RESUMO
BACKGROUND: Lemierre syndrome is characterized by head/neck vein thrombosis and septic embolism usually complicating an acute oropharyngeal bacterial infection in adolescents and young adults. We described the course of Lemierre syndrome in the contemporary era. METHODS: In our individual-level analysis of 712 patients (2000-2017), we included cases described as Lemierre syndrome if these criteria were met: (i) primary site of bacterial infection in the head/neck; (ii) objectively confirmed local thrombotic complications or septic embolism. The study outcomes were new or recurrent venous thromboembolism or peripheral septic lesions, major bleeding, all-cause death and clinical sequelae. RESULTS: The median age was 21 (Q1-Q3: 17-33) years, and 295 (41%) were female. At diagnosis, acute thrombosis of head/neck veins was detected in 597 (84%) patients, septic embolism in 582 (82%) and both in 468 (80%). After diagnosis and during in-hospital follow-up, new venous thromboembolism occurred in 34 (5.2%, 95% CI 3.8-7.2%) patients, new peripheral septic lesions became evident in 76 (11.7%; 9.4-14.3%). The rate of either was lower in patients who received anticoagulation (OR: 0.59; 0.36-0.94), higher in those with initial intracranial involvement (OR: 2.35; 1.45-3.80). Major bleeding occurred in 19 patients (2.9%; 1.9-4.5%), and 26 died (4.0%; 2.7-5.8%). Clinical sequelae were reported in 65 (10.4%, 8.2-13.0%) individuals, often consisting of cranial nerve palsy (n = 24) and orthopaedic limitations (n = 19). CONCLUSIONS: Patients with Lemierre syndrome were characterized by a substantial risk of new thromboembolic complications and death. This risk was higher in the presence of initial intracranial involvement. One-tenth of survivors suffered major clinical sequelae.
Assuntos
Síndrome de Lemierre/complicações , Tromboembolia/etiologia , Trombose Venosa/etiologia , Adolescente , Adulto , Progressão da Doença , Feminino , Humanos , Síndrome de Lemierre/mortalidade , Masculino , Tromboembolia/mortalidade , Trombose Venosa/mortalidadeRESUMO
Hereditary thrombocytopenias (HTPs) constitute a heterogeneous group of diseases characterized by a reduction in platelet count and a potential bleeding risk. As a result of advances in diagnostic methods, HTPs are increasingly being identified, and appear to be less rare than previously thought. Most HTPs do not have effective treatments, except for platelet transfusion when bleeding occurs and in preparation for procedures associated with a risk of bleeding. Preliminary clinical evidence suggests that thrombopoietin receptor agonists (TPO-RAs) with an established use in the treatment of certain acquired thrombocytopenias are well tolerated and provide clinical benefits in patients with some forms of HTP. These drugs may therefore be considered for the treatment of HTPs in clinical practice. However, caution and close monitoring are recommended, owing to the absence of long-term safety data and the potential risks posed by prolonged bone marrow stimulation in certain HTPs. In this review, we summarize the available clinical data on TPO-RAs in the treatment of HTPs, and discuss their use in patients with these disorders. We believe that TPO-RAs will play a major role in the treatment of HTPs, particularly myosin heavy chain 9-related disease, Wiskott-Aldrich syndrome, X-linked thrombocytopenia, and thrombocytopenia caused by THPO mutations.
Assuntos
Benzoatos/uso terapêutico , Hidrazinas/uso terapêutico , Pirazóis/uso terapêutico , Receptores Fc/uso terapêutico , Receptores de Trombopoetina/agonistas , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/tratamento farmacológico , Trombopoetina/uso terapêutico , Benzoatos/efeitos adversos , Benzoatos/farmacologia , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Estudos de Associação Genética , Heterogeneidade Genética , Predisposição Genética para Doença , Neoplasias Hematológicas/etiologia , Humanos , Hidrazinas/efeitos adversos , Hidrazinas/farmacologia , Mielofibrose Primária/etiologia , Púrpura Trombocitopênica Idiopática/complicações , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacologia , Risco , Trombocitopenia/genética , Trombofilia/induzido quimicamente , Trombopoese/efeitos dos fármacos , Trombopoetina/efeitos adversos , Trombopoetina/farmacologiaRESUMO
Essentials Thrombocytopenia 2 (THC2) is an inherited thrombocytopenia (IT) with dysmegakaryopoiesis. Physicians often do not suspect the genetic origin of thrombocytopenia in patients with THC2. We report two THC2 patients misdiagnosed with myelodysplasia and treated with chemotherapy. IT should be always considered in patients with isolated thrombocytopenia and dysmegakaryopoiesis. SUMMARY: Thrombocytopenia 2 (THC2) is an autosomal-dominant disorder caused by point substitutions in the 5'UTR of the ANKRD26 gene. Patients have congenital thrombocytopenia, normal platelet morphology and function, and dysmegakaryopoiesis. Thrombocytopenia is frequently discovered only in adulthood and physicians often do not suspect its genetic origin. We describe two unrelated patients referred to two different institutions for investigation of thrombocytopenia. Based on the finding of dysmegakaryopoiesis at bone marrow examination, patients were diagnosed with myelodysplastic syndrome (MDS) (refractory thrombocytopenia) and treated with several courses of 5-azacytidine. Subsequently, demonstration of thrombocytopenia in their relatives eventually led to molecular diagnosis of THC2 in both families. These cases highlight that patients with THC2 are at risk of being misdiagnosed with MDS and receiving undue myelosuppressive treatments. Because dysmegakaryopoiesis is a feature also of other forms of inherited thrombocytopenia, a genetic disorder must always be considered when a patient presents with isolated thrombocytopenia and dysmegakaryopoiesis.
Assuntos
Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Mutação , Síndromes Mielodisplásicas/diagnóstico , Proteínas Nucleares/genética , Trombocitopenia/congênito , Idoso , Medula Óssea/patologia , Quebra Cromossômica , Transtornos Cromossômicos/patologia , Análise Mutacional de DNA , Erros de Diagnóstico , Predisposição Genética para Doença , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Pessoa de Meia-Idade , Trombocitopenia/diagnóstico , Trombocitopenia/genética , Trombocitopenia/patologia , Trombopoese/genéticaRESUMO
Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world. SUMMARY: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 µL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.
Assuntos
Transtornos Plaquetários/sangue , Transtornos Plaquetários/diagnóstico , Plaquetas/metabolismo , Testes Hematológicos/instrumentação , Testes Hematológicos/métodos , Alelos , Síndrome de Bernard-Soulier/genética , Feminino , Humanos , Imunofenotipagem , Cooperação Internacional , Masculino , Microscopia de Fluorescência , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Fenótipo , Sensibilidade e Especificidade , Trombastenia/genéticaRESUMO
MYH9-related disease (MYH9-RD) is a rare genetic syndromic disorder characterised by congenital thrombocytopenia and is associated with the risk of developing progressive sensorineural hearing loss, nephropathy and presenile cataracts during childhood or adult life. All consecutive patients enrolled in the Italian Registry for MYH9-RD with severe to profound deafness were included in a retrospective study. The study population involved 147 Italian patients with MYH9-RD: hearing loss was identified in 52% of cases and only 4 patients (6%) presented severe to profound deafness at a mean age of 33 years. Deafness was associated with mild spontaneous bleeding in all patients and with kidney involvement in 3 cases. Cochlear implantation was carried out in 3 cases with benefit, and no major complications were observed. Diagnosis was performed about 28 years after the first clinical manifestation of MYH9-RD, which was never suspected by an otolaryngologist. The clinical and diagnostic aspects of 4 patients with severe to profound deafness are discussed with a focus on therapeutic implications.
Assuntos
Surdez/etiologia , Perda Auditiva Neurossensorial/complicações , Trombocitopenia/congênito , Adulto , Surdez/diagnóstico , Feminino , Humanos , Masculino , Estudos Retrospectivos , Índice de Gravidade de Doença , Trombocitopenia/complicaçõesRESUMO
In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.
Assuntos
Glândulas Suprarrenais/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Knowledge in the field of inherited thrombocytopenias (ITs) has greatly improved over the last 15 years. Several new genes responsible for thrombocytopenia have been identified leading to the definition of novel nosographic entities and to a much better characterization of the phenotypes of these diseases. To date, ITs encompass 22 disorders caused by mutations in 24 genes and characterized by different degrees of complexity and great variability in prognosis. Making a definite diagnosis is important for setting an appropriate follow-up, choosing the best treatments and providing proper counseling. Despite the abovementioned progress, diagnosis of ITs remains difficult and these disorders are still underdiagnosed. The purpose of this review is to provide an updated guide to the diagnosis of ITs based on simple procedures. Moreover, the currently available therapeutic options for these conditions are recapitulated and discussed.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/terapia , Trombocitopenia/diagnóstico , Trombocitopenia/terapia , Diagnóstico Diferencial , HumanosRESUMO
MYH9-related disease (MYH9-RD) is a rare autosomal dominant disease caused by mutation of MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (NMMHC-IIA). MYH9-RD patients have macrothrombocytopenia and granulocyte inclusions (pathognomonic sign of the disease) containing wild-type and mutant NMMHC-IIA. During life they might develop sensorineural hearing loss, cataract, glomerulonephritis, and elevation of liver enzymes. One of the MYH9 mutations, p.R705H, was previously reported to be associated with DFNA17, an autosomal dominant non-syndromic sensorineural hearing loss without any other features associated. We identified the same mutation in two unrelated families, whose four affected individuals had not only hearing impairment but also thrombocytopenia, giant platelets, leukocyte inclusions, as well as mild to moderate elevation of some liver enzymes. Our data suggest that DFNA17 should not be a separate genetic entity but part of the wide phenotypic spectrum of MYH9-RD characterized by congenital hematological manifestations and variable penetrance and expressivity of the extra-hematological features.
Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas Motores Moleculares/genética , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Trombocitopenia/congênito , Adolescente , Adulto , Pré-Escolar , Feminino , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Trombocitopenia/diagnóstico , Trombocitopenia/genéticaRESUMO
BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9) L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.
Assuntos
Síndrome de Bernard-Soulier/genética , Plaquetas/metabolismo , Microtúbulos/metabolismo , Polimorfismo de Nucleotídeo Único , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Síndrome de Bernard-Soulier/sangue , Síndrome de Bernard-Soulier/diagnóstico , Plaquetas/efeitos dos fármacos , Cristalografia por Raios X , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Células MCF-7 , Microtúbulos/efeitos dos fármacos , Modelos Moleculares , Fenótipo , Contagem de Plaquetas , Conformação Proteica , Estabilidade Proteica , Índice de Gravidade de Doença , Relação Estrutura-Atividade , Transfecção , Tubulina (Proteína)/química , Moduladores de Tubulina/farmacologiaRESUMO
The chapter of inherited thrombocytopenias has expanded greatly over the last decade and many "new" forms deriving from mutations in "new" genes have been identified. Nevertheless, nearly half of patients remain without a definite diagnosis because their illnesses have not yet been described. The diagnostic approach to these diseases can still take advantage of the algorithm proposed by the Italian Platelet Study Group in 2003, although an update is required to include the recently described disorders. So far, transfusions of platelet concentrates have represented the main tool for preventing or treating bleedings, while haematopoietic stem cell transplantation has been reserved for patients with very severe forms. However, recent disclosure that an oral thrombopoietin mimetic is effective in increasing platelet count in patients with MYH9-related thrombocytopenia opened new therapeutic perspectives. This review summarizes the general aspects of inherited thrombocytopenias and describes in more detail MYH9-related diseases (encompassing four thrombocytopenias previously recognized as separate diseases) and the recently described ANKRD26-related thrombocytopenia, which are among the most frequent forms of inherited thrombocytopenia.
Assuntos
Predisposição Genética para Doença/genética , Peptídeos/uso terapêutico , Transfusão de Plaquetas , Trombocitopenia/congênito , Trombocitopenia/terapia , Anticoagulantes/uso terapêutico , Humanos , Trombocitopenia/diagnósticoRESUMO
BACKGROUND: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. METHODS: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. RESULTS: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. CONCLUSIONS: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.
Assuntos
Comportamento Cooperativo , Países em Desenvolvimento , Testes Genéticos , Testes Hematológicos , Cooperação Internacional , Trombocitopenia/diagnóstico , Adolescente , Adulto , Idoso , Algoritmos , Argentina , Biomarcadores/sangue , Criança , Pré-Escolar , Análise Mutacional de DNA , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Imunofluorescência , Predisposição Genética para Doença , Testes Genéticos/métodos , Acessibilidade aos Serviços de Saúde , Testes Hematológicos/métodos , Hereditariedade , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Proteínas Motores Moleculares/sangue , Cadeias Pesadas de Miosina/sangue , Linhagem , Fenótipo , Contagem de Plaquetas , Testes de Função Plaquetária , Valor Preditivo dos Testes , Prognóstico , Encaminhamento e Consulta , Trombocitopenia/sangue , Trombocitopenia/congênito , Trombospondina 1/sangue , Adulto JovemRESUMO
BACKGROUND: Distinguishing inherited thrombocytopenias from immune thrombocytopenia (ITP) can be difficult, and patients are therefore at risk of misdiagnosis and inappropriate treatments. Although it is known that the most common inherited forms of thrombocytopenia are characterized by increased platelet size, the diagnostic power of this feature has never been investigated. OBJECTIVES: The aim of this study was to test the hypothesis that platelet size can be used to differentiate ITP from inherited macrothrombocytopenias. PATIENTS/METHODS: We measured mean platelet volume (MPV) and mean platelet diameter (MPD), within 2 h of blood sampling, in 35 patients with inherited macrothrombocytopenias [15 MYH9-related disease (MYH9-RD), three biallelic and 17 monoallelic Bernard-Soulier syndrome (BSS)], and 56 with ITP. Using receiving operating characteristic analysis, we searched for the best cut-off values to differentiate between these conditions. RESULTS: As expected, platelets were larger in inherited macrothrombocytopenias than in ITP. An MPD larger than 3.3 mum differentiated MYH9-RD and BSS from ITP with 0.89 sensitivity and 0.88 specificity, and an MPV larger than 12.4 fL had 0.83 sensitivity and 0.89 specificity. Combining MPD with MPV increased sensitivity and specificity to 0.97 and 0.89, respectively. CONCLUSION: Platelet size evaluation by both an appropriate cell counter and blood film examination is useful for differentiating inherited macrothrombocytopenias from ITP.
Assuntos
Plaquetas/patologia , Trombocitopenia/patologia , Síndrome de Bernard-Soulier/diagnóstico , Síndrome de Bernard-Soulier/patologia , Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Transtornos Herdados da Coagulação Sanguínea/patologia , Tamanho Celular , Citodiagnóstico/métodos , Diagnóstico Diferencial , Humanos , Proteínas Motores Moleculares , Cadeias Pesadas de Miosina , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/patologia , Trombocitopenia/diagnósticoRESUMO
BACKGROUND: Although mutations of GPIb alpha 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 GPIb alpha (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, alpha-tubulin distribution within proplatelets was severely deranged. CONCLUSIONS: Megakaryocytes from patients carrying a Bolzano allele of GPIb alpha 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 GPIb alpha in proplatelet formation.
Assuntos
Síndrome de Bernard-Soulier/genética , Plaquetas/patologia , Megacariócitos/patologia , Proteínas de Membrana/genética , Alelos , Síndrome de Bernard-Soulier/patologia , Diferenciação Celular , Forma Celular , Heterozigoto , Humanos , Glicoproteínas de Membrana , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIb-IX de Plaquetas , TrombocitopeniaRESUMO
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 alphaIIbbeta3 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 GPIbalpha 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.
Assuntos
Plaquetas/citologia , Proteínas Sanguíneas/metabolismo , Megacariócitos/citologia , Glicoproteínas de Membrana/metabolismo , Miosina não Muscular Tipo IIA/fisiologia , Adesão Celular , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Humanos , Cinética , Megacariócitos/metabolismo , Microscopia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.
Assuntos
Plaquetas/efeitos dos fármacos , Coagulantes/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Deficiência do Pool Plaquetário/sangue , Receptor PAR-1/agonistas , Trombina/farmacologia , Adolescente , Adulto , Idoso , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Família , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Selectina-P/análise , Linhagem , Fenótipo , Fator Plaquetário 4/análise , Testes de Função Plaquetária , Deficiência do Pool Plaquetário/diagnóstico , Deficiência do Pool Plaquetário/genética , Deficiência do Pool Plaquetário/metabolismo , Deficiência do Pool Plaquetário/patologia , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Síndrome , Trombospondina 1/análise , Tromboxano B2/sangueRESUMO
BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.
Assuntos
Sangue Fetal/citologia , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Trombopoese/fisiologia , Anticorpos Monoclonais/metabolismo , Antígenos CD34/biossíntese , Antígenos CD34/metabolismo , Células Precursoras Eritroides/metabolismo , Citometria de Fluxo , Humanos , Técnicas In Vitro , Microscopia de Fluorescência , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Glicoproteína IIb da Membrana de Plaquetas/química , RNA/química , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.
Assuntos
Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Trombocitopenia/genética , Adolescente , Adulto , Dimerização , Eletroforese em Gel de Poliacrilamida , Saúde da Família , Feminino , GTP Fosfo-Hidrolases/metabolismo , Genes Dominantes , Glicoproteínas/metabolismo , Humanos , Immunoblotting , Masculino , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Mutação , Miosina não Muscular Tipo IIA/química , Polimorfismo Genético , Transdução de SinaisRESUMO
We analysed by immunocytochemistry the expression of p53, bcl-2 and ras proteins in bone marrow blasts from 59 patients with acute leukaemia (AL), 36 myeloid (AML) and 23 lymphoid (ALL), and from 22 patients with myelodysplastic syndrome (MDS); our aim was to examine if abnormalities in their expression were associated with peculiar biological and clinical findings, or with an altered apoptosis rate, as measured by TUNEL technique. The oncoproteins were expressed with extreme variability, without significant differences among the various morphological or immunological AL subtypes. The mean percentages of bcl-2+ blasts were significantly higher in AML than in MDS (p = 0.01), and in MDS with bone marrow blastosis than in the forms without excess of blasts (p = 0.007). The lowest percentages of apoptotic cells were observed in ALL (mean 1%, p = 0.006), whereas in MDS the apoptotic index was higher (16.7%) than in AML (8.6%) and than in the normal controls (10.8%). but the difference tended to be statistically significant only for cases of refractory anaemia. Whereas in AML and MDS the apoptotic rate was independent of the oncoprotein expression, in ALL there was a significant linear relationship between TUNEL and ras positivity (p = 0.01). Among AML patients treated with intensive polychemotherapy, no differences were observed in oncoprotein expression and apoptotic rate between responders and resistant cases. In conclusion, our data are in agreement with the hypothesis that decreased apoptosis and enhanced cell survival are associated with AL, whereas a high level of apoptosis may be responsible for the ineffective hematopoiesis in MDS; abnormal expression of oncoproteins, even if not strictly related to apoptosis level, may influence disease behaviour.
Assuntos
Apoptose/fisiologia , Genes bcl-2 , Genes p53 , Genes ras , Leucemia/genética , Síndromes Mielodisplásicas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/genética , Células da Medula Óssea/patologia , Criança , Pré-Escolar , Feminino , Humanos , Cariotipagem , Leucemia/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
The absolute content of CD34(+) cells in the peripheral blood of 84 patients with myelofibrosis with myeloid metaplasia (MMM) and 23 patients with other Philadelphia-negative (Ph(-)) chronic myeloproliferative disorders (CMDs) was investigated. In MMM, the median absolute number of circulating CD34(+) cells was consistently high (91.6 x 10(6)/L; range, 0-2460 x 10(6)/L). Receiver operating characteristic curve analysis showed that 15 x 10(6)/L as a decision criterion for CD34(+) cells produced an almost complete discrimination between MMM patients out of therapy and other Ph(-) CMDs (positive predictive value, 98.4%; negative predictive value, 85.0%). MMM patients with higher numbers of CD34(+) cells had a significantly longer disease duration (P =.019) and higher spleen volume index (P =.014), liver volume (P =.000), percentage of circulating immature myeloid cells (P =.020), and percentage of myeloid blasts (P =.000). When CD34(+) cells were correlated with the use of Dupriez risk stratification, CD34(+) cells increased significantly from low-risk (median, 68.1 x 10(6)/L) to intermediate-risk (median, 112.8 x 10(6)/L) and high-risk patients (median 666.1 x 10(6)/L) (F = 4.95; P =.009). When CD34(+) cells were correlated with a severity score on the basis of both myeloproliferative and myelodepletive characteristics of the disease, only the myeloproliferation index was significantly associated with CD34(+) cell level (F = 5.7; P =.000). Overall survival and interval to blast transformation from the time of CD34(+) cell analysis were significantly shorter in patients with more than 300 x 10(6)/L CD34(+) cells (P =.005 and.0005, respectively). In conclusion, the absolute number of CD34(+) circulating cells allows MMM to be distinguished from other Ph(-) CMDs; it is strongly associated with the extent of myeloproliferation and predicts evolution toward blast transformation.