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Introduction: Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7') and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3. Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels. Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-ß/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage. Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.
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Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.
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Corantes , Proteínas , Citometria de Fluxo , Linhagem Celular , Microscopia de FluorescênciaRESUMO
Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select for even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell-lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.
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INTRODUCTION: Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AIM: We aimed to study the genetic cause of IPFD mimicking GT. METHODS: During 2017-2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. RESULTS: Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbß3 and αvß3, as well as normal integrin αIIbß3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DISCUSSION: Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. CONCLUSION: Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.
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Erros de Diagnóstico , Fatores de Troca do Nucleotídeo Guanina/genética , Trombastenia/diagnóstico , Trombastenia/genética , Adulto , Feminino , Mutação da Fase de Leitura , Humanos , Lactente , Masculino , Linhagem , Agregação Plaquetária , Mutação Puntual , Sequenciamento do Exoma , Adulto JovemRESUMO
BACKGROUND: In all, 15-30% of pediatric immune thrombocytopenia (ITP) patients will remain chronically thrombocytopenic at 1 year post diagnosis. All attempts to classify patients at diagnosis have proven unsuccessful. We hypothesized that a different pathophysiology is responsible for non-chronic versus chronic pediatric ITP. We aimed to examine differences in the apoptotic markers' presentation at diagnosis between non-chronic and chronic patients. METHODS: Blood samples were collected from 42 pediatric patients with newly diagnosed ITP prior to initiation of treatment. We incubated patients' sera with control platelets and compared the results among three research groups: healthy controls, chronic ITP, and non-chronic ITP patients. We measured apoptotic markers phosphatidylserine (PS) exposure and mitochondrial inner membrane potential (ΔΨm) by flow cytometry and the level of human apoptotic proteins by Human Apoptosis Array. RESULTS: We found increased platelet PS exposure and decreased ΔΨm in response to all ITP patients' sera compared to control subjects. Human Apoptotic Array revealed an increased expression of five apoptotic proteins: BIM, CD40, IGFBP2, P21, and SMAC, following sera incubation of non-chronic pediatric ITP patients, compared to chronic patients' sera, at diagnosis. CONCLUSIONS: Our data contribute to knowledge on apoptosis markers that may aid in predicting the prognosis of children with ITP. IMPACT: The key message of our article is that children with chronic ITP have a different apoptotic profile compared to non-chronic ITP. Addition to existing literature: This is the first study comparing apoptotic markers between children with chronic ITP to non-chronic ITP. IMPACT: Our findings indicate that, in the future, apoptotic markers may help to classify ITP patients into non-chronic versus chronic ones, at diagnosis.
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Apoptose , Púrpura Trombocitopênica Idiopática/patologia , Adolescente , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Progressão da Doença , Feminino , Citometria de Fluxo , Humanos , Lactente , Masculino , Estudos Prospectivos , Púrpura Trombocitopênica Idiopática/sangueRESUMO
Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P-selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live-cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP-selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.
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Plaquetas/efeitos dos fármacos , Calreticulina/genética , Janus Quinase 2/genética , Ativação Plaquetária/efeitos dos fármacos , Trombocitemia Essencial/sangue , Trombofilia/etiologia , Difosfato de Adenosina/farmacologia , Adulto , Cálcio/sangue , Forma Celular , Feminino , Humanos , Leucócitos/patologia , Masculino , Microscopia de Interferência , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Selectina-P/sangue , Receptores de Trombopoetina/metabolismo , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , Trombomodulina/sangue , Trombofilia/genéticaRESUMO
Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compoundâ 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).
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Fenilbutiratos/química , Proteínas/química , Animais , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Fenilbutiratos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína , Proteínas/metabolismo , RatosRESUMO
INTRODUCTION: Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders, but requires large amounts of blood samples and normal platelet count. OBJECTIVES: To compare flow cytometric (FC) platelet function testing to standard LTA in the general population, in patients treated with anti-platelets drugs and in term and preterm neonates. METHODS: Platelet function was assessed with LTA and FC using PAC1 binding and p-selectin expression, as platelet activation markers, in response to agonist activation. A comparison between LTA and FC was performed in a Clopidogrel treated patient, before and after (24 and 72 hours) loading the drug. The platelet activation markers PAC1 and p-selectin, were compared in umbilical cord blood samples of in-term and preterm neonates. RESULTS: ADP-induced platelet aggregation was comparable to p-selectin expression assayed by FC (r=0.79-0.86) as measured before and after Clopidogrel loading. Both tests showed good response to Clopidogrel in 72 hours but not in 24 hours after its loading. Preterm cord blood platelets showed decreased ADP-induced activation in both activation markers: PAC1 and p-selectin, but only p-selectin reached statistical significance. We identified possible platelet activation markers in response to commonly used agonists' stimulation for FC analysis. CONCLUSIONS: FC analysis of platelet function has added value in the diagnosis of impaired platelet function and anti-platelet drug response. Using FC enables us to test platelet function in minimal blood volume and regardless of platelet count. Identification of the unique activation marker for each agonist is prerequisite for FC analysis of platelet function.
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Plaquetas , Citometria de Fluxo , Agregação Plaquetária , Difosfato de Adenosina , Humanos , Inibidores da Agregação Plaquetária , TiclopidinaRESUMO
BACKGROUND: Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder of platelet function caused by mutations in the genes coding for integrin αIIbß3. The aim of this study was to examine the outcome of newborns of GT mothers, with emphasis on thrombocytopenia and bleeding manifestations and their relation to maternal antiplatelet antibodies. PROCEDURE: Medical files of all female patients with GT treated in a single tertiary center from 1999 to 2017 were searched for details on pregnancy and birth. The medical files of their newborns were retrieved, and data on the postnatal course were collected. RESULTS: Nine babies were born to five patients with GT at our center during the study period. Three of the nine newborns had severe thrombocytopenia, and all three were offspring of GT mothers who were positive for antiplatelet antibodies. CONCLUSION: Pregnant GT patients should be examined for platelet antibodies. Assessment and management protocols (including treatment with intravenous immunoglobulins) for fetal and neonatal alloimmune thrombocytopenia should be considered.
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Doenças do Recém-Nascido/etiologia , Complicações na Gravidez , Efeitos Tardios da Exposição Pré-Natal , Trombastenia/etiologia , Trombocitopenia Neonatal Aloimune/etiologia , Autoanticorpos/sangue , Feminino , Humanos , Recém-Nascido , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/imunologia , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/imunologiaRESUMO
BACKGROUND: Glanzmann thrombasthenia (GT) is a disorder of platelet function. Standard therapy includes platelet transfusions, which may be hampered by antiplatelet antibodies. AIMS: To assess potential correlation between bleeding and number of active platelets in GT patients undergoing surgery. Clinical peri- operative patients' hemostasis was compared with flow cytometry analysis (FC), and whole blood clot formation. METHODS: GT patients undergoing surgery were included. Blood counts, platelet activation studies, FC and rotational thromboelastography (ROTEM) were performed as ancillary tests to estimate the effectiveness of treatment. RESULTS: A total of 4 GT patients undergoing 5 surgeries were included. Consecutive FC analysis following platelet transfusions showed gradual decrease of donor platelets with a nadir of 3280 platelets in patients who experienced no post procedural bleeding following minor procedures. After major surgery, bleeding occurred when donor platelets decreased to 2600-4280. Decline in donor platelets was associated with reduced clot firmness as noted by ROTEM. CONCLUSION: Results suggest that very low number of active donor platelets may suffice to achieve proper hemostasis in certain procedures. Our study points to the potential role of consecutive FC examinations to demonstrate the number of donor platelets as an ancillary tool for decision making in GT patients undergoing surgery.
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Assistência Perioperatória/métodos , Transfusão de Plaquetas/normas , Trombastenia/terapia , Doadores de Sangue , Tomada de Decisões , Feminino , Citometria de Fluxo , Hemostasia , Humanos , Masculino , Contagem de Plaquetas , Trombastenia/cirurgiaRESUMO
BACKGROUND: AnWj is a high-incidence blood group antigen associated with three clinical disorders: lymphoid malignancies, immunologic disorders, and autoimmune hemolytic anemia. The aim of this study was to determine the genetic basis of an inherited AnWj-negative phenotype. METHODS: We identified a consanguineous family with two AnWj-negative siblings and 4 additional AnWj-negative individuals without known familial relationship to the index family. We performed exome sequencing in search for rare homozygous variants shared by the two AnWj-negative siblings of the index family and searched for these variants in the four non-related AnWj-negative individuals. RESULTS: Exome sequencing revealed seven candidate genes that showed complete segregation in the index family and for which the two AnWj-negative siblings were homozygous. However, the four additional non-related AnWj-negative subjects were homozygous for only one of these variants, rs114851602 (R320Q) in the SMYD1 gene. Considering the frequency of the minor allele, the chance of randomly finding 4 consecutive such individuals is 2.56 × 10-18 . CONCLUSION: We present genetic and statistical evidence that the R320Q substitution in SMYD1 underlies an inherited form of the AnWj-negative blood group phenotype. The mechanism by which the mutation leads to this phenotype remains to be determined.
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Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Musculares/genética , Fenótipo , Fatores de Transcrição/genética , Adulto , Antígenos de Grupos Sanguíneos/química , Proteínas de Ligação a DNA/química , Eritrócitos/imunologia , Eritrócitos/metabolismo , Evolução Molecular , Feminino , Frequência do Gene , Variação Genética , Genótipo , Humanos , Masculino , Modelos Moleculares , Proteínas Musculares/química , Linhagem , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Fatores de Transcrição/química , Sequenciamento do ExomaRESUMO
Mechanisms of platelet activation are triggered by thrombin, adenosine diphosphate (ADP), epinephrine, thromboxane A2, and other soluble agonists which induce signaling via heterotrimeric Gαq, Gαi, and Gα12/13 proteins. We have undertaken a study addressing the contribution of these G proteins to platelet activation and clot formation in the presence of eptifibatide, thus excluding outside-in signaling provided by integrin αIIbß3-fibrinogen engagement. Selective and combined activation of the G proteins was achieved by using combinations of platelet agonists and inhibitors. Platelet activation in platelet-rich plasma was evaluated by P-selectin expression using flow cytometry. Contribution of platelets to whole blood clotting was assessed by rotation thromboelastometry (ROTEM). Selective signaling of Gαq or Gαi but not Gα12/13 promoted P-selectin expression. Further enhancement of P-selectin expression was achieved by ADP-induced combined signaling of Gαq and Gαi, and to more extent by U46619 at high concentration (1.5 µM) induced combined signaling of Gαq and Gα12/13 while maximal P-selectin expression was achieved by thrombin receptor-activating peptide (TRAP)-induced combined signaling of Gαq, Gαi, and Gα12/13. In ROTEM, selective activation of Gαq, Gαi, or Gα12/13 failed to affect blood clotting. Combined signaling of Gαq and Gαi or Gαq and Gα12/13 or all three G proteins shortened the clotting time and stimulated clot strength. Pretreatment of platelets with acetylsalicylic acid did not change the effect of ADP but inhibited the effect of TRAP. Signaling of Gαq and Gα12/13 triggered by U46619 also stimulated clot formation. Combined signaling of either Gαq and Gαi or Gαq and Gα12/13 is sufficient to stimulate maximal platelet activation and enhanced clot formation in platelets treated with inhibitor of integrin αIIbß3. It could be suggested that outside-in signaling is not necessarily required to fulfill these platelet functions.
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Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Trombose/etiologia , Trombose/metabolismo , Adulto , Biomarcadores , Eptifibatida , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/farmacologia , Testes de Função Plaquetária/métodos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Adulto JovemRESUMO
BACKGROUND: Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease. METHODS: A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s(-) using Impact-R device. RESULTS: Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function. CONCLUSION: The study revealed a differential effect of TIC constituents-hemodilution, hypothermia, fibrinolysis, and acidosis-on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy. LEVEL OF EVIDENCE: A new model of TIC is created. Contribution of various constituents to pathogenesis of TIC and their interactions are evaluated.
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Acidose/fisiopatologia , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/fisiopatologia , Fibrinólise/fisiologia , Hemodiluição/métodos , Hipotermia/fisiopatologia , Ferimentos e Lesões/complicações , Ferimentos e Lesões/fisiopatologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária , TromboelastografiaRESUMO
BACKGROUND: Patients with type 2 diabetes mellitus (DM) display a predisposition for vascular disease. Platelets taken from vasculopathic diabetic patients, show enhanced stimuli-induced activation and aggregation responses. Aspirin remains the cornerstone antiplatelet agent for secondary prevention of vascular complications among diabetic patients, yet evidence of its efficacy and safety in primary prevention are conflicting. Our aim was to assess whether high risk diabetic patients, without previous ischemic events, have abnormal platelet functionality profiles. METHODS: The study included 82 diabetic patients and 86 matched non-diabetic patients without prior ischemic events nor treatment with anti-platelet medications. Blood samples were analyzed for platelet markers of activation, turnover and leukocyte-platelet interactions. RESULTS: Our final analysis included 122 males (74 %), with a mean age of 61 years. Mean platelet volume (MPV) was similar between the diabetic patients and controls (9.2 fL for both). Following activation, PAC-1 binding and P-selectin expression were found comparable between the diabetic patients and controls (83 % versus 81 % and 76 % versus 74 %, respectively). Leukocyte-platelet aggregates (LPAs) were similar between the diabetic patients and controls (18 % versus 17 %, respectively). Neutrophil-platelet aggregates (NPAs) and monocyte-platelet aggregates (MPAs) were also found similar in the diabetic patients and controls. Elevated fasting plasma glucose was associated with increased LPAs rates. CONCLUSIONS: High risk type-2 diabetes mellitus patients, without prior ischemic events, have normal blood platelet functionality profiles.
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Plaquetas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Selectina-P/metabolismo , Agregação Plaquetária , Idoso , Plaquetas/fisiologia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Hidrazonas , Leucócitos , Masculino , Volume Plaquetário Médio , Pessoa de Meia-Idade , Monócitos , Neutrófilos , Piperazinas , Testes de Função PlaquetáriaRESUMO
Immune thrombocytopenia (ITP) in pregnant women can cause neonatal thrombocytopenia by transport of antiplatelet autoantibodies across the placenta. Usually, an infant's platelet count normalizes within 2 months. We observed neonatal thrombocytopenia that persisted more than 4 months and disappeared following discontinuation of breastfeeding. The aim of our study was to discern whether breast milk of ITP mothers contained antiplatelet antibodies causing persistent thrombocytopenia. We collected milk samples from 3 groups of women: ITP group, 7 women who had ITP during pregnancy; R-ITP group, 6 women who recovered from ITP before pregnancy; and 9 healthy controls. We found increased levels of antiplatelet antibodies of the immunoglobulin A type in the milk of ITP patients compared with the other 2 groups. Similar increase was demonstrated for antibodies binding to αIIbß3 expressed in cultured cells. Thus, transfer of antiplatelet antibodies from ITP mothers by breastfeeding can be associated with persistent neonatal thrombocytopenia.
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Autoanticorpos/metabolismo , Plaquetas/imunologia , Leite Humano/imunologia , Complicações Hematológicas na Gravidez/imunologia , Púrpura Trombocitopênica Idiopática/complicações , Trombocitopenia Neonatal Aloimune/etiologia , Adulto , Aleitamento Materno/efeitos adversos , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Imunoglobulina A/metabolismo , Lactente , Recém-Nascido , Troca Materno-Fetal/imunologia , Contagem de Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Gravidez , Púrpura Trombocitopênica Idiopática/imunologia , Trombocitopenia Neonatal Aloimune/imunologiaRESUMO
Mutations in the ITGA2B or ITGB3 genes that encode for the αIIbß3 platelet integrin usually cause Glanzmann thrombasthenia, a severe autosomal recessive bleeding disorder characterized by absence of platelet aggregation, but normal platelet number and size. Several rare mutations cause a Glanzmann-like syndrome which manifests macrothrombocytopenia and usually displays autosomal dominant inheritance. The exact mechanism causing Glanzmann-like syndrome is unknown. One typical example of Glanzmann-like mutations causes deletion of 40 amino acids (p.647-686) in the ß3 ß-tail domain (ßTD_del) that was found in the heterozygous state in Italian and Japanese families. A second example is a missense mutation, C560R, located in the epidermal growth factor-like domain, found in the homozygous state in a French patient. Both mutations cause constitutive activation of αIIbß3, but differ in their surface expression. In the current study, we generated cultured cells expressing ß3-ßTD_del or ß3-C560R mutations along with wild-type αIIb, and examined the cells' ability to create tubulin-dependent protrusions compared to cells expressing wild-type αIIbß3. Unlike cells expressing wild-type αIIbß3, cells harboring each of the mutations exhibited abnormal cytoplasmic extensions on immobilized fibrinogen or Von Willebrand factor, which resembled extensions formed in megakaryocyte leading to proplatelets. Moreover, we showed that formation of abnormal extensions occurred also in wild-type αIIbß3 cells when activated by activating antibody. These results suggest that the active conformation of αIIbß3 can induce cytoskeletal rearrangements that lead to impaired proplatelet formation.
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Citoesqueleto/ultraestrutura , Integrina beta3/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Trombastenia/patologia , Animais , Linhagem Celular , Cricetinae , Citoesqueleto/química , DNA Complementar/genética , Fibrinogênio/metabolismo , Vetores Genéticos , Humanos , Integrina alfa2/genética , Integrina beta3/genética , Megacariócitos/ultraestrutura , Mesocricetus , Microtúbulos/química , Microtúbulos/ultraestrutura , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Trombastenia/sangue , Trombastenia/genética , Tubulina (Proteína)/química , Fator de von Willebrand/metabolismoRESUMO
The platelet integrin αIIbß3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192-200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbß3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbß3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbß3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.
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Sítios de Ligação/genética , Cálcio/metabolismo , Mutação , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genética , Animais , Cálcio/química , Linhagem Celular , Cricetinae , Ácido Edético , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação/genética , Mutação/fisiologia , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
The main interface of the 2 subunits of platelet integrin alphaIIbbeta3 comprises the beta-propeller domain of alphaIIb and the betaA domain of beta3. In the center of the beta-propeller, several aromatic residues interact by cation-pi and hydrophobic bonds with Arg261 of betaA. In this study, we substituted alphaIIb-Trp110 or beta3-Arg261 by residues abundant in other alpha or beta subunits at corresponding locations and expressed them in baby hamster kidney cells along with normal beta3 or alphaIIb, respectively. These mutant cells displayed normal surface expression and fibrinogen binding but grossly impaired outside-in signaling-related functions: adhesion to immobilized fibrinogen, cell spreading, focal adhesion kinase phosphorylation, clot retraction, and reduced alphaIIbbeta3 stability in EDTA (ethylenediaminetetraacetic acid). Expression of mutants with substitutions of Arg261 in beta3 by alanine or lysine with normal alphav yielded normal surface expression of alphavbeta3 and soluble fibrinogen binding as well as normal outside-in signaling-related functions, contrasting findings for alphaIIbbeta3. Structural analysis of alphaIIbbeta3 and alphavbeta3 revealed that alphavbeta3 has several strong interactions between alphav and beta3 subunits that are missing in alphaIIbbeta3. Together, these findings indicate that the interaction between Trp110 of alphaIIb and Arg261 of beta3 is critical for alphaIIbbeta3 integrity and outside-in signaling-related functions.
Assuntos
Integrina beta3/química , Integrina beta3/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Retração do Coágulo , Cricetinae , Primers do DNA/genética , Fibrinogênio/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Integrina alfaV/química , Integrina alfaV/genética , Integrina alfaV/metabolismo , Integrina beta3/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Glicoproteína IIb da Membrana de Plaquetas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transfecção , Fator de von Willebrand/metabolismoRESUMO
In the platelets of a type II Glanzmann thrombasthenia patient, the amount of glycoprotein (GP) IIb and IIIa was significantly reduced. Three novel mutations were identified in the GPIIb gene (c.440C->G/p.Leu116Val, c.1772_1773insG/p.Asp560GlyfsX16 and c.2438C->A/p.His782Asn). p.Leu116Val did not represent a causative mutation. The c.1772_1773insG mutation resulted in an early stop codon and non-sense mediated decay of mRNA. When expressed in transfected BHK cells, the truncated protein was unable to form complex with GPIIIa. The p.His782Asn mutation compromised transport of the pro-GPIIb/IIIa complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression.