RESUMO
SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].
Assuntos
Lúpus Eritematoso Sistêmico , Púrpura Trombocitopênica Idiopática , Trombocitopenia , Autoanticorpos , Plaquetas , Humanos , Lúpus Eritematoso Sistêmico/complicações , Contagem de Plaquetas , Trombocitopenia/complicações , TrombopoeseRESUMO
Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.
Assuntos
Transtornos Plaquetários/genética , Transtornos Plaquetários/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Integrina alfa2/genética , Leucemia Mieloide Aguda/etiologia , Receptores Imunológicos/genética , Transtornos Plaquetários/sangue , Plaquetas/metabolismo , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Megacariócitos/metabolismo , Mutação , Agregação Plaquetária , Testes de Função Plaquetária , Ligação ProteicaRESUMO
The SDF-1-CXCR4 axis plays an essential role in the regulation of platelet production, by directing megakaryocyte (MK) migration toward the vascular niche, thus allowing terminal maturation and proplatelet formation, and also regulates platelet function in an autocrine manner. Inherited thrombocytopenias (IT) comprise a spectrum of diverse clinical conditions caused by mutations in genes involved in platelet production and function. We assessed CXCR4 expression and SDF-1 levels in a panel of well-characterized forms of IT. Decreased surface CXCR4 levels were found in 8 of 27 (29.6%) IT patients by flow cytometry, including 4 of 6 patients with ANKRD26-RT, 3 of 3 patients with GPS and 1 of 6 patients with FPD/AML. Low CXCR4 levels were associated with impaired SDF-1-triggered platelet aggregation, indicating that this decrease is functionally relevant, whereas a normal platelet response was shown in patients harbouring preserved membrane CXCR4. Reduced CXCR4 was not due to decreased gene expression, as platelet RNA levels were normal or increased, suggesting a post-transcriptional defect. Increased ligand-induced receptor internalization was ruled out, as circulating SDF-1 levels were similar to controls. MK CXCR4 expression was normal, indicating that the defect in CXCR4 arises after the step of platelet biogenesis. In conclusion, the finding of defective CXCR4 expression specifically associated with certain IT disorders highlights the fact that abnormalities in several megakaryocytic regulators underlie IT pathogenesis and further reveal the heterogeneous nature of these conditions.
Assuntos
Plaquetas/metabolismo , Quimiocina CXCL12/biossíntese , Regulação da Expressão Gênica , Doenças Genéticas Inatas/sangue , Megacariócitos/metabolismo , Receptores CXCR4/biossíntese , Trombocitopenia/sangue , Adolescente , Adulto , Idoso , Plaquetas/patologia , Criança , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Megacariócitos/patologia , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Agregação Plaquetária/genética , Trombocitopenia/genética , Trombocitopenia/patologiaRESUMO
BACKGROUND: Anagrelide represents a treatment option for essential thrombocythemia, although its place in therapy remains controversial. AIM: To assess the impact of mutational status in response rates and development of adverse events during long-term use of anagrelide. METHODS: We retrospectively evaluated 67 patients with essential thrombocythemia treated with anagrelide during 68 (4-176) months. RESULTS: Mutational frequencies were 46.3%, 28.3%, and 1.5% for JAK2V617F, CALR and MPL mutations. Anagrelide yielded a high rate of hematologic responses, which were complete in 49.25% and partial in 46.25%, without differences among molecular subsets. The rate of thrombosis during treatment was one per 100 patient-years, without excess bleeding. Anemia was the major adverse event, 30.3% at 5-yr follow-up, being more frequent in CALR(+) (P < 0.05). Myelofibrotic transformation developed in 14.9% (12.9%, 21%, and 12.5% in JAK2V617F(+), CALR(+), and triple-negative patients, respectively, P = NS) and those treated >60 months were at higher risk, OR (95% CI) 9.32 (1.1-78.5), P < 0.01, indicating the need for bone marrow monitoring during prolonged treatment. CONCLUSION: Although CALR(+) patients were at higher risk of developing anemia, anagrelide proved effective among all molecular subsets, indicating that mutational status does not seem to represent a major determinant of choice of cytoreductive treatment among essential thrombocythemia therapies.
Assuntos
Calreticulina/genética , Janus Quinase 2/genética , Inibidores da Agregação Plaquetária/administração & dosagem , Quinazolinas/administração & dosagem , Receptores de Trombopoetina/genética , Trombocitemia Essencial/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/etiologia , Anemia/patologia , Calreticulina/imunologia , Criança , Feminino , Seguimentos , Expressão Gênica , Humanos , Janus Quinase 2/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores da Agregação Plaquetária/efeitos adversos , Mielofibrose Primária/etiologia , Mielofibrose Primária/patologia , Quinazolinas/efeitos adversos , Receptores de Trombopoetina/imunologia , Estudos Retrospectivos , Trombocitemia Essencial/genética , Trombocitemia Essencial/imunologia , Trombocitemia Essencial/patologiaRESUMO
The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and ß3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.
Assuntos
Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/imunologia , Trombopoese , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos/imunologia , Apoptose/imunologia , Autoanticorpos/imunologia , Plaquetas/citologia , Humanos , Integrinas/imunologia , Megacariócitos/citologia , Megacariócitos/imunologia , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Trombopoese/imunologia , Adulto JovemRESUMO
BACKGROUND: Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. MATERIALS AND METHODS: Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. RESULTS: In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. CONCLUSION: Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.
Assuntos
Proteína HMGB1 , Lúpus Eritematoso Sistêmico , Humanos , Proteína HMGB1/metabolismo , Ligante de CD40 , Fator de von Willebrand/metabolismo , Receptores Toll-Like/metabolismo , Plaquetas/metabolismo , Inflamação/metabolismo , Receptor Toll-Like 9Assuntos
Autoanticorpos/imunologia , Células da Medula Óssea/imunologia , Proteínas da Matriz Extracelular/imunologia , Megacariócitos/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Adesão Celular/imunologia , Células Cultivadas , Feminino , Humanos , Masculino , Megacariócitos/fisiologia , Pessoa de Meia-Idade , Trombopoese/imunologia , Adulto JovemRESUMO
Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.
Assuntos
Ácidos Nucleicos Livres , Mielofibrose Primária , Humanos , Inflamassomos/metabolismo , Citocinas/metabolismo , Interleucina-18/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mielofibrose Primária/genética , Inflamação/induzido quimicamente , DNA , Progressão da DoençaRESUMO
Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells into in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells in in vitro megakaryopoiesis, thrombopoiesis, and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then cocultured with umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SFs) (all human), or the mouse fibroblast cell line L929. The number of MKs (CD61-positive cells) was increased in the presence of HUVECs and SFs, whereas L929 cells decreased total and mature MK counts. With respect to thrombopoiesis, HUVECs increased proplatelet (PP)-producing MKs, while MSCs, L929 cells, and SFs had the opposite effect (immunofluorescence staining and microscopic analysis). MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVECs and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SFs) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate, and prolyl 4-hydroxylase subunit ß was preserved. These data indicate that each stromal cell type performs distinctive functions that differentially modulate MK growth and platelet production and, at the same time, that MKs also modify stromal cell behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.
Assuntos
Plaquetas , Megacariócitos , Proliferação de Células , Células EstromaisRESUMO
OBJECTIVES: To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). METHODS: Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. RESULTS: MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. CONCLUSIONS: The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.
Assuntos
Plaquetas/metabolismo , Mutação , Mielofibrose Primária/genética , Receptores de Trombopoetina/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Trombocitemia Essencial/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Western Blotting , Criança , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.
Assuntos
Plaquetas/fisiologia , Inflamação/etiologia , Trombocitemia Essencial/complicações , Trombose/etiologia , Receptores Toll-Like/fisiologia , Adulto , Idoso , Quimiocina CCL5/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/fisiologia , Fosforilação , Ativação Plaquetária , Trombocitemia Essencial/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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.
Assuntos
Quimiocina CXCL12/metabolismo , Megacariócitos/metabolismo , Receptores CXCR4/metabolismo , Trombocitemia Essencial/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/química , Plaquetas/metabolismo , Estudos de Casos e Controles , Quimiocina CXCL1/sangue , Quimiocina CXCL12/sangue , Criança , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interleucina-8/sangue , Masculino , Megacariócitos/química , Pessoa de Meia-Idade , Agregação Plaquetária , RNA Mensageiro/análise , Receptores CXCR4/análise , Receptores CXCR4/genética , Receptores de Interleucina-8A/análise , Receptores de Interleucina-8B/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Trombocitemia Essencial/sangueRESUMO
MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils and might develop sensorineural deafness, presenile cataract, and/or progressive nephritis leading to end-stage renal failure. In a family with eight individuals suffering from macrothrombocytopenia and hearing impairment we identified a novel c.Ala95Asp mutation. Affecting the motor domain of the protein, the mutation is likely to be associated with a severe phenotype. Therefore, this family should be carefully monitored to follow-up the renal status even though the affected members do not seem to be at risk of early kidney disease.
Assuntos
Proteínas Motores Moleculares/genética , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Adulto , Alanina/genética , Sequência de Aminoácidos , Animais , Ácido Aspártico/genética , Sequência de Bases , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Modelos Moleculares , Proteínas Motores Moleculares/química , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Linhagem , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.
RESUMO
Megakaryopoiesis and platelet production are driven by transcription factors and cytokines present in bone marrow environment. Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by high platelet count and megakaryocytic hyperplasia. In the present work we evaluated plasmatic levels of cytokines involved in megakaryocytic development in a group of patients with ET that were not on treatment, as well as thrombopoietin (TPO) levels before and during anagrelide treatment. The assays were carried out using ELISA techniques. Among the cytokines mainly involved in proliferation of megakaryocytic progenitors, interleukin 3 (IL-3) levels were found increased in patients compared to normal controls (p = 0.0383). Granulocyte-macrophage colony stimulating factor and stem cell factor levels were normal. Interleukin 6, as well as interleukin 11 and erythropoietin (EPO), cytokines mainly related to megakaryocytic maturation, were normal. Plasma TPO levels before treatment were within the normal range and increased during treatment but the difference was not statistically significant. Patients who displayed spontaneous platelet aggregation had higher plasma TPO levels compared to those who did not (p = 0.049). We did not find any relationship between cytokine levels and clinical or laboratory parameters. The high IL-3 levels seen in some patients with ET could contribute to megakaryocytic proliferation. The simultaneous occurrence of higher TPO levels and elevated platelet count could be a predisposing factor for the development of spontaneous platelet aggregation in ET patients.
Assuntos
Megacariócitos/fisiologia , Trombocitemia Essencial/sangue , Trombopoese/fisiologia , Trombopoetina/sangue , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Humanos , Interleucina-3/sangue , Megacariócitos/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Inibidores da Agregação Plaquetária/uso terapêutico , Quinazolinas/uso terapêutico , Estudos Retrospectivos , Estatísticas não Paramétricas , Fator de Células-Tronco/sangue , Fator de Células-Tronco/efeitos dos fármacos , Trombocitemia Essencial/tratamento farmacológico , Trombocitose/induzido quimicamente , Trombopoese/efeitos dos fármacos , Trombopoetina/efeitos dos fármacosRESUMO
Mechanisms leading to decreased platelet count in immune thrombocytopenia (ITP) are heterogeneous. This study describes increased platelet apoptosis involving loss of mitochondrial membrane potential (ΔΨm), caspase 3 activation (aCasp3) and phosphatidylserine (PS) externalization in a cohort of adult ITP patients. Apoptosis was not related to platelet activation, as PAC-1 binding, P-selectin exposure and GPIb-IX internalization were not increased. Besides, ITP platelets were more sensitive to apoptotic stimulus in terms of aCasp3. Incubation of normal platelets with ITP plasma induced loss of ΔΨm, while PS exposure and aCasp3 remained unaltered. The increase in PS exposure observed in ITP platelets could be reproduced in normal platelets incubated with ITP plasma by adding normal CD3+ lymphocytes to the system as effector cells. Addition of leupeptin -a cathepsin B inhibitor- to this system protected platelets from apoptosis. Increased PS exposure was also observed when normal platelets and CD3+ lymphocytes were incubated with purified IgG from ITP patients and was absent when ITP plasma was depleted of auto-antibodies, pointing to the latter as responsible for platelet damage. Apoptosis was present in platelets from all patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies but was absent in the patient with anti-GPIa-IIa auto-antibodies. Platelet damage inversely correlated with platelet count and decreased during treatment with a thrombopoietin receptor agonist. These results point to a key role for auto-antibodies in platelet apoptosis and suggest that antibody-dependent cell cytotoxicity is the mechanism underlying this phenomenon.
Assuntos
Autoanticorpos/imunologia , Plaquetas/patologia , Púrpura Trombocitopênica Idiopática/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Complexo CD3/metabolismo , Calcimicina/farmacologia , Caspase 3/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo , Plasma , Ativação Plaquetária , Púrpura Trombocitopênica Idiopática/sangue , Adulto JovemRESUMO
The mechanisms underlying increased thrombotic risk in chronic myeloproliferative neoplasms (MPN) are incompletely understood. We assessed whether neutrophil extracellular traps (NETs), which promote thrombosis, contribute to the procoagulant state in essential thrombocythemia, polycythemia vera and myelofibrosis (MF) patients. Although MPN neutrophils showed increased basal reactive oxygen species (ROS), enhanced NETosis by unstimulated neutrophils was an infrequent finding, whereas PMA-triggered NETosis was impaired, particularly in MF, due to decreased PMA-triggered ROS production. Elevated circulating nucleosomes were a prominent finding and were higher in patients with advanced disease, which may have potential prognostic implication. Histone-MPO complexes, proposed as specific NET biomarker, were seldomly detected, suggesting NETs may not be the main source of nucleosomes in most patients, whereas their correlation with high LDH points to increased cell turn-over as a plausible origin. Lack of association of nucleosomes or NETs with thrombosis or activation markers does not support their use as predictors of thrombosis although prospective studies in a larger cohort may help define their potential contribution to MPN thrombosis. These results do not provide evidence for relevant in vivo NETosis in MPN patients under steady state conditions, although availability of standardized NET biomarkers may contribute to further research in this field.
Assuntos
Biomarcadores Tumorais/sangue , Armadilhas Extracelulares/metabolismo , Neoplasias Hematológicas/sangue , Transtornos Mieloproliferativos/sangue , Nucleossomos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Feminino , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Peroxidase/sangue , Espécies Reativas de Oxigênio/sangueRESUMO
OBJECTIVE: In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction. MATERIALS AND METHODS: Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor-α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction. RESULTS: Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal. CONCLUSIONS: Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET.