[The crucial toxic components of ambient fine particles promoted the maturation and differentiation of megakaryocytes].

Objective: To reveal the crucial toxic components of ambient fine particles (PM2.5) that affect the maturation and differentiation of megakaryocytes. Methods: Human megakaryocytes were exposed to the organic fractions, metallic fractions and water-soluble fractions of PM2.5 at two exposure doses (i.e. actual air proportion concentration or the same concentration), respectively. The cell viability was performed to screen the non-cytotoxic levels of toxic components of PM2.5 using the CCK-8 assay. CellTiter-Blue assay, morphological observation, flow cytometry analysis and WGA staining assay were used to evaluate the cell morphological changes, occurrence of DNA ploidy, alteration in the expressions of biomarkers and platelet formation, which were key indicators of the maturation and differentiation of megakaryocytes. Results: Compared to the control group, both metallic and organic components of PM2.5 resulted in a lag in megakaryocytes with an increase in cell volume and the onset of DNA ploidy. Flow cytometry analysis showed that CD33 (the marker of myeloid-specific) decreased and CD41a (a megakaryocyte maturation-associated antigen) increased in metallic and organic components of PM2.5 treatment groups. Moreover, compared to the control group, budding protrusions increased in metallic and organic components of PM2.5 treatment groups. The water-soluble components had no effect on the maturation and differentiation of macrophages. Conclusion: Metallic and organic components of PM2.5 are the crucial toxic components that promote the maturation and differentiation of megakaryocytes.

Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study.

Purpose: Diagnosis of myelodysplastic syndrome (MDS) primarily relies on the detection of morphological dysplasia in bone marrow. It is subjective and many studies have reported lack of interobserver agreement in reporting. Biopsy is preferred specimen for megakaryocyte assessment. We studied 43 bone marrow biopsies from 40 suspected MDS patient having persistent undiagnosed cytopenia. Utility of immunohistochemistry (IHC) with CD61 and p53 in detecting low-grade MDS was analyzed over routine morphology. Method and Results: Total number of megakaryocytes and number of dysplastic megakaryocytes seen on CD61 IHC was significantly higher than that on H and E stain (P value < 0.05) Out of total 43 biopsies, 13 [30.2%] cases showed dysplastic megakaryocytes that were confirmed by interobserver agreement after IHC. From 30 cases with no significant dysplasia on morphology, 21/43 [48.8%] cases showed >10% dysplastic megakaryocytes on CD61 (P value 0.0001). Nine cases showed no significant dysmegakaryopoiesis with either H and E or CD61 IHC. Fourteen cases could meet higher cut off (30%) of dysmegakaryopoiesis with CD 61 IHC. Out of total 34 cases showing significant dysplasia 7 cases (20.6%) showed positivity for p53 on IHC, which is little less than that reported in low-grade MDS. Conclusion: CD61 IHC is helpful in making correct diagnosis of MDS in cases with minimal dysplasia and should be performed before excluding possibility of MDS on morphology in a patient with undiagnosed cytopenia. IHC is cost effective tool for MDS diagnosis in developing world where access to extensive flow cytometery and molecular testing is limited.

The impact of bone marrow fibrosis and JAK2 expression on clinical outcomes in patients with newly diagnosed multiple myeloma treated with immunomodulatory agents and/or proteasome inhibitors.

We determined the impact of bone marrow fibrosis (BMF) on the clinical outcomes of newly diagnosed multiple myeloma (NDMM) patients in the current era of myeloma therapy. A total of 393 MM patients were included in the final analysis. The median followup was 83 months (range: 3.9 to 212 months). BMF was noted in 122 (48.2%) evaluable patients. Median progression free survival (PFS) in patients without BMF was 30.2 (95% CI: 24.7-38.0) months, and 21.1 (95% CI: 18.8-27.5) months in patients with BMF present (P = .024). Median overall survival (OS) was 61.2 (95% CI: 51.5-81.2) months in patients without BMF, and 45.1 (95% CI: 38.7-57.0) months in patients with BMF (P = .0048). A subset of 99 patients had their bone marrow biopsies stained for JAK1 and JAK2 by immunohistochemistry. Of these samples 67 (67.7%) patients had detectable JAK2 expression predominantly noted on bone marrow megakaryocytes. JAK2 expression correlated with myeloma disease stage (P = .0071). Our study represents the largest dataset to date examining the association of BMF with prognosis in the era of novel therapies and widespread use of hematopoietic stem cell transplant (HSCT). Our data suggest that MM patients with BMF (particularly those with extensive BMF) have a poorer prognosis even when treated with immunomodulatory agents and proteasome inhibitors.

Megakaryocyte and Platelet Transcriptomics for Discoveries in Human Health and Disease.

Anucleate platelets, long viewed as merely cell fragments with a limited repertoire of rapid-acting hemostatic functions, are now recognized to have a complex and dynamic transcriptome mirroring that of many nucleated cells. The field of megakaryocyte and platelet transcriptomics has been rapidly growing, particularly with the advent of newer technologies such as next-generation RNA-sequencing. Studies interrogating the megakaryocyte and platelet transcriptome have led to a number of key insights into human health and disease. In this brief focused review, we will discuss some of the recent discoveries made through transcriptome analysis of megakaryocytes and platelets. We will also highlight the utility of integrating ribosome footprint analysis to augment discoveries. Both bulk and single-cell sequencing approaches will be reviewed, along with comparative studies between human and murine platelets under basal healthy settings and during acute systemic inflammatory diseases.

The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis.

Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.

Disrupted filamin A/αIIbß3 interaction induces macrothrombocytopenia by increasing RhoA activity.

Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αIIbß3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbß3-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αIIbß3 interaction.

Engineering human megakaryocytic microparticles for targeted delivery of nucleic acids to hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are important target cells for gene therapy applications. Current genetic modifications of HSPCs rely on viral vectors in vivo or electroporation ex vivo. Here, we developed a nonviral system based on megakaryocytic microparticles (MPs) for targeted delivery of plasmid DNA (pDNA) and small RNAs to HSPCs. We have previously shown that megakaryocytic MPs, the most abundant MPs in blood circulation, target specifically and deliver cargo to HSPCs both in vitro and in vivo. With an optimized electroporation protocol, an average of 4200 plasmid copies per MP were loaded into MP, thus enabling effective delivery of green fluorescent protein (GFP)-encoding pDNA to HSPCs and HSPC nuclei, with up to 81% nuclei containing pDNA. Effective functional small interfering RNA (siRNA) and microRNA (miRNA) delivery were also demonstrated. As patient-specific or generic megakaryocytic MPs can be readily generated and stored frozen, our data suggest that this system has great potential for therapeutic applications targeting HSPCs.

Defective RAB1B-related megakaryocytic ER-to-Golgi transport in RUNX1 haplodeficiency: impact on von Willebrand factor.

Patients with RUNX1 haplodeficiency have thrombocytopenia, platelet dysfunction, and deficiencies of α-granules and dense granules. Platelet expression profiling of a patient with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased RAB1B, which encodes a small G protein. RAB GTPases regulate vesicle trafficking, and RAB1B is implicated in endoplasmic reticulum (ER)-to-Golgi transport in nonhematopoietic cells, but its role in megakaryocytes (MK) is unknown. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and that RAB1B regulates ER-to-Golgi transport in MK cells. Chromatin immunoprecipitation studies and electrophoretic mobility shift assay using phorbol 12-myristate 13-acetate (PMA)-treated human erythroleukemia cells revealed RUNX1 binding to RAB1B promoter region RUNX1 consensus sites, and their mutation reduced the promoter activity. RAB1B promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB1B is a direct RUNX1 target, providing a mechanism for decreased RAB1B in patient platelets. Vesicle trafficking from ER to Golgi in PMA-treated human erythroleukemia cells was impaired along with Golgi disruption on siRNA downregulation of RUNX1 or RAB1B. The effects of RUNX1 knockdown were reversed by RAB1B reconstitution. Trafficking of von Willebrand factor (vWF), an α-granule MK synthesized protein, was impaired with RUNX1 or RAB1B downregulation and reconstituted by ectopic RAB1B expression. Platelet vWF was decreased in patients with RUNX1 mutations. Thus, ER-to-Golgi transport, an early critical step in protein trafficking to granules, is impaired in megakaryocytic cells on RUNX1 downregulation, secondary to decreased RAB1B expression. Impaired RAB1B mediated ER-to-Golgi transport contributes to platelet α-granule defects in RUNX1 haplodeficiency.

Platelet function is modified by common sequence variation in megakaryocyte super enhancers.

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

Transforming growth factor ß- and interleukin 13-producing mast cells are associated with fibrosis in bone marrow.

Although bone marrow fibrosis is a lethal condition, its underlying mechanism is not fully understood. This study aimed to investigate the pathogenesis of fibrosis in the bone marrow through histologic examination of mast cell infiltration and the expression of fibrosis-associated cytokines. We analyzed 22 bone marrows with fibrosis (8 primary myelofibrosis [PMF], 5 post-essential thrombocythemia [ET], myelofibrosis, and 9 myelodysplastic syndrome [MDS] with bone marrow fibrosis [BMF]). Immunohistochemical and immunofluorescence stainings were performed using anti-mast cell tryptase, interleukin (IL) 13, transforming growth factor ß (TGF-ß), CD34, and CD42b antibodies. The number of mast cells in bone marrows with fibrosis was significantly higher than that in controls (P<.0001 for all cases with fibrosis versus control, P=.0470 for PMF versus control, P<.0001 post-ET myelofibrosis versus control, and P=.0005 for MDS with BMF versus control). Moreover, bone marrows with higher fibrotic grades exhibited greater amounts of infiltrating mast cells. Mast cells were positive for TGF-ß and IL-13 in bone marrows with fibrosis of all 3 groups. Megakaryocytes were negative for TGF-ß in post-ET and MDS with BMF, but some megakaryocytes in PMF were weakly positive for TGF-ß. Megakaryocytes were negative for IL-13 in all 3 groups. Blasts were negative for both TGF-ß and IL-13 in all 3 groups. Thus, TGF-ß- and IL-13-producing mast cells might be key players in the development of BMF. Therefore, mast cells could be potential therapeutic targets for the treatment of BMF.

Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms.

AIMS: Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN. METHODS: Immunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and without JAK2(V617F) and CALR mutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12). RESULTS: Myeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases with CALR mutations, had significant reductions in pro-apoptotic BNIP-3. CONCLUSIONS: Uncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation with CALR mutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN with CALR mutations may be due to greater dysregulation of megakaryocyte apoptosis.

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo.

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.

Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte mRNA levels of several genes including those under PPARγ control. Overall, the delivery of PPARγ from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.

Thrombopoietin receptor agonist therapy in primary immune thrombocytopenia is associated with bone marrow hypercellularity and mild reticulin fibrosis but not other stromal abnormalities.

Primary immune thrombocytopenia is an acquired autoimmune disorder characterized by platelet count of <100 × 10(9)/l in the absence of other causes of thrombocytopenia. Primary immune thrombocytopenia is defined as 'chronic' when it has been present for more than 12 months without spontaneous remission or maintenance of complete response to therapy. Recently, thrombopoietin receptor agonists became available for treatment of chronic primary immune thrombocytopenia. Anecdotal reports have raised concerns about a possible association between therapy with thrombopoietin receptor agonists and an increase in bone marrow fibrosis. To investigate this association we studied eight patients with primary immune thrombocytopenia in detail comparing fibrosis and other morphological features in pre-therapy and on-therapy bone marrow biopsies, with the longest follow-up reported to date. A slight but significant increase to MF-1 in reticulin fibrosis was observed during therapy, but collagen was never present. On-therapy bone marrows were hypercellular due to panmyelosis with increased trilineage hematopoiesis. Megakaryocytes were increased in number, with acquisition of evident pleomorphism, nuclear hyperlobulation and tendency in some cases to form clusters. The overall picture of the on-therapy marrows was characterized by myeloproliferative neoplasm-like features, resembling essential thrombocythemia or occasionally early primary myelofibrosis. As thrombopoietin receptor agonists are becoming a mainstream treatment for primary immune thrombocytopenia, general pathologists and especially hematopathologists need to be aware of the characteristic morphological changes associated with use of these therapeutic agents, in order to avoid misdiagnosis of a myeloid neoplasm.

Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm showing aberrant bone marrow remodeling with increased angiogenesis, progressive matrix accumulation, and fibrosis development. Thrombospondins (TSP) are factors sharing pro-fibrotic and anti-angiogenic properties, and have not been addressed in PMF before. We investigated the expression of TSP-1 and TSP-2 in PMF related to the stage of myelofibrosis (n = 51) and in individual follow-up biopsies by real-time PCR, immunohistochemistry, and confocal laser scanning microscopy (CLSM). TSP-1 was significantly overexpressed (p < 0.05) in all stages of PMF when compared to controls. Individual follow-up biopsies showed involvement of TSP-1 during progressive myelofibrosis. TSP-2 was barely detectable but 40% of cases with advanced myelofibrosis showed a strong expression. Megakaryocytes and interstitial proplatelet formations were shown to be the relevant source for TSP-1 in PMF. Stroma cells like endothelial cells and fibroblasts showed no TSP-1 labeling when double-immunofluorescence staining and CLSM were applied. Based on its dual function, TSP-1 in PMF is likely to be a mediator within a pro-fibrotic environment which discriminates from ET cases. On the other hand, TSP-1 is a factor acting (ineffectively) against exaggerated angiogenesis. Both features suggest TSP-1 to be a biomarker for monitoring a PMF-targeted therapy.

Increased Dkk3 protein expression in platelets and megakaryocytes of patients with myeloproliferative neoplasms.

Dickkopf-3 (Dkk3) has been proposed as tumour suppressor gene and a marker for tumour blood vessels. We analysed the expression and function of Dkk3 in platelets and megakaryocytes from healthy controls and patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN). Dkk3 protein and gene expression in platelets was compared with endothelial and other blood cell populations by ELISA, real-time PCR, and immunofluorescence. Moreover, megakaryocytes were isolated from bone marrow aspirates by CD61 microbeads. Immunohistochemical studies of Dkk3 expression were performed in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF) and control reactive bone marrow cases (each n=10). Compared to all other blood cell populations platelets showed the highest concentration of Dkk3 protein (150 ± 19 ng/mg total protein). A strong DKK3 gene and protein expression was also observed in isolated megakaryocytes. Dkk3 co-localised with VEGF in α-granules of platelets and was released similar to VEGF upon stimulation. Addition of recombinant Dkk3 had no influence on blood coagulation (aPTT, INR) and platelet aggregation. Significantly more Dkk3+ megakaryocytes/mm2 could be found in bone marrow biopsies from patients with MPN (ET 40 ± 10, PV 31 ± 4, PMF 22 ± 3) than in controls (15 ± 3). The mean proportion of Dkk3+ megakaryocytes was increased in MPN as well (ET 83% ± 15%; PV 84% ± 12%; PMF 77% ± 8%) compared to controls (53% ± 11%). Dkk3+ megakaryocytes correlated with microvessel density in PV and PMF. We conclude that Dkk3 might be involved in the pathogenesis of MPN.

Development of a downscale sedimentation field flow fractionation device for biological event monitoring.

Classically described as a macroscale size-density based method, Sedimentation field flow fractionation (SdFFF) has been successfully used for cell sorting. The goal of this study was to develop a new SdFFF device for downscale applications, in particular for oncology research to rapidly monitor chemical biological event induction in a cell line. The development of a downscale SdFFF device required reduction of the separation channel volume. Taking advantage of a newly laboratory designed apparatus, channel volume was successfully decreased by reducing both length and breadth. To validate the apparatus and method, we used the well-known model of diosgenin dose-dependent induction of apoptosis or megakaryocytic differentiation in HEL cells. After a minute scale acquisition of a reference profile, the downscale device was able to perform fast, early, significant and reproducible monitoring of apoptosis and differentiation, two important biological mechanisms in the field of cancer research.

Dysregulation of stromal derived factor 1/CXCR4 axis in the megakaryocytic lineage in essential thrombocythemia.

This study investigated the involvement of chemokines including stromal derived factor 1 (SDF-1), interleukin 8 (IL-8), growth-related oncogene alpha (GRO-alpha) and their receptors, CXCR4, CXCR2 and CXCR1 in essential thrombocythemia (ET), a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. Fifty-three ET patients were studied. Plasma levels of SDF-1, IL-8 and GRO-alpha, evaluated by enzyme-linked immunosorbent assay, and flow cytometric analysis of CXCR1 and CXCR2 on the platelet membrane, were found to be normal in ET patients. CXCR4 expression on platelet surface as well as platelet CXCR4 mRNA detected by real-time reverse transcription polymerase chain reaction, were decreased. Platelet CXCR4 internalization rate was normal while SDF-1-induced platelet aggregation was delayed, decreased or absent. Immunohistochemical staining revealed that megakaryocytes were also affected. CXCR4 decrease was not observed either in peripheral white blood cells or in circulating CD34(+) precursors. These results show that CXCR4 is decreased in the megakaryocytic lineage in ET, mainly due to a reduced CXCR4 production, and an abnormal platelet response to SDF-1. This report is the first to describe platelet and megakaryocytic CXCR4 deficiency in a human disease and the presence of this abnormality in a megakaryocytic-related illness highlights the important role of SDF-1/CXCR4 axis in platelet development.