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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology, Brigham and Women's
Hospital, Department of Medicine, Harvard Medical School, Boston, MA.
Arp2/3 complex is believed to induce de novo nucleation of actin
filaments at the edge of motile cells downstream of WASp family
proteins. In this study, the signaling pathways leading to Arp2/3
complex activation, actin assembly, and shape change were investigated
in platelets isolated from patients with Wiskott-Aldrich Syndrome
(WAS), that is, who lack WASp, and in WASp-deficient mouse platelets.
WASp-deficient human and mouse platelets elaborate filopodia, spread
lamellae, and assemble actin, identical to control WASp-expressing
platelets. Human platelets contain 2 µM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the
lamellae and to the Triton X-100-insoluble actin cytoskeleton of
activated WASp-deficient platelets. Furthermore, the C-terminal CA
domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the
actin nucleation capacity of octylglucoside-permeabilized and activated
WAS platelets, similar to its effect in WASp-expressing cells. Along
with WASp, platelets express WAVE-2 as a physiologic activator of
Arp2/3 complex and a small amount of N-WASp. Taken together, our
findings show that platelets activate Arp2/3 complex, assemble actin,
and change shape in the absence of WASp, indicating a more specialized
role for WASp in these cells.
(Blood. 2002;100:2113-2122) Arp2/3 complex is believed to induce de novo
nucleation of actin filaments at the edge of motile cells. This
complex, originally found in Acanthamoeba castellanii,
consists of 2 actin-related proteins, Arp2 and Arp3, and 5 novel
proteins, p41-, p34-, p21-, p20-, and p16-Arc.1,2 Arp2/3
complex initiates the actin-based motility of intracellular parasites
such as Listeria monocytogenes and Shigella
flexneri,3-5 branches actin filaments in
vitro,6,7 and localizes at the leading edge of crawling
cells such as carcinoma cells and Xenopus laevis
keratocytes.8,9
The actin nucleation activity of Arp2/3 complex can be initiated
by WASp family proteins, 5 of which are expressed in mammals: WASp,
N-WASp, and WAVE-1, -2, and -3. WASp is expressed only in hematopoietic
cells,10 notably in monocytes, lymphocytes, and platelets,
whereas N-WASp is widely expressed.11 WAVE-1 (also called
Scar) was first identified in Dictyostelium discoideum, then
in vertebrates,12,13 and 2 homologues, WAVE-2 and -3, were
subsequently cloned.14 WASp and N-WASp are thought to
activate Arp2/3 complex-mediated actin nucleation activity to induce
filopodia formation downstream of the small guanosine triphosphatase
(GTPase) Cdc42,11,15-19 whereas WAVE-1, -2, and -3 are
believed to orchestrate lamellae spreading downstream of
Rac.20 Recently, cortactin and Abp1 (or drebrin)
have also been added to the list of proteins that can activate Arp2/3
complex in mammalian cells.21-25
In vitro observations have elucidated the biochemical and structural
mechanisms by which WASp family proteins activate Arp2/3 complex. The
conserved C-terminal VCA domain of WASp and N-WASp binds and brings
together actin monomers and Arp2/3 complex to stimulate its actin
nucleation activity, whereas the CA domain alone sequesters and
inhibits Arp2/3 complex.26-28 However, WASp and N-WASp are
unable to activate Arp2/3 complex until signaling molecules such as
Cdc42 and polyphosphoinostides (ppIs) release intramolecular
inhibitory interactions to expose the VCA domain.29,30 Listeria ActA resembles the VCA domain of WASp proteins and
activates Arp2/3 complex with some similarity, whereas
Shigella IcsA recruits endogenous N-WASp from the host
cell.31
Mutations in the WASP gene encoding WASp in humans
results in Wiskott-Aldrich Syndrome (WAS), a severe inherited X-linked recessive hematopoietic disorder that is characterized by
thrombocytopenia, immunodeficiency, and eczema.10 WAS
platelets have short circulation times and are generally smaller, a
phenotype that is partially reversed by splenectomy.32,33
Importantly, all described mutations in the WASP gene have
been shown to lead to the complete absence of WASp expression in
platelets.34,35 In this study, aspects of the signaling
pathways leading to Arp2/3 complex activation and actin assembly were
investigated in human WAS platelets and WASp-deficient mouse platelets.
Consistent with previous observations,36,37 WASp-deficient
human and mouse platelets activate Arp2/3 complex and redistribute it
to the cell cortex, and elaborate filopodia, spread lamellae, and
assemble actin normally when activated. Thus, WASp is unnecessary for a
robust actin assembly reaction, suggesting a more specialized role for
WASp in platelets.
Patients
Approval was obtained from the institutional review boards of both
Brigham and Women's Hospital and the Center for Blood Research (Harvard Medical School), and informed consent was approved according to the Declaration of Helsinki.
Animals and reagents
Platelet preparation Blood from healthy volunteers and from WAS patients was collected in 0.1 volume of Aster-Jandl anticoagulant.44 Human platelet-rich plasma (PRP) was prepared by centrifugation of the blood at 100g for 20 minutes. Blood was collected from wild-type and WASp-deficient mice by retro-orbital plexus bleeding and anticoagulated in Aster-Jandl anticoagulant. Mouse PRP was obtained by centrifugation of the blood at 100g for 6 minutes, followed by centrifugation of the supernatant and the buffy coat at 100g for 6 minutes. Human and mouse platelets were isolated from PRP using a metrizamide gradient.45 Platelet concentration was adjusted to 3 × 108/mL, and platelets were allowed to rest for 30 minutes at 37°C before use.DIC light microscopy Attachment and activation of platelets on CRP-coated surface were followed by differential interference contrast (DIC) light microscopy.45 Coverslips were coated with 6 µg/mL CRP in phosphate-buffered saline (PBS) for 2 hours followed by extensive blocking with 0.5% bovine serum albumin (BSA) in PBS. Petri dishes were maintained at 37°C with a Harvard Apparatus (Holliston, MA) temperature controller TC-202. Platelets were imaged on a Zeiss IM-35 inverted microscope with DIC optics and × 100 oil immersion objective. Images were captured with a Hamamatsu C2400 CCD camera (Hamamatsu, Japan), processed for background substraction and frame averaging with a Hamamatsu ARGUS image processor and digitally recorded on Macintosh computer equipped with a SCION frame grabber LG-3 (Frederick, MD).Immunofluorescence analysis Platelets were centrifuged at 280g for 5 minutes onto poly-L-lysine-coated coverslips, fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 0.5% BSA in PBS. Arp2/3 complex sites were exposed by treating the fixed and blocked cytoskeletons with 0.1% sodium dodecyl sulfate (SDS) for 1 minute. Specimens were washed in PBS and labeled with a mixture of rabbit anti-Arp3 and anti-p34-Arc antibodies, each at 4 µg/mL. Images were obtained using a Zeiss Axiovert S100 microscope with a × 100 oil immersion objective. The peptides used to raise the antibodies3,41 were mixed with the anti-Arp2/3 antibodies as controls (each at 50 µg/mL) and blocked all detectable antibody binding (Figure 5B).Electron microscopy and immunogold labeling Platelets isolated from WAS patients were attached to 5-mm round poly-L-lysine-coated coverslips by centrifugation at 280g for 5 minutes at room temperature. Resting platelets isolated from wild-type and WASp-deficient mice were attached to antiglycoprotein Ib (GPIb) antibody-coated coverslips, and
activated with 1 U/mL thrombin for 10 minutes at 37°C. Human and
mouse platelet cytoskeletons were prepared by extraction with 0.75%
Triton X-100 in 60 mM PIPES (piperazine-N,N'-bis-2-ethanesulfonic
acid), 25 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 10 mM EGTA
(ethyleneglycoltetraacetic acid) and 2 mM MgCl2, pH 6.9, containing protease inhibitors and 2 µM phalloidin (PHEM buffer), as
described.46 Extracted platelets were briefly washed in
PHEM buffer and fixed with 1% glutaraldehyde in PHEM buffer for 10 minutes. Unreacted aldehydes were blocked with 1 mg/mL sodium
borohydride in PHEM buffer for 1 minute. Arp2/3 complex sites were
exposed by treating the fixed and blocked cytoskeletons with 0.1% SDS
in PHEM buffer for 1 minute. There was no specific labeling in the
absence of SDS (data not shown), showing the epitopes to be sequestered
when Arp2/3 complex was not denatured. Cytoskeletons were washed with
PHEM buffer and incubated in 1% BSA in 150 mM NaCl, 20 mM Tris (TBS),
pH 8.2. Cytoskeletons were labeled with a mixture of rabbit anti-Arp3
and anti-p34-Arc antibodies, each at 4 µg/mL. Unbound antibodies
were washed away with TBS/1% BSA, pH 8.2, and cytoskeletons were
incubated with 10 nm colloidal gold coated with goat antirabbit IgG for
60 minutes. Coverslips were washed thrice with TBS, then fixed with 1%
glutaraldehyde in PBS for 10 minutes. Controls included omitting the
primary antibody and using nonspecific rabbit antibodies instead
of the affinity-purified anti-Arp3 and anti-p34-Arc antibodies. In the absence of specific primary antibodies there was no detectable signal
(data not shown). Cytoskeletons were washed thrice with double-distilled water just before freezing. Samples were rapid-frozen, freeze-dried, and rotary-shadowed with 1.2 nm tantalum-tungsten followed by 2.5 nm carbon. Specimens were examined and photographed in
a JEOL 1200-EX electron microscope (Tokyo, Japan) using an accelerating
voltage of 100 kV.
SDS-PAGE and immunoblot analysis Platelet proteins were lysed in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 5% -mercaptoethanol. After boiling for 5 minutes, proteins were
separated on 8% or 10% polyacrylamide gels and transferred onto an
Immobilon-P membrane (Millipore, Bedford, MA). Membranes were incubated
overnight in 100 mM NaCl and 20 mM Tris, pH 7.4, containing 1% BSA,
then probed with antibodies directed against proteins of interest.
Detection was performed with an enhanced chemiluminescence system
(Pierce, Rockford, IL).
For the Arp2/3 complex depletion, human platelets were lysed in 1% Igepal CA-630 (also called Nonidet P-40), 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin, and 10 µg/mL aprotinin, as described.47 After centrifugation at 14 000g for 10 minutes at 4°C, the Igepal CA-630-soluble fraction was passed twice over a GST-CA column.40 This procedure resulted in more than 99% depletion of Arp2/3 complex (Figure 3B), as described for neutrophils.48 For isolation of the actin cytoskeleton, platelets were lysed with 0.1% Triton X-100 in PHEM buffer. Filamentous actin (F-actin) was isolated by centrifugation at 100 000g for 30 minutes at 4°C in a Beckman Optima TL ultracentrifuge (Palo Alto, CA) using polycarbonate tubes and a TLA 100 rotor. Triton X-100-insoluble and -soluble fractions were resolved by SDS-PAGE, as described above. Measurement of platelet F-actin content and barbed-end nuclei numbers Resting or activated platelets were fixed in 3.4% formaldehyde and permeabilized with 0.1% Triton X-100 in PHEM buffer in the presence of 2 µM fluorescein isothiocyanate (FITC)-phalloidin.45 Bound FITC-phalloidin was quantitated by FACS analysis using a Becton Dickinson FACSCalibur flow cytometer (Franklin Lakes, NJ). A total of 10 000 events was analyzed for each sample.Platelets were activated and extracted with 0.1% Triton X-100 in PHEM buffer44 or permeabilized with 0.25% octylglucoside (OG) in PHEM buffer and activated.49 The OG-platelet suspension was mixed and the platelets were allowed to extract for 30 seconds at 37°C. When required, GST-CA was added prior to activation, as described.43 The polymerization rate assay started by the addition of 185 µL 100 mM KCl, 2 mM MgCl2, 0.5 mM adenosine triphosphate (ATP), 0.1 mM EGTA, 0.5 mM dithiothreitol, and 10 mM Tris, pH 7.0, to 100 µL platelet extract, and the subsequent addition of 15 µL 20 µM monomeric pyrene-labeled rabbit skeletal muscle actin. Pyrene-actin fluorescence was recorded using a spectrofluorimeter LS50 (Perkin-Elmer Analytical Instruments, Norwalk, CT). Excitation and emission wavelengths were 366 and 386 nm, respectively. The number of barbed-end nuclei was determined as described.49
Platelets change shape normally in the absence of WASp The morphology and organization of the actin cytoskeleton of human WAS platelets and WASp-deficient mouse platelets, activated on a CRP-coated surface, specific for the collagen receptor GPVI,45 were investigated by DIC light microscopy (Figure 1). The attachment and spreading process of WASp-deficient human and mouse platelets were identical to control platelets. Both WASp-expressing and WASp-deficient platelets generated filopodia and lamellae when exposed to CRP-coated surface.
Platelets assemble actin normally in the absence of WASp We addressed whether WASp expression is required for platelet actin assembly. Platelets isolated from WAS patients increased their F-actin content normally after ligation of the thrombin receptor PAR-1 with 25 µM TRAP or of the collagen receptor GPVI with 3 µg/mL CRP (Figure 2A), consistent with previous observations.36,37 Similarly, WASp-deficient mouse platelets showed no defects in actin assembly in response to 1 U/mL thrombin or to 3 µg/mL CRP (Figure 2B).
Human platelets contain 2 µM Arp2/3 complex We determined the amount of Arp2/3 complex expressed in human platelets. By densitometric analysis of immunoblots against the Arp3 and p34-Arc subunits of Arp2/3 complex, using purified platelet Arp2/3 complex as a standard,40 we estimated that human platelets contain 14.3 × 10 21 mol Arp2/3 complex, or
8600 molecules/cell (Figure 3A). To
verify that the presence of other platelet proteins in whole lysates does not affect the Arp2/3 complex signal on immunoblots, purified Arp2/3 complex was added back to platelet lysates depleted of Arp2/3
complex by sequential passages over a GST-CA column. GST-CA removed
more than 99% of Arp2/3 complex from the extract. Similar intensity
signals were obtained with lysates corresponding to 7 × 106 platelets and their depleted counterparts
complemented with 1 × 1013 mol Arp2/3 complex, as
estimated (14.3 × 1021
mol/platelet × 7 × 106 platelets; Figure 3B). Because
the average platelet volume is 7 × 1015 L, human
platelets contain 2 µM Arp2/3 complex.
Normal Arp2/3 complex redistribution in the actin cytoskeleton of WASp-deficient platelets The redistribution of Arp2/3 complex to the actin cytoskeleton of WASp-deficient platelets was evaluated. As is always observed for WASp-expressing platelets (H.F., K.M.H., R.N., et al, submitted manuscript), Arp2/3 complex redistributed to the actin cytoskeleton of WASp-deficient activated platelets. Thirty percent of the total platelet Arp2/3 complex was associated with the Triton X-100-insoluble actin cytoskeleton of resting platelets (Figure 4). This amount increased to 80% after stimulation of human WAS platelets with 25 µM TRAP and 3 µg/mL CRP or after activation of WASp-deficient mouse platelets with 1 U/mL thrombin, identical to normal WASp-expressing platelets. WASp was always Triton X-100 soluble in platelets maintained under nonaggregating conditions (data not shown). WASp redistribution to the actin cytoskeleton requires platelet aggregation50 to cross-link the fibrinogen receptorIIb3,
a condition that is not required for Arp2/3 complex redistribution,
actin assembly, and shape change.
The Arp2/3 complex redistributes to the cell cortex of spread platelets Platelets spread over glass surfaces using large circumferential lamellae, although some filopodia are extended.44 Fluorescence microscopy and immunogold electron microscopy were performed using a mixture of 2 rabbit polyclonal antibodies directed against the Arp3 and p34-Arc subunits of Arp2/3 complex followed by either tetrarhodamine isothiocyanate (TRITC)-labeled antirabbit IgG or 10-nm colloidal gold particles coated with goat antirabbit IgG. In the light microscope, Arp2/3 complex becomes concentrated at the periphery of the active platelet (Figure 5A). Internal labeling is also found, concentrated in a number of foci near the center of the spread platelet.
Because 80% of total Arp2/3 complex is retained in the active cytoskeleton, the localization of Arp2/3 complex was further investigated in the electron microscope in labeled and freeze-dried specimens (Figure 5C). Once again, in the electron microscope, Arp2/3 complex redistributes into lamellae at the cell periphery and is bound predominantly in the short actin filament networks found in these regions (lm). As observed in the light microscope, Arp2/3 complex is also found to rigorously decorate certain dense structures within the cytoskeleton (arrows). One noticeable exception is the tips of filopodia that are poorly labeled with anti-Arp2/3 complex immunogold (f). Actin filaments that enter, and become bundled, in filopodia derive from filaments originating in the cytoskeletal center. Because some Arp2/3 complex labeling is observed throughout the cytoskeleton, as well as the foci of dense labeling that are found internally, some of Arp2/3 complex may be associated with the origins of filaments that eventually become collected in the filopodia. Labeling of Arp2/3 complex was completely dependent on adding denaturating compounds (SDS or methanol) and was not observed in the absence of primary antibodies or with nonspecific primary antibodies substituted (data not shown). Similar to normal platelets, WASp-deficient platelets spread and
elaborate filopodia, fingerlike extensions composed of long actin
fibers, and robustly fill the spaces between the filopodia with
lamellae composed of short actin filaments organized into dense
orthogonal networks (Figures 6 and
7). Arp2/3 complex redistributes normally to the cortex as WASp-deficient human and mouse platelets spread on glass, in identical fashion to that observed in the control
platelets. Once again, some labeling of densities in the cytoskeletal
center was observed and the bundles of actin filaments composing
filopodia were poorly decorated with anti-Arp2/3 complex immunogold.
Arp2/3 complex contributes to the actin nucleation activity of WAS platelets Figure 8A shows that platelets isolated from healthy human volunteers and activated for 1 minute with 25 µM TRAP or 3 µg/mL CRP increase their barbed-end nuclei content from 49 ± 18 (mean ± SD, n = 4) per cell at rest to 509 ± 22 and 326 ± 48, respectively. In resting human WAS platelets, 43 ± 18 barbed-end nuclei were detected per cell (n = 3). This number increased to 471 ± 67 and 348 ± 44 after 1 minute of activation with TRAP and CRP, respectively. Similarly, WASp-deficient mouse platelets showed no defects in barbed-end nuclei production in response to stimulation (Figure 8B), increasing their barbed-end nuclei number from 56 ± 12 (mean ± SD, n = 4) per cell at rest to 408 ± 45 after 1 minute of activation with 1 U/mL thrombin.
To further determine whether Arp2/3 complex participates in the actin nucleation activity of WASp-deficient platelets, the C-terminal CA domain of N-WASp, which sequesters and inhibits Arp2/3 complex,26 was added to WAS platelets permeabilzed with OG (Figure 8C). TRAP (25 µM) induced the exposure of 195 ± 20 and 213 ± 15 barbed-end nuclei/platelet (mean ± SD, n = 3) after 1 minute of activation in normal and WAS platelets, respectively, compared to 42 ± 15 and 41 ± 8 at rest. GST-CA (3 µM) inhibited by approximately 50% barbed-end nuclei production in WAS platelets (134 ± 12) similar to its effect in WASp-expressing cells (134 ± 16).43 Thus, WASp expression is not required for activation of Arp2/3 complex in platelets and Arp2/3 complex contributes in 50% of the barbed-end nuclei in activated OG-permeabilized platelets. Platelet WASp family proteins Because platelets change shape, assemble actin, and activate Arp2/3 complex in the absence of WASp, the expression of other WASp family proteins was investigated by immunoblot analysis in control and WASp-deficient platelets. Figure 9 confirms that human and mouse platelets express WASp as a protein of 64 kDa. WASp was not detected in platelets isolated from WAS patients, as described,34-37 nor in platelets isolated from WASp-deficient mice. Anti-N-WASp antibody recognized a faint polypeptide of slightly higher molecular weight (66 kDa) in human platelets and cross-reacted with WASp, as evidenced by the presence of the 64-kDa band absent in WASp-deficient human and mouse platelets. We and others51 failed to detect N-WASp in human platelets using other available antibodies (data not shown). Human and mouse platelets express WAVE-2 as a protein of apparent 84 kDa. Expression of WAVE-1 and -3 remains to be determined.
De novo nucleation of actin by Arp2/3 complex is a widely recognized mechanism for promoting barbed-end-directed actin filament assembly associated with intracellular propulsion of microorganisms. This system is also thought to be responsible for actin remodeling at the cell edge during spreading, locomotion, and phagocytosis downstream of WASp family proteins. Human platelets contain 2 µM Arp2/3 complex, or 8600 molecules/cell, approximately half the estimated amount of WASp (4.7 µM).52 However, our findings, consistent with those of others,36,37 show that WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, all indistinguishable from WASp-expressing platelets. They also incorporate Arp2/3 complex into the actin cytoskeleton and redistribute it to the edge of the actin-rich lamellae following activation, demonstrating that WASp expression is not required for Arp2/3 complex translocation into the cortex. Human platelets may also express a small amount of N-WASp, as recently described.52 However, because recombinant N-WASp must be added to lysates of human platelets before the motility of Escherichia coli expressing Shigella IcsA initiates,51 there is insufficient N-WASp to have an important physiologic role in platelets. WASp and N-WASp are thought to activate Arp2/3 complex actin nucleation activity to induce filopodia formation downstream of Cdc42 and ppIs, based on in vitro experiments and transfection into cultured cells.11,16,17,26-28 WASp overexpression leads to formation of actin clusters in nonhematopoietic cultured cells,15 and inducible recruitment of WASp to a membrane receptor triggers actin polymerization that results in filopodia formation.18,19 However, endogenous WASp and N-WASp are not required for filopodia formation in platelets, consistent with recent reports that show filopodia formation and actin assembly to be independent of WASp or N-WASp expression in stimulated fibroblasts.53,54 Besides platelets, WASp is expressed in macrophages, dendritic cells, and T and B cells where its deficiency results in impaired podosome formation, cell polarization, motility, and phagocytosis.55-60 WASp may be involved in the formation of supramolecular activation clusters or immunologic synapses, and Arp2/3 complex activation by WASp may be singularly important in such systems for these highly specialized actin polymerization responses. WASp may also have more specialized functions in immune cells, such as in transcriptional activation, proliferative responses, and cytokine production,61,62 functions that are not shared by platelets. Lamellipodial actin assembly is classically thought to be regulated by the small GTPase Rac, rather than Cdc42.63,64 Arp2/3 complex localizes at the edge of the lamellae of platelets spread on glass, as does Rac.65 Pathways leading to Arp2/3 complex from Rac include the WAVE proteins and the activation of phosphoinositide (PI) kinases, particularly PI 5-kinases.49,66 WAVE proteins have been shown to activate Arp2/3 complex downstream of Rac and IRSp53.20 Human and mouse platelets express WAVE-2, consistent with a recent report showing WAVE-2 to be widely expressed, notably in hematopoietic cells.14 Another contributor may also be cortactin. Cortactin is involved in the formation of lamellae downstream of Rac and PAK in cultured cells, where it colocalizes with Arp2/3 complex.67,68 Cortactin activates Arp2/3 complex21-23 and also redistributes to the cortex of spread platelets.69 Rac also mediates the production of ppIs at the cytoplasmic surface of the plasma membrane that recruits actin regulatory proteins and causes the uncapping of the barbed ends of the actin filaments previously severed by Ca++-activated gelsolin in platelets.44,49 These ends, once uncapped, are subsequently amplified by Arp2/3 complex to drive actin assembly (H.F., K.M.H., R.N., et al, submitted manuscript). Hence, Arp2/3 complex activity is potentiated by actin filament barbed-end uncapping at the edge of the platelet lamellae. Recently, the small GTPase Rho has also been shown to activate
and participate in the production of ppIs that regulate actin assembly
required for platelet shape change.70 Rho mediated events
are highlighted in G In conclusion, our findings demonstrate that the actin assembly system remains fully functional in platelets in the absence of WASp, suggesting that proteins other than WASp, probably WAVE-2 or cortactin or both, activate Arp2/3 complex in these cells. The origin of the thrombocytopenia and the short platelet circulation times associated with WAS remains unclear. WASp appears to be required for normal platelet integrity. Thus, WASp deficiency may affect the surface organization of platelets such that clearance is accelerated.
We thank the WAS patients for participating in this study. We thank Drs Thomas P. Stossel, Fred S. Rosen, and Raif S. Geha for valuable discussions and ideas. We thank Dr Joseph E. Italiano Jr for immunofluorescence expertise, our colleagues and collaborators for sharing reagents and animals, and Karen Vengerow for editorial assistance.
Submitted November 13, 2001; accepted May 14, 2002.
Supported by National Institutes of Health grants HL-56949 and HL-56252, Edwin W. Hiam, and the Edwin S. Webster Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hervé Falet, Division of Hematology, Brigham and Women's Hospital, 221 Longwood Ave, LMRC 301, Boston, MA 02115; e-mail: hfalet{at}rics.bwh.harvard.edu.
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© 2002 by The American Society of Hematology.
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  A. Cvejic, C. Hall, M. Bak-Maier, M. V. Flores, P. Crosier, M. J. Redd, and P. Martin Analysis of WASp function during the wound inflammatory response - live-imaging studies in zebrafish larvae J. Cell Sci., October 1, 2008; 121(19): 3196 - 3206. [Abstract] [Full Text] [PDF]
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 A. Di Nardo, G. Cicchetti, H. Falet, J. H. Hartwig, T. P. Stossel, and D. J. Kwiatkowski Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leading-edge actin structure and function PNAS, November 8, 2005; 102(45): 16263 - 16268. [Abstract] [Full Text] [PDF]
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A. Biyasheva, T. Svitkina, P. Kunda, B. Baum, and G. Borisy Cascade pathway of filopodia formation downstream of SCAR J. Cell Sci., February 22, 2004; 117(6): 837 - 848. [Abstract] [Full Text] [PDF]
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H. Falet, K. M. Hoffmeister, R. Neujahr, J. E. Italiano Jr., T. P. Stossel, F. S. Southwick, and J. H. Hartwig Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts PNAS, December 24, 2002; 99(26): 16782 - 16787. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
A mutation in intron 6 at position +5.
Full-length WASp is expressed at 5% to 20% of normal level in his T
cells but is absent in his B cells. Patient P32 presents a mild
phenotype diagnosed as X-linked thrombocytopenia. His WASP
gene has a G291
) and WAS
patient P34 (
) were activated with 25 µM TRAP or 3 µg/mL CRP.
(B) Platelets from wild-type (
) were compared
by anti-Arp3 immunoblot to their Arp2/3 complex-depleted counterparts
complemented or not with 0.5 and 1 × 10
). The amount of Arp2/3 complex was quantified by
densitometric analysis of the immunoblots. The histogram plots the
percentage ± SD of the density relative to total from 3 independent assays.
-chain complex.
Blood.
2000;96:3786-3792