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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 509-518
The Thrombocytopenia of Wiskott Aldrich Syndrome Is Not Related to
a Defect in Proplatelet Formation
By
Elie Haddad,
Elisabeth Cramer,
Christel Rivière,
Philippe Rameau,
Fawzia Louache,
Josette Guichard,
David L. Nelson,
Alain Fischer,
William Vainchenker, and
Najet Debili
From INSERM U 362, PR1, Institut Gustave Roussy, Villejuif, France;
INSERM U474, Hôpital Henri Mondor, Créteil, France; Service
d'Immuno-Hématologie pédiatrique, Hôpital Necker,
Paris, France; and the Metabolism Branch, Division of Clinical Science,
National Cancer Institute, National Institutes of Health, Bethesda, MD.
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ABSTRACT |
The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease
characterized by thrombocytopenia with small platelet size, eczema, and
increased susceptibility to infections. The gene responsible for WAS
was recently cloned. Although the precise function of WAS protein
(WASP) is unknown, it appears to play a critical role in the regulation
of cytoskeletal organization. The platelet defect, resulting in
thombocytopenia and small platelet size, is a consistent finding in
patients with mutations in the WASP gene. However, its exact mechanism
is unknown. Regarding WASP function in cytoskeletal organization, we
investigated whether these platelet abnormalities could be due to a
defect in proplatelet formation or in megakaryocyte (MK) migration.
CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked
thrombocytopenia (XLT) and cultured in serum-free liquid medium
containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor
(SCF) to study in vitro megakaryocytopoiesis. In all cases, under an
inverted microscope, normal MK differentiation and proplatelet
formation were observed. At the ultrastructural level, there was also
no abnormality in MK maturation, and normal filamentous MK were
present. Moreover, the in vitro produced platelets had a normal size,
while peripheral blood platelets of the same patients exhibited an
abnormally small size. However, despite this normal platelet
production, we observed that F-actin distribution was abnormal in MKs
from WAS patients. Indeed, F-actin was regularly and linearly
distributed under the cytoplasmic membrane in normal MKs, but it was
found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In
contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from
WAS patients normally migrated in response to stroma-derived factor-1 (SDF-1), and actin normally polymerized
after SDF-1 or thrombin stimulation. These results suggest that the
platelet defect in WAS patients is not due to abnormal platelet
production, but instead to cytoskeletal changes occuring in platelets
during circulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE WISKOTT-ALDRICH syndrome (WAS) is an
X-linked hereditary disease characterized by thrombocytopenia
with small platelet size, eczema, and increased susceptibility to
infections.1-3 A milder form, designated as hereditary
X-linked thrombocytopenia (XLT), is characterized by isolated
thrombocytopenia with small platelet size.4,5 The gene
responsible for WAS was recently cloned.6 Sequence analysis
identified mutations of the WAS gene in both WAS and XLT, suggesting
that they are two different phenotypes of the same
disease.6-9 The exact function of WAS protein (WASP) is
still unknown, but through its GBD domain that binds to the small
guanosine triphosphatase (GTPase) protein
Cdc4210-12 and through its verprolin and cofilin homology
domains located at its C-terminal region,13 WASP
participates in cytoskeletal organization. Moreover, WASP has been
described to interact with many SH3-containing proteins by its proline
rich domain.14-16 Thus, WASP appears to play an important
role in the regulation of cytoskeletal organization and in signal transduction.
The platelet defect, thrombocytopenia, and small platelet size is a
consistent finding in patients with mutations in the WASP gene. The
exact mechanism of this platelet defect is still unknown. Splenectomy
is generally effective in elevating platelet counts toward
normal,17,18 and the number and morphology of
megakaryocytes (MKs) in WAS bone marrow are normal.3,19
These data suggest peripheral destruction of platelets in WAS. However,
the association of decreased platelet turnover and normal or increased
MK mass suggests ineffective thrombocytopoiesis.3,20
Platelet release by MKs is related to a unique phenomenon called
proplatelet formation. At the end of maturation, MKs extend long
filamentous processes. Constriction areas delineate future platelets,
which are detached after breaking of the processes. Proplatelet
formation is related to a reorganization of the cytoskeleton involving
microtubules and actin filaments. Therefore, it is possible that WASP
is involved in the platelet formation process, ie, development of
platelet demarcation membranes, proplatelet formation, and/or platelet
release from MKs.20,21 In addition, as platelets are not
actually released within the marrow, the transendothelial migration of
MKs is absolutely required for release of platelets into the
circulation.22 This process requires reorganization of
actin filaments and formation of pseudopods. It is worth noting that
Bernard Soulier syndrome, another disease involving changes in actin
organization, is associated with thrombocytopenia, but with large
platelets.23
To study the megakaryocytopoiesis of WAS patients and to examine if
patient MKs demonstrate abnormal proplatelet formation and platelet
release, we performed in vitro cultures of CD34+ cells
isolated from blood and/or bone marrow of 19 patients with WAS or XLT
under conditions that allowed the differentiation of CD34+
cells into platelet producing MKs24,25 and compared them
with CD34+ cells from normal controls. stroma-derived
factor-1 (SDF-1)-induced migration of MKs was also tested. It
was found that WAS megakaryopoiesis and platelet production, as well as
the SDF-1-induced migration of MKs, were normal.
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MATERIALS AND METHODS |
Monoclonal antibodies.
Phycoerythrin (R-PE)-conjugated anti-CD41a monoclonal antibody (MoAb)
(Pharmingen, San Diego, CA), R-PE-conjugated anti-CD34 MoAb (HPCA-2;
Becton Dickinson, Mountain View, CA), and fluorescein isothyocyanate
(FITC)-labeled phalloidin (Sigma Chemical Co, St Louis, MO) were used
for flow cytometric analysis. Mouse anti-CD34 MoAb conjugated to
magnetic beads (Miltenyi Biotech GmbH, Bergisch Gladbuch,
Germany) was used for the Miltenyi technique of purification. Rabbit
anti-von Willebrand factor (vWF) polyclonal antibody (Dako, Glostrup,
Denmark), mouse anti-WASP MoAb,26 FITC-labeled donkey antimouse IgG (Jackson Immunoresearch, West Grove, PA),
tetramethylrhodamine isothiocyanate (TRITC)-conjugated donkey
antirabbit IgG, aminomethylcoumarine (AMCA)-conjugated
donkey antirabbit IgG (Jackson Immunoresearch), FITC-labeled phalloidin
(Sigma) were used for indirect immunofluorescence assays.
Purification of CD34+ cells and cell cultures.
Fresh blood samples (n = 19) and bone marrow aspirates (n = 5) were
harvested from 19 patients with either WAS (n = 14) or XLT (n = 5)
during medical evaluations (blood samples) or under general anesthesia
for splenectomy (marrow aspirates). In 10 patients, the mutation in the
WASP gene was characterized. They consisted of R211stop, Y83stop, R86H,
and a mutation in intron 10 in one patient, respectively. S483FSstop494
was found in two related patients and V75L in four other related
patients. Characterization of the mutation is currently being performed
in the other nine patients.
Cells were separated over a Ficoll-metrizoate gradient (Lymphoprep,
Nycomed Pharma, Oslo, Norway) to obtain an enriched fraction of
mononuclear cells. CD34+ cells were then isolated by the
Miltenyi immunomagnetic bead technique as previously
reported.24,25 Cells were incubated for 30 minutes at
4°C with an anti-CD34 MoAb conjugated to magnetic beads. The
CD34+ cells were retained on the column and were eluted by
pressure using the plunger supplied with the column. Subsequently,
cells were purified by cell sorting. Briefly, cells were labeled with a
R-PE anti-CD34 MoAb and, after one wash, were sorted on a FACSvantage (Becton Dickinson) equipped with an argon ion laser (INNOVA 70-4, Coherent Radiation, Palo Alto, CA) tuned to 488 nm and operating at 500 mW. A "morphological" gate including 80% of the events and all
of the CD34+ cells was determined on two-parameter
histograms (side scatter [SSC] versus forward scatter [FSC]).
Control CD34+ cells were purified by the same procedure
from normal bone marrow of patients undergoing hip surgery or from the
peripheral blood of patients after mobilization by chemotherapy and
granulocyte colony-stimulating factor (G-CSF). Informed consent was
obtained in all cases (patients and controls) in accordance with the
institutional guidelines of the Committee on Human Investigation.
To obtain MKs, purified CD34+ cells were cultured in
serum-free liquid medium containing a recombinant truncated form of
Mpl-L (PEG-rHuMGDF, Amgen, Thousand Oaks, CA, 10 ng/mL) and recombinant human stem cell factor (SCF; Amgen, 50 ng/mL) as previously
described.25
Cultures were observed daily under an inverted microscope to compare
normal and WAS MKs.
Ultrastructural studies.
Cultured cells were studied by electron microscopy. They were washed
twice in Hanks medium at 4°C, fixed in 1.25% glutaraldehyde in
phosphate buffer (0.1 mol/L, pH = 7.4) for 1 hour, and washed twice.
Cells were then fixed with osmium tetroxide, dehydrated, and embedded
in Epon. Thin sections were examined with a Philips CM 10 electron
microscope (Philips, Eindhoven, The Netherlands) after uranyl acetate
and lead citrate staining. Immunolabelling for IIb 3 was performed
on glycol-methacrylate-embedded MK cultures and platelets with
polyclonal anti-IIb3 antibody (a generous gift from D. Pidard,
Institut Pasteur, Paris, France) followed by immunogold (goat
antirabbit IgG coupled to 10 nm colloidal gold) (British
Biocell, Cardiff, UK). The diameter of shed platelets was
measured on electron micrographs.
MK immunofluorescence microscopy.
After 7 to 13 days, cultured cells were fixed in 2% paraformaldehyde
(PFA) for 15 minutes, washed in phosphate-buffered saline (PBS), and
resuspended in PBS at the concentration of 105 cells/100
µL. Cell suspensions (100 µL) were cytocentrifuged at 500 rpm for 2 minutes. Cells were then fixed once more in 2% PFA for 5 minutes,
rehydrated in PBS, permeabilized with 0.1% Triton for 3 minutes, and
washed with PBS before incubation for 30 minutes at room temperature
with a rabbit anti-vWF polyclonal antibody. After three washes with
PBS, cells were incubated at room temperature with TRITC-labeled donkey
antirabbit IgG and FITC-labeled phalloidin. DNA was labeled by 7.5 ng/mL Hoechst 33258 (Hoechst 33528, Sigma) for 15 minutes in the dark.
Cell preparations were analyzed with a fluorescence microscope equipped
with the appropriate filter combinations (Zeiss, Oberkochen, Germany).
For the immunofluorescence studies on adherent MKs, glass coverslips
were coated with poly-L-lysine for 1 hour and gently washed with PBS.
Cultured cells were resuspended in PBS without any fixation at the
concentration of 105/100 µL. The unfixed cell suspension
(100 µL) was pipetted onto the coverslips and allowed to adhere for
1, 5, 15, 30, or 60 minutes. After these different time points, cells
were fixed in PFA and permeabilized with Triton, as described above,
before incubation for 30 minutes at room temperature with a rabbit
anti-vWF polyclonal antibody and, in some experiments, with a mouse
anti-WASP MoAb. After three washes with PBS, cells were incubated with
TRITC- or AMCA-labeled donkey antirabbit IgG, FITC-labeled phalloidin, and TRITC-labeled donkey antimouse IgG. When the AMCA-labeled donkey
antirabbit antibody was not used as secondary antibody, DNA was labeled
by Hoechst, as described above.
MK migration after SDF-1 stimulation.
To analyze MK migration, CD34+ cells were cultured for 7 days. Then, cultured cells were resuspended in serum-free medium at a
final concentration of 2.5 × 106 cells/mL. Migration
assays were performed using 5-µm pore filters (Transwell, 24-well
cell clusters; Costar, Cambridge, MA). Cell suspensions (2.5 × 105 cells in a 100-µL volume) were placed into the upper
chamber, whereas 600 µL of medium with or without recombinant human
SDF-1 (300 ng/mL) (R & D Systems, Minneapolis, MN) was introduced in the lower chamber. The chambers were incubated for 1 hour at 37°C in 5% CO2 and 95% air. The cells in the upper and in the
bottom chamber were recovered separately in the same volume for
counting. The different cell fractions were then labeled with a
PE-anti-CD41a MoAb and analyzed by flow cytometry. All assays were
performed in triplicate. Data are presented as the percentage of
migrating cells (number of cells migrating [lower chamber]/total
number of cells [cells in the lower chamber + remaining cells in the upper chamber]).
MK actin polymerisation after thrombin and SDF-1 stimulation.
After 7 to 13 days, cultured cells were resuspended in three equal
0.1-mL aliquots. The first aliquot was incubated with thrombin (0.1 IU/mL) for 30 seconds with shaking at 37°C. The second
aliquot was incubated with SDF-1 (300 ng/mL) for 30 seconds in the
same conditions. The remaining tube was incubated in the same
conditions without thrombin or SDF-1 to assess baseline activation.
Cells were then fixed with 2% paraformaldehyde (PFA) for
15 minutes, washed in PBS, permeabilized in 0.1% Triton for 3 minutes,
washed, and resuspended in PBS. Cells were then incubated at 4°C
for 30 minutes with PE-labeled anti-CD41a MoAb and FITC-labeled
phalloidin. Cell samples were analyzed on a FacSort (Becton Dickinson).
For each sample, 10,000 cells were acquired in the list mode and
analyzed with the Cellquest software package (Becton Dickinson).
| |
RESULTS |
MK maturation and differentiation under light and electron microscopy.
To study a large series of patients, we developed a purification
technique that allowed the isolation of CD34+ cells from 10 mL of blood. CD34+ cells were first purified by the
Miltenyi technique with one passage on the column. After this first
separation, the purity was between 5% and 10%. Cells were then
further purified by cell sorting, pemitting the recovery of 2,000 to
5,000 CD34+ cells per sample with a purity of over 95%. In
five cases, in addition to blood CD34+ cells, marrow
aspirates could be obtained from patients during general anesthesia for
splenectomy, and the same procedure allowed the purification of 50,000 to 100,000 CD34+ cells per aspirate. The number of purified
CD34+ cells obtained from blood and/or marrow of WAS
patients was the same as obtained with blood and/or marrow from
controls. As previously described, addition of PEG-rHuMGDF and SCF to a
serum-free liquid culture of these cells led to the differentiation of
CD34+ cells into MKs, and eventually to their maturation
into platelet-producing cells. Under these culture conditions, the
number of MKs obtained from cultured CD34+ cells was the
same in WAS as in control cultures (four MKs per CD34+
cell) when cultures were analyzed by flow cytometry after staining with
a PE-CD41a MoAb. All of the steps of differentiation and maturation of
MKs could be observed in the cultures of all patient CD34+
cells (n = 19) and gave identical results, whatever the origin of
CD34+ cells (blood or marrow).
Unexpectedly, MKs from WAS and XLT patients were morphologically
similar to normal MKs, with the same timing of differentiation. Under
light microscopy, round MKs began to deform at day 7 of culture and
formed a pseudopod that progressively elongated to become as long and
thin as that of MKs from normal controls
(Fig 1A and B), and that ultimately gave
rise to detached (pro)platelets. These platelet-producing MKs were as
numerous in patient as in normal cultures (about 10% to 20% of the
MKs being filamentous), and no obvious abnormality could be detected in
their morphologic appearance.
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| Fig 1.
Platelet-producing normal and WAS MKs have the same
morphological aspect. CD34+ cells of WAS patients (A) or
normal controls (B) were purified from blood or bone marrow and were
grown in the presence of PEG-rHuMGDF and SCF. Analysis of cultured
cells under light microscopy by day 8 allowed the identification of
platelet-producing MK characterized by the extension of very long and
thin pseudopods. They give rise to proplatelets by breaking irregularly
at several constriction sites. The frequency and appearance of these
platelet shedding MKs were similar in WAS patients (A) and in normal
controls (B).
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To study the polymerization and distribution of actin in MKs, cultured
cells were costained with anti-vWF antibodies to precisely identify MKs
and with phalloidin to observe actin filaments. F-actin was linearly
located under the cytoplasmic membrane and regularly distributed in
normal MKs (Fig 2A through C). In contrast,
it was found concentrated near the nucleus in the center of 50% of the
WAS MKs (about 60 MKs studied in each patient) (Fig 2D through F). A
part of the F-actin was also distributed under the cytoplasmic membrane. However, in platelet-producing MKs from normals and WAS
patients, the immunofluorescence appearance of F-actin was identical.
Phalloidin staining was linearly distributed all along the pseudopods
that give rise to platelets after breaking
(Fig 3). It is worth
emphasizing that WAS protein, as well as vWF, were present in
pseudopods of normal MKs.
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| Fig 2.
Abnormal F-actin redistribution in WAS MKs. Cultures were
performed as described in Fig 1. At day 8, cells were fixed and, after
cytocentrifugation, were incubated with anti-vWF (TRITC) to localize
the MKs (B and E) and with phalloidin (FITC) to study F-actin
distribution (C and F). DNA was stained by Hoechst dye (A and D). In
normal MKs (A, B, and C), F-actin distribution was located linearly
under the cytoplasmic membrane and regularly distributed (C), while in
WAS MKs (D, E, and F), F-actin was localized in the center of the cell
(F).
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| Fig 3.
Immunofluorescent appearance of a
platelet-producing WAS MK. Cultures were performed and labeled as
described in Fig 2. Phalloidin staining (FITC) was distributed linearly
all along the pseudopod that gives rise to platelets by breaking
irregularly at several constriction sites (A). vWF (TRITC) was present
in this pseudopod (B), while Hoechst staining determined the
localization of the nucleus (C). This immunofluorescent appearance of
platelet-producing WAS MK was similar to that observed in normal
control platelet-producing MKs (not shown).
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Ultrastructural analysis of patient MKs was performed in nine cases. In
all cases, electron microscopic appearance of WAS MKs was similar to
that of normal MKs. The demarcation membrane system was well developed
and normal in distribution in WAS MKs (Fig
4). In only one case, the membrane demarcation system seemed to be more
developed than in normal MKs, but this observation was not confirmed in
the other eight patients. The maturation of WAS MKs during platelet
shedding, consisting of alignment and dilatation of the peripheral
demarcation membranes, proplatelet formation (Fig 4), detachment of
newly formed platelets, and presence of platelets in the supernatant of
the culture medium was also normal in WAS MKs. Surprisingly, the
diameter of in vitro shed platelets measured by electron micrographs in
seven WAS patients (10 platelets in each case) was normal, as compared
with the diameter of shed platelets from normal MKs (2.73 ± 0.9 µm and 2.87 ± 0.9 µm, respectively), whereas the
mean in vivo platelet volume of these patients was abnormally low when
measured in peripheral blood with an electrical impedance particule
counter (5.5 ± 0.3 fL) as compared with mean platelet volume of
normal controls (8.2 ± 1.9 fL).
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| Fig 4.
Electron microscopic view of a cultured mature WAS MK.
This MK is starting the process of platelet shedding. The nucleus (N)
is excentric, the demarcation membrane system (dms) is widening,
delimitating platelet territories (p) and, at the cell periphery,
proplatelets (pp) start to extend. (A, -granules; gly,
glycogen).M= × 7.375. Insert: Normal proplatelet (pp) extend from
mature MK.M= × 2.145.
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Immunofluorescence studies of adherent MKs.
To test the distribution of F-actin during the adhesion process,
unfixed MKs were pipetted onto poly-L-lysine-coated glass coverslips.
Adhesion to poly-L-lysine emphasized the abnormal F-actin distribution
in WAS MKs as compared with normal MKs (Fig 5). After 5 minutes of adhesion, normal MKs began to extend long filopodia that reached their maximum extension after 30 to 60 minutes
(Fig 5A and B). In the same conditions, WAS MKs could extend filopodia,
which however were shorter and less numerous than in normal MKs, even
after 60 minutes of adhesion (Fig 5C and D). In contrast to the
proplatelet filaments, no vWF could be detected in the filopodia of
both normal and WAS MKs. Lamellipodial formations were observed in
normal and WAS MKs and did not seem different (not shown). The defects
observed in F-actin distribution and filopodia formation were equally
represented among WAS and XLT patients. By staining normal MKs with
anti-WASP antibody, we could show that unlike vWF, WASP protein
localized along the filopodia with the same topography as F-actin
(Fig 6A and B). However, in the central
part of the MKs, WASP did not exactly colocalize with F-actin, but was
in a more central position (Fig 6B). WASP protein expression was
studied in two patients with a missense mutation. In both cases, WASP
protein was detected in cultured MKs, but in one of them in contrast to
normal MKs, WASP did not colocalize with F-actin.
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| Fig 5.
Abnormal filopodia formation of WAS MKs after adhesion to
poly-L-lysine. Cultured cells were pipetted onto poly-L-lysine-coated
coverslips. After 30 minutes of adhesion, cells were fixed,
permeabilized, and labeled as described in Fig 3. (A and B) Examination
of a normal MK with a combination of filters allowing simultaneous
visualization of vWF (TRITC), F-actin (FITC), and DNA (Hoechst). The
normal MK displays long, thin, and numerous filopodia that contain
F-actin, but not vWF, which remains localized in the center of the
cell. In contrast, WAS MKs fail to extend long filopodia (C) or extend
very short, but less numerous filopodia (D) than do normal MK.
Examination of WAS MKs with a combination of filters was not
interpretable because of the central localization of F-actin that
overlaps with that of vWF and therefore was not shown in this figure.
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| Fig 6.
Localization of WASP in normal MKs after adhesion on
Poly-L-lysine. Examination of normal MKs was performed as described in
Fig 5. MKs were labeled with anti-vWF polyclonal antibody (AMCA),
phalloidin (FITC), and anti-WASP MoAb (TRITC) after 30 minutes of
adhesion to poly-L-lysine-coated coverslips. Anti-vWF labeling (AMCA)
allowed the localization of MKs. Unlike vWF, WASP is localized in
filopodia (A) with the same topography as F-actin (B).
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Migration of MKs stimulated by SDF-1.
Because it has been shown that SDF-1 can induce MK migration, we
tested migration of MKs to SDF1- in WAS. The baseline percentage of
migrating MKs (no SDF-1 in the lower chamber) was invariably under
0.1% for normal and WAS MKs. After SDF-1 stimulation, the mean
percentage of migrating cells was 21% (± 8%) for WAS MKs and 19%
(± 8%) for normal MKs (P, not significant
[NS]).
Polymerization of actin after stimulation of MKs by thrombin and/or
by SDF-1.
Finally, we investigated whether an abnormality in actin polymerization
was present in WAS MKs after thrombin or SDF-1 stimulation. For this
purpose, the intensity of phalloidin staining was studied by flow
cytometry after double-staining with FITC-phalloidin and R-PE
anti-CD41a MoAb. As illustrated in the representative examples (Fig 7), the same significant increase in
F-actin content after stimulation by thrombin (0.1 IU/mL) or SDF-1
(300 ng/mL) was found in MKs (CD41a+ cells) from normal and
WAS patients. Moreover, kinetic analysis of actin polymerization by the
same method did not show any difference between normal and WAS MKs
after stimulation by thrombin or SDF-1 (data not shown). Baseline
F-actin content was also similar in normal and WAS MKs.
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| Fig 7.
Normal actin polymerization of WAS MKs after stimulation
by SDF-1 and thrombin. After 7 to 13 days, cultured cells were
resuspended and stimulated with thrombin (0.1 IU/mL) or SDF-1 (300 ng/mL) for 30 seconds with shaking at 37°C. Cells were then fixed,
permeabilized, and incubated with PE-labeled anti-CD41a and
FITC-labeled phalloidin. Cell samples were analyzed on a FacSort
(Becton Dickinson). MKs were selected by gating the CD41+
cells (A). Analysis of FITC intensity on CD41+ cells
showed that the baseline content of F-actin and the increase of actin
polymerization after stimulation of MKs by SDF-1 (B and D) and
thrombin (C and E) were the same in normal MKs (B and C) and in WAS MKs
(D and E).
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| |
DISCUSSION |
Thrombocytopenia and small platelet size are two consistent findings in
WAS and XLT. Their precise mechanisms are unknown. In addition to
increased peripheral platelet destruction that probably contributes to
the thrombocytopenia, platelets of WAS patients have several intrinsic
abnormalities in morphology, phenotype, and
function.19,27-29 In addition, defects in the cytoskeletal architecture and organelle organization have been
reported.19,28 By studying autologous platelet survival,
platelet function, and megakaryocyte histology in four WAS patients,
Ochs et al3 demonstrated a decrease in platelet turnover,
which was 30% of the normal value in all four cases. In contrast,
marrow megakaryocyte mass was normal or increased. This discrepancy
between the normal or increased megakaryocyte mass and the decreased
platelet turnover suggests that ineffective thombocytopoiesis does
occur in WAS and may be responsible for thrombocytopenia.20
In some patients, we studied the thrombopoietin (TPO)
level in the plasma by enzyme-linked immunosorbent assay (ELISA) and
found that despite the thrombocytopenia, the TPO level was normal or
slightly elevated (data not shown), as described in immune
thrombocytopenia patients (ITP). This clearly reinforces the contention
that the MK mass is normal or increased in WAS patients, but platelet
numbers are limited by dysmegakaryopoiesis or platelet destruction. The
late stages of megakaryocytopoiesis are accompanied by profound
modifications of the cell structure leading to proplatelet
formation.24,30,31 Thus, we hypothesized that the platelet
abnormalities in WAS could be due to a defect in proplatelet production
as a consequence of an abnormal interaction between WASP and the
cytoskeleton. In favor of this hypothesis, Miki et al21
reported that in MEG-01 cells, a megakaryoblastic cell line, WASP
interaction with actin filaments was indispensable for microvesicle
formation, a process hypothesized to be similar to MK proplatelet formation.
To test this hypothesis, we performed in vitro cultures of
CD34+ cells from 19 patients in conditions supporting the
differentiation of CD34+ cells into platelet-producing
MKs.24 By analyzing F-actin distribution after phalloidin
staining, we confirmed that F-actin distribution was abnormal in WAS
MKs and that this phenomenon was exacerbated during adhesion to
poly-L-lysine. Indeed, we found that WAS MK F-actin was not linearly
distributed at the periphery of the cell, but was concentrated in the
center of the cell, as described recently in WAS patient
lymphocytes.32 After adhesion to poly-L-lysine, WAS MKs
could extend some filopodia, but in general, they were shorter and less
numerous than in normal MKs. One possible explanation for this defect
is the interaction of WASP with Cdc42 through its GBD
domain.10-12 Cdc 42 is a member of the Rho family of
proteins that plays a critical role in cytoskeletal
organization33-35 and is particularly involved in filopodia
formation. Thus, absent or abnormal interaction between WASP and Cdc42
may explain abnormal filopodia formation in WAS MKs. Because this
phenomena was observed in all cases tested except for one (n = 7), this
would mean that WASP interaction with Cdc42 was impaired in all or most
cases. However, of all the mutations reported so
far,20,36,37 none was located in the exon encoding the GBD
domain. This implies that direct interaction of WASP with Cdc42 may be
necesssary, but is not sufficient to allow filopodia formation.
Alternatively it has been reported that the majority of the mutations
alter the quantitative level of WASP expression.38 It is
also possible that the central localization of F-actin does not allow
normal extension of filopodia despite an eventual normal interaction between WASP and Cdc42.
Despite this abnormal F-actin distribution and filipodia formation, all
steps of normal MK differentiation could be observed in vitro in these
patients. Extension of long pseudopods, development and distribution of
the demarcation membrane system, proplatelet formation and detachment
of newly formed platelets, phenomenons that involve profound
reorganization of cytoplasmic structure, were all normal under light
and electron microscopy. Unexpectedly, in vitro produced platelets had
a normal apparent diameter, while platelets from the same patients were
abnormally small in the blood. Recently, it has been shown that Wistar
Furth rat platelets have defective filopodia formation.39
It is noteworthy that in both diseases, characterized by a defective
filopodia formation, a thrombocytopenia is present, but with major
differences in the platelet volume. Indeed, Wistar Furth rats have
thrombocytopenia with large platelets, whereas platelets of WAS
patients are small. This suggests that proplatelet formation and
filopodia formation result from two independent mechanisms.
If one accepts that these in vitro observations are applicable to
normal in vivo platelet production, our results would suggest that
diminution of platelet size occurs after platelet production. It can be
hypothesized that as a consequence of abnormal cytoskeletal organization, platelet deformation occurs in the blood circulation, especially in spleen sinusoids, and leads to diminution of size and
spleen destruction. Alternatively, due to abnormal membrane deformability, WAS platelets of normal size would be preferentially destroyed in the spleen, whereas small platelets would be submitted to
less prononced deformation in the vessels and would thus escape destruction. The fact that the half-life of reinfused, autologous, small-sized platelets was only moderately reduced, ie, 5 ± 1.3 days
compared with 9.5 ± 0.6 days in normal subjects3 argues for the second hypothesis. This may also explain why platelet size
increases after splenectomy.18 In five patients who
underwent splenectomy in the course of this study, platelet volume also increased, but slightly without reaching normal values. It is worth
emphasizing that WASP-deficient mice have only modest alterations in
their platelet numbers (five of seven wild-type mice), with normal
platelet size.40 The absence of a marked thrombocytopenia in mice may be related to the much smaller platelet size in comparison to human platelets, allowing normal passage through the spleen.
In vitro chemotaxis of WAS neutrophils and monocytes has been shown to
be deficient.3,41,42 It was recently reported that SDF-1
could induce the migration of MKs through endothelial cells43 or directly through a 5-µm pore size
transwell.44,45 Because migration involves a profound
reorganization of the cytoskeleton with polarization of actin
filaments, we tested to see if SDF-1-induced migration of MKs was
altered in WAS. However, in all cases, normal migration through
transwells was observed. Because SDF-1 has been described to induce
actin polymerization in lymphocytes46 and
MKs,44 we also tested the effects of SDF-1 on induction of actin polymerization in WAS. No differences were found between normal and WAS MKs in the induction of actin polymerization by SDF-1
or thrombin. These observations demonstrate that the abnormal F-actin
distribution in WAS MKs does not impair migration and actin
polymerization in vitro. These results also demonstrate that the
machinery necessary for migration is functional in cultured MKs. This
normal in vitro MK migration does not definitively exclude a defect in
in vivo transmigration of WAS MKs through the endothelial barrier.
Indeed, the in vivo process also requires that MKs normally adhere to
the extracellular matrix and to endothelial cells. Hamada et
al43 demonstrated that a neutralizing MoAb against
E-selectin blocked the SDF-1-induced transmigration of MKs by 50%,
suggesting that cellular interaction of MKs with bone marrow
endothelial cells was critical for MK migration. Furthermore, it was
recently reported that, in human platelets, WASP is
tyrosine-phosphorylated after stimulation by collagen,47 a
molecule that may be involved in the interaction between MKs and
endothelial cells. Thus, one cannot exclude that abnormal interaction
between WAS MKs and endothelial cells through adhesion signals other
than SDF-1 may have a direct impact on platelet formation. Further
analyses investigating the interactions between bone marrow endothelial
cells, collagen, and WAS MKs will be necessary to resolve this issue.
Taken together, these results confirm that the abnormalities of
cytoskeletal reorganization are probably very complex in WAS and may
vary among the different hematopoietic lineages, as already observed
for B and T lymphocytes. The fact that all of the steps of maturation
of MKs, including the size of shed platelets, are normal in vitro
suggest that the platelet defect is probably related to a peripheral
mechanism. However, these studies are limited by the number of MKs that
can be isolated from a small volume of blood and/or marrow of patients.
Moreover, all of the tests were performed on MKs cultured in vitro
under stimulation by Mpl-L and SCF. One cannot completely exclude that
in vitro stimulation by these cytokines does not change the behavior of
MKs. The recent description of WASP-deficient mice should make it
possible to obtain enough material to study platelets and MKs in vitro
and in vivo and to determine the precise mechanisms of cytoskeletal reorganization regulated by WASP during adhesion and MK migration.
| |
ACKNOWLEDGMENT |
The authors are grateful to J.-L. Nichol (Amgen, Thousand Oaks, CA) and
C. Caillot (Amgen, Neuilly, France) for providing SCF and PEG-rHuMGDF.
We thank Pr G. Tchernia (Hôpital Kremlin Bicêtre,
Bicêtre, France), Pr J.P. Fermand (Hôpital St Louis, Paris,
France), and Pr B. Varet, Pr S. Blanche (Hôpital Necker-Enfants Malades, Paris, France), Dr M. Michaux (Hôpital Pellegrin,
Bordeaux, France), and Dr Mazingue (Hôpital Claude Huriez, Lille,
France). We thank Klaus Schwarz (Ulm, Germany) for having performed
mutation analysis. We thank J.-M. Massé for photographic
assistance and Pr B. Forget for improving the English of this manuscript.
| |
FOOTNOTES |
Submitted November 19, 1998; accepted March 11, 1999.
Supported by grants from the INSERM, Institut Gustave Roussy, and the
Association pour la Recherche contre le Cancer (to N.D.).
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 Elie Haddad, MD, INSERM U 362, PR1, Institut Gustave Roussy, Villejuif 94805, France.
| |
REFERENCES |
1.
Wiskott A:
Familiärer, angeborener morbus Werlhofii?
Monatschrift Kinderheil
68:212, 1936
2.
Aldrich R, Steinberg A, Campbell D:
Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea.
Pediatrics
13:133, 1954[Abstract/Free Full Text]
3.
Ochs H, Slichter S, Herker L, Von Behrens W, Clark R, Wedgwood R:
The Wiskott-Aldrich syndrome: Studies of lymphocytes, granulocytes and platelets.
Blood
55:243, 1980[Free Full Text]
4.
Canales M, Mauer A:
Sex-linked hereditary thrombocytopenia as a variant of Wiskott-Aldrich syndrome.
N Engl J Med
277:899, 1967
5.
Notarangelo N, Parolini O, Faustini R, Porteri V, Albertini A:
Presentation of Wiskott-Aldrich syndromes as isolated thrombocytopenia.
Blood
77:1125, 1991[Free Full Text]
6.
Derry J, Ochs H, Francke U:
Isolation of a novel gene mutated in Wiskott-Aldrich syndrome.
Cell
78:635, 1994[Medline]
[Order article via Infotrieve]
7.
Derry J, Kerns J, Weinberg K, Ochs H, Volpini V, Estivill X, Walker A, Francke U:
WASP gene mutations in Wiskott-Aldrich syndrome and X-linked thrombocytopenia.
Hum Mol Genet
4:1127, 1995[Abstract/Free Full Text]
8.
Kolluri R, Shehabeldin A, Peacocke M, Lamhonwah A, Teichert-Kuliszewska K, Weissman S, Siminovitch K:
Identification of WASP mutations in patients with Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus.
Hum Mol Genet
4:1119, 1995[Abstract/Free Full Text]
9.
Villa A, Notarangelo L, Macchi P, Mantuano E, Cavagni G, Brugnoni D, Strina D, Patrosso M, Ramenghi U, Sacco M, Ugazio A, Vezzoni P:
X-linked thrombocytopenia and Wiskott-Aldrich syndrome are allelic diseases with mutations in the WASP gene.
Nat Genet
9:414, 1995[Medline]
[Order article via Infotrieve]
10.
Aspenström P, Lindberg U, Hall A:
Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome.
Curr Biol
6:70, 1996[Medline]
[Order article via Infotrieve]
11.
Kolluri R, Tolias K, Carpenter C, Rosen F, Kirchhausen T:
Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.
Proc Natl Acad Sci USA
93:5615, 1996[Abstract/Free Full Text]
12.
Symons M, Derry J, Karlak B, Jiang S, Lemahieu V, McCormick F, Francke U, Abo A:
Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell
84:723, 1996[Medline]
[Order article via Infotrieve]
13.
Miki H, Miura K, Takenawa T:
N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeleton rearrangement in a PIP2-dependent manner downstream of tyrosine kinases.
EMBO J
15:5326, 1996[Medline]
[Order article via Infotrieve]
14.
Rivero-Lezcano O, Macilla A, Sameshima J, Robbins K:
Wiskott-Aldrich syndrome protein physically associates with Nck through Src Homology domains.
Mol Cell Biol
15:5725, 1995[Abstract]
15.
Banin S, Truong O, katz D, Waterfield M, Brickell P, Gout I:
Wiskott-Aldrich syndrome protein (WASp) is a binding partner for the c-Src family protein-tyrosine kinases.
Curr Biol
6:981, 1996[Medline]
[Order article via Infotrieve]
16.
Finan P, Soames C, Wilson L, Nelson D, Stewart D, Truong O, Hsuan J, Kellie S:
Identification of regions of the Wiskott-Aldrich syndrome protein responsible for association with selected Src homology 3 domains.
J Biol Chem
271:26291, 1996[Abstract/Free Full Text]
17.
Lum L, Tubergen D, Corash L, Blaese R:
Splenectomy in the management of the thrombocytopenia of the Wiskott-Aldrich syndrome.
N Engl J Med
302:892, 1980[Abstract]
18.
Corash L, Shafer B, Blaese R:
Platelet-associated immunoglobulin, platelet size, and the effect of splenectomy in the Wiskott-Aldrich syndrome.
Blood
65:1439, 1985[Abstract/Free Full Text]
19.
Gröttum K, Hovig T, Holmsen H, Abrahamsen A, Jeremic M, Seip M:
Wiskott-Aldrich syndrome: Qualitative platelet defects and short platelet survival.
Br J Haematol
17:373, 1969[Medline]
[Order article via Infotrieve]
20.
Ochs H:
The Wiskott-Aldrich syndrome.
Springer Semin Immunopathol
19:435, 1998[Medline]
[Order article via Infotrieve]
21.
Miki H, Nonoyama S, Zhu Q, Aruffo A, Ochs H, Takenawa T:
Tyrosine kinase signaling regulates Wiskott-Aldrich syndrome protein function, which is essential for megakaryocyte differentiation.
Cell Growth Differ
8:195, 1997[Abstract]
22.
Tavassoli M, Aoki M:
Migration of entire megakaryocyte through the marrow-blood barrier.
Br J Haematol
48:25, 1981[Medline]
[Order article via Infotrieve]
23.
Cunningham J, Meyer S, Fox J:
The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.
J Biol Chem
271:11581, 1996[Abstract/Free Full Text]
24.
Cramer E, Norol F, Guichard J, Breton-Gorius J, Vainchenker W, Debili N:
Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl Ligand.
Blood
89:2336, 1997[Abstract/Free Full Text]
25.
Debili N, Wendling F, Katz A, Guichard J, Breton-Gorius J, Hunt P, Vainchenker W:
The Mpl-Ligand, or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86:2516, 1995[Abstract/Free Full Text]
26.
Stewart D, Treiber-Held S, Kurman C, Facchetti F, Notarangelo L, Nelson D:
Studies of the expression of the Wiskott-Aldrich syndrome protein (WASP).
J Clin Invest
97:2627, 1996[Medline]
[Order article via Infotrieve]
27.
Kuramoto A, Steiner M, Baldini M:
Lack of platelet response to stimulation in the Wiskott-Aldrich syndrome.
N Engl J Med
282:475, 1971
28.
Baldini M:
Nature of the platelet defect in the Wiskott-Aldrich syndrome.
Ann N Y Acad Sci
201:437, 1972[Medline]
[Order article via Infotrieve]
29.
Remold-O'Donnell E, Rosen F, Kenney D:
Defects in Wiskott-Aldrich syndrome blood cells.
Blood
87:2621, 1996[Free Full Text]
30.
Thiery JB, Bessis M:
La genèse des plaquettes à partir des mégacaryocytes observés sur la cellule vivante.
C R Acad Sci
242:290, 1956
31.
Radley JM, Scurfield G:
The mechanism of platelet release.
Blood
56:996, 1980[Abstract/Free Full Text]
32.
Gallego M, Santamaria M, Pena J, Molina I:
Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation.
Blood
90:3089, 1997[Abstract/Free Full Text]
33.
Kozma R, Ahmed S, Best A, Lim L:
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
Mol Cell Biol
15:1942, 1995[Abstract]
34.
Nobes C, Hall A:
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:53, 1995[Medline]
[Order article via Infotrieve]
35.
Miki H, Sasaki T, Takai Y, Takenawa T:
Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP.
Nature
391:93, 1998[Medline]
[Order article via Infotrieve]
36.
Schwartz K, Nonoyama S, Peitsch M, de Saint Basile G, Espanol T, Fasth A, Fischer A, Freitag K, Friedrich W, Fugmann S, Hossle H, Jones A, Kinnon C, Meindl A, Notarangelo L:
WASPbase: A database of WAS- and XLT-causing mutations.
Immunol Today
17:496, 1996[Medline]
[Order article via Infotrieve]
37.
Zhu Q, Watanabe C, Liu T, Hollenbaugh D, Blaese R, Kanner S, Aruffo A, Ochs H:
Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP mutations, protein expression, and phenotype.
Blood
90:2680, 1997[Abstract/Free Full Text]
38.
MacCarthy-Morrogh L, Gaspar H, Wang Y, Katz F, Thompson L, Layton M, Jones A, Kinnon C:
Absence of expression of the Wiskott-Aldrich syndrome protein in peripheral blood cells of Wiskott-Aldrich syndrome patients.
Clin Immunol Immunopathol
88:22, 1998[Medline]
[Order article via Infotrieve]
39.
Stenberg P, Barrie R, Pestina T, Steward S, Arnold J, Murti A, Hutson N, Jackson C:
Prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth rat.
Blood
91:1599, 1998[Abstract/Free Full Text]
40.
Snapper S, Rosen F, Mizoguchi E, Cohen P, Khan W, Liu C, Hagemann T, Kwan S, Ferrini R, Davidson L, Bhan A, Alt F:
Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation.
Immunity
9:81, 1998[Medline]
[Order article via Infotrieve]
41.
Zicha D, Allen W, Brickell P, Kinnon C, Dunn G, Jones G, Thrasher A:
Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome.
Br J Haematol
101:659, 1998[Medline]
[Order article via Infotrieve]
42.
Badolato R, Sozzani S, Malacarne F, Bresiani S, Fiorini M, Borsatti A, Albertini A, Mantovani A, Ugazio A, Notarangelo L:
Monocytes from Wiskott-Aldrich patients display reduced chemotaxis and lack of cell polarization in response to monocyte chemoattractant protein-1 and formyl-methionyl-leucyl-phenylalanine.
J Immunol
161:1026, 1998[Abstract/Free Full Text]
43.
Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman R, Moore M, Rafii S:
Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation.
J Exp Med
188:539, 1998[Abstract/Free Full Text]
44.
Rivière C, Subra F, Cohen-Solal K, Cordette-Lagarde V, Letestu R, Auclair C, Vainchenker W, Louache F:
Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.
Blood
93:1511, 1999[Abstract/Free Full Text]
45.
Wang J, Liu Z, Groopman J:
The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion.
Blood
92:756, 1998[Abstract/Free Full Text]
46.
Bleul C, Fuhlbrigge R, Casasnovas J, Aiuti A, Springer T:
A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor (SDF-1).
J Exp Med
184:1101, 1996[Abstract/Free Full Text]
47.
Oda A, Ochs H, Drucker B, Ozaki K, Watanabe C, Handa M, Miyakawa Y, Ikeda Y:
Collagen induces tyrosine phosphorylation of Wiskott-Aldrich syndrome protein in human platelets.
Blood
92:1852, 1998[Abstract/Free Full Text]
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TOP
ABSTRACT
INTRODUCTION