A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) ...are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.
Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To ...analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood‐derived CD34+ cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time‐dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin αIIbβ3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb‐IX‐V, or upon cleavage of GPIbα by the metalloproteinase mocarhagin. Membrane‐associated VWF was detected exclusively on proplatelet‐forming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio‐temporal regulation orchestrated by adhesive proteins, GPIb‐IX‐V and myosin IIA.
Background: Although mutations of GPIbα are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly ...defined. Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIbα (Bolzano mutation). Methods: Human megakaryocytes were obtained by differentiation of patient cord blood‐derived CD34+ cells and peripheral blood‐derived CD45+ cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients’ blood platelets. Moreover, α‐tubulin distribution within proplatelets was severely deranged. Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIbα display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIbα in proplatelet formation.
Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes ...leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm that arises from clonal proliferation of hematopoietic stem cells and leads to progressive bone marrow (BM) fibrosis. While cellular ...mutations involved in the development of PMF have been heavily investigated, noteworthy is the important role the extracellular matrix (ECM) plays in the progression of BM fibrosis. This review surveys ECM proteins contributors of PMF, and highlights how better understanding of the control of the ECM within the BM niche may lead to combined therapeutic options in PMF.
OBJECTIVE:Epoxyeicosatrienoic acids (EETs) act as vasodilators activating high conductance calcium-operated potassium (K) channels (Kca1.1, also named BK, MAXI-K). We found expression of MAXI-K ...channel in platelets. The present study aimed at defining its functionality in platelet using an in vitro model of platelet thrombosis.
DESIGN AND METHOD:We tested the effects of 5 μmol/L 11,12-EET and the pharmacological modulation of MAXI-K channel (agonists5 and 20 μmol/L BMS 191011, 5 μmol/L NS1619, 5 μmol/L NS11021). Platelet rich plasma was used to assess adhesion-induced thrombi formation under flow by microfluidics technology with collagen-coated microchips mimicking arterial blood flow. The kinetic of platelet responses to scalar doses of 0.3–10 μmol/L ADP, 0.05–2 μmol/L U46619 0.5–10 μg/mL collagen was determined by paired analysis using Born aggregometry. Flow-cytometry was used to analyse the expression of active fibrinogen receptor and P-selectin in stimulated platelets. The effects of 100 μmol/L aspirin and 1 μmol/L ticagrelor were also assessed.
RESULTS:In vitro thrombi formation was halved (expressed as platelet-covered area) by pre-treatment with either 11,12-EET (−45 ± 11%, n = 5, P < 0.001 vs control, Mean ± SD), aspirin (−66 ± 8%, n = 4, P < 0.001) or ticagrelor (−55 ± 8%, n = 4, P < 0.001). Similar results were obtained using BMS 5 μmol/L (−54 ± 17%, n = 6, P < 0.001), NS1619 (−50 ± 19%, n = 9, P < 0.001), NS11021 (−60 ± 21%, n = 6, P < 0.001). The addition of 20 μmol/L BMS191011 prior to platelet aggregation (EC502.67 μmol/L, 95%CI0.97–7.29, n = 36) significantly shifted to the right the dose response-curve to ADP (EC500.91 μmol/L, 0.43–1.92, n = 36). Platelet aggregation was further blunted by the addition of aspirin to BMS191011 (EC506.18 μmol/L, 2.11–18.09, n = 36). U46619- and collagen-induced aggregation was not altered. ADP-induced activation of the fibrinogen receptor (−48 ± 14% to −62 ± 10%, n = 7, P < 0.05) and P-selectin expression (−37 ± 15% to −41 ± 13%, n = 7, P < 0.01) were blunted by the activators of MAXI-K channel.
CONCLUSIONS:Activation of the MAXI-K channel by 11,12-EET and all the tested synthetic compounds is associated with reduced sensitivity to ADP and reduced thrombus generation through the inhibition of the amplificatory phase of platelet activation. The present results reveal new mechanisms of platelet activation and suggest that targeting MAXI-K might be of potential pharmacological interest for the prevention of atherothrombosis.
Purpose
The AMPK-activator AICAR recently raised great interest for its anti-cancer properties. With specific regard to thyroid cancer, AICAR reduces cancer cell growth, invasion and metastasis. ...CXCL8, a chemokine with several recognized tumorigenic effects, is abundantly secreted in thyroid cancer microenvironment. The aim of this study was to investigate if AICAR could inhibit the basal and the TNFα-induced CXCL8 secretion in normal human thyroid cells (NHT) and in thyroid cancer cell lines TPC-1 and BCPAP (RET/PTC and BRAFV600e mutated, respectively).
Methods
The effect of AICAR on basal and CXCL8-induced cell migration was assessed. Cells were incubated with AICAR (0.05, 0.5, 1, 2 mM) alone or in combination with TNF-α (10 ng/ml) for 24 h. CXCL8 concentrations were measured in cell supernatants. Transwell migration assays were performed in NHT, TPC-1 and BCPAP, basally and after treatment with AICAR (2 mM) and rh-CXCL8 (50 ng/ml) alone or in combination.
Results
AICAR dose dependently inhibited the basal secretion of CXCL8 in TPC-1 (
F
= 4.26;
p
< 0.007) and BCPAP (
F
= 6.75;
p
< 0.0001) but not in NHT. TNFα-induced CXCL8 secretion was dose dependently reduced by AICAR in NHT (
F
= 9.99;
p
< 0.0001), TPC-1 (
F
= 9.25;
p
< 0.0001) and BCPAP (
F
= 6.82;
p
< 0.0001). AICAR significantly reduced the basal migration of TPC-1 and BCPAP but not of NHT.
Conclusions
CXCL8-induced cell migration was inhibited in NHT, TPC-1 and BCPAP. This is the first demonstration of the inhibition of CXCL8 secretion exerted by AICAR in TPC-1 and BCPAP indicating that the anti-cancer properties of AICAR are, at least in part, mediated by its ability to reduce the pro-tumorigenic effects of CXCL8.
In vitro generation of platelets: Where do we stand? Di Buduo, C.A.; Kaplan, D.L.; Balduini, A.
Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine,
September 2017, 2017-Sep, 2017-09-00, Letnik:
24, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Millions of platelets, specialized cells that participate in haemostatic and inflammatory functions, are transfused each year worldwide, but their supply is limited. Platelets are produced by ...megakaryocytes by extending proplatelets, directly into the bloodstream. Bone marrow structure and extracellular matrix composition together with soluble factors (e.g. Thrombopoietin) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet release. Despite this knowledge, the scarcity of clinical cures for life threatening platelet diseases is in a large part due to limited insight into the mechanisms that control the developmental process of megakaryocytes and the mechanisms that govern the production of platelets within the bone marrow. To overcome these limitations, functional human tissue models have been developed and studied to extrapolate ex vivo outcomes for new insight on bone marrow functions in vivo. There are many challenges that these models must overcome, from faithfully mimicking the physiological composition and functions of bone marrow, to the collection of the platelets generated and validation of their viability and function for human use. The overall goal is to identify innovative instruments to study mechanisms of platelet release, diseases related to platelet production and new therapeutic targets starting from human progenitor cells.
Des millions de plaquettes, cellules spécialisées qui participent aux fonctions hémostatiques et inflammatoires, sont transfusées chaque année dans le monde entier, mais leur offre est limitée. Les plaquettes sont produites par les mégacaryocytes en émettant les pro-plaquettes directement dans la circulation sanguine. La structure de la moelle osseuse et la composition de la matrice extracellulaire ainsi que les facteurs solubles (par exemple la thrombopoïétine) sont des régulateurs clés de la mégacaryopoïèse en favorisant la différenciation cellulaire et la libération de plaquettes. Malgré cette connaissance, la rareté des traitements cliniques à vie des maladies des plaquettes est en grande partie due à une connaissance limitée quant aux mécanismes qui contrôlent le processus de développement des mégacaryocytes et des mécanismes qui régissent la production de plaquettes dans la moelle osseuse. Pour surmonter ces limites, des modèles de tissus humains ont été développés et étudiés pour extrapoler ex vivo des résultats pour une nouvelle vision sur les fonctions de la moelle osseuse in vivo. Il y a beaucoup de défis que ces modèles doivent surmonter, notamment : imiter fidèlement la composition physiologique et les fonctions de la moelle osseuse, collecter les plaquettes générées et valider de leur viabilité et fonctions pour l’usage humain. L’objectif global est d’identifier des instruments innovants pour étudier les mécanismes de libération de plaquettes, les maladies liées à la production de plaquettes et de nouvelles cibles thérapeutiques à partir de cellules progénitrices humaines.
Eltrombopag is a small, non-peptide thrombopoietin mimetic that has been approved for increasing platelet count not only in immune thrombocytopenia and Hepatitis C virus-related thrombocytopenia, but ...also in aplastic anemia. Moreover, this drug is under investigation for increasing platelet counts in myelodysplastic syndromes. Despite current clinical practice, the mechanisms governing eltrombopag's impact on human hematopoiesis are largely unknown, in part due to the impossibility of using traditional in vivo models. To investigate eltrombopag's impact on megakaryocyte functions, we employed our established in vitro model for studying hematopoietic stem cell differentiation combined with our latest 3-dimensional silk-based bone marrow tissue model. Results demonstrated that eltrombopag favors human megakaryocyte differentiation and platelet production in a dose-dependent manner. These effects are accompanied by increased phosphorylation of AKT and ERK1/2 signaling molecules, which have been proven to be crucial in regulating physiologic thrombopoiesis. These data further clarify the different mechanisms of action of eltrombopag when compared to romiplostim, which, as we have shown, induces the proliferation of immature megakaryocytes rather than platelet production, due to the unbalanced activation of AKT and ERK1/2 signaling molecules. In conclusion, our research clarifies the underlying mechanisms that govern the action of eltrombopag on megakaryocyte functions and its relevance in clinical practice.
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