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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4166-4176
Regulation and Function of WASp in Platelets by the Collagen Receptor,
Glycoprotein VI
By
Barbara S. Gross,
Jonathan I. Wilde,
Lynn Quek,
Helen Chapel,
David L. Nelson, and
Steve P. Watson
From the Department of Pharmacology, University of Oxford, Oxford,
UK; the Metabolism Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD; and the Department of Immunology,
John Radcliffe Hospital, Headington, Oxford, UK.
 |
ABSTRACT |
Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder
associated with abnormalities in platelets and lymphocytes giving rise
to thrombocytopenia and immunodeficiency. WAS is caused by a mutation
in the gene encoding the cytoskeletal protein (WASp). Despite its
importance, the role of WASp in platelet function is not established.
WASp was recently shown to undergo tyrosine phosphorylation in
platelets after activation by collagen, suggesting that it may play a
selective role in activation by the adhesion molecule. In the present
study, we show that WASp is heavily tyrosine phosphorylated by a
collagen-related peptide (CRP) that binds to the collagen receptor
glycoprotein (GP) VI, but not to the integrin  2 1.
Tyrosine phosphorylation of WASp was blocked by Src family kinase
inhibitors and reduced by treatment with wortmannin and in patients
with X-linked agammaglobulinemia (XLA), a condition caused by a lack of
functional expression of Btk. This indicates that Src kinases,
phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to
the regulation of tyrosine phosphorylation of WASp. The functional
importance of WASp was investigated in 2 WAS brothers who show no
detectable expression of WASp. Platelet aggregation and secretion from
dense granules induced by CRP and thrombin was slightly enhanced in the
WAS platelets relative to controls. Furthermore, there was no apparent
difference in morphology in WAS platelets after stimulation by these
agonists. These observations suggest that WASp does not play a critical
role in intracellular signaling downstream of tyrosine kinase-linked
and G protein-coupled receptors in platelets.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WISKOTT ALDRICH syndrome (WAS), resulting
from mutations in the WAS protein (WASp), is an X-linked recessive
disorder associated with severe thrombocytopenia, eczema,
immunodeficiency (recurrent infection), and an increased susceptibility
to lymphoid malignancies. The clinical and immunological course of the
disease varies from one patient to another. In its most mild form, it is known as X-linked thrombocytopenia and patients have minimal impairment of their immune response.1
WASp is composed of 502 amino acids and has a molecular weight of 64 kD.2 Its expression is limited to hematopoietic cells, where it is found in all lineages and at all stages of
development.3 WASp can be divided into a number of protein
domains. It has a pleckstrin homology (PH domain) at its N-terminus; a
WASp homology (WH) domain, WH1; a G protein binding domain (GBD; also
known as CDC42/Rac interactive binding region [CRIB]); a proline-rich domain; a second WH domain, WH2; a verprolin-like sequence; a cofilin
homology sequence; and an acidic region. WASp lacks enzymatic activity,
and its major role is thought to be as a scaffold or adapter protein.
WASp has been shown to interact with a number of important signaling
proteins, although the significance of these interactions is not clear.
WASp interacts with the SH3 domain of a number of proteins in vitro,
including Btk, Cbl, Fgr, Lyn, and phospholipase C 1
(PLC1).4-7 However, a reduced number of interactions
have been shown to occur in vivo, including binding to the adapters Nck8,9 and Grb210 and to the tyrosine kinases
Fyn5 and Btk.7
Mounting evidence suggests that WASp is involved in the regulation of
the cytoskeleton downstream of members of the Rho family of small
molecular weight G proteins. WASp has been shown to interact directly
with Cdc42, a member of the Rho family of G proteins, via its GBD
domains.11 The overexpression of WASp leads to formation of
extended clusters of WASp-rich particles that are highly enriched in
polymerized actin.11 WASp has also been shown to interact in vivo with the cytoskeletal associated proteins PSTPTP,12 WIP,13,14 and the Arp2/3 complex.15 WIP has
been shown to induce actin polymerization in lymphoid cells through
association with the actin binding protein profilin. The Arp2/3
complex, which comprises 7 proteins, is thought to be a major regulator
of actin polymerization.16 Other members of the WASp family
of proteins, namely N-WASp, WAVE, and the recently discovered Scar
proteins, have also been shown to regulate the
cytoskeleton.17-19
More than 100 mutations in WASp have been reported. These are found
throughout the length of the molecule, although the majority occur in
the N-terminal portion covering the PH and WH1 domains; however, there
is no specific grouping of mutations to suggest the loss of a
particular function of the protein. Instead, a number of reports have
shown a correlation between the presence or absence of WASp and the
clinical phenotype.20-22 For example, Zhu et
al20 determined WASp gene mutations in 48 unrelated WAS
families and showed that mutations that permitted WASp expression,
albeit at a reduced level, caused mild disease, whereas mutations that
resulted in classic WAS were associated with a lack of protein. A more recent study has shown that 8 different mutations resulted in lack of
expression of WASp in peripheral mononuclear cells and in
B-lymphoblastoid cell lines, at least to a level of less than 0.5% of
that found in normal donors, and were associated with classical
WAS.22 The lack of protein expression may be the major cause of classic WAS, rather than the expression of a mutated form of WASp.
The absence of WASp results in the alteration of responses in a number
of cell types. WASp appears to play an important role in signaling
downstream of the T-cell antigen receptor. There is decreased
proliferation of WAS T cells in response to antigen challenge,23 and a similar result has been reported in T
cells from WASp-deficient mice.24 The role of WASp in B
cells is unclear, because both normal and defective responses have been
reported. For example, in WASp-knockout mice, B-cell function is
normal.24 It has been suggested that the lack of WASp may
result in disturbances in cell motility of neutrophils and macrophages,
which may contribute to the immunopathology of WAS.25
WAS platelets are characterized by a reduction in cell number
(thrombocytopenia) and size. Bleeding disorders such as intestinal intraluminal bleeding are often described in WAS
patients.9,26 After splenectomy, platelet number and size
return towards normal levels.27 The number of reticulated
(young) platelets is also relatively normal in WAS.28 In a
recent report, it has been shown that, despite some cytoskeletal
defects in WAS megakaryocytes, their ability to produce platelets is
not affected.29 Together, these results suggest that the
major defect in WAS is increased platelet removal by the spleen rather
than impaired production. However, a reduction in density of surface
proteins such as GPIIb-IIIa and GPIV is also seen in WAS platelets,
suggesting that there may be a defect in development.28
A number of early studies reported defective platelet responses,
notably aggregation, in WAS platelets to a number of agonists, including collagen, ADP, adrenaline, and thrombin.30-34
However, other studies reported normal platelet aggregation in WAS
patients as estimated from changes in optical transmission after the
addition of ADP, adrenaline, and collagen.35 These studies
were performed in the absence of a detailed understanding of the
mechanism of signaling by these agonists and before the genetic basis
of WAS was known. This may explain their inconclusive and contradictory nature. It is therefore necessary to reassess the response of WAS-deficient platelets in light of this increased knowledge. For
example, a recent study was unable to confirm the defect in aggregation
to ADP in WAS platelets, although that to thrombin was
reduced.28
Tyrosine phosphorylation of WASp was recently reported in platelets in
response to collagen,36 in mast cells stimulated by
Fc RI,37 and B cells stimulated through the B-cell
antigen receptor.7 These 3 sets of stimuli signal through a
similar pathway that involves tyrosine phosphorylation of an
immunoreceptor tyrosine-based activation motif (ITAM), the tyrosine
kinase Syk, and PLC2. The functional consequence of tyrosine
phosphorylation of WASp is not known. In the present study, we have
investigated the mechanism of tyrosine phosphorylation of WASp in
platelets stimulated by the collagen receptor, glycoprotein (GP)
VI. In addition to collagen, we have used a
collagen-related peptide (CRP) that activates GPVI but is unable to
bind the collagen adhesion receptor, the integrin
 21.38,39 The role of WASp in platelets has been
investigated through the study of platelets from 2 WAS brothers with
the same genetic defect that results in a lack of detectable expression
of the protein.
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MATERIALS AND METHODS |
Reagents.
A CRP [GCP*(GPP)10GCP*G; single amino acid code P* = hydroxyproline; the monomer is cross-linked through the N- and
C-terminals] was cross-linked via cysteine residues as described
previously38; CRP was kindly donated by Drs M. Barnes, R.W.
Farndale, and G. Knight (Department of Biochemistry, Cambridge
University, Cambridge, UK). Collagen (native collagen fibrils from
equine tendons) was from Nycomed (Munich, Germany). FcRII specific
monoclonal antibody (MoAb) was purchased from Medarex Inc (Annandale,
NJ). Sheep F(ab')2 raised against mouse IgG (M-1522)
and thrombin were purchased from Sigma (Poole, Dorset, UK). Monoclonal
antiphosphotyrosine antibody 4G10 and p85 anticortactin polyclonal
antibody was purchased from Upstate Biotechnology (TCS Biologicals Ltd,
Botolph Claydon, Bucks, UK). GST-Grb2 and GST-PLC-2-SH3 fusion
proteins were expressed in bacteria as previously
described.40,41 GST-Btk-SH3 fusion protein construct was a
kind gift from Dr C. Kinnon (Institute of Child Health, University
College London, London, UK). WASp monoclonal and polyclonal antibodies
were raised as described.42 Annexin V-fluorescein
isothiocyanate (FITC) was purchased from Pharmingen (Becton Dickinson,
Oxford, UK). PP1
(4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine)43 and PD173956 were kinds gifts from Dr J. Hanke (Pfizer Central Research, Groton, CT) and Dr A.J.
Kraker (Parke-Davis, Ann Arbor, MI), respectively.
FITC-annexin V was from Becton Dickinson (Oxford, UK).
[3H]5-hydroxytryptamine (5-HT) was from New England
Nuclear (Herts, UK). Other reagents were from previously described
sources or of analar grade.
Platelet preparation and stimulation.
Human platelets were isolated from blood taken on the day of experiment
using citrate as anticoagulant. Platelet-rich plasma was collected
after centrifugation at 200g for 20 minutes, pooled, and, after
the addition of prostacyclin (100 nmol/L), recentrifuged at
1,000g for 10 minutes. The platelet-poor plasma was discarded and the platelet pellet was resuspended in 20 mL of Tyrodes-HEPES buffer (134 mmol/L NaCl, 0.34 mmol/L Na2HPO4,
2.9 mmol/L NaHCO3, 20 mmol/L HEPES, 5 mmol/L glucose, and 1 mmol/L MgCl2, pH 7.3) containing 1 mmol/L EGTA and 10 µmol/L indomethacin, as described in the text. Prostacyclin was added
(100 nmol/L) and the platelet suspension was centrifuged for a further
10 minutes. The supernatant was discarded and platelets were
resuspended at a concentration of 4 × 108 cells/mL,
unless stated (see studies on WAS platelets). Experiments were
performed at 37°C in an aggregometer (Chrono-Log Corp, Havertown, PA) with continuous stirring at 1,200 rpm. Stimulation of platelets with CRP, collagen, and thrombin was performed at 37°C for the times shown. Platelets were stimulated via FcRIIA using MoAb IV.3 (1 µg/mL) for 1 minute and then the cross-linker
F(ab')2 antimouse IgG (30 µg/mL) for 90 seconds.
