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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Blood Research and the Department
of Pediatrics, Harvard Medical School, Boston, MA; the Research
Institute for Pediatric Hematology, Moscow, Russia; and the Department
of Biochemistry, Institute of Medical Science, University of Tokyo,
Japan.
Mutations of Wiskott-Aldrich syndrome protein (WASP) underlie the
severe thrombocytopenia and immunodeficiency of the Wiskott-Aldrich syndrome. WASP, a specific blood cell protein, and its close homologue, the broadly distributed N-WASP, function in dynamic actin
polymerization processes. Here it is demonstrated that N-WASP is
expressed along with WASP, albeit at low levels, in human blood cells.
The presence of approximately 160 nmol/L rapidly acting N-WASP
molecules may explain the normal capacity of WASP-negative patient
platelets for early agonist-induced aggregation and filopodia
formation. Ex vivo experiments revealed a significant difference
between WASP and N-WASP in sensitivity to calpain, the
Ca++-dependent protease activated in agonist-stimulated
platelets. Through the use of a series of calpain-containing broken
cell systems, it is shown that WASP is cleaved in a
Ca++-dependent reaction inhibitable by calpeptin and E64d
and that N-WASP is not cleaved, suggesting that the cleavage of WASP by calpain functions in normal platelets as part of a
Ca++-dependent switch mechanism that terminates the surface
projection phase of blood cell activation processes.
(Blood. 2001;98:2988-2991) Wiskott-Aldrich syndrome protein (WASP) and
its close homologue, the broadly distributed N-WASP, members of the
WASP/SCAR protein family (reviewed in1), are cytoplasmic
proteins thought to mediate dynamic cytoskeletal rearrangements by
nucleating actin filament formation in response to specific
stimuli.2 N-WASP, which was discovered in brain, has broad
tissue distribution.3 WASP, in contrast, is exclusive to
blood cells.4,5 Mutations of WASP lead to the
Wiskott-Aldrich syndrome (WAS), a severe blood cell disease that
includes immunodeficiency of variable severity and profound
thrombocytopenia (reviewed in6). A recent
genotype-phenotype study established that WAS patients with severe
immune disease (nonsense and frameshift mutations) have WASP-negative
leukocytes and that patients with mild immune disease (missense
mutations) have decreased WASP levels in leukocytes.7 In
contrast, 18 patients with diverse mutations all had WASP-negative
platelets, providing an apparent explanation for the uniform severity
of platelet dysfunction in this disease.
Although substantial inroads have been made in delineating the
biochemistry, regulation, and signaling capability of N-WASP and WASP,
the pathologic steps leading to the dysfunction of WAS blood cells
remain undefined. Neither is it known how blood cells of patients
retain a substantial number of normal cytoarchitectural responses
despite the absence or deficit of WASP. For example, WAS patient
platelets, though hypersusceptible to late-phase activation events such
as phosphatidylserine exposure and microparticle release,8 are nonetheless capable of normal early responses, including actin polymerization, shape change, and others.8-10 To determine
whether N-WASP might contribute to early cytoarchitectural
reorganization events, we examined blood cells from healthy control
subjects and from patients for the content of this protein. We
also compared WASP and N-WASP for stability under conditions of
platelet activation.
Patients
Cells
Epstein-Barr virus (EBV)-transformed cell lines from patients with WAS and healthy control subjects11 were grown in RPMI 1640 with 10% fetal calf serum, penicillin, and streptomycin. HeLa epithelial carcinoma cells strain S3 were grown as adherent cells in Dulbecco minimum essential high-glucose medium with the same additives and were detached for harvest by 10-minute incubation in 25 mM EDTA in phosphate-buffered saline at 37°C. Western blots After a final wash of the cells in the presence of 2 mM diisopropylfluorophosphate and 25 µg/mL leupeptin (Sigma), lysates were prepared as described7 of 15 × 106/mL PBMCs, 15 × 106/mL neutrophils, 5 × 108/mL platelets, and 10 × 106/mL HeLa cells in sodium dodecyl sulfate (SDS) containing the same protease inhibitors. Lysates were electrophoresed (reducing conditions) and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) for antibody staining as described.7,11 Reactive bands detected by iodine 125-labeled secondary antibodies were quantified with the PhosphorImager Storm 860 and Image Quant v1.1 program (Molecular Dynamics, Sunnyvale, CA).Antibodies and proteins The previously described N-WASP antibodies were generated in rabbits immunized with amino acids 388-501 (antibody [Ab]-1)12 (see also 13,14) or full-length (Ab-2)15 recombinant N-WASP. On Western blots, both antibodies recognized N-WASP and cross-reacted with WASP (shown in "Results"). Ab-1 also cross-reacted with WAVE/SCAR-1, -2, and -3 (not shown). WASP antibodies (W485) were raised in rabbits immunized with amino acids 485-502 (C-terminus) of human WASP.11 B27D8 monoclonal antibody is a mouse IgG1 that recognizes µ-calpain heavy chain.16 Affinity-purified goat anti-rabbit and rabbit anti-mouse immunoglobulin (Ig) was from Cappel (Durham, NC) and Pierce Chemical (Rockford, IL), respectively. Recombinant full-length rat N-WASP was generated in insect cells and purified.15 Glutathione S-transferase (GST) fusion proteins of the VCA regions of rat N-WASP (amino acids 392-505) and human WASP (amino acids 414-502) were generated in Escherichia coli and purified on glutathione-Sepharose as described.3,15 Calpain (porcine erythrocyte) was from Calbiochem.Platelet activation Platelets (5 × 108) in 1 mL platelet buffer with 2 mM CaCl2 were preincubated with calpeptin (50 µg/mL; Calbiochem), E64d (50 µg/mL; Sigma), or diluent (1% dimethyl sulfoxide) without stirring for 30 minutes at 37°C. A23187 (1 µM; Sigma) was added, and incubation was continued with stirring for 5 to 20 minutes at 37°C. Reactions were terminated by the addition of 2 × SDS solution and heating at 100°C. In some experiments, the reaction was stopped by the addition of EGTA and leupeptin, and platelets were pelleted in a microcentrifuge at 4000 rpm for 8 minutes, resuspended in one-fourth original volume platelet buffer, and lysed by heating with 2 × SDS.Calpain reactions in broken cell preparations Platelets at 109/mL in Ca++/Mg2+-free HBSS containing 2 mM EGTA and 2 mM mercaptoethanol were lysed by sonication at 4°C with a macrotip preparative probe at 50% duty cycle with 96 bursts of 1 second each delivered over 2 minutes (model W-225 Sonicator; Heat Systems, Ultrasonics, Farmingdale, NY), clarified by centrifugation, and stored in aliquots at 80°C. HeLa cells (15 × 106/mL) were
lysed in 0.5% NP-40, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM
diisopropylfluorophosphate, 25 µg/mL leupeptin, and 2 mM EGTA and
were clarified by centrifugation. CaCl2 (5 mM) was added to
platelet sonicates or HeLa cell lysates immediately before incubation
at approximately 22°C for 0 to 30 minutes. Reactions were terminated
by the addition of 2 × SDS solution and incubation at
100°C.
Immunoprecipitation Four hundred microliters platelet sonicate was incubated with 2 µg N-WASP Ab-2 or normal rabbit IgG at 4°C overnight. Washed A/G Plus agarose (100 µL; Santa Cruz Biotechnology, Santa Cruz, CA) was added, and incubation continued for 2 hours at 4°C. The resin was centrifuged for 10 seconds in a microcentrifuge and washed, and the proteins were eluted with 40 µL SDS solution at 100°C for 2 minutes.
Western blots of HeLa cells with Ab-1 to the N-WASP
carboxy-terminal VCA region stained N-WASP as the major band (Figure
1A, lane 1) and cross-reacted with bands
of 74 to 80 kd, which are apparently the 3 SCAR/WAVE
proteins,17,18 known to be expressed in HeLa cells and
detectable with Ab-1 (data not shown). In PBMCs, Ab-1 stained bands in
the 74- to 80-kd range (which were not further investigated), and the
major band WASP and N-WASP at low levels (Figure 1A, lane 2). The
N-WASP band was detected in PBMCs of additional healthy control
subjects (N) and WASP patients without (P1) and with
(P2) low levels of WASP expression (Figure 1C, lanes 1-3).
The N-WASP band was also detected in the neutrophils of control
subjects and WAS patients and in EBV-transformed B-cell lines (Figure
1C, upper panel) and platelets (lower panel). As previously
described,7 WAS patient platelets lack WASP (lower panel).
N-WASP levels were not detectably different in paired patient and
healthy cells (Figure 1C), indicating that WASP-deficient patient cells
do not have compensatory up-regulation of N-WASP.
To confirm the identity of the N-WASP band in blood cells, we performed immune precipitation of normal platelet lysate with an independent antibody generated to full-length N-WASP. N-WASP and WASP, but not the 74- to 80-kd bands, were specifically precipitated by Ab-2 and detected by Ab-1 (Figure 1B). Control rabbit IgG failed to precipitate WASP or N-WASP. N-WASP was quantified in normal platelets by phosphor imaging of
Western blots in which dilutions of recombinant N-WASP from insect
cells were used as standard. N-WASP content was determined as 30 ng/mg
platelet protein (Figure 2A), which, with
a volume of 7 × 10
For contrast, we examined normal platelets for WASP content, previously reported as 700 ng/mg total protein.20 Phosphor imaging of Western blots in which dilutions of WASP GST-VCA region were used as standard revealed the presence of approximately 900 ng WASP/mg total platelet protein (Figure 2B), corresponding to 4.7 µM. To examine the sensitivity of WASP and N-WASP to protease, platelets
were activated with Ca++ ionophore A23187, a potent agonist
that induces various events of platelet activation, including calpain
activation.21 Western blots showed time-dependent cleavage
of WASP during ionophore-induced platelet activation (Figure
3A, lanes 1-4), which confirms previous results.22 Cleavage of WASP in activated platelets was
abrogated by calpeptin, a specific cell permeant calpain
inhibitor23 (lane 5), and by E64d (lane 6), a chemically
unrelated calpain inhibitor.24 N-WASP, in contrast,
remained uncleaved (Figure 3B).
To further compare WASP and N-WASP, we examined these molecules in broken cell preparations in which calpain activation was induced by the addition of Ca++. Although WASP underwent time- and Ca++-dependent calpeptin-inhibitable cleavage in platelet lysates (Figure 3C), N-WASP in HeLa cells lysates was not cleaved despite calpain activation (Figure 3D). Because platelet and HeLa cell lysates are not comparable environments, WASP and N-WASP were also exposed to calpain when present at matched concentrations in the same incubation. In combined lysates of HeLa cells and platelets, the addition of Ca++ induced the activation of calpain and the cleavage of WASP but not of N-WASP (Figure 3E). In addition, recombinant N-WASP was not cleaved when incubated with endogenous WASP in Ca++-supplemented platelet lysates (Figure 3F).
A major finding of this study is the demonstration that N-WASP is present along with WASP in normal human leukocytes and platelets. This finding contrasts with previous studies that failed to detect N-WASP in platelets.10,25 The source of the conflicting findings is unknown, though the use of detection systems with different sensitivity is a possibility because platelet N-WASP levels are low. We also showed that WASP, but not N-WASP, is cleaved by calpain, which is known to be activated as a late-phase event in platelets.21,26 The difference in calpain sensitivity of WASP and N-WASP is surprising given the high degree of sequence and structural homology of these proteins and their shared in vitro functions. These findings strongly suggest that calpain sensitivity of WASP is physiologically relevant. In WASP family proteins, the N-terminal and C-terminal regions have different functions. The acidic (A) region at the extreme C-terminus is responsible for binding the Arp2/3 complex, and the verprolin-cofilin-acidic (VCA) region (429-502 in WASP, also called the WA region) functions to dramatically enhance Arp2/3-dependent actin filament nucleation in vitro.15,27 N-terminal regions, and especially the GBD domain (230-288 in WASP), respond to cell signaling events that recruit N-WASP/WASP to the surface membrane and convert the dormant auto-inhibited folded structures to the activated open conformation.13,28,29 This unfolding and activation reaction is induced in both WASP and N-WASP by the binding of phosphatidylinositol-4,5-bisphosphate and Cdc42-GTP (reviewed in 1,2). There are, however, notable differences between WASP and N-WASP in their responsiveness to these mediators.15,30 The severe platelet defect in WAS is characterized by small platelet
size and low number, typically approximately 10% of normal, primarily
caused by enhanced platelet destruction (reviewed in 6).
Surprisingly, several laboratories found that early events of platelet
activation What then is the function of platelet WASP? In contrast to the normal early events, late-phase events are abnormal in the WASP-negative patient platelets. These include altered procalpain levels,31 increased resting Ca++ levels, increased amplitude and duration of Ca++ transients, and hyperreactivity of Ca++-dependent late-phase events including phosphatidylserine exposure and microparticle release.8 These findings suggest that the role of WASP is to indirectly modulate Ca++ transients in the filopodia-lamellipodia phase and, through calpain cleavage, to contribute to the dissolution of surface projections in response to high Ca++ levels. Based on transfection studies,17 a cleavage pattern that releases an intact VCA region is expected to disrupt Arp2/3-dependent nucleation. Cleavage within the VCA region is expected to end actin nucleation and to allow decay of surface projections. We speculate that N-WASP rather than WASP is largely responsible for the initial agonist-induced rapid generation of platelet filopodia, with the more abundant and possibly slower-acting WASP molecules sustaining actin nucleation and the filopodia- lamellipodia phase of platelet activation.
We thank Drs Hsin-yi Henry Ho, Rajat Rohatgi, and Marc W. Kirschner (Department of Cell Biology, Harvard Medical School, Boston, MA) for providing advice and reagents and Drs John Hartwig and Herve Falet (Division of Hematology, Brigham and Women's Hospital, Boston, MA) for discussing their findings before publication.
Submitted February 27, 2001; accepted July 13, 2001.
Supported by National Institutes of Health grants AI39574 and HL59561.
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: Eileen Remold-O'Donnell, Center for Blood Research, 800 Huntington Ave, Boston, MA 02115; e-mail: remold{at}cbr.med.harvard.edu.
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© 2001 by The American Society of Hematology.
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  K. Eto, H. Nishikii, T. Ogaeri, S. Suetsugu, A. Kamiya, T. Kobayashi, D. Yamazaki, A. Oda, T. Takenawa, and H. Nakauchi The WAVE2/Abi1 complex differentially regulates megakaryocyte development and spreading: implications for platelet biogenesis and spreading machinery Blood, November 15, 2007; 110(10): 3637 - 3647. [Abstract] [Full Text] [PDF]
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 S. Dewitt and M. Hallett Leukocyte membrane "expansion": a central mechanism for leukocyte extravasation J. Leukoc. Biol., May 1, 2007; 81(5): 1160 - 1164. [Abstract] [Full Text] [PDF]
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 A. Konno, M. Kirby, S. A. Anderson, P. L. Schwartzberg, and F. Candotti The expression of Wiskott-Aldrich syndrome protein (WASP) is dependent on WASP-interacting protein (WIP) Int. Immunol., February 1, 2007; 19(2): 185 - 192. [Abstract] [Full Text] [PDF]
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 M. A. de la Fuente, Y. Sasahara, M. Calamito, I. M. Anton, A. Elkhal, M. D. Gallego, K. Suresh, K. Siminovitch, H. D. Ochs, K. C. Anderson, et al. WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP) PNAS, January 16, 2007; 104(3): 926 - 931. [Abstract] [Full Text] [PDF]
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A. Oda, H. Miki, I. Wada, H. Yamaguchi, D. Yamazaki, S. Suetsugu, M. Nakajima, A. Nakayama, K. Okawa, H. Miyazaki, et al. WAVE/Scars in platelets Blood, April 15, 2005; 105(8): 3141 - 3148. [Abstract] [Full Text] [PDF]
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C. Lacout, E. Haddad, S. Sabri, F. Svinarchouk, L. Garcon, C. Capron, A. Foudi, R. Mzali, S. B. Snapper, F. Louache, et al. A defect in hematopoietic stem cell migration explains the nonrandom X-chromosome inactivation in carriers of Wiskott-Aldrich syndrome Blood, August 15, 2003; 102(4): 1282 - 1289. [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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Z. Li, E. S. Kim, and E. L. Bearer Arp2/3 complex is required for actin polymerization during platelet shape change Blood, May 29, 2002; 99(12): 4466 - 4474. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
15 L/platelet,
including shape change, formation of filopodia and
lamellipodia, polymerization of actin, aggregation, and alpha granule
release