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1.
Life Sci ; 241: 117103, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31783053

RESUMO

AIMS: Glycoprotein VI (GPVI) is an important platelet membrane receptor. The expression of GPVI on platelet membranes is increased in patients with coronary heart disease (CHD). DNA methylation is one of the most common post-replication and pre-transcriptional modifications and plays a critical role in the regulation of gene expression. Here, we aimed to reveal how methylation regulates GPVI expression. MAIN METHODS: Pyrosequencing was used to determine whether the GPVI promoter region in leukocytes from CHD patients is hypomethylated. The expressions of GPVI in CHD patients were detected using qRT-PCR and Western blot. The effect of methylation of the GPVI promoter region on regulating its transcriptional activity was analyzed using in vitro luciferase assay. The expression of P-selectin in platelet-like particles was determined using flow cytometry, and SYK phosphorylation was observed using Western blot. KEY FINDINGS: We found that the GPVI promoter region in leukocytes from CHD patients was hypomethylated and the expression of GPVI at the mRNA and protein level was elevated in CHD patients. We also found that the hypermethylation of GPVI promoter region inhibited the expression of GPVI in the -322 to +75, -539 to +75, and -937 to +75 regions in Dami cells. Moreover, the data showed that the methylation or demethylation regulated the GPVI expression and platelet-like particle activation in Dami cells. SIGNIFICANCE: Taken together, these results indicate that DNA methylation regulates GPVI expression and that CpG methylation levels in the promoter region of the GPVI gene may be a biomarker of CHD.


Assuntos
Doença das Coronárias/genética , Metilação de DNA , Epigênese Genética , Glicoproteínas da Membrana de Plaquetas/genética , Estudos de Casos e Controles , Células Cultivadas , Doença das Coronárias/sangue , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Ftalimidas/farmacologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Regiões Promotoras Genéticas , Acetato de Tetradecanoilforbol/farmacologia , Triptofano/análogos & derivados , Triptofano/farmacologia
2.
PLoS One ; 14(8): e0216839, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398205

RESUMO

The two main collagen receptors on platelets, GPVI and integrin α2ß1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2ß1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.


Assuntos
Plaquetas/citologia , Deleção de Genes , Integrina alfa2beta1/deficiência , Integrina alfa2beta1/genética , Megacariócitos/citologia , Glicoproteínas da Membrana de Plaquetas/deficiência , Glicoproteínas da Membrana de Plaquetas/genética , Animais , Camundongos , Trombopoese/genética
3.
Food Funct ; 10(8): 4661-4673, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292579

RESUMO

Hydroxysafflor yellow A (HSYA) is the main active ingredient of edible plant safflower. HSYA has demonstrated anti-inflammatory effects. The inflammatory response is the key mechanism responsible for asthma, and the pro-inflammatory platelet-activating factor (PAF) is known to play a role in the pathology of bronchial asthma. In this study, we stimulated human bronchial smooth muscle cells (HBSMCs) with PAF and examined the effects of HSYA on the resulting asthma-related process. PAF stimulation induced HBSMC activation, induced proliferation, increased expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α, and activated asthma-related signaling pathways. All these effects were significantly inhibited by treatment with HSYA (9, 27, 81 µmol L-1). The effects of HSYA were prevented by the addition of a PAF receptor (PAFR) antagonist or by PAFR gene silencing with small interfering RNA. These results suggest that HSYA may inhibit PAF-induced activation of HBSMCs by targeting the PAFR. Overall, these findings provide evidence that HSYA can be applied as a potential therapeutic agent in the treatment of bronchial asthma.


Assuntos
Brônquios/efeitos dos fármacos , Chalcona/análogos & derivados , Músculo Liso/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Quinonas/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Brônquios/metabolismo , Chalcona/farmacologia , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Músculo Liso/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Ativação de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
4.
Food Funct ; 10(6): 3379-3385, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31107473

RESUMO

Studies have shown that gelatin is not only a good hemostatic material, but also a food additive with potentially broad use. Yak skin gelatin is a new gelatin resource, but its oral coagulant effects have not been studied. Given the central role of platelets in hemostasis, in this study we examined the pharmacodynamical differences between different molecular Yak skin gelatins on platelet activation. The hemostatic effects of Yak skin gelatins with different molecular weight distributions were evaluated for bleeding time (BT), clotting time (CT), and platelet activity by measuring the contents of P-selectin, platelet membrane glycoprotein Ia/IIa (GP Ia/IIa), platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa), and platelet membrane glycoprotein IV (GP IV). Intragastric administration of Yak skin gelatin resulted in a significant reduction in CT and BT, and an increase in the contents of P-selectin, GP Ia/IIa, GP IIb/IIIa, and GP IV in all groups in comparison with the control group. The strongest activation of platelets by Yak skin gelatin was observed with size between 0.1 µm and 0.22 µm, and activation may have been in response to improving GP IIb/IIIa and GP IV levels. When measuring the levels of an established indicator of platelet activation, platelet activation-dependent granule membrane protein (CD62P), its promotion was observed for all molecular weight ranges of Yak skin gelatins. In brief, Yak skin gelatin has hemostatic effects, and Yak skin gelatin fractions between 0.1 µm and 0.22 µm are the primary effectors of hemostasis via promoting platelet membrane glycoprotein activities and strengthening platelet function.


Assuntos
Plaquetas/efeitos dos fármacos , Gelatina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Pele/química , Animais , Plaquetas/fisiologia , Bovinos , Feminino , Gelatina/química , Hemostasia/efeitos dos fármacos , Masculino , Camundongos , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Platelets ; 30(6): 708-713, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068042

RESUMO

Platelet membrane glycoprotein VI (GPVI) is increasingly recognized as an important receptor for thrombus formation and growth. Numerous arguments have been published indicating that GPVI plays a major role in thrombosis without being essential for physiological hemostasis. In humans, GPVI deficiencies are rarely reported. These are most often deficiencies occurring in the context of autoimmunity and, more rarely, genetic deficits. The purpose of this review is to compile data on the quantitative and qualitative genetic abnormalities of GPVI.


Assuntos
Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Humanos , Glicoproteínas da Membrana de Plaquetas/deficiência
6.
Nutrients ; 11(3)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866528

RESUMO

Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P ⁺ hypereosinophilic syndrome.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Leucemia/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
7.
Thromb Haemost ; 119(3): 431-438, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30597490

RESUMO

Sepsis triggers a complex series of pathophysiologic events involving inflammatory responses and coagulation abnormalities. While circulating blood platelets are well-characterized for their contributions to coagulation, increasingly platelet-dependent effects on inflammation are being recognized. Here, we focus on the platelet membrane receptor, glycoprotein VI (GPVI), and its role in platelet microparticle (pMP) release. The GPVI receptor is a platelet-specific collagen membrane receptor that, upon ligand binding, facilitates the release of pMPs. As membrane-bound platelet fragments of less than 1 µm, pMPs are known to have both pro-inflammatory and pro-coagulant properties. Thus, pMPs are potentially impacting sepsis at multiple stages of the inflammatory response. Studies are presented documenting the impact of the most common GPVI haplotypes, GPVIa and GPVIb, on pMP levels and release in healthy individuals (n = 49). The GPVIa haplotype corresponds to an approximately twofold increase in circulating pMPs as a percentage of total microparticles in healthy individuals along with a heightened in vitro release of pMPs. Additionally, patients admitted to a paediatric intensive care unit (ICU) (n = 73) with an initial diagnosis of sepsis were recruited and their GPVI haplotypes determined. Septic patients of the GPVIa haplotype (n = 59) were statistically more likely to present with a diagnosis of severe sepsis or septic shock, as compared with GPVIb individuals (n = 14). Independent disease classification via PELOD-2 and Pediatric Risk of Mortality III scores confirmed individuals with the GPVIa haplotype were more likely to have significant organ failure. Thus, GPVI haplotypes influence pMP levels in the circulation and are predictive of sepsis severity when presenting to the ICU.


Assuntos
Plaquetas , Micropartículas Derivadas de Células/genética , Haplótipos , Glicoproteínas da Membrana de Plaquetas/genética , Sepse/genética , Adolescente , Idade de Início , Plaquetas/metabolismo , Estudos de Casos e Controles , Micropartículas Derivadas de Células/metabolismo , Criança , Pré-Escolar , Progressão da Doença , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Fatores de Risco , Sepse/sangue , Sepse/diagnóstico , Índice de Gravidade de Doença
8.
Transfusion ; 59(1): 396-404, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30488955

RESUMO

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. STUDY DESIGN AND METHODS: The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. RESULTS: Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. CONCLUSION: This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.


Assuntos
Integrina beta3/genética , Polimorfismo Genético/genética , Trombocitopenia Neonatal Aloimune/genética , Animais , Animais Recém-Nascidos , Antígenos de Plaquetas Humanas/genética , Células HEK293 , Humanos , Recém-Nascido , Isoantígenos/genética , Glicoproteínas da Membrana de Plaquetas/genética , Trombocitopenia Neonatal Aloimune/patologia
9.
J Steroid Biochem Mol Biol ; 187: 152-159, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30476590

RESUMO

Human rhinoviruses commonly cause upper respiratory infections, which may be complicated by secondary bacterial infection. Vitamin D replacement reduces risk of acute respiratory infections in vitamin D-deficient individuals, but the mechanisms by which such protection is mediated are incompletely understood. We therefore conducted experiments to characterise the influence of the major circulating metabolite 25-hydroxyvitamin D (25[OH]D) and the active metabolite 1,25-dihydroxyvitamin D (1,25[OH]2D) on responses of a respiratory epithelial cell line (A549 cells) to infection with a major group human rhinovirus (RV-16). Pre-treatment of A549 respiratory epithelial cells with a physiological concentration (10-7M) of 25(OH)D induced transient resistance to infection with RV-16 and attenuated RV-16-induced expression of the genes encoding intercellular adhesion molecule 1 (ICAM-1, a cell surface glycoprotein that acts as the cellular receptor for major group rhinoviruses) and platelet-activating factor receptor (PAFR, a G-protein coupled receptor implicated in adhesion of Streptococcus pneumoniae to respiratory epithelial cells). These effects were associated with enhanced expression of the genes encoding the NF-κB inhibitor IκBα and the antimicrobial peptide cathelicidin LL-37. Our findings suggest possible mechanisms by which vitamin D may enhance resistance to rhinovirus infection and reduce risk of secondary bacterial infection in vitamin D-deficient individuals.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Infecções por Picornaviridae/prevenção & controle , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/genética , Rhinovirus/efeitos dos fármacos , Vitamina D/análogos & derivados , Vitaminas/farmacologia , Células A549 , Calcitriol/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Infecções por Picornaviridae/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Rhinovirus/fisiologia , Vitamina D/farmacologia
10.
J Lipid Res ; 59(11): 2063-2074, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30139761

RESUMO

Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Azepinas/farmacologia , Cromatografia Líquida , Feminino , Voluntários Saudáveis , Lisofosfatidilcolinas/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/sangue , Fosfolipídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Triazóis/farmacologia
11.
J Invest Dermatol ; 138(11): 2461-2469, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29857067

RESUMO

Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line in vitro, human skin explants ex vivo, as well as in murine skin in vivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.


Assuntos
Queimaduras/imunologia , Etanol/metabolismo , Queratinócitos/fisiologia , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/genética , Doença Aguda , Intoxicação Alcoólica , Animais , Linhagem Celular , Citocinas/metabolismo , Feminino , Temperatura Alta , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima
12.
Clin Chim Acta ; 484: 87-90, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29802830

RESUMO

Platelets have various functions and participate in primary hemostasis, inflammation, and immune responses. Human platelet antigens (HPAs) are alloantigens expressed on the platelet membrane. Each HPA represent one of six platelet glycoproteins GPIIb, GPIIIa, GPIa, GPIbα, GPIbß, and CD109, and six biallelic systems are grouped. A single nucleotide polymorphism (SNP) in the gene sequence causes a single amino acid substitution of relevant platelet glycoprotein with the exception of HPA-14bw. High-throughput next-generation sequencing-based method has been developed, which enable accurately identification of HPA polymorphisms. The roles of HPA in disease were reviewed. HPAs mediate platelet-microorganism and platelet-malignant cell interactions, and they also participate in pathogenesis of hemorrhagic fever with renal syndrome and infective endocarditis. The exploration of HPA polymorphisms in association with disease susceptibility of individuals will benefit prevention or management of disease.


Assuntos
Antígenos de Plaquetas Humanas/genética , Endocardite/genética , Febre Hemorrágica com Síndrome Renal/genética , Substituição de Aminoácidos/genética , Humanos , Glicoproteínas da Membrana de Plaquetas/genética , Polimorfismo de Nucleotídeo Único/genética
13.
Haematologica ; 103(5): 898-907, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29472360

RESUMO

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.


Assuntos
Plaquetas/fisiologia , Fibrinogênio/metabolismo , Leucemia Basofílica Aguda/patologia , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Humanos , Leucemia Basofílica Aguda/genética , Leucemia Basofílica Aguda/metabolismo , Camundongos , Adesividade Plaquetária , Glicoproteínas da Membrana de Plaquetas/genética , Ratos , Quinase Syk/genética , Quinase Syk/metabolismo , Trombose , Células Tumorais Cultivadas
15.
Blood ; 131(10): 1122-1144, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29301754

RESUMO

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.


Assuntos
Plaquetas/metabolismo , Homeostase , Trombose/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Animais , Plaquetas/patologia , Camundongos , Camundongos Knockout , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Trombose/genética , Quinases da Família src/genética
16.
Reprod Sci ; 25(3): 384-394, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28631554

RESUMO

It is widely hypothesized that menstrual pain is triggered by prostaglandin synthesis that evokes high-pressure uterine contractions and ischemia. However, the effects of molecules implicated in menstrual pain on uterine contractility, perfusion, and oxygenation in vivo have been rarely demonstrated. Studies in women that do not respond to nonsteroidal anti-inflammatory drugs (NSAIDs) have reported elevated levels of platelet-activating factor (PAF). To establish in vivo evidence of PAF's capability to impair uterine homeostasis and to elicit visceral pain, we examined the effects of the PAF receptor agonist (carbamyl PAF [CPAF]) in comparison to other molecules hypothesized to play a role in uterine pain in mice. Uterine pressure was increased by oxytocin, prostaglandin F2α (PGF2α), and CPAF. Even in the absence of inflammatory molecules, uterine contractions reduced uterine oxygenation by 38%. CPAF reduced uterine perfusion by 40% ± 8% and elicited further oxygen desaturation approaching hypoxia (9.4 ± 3.4 mm Hg Pao2). Intraperitoneal injections of CPAF and PGF2α evoked visceral pain and pelvic hyperalgesia in awake wild-type mice. However, pain was not observed in identically injected PAF-receptor knockout mice. Thus, our model provides a demonstration that a molecule implicated in NSAID-resistant dysmenorrhea has a detrimental effect on uterine homeostasis and is capable of causing visceral pain. Our results support the general hypothesis that menstrual cramps are caused by uterine contractions, impaired perfusion, and reduced oxygenation. Since this study was limited to mice, confirmation of these results in humans would be valuable for development of novel therapeutics targeted at inflammatory precursors, contractility, perfusion, and tissue oxygenation.


Assuntos
Hiperalgesia/metabolismo , Hipóxia/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Contração Uterina/efeitos dos fármacos , Útero/efeitos dos fármacos , Dor Visceral/metabolismo , Animais , Dinoprosta/farmacologia , Feminino , Camundongos , Camundongos Knockout , Ocitocina/farmacologia , Perfusão , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/genética
17.
Clin Appl Thromb Hemost ; 24(1): 63-69, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28041267

RESUMO

Disequilibrium of hemostasis is central to the pathogenesis of all thromboses, and platelets are essential for primary hemostasis. The platelet membrane glycoprotein receptor is involved in the clot formation in blood; therefore, the changes in related genes could impair platelet aggregation in patients with sticky platelet syndrome (SPS). Patients with SPS who experienced fetal loss were shown to harbor a risk haplotype at GP6 locus. The aim of the study was to examine the genetic linkage of this selected risk haplotype with single nucleotide variations (SNVs) in the coding sequence of the GP6 gene in order to identify possible functional SNVs in association with SPS and fetal loss. A total of 37 patients with SPS manifested fetal loss, and 42 healthy controls were enrolled in the study. The SPS was diagnosed with platelet aggregometry. The SNVs were determined by dideoxy sequencing and high-resolution melting analysis. The missense variations were detected in patients with risk haplotype only. The association analysis showed association of the minor alleles with the SPS manifested by fetal loss as follows-rs1671152 (odds ratio [OR]: 4.667, 95% confidence interval [CI]: 1.462-14.89, P = .006), rs2304167 (OR: 5.085, 95% CI: 1.605-16.10, P = .003), and rs1654416 (OR: 5.085, 95% CI: 1.605-16.10, P = .003). Using the Expectation-Maximization (EM) algorithm, the estimated minor haplotype with predicted protein residue PEAN was significantly associated with the given phenotype (OR: 4.746, 95% CI: 1.486-15.15, P = .005). We have shown that haplotype PEAN associated with SPS and manifested by fetal loss and suggest that the mechanism involved in the action of GPVI has significant effect on GPVI-mediated signal transduction through Syk-phosphorylation.


Assuntos
Aborto Habitual , Transtornos Plaquetários , Doenças Genéticas Inatas , Haplótipos , Desequilíbrio de Ligação , Mutação de Sentido Incorreto , Agregação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas , Aborto Habitual/sangue , Aborto Habitual/genética , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Feminino , Doenças Genéticas Inatas/sangue , Doenças Genéticas Inatas/genética , Humanos , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Síndrome
18.
Biochem Biophys Res Commun ; 495(4): 2475-2481, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29278700

RESUMO

Myocardial ischemia/reperfusion (I/R) still have high morbidity and mortality worldwide. Platelet activating factor (PAF) is a potent phospholipid regulator of inflammation. PAF acts on a single receptor (PAFR), which is expressed on cellular and nuclear membranes of various cell types. The study is aimed to explore if PAFR could modulate myocardial I/R injury in mice. PAFR expressions began to up-regulate at 1 h, and reached peak at 24 h. PAFR deletion markedly attenuated myocardial I/R injury, evidenced by the reduced infarct size and the improved cardiac function. Furthermore, PAFR-knockout inhibited inflammatory response, as demonstrated by down-regulated pro-inflammatory cytokines and chemokine, as well as the inactivation of nuclear factor κB (NF-κB). Additionally, PAFR-absence ameliorated oxidative stress induced by myocardial I/R, associated with the up-regulation of superoxide dismutase (SOD) and nuclear respiratory factor 2 (Nrf-2) activity. Finally, PAFR-deficiency impeded apoptosis, which was proved by the decreasing in terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL)-positive myocytes, and Caspase-3 cleavage. And the activation of Janus kinase 1-signal transducer and activator of transcription 1 (JAK1/STAT1) pathway was also suppressed by PAFR-knockout. The findings above were confirmed in lipopolysaccharide (LPS)-incubated cardiomyocytes with or without PAFR expressions in vitro. In summary, we supposed that inhibiting PAFR reduced inflammation, oxidative stress and apoptosis, and thus might be a promising therapeutic strategy to alleviate myocardial I/R injury.


Assuntos
Apoptose/imunologia , Traumatismo por Reperfusão Miocárdica/imunologia , Miocardite/imunologia , Estresse Oxidativo/imunologia , Animais , Citocinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/patologia , Miocardite/patologia , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/imunologia , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia
19.
J Thromb Haemost ; 16(2): 389-404, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29210180

RESUMO

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbß3. SUMMARY: Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbß3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbß3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbß3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.


Assuntos
Plaquetas/metabolismo , Colágeno/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombastenia/sangue , Trombose/sangue , Sítios de Ligação , Estudos de Casos e Controles , Fibrina/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Adesividade Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Trombastenia/genética , Trombose/genética
20.
Brain Res Bull ; 137: 71-78, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29122692

RESUMO

Accumulating evidence suggests that neuroinflammation is one of the important etiologic factors of abusive and neuropsychiatric disorders. Platelet-activating factor (PAF) is potent proinflammatory lipid mediat1or and plays a pivotal role in neuroinflammatory disorders through the specific PAF receptor (PAF-R). Phencyclidine (PCP) induces a psychotomimetic state that closely resembles schizophrenia. Here, we investigated the role of PAF-R in the abnormal behaviors induced by PCP in mice. Repeated treatment with PCP resulted in a significant increase in PAF-R gene expression in the prefrontal cortex (PFC) and in the hippocampus. This increase was more pronounced in the PFC than hippocampus. Treatment with PCP resulted in a significant increase in nuclear translocation of the nuclear factor kappa beta (NF-κB) p65 and DNA binding activity, indicating that the proinflammatory molecule NF-κB was increased through up-regulation of PAF-R. Consistently, NF-κB activation was significantly protected by the PAF-R antagonist, ginkgolide B (Gink B), in PAF-R knockout mice and by the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). In addition, PCP-induced abnormal behaviors (i.e., reduced sociability, depression, cognitive impairment, and behavioral sensitization) were significantly attenuated by Gink B, in PAF-R knockout mice, and by PDTC. Importantly, PDTC did not significantly alter the attenuations observed in Gink B-treated mice or PAF-R knockout mice, indicating that NF-κB is a critical target for neuropsychotoxic modulation of PAF-R. Therefore, the results suggest that PAF-R mediates PCP-induced neuropsychotoxicity via a NF-κB-dependent mechanism, and that up-regulation of PAF-R may be associated with schizophrenia-like behavior in animal models.


Assuntos
Antipsicóticos/farmacologia , Ginkgolídeos/farmacologia , Lactonas/farmacologia , NF-kappa B/metabolismo , Fenciclidina/toxicidade , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Psicoses Induzidas por Substâncias/tratamento farmacológico , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Psicoses Induzidas por Substâncias/metabolismo , Psicoses Induzidas por Substâncias/patologia , Psicoses Induzidas por Substâncias/psicologia , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Tiocarbamatos/farmacologia
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