For studies involving measurement of 5-HT secretion, platelets were
labeled in platelet-rich plasma with [3H]5-HT (1 µCi/mL) for 60 minutes. The secretion of [3H]5-HT was
measured as previously described.44
Flow cytometry analysis of annexin V binding.
Platelets (2.5 × 106 cells) were stimulated by CRP or
thrombin for 3 minutes at room temperature in a volume of 100 µL in
Tyrodes-HEPES buffer. Ca2+ (1 mmol/L) was present at every
stage. Platelets were incubated with annexin V-FITC for 10 minutes. The
final volume was adjusted to 500 µL by the addition of Tyrodes-HEPES
buffer and analyzed by flow cytometry using a Becton Dickinson FACScan
flow cytometer. Excitation was at 488 nm, with emission measured at 530 nm. Ten thousand events were analyzed per sample. Platelets were gated and results were presented as a percentage of cells positive for annexin V.
Patients.
Blood was taken from 2 brothers with WAS or patients with X-linked
agammaglobulinemia (XLA) on the day of the experiment. All patients
denied having taken aspirin in the preceding 2 weeks. The work was
performed with parental consent and approval of the Central Oxford
Research Ethics Committee.
The study on the WAS brothers was completed over a period of 2 years
and by the end of this time the boys were 12 and 14 years of age. The
oldest was born by Caesarian section (breech presentation) and was
found to have bruising, petichiae, and jaundice at birth. He had
persistent thrombocytopenia with bruising, but no major bleeding. His
infection was a severe episode of bacterial tonsillitis at the age of
18 months for which he was admitted to hospital. He had recurrent
otitis media requiring insertion of grommets at 4 years of age. The
diagnosis of WAS was made when he was 5 years of age. He began
receiving prophylactic cotrimoxazole 1 year later and intravenous
immunoglobulin at 7 years of age; he has remained free of bacterial
infections since this time. Splenectomy was performed in 1993 for
recurrent bruising and epistaxes. His platelet count increased from 20 × 109/L to a resting level of 120 × 109/L. He has had only small infrequent patches of eczema.
In view of his family history, his brother had his platelet count
measured at birth; this was low, and an initial diagnosis of autosomal recessive congenital thrombocytopenia was made. He had recurrent bruising and minor epistaxes throughout childhood. He developed otitis
media and chest infections at 10 months of age. WAS was diagnosed at
the same time as for his elder brother. Infections were prevented by
prophylactic cotrimoxazole and intravenous Ig as described above.
Unlike his older brother, he was referred at 6 years of age for
investigation of developmental delay with autistic features and,
although no definite diagnosis was made, fragile X syndrome has been
ruled out. He also had a splenectomy with similar effect in 1993, after
which he had an episode of fever, cervical lymphadenopathy, and a
presumptive diagnosis of Epstein-Barr virus infection from which he
made a quick recovery. His platelet count is similar to that of his
older brother. He remains well and has had only occasional small
patches of eczema. The level of expression of WASp in the brothers was
measured by Western blotting in peripheral mononuclear
cells and in B-lympho- blastoid cell lines. There was no detectable
expression. The limit of this assay is 0.5% of that in
normal donors. Because of the small vol- ume of blood that could be
taken from WAS donors, experiments were performed on a platelet
concentration of between 0.7 and 2.0 × 108/mL.
Platelets were taken from 3 different donors with XLA. Although the
mutations that give rise to the XLA syndrome have not been identified
in these patients, none was found to express Btk in their platelets as
measured by Western blotting. We have previously reported that the
platelets from all 3 of these donors show impaired activation by
collagen and CRP.45
Scanning electron microscopy.
Scanning electron microscopy was performed as described.46
Basal or stimulated platelets (300 µL), at a concentration of 5 × 107 platelets/mL, were mixed with an equal volume
of 4% glutaraldehyde in 0.15 mol/L NaCl, 50 mmol/L phosphate buffer,
pH 7.4 (prewarmed to 37°C). The platelets were collected with
gentle suction onto 0.6-µm pore size polycarbonate filters (Whatman,
Maidstone, UK) that had been prerinsed with 2%
glutaraldehyde in 0.15 mol/L NaCl buffered to pH 7.4 in 50 mmol/L
phosphate buffer. Filters were transferred to vials and rinsed once
with 0.15 mol/L NaCl and twice with distilled water for removal of
glutaraldehyde. Dehydration of filters was accomplished by washing with
10%, 25%, 50%, 75%, 95%, and 100% ethanol. The filters were
subjected to critical point drying (on a Polaron CPD7501 critical point
drier; Agar Scientific Ltd, Stansted, UK), coated with
gold (using a Nanotech Semprep 2 sputter coater; Emitech
Ltd, Ashford, UK), and analyzed on a Philips 515 scanner (FEI UK Ltd,
Cambridge, UK).
