5). Altogether, these results indicate that
GPVI is expressed at low levels during megakaryocyte differentiation
and might be functional.
Megakaryocyte Express GPVI as a 55-kDa Protein Associated
with the FcR
In platelets, FcR
These results indicate that megakaryocytes express GPVI as a complex
with its signaling subunit FcR Megakaryocyte Adhesion to Immobilized Collagen or Convulxin
Involves GPVI and Integrin
At day 6 of culture, the number of megakaryocytes that adhered to
immobilized collagen or convulxin was very low, only slightly above the
level of nonspecific binding to immobilized BSA (Fig. 8A). Later in the maturation,
adhesion of megakaryocytes to immobilized collagen or convulxin
increased (12- and 22-fold, respectively, at day 9; 18- and 30-fold at
day 14; Fig. 8, B and C). Adhesion to immobilized
convulxin was inhibited in part by the polyclonal anti-GPVI Fab
fragments (data not shown) as already observed for platelet adhesion to
immobilized convulxin (4). Megakaryocyte adhesion to immobilized
collagen was reduced in the presence of the anti-integrin
Collagen and Convulxin Trigger Protein Tyrosine
Phosphorylations in Megakaryocytes--
GPVI dimerization by its
ligands is coupled to a very potent signaling pathway, which involves
activation of a cascade of tyrosine kinases and which culminates in the
phosphorylation and activation of PLC
The pattern of phosphorylation obtained with megakaryocytes from
the same culture at different stages of maturation is shown in Fig.
9B. With equal sample loading (5 × 105
cells in each sample) and exposure, differences in the intensity of
phosphorylated bands were observed according to maturation. Convulxin-
or collagen-induced protein tyrosine phosphorylations were very faint
at day 6. The intensity of the signal greatly increased at day 9 parallel to GPVI expression but decreased at day 14 (Fig.
9C), suggesting that the efficiency of GPVI-coupled signals
decreases at this late stage of maturation. In platelets, p72Syk and PLC Megakaryocyte maturation occurs in the marrow, whereas
proplatelet formation and platelet release takes place in the blood stream either in marrow sinusoids or in the general circulation (33).
It is thus likely that specific mechanisms exist for the adhesion of
progenitors to bone marrow stroma cells or to extracellular matrix
proteins during maturation. On the other hand, to release platelets,
megakaryocytes have to migrate through the endothelial barrier and
cross the extracellular matrix, where they may interact with adhesive
proteins such as fibronectin and collagen. In platelets, the synergy of
the two collagen receptors Based on the response to collagen-related peptides, GPVI
signaling has been proposed to occur only in mature megakaryocytes (34-36). Because FcR Studies of the promoter regions and of transgenic mice have shown that
platelet-restricted genes are controlled by two types of molecular
regulation (37-39). The main platelet-restricted genes such those of
the GPIb complex, GPIIb and PF4, contain binding sites in their
promoters for GATA and Ets transcription factors. Expression of
megakaryocyte-specific genes is down-regulated in GATA-1 Polymorphisms in the Platelet membrane GPVI migrates with an apparent molecular mass of 58 kDa under nonreducing conditions. The human protein contains one
N-glycosylation site and several presumptive sites for
O-glycosylation on its extracellular domain (5, 7), and the
predicted molecular mass of the core protein is ~40 kDa. GPVI
detected in megakaryocyte lysates by immunoblotting or ligand blotting
migrated with an apparent molecular mass of 55-56 kDa, which is
slightly lower than the mass of platelet GPVI but slightly higher than
the mass of N-deglycosylated GPVI (54 kDa) when runned in
parallel (data not shown); this suggests that GPVI undergoes a final
processing step at the end of megakaryocyte maturation.
Despite the fact that the GPVI molecular mass is slightly different in
megakaryocytes than in platelets, it associates with the FcR
In conclusion, our results indicate that collagen receptors like GPVI
and CD49b are expressed by megakaryocytes at an early stage of
maturation and that they are fully functional later in differentiation.
The characterization of their role in megakaryocyte interactions with
collagen should allow a better comprehension of diseases such as
platelet production defects.
chain (FcR
). The FcR
chain was detected
at the RNA and protein level at all stages of megakaryocyte maturation
preceding the expression of GPVI. The other collagen receptor,
2
1 integrin (CD49b/CD29), had a pattern
of expression similar to GPVI. Megakaryocytic GPVI was recognized as a
55-kDa protein by immunoblotting and ligand blotting, and thus it
presented a slightly lower apparent molecular mass than platelet GPVI
(58 kDa). Megakaryocytes began to adhere to immobilized convulxin via
GPVI after only 8-10 days of culture, at a time when megakaryocytes
were maturing. At this stage of maturation, they also adhered to
immobilized collagen by
2
1
integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in
megakaryocytes and platelets including Syk, FcR
, and
PLC
2. Our results showed that GPVI is
expressed early during megakaryocytic differentiation but functionally
allows megakaryocyte adherence to collagen only at late stages of
differentiation when its expression increases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 (CD49b/CD29),
GPIV1 (CD36), GPVI, p65, and
a recently cloned type III collagen receptor (1, 2). The direct
interaction between collagen and integrin
2
1 adheres platelets at the collagen
surface, but platelet activation and thrombus formation and attachment
are subsequently mainly supported by GPVI. In addition to collagen, two
GPVI-specific ligands have been described: collagen-related peptides
(3) and convulxin, a snake venom protein (4). GPVI has recently been
characterized as a new member of the immunoglobulin superfamily of
cell-associated receptors, in which expression is restricted to the
hematopoietic lineage (5-7). It is coexpressed as a noncovalent complex with the common immunoglobulin receptor
(FcR
) chain, which acts as the signaling subunit of the complex (8, 9) As a
consequence, signaling pathways coupled to GPVI dimerization are
identical to those coupled to other immune receptors; tyrosine phosphorylation of the FcR
chain on its immunoreceptor
tyrosine-based motif (ITAM) by a Src familly kinase (8, 10, 11) allows recruitment by the SH2 (Src homology 2) domain of P72Syk
that is in turn phosphorylated and activated. Downstream of Syk, PLC
2 is activated, leading to platelet aggregation and dense granule secretion (12).
chain mRNA was studied by RT-PCR. Functional characterization was performed by measuring adhesion to immobilized collagen or convulxin and by studying the signals coupled to GPVI. Our
results indicate that GPVI is expressed at an early stage of
megakaryocyte differentiation but at low levels and that it is
functional in mature megakaryocytes, suggesting that GPVI may regulate
thrombopoiesis and platelet release in vivo through its interactions with collagen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 monoclonal antibody was kindly
provided by Dr. B Coller (Mount Sinai Medical Center, New York, NY)
(22).
2; clone Gi9), R-PE anti-CD41 mAb, R-PE-Cy5 anti-CD34 mAb and FITC anti-CD42b mAbs. The anti-PY 4G10 was obtained from Upstate Biotechnology (Euromedex, Souffelweyersheim, France) as the mAb
directed against the FcR
chain that was conjugated to FITC.
Anti-phosphotyrosine (PY20), anti-Syk (LR Syk), and
anti-PLC
2 (Q20) mAbs were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). The peroxidase-coupled sheep
anti-mouse IgG and the chemiluminescent reagent ECL were from Amersham
Pharmacia Biotech. Phenylarsine oxide, vanadate, leupeptin, PMSF,
aprotinin, and Nonidet P-40 were obtained from Sigma, and
polyvinylidene difluoride (PVDF) membranes from Millipore (Bedford, MA).
,
CD34+CD41a+CD42a
,
CD34
CD41a+CD42a
,
CD34
CD41a+CD42a+, and the entire
CD41high cells corresponding to mature megakaryocytes. Each
cell fraction except the CD41high one was re-sorted to
ensure a purity level greater than 97%.
chain, or
2 microglobulin
cDNA. The sequences of the specific primers for GPVI, FcR
chain, and
2 microglobulin were:
5'-TTCTGTCTTGGGCTGTGTCTG-3', 5'-AGATGATTCCAGCAGTGGTCTT-3', and
5'-CCTGAAGCTGACAGCATTCGG-3' for sense primers, respectively, and
5'-CCCGCCAGGATTATTAGGATC-3', 5'-TCTTTAACAGGGAGGAGGAACC-3', and
5'-CTCCTAGAGCTACCTGTGGAG-3' for antisense primers, respectively.
chain amplifications and 10 pmol of primers for
2
microglobulin amplification) in ATGC buffer. The reaction mixture was
subjected to denaturation for 5 min at 95 °C and then amplified by
35 cycles as previouly reported (7). PCR products (9 µl)
were electrophoresed on a 2% agarose gel. Fragments were visualized by
illumination after ethidium bromide staining. MassRuler DNA Ladder, low
range (MBI, Fermentas Vilnius, Lithuania) was used as a size marker.
chain, cells were
incubated with R-PE CD41, washed, fixed by 0.5% paraformaldehyde for
15 min, and then permeabilized by 0.1% Triton X-100, washed, and
incubated with the FITC anti-FcR
chain antibody. Cells were then
analyzed by flow cytometry using a FACSort flow cytometer (Becton
Dickinson, Franklin Lakes, NJ).
chain mAbs. IgGs were
detected with peroxidase-coupled protein A for the polyclonal antibody
or peroxidase-coupled goat anti-mouse IgG for the mAbs and revealed by
chemiluminescence. Ligand blotting was performed as previously reported
(4, 7) using 125I-labeled convulxin and autoradiography.
-mercaptoethanol, 4.2 mM EDTA, 24 mM
vanadate, 4 µM PAO, 0.25 mg/ml pefabloc, 1.5 mM PMSF, 500 KIU/ml aprotinin, and 5 µg/ml leupeptin
(Sigma). Proteins were separated by SDS- polyacrylamide gel
electrophoresis and transferred to PVDF membranes. Membranes were
incubated with the anti-phosphotyrosine mAb PY20 followed by
peroxidase-coupled anti-mouse IgGs and chemiluminescence.
2 mAb, anti-Syk mAb, or 3J24-2 mAb
overnight at 4 °C and then incubated with the protein A/G-Sepharose
for 2 h at 4 °C. Immunoprecipitated proteins were solubilized
by 2% (w/v) SDS, reduced with 5% (v/v) 2-mercaptoethanol, except for
GPVI immunoprecipitation, and finally transferred to PVDF.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1integrin), and with
GPIb
.
View larger version (54K):
[in a new window]
Fig. 1.
Expression of GPVI and
2
1
integrin using flow cytometry on mobilized peripheral blood
CD34+. Cells were analyzed on a FACSort flow cytometer
at day 5 or 10 in in vitro culture. Cells (100,000 cells/50
µl) were incubated for 30 min at 4 °C with R-PE-Cy5 anti-CD34 mAb,
R-PE anti-CD41 mAb, FITC anti-CD42 mAb, FITC anti-CD49b mAb, or FITC
anti-GPVI mAb as described under "Experimental Procedures," and
controls were performed with R-PE-Cy5 IgG1 mouse antibody,
R-PE IgG1 mouse antibody, or FITC IgG1 mouse
antibody. Histograms of fluorescence were determined in CD41 negative
cells (CD41
), immature megakaryocytic cells
(CD41+), and more mature megakaryocytic cells
(CD412+). FITC anti-CD42 labeling (green
line), FITC anti-CD49b labeling (blue line), and FITC
anti-GPVI labeling (red line) were compared with control
FITC IgG1 mouse antibody (black line). The
results are representative of five independent experiments.
View larger version (54K):
[in a new window]
Fig. 2.
Expression of GPVI and
2
1
integrin using flow cytometry on cord blood CD34+.
Experiments were performed as described in Fig. 1. Cultures were
performed from cord blood CD34+ cells and analyzed at days
5 and 10. Histograms of fluorescence were determined in
CD41
, CD41+, and CD412+ cells.
FITC anti-CD42 labeling (green line), FITC anti-CD49b
labeling (blue line), and FITC anti-GPVI labeling (red
line) were compared with control FITC IgG1 mouse
antibody (black line). The results are representative of
three independent experiments.
View larger version (54K):
[in a new window]
Fig. 3.
Differences in CD49b expression among
patients. Similar experiments as in Fig. 1 were performed on the
cells from another donor. A higher level of CD49b expression is
observed, probably reflecting integrin 2
1
polymorphisms (29). Of seven individuals, CD49b expression was only
found highly expressed in two.
cell fraction (Fig.
4A).
View larger version (78K):
[in a new window]
Fig. 4.
Expression of GPVI using RT-PCR.
Megakaryocytes at different stage of differentiation were obtained
after 6 days of culture and were sorted according to their
immunophenotype into five different populations: 1)
CD34+CD41a+CD42 , 2)
CD34
CD41a+CD42a
, 3)
CD34
CD41a2+CD42a2+, 4)
CD34+CD41a
, and 5) the entire
CD412+ cell population, corresponding to mature
megakaryocytes. GPVI and
2 microglobulin
(
2) transcripts were amplified in panel
A. The high molecular weight fragment (830 bp) is generated from
GPVI primers, and the low molecular weight fragment (603 bp) is
generated from the
2 microglobulin primers. FcR
chain and
2 microglobulin transcripts were amplified in
panel B. The high molecular weight fragment (603 bp) is generated from the
2 microglobulin primers, and
the low molecular weight fragment (429 bp) is generated from the FcR
chain primers.
View larger version (54K):
[in a new window]
Fig. 5.
Expression of GPVI by mobilized
CD34+ using FITC-convulxin.
Cells (100,000 cells/50 µl) were incubated for 30 min at 4 °C with
either R-PE anti-CD34 mAb or R-PE anti-CD41 mAb and FITC-convulxin.
Controls were performed with R-PE-IgG1 mouse antibody or
with FITC-bothrojaracin. Cells were analyzed at day 5 or day 8 of
mobilized blood CD34+ cell cultures.
Chain--
Expression of GPVI by
megakaryocytes was further characterized by immunoblotting and ligand
blotting and by immunoprecipitation. As previously reported, platelet
GPVI was detected as a 58-kDa band protein with both the anti-GPVI mAb
(Fig. 6, lane a) and 125I-convulxin (Fig. 6, lane d). The anti-GPVI
mAb (Fig. 6, lane b) as well as 125I-convulxin
(Fig. 6, lane e) both labeled
a 55-kDa band in megakaryocyte lysates, suggesting that GPVI expressed
by megakaryocytes has a slightly lower molecular mass than platelet
GPVI. One characteristic behavior of platelet GPVI as of recombinant
GPVI in immunoblotting experiments is that convulxin blocks the binding
of polyclonal anti-GPVI antibodies. (4, 7) The same observation is made for megakaryocyte GPVI (Fig. 6, lanes g-h). At least,
3J24.2 precipitated a 56-kDa protein from megakaryocyte lysates, which
was recognized by 125I-convulxin (Fig. 6, lanes
c and d). Together, these results indicate that
megakaryocytes produce GPVI.
View larger version (34K):
[in a new window]
Fig. 6.
Expression of GPVI using immunoblotting,
immunoprecipitation, and ligand blotting. Megakaryocyte
(lanes b and f-h) and platelet
(lanes a and e) proteins were
solubilized in SDS, separated on 10% acrylamide slab gels, and
transferred to PVDF membranes. Alternatively, megakaryocyte proteins
were immunoprecipitated by the anti-GPVI mAb 3J24.2 before
electrophoresis (lanes c and d).
Proteins were detected by immunoblotting with 3J24.2 (lanes
a-c), ligand blotting with 125I-convulxin
(lanes d-f), or immunoblotting with the
polyclonal anti-GPVI antibody in the absence (lane g) or
presence of cold convulxin (lane h). Detection was performed
using peroxidase-coupled anti-mouse IgGs for 3J24.2, peroxidase-coupled
protein A for the polyclonal antibody, and chemiluminescence or direct
autoradiography for 125I-convulxin.
chain is associated as a noncovalent complex with
GPVI and acts as the signaling chain (8, 11). We checked whether the
FcR
chain is also expressed by adult or neonate
CD34+-derived megakaryocytes. Using FITC-coupled antibody,
which binds to the intracellular domain of the
chain and
permeabilized cells in flow cytometry studies, FcR
chain was
detected in all cells, i.e. in CD41
cells as
well as in CD41+ cells (Fig.
7). Accordingly, FcR
chain mRNA
was detected by RT-PCR in megakaryocytes, whatever their stage of
maturation, as well as in CD34+CD41
cells. By
immunoblotting with the anti-FcR
chain mAb, an intense doublet
corresponding to FcR
was detected in megakaryocyte lysates (Fig. 7).
Furthermore, as already observed with platelet lysates (9) or for
recombinant GPVI (7), FcR
was coprecipitated with GPVI, indicating
that both proteins are noncovalently associated in megakaryocytes (Fig.
7)
View larger version (36K):
[in a new window]
Fig. 7.
Expression of the FcR chain using flow cytometry on mobilized CD34+ and
immunoblotting. Cells (100,000 cells/50 µl) were incubated for
30 min at 4 °C with FITC anti-FcR
polyclonal antibody after
permeabilization with 0.1% Triton X-100. Controls were performed with
FITC IgG1 mouse antibody. Mobilized blood CD34+
cells were analyzed at day 10 of culture. Three experiments were
performed, which gave identical results. Lower left,
megakaryocyte (MK) and platelet (Pl) proteins
were separated on 12% acrylamide gels, transferred onto PVDF
membranes, and probed with the polyclonal anti-FcR
chain antibody.
Proteins were revealed using a peroxidase-coupled secondary antibody
and chemiluminescence. FcR
chain migrates as a doublet.
Lower right, GPVI was immunoprecipitated
(IP) from platelet or megakaryocyte lysates using 3J24.2,
and precipitated proteins were immunoblotted (IB) using the
anti-FcR
chain antibody.
chain. Thus, in subsequent experiments we investigated whether the signaling pathway coupled to
GPVI was functional in megakaryocytes.
2
1--
GPVI
is responsible for platelet adhesion to immobilized convulxin and
contributes to the Mg2+-independent platelet adhesion to
collagen in a static model of adherence (27, 28), whereas
2
1 integrin serves as a
Mg2+-dependent platelet receptor for type I
collagen both in static conditions and in flow (22, 29).
2
1 mAb 6F1 (data not shown), suggesting
that the integrin is involved in adhesion (22). In fact, EDTA also reduced megakaryocyte adhesion to collagen, and its inhibitory effect
increased with maturation increasing from 60% at day 9 to 80% at day
14 of culture. Together, these results suggest that the combined
expression of GPVI and
2
1 integrin is too
low at the earlier stage of maturation to allow the multiple
interactions required for adhesion to the immobilized collagen. Later
in the maturation, adhesion of megakaryocytes to convulxin indicates that GPVI is functional and adhesion to collagen may be via either GPVI
or
2
1. However, as shown by the greater
inhibitory effect of EDTA at day 14 compared with day 9,
2
1 integrin is the major contributor to
megakaryocyte adhesion to collagen later in differentiation.
View larger version (16K):
[in a new window]
Fig. 8.
Adhesion of cultured megakaryocytes to
immobilized convulxin and immobilized collagen. Megakaryocytes
(200,000 cells/ml) obtained from blood-mobilized CD34+
cells cultured in the presence of TPO at day 6 (A), day 9 (B), or day 14 (C) were added to microtitration
plates coated with convulxin or collagen type I. After washing,
detection of adherent megakaryocytes was performed by measuring
alkaline phosphatase activity. Nonspecific adhesion was detected on
BSA-coated wells (lane a). Megakaryocytes were
incubated with immobilized convulxin (lane b) or with
immobilized-collagen in the absence (lane c) or presence of
EDTA (lane d) for 1 h at 37 °C. The results are the
mean ± S.E. of triplicate experiments.
2
leading to platelet activation (8, 30, 31). To further characterize the
functional state of GPVI in megakaryocytes, we analyzed protein
tyrosine phosphorylation in response to convulxin or collagen. Adult
CD34+-derived megakaryocytes of the same culture were
studied sequentially. This approach allowed us to study the pattern of
tyrosine phosphorylation according to maturation because cultures are
partially synchronized (see Figs. 1 and 2). The addition of convulxin
or collagen to the cell suspension at 37 °C with stirring resulted
in a slight transient increase in light transmission comparable with
the light transmission change observed during platelet shape change
(not shown). The pattern of tyrosyl-phosphorylated proteins from day 9 cultured megakaryocytes and from platelets is shown in Fig. 9A. Before activation, some
proteins were phosphorylated in both platelets (lane 4) and
megakaryocytes (lane 1), most notably a doublet at 55 kDa,
and an additional low molecular mass protein (~30 kDa) was detected
in megakaryocytes. In both cell types, additional proteins were
phosphorylated after incubation with convulxin to give bands at 145, 120, 105, and 72 kDa. However, a phosphorylated 38-kDa protein was
detected in platelets as in previous studies (25, 30) but not in
megakaryocytes. Megakaryocyte incubation with collagen also resulted in
the tyrosine phosphorylation of these proteins but to a much lower
extent than convulxin, as already reported for platelets (25).
View larger version (50K):
[in a new window]
Fig. 9.
Convulxin- and collagen-triggered protein
tyrosine phosphorylation in human megakaryocytes. In A,
megakaryocytes obtained from CD34+ cells cultured in the
presence of TPO for 9 days were stimulated with 10 nM
convulxin or 100 µg/ml collagen for 6 min at 37° with agitation.
Proteins from nonstimulated (lane 1) or convulxin
(lane 2)- or collagen-stimulated (lane 3)
megakaryocytes were separated using 8.5% acrylamide gels, transferred
on PVDF membranes, and probed by immunoblotting with the
anti-phosphotyrosine antibody PY20. Detection was performed using a
peroxidase-coupled anti-mouse IgG antibody and chemiluminescence. In
lanes 4 and 5, proteins from nonstimulated or
convulxin-stimulated platelets were analyzed using the same method. In
B, changes in convulxin- or collagen-induced phosphorylation
were studied according to megakaryocyte maturation.
CD34+-derived cells were cultured in the presence of TPO
(5 × 105 cells/sample) for 6 (lanes 1, 2,
and 3), 9 (lanes 4, 5, and 6), or 14 days (lanes 7, 8, and 9) and were not stimulated
(lanes 1, 4, and 7) or stimulated with 10 nM convulxin (lanes 2, 5, and 8) or
100 µg/ml collagen (lanes 3, 6, and 9). Samples
were prepared and analyzed as described in A.
2 are
tyrosine-phosphorylated upon GPVI activation by convulxin (25, 30, 32).
Among the different proteins phosphorylated upon incubation of
megakaryocytes with collagen or convulxin, bands with the same
migration as p72Syk and PLC
2
were also observed at 72 and 145 kDa, respectively. p72Syk
and PLC
2 were immunoprecipitated from megakaryocytes (day 10 culture) or from platelets, before or after stimulation by convulxin or collagen, and analyzed for their content in
phosphotyrosine (Fig. 10).
PLC
2 was tyrosine-phosphorylated only upon
megakaryocyte and platelet activation by collagen or convulxin.
p72Syk was very slightly phosphorylated in nonactivated
megakaryocytes and platelets, but the intensity of the band increased
upon incubation with collagen or convulxin. In addition, a
phosphorylated low molecular weight protein corresponding to FcR
was
coprecipitated with Syk upon activation of megakaryocytes and platelets
by collagen or by convulxin, indicating that Syk is recruited by
phosphorylated FcR
chain upon GPVI activation. Together, these
results indicate that the signaling pathway coupled to GPVI is
functional in megakaryocytes.
View larger version (19K):
[in a new window]
Fig. 10.
Convulxin-induced
PLC 2, Syk, and FcR
chain phosphorylation in human
megakaryocytes. Proteins from megakaryocytes (lanes A)
or platelets (lanes B), nonstimulated or stimulated by
collagen or convulxin, were immunoprecipitated using
anti-PLC
2 or anti-Syk antibodies. After
SDS-polyacrylamide gel electrophoresis, proteins were transferred to
PVDF membranes. Membranes were probed with the
anti-phosphotyrosine 4G10 antibody, stripped, and reprobed with
the anti-PLC
2 antibody or the anti-Syk
antibody, respectively. Detection was performed using secondary
peroxidase-coupled antibodies and chemiluminescence.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 and GPVI is
important in mediating adhesion and in subsequent platelet secretion
and aggregation (31). The precise expression of these two proteins and
their function during megakaryocyte differentiation are poorly understood. Recently, GPVI has been cloned, and this collagen receptor
seems to be specific to the megakaryocyte/platelet lineage (5, 7).
Thus, it is now possible to study the expression and function of GPVI
during megakaryocyte maturation.
chain is expressed in several megakaryocytic cell lines in which GPVI has not been detected, it was suggested that
GPVI could be a late marker of megakaryocyte differentiation in
contrast to FcR
chain (37). Here we have directly studied the
expression of GPVI during megakaryocytic differentiation using a
culture system from CD34+ cell. By flow cytometry, the
pattern of GPVI expression partially mimicked that of CD41. Indeed,
GPVI expression was found only in cells expressing CD41 including
CD41low cells. However, expression was weak, just at the
threshold of detection, in these immature cells, and expression of GPVI
is seen clearly only in the brightest CD41+ cells
corresponding to mature megakaryocytes. The fact that GPVI was
expressed at a low level in immature megakaryocytic cells, including
those expressing CD34, was confirmed by RT-PCR after cell sorting.
Interestingly, expression of GPVI was quite similar to that of CD49b,
which was also considered as a late marker of megakaryocyte
differentiation. There is increasing evidence that the
CD41low cell population is not committed to the
megakaryocytic lineage. Thus, it is possible that the GPVI expression
is not entirely specific to the megakaryocytic lineage and might be
expressed at low level on nonmegakaryocytic progenitor cells. However,
preliminary results suggest that the majority of the
CD34+GPVI+ cells are late megakaryocyte
progenitors.2
/
mice (40). A second set of genes,
which includes thromboxane synthase and
1 tubulin, is
regulated by p45NF-E2 (41-43). Expression of these genes
is totally inhibited in the p45NF-E2
/
megakaryocytes leading to the absence of proplatelet formation (38, 42,
43). It has been suggested that this set of genes is involved in
platelet functions and is expressed at late stages of differentiation.
Recently, we investigated the expression of thomboxane synthase during
human megakaryocyte differentiation and also found that its expression
started early during differentiation in contrast to
1 tubulin (44).
The pattern of thromboxane synthase, GPVI, and CD49b expression in
megakaryocyte differentiation has many similarities because the level
of these proteins markedly increased in the more mature megakaryocytes.
It has been shown that megakaryocyte-specific enhancer of the
2 integrin gene contains two tandem AP1 binding sites
that are consensus sites for p45NF-E2 (45, 46). However,
presently there is no evidence that p45NF-E2 regulates
transcription of the
2 integrin gene. The promoter of
the human GPVI gene is still unknown, and the identity of the regulatory factors remains to be determined.
2 gene have been identified and
shown to result in a variability of
2
1
integrin density at the platelet surface (26, 47). Polymorphisms are
obvious even in megakaryocytes, as indicated by the heterogeneous
expression of CD49b by megakaryocytes from the different donors that we
have analyzed.
chain and is functional in megakaryocytes. Expression of GPVI
allows megakaryocytes to bind to convulxin in solution and to adhere to
immobilized convulxin and immobilized collagen in a
Mg2+-independent manner. Furthermore, collagen and
convulxin trigger GPVI-coupled signals in megakaryocytes. This finding
is in agreement with the results of Melford et al. (34) and
Mountford et al. (36) showing that collagen-related peptides
induces a rise in intracellular [Ca2+] in mature murine
megakaryocytes and protein tyrosine phosphorylation in human
megakaryocytes. At the earlier stages of maturation (day 5-6), the
amount of GPVI expressed on megakaryocyte surface is probably too low
for stable adhesion to immobilized convulxin or collagen.
However, it signifies that several proteins are
tyrosine-phosphorylated upon incubation with convulxin in solution,
indicating that GPVI dimerization occurs in these conditions. Thus, our
results indicate that, as soon as it is expressed, GPVI has the
potential to bind to the ubiquitously expressed FcR
chain and to
trigger signaling. Later in differentiation (day 9), an increase in
GPVI expression allows megakaryocyte adhesion to immobilized convulxin.
At the end of maturation, the decreased level of GPVI-coupled protein tyrosine phosphorylation suggests that GPVI might be uncoupled from its
signaling pathway by a mechanism that remains to be determined. At the
same moment, the role of
2
1 integrin
appears to become predominant in cellular interaction with
collagen. These results are in agreement with the respective role of
2
1 integrin and GPVI in platelets. It has
been already shown that
2
1 integrin plays
a major role in mature megakaryocyte (48) and megakaryocytic cell line
adhesion (46). In mature mouse megakaryocytes, the cross-linking of
2
1 integrin has been reported to increase
intracellular [Ca2+] via calcium influx and independently
of Src and Syk activation (35). From these results, it was suggested
that collagen triggers [Ca2+]i increase in mature
megakaryocytes via multiple receptors including GPVI, causing
calcium mobilization and
2
1 integrin, which stimulates an influx of extracellular calcium (35). Our observations that
2
1 integrin involvement
in collagen-mediated megakaryocytes adhesion increases with maturation
and that simultaneously GPVI-triggered signals decrease at the latest
stages of maturation provide further evidence that
2
1 integrin may be more important than
GPVI for mature megakaryocyte adhesion. Collagen interaction with
megakaryocytes and platelets may have different physiological consequences. Although collagen interaction with platelets is associated with activation, leading them to an irreversible process, collagen interactions with megakaryocytes may have a different role
depending on the stage of maturation. One can hypothesize that collagen
contributes to localizing the immature progenitors in bone marrow at
sites where conditions for proliferation and maturation are optimal.
Alternatively, at the end of maturation, collagen could contribute to
cell migration of mature megakaryocytes and to their subsequent exit
into circulation. Whatever its role, when platelets are released they
must be fully functional, which means that no activation processes
between GPVI and collagen should happen during their formation. How
this occurs remains to be understood. Some types of collagen do
not activate platelets, and they may trigger adhesion without
activation in the sites of platelet release. Alternatively, the
signaling pathway of the GPVI·FcR
complex may be inefficient or
inhibited in megakaryocytes and young platelets.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the INSERM, Association de Recherche Contre le Cancer Grant 9472, and University Paris 7.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-1-42-11-4233; Fax: 33-1-42-11-5240; E-mail: verpre@igr.fr.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M009117200
2 A.-H. Lagrue-Lak-Hal, N. Debili, G. Kingbury,, C. Lecut, J.-P. Le Couedic, J.-L. Villeval, M. Jandrot-Perrus, and W. Vainchenker, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GP, glycoprotein;
FcR Fc receptor
chain, PCR, polymerase chain
reaction;
RT-PCR, reverse transcriptase-PCR;
BSA, bovine serum albumin;
mAb, monoclonal antibody;
FITC, fluorescein isothiocyanate;
PEG, polyethylene glycol;
PMSF, phenylmethylsulfonyl fluoride;
PVDF, polyvinylidene difluoride;
TPO, thrombopoietin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Barnes, M. J., Knight, C. G., and Farndale, R. W. (1998) Curr. Opin. Hematol. 5, 314-320[Medline] [Order article via Infotrieve] |
2. |
Monnet, E.,
and Fauvel-Lafeve, F.
(2000)
J. Biol. Chem.
275,
10912-10917 |
3. |
Asselin, J.,
Gibbins, J. M.,
Achison, M.,
Lee, Y. H.,
Morton, L. F.,
Farndale, R. W.,
Barnes, M. J.,
and Watson, S. P.
(1997)
Blood
89,
1235-1242 |
4. |
Jandrot-Perrus, M.,
Lagrue, A. H.,
Okuma, M.,
and Bon, C.
(1997)
J. Biol. Chem
272,
27035-27041 |
5. |
Clemetson, J. M.,
Polgar, J.,
Magnenat, E.,
Wells, T. N.,
and Clemetson, K. J.
(1999)
J. Biol. Chem.
274,
29019-29024 |
6. | Miura, Y., Okuma, M., Jung, S. M., and Moroi, M. (2000) Thromb. Res. 98, 301-309[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Jandrot-Perrus, M.,
Busfield, S.,
Lagrue, A.-H.,
Xiong, X.,
Debili, N.,
Chickering, T.,
Le Couedic, J.-P.,
Goodearl, A.,
Dussault, B.,
Fraser, C.,
Vainchenker, W.,
and Villeval, J. L.
(2000)
Blood
96,
1798-1807 |
8. | Gibbins, J. M., Okuma, M., Farndale, R., Barnes, M., and Watson, S. P. (1997) FEBS Lett. 413, 255-259[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Tsuji, M.,
Ezumi, Y.,
Arai, M.,
and Takayama, H.
(1997)
J. Biol. Chem.
272,
23528-23531 |
10. |
Ezumi, Y.,
Shindoh, K.,
Tsuji, M.,
and Takayama, H.
(1998)
J. Exp. Med.
188,
267-276 |
11. |
Poole, A.,
Gibbins, J. M.,
Turner, M.,
van Vugt, M. J.,
van de Winkel, J. G.,
Saito, T.,
Tybulewicz, V. L.,
and Watson, S. P.
(1997)
EMBO J.
16,
2333-2341 |
12. | Watson, S. P., and Gibbins, J. (1998) Immunol. Today 19, 260-265[CrossRef][Medline] [Order article via Infotrieve] |
13. | Rabellino, E. M., Nachman, R. L., Williams, N., Winchester, R., and Ross, G. D. (1979) J. Exp. Med. 149, 1273-1287[Abstract] |
14. | Vinci, G., Tabilio, A., Deschamps, J.-F., Van Haeke, D., Henri, A., Guichard, J., Tetteroo, P., Lansdorp, P. M., Hercend, T., Vainchenker, W., and Breton-Gorius, J. (1984) Br. J. Haematol. 56, 589-605[Medline] [Order article via Infotrieve] |
15. | Murray, L. J., Mandich, D., Bruno, E., DiGiusto, R. K., Fu, W. C., Sutherland, D. R., Hoffman, R., and Tsukamoto, A. (1996) Exp. Hematol. 24, 236-245[Medline] [Order article via Infotrieve] |
16. | Tronik-LeRoux, D., Roullot, V., Schweitzer, A., Berthier, R., and Marguerie, G. (1995) J. Exp. Med. 181, 2141-2151[Abstract] |
17. | Debili, N., Issaad, C., Masse, J. M., Guichard, J., Katz, A., Breton-Gorius, J., and Vainchenker, W. (1992) Blood 80, 3022-3035[Abstract] |
18. |
Lepage, A.,
Uzan, G.,
Touche, N.,
Morales, M.,
Cazenave, J. P.,
Lanza, F.,
and de la Salle, C.
(1999)
Blood
94,
3366-3380 |
19. | Francischetti, I. M., Saliou, B., Leduc, M., Carlini, C. R., Hatmi, M., Randon, J., Faili, A., and Bon, C. (1997) Toxicon 35, 1217-1228[CrossRef][Medline] [Order article via Infotrieve] |
20. | Kilpatrick, K. E., Cutler, T., Whitehorn, E., Drape, R. J., Macklin, M. D., Witherspoon, S. M., Singer, S., and Hutchins, J. T. (1998) Hybridoma 17, 569-576[Medline] [Order article via Infotrieve] |
21. | Sugiyama, T., Okuma, M., Ushikubi, F., Sensaki, S., Kanaji, K., and Uchino, H. (1987) Blood 69, 1712-1720[Abstract] |
22. | Coller, B., Beer, J., Scudder, L., and Steinberg, M. (1989) Blood 74, 182-192[Abstract] |
23. |
Debili, N.,
Wendling, F.,
Katz, A.,
Guichard, J.,
Breton-Gorius, J.,
Hunt, P.,
and Vainchenker, W.
