Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Br J Haematol. 2009 Oct 27;148(3):416–427. doi: 10.1111/j.1365-2141.2009.07959.x

WASP plays a novel role in regulating platelet responses dependent on αIIbβ3 integrin outside-in signaling

Anna Shcherbina 1,2, Jessica Cooley 1, Maxim I Lutskiy 1, Charaf Benarafa 1, Gary E Gilbert 3, Eileen Remold-O’Donnell 1
PMCID: PMC2810352  NIHMSID: NIHMS163771  PMID: 19863535

Summary

The most consistent feature of Wiskott-Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WASP (WAS protein), which functions in leukocytes as an actin filament nucleating agent – yet – actin filament nucleation proceeds normally in patient platelets in shape change, filopodia and lamellipodia generation. Because WASP localizes in platelets in the membrane skeleton and is mobilized by αIIbβ3 integrin outside-in signaling, we questioned whether its function might be linked to integrin. Agonist-induced αIIbβ3 activation, detected by PAC-1, was normal for patient platelets, indicating normal integrin inside-out signaling. Likewise, fibrinogen and JON/A binding were normal for agonist-treated wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in dependent response. Another integrin outside-in dependent response, fibrin clot retraction, which requires contraction of the post-aggregation actin cytoskeleton, was decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilization of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signaling.

Keywords: Wiskott-Aldrich syndrome, platelet disorder, integrins, fibrin clot retraction, phosphatidylserine exposure

Wiskott-Aldrich syndrome (WAS) is an X-linked combined immunodeficiency and platelet disease. The immune defects, opportunistic infections, eczema and autoimmune disorders, are of varying clinical severity (Sullivan, et al 1994). The platelet component consisting of profound thrombocytopenia (5,000 to 50,000 platelets per µL) with characteristic small platelet size (effective diameter of 1.82 ± 0.12 µm compared with normal platelet diameter of 2.3 ± 0.12 µm) are highly reliable disease features (Kenney 1990, Sullivan, et al 1994, Oda and Ochs 2000).

WASP, the affected gene, encodes for a scaffolding molecule exclusive to blood cells that functions to integrate cellular activation and cytoskeletal rearrangements. In leukocytes, WASP is an autoinhibited cytosolic protein that is recruited to sites of activation where ligand binding releases autoinhibition. This allows WASP to bind monomeric actin and simultaneously bind and activate actin-related protein complex 2/3 (Arp2/3), a process that nucleates the generation of new actin filaments (Higgs and Pollard 2000, Kim, et al 2000). In keeping with this actin nucleation mechanism, leukocytes of WAS patients and wasp-deficient mice are impaired in physiological responses that require actin filament remodeling such as cell orientation, directed migration, immune synapse formation and proliferative response through T cell receptor mediated signaling.

The fact that thrombocytopenia and small platelets are universally present in WAS, even in newborn patients, speaks for a non-redundant role for WASP in platelets (Sullivan, et al 1994). Studies conducted several decades ago in which patients were transfused with either autologous or donor platelets showed that patient platelets, but not normal platelets, are lost from the circulation at a greatly accelerated rate (Grottum, et al 1969, Murphy, et al 1969, Baldini 1972) (see also Discussion). The etiology of platelet loss remains unknown. Accelerated platelet loss occurred even when patient platelets were transfused to normal recipients (Murphy, et al 1969, Baldini 1972), strongly indicating that patient platelets are intrinsically defective. Because of the well-characterized function of leukocyte WASP as an actin filament nucleating factor, it came as a surprise when one after another of platelet responses that require actin filament remodeling were found to be normal in patient platelets (Gross, et al 1999, Rengan and Ochs 2000, Falet, et al 2002) These include agonist-induced shape change, actin polymerization, elaboration of filopodia, generation of lamellipodia, and even activation of Arp2/3, which, as noted above, partners with WASP in actin filament nucleation in leukocytes. The cumulative results indicate that the critical function of WASP in platelets has not yet been identified.

The hypothesis of this study is that the non-redundant function of WASP in platelets is linked to that of αIIbβ3. This prevalent protein also known as glycoprotein IIb–IIIa (GPIIb-IIIa) complex, belongs to the integrin family of cell adhesion and signaling receptors with capacity to transduce signals bidirectionally (reviewed in (Coller and Shattil 2008)). In response to agonist-initiated interactions that culminate at the cytoplasmic face of the membrane, αIIbβ3 undergoes conformational changes that increase its affinity for ligand fibrinogen, a process known as integrin “inside-out” signal transduction. Integrins can also transmit bound ligand dependent signals into the cytoplasm to initiate biochemical and cytoskeletal changes, a process referred to as “outside-in” integrin signal transduction. Functional linkage of WASP and αIIbβ3 was suggested by the finding that a subset of WASP molecules are localized in platelets in the membrane skeleton (Lutskiy, et al 2007), the spectrin-rich structure underlying the lipid bilayer that stabilizes the surface membrane and is thought to preposition molecules involved in integrin responses (Hartwig and DeSisto 1991, Fox 1993). In response to activating conditions that initiate integrin outside-in signaling (thrombin plus stirring), this pool of WASP participates along with integrin αIIbβ3 in remodeling of the combined membrane skeleton and cortical cytoskeleton, and these changes fail to occur in αIIbβ3-deficient platelets or on inhibition of outside-in integrin signaling (Lutskiy, et al 2007).

To test the hypothesized function, we compared WASP-expressing normal platelets and WASP-deficient patient platelets for inside-out αIIbβ3 signaling and for a series of functional responses that require αIIbβ3 integrin outside-in signaling. We also subjected platelets of wasp gene-targeted mice to similar analyses, proceeding cautiously - bearing in mind that these animals lack key features of the patients’ platelet disease. Whereas the patients are subject to hemorrhage syndrome due to profound thrombocytopenia, wasp gene targeted mice are thought to have modestly decreased platelet number and do not have a bleeding tendency (Snapper, et al 1998, Zhang, et al 1999). Also, they lack reduced platelet size, the hallmark of the human disease. We demonstrate here that patient platelets and wasp-deficient murine platelets, when isolated and examined ex vivo, have comparable functional defects, and these defects relate to integrin outside-in dependent physiological responses.

Methods

Platelets

Blood was collected under protocols including written informed consent approved by the Institutional Review Boards of the Immune Disease Institute (formerly the Center for Blood Research) (Boston, MA) and the Research Institute for Paediatric Haematology (Moscow, Russia). WAS diagnosis was based on male sex, thrombocytopenia with small platelets, eczema and, in all but one case, DNA sequencing (Table 1). Healthy adults, primarily males, served as control population for adult patients and a minority of pediatric patients. Control blood for most pediatric patient samples consisted of “discarded materials”, i.e., portions of blood samples from normal boys that remained on completion of clinical studies. All patients had undergone splenectomy, an intervention that increases platelet number and size toward normal (Corash, et al 1985), except for 2 of the 6 patients studied for αIIbβ3 expression and 1 of 7 studied for procoagulant response.

Table I.

Summary of patient characteristics

Patient Age
(years)		 Mutation Splenectomy Platelet count
(×109/L)
W1 20 years Exon 2, G291 to A, Arg86 to His + 169
W2 7 years Intron 3, t insertion (+2) + 206
W3 0.83 year Intron 3, 323 bp deletion at (+15) −/+ *n.a.
W4 8 years Exon 4, A insert 476 − 8
W5 48 years Intron 6, g to a (+5) + 211
W6 12 years Intron 8, g to a (+1) + 118
W7 3 years Intron 8, del (+3–6) − 40
W8 26 years Exon 10, G1305 delete + 253
W9 16 years Exon 10, G1487 to A + 180
W10 17 years Exon 10, G1487 to A + 240
W11 6 years Unknown + 210
Open in a new tab
*

n.a. = not available

Fresh blood samples collected in 1/6 volume of acid-citrate-dextrose (ACE) were centrifuged at 200 g for 15 min. Platelet-rich plasma (PRP) was combined with ACD (1/3 volume) and prostacyclin (1 µg/ml). The platelets were pelleted, resuspended in modified TES/Tyrodes (10 mmol/l TES, pH 7.2, 136 mmol/l NaCl, 2.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 2 mmol/l MgCl2, 0.1% glucose) with 0.1% bovine serum albumin (Tes/Tyrodes/BSA) and, after addition of ACD (1/5 volume) and prostacyclin (1 µg/ml), were repelleted. Unless otherwise indicated, the platelets were resuspended at 2–5 × 108 /ml in TES/Tyrodes/BSA, counted on a Coulter model AcT diff2 (Beckman Coulter, Fullertown, CA), and incubated at 37°C for 1 h to ensure resting state. Murine blood was collected by heparinized capillary puncture of the retro-orbital venous sinus of anesthesized mice (ketamine/xylazine) into measured ACD. The blood was diluted with TES/Tyrodes/BSA containing ACD and centrifuged to obtain PRP and processed as described above. Mean volume of murine platelets was determined by whole blood analysis on a Hemavet 950FS (Drew Scientific Inc, Waterbury, CT).

Mice

Wasp gene-targeted mice generated on the 129Sv background (Snapper, et al 1998) and backcrossed to C57BL/6J for 5 generations (Strom, et al 2002) were provided by Drs. John Cunningham and Arthur Nienhuis (St. Jude Children’s Research Hospital, Memphis, TN). Backcrossing to C57BL/6J was continued to generation 12. Male and female mice were studied for platelet count and size (Fig 1A); all other studies were performed with wasp gene-targeted male mice (hemizogotes) (wasp−/ ) compared to same age wild type males (wasp+/ ). The symbol wasp− is used throughout to indicate mice and platelets lacking the wasp gene.

Fig 1.

Fig 1

Open in a new tab

WASP deficiency in mice is associated with decreased platelet number, but has no significant effect on inside-out regulation of integrin αIIbβ3. (A) Platelet count of whole blood of wasp− and wasp+ mice, 5–10 weeks old, in the C57BL/6 background. Mean ±SEM for 24 mice each genotype. (B,C) Inside-out αIIbβ3 signaling. Pooled platelets of wasp− and wasp+ mice were treated with the indicated agonists, and surface densities of activated αIIbβ3 were quantified by binding of (B) Alexa Fluor-488-labeled fibrinogen or (C) PE-JON/A antibody to active αIIbβ3. The data are the mean ± SEM of 4 experiments in which pooled platelets of the two genotypes were compared. P >0.05 for all comparisons between genotypes.

Inside-out regulation of integrin activation

Isolated rested platelets (106) in TES/Tyrodes/BSA with 1 mmol/l CaCl2 were combined with Alexa Fluor-488 coupled fibrinogen (20 µg/l) (Invitrogen, Carlsbad, CA), or phycoerythrin (PE) coupled JON/A monoclonal antibody (mAb) (Emfret, Eibelstadt, Germany), or fluorescein isothiocyanate (FITC) conjugated PAC-1 mAb (BD Biosciences, San Jose, CA). Platelet activation was initiated with thrombin (bovine) (Sigma, St Louis, MO) or ADP plus epinephrine (Sigma) or murine protease-activated receptor 4 (PAR4) activating peptide (AYPGKF-NH2) (Bachem, King of Prussia, PA) (Faruqi, et al 2000), or (human) thrombin receptor activating peptide (TRAP) (SFLLRN-NH2) (Sigma) (Vu, et al 1991). After gentle mixing, the reactions were incubated for 10 (human) or 30 (mouse) min at ~22°C, conditions established as limiting in preliminary experiments. The reactions were stopped with 2% paraformaldehyde, and the fixed platelets were stored at 4°C until analyzed by flow cytometry.

Platelet adhesion and spreading on fibrinogen

Washed platelets (1.5 × 107/ml) in TES/Tyrodes were allowed to rest in the presence of 10 µM indomethacin (Sigma) for 1 h at 37°C. After addition of apyrase (2 U/mL)(Sigma), the platelets were added to two-chamber slides that had been coated with human fibrinogen (Enzyme Research Laboratories, South Bend, IN) (100 µg/ml) for 2 h at ~22°C and blocked with 2% fetal bovine serum. The platelets were allowed to adhere and spread for 20 – 60 min at ~22°C. The slides were washed with PBS, fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 at ~22°C. The adherent platelets were incubated with phalloidin conjugated with FITC or Alexa Fluor-594 (Invitrogen) for 20–30 min at ~22°C, washed, and air dried briefly. Coverslips were affixed with Gel/Mount (Biomeda, Foster City, CA) and the platelets were stored at 4°C until examined by fluorescent microscopy on the Zeiss Axioplan (Zeiss, Thornwood, NY). Images of platelets were obtained with a 100× objective. Adherent platelets were enumerated using Zeiss Axiovision software.

To assess spreading, individual on-screen images of platelets in a total of 8 fields (area/field of 5200 µm2) were visually evaluated and scored as “non-spread” (round or discoid platelets with zero or one pseudopodial projection and no evidence of flattening) vs. “spread” (platelets with multiple filopodial and/or lamellipodial protrusions or platelets that were partially or fully flattened and spread (Goodman 1999). Independent blind scoring by a second evaluator produced similar scores. In an additional series, murine wasp− and wild type platelets were allowed to adhere to fibrinogen slides in absence of agonist or presence of PAR4 activating peptide (0.5 mM) or ADP (50 µM). Indomethacin and apyrase were omitted from agonist-treated platelets. Platelet adherence/spreading was evaluated as surface area covered per field (8 fields were evaluated) as determined by image analysis using Image-J Software (NIH, Bethesda, MD).

Flow cytometry

To measure αIIbβ3 surface density, washed platelets (human) or whole blood (mouse) were incubated with antibodies to human (BD Biosciences) or murine (Emfret) CD41a (αIIb) or CD42a (an internal control) (5 µg/ml) for 20 min at ~22°C. Platelet samples were acquired on a FACS-Calibur flow cytometer (BD Biosciences) and analyzed with Flowjo software (Tree Star Inc, Ashland, OR). The lower limit of the platelet gate was defined on the forward scatter profile of resting platelets, or for detecting FITC-annexin V, platelets were further gated on fluorescence of bound PE-CD41 mAb.

Clot retraction

CaCl2 was added (2 mM) was added to isolated rested platelets at 2 × 108/l in TES/Tyrodes/BSA, and 250 µl was prewarmed to 37°C in flat-bottom borosilicate tubes (National Scientific, Thermo Scientific) containing a cushion of 7.5% polyacrylamide (Osdoit and Rosa 2001, Racanelli, et al 2003). Clotting was initiated by adding fibrinogen (human) (5 µL of 50 mg/mL) (Enzyme Research Laboratories, South Bend, IN) and thrombin (bovine) (250 l of 0.2 unit/ml) in TES/Tyrodes followed by brief pipetting. Incubation at 37°C was continued for 2 h. The clots were photographed at intervals, and two-dimensional clot size was determined by Image-J image Analysis Software.

Bleeding/rebleeding time

All male pups born to heterozygous (wasp+/−) female mice were studied at 4 to 8 weeks prior to genotyping so that the tails would be intact. The mice were sedated with ketamine/xylazine, and 3-mm of the distal tip of the tails was cut with a scalpel, and the tails were immediately immersed in isotonic saline at 37°C. The time for the flow of blood to cease was recorded as the bleeding time. The mice were monitored for an additional 10 min, and if tail bleeding restarted, incidence was recorded. Tail tips were used to genotype the mice.

Phosphatidylserine (PS) exposure

Isolated rested platelets (107 in 200 µl) in TES/Tyrodes/BSA with 2 mmol/l CaCl2 were placed in siliconized 7× 45 mm glass cuvettes (Sienco, Arvada, CO) under stirring. Thrombin (bovine, 1 U/ml) plus collagen (20 µg/ml) (Collagen reagent Horn, Nycomed Pharma, Munich) were added, and after an initial mixing, incubation was continued without stirring at 37° C. After varying time, 50 µl aliquots were transferred to a ~22°C bath and, after 1 min, combined with FITC-conjugated annexin V (1 µg/ml, Santa Cruz, Santa Cruz, CA) and PE-conjugated CD41a (GPIIb) mAb (BD Pharmingen) and incubated for 10 min. Samples were diluted 5-fold with TES/Tyrodes/BSA with 2 mmol/l CaCl2 for immediate flow cytometry.

For spontaneous PS exposure, isolated murine platelets at 5 × 107/ml in TES/Tyrodes/BSA were incubated without stimulating agent at ~37°C for varying time. Aliquots (10 µl) were incubated with Alexa Fluor-488 coupled lactadherin (Shi, et al 2008b) (60 ng/ml) for 10 min at ~22°C, diluted 10-fold, and analyzed by flow cytometry. To correct for autofluorescence, MFI of platelets incubated without lactadherin was subtracted. Data from multiple experiments were normalized by considering MFI values of wasp− platelets at 3 h as 100 relative units.

Data Analysis

Results of 3 or more experiments are presented as mean ± standard error of the mean (SEM). Two-tailed Student t test was used to compare the difference between groups, except for time course studies for which differences between groups were evaluated by 2 way ANOVA, and rebleeding frequency for which difference between groups was assessed by the χ2 test. A probability value (P) of 0.05 or less was considered statistically significant.

Results

Deficiency of murine wasp results in decreased platelet number, but has no significant effect on inside-out regulation of integrin αIIbβ3

Wasp gene-targeted (wasp−) mice were described a decade ago (Snapper, et al 1998, Zhang, et al 1999), but their integrin mediated platelet responses have not been directly examined. Because the long term goal of studying platelet wasp is to understand its connection to the thrombocytopenia and small platelets of patients, we first measured platelet number and size of the gene-targeted mice. For wasp− mice in C57BL/6 background, platelet number was 380,000 ± 25,000/µL compared to 779,000 ± 54,000/µL for wild type mice (Fig 1A). The extent of the platelet deficit (51% decrease), which is similar to a recent report (Prislovsky, et al 2008), is substantially greater than that of wasp− mice on 129Sv background from which this colony was derived (29% decrease) (Snapper, et al 1998). In this respect, C57BL/6 wasp− mice come closer to replicating the human platelet defect. In contrast, platelet size did not differ between the genotypes (4.5 ± 0.1 fL for wasp− platelets; 4.5 ± 0.2 fL for wild type, n=11 each genotype).

To evaluate inside-out signaling, we first examined αIIbβ3 expression. Surface density, measured by antibody binding, was not different between wasp− and wild type platelets for αIIb subunit (average MFI of 432 vs. 427) or the internal reference protein GPIX (average MFI of 187 vs.181). Accordingly, wasp absence does not affect expression of αIIbβ3 in murine platelets. To measure inside-out αIIbβ3 signaling, platelets were incubated with epinephrine plus ADP or thrombin receptor PAR4 activating peptide (PAR4p) at ambient temperature in the absence of stirring. Levels of bound fibrinogen, a measure of active αIIbβ3, were increased in response to both agonists, but did not differ between the two genotypes (Fig 1B). Likewise, no difference was found between wasp− and wild type platelets when JON/A antibody was used to quantify active αIIbβ3, and thrombin was tested as an additional agonist (Fig 1C). Incubation at higher temperature (37°C) caused greater inside-out signaling as shown by increased activated αIIbβ3 (Fig 1B, C), verifying the limiting conditions. The findings indicate that inside-out αIIbβ3 integrin signaling is not significantly altered by wasp absence in murine platelets, a finding that contrasts with published findings of decreased inside-out αIIbβ3 integrin signaling for WAS patient platelets (Tsuboi, et al 2006).

Absence of murine wasp decreases integrin αIIbβ3 outside-in dependent responses initiated by immobilized fibrinogen

To determine the functional consequences of wasp deletion on integrin αIIbβ3 outside-in initiated events, platelets were evaluated for binding and spreading on fibrinogen-coated surfaces. Immobilized fibrinogen, unlike soluble fibrinogen, binds non-activated αIIbβ3 on the platelet surface and initiates outside-in signal transduction resulting in increased platelet adherence and spreading (adhesion strengthening), which occur in the absence of agonist-induced changes (Savage and Ruggeri 1991). Precautions to ensure specificity for fibrinogen-initiated events included blocking of secondary agonists thromboxane A2 and ADP with indomethacin and apyrase, respectively. Fluorescence microscopy revealed a role for wasp evidenced by the decreased number of adherent wasp− platelets compared to wild type (Fig 2A, B). To assess spreading, platelets were scored as “non-spread” vs. “spread” (Methods); the rationale for this approach is outlined in the Discussion. The frequency of spread wasp− platelets (mean 69–72%) was decreased compared to wild type (mean 84–87%). The significant role for wasp in integrin outside-in dependent platelet adhesion strengthening is evidenced by the combined effect, i.e., the decreased number of spread wasp− platelets compared to wild type (Fig 2C). Spread platelets were also quantified by an independent method, surface area imaging. The area covered by adherent platelets was decreased for wasp− platelets compared to wild type platelets (Fig 2 D, E, left). The findings indicate that wasp is required for platelet adhesion strengthening initiated by fibrinogen-mediated outside-in signaling through αIIbβ3. In the presence of agonists PAR4 activating peptide or ADP, surface area covered was increased, and there was no difference between wasp− and wild type platelets (Fig 2D, E, middle and right), consistent with the earlier finding that wasp is dispensable for PAR4 peptide-induced or ADP-induced activation of αIIbβ3.

Fig 2.

Fig 2

Open in a new tab

WASP deficiency of murine platelets decreases integrin αIIbβ3 outside-in dependent platelet adhesion and spreading initiated by immobilized fibrinogen. (A–C) Wasp− and wasp+ platelets adherent on fibrinogen monolayers (in the absence of agonist) were stained with Alexa Fluor-594-phalloidin. Bars = 10 µm. (B) Adherent platelets per fixed area enumerated by Zeiss Axiovision software. (C) Spread platelets per fixed area (adherent platelets × percent spread). The data are the mean ± SEM of 3 experiments in which pooled platelets of the two genotypes were compared. (D, E) Wasp− and wasp+ platelets were allowed to adhere to fibrinogen for 45 min without agonist (left) or with PAR4 peptide (PAR4p)(middle) or ADP (right). (E). Surface area covered by platelets (µm2) determined by Image J software. The data are the mean ± SEM of 3 experiments in which pooled platelets of the two genotypes were compared except for ADP-treated platelets, which are the average of two experiments. *P = 0.01 for the comparison of wasp− and wasp+ platelets in the absence of agonist.

WASP deficiency in patient platelets decreases αIIbβ3 integrin outside-in dependent responses initiated by immobilized fibrinogen, but has no significant effect on inside-out regulation of this integrin

To examine patient platelets, we first verified that αIIbβ3 surface levels are not different between patient and normal platelets; MFI for αIIb was 366 ± 121 for 6 patients compared to 348 ± 108 for 6 normal individuals. Information on the WAS patients studied is in Table I. Platelets were then evaluated for integrin inside-out signaling by incubation with limiting dose of various agonists in the presence of fluorescently labeled PAC-1 antibody specific for active αIIbβ3. Levels of bound PAC-1 were increased in response to thrombin, epinephrine plus ADP, and thrombin receptor activating peptide (TRAP), but did not differ between the genotypes (Fig 3A). Hence, despite the observations by others (Tsuboi, et al 2006), we found that inside-out integrin αIIbβ3 signal transduction is normal in patient platelets.

Fig 3.

Fig 3

Open in a new tab

WASP deficiency of patient platelets has no significant effect on regulation of αIIbβ3 integrin, but decreases outside-in dependent platelet adhesion and spreading initiated by immobilized fibrinogen. (A) Inside-out αIIbβ3 signaling. Washed platelets of WAS patients and normal control individuals were treated with the indicated agonists, and activated αIIbβ3 was quantified by binding of FITC-PAC-1 antibody. The data are the mean ± SEM of 4 experiments performed with platelets from 4 splenectomized patients and 4 normal control individuals. P >0.05 for all comparisons between genotypes. (B–D) Platelet adhesion and spreading on immobilized fibrinogen. (B) Platelets that adhered on fibrinogen-coated slides (in the absence of agonist) stained with FITC-phalloidin. Bars = 10 µm. (C) Adherent platelets per fixed area enumerated by Axiovision software. (D) Spread platelets per fixed area quantified as described in text. The data are the mean µ SEM of 3 experiments performed with platelets from 3 splenectomized patients and 3 normal control individuals.

The effect of WASP deficiency of human platelets on outside-in integrin dependent responses was evaluated by platelet adhesion on immobilized fibrinogen. Although the difference between the genotypes was less robust than in the murine system, a role for WASP in human platelet adhesion strengthening is evidenced by the decreased number of adherent patient platelets compared to normal platelets (Fig 3 B, C). The frequency of spread patient platelets determined by visual scoring (mean 68–74%) was decreased compared to normal platelets (mean 80–85%). This method does not account for the extensive spreading of individual normal platelets and thus may underestimate the spreading defect. Nonetheless, the role for WASP in outside-in integrin transduced adhesion strengthening of human platelets is demonstrated by the decreased number of spread patient platelets compared to normal platelets (Fig 3D).

Retraction of fibrin clots is impaired in WASP-deficient human and murine platelets

To complement the studies of integrin outside-in responses initiated by immobilized fibrinogen, we examined the impact of WASP deficiency on more complex physiological responses. Fibrin clot retraction, a classical integrin outside-in dependent function important in thrombus consolidation and stabilization, is driven by myosin-mediated contraction of the actin (Schoenwaelder, et al 1997, Jenkins, et al 1998). Washed platelets were prepared and coagulation was induced by adding soluble fibrinogen and thrombin. Fibrin clots formed rapidly for both genotypes, and retraction was monitored over time. Whereas normal platelets efficiently retracted the fibrin clot, retraction was less efficient for platelets of WAS patients (Fig 4A–D). To eliminate the possibility that decreased clot retraction by patient platelets is due to their smaller size, same-size wasp− and wild type murine platelets, were also examined. Clot retraction was also less robust for wasp− platelets compared to wild type (Fig 4E, F). On extended incubation, retraction of all fibrin clots approached completion (not shown), indicating that the impairment of retraction is due to an effect on rate. Because the studies were performed with washed platelets, potential effects of plasma components including fibrinogen were eliminated. Consequently, the results indicate that the decreased rate of clot retraction associated with WASP-deficiency is an intrinsic platelet defect.

Fig 4.

Fig 4

Open in a new tab

WASP deficiency impairs fibrin clot retraction and is associated with increased frequency of rebleeding in a murine tail cut model. (A–F) Fibrin clot retraction. (A,C) Retraction over time of platelet-containing fibrin clots of 2 splenectomized WAS patients and 2 normal control individuals. (B,D) Percent retraction of the clots in A and C (plasma area/original clot area × 100). (E,F) Clot retraction of platelet-containing fibrin clots of wasp− and wasp+ mice. (E) Representative experiment. (F) Mean percent clot retraction ± SEM for 4 experiments in which pooled platelets of the two genotypes were compared. (G,H) Bleeding time and frequency of rebleeding in a mouse tail cut model. The distal tip of the tail (3 mm) was severed, and the tail was immersed in saline and monitored until the flow of blood ceased. (G) Bleeding time for wasp− male mice (n =19), 5–8 weeks old, and wasp+ male littermates (n=30). Symbols indicate values for individual mice; horizontal lines indicate the mean. (H) Frequency of rebleeding. The mice were monitored for an additional 10 min and incidence of rebleeding recorded (P =0.006, χ2 test).

Primary hemostasis is normal, but stabilization of the platelet clot is impaired in wasp− mice

The murine system was next used to evaluate the impact of wasp deletion on primary hemostasis and stabilization of the primary platelet plug. The latter process is an integrin outside-in transduced response (Law, et al 1999). The time to hemostasis following tail cut (bleeding time) was not different between wasp− and wild type mice (Fig 4G), consistent with reports that wasp− mice do not have a bleeding tendency (Snapper, et al 1998, Zhang, et al 1999). On the other hand, rebleeding was more frequent for wasp− mice (14 of 19 mice) compared to wild type mice (14 of 30) (Fig 4H), suggesting that deficiency of murine wasp impairs stabilization of the primary platelet plug.

Procoagulant response is increased in WASP-deficient human and murine platelets

We reported earlier an effect of WASP absence on the procoagulant response of patient platelets. We could show that both spontaneous, and calcium ionophore-induced, exposure of phosphatidylserine (PS) and release of microparticles were increased for platelets of WAS patients compared to normal platelets (Shcherbina, et al 1999). To better understand the role of WASP in procoagulant responses, we re-examined patient platelets - this time using the physiological agonist thrombin plus collagen. Exposure of PS was assessed by annexin V binding. As previously reported for the calcium-ionophore induced process, the conversion of platelets from annexin V-negative to annexin V-positive following thrombin plus collagen addition occurred more rapidly and was more extensive for patient platelets compared to normal platelets (Fig 5A). PS exposure in response to thrombin plus collagen was also more rapid and more extensive for wasp− murine platelets compared to wild type (Fig 5B). For the murine system, we also examined spontaneous low level exposure of PS in the absence of stimulating agent. In this case, exposed PS was detected by lactadherin, a sensitive reagent that binds to platelets in proportion to the amount of PS exposed (Shi, et al 2008a). Lactadherin binding was greater for wasp− platelets than wild type platelets on incubation for 3 h without stimulating agent (Fig 5C,D), indicating that spontaneous PS exposure is increased in wasp− murine platelets as previously shown for platelets of WAS patients.

Fig 5.

Fig 5

Open in a new tab

WASP deficiency enhances platelet phosphatidylserine exposure. (A,B) Exposure of PS on WASP-deficient and control platelets treated with thrombin plus collagen for the indicated time. Exposed PS was assessed by binding of FITC-annexin V. (A) WAS patient platelets compared to normal platelets. Mean percent annexin-V+ platelets ± SEM for platelets of 7 patients and 7 normal control individuals. (B) Wasp− murine platelets compared to wasp+ platelets. Mean percent annexin-V+ platelets ± SEM for 6 experiments in which pooled platelets of the two genotypes were compared. (C,D) Spontaneous PS exposure for murine wasp− and wasp+ platelets. Washed platelets of each genotype were incubated for 0–3 h in the absence of stimulating agent. Exposed PS was assessed by subsequent incubation with Alexa Fluor-488 coupled lactadherin. (C) Histogram of pooled wasp− and wasp+ platelets at 0 and 3 h from a representative experiment. (D) Lactadherin binding expressed as mean relative MFI ± SEM for 8 comparisons of pooled platelets of the two genotypes.

Discussion

Activation of αIIbβ3, detected by binding of conformation-specific antibody or fibrinogen (murine platelets) was not different for WAS patient platelets compared to platelets of normal healthy donors or wasp− murine platelets compared to wild type platelets on treatment with thrombin, or epinephrine plus ADP, or thrombin receptor activating peptide. These findings indicate that integrin αIIbβ3 inside-out signaling is normal in the absence of WASP. In contrast, a direct integrin αIIbβ3 outside-in initiated response, platelet adhesion and spreading on immobilized fibrinogen, was decreased for patient platelets and wasp− murine platelets compared to respective control platelets. A more complex outside-in dependent response, retraction of fibrin clots, was also decreased for patient platelets and wasp− murine platelets compared to control platelets. Frequency of rebleeding from tail cuts was increased for wasp− mice, suggesting decreased stability of the primary platelet plug. In contrast to these deficient responses, PS exposure induced by thrombin plus collagen was enhanced for WASP-deficient patient and murine platelets compared to control platelets. Cumulatively, the findings demonstrate that integrin αIIbβ3 outside-in transduced pro-aggregatory and pro-coagulant responses are altered when WASP is absent, indicating that WASP plays a non-redundant role in regulating alterations of platelets post integrin ligation.

The normal agonist-induced αIIbβ3 activation of WASP-deficient platelets reported here is consistent with previous findings of normal shape change, actin polymerization, elaboration of filopodia, generation of lamellipodia, and activation/deposition of Arp2/3 (Gross, et al 1999, Rengan and Ochs 2000, Falet, et al 2002). The cumulative results strongly suggest that WASP is not required for actin nucleation events that are independent of integrin outside-in signal transduction. As noted, the normal integrin inside-out signaling reported here differs from a previous report for patient platelets treated with ADP and thrombin (Tsuboi, et al 2006). A possible explanation for the discrepant findings between the two studies might be methodological differences.

The current findings place WASP within the overall platelet activation process in temporal terms by showing that its non-redundant role occurs post-integrin ligand binding. The issue of location (subcellular site) was addressed in an earlier study in which activation of normal platelets under aggregating conditions mobilized the subset of WASP molecules that are localized in the membrane skeleton (Lutskiy, et al 2007). The latter is the spectrin-rich structure that underlies the surface membrane lipid bilayer, connects with the cortical cytoskeleton, and provides mechanical stability to the platelet surface (Hartwig and DeSisto 1991, Fox 1993). In response to thrombin plus stirring, membrane skeletal WASP molecules participate along with αIIbβ3 in remodeling of the combined membrane skeleton-cortical cytoskeleton network leading to altered distribution of both molecules, changes that failed to occur in αIIbβ3-deficient platelets or in normal platelets on inhibition of outside-in integrin signaling (Lutskiy, et al 2007).

Outside-in signaling denotes reactions initiated by integrin ligation and clustering that lead to an incompletely-delineated pathway(s) allowing extracellular matrix proteins to communicate via integrins with intracellular cytoskeletal and signaling components. Early molecular events include autophosphorylation and activation of Src tyrosine kinase molecules bound in resting platelets to integrin β3 cytoplasmic tails (Obergfell, et al 2002, Arias-Salgado, et al 2003). The role of platelet WASP at the molecular level remains to be addressed; however, it is known that integrin outside-in initiated events include the activation of WASP by tyrosine phosphorylation (Soriani, et al 2006). The cumulative evidence suggests a model whereby WASP of resting platelets is primed for activation by its localization in the membrane skeleton and, once activated by outside-in signaling, functions by generating actin filaments, possibly linking the plasma membrane to the cortical cytoskeleton. We speculate that these WASP-dependent plasma membrane-cortical cytoskeleton linkages strengthen and sustain platelet adhesiveness and support platelet spreading.

The finding of parallel aberrancies of patient platelets and wasp− murine platelets is significant because the gene-targeted mice, which lack the hemorrhage syndrome and small platelet size, are at best an imperfect disease model. A recent study of wasp− mice identified both accelerated platelet clearance and impaired platelet production (Prislovsky, et al 2008). Compelling evidence for accelerated clearance of patient platelets is provided by the beneficial effects of splenectomy on platelet number (Corash, et al 1985) and the high density of patient platelets in close association with splenic macrophages (Shcherbina, et al 1999). Also, greatly accelerated platelet clearance was detected in three studies of transfused patient platelets (Grottum, et al 1969, Murphy, et al 1969, Baldini 1972), but a fourth study found consumption only modestly accelerated, suggesting a role for inefficient platelet production (Ochs, et al 1980) (reviewed in ref (Strom 2009)). Impaired platelet production is suggested for wasp− mice because their megakaryocytes release proplatelets prematurely within the bone marrow space (Sabri, et al 2006). Interestingly, this defect is caused by defective regulation by the collagen receptor α2β1 (Sabri, et al 2006), another aberrant integrin outside-in dependent response. Although it remains unclear which disease events are replicated in wasp− mice, we conclude on the basis of the current findings that, at least for a range of ex vivo responses, platelets of these mice provide a validated disease model.

Platelet size was a significant factor in study design because of the difficulty of eliminating small size of patient platelets as the cause of functional defects. The potential impact of size was minimized first of all by relying almost exclusively on splenectomized patients with platelet size closer to normal. For platelet spreading studies where size discrepancy would have a major impact in conventional assays, we relied on a “yes-no” visual assessment to distinguish spread and non-spread platelets. We validated this assay by applying it to murine platelets, which were independently evaluated by conventional surface area imaging. The difference of adherence/spreading between the two genotypes of murine platelets were similar for the two methods (Fig 2). Lastly, the finding of parallel aberrancies for wasp− murine platelets compared to wild type and patient platelets compared to normal platelets strongly indicates that these defects are due to absence of WASP.

An in vivo function, time to cessation of bleeding from a tail cut, was not altered in wasp− mice. However, spontaneous rebleeding was more frequent in the absence of wasp. Increased rebleeding frequency reflecting decreased stabilization of the initial platelet plug has been linked to deficient outside-in signaling, e.g., for mice with tyrosine→phenylalanine mutations in the β3-integrin cytoplasmic tail (Law, et al 1999) and this may be the cause of more frequent rebleeding of wasp− mice. However, because platelet number is substantially decreased in these wasp− mice, we can’t rule out a contributory role for low platelet number.

In the fibrin clot studies, patient platelets and wasp− murine platelets were be less efficient than control platelets at retracting clots. In this process, essentially myosin-mediated contraction of the actin cytoskeleton, αIIbβ3 in the surface membrane interacts simultaneously with extracellular fibrin and with the cortical cytoskeleton. Successful fibrin clot retraction requires that the dynamic post-aggregation platelet cytoskeletal network develop sufficient tension on the fibrin clot to cause retraction (Schoenwaelder, et al 1997, Jenkins, et al 1998). Accordingly, the lower rate of fibrin clot retraction by WASP-deficient platelets is consistent with the postulated function for WASP in regulating post-aggregation dynamic remodeling of the membrane skeleton-cortical cytoskeleton network.

Exposure of PS, the final response studied, involves translocation of this anionic lipid from the inner to the outer bilayer of the platelet surface membrane, creating binding sites for coagulation factors that support thrombin generation (Bevers, et al 1982, Heemskerk, et al 2002). Reports linking exposure of platelet PS to integrin engagement include decreased tissue factor-induced thrombin generation on blockage of integrins (Reverter, et al 1996). Whereas the integrin outside-in dependent responses discussed thus far were attenuated when WASP was absent, accumulation of PS on the outer membrane surface was more robust for patient platelets and wasp− murine platelets than control platelets, both spontaneously and on stimulation with agonist. The finding of enhanced exposure of PS by WASP-deficient platelets challenged with thrombin plus collagen extends a previous finding for patient platelets treated with Ca2+ ionophore (Shcherbina, et al 1999). In that study, microparticle release, a closely linked integrin outside-in dependent procoagulant response (Gemmell, et al 1993), was also increased. The conversion of pro-aggregatory platelets to the procoagulant state is a striking functional platelet response, the regulation of which is incompletely understood. The findings reported here indicate that, under certain conditions of outside-in signaling, the transition from proaggregatory phenotype to procoagulant phenotype is facilitated in platelets that lack WASP.

To interpret the cumulative findings, we propose that integrin αIIbβ3 outside-in dependent responses that require strengthened plasma membrane-cortical cytoskeleton interactions, i.e., platelet adhesion, spreading, clot retraction and platelet plug stabilization, are deficient in platelets that lack WASP. The corollary is that platelet responses that are negatively regulated by stable actin filament linkages between the plasma membrane and the cortical cytoskeleton, e.g., release of PS-expressing microparticles (Fox, et al 1990, Cauwenberghs, et al 2006, Flaumenhaft 2006, Kulkarni, et al 2007), are facilitated when WASP is absent. This interpretation is consistent with a novel regulatory role for WASP post-integrin ligation in generating plasma membrane-cortical cytoskeleton linkages that strengthen and sustain platelet adhesion and spreading and delay the conversion of platelets to the procoagulant phenotype.

Acknowledgements

We thank Drs. John Cunningham and Arthur Nienhuis (St. Jude Children’s Research Hospital, Memphis, TN) for wasp− mice, Dr. Susanna Remold for advice on statistical analysis, and Tessa LeCuyer and Michael Stolley for animal husbandry. We are grateful to the patients and control blood donors and to Drs. Diana Beardsley, Francisco Bonilla, Raif Geha, Erwin Gelfand and the late Fred S. Rosen for procuring blood specimens. This work was supported by National Institutes of Health grants AI039574, HL59561, HL081407 and the Jeffrey Modell Foundation.

Footnotes

Author contributions

AS, JC, ER designed experiments, AS, JC, ML, CB performed research and collected data; GG contributed vital new reagent, AS, JC, CB, GG, ER analyzed and interpreted data, ER wrote the paper.

References

  1. Arias-Salgado EG, Lizano S, Sarkar S, Brugge JS, Ginsberg MH, Shattil SJ. Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc Natl Acad Sci U S A. 2003;100:13298–13302. doi: 10.1073/pnas.2336149100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldini MG. Nature of the platelet defect in the Wiskott-Aldrich syndrome. Ann N Y Acad Sci. 1972;201:437–444. doi: 10.1111/j.1749-6632.1972.tb16316.x. [DOI] [PubMed] [Google Scholar]
  3. Bevers EM, Comfurius P, van Rijn JL, Hemker HC, Zwaal RF. Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem. 1982;122:429–436. doi: 10.1111/j.1432-1033.1982.tb05898.x. [DOI] [PubMed] [Google Scholar]
  4. Cauwenberghs S, Feijge MA, Harper AG, Sage SO, Curvers J, Heemskerk JW. Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton. FEBS Lett. 2006;580:5313–5320. doi: 10.1016/j.febslet.2006.08.082. [DOI] [PubMed] [Google Scholar]
  5. Coller BS, Shattil SJ. The GPIIb/IIIa (integrin alphaIIbbeta3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood. 2008;112:3011–3025. doi: 10.1182/blood-2008-06-077891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Corash L, Shafer B, Blaese RM. Platelet-associated immunoglobulin, platelet size, and the effect of splenectomy in the Wiskott-Aldrich syndrome. Blood. 1985;65:1439–1443. [PubMed] [Google Scholar]
  7. Falet H, Hoffmeister KM, Neujahr R, Hartwig JH. Normal Arp2/3 complex activation in platelets lacking WASp. Blood. 2002;100:2113–2122. [PubMed] [Google Scholar]
  8. Faruqi TR, Weiss EJ, Shapiro MJ, Huang W, Coughlin SR. Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J Biol Chem. 2000;275:19728–19734. doi: 10.1074/jbc.M909960199. [DOI] [PubMed] [Google Scholar]
  9. Flaumenhaft R. Formation and fate of platelet microparticles. Blood Cells Mol Dis. 2006;36:182–187. doi: 10.1016/j.bcmd.2005.12.019. [DOI] [PubMed] [Google Scholar]
  10. Fox JE. The platelet cytoskeleton. Thromb Haemost. 1993;70:884–893. [PubMed] [Google Scholar]
  11. Fox JE, Austin CD, Boyles JK, Steffen PK. Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane. J Cell Biol. 1990;111:483–493. doi: 10.1083/jcb.111.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gemmell CH, Sefton MV, Yeo EL. Platelet-derived microparticle formation involves glycoprotein IIb–IIIa. Inhibition by RGDS and a Glanzmann's thrombasthenia defect. J Biol Chem. 1993;268:14586–14589. [PubMed] [Google Scholar]
  13. Goodman SL. Sheep, pig, and human platelet-material interactions with model cardiovascular biomaterials. J Biomed Mater Res. 1999;45:240–250. doi: 10.1002/(sici)1097-4636(19990605)45:3<240::aid-jbm12>3.0.co;2-c. [DOI] [PubMed] [Google Scholar]
  14. Gross BS, Wilde JI, Quek L, Chapel H, Nelson DL, Watson SP. Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI. Blood. 1999;94:4166–4176. [PubMed] [Google Scholar]
  15. Grottum KA, Hovig T, Holmsen H, Abrahamsen AF, Jeremic M, Seip M. Wiskott-Aldrich syndrome: qualitative platelet defects and short platelet survival. Br J Haematol. 1969;17:373–388. doi: 10.1111/j.1365-2141.1969.tb01383.x. [DOI] [PubMed] [Google Scholar]
  16. Hartwig JH, DeSisto M. The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments. J Cell Biol. 1991;112:407–425. doi: 10.1083/jcb.112.3.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heemskerk JW, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost. 2002;88:186–193. [PubMed] [Google Scholar]
  18. Higgs HN, Pollard TD. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J Cell Biol. 2000;150:1311–1320. doi: 10.1083/jcb.150.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jenkins AL, Nannizzi-Alaimo L, Silver D, Sellers JR, Ginsberg MH, Law DA, Phillips DR. Tyrosine phosphorylation of the beta3 cytoplasmic domain mediates integrin-cytoskeletal interactions. J Biol Chem. 1998;273:13878–13885. doi: 10.1074/jbc.273.22.13878. [DOI] [PubMed] [Google Scholar]
  20. Kenney D. Wiskott-Aldrich syndrome and related X-linked thrombocytopenia. Current Opinions in Pediatrics. 1990;2:932–935. [Google Scholar]
  21. Kim AS, Kakalis LT, Abdul-Manan N, Liu GA, Rosen MK. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature. 2000;404:151–158. doi: 10.1038/35004513. [DOI] [PubMed] [Google Scholar]
  22. Kulkarni S, Woollard KJ, Thomas S, Oxley D, Jackson SP. Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading. Blood. 2007;110:1879–1886. doi: 10.1182/blood-2006-08-040980. [DOI] [PubMed] [Google Scholar]
  23. Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature. 1999;401:808–811. doi: 10.1038/44599. [DOI] [PubMed] [Google Scholar]
  24. Lutskiy MI, Shcherbina A, Bachli ET, Cooley J, Remold-O'Donnell E. WASP localizes to the membrane skeleton of platelets. Br J Haematol. 2007;139:98–105. doi: 10.1111/j.1365-2141.2007.06745.x. [DOI] [PubMed] [Google Scholar]
  25. Murphy S, Oski FA, Gardner FH. Hereditary thrombocytopenia with an intrinsic platelet defect. N Engl J Med. 1969;281:857–862. doi: 10.1056/NEJM196910162811601. [DOI] [PubMed] [Google Scholar]
  26. Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ. Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton. J Cell Biol. 2002;157:265–275. doi: 10.1083/jcb.200112113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ochs HD, Slichter SJ, Harker LA, Von Behrens WE, Clark RA, Wedgwood RJ. The Wiskott-Aldrich syndrome: studies of lymphocytes, granulocytes, and platelets. Blood. 1980;55:243–252. [PubMed] [Google Scholar]
  28. Oda A, Ochs HD. Wiskott-Aldrich syndrome protein and platelets. Immunol Rev. 2000;178:111–117. doi: 10.1034/j.1600-065x.2000.17808.x. [DOI] [PubMed] [Google Scholar]
  29. Osdoit S, Rosa JP. Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation. J Biol Chem. 2001;276:6703–6710. doi: 10.1074/jbc.M008945200. [DOI] [PubMed] [Google Scholar]
  30. Prislovsky A, Marathe B, Hosni A, Bolen AL, Nimmerjahn F, Jackson CW, Weiman D, Strom TS. Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets. Exp Hematol. 2008;36:609–623. doi: 10.1016/j.exphem.2007.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Racanelli AL, Gibbs SK, Zondlo SC, Seiffert DA, Friedman SM, Feuerstein GZ. Differential effect of the GPIIb/IIIa antagonist orbofiban on human platelet aggregate formation in vitro. Thromb Res. 2003;110:99–105. doi: 10.1016/s0049-3848(03)00337-2. [DOI] [PubMed] [Google Scholar]
  32. Rengan R, Ochs HD. Molecular biology of the Wiskott-Aldrich syndrome. Rev Immunogenet. 2000;2:243–255. [PubMed] [Google Scholar]
  33. Reverter JC, Beguin S, Kessels H, Kumar R, Hemker HC, Coller BS. Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis". J Clin Invest. 1996;98:863–874. doi: 10.1172/JCI118859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sabri S, Foudi A, Boukour S, Franc B, Charrier S, Jandrot-Perrus M, Farndale RW, Jalil A, Blundell MP, Cramer EM, Louache F, Debili N, Thrasher AJ, Vainchenker W. Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment. Blood. 2006;108:134–140. doi: 10.1182/blood-2005-03-1219. [DOI] [PubMed] [Google Scholar]
  35. Savage B, Ruggeri ZM. Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb–IIIa on nonactivated platelets. J Biol Chem. 1991;266:11227–11233. [PubMed] [Google Scholar]
  36. Schoenwaelder SM, Yuan Y, Cooray P, Salem HH, Jackson SP. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem. 1997;272:1694–1702. doi: 10.1074/jbc.272.3.1694. [DOI] [PubMed] [Google Scholar]
  37. Shcherbina A, Rosen FS, Remold-O'Donnell E. Pathological events in platelets of Wiskott-Aldrich syndrome patients. Br J Haematol. 1999;106:875–883. doi: 10.1046/j.1365-2141.1999.01637.x. [DOI] [PubMed] [Google Scholar]
  38. Shi J, Pipe SW, Rasmussen JT, Heegaard CW, Gilbert GE. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure. J Thromb Haemost. 2008a;6:1167–1174. doi: 10.1111/j.1538-7836.2008.03010.x. [DOI] [PubMed] [Google Scholar]
  39. Shi J, Pipe SW, Rasmussen JT, Heegaard CW, Gilbert GE. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure. J Thromb Haemost. 2008b doi: 10.1111/j.1538-7836.2008.03010.x. [DOI] [PubMed] [Google Scholar]
  40. Snapper SB, Rosen FS, Mizoguchi E, Cohen P, Khan W, Liu CH, Hagemann TL, Kwan SP, Ferrini R, Davidson L, Bhan AK, Alt FW. Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation. Immunity. 1998;9:81–91. doi: 10.1016/s1074-7613(00)80590-7. [DOI] [PubMed] [Google Scholar]
  41. Soriani A, Moran B, de Virgilio M, Kawakami T, Altman A, Lowell C, Eto K, Shattil SJ. A role for PKCtheta in outside-in alpha(IIb)beta3 signaling. J Thromb Haemost. 2006;4:648–655. doi: 10.1111/j.1538-7836.2006.01806.x. [DOI] [PubMed] [Google Scholar]
  42. Strom TS. The thrombocytopenia of WAS: a familial form of ITP? Immunol Res. 2009;44:42–53. doi: 10.1007/s12026-008-8069-2. [DOI] [PubMed] [Google Scholar]
  43. Strom TS, Li X, Cunningham JM, Nienhuis AW. Correction of the murine Wiskott-Aldrich syndrome phenotype by hematopoietic stem cell transplantation. Blood. 2002;99:4626–4628. doi: 10.1182/blood-2001-12-0319. [DOI] [PubMed] [Google Scholar]
  44. Sullivan KE, Mullen CA, Blaese RM, Winkelstein JA. A multiinstitutional survey of the Wiskott-Aldrich syndrome. J Pediatr. 1994;125:876–885. doi: 10.1016/s0022-3476(05)82002-5. [DOI] [PubMed] [Google Scholar]
  45. Tsuboi S, Nonoyama S, Ochs HD. Wiskott-Aldrich syndrome protein is involved in alphaIIb beta3-mediated cell adhesion. EMBO Rep. 2006;7:506–511. doi: 10.1038/sj.embor.7400665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991;64:1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  47. Zhang J, Shehabeldin A, da Cruz LA, Butler J, Somani AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy A, Grinstein S, Penninger JM, Siminovitch KA. Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. J Exp Med. 1999;190:1329–1342. doi: 10.1084/jem.190.9.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES