Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO.

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands in vitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells in vitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, in vivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

HCMV modulates c-Mpl/IEX-1 pathway-mediated megakaryo/thrombopoiesis via PDGFRα and αvß3 receptors after allo-HSCT.

Thrombocytopenia is a common complication of human cytomegalovirus (HCMV) infection in immunocompromised hosts, which contributes to poor prognosis even in patients receiving antiviral treatment. Here, we investigated the megakaryo/thrombopoiesis process, including the involvement of the c-Mpl/IEX-1 pathway, after HCMV infection, identified receptors mediating the interaction between megakaryocytes (MKs) and HCMV, and explored novel therapeutic targets. Our data shows that HCMV directly infects megakaryocytes in patients with HCMV DNAemia and influences megakaryopoiesis via the c-Mpl/IEX-1 pathway throughout megakaryocyte maturation, apoptosis, and platelet generation in vivo and in vitro. After treatment with inhibitors of PDGFRα and αvß3, the HCMV infection rate in MKs was significantly reduced, suggesting that IMC-3G3 and anti-αvß3 are potential therapeutic alternatives for viral infection. In summary, our study proposes a possible mechanism and potential treatments for thrombocytopenia caused by HCMV infection and other viral diseases associated with abnormal hemostasis.

C-Cbl regulates c-MPL receptor trafficking and its internalization.

Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c-MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c-Cbl which was previously shown to negatively regulated c-MPL signalling. We developed a new conditional mouse model using c-Cblfl/fl Pf4Cre mice and demonstrated that platelet-specific knockout of c-Cbl led to severe microthrombocytosis and impaired uptake of TPO and c-MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c-Cbl KO platelets. This study identified c-Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.

c-MPL Is a Candidate Surface Marker and Confers Self-Renewal, Quiescence, Chemotherapy Resistance, and Leukemia Initiation Potential in Leukemia Stem Cells.

Acute myeloid leukemia (AML) is initiated and maintained by a unique, small subset of leukemia cells known as leukemia stem cells (LSCs). Self-renewal, quiescence, and chemotherapy resistance are key stemness properties of LSCs that are essential for poor clinical responses to conventional therapies. Identifying LSC surface markers and targeting LSCs are important for the development of potential therapies. In this study, application of chemotherapy treatment in AML-ETO9a (AE9a) leukemia mice led to the enrichment of a chemotherapy-resistant cell population identified as Lin- c-Kit+ c-MPL+ . In addition, this c-MPL-positive cell population within Lin- c-Kit+ leukemia cells included a high percentage of cells in a quiescent state, enhanced colony formation ability, and increased homing efficiency. Serial transplantation demonstrated that Lin- c-Kit+ c-MPL+ cells displayed a significantly high potential for leukemia initiation. Furthermore, it was demonstrated that in AML patients, c-MPL was expressed on the majority of CD34+ leukemia cells and that the proportion of c-MPL+ cells in CD34+ leukemia cells is associated with poor prognosis. Finally, AMM2, an inhibitor of c-MPL, was shown to significantly enhance the survival of AE9a leukemia mice when combined with chemotherapeutic agent. These results indicate that c-MPL is a candidate LSC surface marker that may serve as a therapeutic target for the elimination of LSCs. Stem Cells 2018;36:1685-1696.

Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018;36:943-950.

A Case of Acquired Amegakaryocytic Thrombocytopenia with Anti-c-mpl Autoantibody: Comparison with Idiopathic Thrombocytopenic Purpura.

Acquired amegakaryocytic thrombocytopenia (AAMT) is a rare disease that causes severe bleeding. The pathogenesis and treatment of AAMT have not yet been defined. We report the case of a 60-year-old woman diagnosed with AAMT, who presented with severe thrombocytopenia, gastroin-testinal bleeding, and significantly reduced bone marrow megakaryocytes. The patient was treated with methylprednisolone, cyclosporin, and intravenous immunoglobulin. After 2 weeks of treatment, her platelet count started to increase, and her bone marrow megakaryocyte count had normalized 3 months after diagnosis. At the time of diagnosis, the patient was seropositive for anti-c-mpl antibody but was seen to be seronegative once the platelet count recovered. In contrast, anti-c-mpl antibodies were not detected in the serum of 3 patients with idiopathic thrombocytopenic purpura. This case study suggests that anti-c-mpl antibody plays an important role in the development of AAMT, and that intensive immunosuppressive treatment is required for autoantibody clearance and recovery of megakaryocyte count.

Novel thrombopoietin mimetic peptides bind c-Mpl receptor: Synthesis, biological evaluation and molecular modeling.

Thrombopoietin (TPO) acts in promoting the proliferation of hematopoietic stem cells and by initiating specific maturation events in megakaryocytes. Now, TPO-mimetic peptides with amino acid sequences unrelated to TPO are of considerable pharmaceutical interest. In the present paper, four new TPO mimetic peptides that bind and activate c-Mpl receptor have been identified, synthesized and tested by Dual-Luciferase reporter gene assay for biological activities. The molecular modeling research was also approached to understand key molecular mechanisms and structural features responsible for peptide binding with c-Mpl receptor. The results presented that three of four mimetic peptides showed significant activities. In addition, the molecular modeling approaches proved hydrophobic interactions were the driven positive forces for binding behavior between peptides and c-Mpl receptor. TPO peptide residues in P7, P13 and P7' positions were identified by the analysis of hydrogen bonds and energy decompositions as the key ones for benefiting better biological activities. Our data suggested the synthesized peptides have considerable potential for the future development of stable and highly active TPO mimetic peptides.

Promoter motifs required for c-mpl gene expression induced by thrombopoietin in CMK cells.

Thrombopoietin (TPO) and its receptor, c-Mpl, are the central regulators of megakaryocyte development and platelet production and are also crucial to regulate megakaryocytopoiesis. TPO remarkably elevated c-mpl promoter activity, while the protein kinase C (PKC) inhibitors, GF109203, H7 and Calphostin C, clearly reduced the steady level of its promoter activity.  In the present study, motifs crucial for c-mpl promoter activity induced by TPO treatment have been analyzed using a human megakaryoblastic cell line, CMK. Destruction of the -107Sp1 and the -57Sp1 sites in the c-mpl promoter enhancer region resulted in decrease of the promoter activity by 53.1% and 64.4%, respectively, and destruction of -69Ets and -28Ets elements dramatically decreased the promoter activity by 96.4% and 87.8%, respectively, while mutation of -77GATA moderately reduced the activity by 31.4%. The result was in agreement with our previous report that showed the crucial motifs in the c-mpl promoter for the promoter activity induced by PMA-treatment. This indicates that TPO-induced activation of the c-mpl promoter activity is fully modulated by transcription through a PKC-dependent pathway and the two Sp1 and two Ets motifs are crucial for the activation of the c-mpl promoter activity rather than a GATA motif in the c-mpl promoter of CMK cells.

C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis.

C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.

C-Mannosylation of thrombopoietin receptor (c-Mpl) regulates thrombopoietin-dependent JAK-STAT signaling.

The thrombopoietin receptor, also known as c-Mpl, is a member of the cytokine superfamily, which regulates the differentiation of megakaryocytes and formation of platelets by binding to its ligand, thrombopoietin (TPO), through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. The loss-of-function mutations of c-Mpl cause severe thrombocytopenia due to impaired megakaryocytopoiesis, and gain-of-function mutations cause thrombocythemia. c-Mpl contains two Trp-Ser-Xaa-Trp-Ser (Xaa represents any amino acids) sequences, which are characteristic sequences of type I cytokine receptors, corresponding to C-mannosylation consensus sequences: Trp-Xaa-Xaa-Trp/Cys. C-mannosylation is a post-translational modification of tryptophan residue in which one mannose is attached to the first tryptophan residue in the consensus sequence via C-C linkage. Although c-Mpl contains some C-mannosylation sequences, whether c-Mpl is C-mannosylated or not has been uninvestigated. We identified that c-Mpl is C-mannosylated not only at Trp(269) and Trp(474), which are putative C-mannosylation site, but also at Trp(272), Trp(416), and Trp(477). Using C-mannosylation defective mutant of c-Mpl, the C-mannosylated tryptophan residues at four sites (Trp(269), Trp(272), Trp(474), and Trp(477)) are essential for c-Mpl-mediated JAK-STAT signaling. Our findings suggested that C-mannosylation of c-Mpl is a possible therapeutic target for platelet disorders.

The Reconstitution of T-cells after Allogeneic Hematopoietic Stem Cell Transplant in a Pediatric Patient with Congenital Amegakaryocytic Thrombocytopenia (CAMT).

BACKGROUND: Congenital amegakaryocytic thrombocytopenia (CAMT) is a bone marrow failure syndrome with autosomal recessive inheritance characterized by the lack of megakaryocytes and thrombocytopenia. The cause of the disease is a mutation in the c-Mpl gene, which encodes the thrombopoietin (TPO) receptor. The main treatment for this genetic disorder is an allogeneic hematopoietic stem cell transplant (allo-HSCT). However, transplant-related mortality, development of acute and chronic graft-versushost disease (GvHD), and susceptibility to opportunistic infections are major barriers to transplantation. Delay in the reconstitution of T cells and imbalance in the regeneration of distinct functional CD4 and CD8 T-cell subsets mainly affect post-transplant complications. We report a case of CAMT, who developed acute GvHD but had no signs and symptoms of chronic GvHD following allo-HSCT. CASE PRESENTATION: At the age of four, she presented with petechiae and purpura. In laboratory investigations, pancytopenia without organomegaly, and cellularity less than 5% in bone marrow biopsy, were observed. A primary diagnosis of idiopathic aplastic anemia was made, and she was treated with prednisolone, cyclosporine, and anti-thymocyte globulin (ATG), which did not respond. Genetic analysis revealed the mutation c.1481T>G (p. L494W) in exon 10 of the c-Mpl gene, and the diagnosis of CAMT was confirmed. The patient underwent allo-HSCT from a healthy sibling donor. Alloimmunization reactions and immune disorders were present due to long-term treatment with immunosuppressive medications and repeated blood and platelet transfusions. Hence, the regeneration of T-lymphocytes after allo-HSCT was evaluated. CONCLUSION: Successful treatment of acute GvHD prevented advancing the condition to chronic GvHD, and this was accompanied by delayed T-cell reconstitution through an increase in Treg:Tcons ratio.

Spatiotemporal expression profiles of c-Mpl mRNA in the tooth germ: Comparative expression dynamics of vascularization-related genes.

BACKGROUND: Vascularization is an essential event for both embryonic organ development and tissue repair in adults. During mouse tooth development, endothelial cells migrate into dental papilla during the cap stage, and form blood vessels through angiogenesis. Megakaryocytes and/or platelets, as other hematopoietic cells, express angiogenic molecules and can promote angiogenesis in adult tissues. However, it remains unknown which cells are responsible for attracting and leading blood vessels through the dental papilla during tooth development. METHODS: Here we analyzed the spatiotemporal expression of c-Mpl mRNA in developing molar teeth of fetal mice. Expression patterns were then compared with those of several markers of hematopoietic cells as well as of angiogenic elements including CD41, erythropoietin receptor, CD34, angiopoietin-1 (Ang-1), Tie-2, and vascular endothelial growth factor receptor2 (VEGFR2) through in situ hybridization or immunohistochemistry. RESULTS: Cells expressing c-Mpl mRNA was found in several parts of the developing tooth germ, including the peridental mesenchyme, dental papilla, enamel organ, and dental lamina. This expression occurred in a spatiotemporally controlled fashion. CD41-expressing cells were not detected during tooth development. The spatiotemporal expression pattern of c-Mpl mRNA in the dental papilla was similar to that of Ang-1, which preceded invasion of endothelial cells. Eventually, at the early bell stage, the c-Mpl mRNA signal was detected in morphologically differentiating odontoblasts that accumulated in the periphery of the dental papilla along the inner enamel epithelium layer of the future cusp region. CONCLUSION: During tooth development, several kinds of cells express c-Mpl mRNA in a spatiotemporally controlled fashion, including differentiating odontoblasts. We hypothesize that c-Mpl-expressing cells appearing in the forming dental papilla at the cap stage are odontoblast progenitor cells that migrate to the site of odontoblast differentiation. There they attract vascular endothelial cells into the forming dental papilla and lead cells toward the inner enamel epithelium layer through production of angiogenic molecules (e.g., Ang-1) during migration to the site of differentiation. C-Mpl may regulate apoptosis and/or proliferation of expressing cells in order to execute normal development of the tooth.

A Review on Romiplostim Mechanism of Action and the Expressive Approach in E. coli.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder determined by immune-mediated platelet demolition and reduction of platelet production. Romiplostim is a new thrombopoiesis motivating peptibody that binds and stimulates the human thrombopoietin receptor the patent of which was registered in 2008. It is used to treat thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Romiplostim is a 60 kDa peptibody designed to inhibit cross-reacting immune responses. It consists of four high-affinity TPO-receptor binding domains for the Mpl receptor and one human IgG1 Fc domain. Escherichia coli is a good host for the fabrication of recombinant proteins such as romiplostim. The expression of a gene intended in E. coli is dependent on many factors such as a protein's inherent ability to fold, mRNA's secondary structure, its solubility, its toxicity preferential codon use, and its need for post-translational modification (PTM). This review focuses on the structure, function, mechanism of action, and expressive approach to romiplostim in E. coli.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

c-Mpl and TPO expression in the human central nervous system neurons inhibits neuronal apoptosis.

Thrombopoietin (TPO) is a growth factor for the megakaryocytic/platelet lineage. In this study, we investigated the expression of TPO and its receptor, c-Mpl, in the human central nervous system (CNS) and their roles after a neural insult. Our results demonstrate that both TPO and c-Mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models. In these rat models, treatment with TPO reduced brain damage and improved sensorimotor functions. In addition, TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an antiapoptotic effect by suppressing mitochondrial membrane potentials. Taken together, our results indicate that TPO is neuroprotective in the CNS.

Thrombopoietin and its receptor expression in pediatric patients with chronic immune thrombocytopenia.

OBJECTIVES: Chronic immune thrombocytopenia (cITP) is common in children. However, the pathogenesis has not been fully elucidated. This study aimed to determine whether thrombopoietin (TPO) and its receptor c-mannosylation of the TPO receptor (c-Mpl) have an impact on childhood cITP. METHODS: Sixty-four patients with newly diagnosed ITP (nITP), 64 patients with persistent ITP, 80 patients with cITP, and 64 healthy children (control) were enrolled in this study. Plasma TPO was measured with an ELISA, and c-Mpl was determined by flow cytometry. RESULTS: Plasma TPO levels showed differences among the four groups (p = 0.001). TPO levels in the cITP group were significantly decreased compared to those in the nITP group (p < 0.05). The mean fluorescence intensity (MFI) of c-Mpl was significantly different among the four groups (p = 0.0275). c-Mpl MFI was lower in the cITP group than in the nITP group(p < 0.05). Quantitative real-time PCR analysis showed that TPO mRNA expression was higher in the control group than in the ITP groups (p < 0.0001). The c-Mpl mRNA levels also showed significant differences among the four groups (p = 0.023). The control group, compared with the other groups, had lower levels of c-Mpl mRNA. CONCLUSIONS: The expression of TPO and c-Mpl was significantly decreased in the cITP group compared to the nITP group, suggesting that TPO and its receptor may play important roles in childhood cITP pathogenesis.

Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors.

Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

Anti-c-Mpl antibodies in immune thrombocytopenia suppress thrombopoiesis and decrease response to rhTPO.

INTRODUCTION: Anti-TPO receptor (anti-c-Mpl) antibodies exist and could affect rhTPO treatment in ITP. MATERIALS AND METHODS: Anti-c-Mpl autoantibodies and TPO levels were measured in serum from 187 ITP patients and 59 healthy controls. The anti-c-Mpl-antibody-positive (anti-c-Mpl+) and anti-c-Mpl-antibody-negative (anti-c-Mpl-) IgG from ITP patients were assessed on megakaryocyte proliferation, polyploidy, apoptosis, and platelet release with rhTPO treatment. RESULTS: Anti-c-Mpl antibodies were detected in 54/187 ITP patients but in none of the controls. ITP patients with anti-c-Mpl antibodies had higher serum TPO levels, but lower megakaryocyte and platelet counts compared with anti-c-Mpl- patients. Antibodies targeting platelet glycoprotein (GP) were also more frequently identified in anti-c-Mpl+ ITP patients. Moreover, patients with anti-c-Mpl antibodies were less responsive to rhTPO treatment than patients without anti-c-Mpl antibodies. Additionally, the anti-c-Mpl antibody titer decreased in ITP patients following treatment. In vitro study, lower megakaryopoiesis, platelet generation and percent of polyploidy were observed in anti-c-Mpl+ group compared with the anti-c-Mpl- group in the presence of rhTPO. CONCLUSIONS: Our findings demonstrated that the anti-c-Mpl antibody represents a novel indicator of poor prognosis and may be a potential therapeutic target in ITP.