(1995)
Blood
86,
2516-2525 |
24. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
25. | Lagrue, A. H., Francischetti, I. M., Guimaraes, J. A., and Jandrot-Perrus, M. (1999) FEBS Lett. 448, 95-100[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kunicki, T. J., Orchekowski, R., Annis, D., and Honda, Y. (1993) Blood 82, 2693-2703[Abstract] |
27. |
Nakamura, T.,
Jamieson, G. A.,
Okuma, M.,
Kambayashi, J.,
and Tandon, N. N.
(1998)
J. Biol. Chem.
273,
4338-4344 |
28. |
Nakamura, T.,
Kambayashi, J.,
Okuma, M.,
and Tandon, N. N.
(1999)
J. Biol. Chem.
274,
11897-11903 |
29. | Santoro, S. A. (1986) Cell 46, 913-920[Medline] [Order article via Infotrieve] |
30. |
Polgar, J.,
Clemetson, J. M.,
Kehrel, B. E.,
Wiedemann, M.,
Magnenat, E. M.,
Wells, T. N.,
and Clemetson, K. J.
(1997)
J. Biol. Chem.
272,
13576-13583 |
31. | Watson, S. P. (1999) Thromb. Haemost. 82, 365-376[Medline] [Order article via Infotrieve] |
32. | Faili, A., Randon, J., Francischetti, I. M. B., Vargaftig, B. B., and Hatmi, M. (1994) Biochem. J. 298, 87-92[Medline] [Order article via Infotrieve] |
33. | Tavassoli, M., and Aoki, M. (1981) Br. J. Haematol. 48, 25-29[Medline] [Order article via Infotrieve] |
34. |
Melford, S. K.,
Turner, M.,
Briddon, S. J.,
Tybulewicz, V. L.,
and Watson, S. P.
(1997)
J. Biol. Chem.
272,
27539-27542 |
35. |
Briddon, S. K.,
Turner, M.,
Tybulewicz, V.,
and Watson, S. P.
(1999)
Blood
93,
3847-3855 |
36. | Mountford, J. C., Melford, S. K., Bunce, C. M., Gibbins, J., and Watson, S. P. (1999) Thromb. Haemost. 82, 1153-1159[Medline] [Order article via Infotrieve] |
37. | Lemarchandel, V., Ghysdael, J., Mignotte, V., Rahuel, C., and Roméo, P. H. (1993) Mol. Cell. Biol. 13, 668-676[Abstract] |
38. | Shivdasani, R. A., Rosenblatt, M. F., Zucker-Franklin, D., Jackson, C. W., Hunt, P., Saris, C. J. M., and Orkin, S. H. (1995) Cell 81, 695-704[Medline] [Order article via Infotrieve] |
39. |
Shivdasani, R. A.,
Fujiwara, Y.,
McDevitt, M. A.,
and Orkin, S. H.
(1997)
EMBO J.
16,
3965-3973 |
40. |
Vyas, P.,
Ault, K.,
Jackson, C. W.,
Orkin, S. H.,
and Shivdasani, R. A.
(1999)
Blood
93,
2867-2875 |
41. |
Deveaux, S.,
Cohen-Kaminsky, S.,
Shivdasani, R. A.,
Andrews, N. C.,
Filipe, A.,
Kuzniak, I.,
Orkin, S. H.,
Roméo, P. H.,
and Mignotte, V.
(1997)
EMBO J.
16,
5654-5661 |
42. |
Lecine, P.,
Villeval, J. L.,
Vyas, P.,
Swencki, B.,
Xu, Y.,
and Shivdasani, R. A.
(1998)
Blood
92,
1608-1616 |
43. |
Lecine, P.,
Italiano, J. E.,
Kim, S. W.,
Villeval, J. L.,
and Shivdasani, R. A.
(2000)
Blood
96,
1366-1373 |
44. | Vitrat, N., Letestu, R., Massé, A., Lazar, V., Vainchenker, W., and Debili, N. (2000) Thromb. Haemost. 83, 759-768[Medline] [Order article via Infotrieve] |
45. |
Zutter, M. M.,
Painter, A. D.,
and Yang, X.
(1999)
Blood
93,
1600-1611 |
46. |
Zutter, M. M.,
Painter, A. A.,
Staatz, W. D.,
and Tsung, Y. L.
(1995)
Blood
86,
3006-3014 |
47. |
Kunicki, T. J.,
Kritzik, M.,
Annis, D. S.,
and Nugent, D. J.
(1997)
Blood
89,
1939-1943 |
48. | Mossuz, P., Schweitzer, A., Molla, A., and Berthier, R. (1997) Br. J. Haematol. 98, 819-827[Medline] [Order article via Infotrieve] |