RESUMO
OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbß3 (αIIbß3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbß3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbß3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbß3, and P-selectin.
Assuntos
Plaquetas , Doenças do Cão/sangue , Insuficiência Renal Crônica/veterinária , Animais , Doenças do Cão/fisiopatologia , Cães , Fibrinogênio/metabolismo , Citometria de Fluxo/veterinária , Selectina-P/biossíntese , Tempo de Tromboplastina Parcial , Ativação Plaquetária , Testes de Função Plaquetária/veterinária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Tempo de Protrombina/veterinária , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologia , Tromboelastografia/veterinária , Trombofilia/veterináriaRESUMO
A significant percentage of cancer patients develop secondary lymphedema after surgery or radiotherapy. The preferred treatment of secondary lymphedema is complex physical therapy. Pharmacotherapy, for example with diuretics, has received little attention, because they were not effective and only offered short-term solutions. Sodium selenite showed promise as a cost-effective, nontoxic anti-inflammatory agent. Treatment with sodium selenite lowers reactive oxygen species (ROS) production, causes a spontaneous reduction in lymphedema volume, increases the efficacy of physical therapy for lymphedema, and reduces the incidence of erysipelas infections in patients with chronic lymphedema. Besides biological effects in reducing excessive production of ROS, sodium selenite also displays various pharmacological effects. So far the exact mechanisms of these pharmacological effects are mostly unknown, but probably include inhibition of adhesion protein expression.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Linfedema/tratamento farmacológico , Selenito de Sódio/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Humanos , Linfedema/metabolismo , Linfedema/patologia , Modalidades de Fisioterapia , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Selenito de Sódio/química , Selenito de Sódio/uso terapêuticoRESUMO
Quorum-sensing molecules, also known as autoinducer, are essential for bacterial biofilm formation. Our focus is on N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), because it is also known as an 'interkingdom signalling molecule', which means that it also interacts with mammalian cells. AHL-12 activates defence-relevant functions of phagocytic cells, including enhancement of phagocytosis, increased expression of adhesion receptors and induction of chemotaxis. This leads to the hypothesis that early recognition of developing biofilms might be the key to a successful host defence against biofilm infection. In that context we studied activation of phagocytic cells by AHL-12, and found that phagocytes are activated via a rather specialized receptor that was not previously described on myeloid cells, the bitter taste receptor T2R38. Taste receptors are commonly associated with cells of the gustatory system. The extragustatory expression, however, suggests an additional role, namely the sensing of the onset of bacterial biofilm infection.
Assuntos
4-Butirolactona/análogos & derivados , Biofilmes/crescimento & desenvolvimento , Homosserina/análogos & derivados , Macrófagos/imunologia , Neutrófilos/imunologia , Percepção de Quorum/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , 4-Butirolactona/metabolismo , Linhagem Celular Tumoral , Quimiotaxia/fisiologia , Células HL-60 , Homosserina/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Fagocitose/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Células U937RESUMO
The aim of this study was to assess platelet reactivity in patients after ischemic stroke and to investigate the influence of hyperlipidemia (HL) on platelet activity markers. A total of 41 patients after ischemic stroke were divided into the following 2 groups: patients with HL and patients with normolipidemia. Expression of CD42b on resting, thrombin-activated blood platelets, and fibrinogen level was assessed. The CD42b-positive platelets were analyzed using the flow cytometer, anti-CD61, and anti-CD42b monoclonal antibodies. The results confirmed increased platelet reactivity to thrombin in all patients after ischemic stroke manifested by significantly lower CD42b expression and percentage of CD42b(+) platelets after activation by thrombin. The influence of HL on the expression of CD42b on resting and thrombin-activated platelets was not found. However, increased level of fibrinogen but no influence of HL on fibrinogen concentration was observed in patients after ischemic stroke. Increased susceptibility to platelet agonists was found in patients after ischemic stroke in the convalescent phase.
Assuntos
Plaquetas/metabolismo , Isquemia Encefálica/sangue , Hiperlipidemias/sangue , Ativação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Acidente Vascular Cerebral/sangue , Idoso , Idoso de 80 Anos ou mais , Plaquetas/patologia , Isquemia Encefálica/patologia , Feminino , Fibrinogênio/metabolismo , Seguimentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hiperlipidemias/patologia , Integrina beta3/biossíntese , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/patologia , Trombina/farmacologiaRESUMO
We report that phytosphingosine, a sphingolipid found in many organisms and implicated in cellular signaling, promotes megakaryocytic differentiation of myeloid leukemia cells. Specifically, phytosphingosine induced several hallmark changes associated with megakaryopoiesis from K562 and HEL cells including cell cycle arrest, cell size increase and polyploidization. We also confirmed that cell type specific markers of megakaryocytes, CD41a and CD42b are induced by phytosphingosine. Phospholipids with highly similar structures were unable to induce similar changes, indicating that the activity of phytosphingosine is highly specific. Although phytosphingosine is known to activate p38 MAPK-mediated apoptosis, the signaling mechanisms involved in megakaryopoiesis appear to be distinct. In sum, we present another model for dissecting molecular details of megakaryocytic differentiation which in large part remains obscure.
Assuntos
Leucemia Mieloide/patologia , Megacariócitos/efeitos dos fármacos , Esfingosina/análogos & derivados , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Hematopoese , Humanos , Células K562 , Leucemia Mieloide/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Transdução de Sinais , Esfingosina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Animal models have been instrumental in defining thrombus formation, including the role of platelet surface glycoprotein (GP) receptors, in acute ischemic stroke (AIS). However, the involvement of GP receptors in human ischemic stroke pathophysiology and their utility as biomarkers for ischemic stroke risk and severity requires elucidation. AIMS: To determine whether platelet GPIb and GPIIb/IIIa receptors are differentially expressed in patients with AIS and chronic cerebrovascular disease (CCD) compared with healthy volunteers (HV) and to identify predictors of GPIb and GPIIb/IIIa expression. METHODS: This was a case-control study of 116 patients with AIS or transient ischemic attack (TIA), 117 patients with CCD, and 104 HV who were enrolled at our University hospital from 2010 to 2013. Blood sampling was performed once in the CCD and HV groups, and at several time points in patients with AIS or TIA. Linear regression and analysis of variance were used to analyze correlations between platelet GPIb and GPIIb/IIIa receptor numbers and demographic and clinical parameters. RESULTS: GPIb and GPIIb/IIIa receptor numbers did not significantly differ between the AIS, CCD, and HV groups. GPIb receptor expression level correlated significantly with the magnitude of GPIIb/IIIa receptor expression and the neutrophil count. In contrast, GPIIb/IIIa receptor numbers were not associated with peripheral immune-cell sub-population counts. C-reactive protein was an independent predictor of GPIIb/IIIa (not GPIb) receptor numbers. CONCLUSIONS: Platelet GPIb and GPIIb/IIIa receptor numbers did not distinguish between patient or control groups in this study, negating their potential use as a biomarker for predicting stroke risk.
Assuntos
Plaquetas/metabolismo , Isquemia Encefálica/metabolismo , Regulação da Expressão Gênica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Acidente Vascular Cerebral/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Glycoprotein Ibα (GpIbα), a family of LRR (leucine-rich repeat) proteins, is a membrane protein on the platelet, and plays an important role in atherothrombotic events. The complex formation of GpIbα with the von Willebrand Factor (vWF) has been revealed to lead to acute coronary syndrome (ACS) or stroke. A considerable attention has been paid to understand the biological functions of GpIbα and its regulation. However, difficulty with the soluble expression of human GpIbα in bacteria has hampered the relevant research. Herein, we present a soluble expression of GpIbα in Escherichiacoli by replacing the N-terminal capping domain of GpIbα with that of Internalin B using a computational approach. The resulting protein was expressed as a soluble form in E. coli, maintaining its structural feature and binding property for vWF. The present approach can be broadly used for the soluble expression of human LRR proteins in E. coli.
Assuntos
Escherichia coli/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Proteínas Recombinantes de Fusão/genética , Fator de von Willebrand/química , Anticorpos/imunologia , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Humanos , Proteínas de Membrana/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Although the lung expresses procoagulant proteins under inflammatory conditions, underlying mechanisms remain unclear. Here, we addressed lung endothelial expression of tissue factor (TF), which initiates the coagulation cascade and expression of which signifies development of a procoagulant phenotype in the vasculature. To establish the model of acid-induced acute lung injury (ALI), we intranasally instilled anesthetized mice with saline or acid. Then 2 h later, we isolated pulmonary vascular cells for flow cytometry and confocal microscopy to detect the leukocyte antigen, CD45 and the endothelial markers VE-cadherin and von Willebrand factor (vWf). Acid increased both the number of vWf-expressing cells as well as TF and P-selectin expressions on these cells. All of these effects were markedly inhibited by treating mice with antiplatelet serum, suggesting the involvement of platelets. The increased expressions of TF, vWf, and P-selectin in response to acid also occurred in platelets. Moreover, the effects were replicated in endothelial cells derived from isolated, blood-perfused lungs. However, the effect was inhibited completely in lungs perfused with platelet-depleted and, to a lesser extent, with leukocyte-depleted blood. Acid injury increased endothelial expressions of the platelet proteins, CD41 and CD42b, providing evidence that platelet proteins were transferred to the vascular surface. Reactive oxygen species (ROS) were implicated in these responses, in that the endothelial and platelet protein expressions were inhibited. We conclude that acid-induced ALI causes NOX2-mediated ROS generation that activates platelets, which then generate a procoagulant endothelial surface.
Assuntos
Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/induzido quimicamente , Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tromboplastina/biossíntese , Animais , Antígenos CD/biossíntese , Coagulação Sanguínea , Plaquetas/imunologia , Caderinas/biossíntese , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácido Clorídrico/efeitos adversos , Ácido Clorídrico/toxicidade , Antígenos Comuns de Leucócito/biossíntese , Pulmão/imunologia , Pulmão/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Selectina-P/biossíntese , Selectina-P/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Tromboplastina/metabolismo , Fator de von Willebrand/biossíntese , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Atrial fibrillation (AF) is associated with a prothrombotic state. The aim of this study was to analyze platelet activation and tissue factor (TF) induction in mononuclear cells (MNCs) and granulocytes downstream of cell-cell interactions in AF patients. METHODS: Blood samples were obtained from patients with paroxysmal AF (n=14) at sinus rhythm and at 15 min after induction of AF during an electrophysiological study, and from control subjects (n=13) and patients with chronic AF (n=14) in the outpatient clinic. The expression of CD41a, CD42b, P-selectin, and P-selectin glycoprotein ligand-1 (PSGL-1) on platelets and microparticles in platelet-rich plasma (PRP), and on MNCs and granulocytes in whole blood were examined by flow cytometry. MNC-platelet interaction was investigated ex vivo. RESULTS: The expression of CD41a and CD42b on platelets and microparticles was comparable between the control and chronic AF groups, and unchanged after AF induction. Acute induction of AF significantly increased the expression of P-selectin on platelets and microparticles, and to a similar extent, P-selectin-positive MNCs and granulocytes and P-selectin/PSGL-1-double positive MNCs. However, AF induction had no effect on platelet-MNC interactions ex vivo or TF expression on MNCs and granulocytes. Only patients with chronic AF showed platelet-MNC interaction ex vivo and TF overexpression on MNCs. CONCLUSIONS: Acute-onset AF activates platelets within minutes to initiate platelet-MNC interaction. The subsequent platelet binding induced TF expression in patients with chronic AF. These findings support the efficacy of anticoagulant therapeutics in chronic AF and suggest the underlying utility of antiplatelet therapeutics in early phase of AF occurrence.
Assuntos
Fibrilação Atrial/sangue , Plaquetas/metabolismo , Ativação Plaquetária/fisiologia , Doença Aguda , Estudos de Casos e Controles , Comunicação Celular/fisiologia , Doença Crônica , Feminino , Citometria de Fluxo , Granulócitos/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Selectina-P/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/sangue , Tromboplastina/metabolismoRESUMO
BACKGROUND/AIMS: Numerous studies conducted on Caucasian patients have reported that individual responsiveness to clopidogrel varies widely, whereas there are only a few published studies on the antiplatelet effect of clopidogrel therapy in Chinese patients undergoing percutaneous coronary intervention. The present study aimed to evaluate clopidogrel antiplatelet effects and their correlation with early recurrent cardiovascular (CV) events. METHODS: Platelet aggregation (with 5 and 20 µmol/l ADP) and the expression of glycoprotein Ib and P-selectin were measured at baseline and 12 and 36 h after the clopidogrel loading dose in 111 consecutive patients. The primary outcome was a definite CV event. RESULTS: There was marked interindividual variability in the drug response, as measured by platelet aggregation and P-selectin expression. The proportions of nonresponders at 12 and 36 h were 32 and 19%, respectively, with 5 µmol/l ADP, 38 and 28% with 20 µmol/l ADP, and 27 and 17% according to P-selectin expression. The maximal aggregation rates stimulated by 5 µmol/l ADP in nonresponders were significantly higher than those of the responders at 12 h (57.53 ± 14.24% vs. 33.91 ± 10.79%; p < 0.0001) and at 36 h (48.65 ± 15.46% vs. 30.31 ± 16.04%; p < 0.0001). During the 3-month follow-up period, 11 patients (32.4%) among the nonresponders, 2 patients (7.1%) among the low responders and none of the responders suffered a recurrent CV event (p < 0.0001). CONCLUSIONS: The antiplatelet effectiveness of clopidogrel has a wide interindividual variation, and nonresponsiveness to clopidogrel is associated with an increased risk of early recurrent CV events.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Inibidores da Agregação Plaquetária/administração & dosagem , Ticlopidina/análogos & derivados , Idoso , Angioplastia Coronária com Balão/métodos , Povo Asiático , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/cirurgia , Clopidogrel , Feminino , Humanos , Individualidade , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Stents , Ticlopidina/administração & dosagem , Resultado do TratamentoRESUMO
Glanzmann's Thrombasthenia (GT) is a rare inherited autosomal recessive platelet disorder caused by a deficiency or dysfunction of the GPIIb-IIIa receptor on platelets, which is characterized by a lack of platelet aggregation in response to multiple physiologic agonists and a life-long bleeding disorder. Flow cytometry is a rapid and highly sensitive method that can detect reduced levels of receptors, as well as absolute deficiency. The aim of this study was to classify Iranian GT patients by a flow cytometric method, and to correlate these findings with the severity of clinical bleeding. The expression of GPIIb-IIIa on the platelet surface was assessed in 123 GT patients using quantitative flow cytometry to determine the most common subtype among these patients. We used a panel of antibodies to detect the expression of glycoproteins GPIb, GPIIb, GPIIIa, as well as Integrin αv. Patients were also interviewed with regard to the severity and frequency of bleeding, according to history and gender, in order to evaluate the nature of their bleeding phenotype, and classify them as mild, moderate or severe bleeders, in accordance with the Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. In the detailed analysis of the results of our investigation, 95 out of 123 (77.5%) were classified as type I; 20 (16%) as type II with residual GPIIb-IIIa, and eight (6.5%) as GT variants. The variant type was diagnosed by the inability of GPIIb-IIIa to bind fibrinogen, as evidenced by the absence of platelet aggregation in response to physiologic agonists. There was no significant correlation between bleeding severity and different subtypes of GT. This study demonstrates that GT type I is the most common subtype among Iranian patients. There was no correlation between severity of symptoms and cytometric phenotype of the disease. The identification of families at risk may significantly decrease the incidence of the severe form of the disorder if genetic counseling is provided.
Assuntos
Plaquetas/metabolismo , Citometria de Fluxo/métodos , Integrina beta3/biossíntese , Tipagem Molecular/métodos , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Trombastenia , Adolescente , Adulto , Anticorpos Monoclonais/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Fibrinogênio/metabolismo , Hemorragia , Humanos , Lactente , Integrina alfaV/biossíntese , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Ligação Proteica , Estudos Retrospectivos , Índice de Gravidade de Doença , Trombastenia/classificação , Trombastenia/diagnóstico , Trombastenia/genéticaRESUMO
BACKGROUND: Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5-7 days). STUDY DESIGN AND METHODS: Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase-3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. RESULTS: Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase-3/7 activity with other apoptosis markers. CONCLUSION: We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.
Assuntos
Apoptose/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Preservação de Sangue/métodos , Ativação Plaquetária/efeitos dos fármacos , Soluções/farmacologia , Trifosfato de Adenosina/sangue , Adulto , Biomarcadores , Plaquetas/citologia , Plaquetas/metabolismo , Glicólise , Humanos , Selectina-P/biossíntese , Plasma , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Temperatura , Fatores de TempoRESUMO
The aim of this study was to construct Chinese Hamster Ovary (CHO) cell models expressing recombinant wild-type GPIb-IX and mutant GPIb-IX complex, so as to provide the platform to study the related physiologic functions of GPIb-IX. The plasmids were extracted from E.coli expressing wild-type or deletion mutant GPIbalpha and were identified by digestion with EcoR I. Three plasmids containing GPIbalpha, GPIbbeta, and GPIX genes were co-transfected into CHO cells, and then the expression of GPIb-IX complex was detected by immune coprecipitation, Western blot and flow cytometry. The results showed that the expression of GPIb-IX complex could be detected in the lysate and on the surface of CHO cells at 48 hours after transfection. In conclusion, CHO cell models expressing recombinant wild-type or mutation GPIb-IX complex has been successfully constructed.
Assuntos
Células CHO , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Animais , Cricetinae , Cricetulus , Mutação , Plasmídeos , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVE: To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression. METHODS: Washed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer. RESULTS: After PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor. CONCLUSION: PKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.
Assuntos
Plaquetas/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Calpaína/metabolismo , Citometria de Fluxo , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/biossínteseRESUMO
OBJECTIVE: A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations. MATERIALS AND METHODS: Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. RESULTS: The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. CONCLUSION: In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.
Assuntos
Plaquetas/citologia , Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/transplante , Animais , Antígenos CD34/análise , Células Sanguíneas/citologia , Células da Medula Óssea/citologia , Meios de Cultura/farmacologia , Citocinas/farmacologia , Sangue Fetal/citologia , Sobrevivência de Enxerto , Humanos , Integrina beta3/biossíntese , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Quimera por Radiação , TrombopoeseRESUMO
OBJECTIVE: Megakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells. MATERIALS AND METHODS: A preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days. RESULTS: For both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell-derived factor 1alpha (SDF1alpha) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes. CONCLUSION: This report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed.
Assuntos
Plaquetas/citologia , Células da Medula Óssea/enzimologia , Quimiotaxia/fisiologia , Fígado/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Megacariócitos/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Trombopoese/fisiologia , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Células da Medula Óssea/citologia , Células Cultivadas/citologia , Células Cultivadas/enzimologia , Quimiocina CXCL12/farmacologia , Quimiotaxia/efeitos dos fármacos , Fígado/embriologia , Fígado/enzimologia , Megacariócitos/citologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Trombopoetina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/análiseRESUMO
BACKGROUND: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. OBJECTIVE: The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. RESULTS: In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. CONCLUSIONS: Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.
Assuntos
Proteínas 14-3-3/química , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Deleção de Genes , Hemostasia , Humanos , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Ligação Proteica , Isoformas de Proteínas , Trombose/sangue , Trombose/genéticaRESUMO
Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149-154 in the cytoplasmic domain of the GPIbbeta subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbbeta by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbbeta cytoplasmic domain in biosynthesis of the GPIb-IX complex.
Assuntos
Membrana Celular/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Dados de Sequência Molecular , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Estrutura Terciária de Proteína/genética , TransfecçãoRESUMO
We retrospectively investigated the association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) in 17 patients with immune thrombocytopenia (ITP). Platelet-associated antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. A response occurred in 7 of 7 patients without anti-GPIb/IX, but in only 3 of 10 patients with anti-GPIb/IX (p<0.01). There was no difference in the response rates in patients with or without anti-GPIIb/IIIa or anti-GPIa/IIa. We conclude that ITP patients with anti-GPIb/IX may be less responsive to IVIG.
Assuntos
Autoanticorpos/imunologia , Plaquetas/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/uso terapêutico , Trombocitopenia/sangue , Trombocitopenia/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Infusões Intravenosas , Integrina alfa2beta1/sangue , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Estudos RetrospectivosRESUMO
OBJECTIVE: To investigate the generation of platelet antibodies in hepatitis C virus (HCV)-infected individuals and their relation to the development to thrombocytopenia with the aim of using their detection as a diagnostic aid of immune thrombocytopenia in these patients. MATERIALS AND METHODS: We tested by the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA) for the presence of platelet antibodies against specific glycoprotein (GP) targets (GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIIIb, GPV, and FcRgammaIIa) in 48 HCV-infected individuals of various stages of disease and compared the results with those from 35 patients with alcoholic liver cirrhosis. RESULTS: Thirty-two HCV-infected individuals (66%) had detectable platelet antibodies. The most common target was GPIIb/IIIa, but all other GP were also targets. Results were not different from patients with alcoholic liver cirrhosis. There was no correlation between antibodies and platelet counts, or the stage of disease, or the viral genotype, or a discernible influence of treatment with alpha-interferon. CONCLUSION: While platelet autoantibodies are common in individuals with HCV infection, their detection does not assist in the diagnosis of immune thrombocytopenia.