AbstractSeveral patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia.
Integrin αIIbβ3 (GPIIb/IIIa), the main platelet receptor, is a heterodimeric calcium-dependent cell-surface glycoprotein expressed on platelets and megakaryocytes that plays a central role in platelet aggregation and thrombus formation.1 Recent observations suggest that αIIbβ3 is also involved in proplatelet formation.32 Integrin αIIbβ3 function depends on two different signal transduction pathways: inside-out and outside-in signaling. Under resting conditions, αIIbβ3 is expressed on platelets in a bent, inactive conformation. Upon platelet activation, inside-out signaling causes αIIbβ3 extension and headpiece opening, leading the receptor to assume an active conformation, to acquire the ability to bind its ligands, mainly fibrinogen, and to initiate platelet aggregation.4 On the other hand, ligand binding leads αIIbβ3 complexes to cluster, thus triggering outside-in signaling, with phosphorylation of the β3 cytoplasmic tail, activation of signaling proteins (e.g. Src-family kinases and focal adhesion kinase), and ultimately reorganization of the actin cytoskeleton.5 Cytoskeletal remodeling, with actin polymerization and depolymerization, is a finely regulated event,6 and when altered it may lead to impaired platelet function and formation, as observed in patients with mutations of filamin A or with MYH9-RD.87
Mutations of ITGA2B and ITGB3, the genes coding for integrins αIIb and β3, generate Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder characterized by absent platelet aggregation and a normal platelet count and volume, due to quantitative or qualitative defects of αIIbβ3. Heterozygous carriers of GT are usually asymptomatic because 50% of normal αIIbβ3 is sufficient for platelet aggregation,9 but rare autosomal dominant variants of GT, with platelet dysfunction and macrothrombocytopenia, have been associated with gain-of-function mutations of ITGA2B or ITGB3 leading to reduced expression and constitutive activation of αIIbβ3103
We have previously described a hereditary Glanzmann-like platelet disorder transmitted in an autosomal dominant way, associated with macrothrombocytopenia and a bleeding diathesis, due to a heterozygous G>C transversion (c.2134+1G>C) of ITGB3 leading to the deletion of exon 13 and to the loss of 40 amino acids (del647-686) of integrin β3.11 This mutation was the first-described, naturally-occurring deletion of the β-tail domain (β-TD) of integrin β3, the membrane-proximal portion of the extracellular domain of the protein. β-TD contributes to maintain the bent, inactive conformation of αIIbβ312; in fact, the loss of its disulfide bonds causes constitutive activation of αIIbβ3 1513, but no information is available on its role in outside-in signaling. Recently, a Japanese family carrying a heterozygous ITGB3 c.2134+1G>A transversion leading to the same integrin β3 deletion and a similar phenotype was reported.16
A few other heterozygous patients with gain-of-function mutations of ITGB3, mucocutaneous bleeding and macrothrombocytopenia have been reported1917 suggesting that, independently of the mutation, constitutive activation of αIIbβ3 leads to platelet dysfunction and macrothrombocytopenia by a common mechanism that, however, has never been addressed.
Here we show that constitutive activation of integrin αIIbβ3 decreases surface expression of the complex through receptor internalization and permanently triggers outside-in signaling, which leads to altered cytoskeletal reorganization that is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in autosomal dominant GT variant forms.
Blood samples were taken from healthy volunteers and from two patients carrying the ITGB3c.2134+1G>C mutation.11 All subjects gave written informed consent in accordance with the Declaration of Helsinki. The study was approved by the ethical committee of the University of Perugia.
Construction of the expression vectors, mutagenesis and transfection
Expression vectors were obtained20 and Chinese hamster ovary (CHO) cells were transfected as described in the Online Supplementary Data. All experiments were performed 2 days after transfection.
Surface biotinylation, β3 immunoprecipitation and western blotting
Surface proteins on CHO cells and platelets were biotinylated, and integrin β3 was immunoprecipitated and analyzed by western blotting as described in the Online Supplementary Data.
αIIbβ3 expression and internalization, and αvβ3 expression
αIIbβ3 expression on resting or activated platelets or CHO cells21 was assessed by flow cytometry22 or by western blotting after biotinylation of membrane proteins and β3 immunoprecipitation. αvβ3 expression was assessed by flow cytometry.22 Internalization of αIIbβ3 was assessed by flow cytometry as described elsewhere23. Platelet fibrinogen content was quantified by enzyme-linked immunosorbent assay (GenWay Biotech. San Diego, CA, USA). Details are given in the Online Supplementary Data.
CHO cells and platelets were layered on human fibrinogen. Platelets were also layered on human von Willebrand factor after treatment with 0.1 U/mL human α-thrombin.2418 Details are given in the Online Supplementary Data.
CHO cells and platelets were plated on human fibrinogen and protein phosphorylation was assessed as described in the Online Supplementary Data.
Clot retraction was assessed with CHO cells and platelet-rich plasma25 as described in the Online Supplementary Data.
Actin polymerization in CHO cells and platelets was assessed by flow cytometry as described in the Online Supplementary Data.
Analysis of cytoskeletal proteins
The cytoskeleton was extracted from either resting platelets or platelets that had been stimulated with thrombin as described elsewhere26. Cytoskeletal proteins were separated on an acrylamide-gel and stained with Comassie-blue26. For details see the Online Supplementary Data.
Protein digestion and peptide analysis were performed as reported previously.27 Databases were searched using MASCOT v.2.2 and MyriMatch v.126.96.36.199 Protein assembly for MyriMatch analysis was made using IDPicker v.3.0.29 Spectral-counts were used for semiquantitative comparisons between controls and patients. For details see the Online Supplementary Data.
Effect of the perturbation of actin polymerization on platelet function
αIIbβ3 activation, platelet aggregation,30 clot retraction and spreading on fibrinogen30 were assessed in platelets treated with jasplakinolide to induce actin polymerization31 as described in the Online Supplementary Data.
Construction of retroviral vectors, retrovirus production and megakaryocyte infection
The bicistronic pMYs-IRES-GFP/αIIb, pRetroX-IRES-DsRedExpress/β3 and pRetroX-IRES-DsRedExpress/β3del647-686 expression vectors were obtained, retroviruses produced and megakaryocytes double-infected32 as described in the Online Supplementary Data.
Megakaryocyte spreading, proplatelet formation and morphology
Spreading and proplatelet formation in murine megakary-ocytes32 were evaluated by immunofluorescence.3 Proplatelet morphology in human blood was analyzed by immunofluorescence as described elsewhere.333 For details see the Online Supplementary Data.
Effect of the perturbation of actin polymerization on proplatelet formation
Human megakaryocyte cultures3 were treated with jasplakinolide (1 µM) for 10 min and then cultured for 16 h. Proplatelet formation, spreading on fibrinogen, proplatelet tip number and diameter were assessed as described above.
Structural consequences of del647-686
To unravel the possible structural consequences of del647-686 the three-dimensional structure of the β-TD was derived from the αvβ3 structure (PDBID-code 4G1E) and visualized using PyMOL (DeLano Scientific, San Carlos, CA, USA).34
Data are expressed as means ± standard deviation. An unpaired t-test or the two-way ANOVA with the Bonferroni post-test was applied, where appropriate, using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant when P<0.05.
β3 mutations and αIIbβ3 activation
CHO cells transfected with normal αIIb and with four different mutant β3 subunits express a constitutively activated αIIbβ3 receptor on their surface, i.e. a receptor able to bind PAC-1 and fibrinogen under resting conditions (Online Supplementary Figure S1A,B), confirming previous findings.19173
Gain-of-function mutant β3 reduces surface expression of αIIbβ3 by enhancing its internalization
Western blotting and flow-cytometry showed decreased surface expression of β3 but a normal amount of β3 in cell lysates, both in CHO cells expressing del647-686 integrin β3 and in heterozygous patients’ platelets (Figures 1A,B). Confocal microscopy of integrin β3-transfected CHO cells showed that mutant β3 is localized predominantly in the cytoplasm, while normal β3 is localized predominantly on the cell surface (Online Supplementary Figure S2). Mutant αIIbβ3 co-localized with concavalin A and with WGA as shown by fluorescence microscopy, confirming normal synthesis and maturation (Online Supplementary Figure S3). Moreover, in patients’ platelets, cytoplasmic αIIbβ3 co-localized with P-selectin in α-granules but also localized in other cytoplasmic structures, probably the open canalicular and dense tubular systems35 (Online Supplementary Figure S4).
The fraction of internalized αIIbβ3 was significantly higher in unstimulated CHO cells expressing mutant β3 than in cells expressing wild-type β3 (Figure 1C). Similar findings were observed with resting patients’ platelets as compared with control platelets (Figure 1D). In accordance, patients’ platelets contained more fibrinogen than control platelets (Figure 1E) and mutant αIIbβ3-expressing CHO cells internalized fibrinogen under resting conditions differently from CHO cells expressing normal αIIbβ3 that required activation with DTT to internalize it (Online Supplementary Figure S1C). However, upon platelet stimulation with ADP or TRAP-6, the internal pool of αIIbβ3 of patient’s platelets externalized regularly showing that it is correctly recycled after internalization (Figure 1F).
αvβ3 expression on patient’s platelets was comparable to that on control platelets (control 34.9±8.0% versus patients 37.6±7.1%, P=ns).
Constitutively activated αIIbβ3 triggers outside-in signaling
CHO cells expressing mutant β3 spread faster on fibrinogen than wild-type cells (Figure 2A). Spreading of patients’ platelets was initially faster (Figure 2B), but after 30 min became defective11. Moreover, patients’ platelets spread spontaneously on von Willebrand factor, unlike control platelets that required αIIbβ3 activation to undergo full spreading2418 (Figure 2C).
Integrin β3 Tyr773 and Tyr785, as well as focal adhesion kinase, were constitutively phosphorylated in mutant CHO cells, while they were phosphorylated only after spreading on fibrinogen in control cells. Similarly, constitutive β3 and focal adhesion kinase phosphorylation was observed in resting patients’ platelets but not in control platelets (Figure 3A–C).
Constitutive αIIbβ3 activation leads to impaired cytoskeletal reorganization
The content of polymerized actin (F-actin) of CHO cells expressing each of the heterozygous, β3-activating mutants described so far191711 together with normal αIIb was higher than that of CHO cells expressing normal αIIbβ3, and it did not increase after stimulation with DTT (Figure 4A). Similarly, F-actin content was significantly higher in resting patients’ platelets as compared with con trol platelets. Consistently, stimulation with ADP did not significantly increase F-actin content of patients’ platelets while it doubled it in control platelets (Figure 4B).
Cytoskeletal protein content was higher in resting patients’ platelets than in resting control platelets. Moreover, electrophoresis showed three overexpressed bands in patients’ platelets when compared to resting control platelets. One had a molecular weight of about 70 kDa (band A) while the other two bands migrated at 55 kDa (bands B and C) (Figure 4C). Mass spectrometry showed that band A was mainly composed of fibrinogen α-chain, band B of fibrinogen β-chain, and band C of a fragment of fibrinogen γ-chain (Online Supplementary Tables S1 and S2, Online Supplementary Figure S5).
Perturbed cytoskeletal reorganization causes platelet dysfunction
To induce actin polymerization in control platelets we used jasplakinolide, a natural cyclic peptide that induces actin polymerization and stabilizes actin filaments.31 We first determined that jasplakinolide is able to induce actin polymerization in control platelets (Online Supplementary Figure S6).
Pre-incubation with jasplakinolide impaired, dose-dependently, ADP-induced platelet aggregation (Figure 5C), clot retraction (Figure 5D) and spreading on fibrinogen (Figure 5E). Platelets treated with jasplakinolide (1 µM) resembled patients’ platelets with regards to their spreading morphology11 (Figure 5F). These effects were not a consequence of cell death because platelets were still able to expose P-selectin on their surface upon stimulation with 10 µM ADP (resting platelets 2.92±1.33%; ADP-stimulated platelets 55.03±3.1%; jasplakinolide-treated resting platelets 4.52±1.8%; jasplakinolide-treated ADP-stimulated platelets 57.21±7.9%).
Constitutively activated αIIbβ3 impairs proplatelet formation
The proportion of mutant-β3-transduced megakaryocytes extending proplatelets in suspension was not different from that of wild-type megakaryocytes (wild-type megakaryocytes=73.8±19.3%, mutant megakaryocytes=61.4±20.7%; n=5, P=ns). However, proplatelet number was reduced and tip diameter was larger in mutant megakaryocytes (Figure 6A,B). Moreover, barbell-proplatelets were significantly more asymmetrical (difference between tips: wild-type megakaryocytes=1±0.9 mm, mutant megakaryocytes=2.5±1.1 mm; n=5, P<0.01). Similarly, barbell-proplatelets circulating in peripheral blood of our patient were more asymmetrical than in normal controls (difference between tips: controls=0.5±0.4 µm; patient=1.2±0.8 mm, n=5, P<0.05) (Figure 6C).
Mutant β3-transduced megakaryocytes adhered normally to fibrinogen (wild-type megakaryocytes=51.2±15.3%, mutant megakaryocytes=44.6±20.1%; n=5, P=ns) but displayed abnormal spreading, reminiscent of what was previously observed with patient’s megakaryocytes3 (Figure 6D). In contrast, when megakaryocytes were plated on von Willebrand factor, spreading was not different from that of controls (wild-type megakaryocytes=47.9±19.3%, mutant megakaryocytes=50.2±22.6%; n=5, P=ns), as already observed with patient’s megakaryocytes.3
Perturbed actin polymerization impairs proplatelet formation
The proportion of jasplakinolide-treated human megakaryocytes extending proplatelets in suspension was not different from that of control megakaryocytes (vehicle=26.3±2.6%; jasplakinolide=26.9±9.3%, n=3, P=ns); however, proplatelet formation was abnormal, with most megakaryocytes displaying a reduced number of proplatelet tips (vehicle=13.9±6.1 tips; jasplakinolide=1.4±1.4 tips, n=3, P<0.01) and a reduced tip diameter (vehicle=2.9±0.9 µm; jasplakinolide=1.7±0.8 µm, n=3, P<0.01) (Figure 7). Adhesion to fibrinogen of jasplakinolide-treated megakaryocytes was strongly impaired (vehicle=41.4±5.7%; jasplakinolide=4.6±0.5%, n=3, P<0.01) and the few adhering megakaryocytes did not spread (Figure 7).
Structural consequences of del647-686
The β-TD connects the lower β-leg of integrin β3 to the transmembrane portion and consists of an amphipathic α-helix lying across a single β-sheet with four β-strands and four disulfide bonds; it non-covalently associates with the calf-2 domain of αIIb (Figure 8). Residues spanning from Lys532 through Gly690 in the lower β-leg stabilize the interactions with integrins αv and αIIb in the bent conformation, and mutations of these residues activate αIIbβ3 and trigger fibrinogen binding.36 Del647-686 removes the 2, 3, and 4 β-strands, which represent a large interaction interface with the calf-2 domain in both αIIbβ312 and αvβ334, thus destroying the β-TD structure (Figure 8), hindering the adoption of the bent conformation. The observation that, despite a 40-amino acid deletion, αIIbβ3 is still synthesized and expressed on the surface, confirms that β-TD is a domain important for integrin function/activation but not for β3 synthesis or dimerization with αIIb.12
Del647-686 also removes Cys-655 and 663, partners of Cys-608 and 687 in the formation of disulfide bonds. Whether the remaining cysteines would then disulfide-link to one another, exchange with the two α1-helix-loop disulfides, or remain as free sulfhydryls is unknown. However, previous site-directed mutagenesis of Cys-655 and 663 leading to the loss of disulfide bonds, resulted in constitutive activation of αIIbβ31513, suggesting that the same mechanism is responsible for the constitutive αIIbβ3 activation associated with del647-686.
Our results show that constitutive activation of αIIbβ3 due to gain-of-function mutations of ITGB3, and the consequent permanent triggering of αIIbβ3-mediated outside-in signaling, induce a perturbation of cytoskeletal remodeling that leads to platelet dysfunction and impaired proplatelet formation. Starting from the study of platelets from two patients carrying the heterozygous ITGB3 del647-686 mutation11 we extended our observations to three additional ITGB3 gain-of-function mutations1917 in order to describe a general mechanism that leads to bleeding in patients with dominant variants of GT.
We show that constitutive activation of αIIbβ3 leads to permanent triggering of αIIbβ3-mediated outside-in signaling, as shown by the phosphorylation of β3 Tyr773, Tyr785 and focal adhesion kinase in patients’ resting platelets and in CHO cells expressing del647-686 β3, by faster spreading on fibrinogen and spontaneous spreading on von Willebrand factor of patients’ platelets, contrarily to control platelets that require αIIbβ3 activation for spreading on von Willebrand factor.2418
Activation of αIIbβ3 is physiologically followed by receptor internalization, a way for limiting platelet aggregation.3837 We show here that enhanced αIIbβ3 internalization is the common mechanism leading to reduced surface expression of αIIbβ3 in patients with GT-like syndromes associated with constitutive activation of αIIbβ3.10 In fact, we observed enhanced αIIbβ3 internalization in CHO cells expressing several different ITGB3 gain-of-function mutations1917 as well as in platelets of del647–686 β3 patients.
We have previously shown, by flow cytometry using a set of clones directed toward different epitopes of αIIbβ3, that our patients’ platelets express on average 40% residual αIIbβ3 on their surface if compared with control platelets.11 We show here that in patients’ platelets the ratio between normal and mutant β3 is maintained on the platelet surface (Figure 1A). The same is observed in CHO cells transfected with both wild-type and mutant β3 (heterozygous cell model) which express an approximately equal amount of αIIbβ3 to that of CHO cells transfected with only mutant β3 (homozygous cell model) (Figure 1A,B). It can, therefore, be speculated that normal αIIbβ3 is passively internalized along with the mutant one.
Confocal microscopy revealed that in patients’ platelets the internal pool of integrin β3 localizes in α-granules and in other cytoplasmic compartments, probably corresponding to the open canalicular and dense tubular systems,35 similar to control platelets. Moreover, we show that upon platelet activation, mutant αIIbβ3 externalizes as well as normal αIIbβ3, and is, therefore, presumably correctly recycled after internalization.
Enhanced αIIbβ3 internalization was associated with an increased platelet content of fibrinogen. The intra-platelet fibrinogen pool is largely generated by its internalization mediated by activated αIIbβ34039, although internalization by inactive αIIbβ3 has also been reported.41 Increased fibrinogen content in the patients’ platelets is, therefore, in accordance with constitutively activated αIIbβ3.
Given that integrin β3 is a component of the vitronectin receptor αvβ3, we measured αvβ3 expression on patients’ platelets and found it to be comparable to that on control platelets. Therefore, our mutation, like others previously reported,9 influences αvβ3 and αIIbβ3 expression differently.
In normal conditions, αIIbβ3-mediated outside-in signaling leads to a reorganization of the cytoskeleton that is required for platelet aggregation, clot retraction and spreading. Cytoskeletal actin reorganization is a finely regulated process with consecutive phases including actin polymerization, which enhances the cytoskeletal rigidity required for platelet shape change, and then depolymerization, which restores the cytoskeletal plasticity required for aggregation and clot retraction.42 An alteration of actin dynamics and cytoskeletal remodeling can, therefore, impair platelet function.87
We show here that CHO cells expressing ITGB3 gain-of-function mutations, as well as del647–686 β3-bearing platelets, have an increased F-actin content under resting conditions and show impaired clot retraction, suggesting that actin turnover is arrested at the stage of polymerization due to permanently triggered outside-in signaling. This is in line with the earlier spreading on fibrinogen of mutant platelets becoming defective at later time points, because actin turnover is arrested at the stage of polymerization and further cytoskeletal remodeling is no longer possible. Moreover, patients’ platelets had an enhanced cytoskeletal-associated protein content as compared with control platelets, and fibrinogen α, β and γ chains were found to be associated with the cytoskeleton.
In order to assess whether impaired cytoskeletal remodeling recapitulates the platelet features associated with αIIbβ3-activating ITGB3 mutations we used jasplakinolide, a natural cyclic peptide that induces actin polymerization by promoting actin nucleation and by preventing depolymerization of stress fibers.31 Jasplakinolide-treated control platelets showed impaired αIIbβ3 activation, reduced aggregation, altered spreading on fibrinogen and clot retraction, a phenotype resembling that of del647–686 platelets.11 Altogether, these data are compatible with a model in which αIIbβ3 constitutive activation causes the arrest of cytoskeletal remodeling at the stage of polymerization, thus impairing platelet aggregation, clot retraction and spreading. On the other hand, reduced αIIbβ3 surface expression does not seem to contribute significantly to platelet dysfunction because incubation of normal platelets with jasplakinolide induced the same platelet dysfunction observed in patients with variant GT without affecting αIIbβ3 surface expression. Moreover, αIIbβ3 internalization is not triggered by actin polymerization but depends on the constitutive activation of the receptor.
Cytoskeletal remodeling is also crucial for proplatelet formation: in fact actin polymerization leads to the amplification of proplatelet ends, thus increasing tip number,43 and regulates platelet size.3635 Impaired actin remodeling in megakaryocytes may thus perturb platelet formation, and in fact defects in actin-related proteins were shown to lead to thrombocytopenia and to the production of platelets with altered dimensions.4744 We induced actin polymerization by incubating control megakaryocytes with jasplakinolide and indeed we observed impaired proplatelet formation.
Murine megakaryocytes transduced with del647–686 human β3 and wild-type human αIIb showed impaired pro-platelet formation, with a reduced number of abnormally large proplatelet tips. These data confirm our previous observations with peripheral blood CD34-derived patients’ megakaryocytes3 and prove that the 2134+1 G>C mutation is the cause of macrothrombocytopenia. We also observed that barbell-proplatelets produced by transduced murine megakaryocytes are asymmetrical, similar to those found in patients’ peripheral blood. This is therefore probably responsible for the platelet anisocytosis observed in patients with the del647–686 mutation.11
Our observations confirm that αIIbβ3 plays a role in pro-platelet formation48183 and show that when outside-in signaling is constitutively triggered, cytoskeletal reorganization is disturbed and altered proplatelet formation and pre-platelet maturation occur.
In conclusion, we show that gain-of-function mutations of ITGB3 generating constitutive activation of αIIbβ3 lead to receptor internalization and to the arrest of cytoskeletal remodeling in platelets and megakaryocytes which, in turn, generate platelet dysfunction and perturb the finely regulated process of proplatelet formation leading to macrothrombocytopenia. Reduced platelet number and platelet dysfunction in patients with dominant variants of GT are both consequent to the cytoskeletal perturbation induced by the constitutive αIIbβ3-mediated outside-in signaling.
The authors thank Ildo Nicoletti and Alessandra Balduini for help with confocal microscopy, and Silvia Giannini, Teresa Corazzi, Luca Cecchetti, Anna Maria Mezzasoma, Giuseppe Guglielmini and Viviana Appolloni for help with some experiments and discussion of results. W. Vainchenker (Université Paris-Sud, Villejuif, France) kindly gave the GP+E-86 TPO-secreting cell line, and F. Grignani (Department of Experimental Medicine, University of Perugia) the Phoenix cell line. We also thank Francesca for her kind and continuous collaboration.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/1/46
- FundingThis work was supported by a Telethon grant (GGP10155) to PG.
- Received May 18, 2015.
- Accepted October 1, 2015.
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