Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin alpha2beta1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.     

The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34(+) cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1alpha (SDF-1alpha), and actin normally polymerized after SDF-1alpha or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.     

The WAVE2/Abi1 complex differentially regulates megakaryocyte development and spreading: implications for platelet biogenesis and spreading machinery

Actin polymerization is crucial in throm-bopoiesis, platelet adhesion, and mega-karyocyte (MK) and platelet spreading. The Wiskott-Aldrich syndrome protein (WASp) homolog WAVE functions downstream of Rac and plays a pivotal role in lamellipodia formation. While MKs and platelets principally express WAVE1 and WAVE2, which are associated with Abi1, the physiologic significance of WAVE isoforms remains undefined. We generated WAVE2(-/-) embryonic stem (ES) cells because WAVE2-null mice die by embryonic day (E) 12.5. We found that while WAVE2(-/-) ES cells differentiated into immature MKs on OP9 stroma, they were severely impaired in terminal differentiation and in platelet production. WAVE2(-/-) MKs exhibited a defect in peripheral lamellipodia on fibrinogen even with phorbol 12-myristate 13-acetate (PMA) costimulation, indicating a requirement of WAVE2 for integrin alpha(IIb)beta(3)-mediated full spreading. MKs in which expression of Abi1 was reduced by small interfering RNA (siRNA) exhibited striking similarity to WAVE2(-/-) MKs in maturation and spreading. Interestingly, the knockdown of IRSp53, a Rac effector that preferentially binds to WAVE2, impaired the development of lamellipodia without affecting proplatelet production. In contrast, thrombopoiesis in vivo and platelet spreading on fibrinogen in vitro were intact in WAVE1-null mice. These observations clarify indispensable roles for the WAVE2/Abi1 complex in alpha(IIb)beta(3)-mediated lamellipodia by MKs and platelets through Rac and IRSp53, and additionally in thrombopoiesis independent of Rac and IRSp53.     

Normal Arp2/3 complex activation in platelets lacking WASp

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 microM 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.     

Wiskott-Aldrich syndrome protein deficiency leads to reduced B-cell adhesion, migration, and homing, and a delayed humoral immune response

The Wiskott-Aldrich syndrome protein (WASp) is mutated in the severe immunodeficiency disease Wiskott-Aldrich syndrome (WAS). The function of B cells and the physiologic alterations in WAS remain unclear. We show that B cells from WAS patients exhibited decreased motility and had reduced capacity to migrate, adhere homotypically, and form long protrusions after in vitro culture. WASp-deficient murine B cells also migrated less well to chemokines. Upon antigen challenge, WASp-deficient mice mounted a reduced and delayed humoral immune response to both T-cell-dependent and -independent antigens. This was at least in part due to deficient migration and homing of B cells. In addition, the germinal center reaction was reduced in WASp-deficient mice. Thus, WASp is crucial for optimal B-cell responses and plays a pivotal role in the primary humoral immune response.     

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.     

WASP plays a novel role in regulating platelet responses dependent on αIIbβ3 integrin outside‐in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent –yet– actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by αIIbβ3 integrin outside‐in signalling, we questioned whether its function might be linked to integrin. Agonist‐induced αIIbβ3 activation (PAC‐1 binding) was normal for patient platelets, indicating normal integrin inside‐out signalling. Inside‐out signalling (fibrinogen, JON/A binding) was also normal for 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 responses. Another integrin outside‐in dependent response, fibrin clot retraction, involving contraction of the post‐aggregation actin cytoskeleton, was also decreased for patient platelets and wasp‐deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp‐deficient mice, suggesting decreased stabilisation 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 signalling.     

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2–3) and extensive emperipolesis, with many (36–65%) MKs containing 2–4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.     

The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis

To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp(+) murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp(+) human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease.     

Spirochete-platelet attachment and thrombocytopenia in murine relapsing fever borreliosis

Thrombocytopenia is common in persons infected with relapsing fever Borreliae. We previously showed that the relapsing fever spirochete Borrelia hermsii binds to and activates human platelets in vitro and that, after platelet activation, high-level spirochete-platelet attachment is mediated by integrin alpha IIb beta 3, a receptor that requires platelet activation for full function. Here we established that B hermsii infection of the mouse results in severe thrombocytopenia and a functional defect in hemostasis caused by accelerated platelet loss. Disseminated intravascular coagulation, immune thrombocytopenic purpura, or splenic sequestration did not play a discernible role in this model. Instead, spirochete-platelet complexes were detected in the blood of infected mice, suggesting that platelet attachment by bacteria might result in platelet clearance. Consistent with this, splenomegaly and thrombocytopenia temporally correlated with spirochetemia, and the severity of thrombocytopenia directly correlated with the degree of spirochetemia. Activation of platelets and integrin alpha IIb beta 3 were apparently not required for bacterium-platelet binding or platelet clearance because the bacterium-bound platelets in the circulation were not activated, and platelet binding and thrombocytopenia during infection of beta 3-deficient and wild-type mice were indistinguishable. These findings suggest that thrombocytopenia of relapsing fever is the result of platelet clearance after beta 3-independent bacterial attachment to circulating platelets.     

Accelerated death of megakaryocytes from Wiskott–Aldrich syndrome patients

Wiskott–Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients.     

The tetraspanin superfamily member CD151 regulates outside-in integrin alphaIIbbeta3 signaling and platelet function

The tetraspanin family member CD151 forms complexes with integrins and regulates cell adhesion and migration. While CD151 is highly expressed in megakaryocytes and to a lesser extent in platelets, its physiologic role in platelets is unclear. In this study, we investigate the physical and functional importance of CD151 in murine platelets. Immunoprecipitation/Western blot studies reveal a constitutive physical association of CD151 with integrin alpha(IIb)beta(3) complex under strong detergent conditions. Using CD151-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta(3) signaling with defective platelet aggregation responses to protease-activated receptor 4 (PAR-4) agonist peptide, collagen, and adenosine diphosphate (ADP); impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro. This functional integrin alpha(IIb)beta(3) defect could not be attributed to altered expression of integrin alpha(IIb)beta(3). CD151(-/-) platelets displayed normal platelet alpha granule secretion, dense granule secretion, and static platelet adhesion. In addition, CD151(-/-) platelets displayed normal "inside-out" integrin alpha(IIb)beta(3) signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol 1,4,5 triphosphate (IP(3)) levels. This study provides the first direct evidence that CD151 is essential for normal platelet function and that disruption of CD151 induced a moderate outside-in integrin alpha(IIb)beta(3) signaling defect.     

Rapid platelet turnover in WASP(-) mice correlates with increased ex vivo phagocytosis of opsonized WASP(-) platelets

OBJECTIVE: Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). MATERIALS AND METHODS: Consumption rates of WAS protein (WASP)(-) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(-) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. RESULTS: CMFDA-labeled WASP(-) platelets are consumed more rapidly than WT platelets in either WT or WASP(-) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(-) reticulated platelets. The number of reticulated platelets is reduced in WASP(-) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(-) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(-) platelets are more accelerated by opsonization than are those of WT platelets. CONCLUSION: Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(-) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies.     

Flow cytometric analysis of platelets from childrenwith the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.     

WASP localizes to the membrane skeleton of platelets

Patients with Wiskott-Aldrich syndrome (WAS), an X-linked blood cell disease, suffer from severe thrombocytopenia due to accelerated loss of defective platelets. The affected gene encodes WASP, an actin regulatory protein thought to reside in the cytoplasm of resting leucocytes. In contrast, this study showed that, for platelets, one-quarter of WASP molecules fractionate in the detergent-insoluble high speed pellet characterized as the membrane skeleton, the scaffold structure that underlies the lipid bilayer and stabilizes the surface membrane. Following treatment of platelets with thrombin and stirring, which induces cytoarchitectural remodelling, WASP and other membrane skeletal components sedimented at lower g force and partitioned in the low-speed pellet. Thrombin and stirring also induced WASP tyrosine phosphorylation, a rapid activating reaction, and proteolytic inactivation by cysteine protease calpain. Both the alteration of the sedimentation profile and the proteolytic inactivation were specific for the membrane skeletal pool of WASP and were abrogated in alphaIIb beta3 integrin-deficient platelets and in normal platelets treated with an integrin antagonist. The findings demonstrate that WASP is a component of the resting platelet membrane skeleton and participates in membrane skeletal rearrangements downstream of integrin outside-in signalling. The possible implications for the platelet defect in WAS are discussed.     

The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3

The involvement of the small GTPase Rap1b in platelet integrin alpha2beta1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin alpha2beta1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)-derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-alpha2beta1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin alpha2beta1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCgamma2. In addition, CalDAG-GEFI-deficient platelets showed defective integrin alpha2beta1-dependent adhesion and spreading. We found that outside-in signaling through integrin alpha2beta1 triggered inside-out activation of integrin alphaIIbbeta3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors.     

Dasatinib enhances megakaryocyte differentiation but inhibits platelet formation

Dasatinib is a novel, potent, ATP-competitive inhibitor of Bcr-Abl, cKIT, and Src family kinases that exhibits efficacy in patients with imatinib-resistant chronic myelogenous leukemia. Dasatinib treatment is associated with mild thrombocytopenia and an increased risk of bleeding, but its biological effect on megakaryocytopoiesis and platelet production is unknown. In this study, we show that dasatinib causes mild thrombocytopenia in mice without altering platelet half-life, suggesting that it inhibits platelet formation. Conversely, the number of megakaryocytes (MKs) in the bone marrow of dasatinib-treated mice was increased and the ploidy of MKs derived from bone marrow progenitor cells in vitro was elevated in the presence of dasatinib. Furthermore, a significant delay in platelet recovery after immune-induced thrombocytopenia was observed in dasatinib-treated mice even though the number of MKs in the bone marrow was increased relative to controls at all time points. Interestingly, the migration of MKs toward a gradient of stromal cell-derived factor 1α (SDF1α) and the formation of proplatelets in vitro were abolished by dasatinib. We propose that dasatinib causes thrombocytopenia as a consequence of ineffective thrombopoiesis, promoting MK differentiation but also impairing MK migration and proplatelet formation.     

A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23 [see comments]

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha- granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.