RESUMO
HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.
Assuntos
Coagulação Sanguínea/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Animais , Biomarcadores/metabolismo , Plaquetas/metabolismo , Deleção de Genes , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína IIb da Membrana de Plaquetas/genéticaRESUMO
Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.
Assuntos
Mutação de Sentido Incorreto , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Modelos Moleculares , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in â¼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.
Assuntos
Adesões Focais/metabolismo , Integrina beta3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Animais , Linhagem Celular , Cricetinae , Dissulfetos/análise , Adesões Focais/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta3/química , Integrina beta3/genética , Simulação de Dinâmica Molecular , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genéticaRESUMO
Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche-related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented â¼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.
Assuntos
Camundongos/genética , Análise de Célula Única , Trombopoese , Transcriptoma , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos/fisiologia , Camundongos Endogâmicos C57BL , Glicoproteína IIb da Membrana de Plaquetas/análise , Glicoproteína IIb da Membrana de Plaquetas/genética , PloidiasRESUMO
We previously demonstrated that the percentage of blood eosinophils that are associated with platelets and thus positive for CD41 (integrin αIIb-subunit) correlates with and predicts peak eosinophil count (PEC) in biopsies of eosinophilic esophagitis (EoE) patients after treatment. Thus, flow cytometric determination of CD41+ eosinophils is a potential measure of EoE disease activity. Determinants of association of platelets with eosinophils and other leukocytes in EoE are largely unknown. The objectives of this study were to test the hypotheses that platelets associate with blood leukocytes other than eosinophils in EoE and that such associations also predict EoE activity. Whole blood flow cytometry was performed on samples from 25 subjects before and after two months of standard of care EoE treatment. CD41 positivity of cells within gates for eosinophils, neutrophils, monocytes, lymphocytes, and natural killer cells was compared. We found that percent CD41+ neutrophils, monocytes, and eosinophils correlated with one another such that principal component analysis of the five cell types identified "myeloid" and "lymphoid" factors. Percent CD41+ neutrophils or monocytes, or the myeloid factor, like CD41+ eosinophils, correlated with PEC after treatment, and CD41+ neutrophils or the myeloid factor predicted PEC < 6/high power field after treatment, albeit with lower area under the curve than for CD41+ eosinophils. We conclude that the processes driving platelets to associate with eosinophils in EoE also drive association of platelets with neutrophils and monocytes and that association of platelets with all three cell types is related to disease activity. Clinicaltrials.gov identifier: NCT02775045.
Assuntos
Plaquetas/metabolismo , Esofagite Eosinofílica/sangue , Eosinófilos/patologia , Citometria de Fluxo , Adulto , Anticorpos/imunologia , Plaquetas/patologia , Esofagite Eosinofílica/patologia , Eosinófilos/metabolismo , Feminino , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Contagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/patologia , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Análise de Componente PrincipalRESUMO
We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.
Assuntos
Interferon-alfa/uso terapêutico , Janus Quinase 2/genética , Megacariócitos/metabolismo , Transtornos Mieloproliferativos/genética , Animais , Técnicas de Introdução de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Megacariócitos/citologia , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/tratamento farmacológico , Glicoproteína IIb da Membrana de Plaquetas/genética , Mutação Puntual/efeitos dos fármacosRESUMO
Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.
Assuntos
NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Linhagem Celular Tumoral , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Lipopolissacarídeos/farmacologia , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/química , Zimosan/farmacologiaRESUMO
Soluble ligand and conformation-dependent antibody binding assay of integrins on the cell surface is an effective approach to evaluate the activation status of integrins in live cells. The ligands or antibodies are usually labeled with biotin or a fluorescent dye and incubated with integrin-expressing cells in suspension. The cell-bound ligands and antibodies are then detected by flow cytometry. Here we describe the detailed protocols of soluble ligand or antibody binding assay for αIIbß3, αVß3, α5ß1, and αLß2 integrins that are transiently or stably expressed in the model cell lines such as HEK293 or CHO-k1 cells.
Assuntos
Bioensaio , Integrina alfaVbeta3/química , Antígeno-1 Associado à Função Linfocitária/química , Glicoproteína IIb da Membrana de Plaquetas/química , Receptores de Vitronectina/química , Coloração e Rotulagem/métodos , Animais , Anticorpos/química , Anticorpos/metabolismo , Células CHO , Adesão Celular , Cricetulus , Citometria de Fluxo , Corantes Fluorescentes/química , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Molécula 1 de Adesão Intercelular , Ligantes , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Platelets are small, anucleate cells that play oversized roles in hemostasis, immunity, and inflammation. An important mediator of platelet function is integrin αIIbß3, which is required for fibrinogen-dependent platelet aggregation during hemostasis. This platelet response is dependent on conformational changes in the integrin induced by "inside-out" biochemical signals that are triggered by platelet agonists. In turn, fibrinogen binding to αIIbß3 initiates "outside-in" biochemical and mechanical signals that regulate the platelet cytoskeleton and help to promote full platelet aggregation and secretory responses. Without a nucleus, there is a limited range of experimental manipulations that are possible with human platelets to study the molecular basis of integrin signaling in these primary cells. Consequently, many studies of αIIbß3 function use genetic approaches that rely on heterologous expression systems or platelets from gene-targeted mice, sometimes with uncertain applicability to human platelets. This chapter will detail a method for genetic manipulation of megakaryocytes and platelets derived from human induced pluripotent stem cells for molecular studies of αIIbß3 signaling and for modeling of human platelet functions potentially relevant to hemostasis, immunity, and inflammation.
Assuntos
Plaquetas/metabolismo , Engenharia Celular/métodos , Megacariócitos/metabolismo , Agregação Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/genética , Plaquetas/citologia , Diferenciação Celular , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Fibrinogênio/genética , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Hemostasia/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/citologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.
Assuntos
Ataxina-2/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras de Megacariócitos/citologia , Animais , Antígenos CD34/genética , Ataxina-2/metabolismo , Diferenciação Celular , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.
Assuntos
Plaquetas/metabolismo , Cadeias beta de Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Colágeno/farmacologia , AMP Cíclico/metabolismo , Epoprostenol/farmacologia , Integrina alfa2/genética , Integrina alfa2/metabolismo , Cadeias beta de Integrinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Ligação Proteica , Transporte Proteico , Trombina/farmacologiaRESUMO
INTRODUCTION: Glanzmann thrombasthenia (GT) is a rare bleeding disorder. The disease is caused by the lack or dysfunction of platelet membrane glycoprotein IIb/IIIa (integrin αIIbß3) which is essential for platelet aggregation. Bleeding episodes are usually managed by platelet transfusions. Recombinant activated factor VII (rFVIIa) is a common adjunct or alternative treatment option. OBJECTIVE: This article evaluates GT patients' response to increasing concentrations of rFVIIa using an ex vivo thrombin generation assay to elaborate the knowledge in which rFVIIa treats a platelet dysfunction for bleeding episodes and preoperative management. MATERIALS AND METHODS: Twenty-four GT patients in a non-bleeding state were enrolled into the study. Thrombin generation was measured in platelet-rich plasma (PRP) in the presence of 0.7 to 7.0 µg/mL rFVIIa. Clinical data of rFVIIa used to treat GT patients' bleeding episodes was collected, and patients' follow-up course was documented. RESULTS: Thrombin generation was significantly decreased in GT patients compared with controls. An individual response to rFVIIa spiking was noted in GT patients' PRP. In the majority of patients, a significant improvement in thrombin generation was already demonstrated with low concentrations (0.7 µg/mL) of rFVIIa. CONCLUSION: Thrombin generation is improved in the majority of GT patients following ex vivo spiking with rFVIIa. The magnitude of this improvement is individual and was noted at low concentrations of rFVIIa. There is a need for a prospective clinical trial to find the optimal doses or rFVIIa for treatment of GT patients.
Assuntos
Fator VIIa/administração & dosagem , Trombastenia/sangue , Trombastenia/tratamento farmacológico , Trombina/química , Transtornos Plaquetários/etiologia , Relação Dose-Resposta a Droga , Feminino , Hemorragia , Humanos , Masculino , Mutação , Fenótipo , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Transfusão de Plaquetas , Proteínas Recombinantes/administração & dosagemRESUMO
Immune thrombocytopaenia (ITP) is the most common autoimmune bleeding disorder, where platelets are destroyed by auto-antibodies and/or cell-mediated mechanisms. To understand the pathogenesis of ITP and explore novel therapeutics, three types of animal models have been used: passive ITP, secondary ITP and platelet-induced ITP. However, the first two are not ideal for chronic ITP pathophysiology where both T cell and B cell play important roles in platelet destruction. The most efficient model to mimic chronic ITP is developed by Chow et al through transferring splenocytes from platelet-immune CD61-knockout (KO) mice into mice with severe combined immunodeficiency (SCID). However, placental defects are evident in 25% of CD61-KO females and post-natal haemorrhage does occur, reducing the survival rate of embryos and pups. Compared with CD61-KO mice, CD41-KO ones do not present such problems. In our study, we employ CD41-KO mice as another source of immunized spleen cells. We evaluated our model with existing standards. Transferred SCID mice presented typical features of ITP, such as reduced platelet counts in the peripheral blood, increased anti-platelet antibody levels in the serum and reduced mature megakaryocytes in the bone marrow. What is more, lymphocyte-depletion experiments showed the role of CD8+ T cells in mature megakaryocyte decrease and thrombocytopaenia. And we confirmed the antibody-mediated platelet destruction by phagocytosis in the spleen. Our study develops another efficient murine ITP model through immunized CD41-KO mice.
Assuntos
Transferência Adotiva , Plaquetas/imunologia , Glicoproteína IIb da Membrana de Plaquetas/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Baço/imunologia , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Plaquetas/metabolismo , Plaquetas/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Megacariócitos/imunologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Fagocitose , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Transfusão de Plaquetas , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/metabolismo , Baço/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Studies on the inherited bleeding disorder, Glanzmann thrombasthenia (GT), have helped define the role of the αIIbß3 integrin in platelet aggregation. Stable bent αIIbß3 undergoes conformation changes on activation allowing fibrinogen binding and its taking an extended form. The αIIb genu assures the fulcrum of the bent state. Our goal was to determine how structural changes induced by missense mutations in the αIIb genu define GT phenotype. METHODS: Sanger sequencing of ITGA2B and ITGB3 in the index case followed by in silico modeling of all known GT-causing missense mutations extending from the lower part of the ß-propeller, and through the thigh and upper calf-1 domains. RESULTS: A homozygous c.1772A>C transversion in exon 18 of ITGA2B coding for a p.Asp591Ala substitution in an interconnecting loop of the lower thigh domain of αIIb in a patient with platelets lacking αIIbß3 led us to extend our in silico modeling to all 16 published disease-causing missense variants potentially affecting the αIIb genu. Modifications of structuring H-bonding were the major cause in the thigh domain although one mutation gave mRNA decay. In contrast, short-range changes induced in calf-1 appeared minor suggesting long-range effects. All result in severe to total loss of αIIbß3 in platelets. The absence of mutations within a key Ca2+-binding loop in the genu led us to scan public databases; three potential single allele variants giving major structural changes were identiffied suggesting that this key region is not protected from genetic variation. CONCLUSIONS: It appears that the αIIb genu is the object of stringent quality control to prevent platelets from circulating with activated and extended integrin.
Assuntos
Integrina alfa2/genética , Trombastenia/genética , Adulto , Alelos , Plaquetas/metabolismo , Plaquetas/fisiologia , Simulação por Computador , Éxons , Feminino , Humanos , Integrina alfa2/metabolismo , Integrina beta3/genética , Articulação do Joelho , Agregação Plaquetária , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genética , Trombastenia/metabolismoRESUMO
BACKGROUND & OBJECTIVES: Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbß3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. METHODS: Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. RESULTS: Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). INTERPRETATION & CONCLUSIONS: Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.
Assuntos
Hemorragia/sangue , Integrina beta3/genética , Glicoproteína IIb da Membrana de Plaquetas/genética , Trombastenia/genética , Adulto , Plaquetas/metabolismo , Plaquetas/patologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Glicoproteínas/sangue , Hemorragia/genética , Hemorragia/patologia , Humanos , Integrina beta3/sangue , Masculino , Pessoa de Meia-Idade , Fenótipo , Agregação Plaquetária/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/sangue , Trombastenia/sangue , Trombastenia/patologiaRESUMO
The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and ß3 (CD61) were found to be induced. Signaling focal contacts via ITGß3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.
Assuntos
Células da Medula Óssea/metabolismo , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Integrina beta3/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/genética , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Células da Medula Óssea/citologia , Adesão Celular , Comunicação Celular , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Microambiente Celular , Matriz Extracelular/ultraestrutura , Adesões Focais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Integrina alfaV/genética , Integrina alfaV/metabolismo , Integrina beta3/genética , Células-Tronco Mesenquimais/citologia , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Alicerces TeciduaisRESUMO
Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Animais , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Transgênicos , Glicoproteína IIb da Membrana de Plaquetas/genéticaRESUMO
Glanzmann thrombasthenia (GT) is a rare monogenic hemorrhagic disorder involving aggregation defect of non-nuclear platelets. In this study we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of a GT patient with complex heterogeneous mutations of ITGA2B gene. GT-iPSCs could be successfully differentiated into platelets (GT-iPS-platelets). GT-iPS-platelets were CD41-/CD42b+/CD61- and were platelet activation marker (PAC-1) negative after adenosine diphosphate (ADP) activation. Furthermore, GT-iPS-platelets were defective in platelet aggregation tests in vitro. Moreover, exogenous expression of the wild-type ITGA2B gene in GT-iPS platelets restored CD41 expression and normal platelet aggregation. Our study suggested that patient-specific iPSCs could be a potential target of stem cell based gene therapy for platelet diseases.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Trombastenia/patologia , Animais , Sequência de Bases , Plaquetas/citologia , Plaquetas/metabolismo , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Fosfatase 2 de Especificidade Dupla/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Integrina alfa2/genética , Cariótipo , Camundongos , Camundongos Nus , Agregação Plaquetária , Glicoproteína IIb da Membrana de Plaquetas/genética , Polimorfismo Genético , Teratoma/metabolismo , Teratoma/patologia , Trombastenia/genética , Trombastenia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Glycoprotein IIb/IIIa (GPIIb/IIIa) is the most abundant platelet surface receptor for fibrinogen and von Willebrand factor. Polymorphism PlA1/A2 in the gene of GPIIb/IIIa is among the risk factors for the development of arterial and venous thrombosis. The aim of this study is to evaluate the effect of the carriage of PlA1/A2 on the size, topographic features, and membrane stiffness of platelets from healthy controls and patients with deep venous thrombosis (DVT). Atomic force microscopy (AFM) imaging and nanoindentation (force-distance curves) were applied to investigate the morphological and nanomechanical properties (Young's modulus) of platelets immobilized on glass surface. The surface roughness ( Ra) and height ( h) of platelets from patients with DVT, carriers of mutant allele PlA2 ( Ra = 30.2 ± 6 nm; h = 766 ± 182 nm) and noncarriers ( Ra = 28.6 ± 6 nm; h = 865 ± 290 nm), were lower than those of healthy carriers of allele PlA2 ( Ra = 48.1 ± 12 nm; h = 1072 ± 338 nm) and healthy noncarriers ( Ra = 49.7 ± 14 nm; h = 1021 ± 433 nm), respectively. Platelets isolated from patients with DVT, both carriers and noncarriers, exhibit much higher degree of stiffness at the stage of spreading ( E = 327 ± 85 kPa and 341 ± 102 kPa, respectively) compared to healthy noncarriers ( E = 198 ± 50 kPa). In addition, more pronounced level of platelet activation was found in polymorphism carriers. In conclusion, the carriage of PlA2 allele modulates the activation state, morphology, and membrane elasticity of platelets.
Assuntos
Plaquetas , Integrina beta3 , Microscopia de Força Atômica , Glicoproteína IIb da Membrana de Plaquetas , Polimorfismo Genético , Trombose Venosa , Adulto , Idoso , Alelos , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Módulo de Elasticidade , Feminino , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Masculino , Pessoa de Meia-Idade , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Trombose Venosa/genética , Trombose Venosa/metabolismo , Trombose Venosa/patologiaRESUMO
Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.