Immunoprecipitation, GST precipitation, and immunoblotting.
Platelets were lysed with an equal volume of lysis buffer (2% NP-40,
300 mmol/L NaCl, 20 mmol/L Tris, 10 mmol/L EDTA, 2 mmol/L Na3VO4, 1 mmol/L phenylmethylsulphonyl
fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 1 µg/mL
pepstatin A, pH 7.3). Insoluble cell debris was removed by
centrifugation. Cell lysates were precleared with glutathione-agarose
or protein A-sepharose for GST precipitation and immunoprecipitation,
respectively. For GST precipitation, lysates were incubated with 5 to
10 µg of fusion protein immobilized on agarose. Endogenous WASp was
immunoprecipitated using 5 µL of anti-WASp polyclonal antibody.
Resulting protein complexes and immunoprecipitates were resolved by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to polyvinylidene difluoride membranes. Immunoblotting
was performed using an MoAb to WASp,42 with detection by
enhanced chemiluminescence (ECL; Amersham, Bucks, UK).
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RESULTS |
WASp is tyrosine phosphorylated in CRP-stimulated platelets.
Although collagen has recently been reported to stimulate tyrosine
phosphorylation of WASp in platelets, the surface receptor mediating
this effect is not known. We have addressed this in the present study
using the GPVI-selective ligand CRP that does not bind to the collagen
adhesion receptor 21.38,39
WASp is weakly tyrosine phosphorylated under basal conditions and
undergoes a marked increase in phosphorylation after stimulation by CRP (3 µg/mL; Fig 1A). In comparison,
collagen (10 µg/mL) and thrombin (1 U/mL) induce a much lower
increase in tyrosine phosphorylation. Cross-linking of FcRIIA
induces an increase in tyrosine phosphorylation similar to that induced
by collagen (not shown). Oda et al36 reported that collagen
induced a greater increase in tyrosine phosphorylation of WASp relative
to thrombin, with the latter inducing only marginal phosphorylation. We
observed a reproducible increase in phosphorylation of WASp in response
to thrombin in all studies, although concentrations of collagen similar
to those used by Oda et al36 consistently gave a slightly
larger response than that to the protease. The response to collagen was
always smaller than that to CRP, which is a more powerful stimulus of protein tyrosine phosphorylation in platelets, eg, Asselin et al.39
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| Fig 1.
WASp is phosphorylated on tyrosine in platelets; effect
of inhibitors. (A) Tyrosine phosphorylation of WASp in stimulated
platelets. Western blots of WASp immunoprecipitates (10% SDS-PAGE)
were probed with antiphosphotyrosine (4G10; upper panel) and, after
stripping of the blot, anti-WASp antibodies (lower panel). WASp was
tyrosine phosphorylated under basal conditions (lane 1) and underwent
an increase in phosphorylation after stimulation by collagen (10 µg/mL) for 90 seconds (lane 2); CRP (3 µg/mL) for 90 seconds (lane
3); and thrombin (1 U/mL) for 30 seconds (lane 4). (B) The effect of
inhibition of PLC activity on tyrosine phosphorylation of WASp. Western
blots of WASp immunoprecipitates were probed with antiphosphotyrosine
(4G10; upper panel) and, after stripping of the blot, anti-WASp (middle
panel) and anticortactin (lower panel) antibodies. Basal conditions are
shown in lane 1 and platelets stimulated with CRP (3 µg/mL, 90 seconds) are shown in lane 2. No significant effect on tyrosine
phosphorylation induced by CRP occurred when platelets were
preincubated with 5 µmol/L Ro31-8220 for 5 minutes and 40 µmol/L
BAPTA-AM for 5 minutes (lane 3). (C) The effect of PI 3-kinase
inhibitors on tyrosine phosphorylation of WASp. (D) The effect of
tyrosine kinase inhibitors on phosphorylation of WASp. In (C) and (D),
conditions are as in (A), with the exception that the probe for
cortactin is not shown. One experiment is shown that is representative
of 3.
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A tyrosine phosphorylated band of 70 kD that was resolved as a doublet
in some experiments coimmunoprecipitated with WASp in CRP-stimulated
platelets, but was absent in collagen-stimulated samples, possibly
because it is a weaker stimulus. This band was shown to contain
cortactin by reprobing with a specific antibody (Fig 1B).
Immunoprecipitation of cortactin from platelets confirmed that CRP
stimulates an increase in tyrosine phosphorylation of cortactin (not
shown). A very low level of association of cortactin with WASp was
detected under basal conditions that increased upon stimulation by CRP,
suggesting that the interaction is dependent on tyrosine
phosphorylation of at least 1 of the 2 proteins. This interaction may
be similar to that observed between WASp and PSTPIP, because cortactin
and PSTPIP show homology in the location and sequence of their SH3 and
proline-rich regions.
CRP was used in further biochemical studies in preference to collagen,
because it gives a stronger, more reproducible increase in tyrosine
phosphorylation of WASp and is also selective to GPVI. CRP induced
rapid tyrosine phosphorylation of WASp and this was maintained for up
to 600 seconds (not shown). Tyrosine phosphorylation of WASp was
detected within 30 seconds, making it one of the earliest proteins to
show an increase in phosphorylation upon stimulation by CRP, suggesting
that it may play an early role in CRP-induced signaling. A similar time
course of tyrosine phosphorylation of WASp by CRP was observed by
monitoring its association to GST-PLC2-SH3 (see later).
All of the phosphorylation studies were performed in the presence of
indomethacin to prevent formation of thromboxanes. The effect of
additional inhibitors on tyrosine phosphorylation of WASp by CRP was
examined to further investigate the basis of its regulation. Tyrosine
phosphorylation of WASp by CRP was not altered significantly in the
presence of both Ro 31-8220 and BAPTA-AM, which together prevent the
action of the second messengers, 1,2-diacylglycerol/protein kinase C
and inositol 1,4,5-trisphosphate/Ca2+, demonstrating that
it is independent of activation of phospholipase C (Fig 1B). The ADP
scavenger, apyrase, and RGDS, which inhibits activation of the
fibrinogen receptor, GPIIb-IIIa, did not alter tyrosine phosphorylation
of WASp. Maximally effective concentrations of the 2 structurally
distinct inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase),
wortmannin and LY294002, gave a partial but incomplete reduction in
tyrosine phosphorylation of WASp (Fig 1C). Submaximal concentrations or
shorter incubations with wortmannin and LY294002 gave a lower level of
inhibition (Fig 1C). In contrast, tyrosine phosphorylation of WASp was
completely inhibited under stimulated conditions in the presence of the
2 structurally distinct Src family kinase inhibitors PP1 (10 µmol/L)
and PD173956 (10 µmol/L; Fig 1D). It is not clear from this study
whether Src kinases play a direct role in phosphorylation of WASp,
because they also have a critical early role in signaling by GPVI
upstream of phosphorylation of Fc receptor -chain.47,48
Oda et al36 reported complete inhibition of WASp
phosphorylation by collagen in the presence of wortmannin, which is in contrast to the observation given above that tyrosine phosphorylation of WASp by CRP is only partially reduced in the presence of wortmannin and LY294002. However, we were also unable to confirm the observation of Oda et al36 that tyrosine phosphorylation of WASp
induced by collagen is completely inhibited in the presence of
wortmannin (n = 4; not shown).
WASp associates with PLC2, Btk, and Grb2.
WASp has been shown to bind to the SH3 domains of a number of proteins
through its proline-rich region in vitro. In the present study, we show
that this can be extended to the SH3 domain of PLC-2, the major
isoform of PLC- in platelets. GST-PLC2-SH3, the fusion protein
for the SH3 domain of PLC-2, associates with 2 major tyrosine
phosphorylated bands of 125 and 64 kD and 2 minor bands of 70 and 130 kD in CRP-stimulated platelets (Fig 2A).
The tyrosine phosphorylated bands are detected 30 seconds after
stimulation with CRP and phosphorylation is maintained for 30 minutes
(Fig 2A). The 64-kD protein was shown to contain WASp by
immunoblotting. Because a similar level of WASp associates with
GST-PLC2-SH3 in basal and CRP-stimulated platelets, the interaction
is not regulated by tyrosine phosphorylation of WASp.
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| Fig 2.
Association of WASp with Btk, PLC2, and Grb2. Lysate
from resting or CRP (3 µg/mL)-stimulated platelets were incubated
with GST linked to the SH3 domains of Btk (10 µg) and PLC2 (5 µg) and full-length Grb2 (10 µg). Proteins were separated on 10%
SDS-PAGE and electroblotted to PVDF membranes. Membranes were
immunoblotted using the antiphosphotyrosine MoAb 4G10 (upper panel).
Membranes were stripped and reprobed with anti-WASp MoAb (lower panel).
(A) Time course of WASp association to GST-PLC2-SH3. Several
tyrosine phosphorylated proteins bind to GST-PLC2-SH3 but not to GST
(90-second time point shown) from CRP-stimulated platelets. The 64-kD
band was shown to contain WASp by stripping the blot and reprobing. (B)
WASp association to GST-Btk-SH3. (C) WASp association to GST-Grb2. The
gels are representative of 3 to 5 experiments.
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WASp has been reported to bind to the SH3 domains of Btk in
vitro6 and to be a substrate for the tyrosine kinase when
overexpressed in baby hamster kidney (BHK-21) cells.37 In
the present study, we have investigated whether tyrosine
phosphorylation of WASp interferes with its ability to interact with
the Btk SH3 domain. GST-Btk-SH3 precipitated a tyrosine phosphorylated
band of 64 kD in resting platelets that, along with a second band of 75 kD, underwent a marked increase in tyrosine phosphorylation upon
stimulation by CRP (Fig 2B). WASp was identified as a component of the
64-kD phosphotyrosyl band by immunoblotting and was present at a
similar level in resting and stimulated platelets. The interaction with GST-Btk-SH3 is therefore not altered by tyrosine phosphorylation of WASp.
Oda et al36 recently observed that binding of WASp to
GST-Grb2 is reduced in platelets stimulated by collagen. This was also
observed in the present study in CRP-stimulated platelets (Fig 2C). The
reduction in binding suggests that Grb2 has a decreased affinity for
tyrosine phosphorylated WASp. This may reflect an important
physiological role of tyrosine phosphorylation of WASp in the platelet.
A number of other tyrosine phosphorylated proteins associate with
GST-Grb2 in CRP-stimulated platelets, including bands of 120, 75, 53, 36, and 30 kD (Fig 2C).
Regulation of tyrosine phosphorylation of WASp.
WASp has recently been shown to be a potential substrate for the
tyrosine kinases Btk and Lyn but not Syk. Coexpression of Btk or Lyn in
BHK-21 cells leads to an increase in tyrosine phosphorylation of WASP
and both tyrosine kinases coimmunoprecipitated with the cytoskeletal
protein, with the interaction with Btk being dependent on its SH3
domain.37
To address whether WASp is a substrate for Btk in platelets, studies
were performed on cells from patients with the immunodeficiency syndrome, XLA, caused by mutation of the gene encoding Btk. This approach differs from that of Guinamard et al37 in that the role of an endogenous kinase is investigated rather than an enzyme that
has been introduced through transfection and may therefore be present
at a nonphysiological level.
In XLA platelets, tyrosine phosphorylation of WASp was slightly
elevated in nonstimulated cells relative to controls. CRP stimulated a
small increase in tyrosine phosphorylation of WASp in XLA platelets,
although this was considerably weaker than in controls
(Fig 3A). This suggests that Btk, and
probably a second kinase, lies upstream of WASp phosphorylation in
CRP-stimulated platelets, but that phosphorylation under basal
conditions is independent of Btk. Candidates for the second kinase
include members of the Src family of tyrosine kinases, of which several
are expressed in platelets, including Lyn and Fyn, and Tec, another
member of the Btk family expressed in platelets.
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| Fig 3.
Tyrosine phosphorylation of WASp in XLA platelets
stimulated with CRP. (A) WASp was immunoprecipitated from resting or
CRP (3 µg/mL)-stimulated platelets. Proteins were resolved on 10%
SDS-PAGE, transferred, and immunoblotted using the antiphosphotyrosine
MoAb 4G10 (upper panel). Reprobing with the anti-WASp MoAb confirmed
that a similar level of WASp was present in control and XLA platelets
(bottom panel). One experiment is shown that is representative of 3. (B) WASp was immunoprecipitated from resting platelets from control
(WT) and WAS patients. Proteins were resolved on 10% SDS-PAGE,
transferred, and immunoblotted using the WASp MoAb.
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Studies in WAS platelets.
To investigate the role of WASp in platelets after cross-linking of
GPVI, a series of studies were performed on 2 brothers who have been
shown to have a missense mutation in which valine 75 is substituted by
a methionine, resulting in the absence of detectable expression (to a
level less than 0.5% of controls) of WASp in B cells.22
Neither brother had a detectable level of WASp as measured by Western
blotting using a specific antibody (Fig 3B). There was no apparent
difference in response between the platelets from the 2 brothers.
Although a number of studies have been published describing impairment
in aggregation in WAS platelets to a number of platelet agonists,
including collagen, there is a need for confirmation of these findings,
because the majority of these studies were performed before the cloning
of WASp and the establishment of the mechanism of platelet activation
by the majority of cell surface receptors (see introduction). Platelets
were challenged with collagen, CRP, thrombin, and FcRIIA
cross-linking and responses were compared with those of volunteer
donors. All platelets were prepared in the same way and suspended at
the same concentration. Five separate studies were performed on the 2 sets of WASp-deficient platelets in comparison to platelets from 8 normal donors and also from the boys' mother. The pattern of shape
change and aggregation was similar in all cases, although the rate of
response to all agonists was slightly increased in the WAS donors for
all 4 agonists (Fig 4). Potentiation of
aggregation was seen over the length of the concentration response
curves for CRP (Fig 4E) and thrombin (not shown).
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| Fig 4.
Aggregation in WAS platelets. Platelets from control and
WAS patients were prepared as described in Materials and Methods. Care
was taken to ensure that platelets were prepared in exactly the same
way and used at the same concentration. The experiments in (A) through
(C) were performed on the same day in the absence of indomethacin at a
platelet concentration of 2 × 108/mL. Collagen is unable
to stimulate aggregation in the presence of indomethacin; similar
results were observed for thrombin in the presence of indomethacin. The
experiments in (D) and (E) were performed on a separate donor in the
presence of indomethacin and at a lower platelet concentration of 0.7 × 108/mL. The mean aggregation over the length of the
concentration response curve to collagen was measured. The results are
representative of between 2 and 5 separate experiments.
|
|
Platelet activation is associated with the movement of
phosphatidylserine from the inner leaflet of the plasma membrane to the
outer layer, forming a catalytic surface to support coagulation reactions (procoagulant activity). Annexin V has high affinity and a
strict specificity for aminophospholipids at physiological Ca2+ concentrations, enabling it to be used in flow
cytometry for measurement of procoagulant activity when conjugated to
FITC. The magnitude of response and concentration response
relationships for thrombin and CRP were similar in the control and
WAS-platelets (Fig 5A). Similar studies
could not be performed with collagen because of the interference of
adhesion with flow cytometry.
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| Fig 5.
Aminophospholipid exposure and 5-HT secretion in WAS
platelets. Platelets from control and WAS patients were prepared as
described in Materials and Methods. Care was taken to ensure that
platelets were prepared in exactly the same way and used at the same
concentration. Studies were performed on the 2 WAS platelets in
comparison with platelets from 2 controls. Data have been pooled and
are shown as the mean ± range. (A) Procoagulant activity. Platelets
were stimulated with CRP or thrombin for 3 minutes and annexin V
binding was measured by flow cytometry as described in Materials and
Methods. The number of positive cells is shown in the y-axis; (B) 5-HT
secretion. Platelets were stimulated with CRP (90 seconds) or thrombin
(30 seconds) and 5-HT secretion was measured as described in Materials
and Methods. The results are representative of 3 experiments.
|
|
WASp-deficient platelets were analyzed for dense granule secretion by
prelabeling with [3H]5-HT. The concentration response
curve to CRP was similar in control and WAS platelets, although
secretion was slightly enhanced in the latter group (Fig 5B). A similar
result was seen for the G protein receptor agonist thrombin (Fig 5B and
data not shown). WAS platelets were also measured for -granule
secretion by measurement of P-selectin expression by flow cytometry
after stimulation with CRP and thrombin. The maximal response to CRP
and thrombin was reduced by approximately 40% in the WASp-deficient
platelets (not shown), although this may be a consequence of the
smaller size of the WAS platelets rather than of a change in reactivity.
The pattern of increase in protein tyrosine phosphorylation in whole
cell lysates was similar in control and WAS platelets challenged with
collagen, FcRIIA, and thrombin (Fig 6).
A similar result was seen in platelets stimulated with CRP (not shown). This approach only monitors gross changes in protein tyrosine phosphorylation and could have missed changes in phosphorylation of
specific proteins. Although it is beyond the scope of this study to
immunoprecipitate all of the proteins that undergo increases in
tyrosine phosphorylation upon platelet activation, it is noteworthy that there was no apparent alteration in tyrosine phosphorylation of
PLC2 in CRP-stimulated platelets (not shown). This result is in
contradiction to the observation of Simon et al49 of
reduced tyrosine phosphorylation of PLC1 in transformed B
lymphocytes from patients with WAS.
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| Fig 6.
Tyrosine phosphorylation of total cell protein is not
altered in WAS platelets. WASp-deficient platelets and control
platelets were incubated in Tyrodes-HEPES buffer and stimulated by the
addition of collagen (3 µg/mL) for 90 seconds and thrombin (1 U/mL)
for 60 seconds and through cross-linking of FcRIIA for 90 seconds
with 1 µg/mL MoAb IV.3 for 60 seconds, followed by the addition of
F(ab')2 (30 µg/mL). Stimulation was stopped by the
addition of equal volume of 2× loading buffer. Proteins were
separated by 10% SDS-PAGE, electroblotted onto PVDF membranes, and
then immunoblotted using the antiphosphotyrosine MoAb 4G10. The same
concentration of platelets (1 × 108/mL) was used in
samples from the WAS patients and controls. The gel is representative
of 2 experiments.
|
|
A possible role of WASp in cytoskeletal remodeling was investigated
using scanning electron microscopy in platelets that had been
stimulated by collagen, CRP, and thrombin. No major morphological differences were observed between normal and WAS platelets, with cells
showing characteristic rounding and formation of pseudopodia (Fig 7).
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| Fig 7.
Formation of filopodia in WASp-deficient platelets. The
presence of filopodia on control and platelets stimulated with CRP (5 µg/mL), thrombin (1 U/mL), and collagen (30 µg/mL) from WAS and
control donors was assessed by scanning electron microscopy (SEM).
Original magnification × 5,200. Results are representative of 6 independently analyzed fields.
|
|
| |
DISCUSSION |
Collagen activates platelets through a tyrosine kinase-linked pathway
that shares many features with signaling by immune receptors. This has
lead to the realization that the adhesion molecule is a unique platelet
agonist in that it induces activation of platelets through a mechanism
distinct from that used by other agonists at sites of damage to the
vasculature. In consideration of this, we investigated the ability of
collagen and a CRP that is selective to GPVI to stimulate tyrosine
phosphorylation of a number of proteins in platelets. We show in this
study that WASp undergoes tyrosine phosphorylation after stimulation by
CRP to a much greater extent than induced by the G protein-coupled
receptor agonist thrombin. Oda et al36 have also recently
published that WASp is selectively phosphorylated by collagen in
platelets. This observation suggested the possibility that WASp may
play a unique role in platelet activation by collagen. The present
study was therefore undertaken to investigate the role and regulation
of WASp in platelets stimulated by ligation of the collagen receptor
GPVI in comparison with results obtained in platelets stimulated by thrombin.
We have characterized the mechanism of regulation of tyrosine
phosphorylation of WASp in human platelets stimulated by CRP using a
variety of pharmacological inhibitors and by studies on platelets from
patients deficient in the tyrosine kinase Btk. We were unable to extend
this work to murine platelets from knockout animals to take advantage
of these genetically modified cells, because WASp undergoes little or
no increase in tyrosine phosphorylation in platelets of this species
after stimulation by CRP (not shown). GPVI receptor signaling is
believed to be mediated by activation of a Src family kinase, probably
Fyn or Lyn, which leads to phosphorylation of the Fc
receptor -chain.47,48 The observation that the Src family kinase inhibitors PP1 and PD173956 blocked the tyrosine phosphorylation of WASp is consistent with its regulation downstream of
GPVI. We observed partial inhibition of tyrosine phosphorylation of
WASp in the presence of the PI 3-kinase inhibitors Ly294002 and
wortmannin. This is in contrast to the results obtained by Oda et
al,36 who observed complete inhibition; the explanation for
this is not known. A marked reduction in tyrosine phosphorylation of
WASp in XLA platelets was also observed, demonstrating that this event
is mediated downstream of Btk and suggesting that WASp is a substrate
for the Tec family kinase. Guinamard et al37 and Baba et
al7 have also recently shown that WASp is a substrate for
Btk downstream of FcRI and B-cell antigen receptors, respectively, both of which signal via an ITAM. Btk has a PH domain that has high
selectivity for the product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate. The inhibition of PI 3-kinase activity has been
shown to cause a partial inhibition of Btk activation (Quek and Watson,
unpublished observation), suggesting that WASp may be
regulated downstream of PI 3-kinase through Btk.
The functional role of WASp in GPVI receptor signaling and platelet
function was investigated by studies in 2 WAS brothers who share the
same genetic defect. In contrast to earlier results, the present
studies did not show impairments in aggregation to a number of
agonists, including collagen, CRP, and thrombin. The only differences
seen between WASp platelets and those of controls was a slightly
increased rate of aggregation and dense granule secretion in response
to all agonists. The increase in these responses in the WAS platelets
may be analogous to the effect of low concentrations of the actin
polymerization inhibitor, cytochalasin. Previous studies have suggested
that incubation of platelets with a low dose of cytochalasin before the
addition of phorbol ester resulted in the increased secretion of
5-HT.50 Furthermore CRP-, collagen-, and
thrombin-induced aggregation was enhanced by a low concentration of
cytochalasin (unpublished observation). The pattern of
protein tyrosine phosphorylation was similar in WASp-deficient donors in response to GPVI activation, including tyrosine phosphorylation of
PLC2.
There was also no major difference in overall morphology of the WASp
platelets upon stimulation by collagen or thrombin, although a more
detailed analysis of the cytoskeletal remodeling may show subtle
alterations. The absence of major cytoskeletal defects in WAS platelets
may be due to functional redundancy between WASp family proteins. In
particular, N-WASp has been shown to be expressed in a number of
tissues17 and is capable of inducing filopodia production18 and, like WASp, actin polymerization via an
interaction with the Arp2/3 complex.51
In conclusion, this study has shown that, in platelets, WASp is
tyrosine phosphorylated downstream of the collagen receptor GPVI, but
not the G protein-coupled thrombin receptor via a pathway that involves
PI 3-kinase and the tyrosine kinase Btk. However, this study has failed
to find evidence for a specific role of WASp in aggregation, dense
granule secretion, and shape change, suggesting that tyrosine
phosphorylation does not appear to confer a unique role on WASp in GPVI
receptor signaling in these responses.
| |
ACKNOWLEDGMENT |
The authors are grateful to Nicky Brennan and Nicola Salome-Bentley,
specialist immunology nurses, for their considerable help with the
patient samples; to Jenny Corrigan (Department of Zoology, University
of Oxford) for final preparation and analysis of scanning electron
microscopy samples; to Prof Adrian Gear (University of Virginia) for
help in setting up the scanning electron microscopy procedure; to Dr
Jean-max Pasquet for help with the flow cytometry studies; and to Dr A. Thresher and Prof P. Brickell for useful discussions. The Department of
Molecular Immunology, Institute of Child Health, London, kindly
performed the initial WAS protein and gene studies.
| |
FOOTNOTES |
Submitted February 17, 1999; accepted August 11, 1999.
B.S.G. and J.I.W. contributed equally to this work.
Supported by the Wellcome Trust and British Heart Foundation (BHF).
S.P.W. is a BHF Senior Research Scientist. B.S.G. is a Wellcome Prize
Student. L.Q. holds a BHF Studentship.
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 Steve P. Watson, PhD,
Department of Pharmacology, University of Oxford, Mansfield Road,
Oxford OX1 3QT, UK; e-mail: steve.watson{at}pharm.ox.ac.uk.
| |
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A. C. Pearce, J. I. Wilde, G. M. Doody, D. Best, O. Inoue, E. Vigorito, V. L. J. Tybulewicz, M. Turner, and S. P. Watson
Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C
Blood,
November 15, 2002;
100(10):
3561 - 3569.
[Abstract]
[Full Text]
[PDF]
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H. Falet, K. M. Hoffmeister, R. Neujahr, and J. H. Hartwig
Normal Arp2/3 complex activation in platelets lacking WASp
Blood,
August 28, 2002;
100(6):
2113 - 2122.
[Abstract]
[Full Text]
[PDF]
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D. Tulasne, B. A. Judd, M. Johansen, N. Asazuma, D. Best, E. J. Brown, M. Kahn, G. A. Koretzky, and S. P. Watson
C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor gamma -chain-associated signaling pathway and by agglutination
Blood,
December 1, 2001;
98(12):
3346 - 3352.
[Abstract]
[Full Text]
[PDF]
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A. Oda, H. D. Ochs, L. A. Lasky, S. Spencer, K. Ozaki, M. Fujihara, M. Handa, K. Ikebuchi, and H. Ikeda
CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk
Blood,
May 1, 2001;
97(9):
2633 - 2639.
[Abstract]
[Full Text]
[PDF]
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K. M. Hoffmeister, H. Falet, A. Toker, K. L. Barkalow, T. P. Stossel, and J. H. Hartwig
Mechanisms of Cold-induced Platelet Actin Assembly
J. Biol. Chem.,
June 29, 2001;
276(27):
24751 - 24759.
[Abstract]
[Full Text]
[PDF]
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J.-F. Cote, P. L. Chung, J.-F. Theberge, M. Halle, S. Spencer, L. A. Lasky, and M. L. Tremblay
PSTPIP Is a Substrate of PTP-PEST and Serves as a Scaffold Guiding PTP-PEST Toward a Specific Dephosphorylation of WASP
J. Biol. Chem.,
January 18, 2002;
277(4):
2973 - 2986.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION