RESUMO
Human Leukocyte Antigen-G (HLA-G) prevents the activity of immune cells and is decreased in women with preeclampsia. We aimed to investigate the associations between circulating soluble HLA-G (sHLA-G) and 92 cardiovascular disease-related biomarkers from a previously published multiplex study in women with preeclampsia and controls. We found 15 markers significantly associated with circulating sHLA-G in univariate analyses. After multivariable adjusted regression, only proto-oncogene tyrosine-protein kinase Src (SRC) and vascular endothelial growth factor D were significantly associated with sHLA-G. Low SRC, previously observed in the circulation of preeclamptic women, may be regulated by low sHLA-G, and reflect decreased trophoblast differentiation and syncytical formation.
Assuntos
Antígenos HLA-G/sangue , Pré-Eclâmpsia/epidemiologia , Trofoblastos/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Diferenciação Celular/imunologia , Cesárea , Feminino , Antígenos HLA-G/imunologia , Fatores de Risco de Doenças Cardíacas , Humanos , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/imunologia , Gravidez , Medição de Risco/métodos , Fator D de Crescimento do Endotélio Vascular/sangue , Quinases da Família src/sangueRESUMO
The Src-family protein tyrosine kinases (SFKs), a subfamily of non-receptor tyrosine kinases, are ubiquitously expressed in various cell types. Numerous studies have suggested that SFKs are related to signal transduction in major cardiac physiological and pathological processes, it is the activity of SFKs that is connected with the maintenance of cardiovascular homeostasis. Upon stimulation of various injury factors or stress, the phosphorylation state of SFKs is changed, which has been found to modulate different cardiac pathological conditions, such as hypertension, coronary heart disease, ischemic heart disease, myocardial ischemia-reperfusion injury, arrhythmia and cardiomyopathy via regulating cell growth, differentiation, movement and function, electrophysiologic signals. This review summarizes the basic information about SFKs, updates its role in the different processes underlying the development of multiple cardiovascular diseases (CVDs), and highlights their potential role as disease biomarkers and therapeutic targets, which would help understand the pathophysiology of CVDs and promote the further potential clinical adhibition.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases da Família src/antagonistas & inibidores , Biomarcadores/sangue , Biomarcadores/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/sangue , Quinases da Família src/metabolismoRESUMO
Previous studies are inconclusive on the relationships between BLK gene polymorphisms and clinical features of systemic lupus erythematosus (SLE). The present study aimed to estimate association between BLK loci and SLE clinical features in Chinese population. Associations between BLK single-nucleotide polymorphisms (SNPs) and susceptibility to SLE in this study were estimated using data of 1205 health controls previously reported in the same population. And a total of 814 SLE patients recruited from two different sources according with ACR criteria were analyzed for genotype-phenotype associations. A meta-analysis was conducted of the associations between BLK loci and renal disorder in SLE. The expression quantitative trait locus (eQTL) data were also extracted from the public databases for the selected SNPs. Significant associations were observed between these SNPs and susceptibility to SLE. In addition, the data showed that rs2618479 and rs7812879 were associated with renal disorder [OR = 1.51 (95% CI: 1.15, 1.99) and 1.61 (95% CI: 1.21, 2.14), Pcorr = 0.033 and 0.011, respectively] and proteinuria [OR = 1.47 (95% CI: 1.12, 1.95) and 1.52 (95% CI: 1.14, 2.03), Pcorr = 0.048 and 0.040, respectively]. The consistent associations were observed in two independent centers as well as new cases group. The result of meta-analysis for rs2618479 was also significant [OR = 1.35 (95% CI: 1.12, 1.62)]. In addition, bioinformatics analysis demonstrated that the two SNPs were significantly associated with the expression of BLK in whole blood and several immune cells. Our data support that variant loci of BLK are associated with presence of renal disorder in patients with SLE.
Assuntos
Nefropatias/genética , Lúpus Eritematoso Sistêmico/genética , Quinases da Família src/genética , Adulto , Povo Asiático/genética , Feminino , Estudos de Associação Genética , Humanos , Nefropatias/complicações , Nefropatias/fisiopatologia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Quinases da Família src/sangue , Quinases da Família src/metabolismoRESUMO
This study aimed to detect serum miR-203 expression levels in AML and explore its potential clinical significance. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to measure the serum miR-203 levels in 134 patients with AML and 70 healthy controls. The results demonstrated that serum miR-203 expression was significantly reduced in AML patients compared with healthy controls. Receiver operating characteristic curve (ROC) analysis revealed miR-203 could distinguish AML cases from normal controls. Low serum miR-203 levels were associated with worse clinical features, as well as poorer overall survival and relapse free survival of AML patients. Moreover, multivariate analysis confirmed low serum miR-203 expression to be an independent unfavorable prognostic predictor for AML. The bioinformatics analysis showed that the downstream genes and pathways of miR-203 was closely associated with tumorigenesis. Downregulation of miR-203 in AML cell lines upregulated the expression levels of oncogenic promoters such as CREB1, SRC and HDAC1. Thus, these findings demonstrated that serum miR-203 might be a promising biomarker for the diagnosis and prognosis of AML.
Assuntos
Biomarcadores Tumorais/genética , Carcinogênese/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Antagomirs/genética , Antagomirs/metabolismo , Biomarcadores Tumorais/sangue , Carcinogênese/metabolismo , Carcinogênese/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Biologia Computacional/métodos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/sangue , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Perfilação da Expressão Gênica , Ontologia Genética , Histona Desacetilase 1/sangue , Histona Desacetilase 1/genética , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/sangue , Anotação de Sequência Molecular , Análise Multivariada , Proteínas de Neoplasias/sangue , Prognóstico , Curva ROC , Recidiva , Transdução de Sinais , Análise de Sobrevida , Quinases da Família src/sangue , Quinases da Família src/genéticaRESUMO
OBJECTIVE: To determine the extent of dasatinib uptake and effect on Src kinase activity in tumor, normal adjacent tissue, and blood in newly diagnosed endometrial cancer patients. METHODS: Dasatinib was dosed at 100 or 200 mg PO BID at 32 and 8 h preoperatively. Blood and tissue were collected pre-treatment and at surgery to assess active (pY419) and total Src protein (pharmacodynamics [PD]) and pharmacokinetics (PK). Plasma PK and PD were also analyzed at 2, 4 and 8 h following the second dose. RESULTS: Ten patients completed the study, 5 at each dose level (DL). Average (median, standard deviation, range) 2 h plasma concentration of drug was 119 (121, 80, 226) and 236 (162, 248, 633) ng/mL, for the 100 and 200 mg DL, respectively. Average ratio of 8 h normal and tumor tissue to plasma concentration overall was 3.6 (2.3, 3.4, 9.6) and 8.3 (3.2, 11.9, 38.7), respectively. Dasatinib concentration in tumor was higher than in plasma for both DL. Four patients displayed significant reductions in pTyr419Src at ≥ 1 time points in blood, and four patients satisfied the PD activity criteria in tissue, with reductions in pTyr419Src of ≥ 60%. CONCLUSIONS: This is the first study to show PK and PD effects of dasatinib in tumor tissue, allowing evaluation of tissue PD markers as a function of tumor dasatinib concentration. Dasatinib tissue concentrations at 8 h after dosing were associated with modulation of pTyr419Src, total Src protein, and pTyr419Src/Src ratio. All patients had reduction in at least one Src parameter in either tissue or blood.
Assuntos
Biomarcadores Tumorais/sangue , Dasatinibe/farmacologia , Neoplasias do Endométrio/terapia , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/sangue , Administração Oral , Idoso , Biomarcadores Tumorais/metabolismo , Biópsia , Linhagem Celular Tumoral , Dasatinibe/uso terapêutico , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/patologia , Endométrio/patologia , Endométrio/cirurgia , Feminino , Humanos , Histerectomia , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Salpingo-Ooforectomia , Fatores de Tempo , Distribuição Tecidual , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Several protein tyrosine phosphatase non-receptor 22 (PTPN22) single-nucleotide polymorphisms (SNPs) have been significantly related with rheumatoid arthritis (RA) susceptibility. Nevertheless, its potential influence on PTPN22 expression in RA has not been completely elucidated. Furthermore, PTPN22 binds to C-Src tyrosine kinase (CSK) forming a key complex in autoimmunity. However, the information of CSK gene in RA is scarce. In this study, we analyzed the relative PTPN22 and CSK expression in peripheral blood from 89 RA patients and 43 controls to determine if the most relevant PTPN22 (rs2488457, rs2476601 and rs33996649) and CSK (rs34933034 and rs1378942) polymorphisms may influence on PTPN22 and CSK expression in RA. The association between PTPN22 and CSK expression in RA patients and their clinical characteristics was also evaluated. Our study shows for the first time a marked down-regulation of PTPN22 expression in RA patients carrying the risk alleles of PTPN22 rs2488457 and rs2476601 compared to controls (p = 0.004 and p = 0.007, respectively). Furthermore, CSK expression was significantly lower in RA patients than in controls (p < 0.0001). Interestingly, a reduced PTPN22 expression was disclosed in RA patients with ischemic heart disease (p = 0.009). The transcriptional suppression of this PTPN22/CSK complex may have a noteworthy clinical relevance in RA patients.
Assuntos
Artrite Reumatoide/sangue , Regulação para Baixo , Proteína Tirosina Fosfatase não Receptora Tipo 22/sangue , Quinases da Família src/sangue , Adulto , Idoso , Artrite Reumatoide/genética , Biomarcadores/sangue , Proteína Tirosina Quinase CSK , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Quinases da Família src/genéticaRESUMO
OBJECTIVE: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif-containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif-containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1-deficient mice. APPROACH AND RESULTS: Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1-deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI-specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI-FcRγ-chain and integrin αIIbß3 in megakaryocytes because of enhanced Src family kinase activity. CONCLUSIONS: Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein-coupled receptors.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Ativação Plaquetária , Receptores Imunológicos/deficiência , Trombocitose/sangue , Trombose/sangue , Animais , Plaquetas/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Células Cultivadas , Cloretos , Modelos Animais de Doenças , Ativação Enzimática , Compostos Férricos , Predisposição Genética para Doença , Megacariócitos/efeitos dos fármacos , Camundongos Knockout , Peptídeos/farmacologia , Fenótipo , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de IgG/sangue , Receptores Imunológicos/genética , Transdução de Sinais/efeitos dos fármacos , Trombocitose/genética , Trombose/induzido quimicamente , Trombose/genética , Quinases da Família src/sangueRESUMO
ADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/ß inhibitor and in PKC α or ß knockout murine platelets compared to controls. Furthermore, we show that PKC α/ß inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/ß regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.
Assuntos
Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Proteína Quinase C/sangue , Agonistas do Receptor Purinérgico P2Y/farmacologia , Tromboxano A2/sangue , Animais , Plaquetas/enzimologia , Humanos , Isoenzimas , Camundongos Knockout , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Inibidores de Proteínas Quinases/farmacologia , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Receptores Purinérgicos P2Y1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/sangue , Quinases da Família src/genéticaRESUMO
RATIONALE: Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus. OBJECTIVE: To test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. METHODS AND RESULTS: By using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. CONCLUSIONS: TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.
Assuntos
Plaquetas/enzimologia , Transdução de Sinais , Trombose/enzimologia , Timidina Fosforilase/metabolismo , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Transplante de Medula Óssea , Cloretos , Inibidores Enzimáticos/farmacologia , Compostos Férricos , Haploinsuficiência , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Fosforilação , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Transfusão de Plaquetas , Proteínas Proto-Oncogênicas c-akt/sangue , Proteínas Proto-Oncogênicas c-fyn/sangue , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-yes/sangue , Selenoproteína P/sangue , Transdução de Sinais/efeitos dos fármacos , Trombose/sangue , Trombose/induzido quimicamente , Trombose/prevenção & controle , Timidina Fosforilase/antagonistas & inibidores , Timidina Fosforilase/sangue , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Fatores de Tempo , Quinases da Família src/sangue , Quinases da Família src/genéticaRESUMO
RATIONALE: A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance of altered-self ligands in circulation that can be recognized by Toll-like receptors (TLRs). Platelets express a number of TLRs, including TLR9; however, the role of TLR in platelet function and thrombosis is poorly understood. OBJECTIVE: To investigate the biological activities of carboxy(alkylpyrrole) protein adducts, an altered-self ligand generated in oxidative stress, on platelet function and thrombosis. METHODS AND RESULTS: In this study we show that carboxy(alkylpyrrole) protein adducts represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that carboxy(alkylpyrrole) protein adducts promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and it is Src kinase-dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. CONCLUSIONS: Our study demonstrates that platelet TLR9 is a functional platelet receptor that links oxidative stress, innate immunity, and thrombosis.
Assuntos
Plaquetas/metabolismo , Ativação Plaquetária , Albumina Sérica/metabolismo , Trombose/sangue , Receptor Toll-Like 9/sangue , Animais , Plaquetas/imunologia , Antígenos CD36/deficiência , Antígenos CD36/genética , Linhagem Celular , Modelos Animais de Doenças , Genes Reporter , Humanos , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/sangue , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/sangue , Fosforilação , Agregação Plaquetária , Proteínas Proto-Oncogênicas c-akt/sangue , Receptores Depuradores Classe B/deficiência , Receptores Depuradores Classe B/genética , Transdução de Sinais , Trombose/genética , Trombose/imunologia , Fatores de Tempo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 6 Toll-Like/deficiência , Receptor 6 Toll-Like/genética , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Transfecção , Quinases da Família src/sangueRESUMO
The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.
Assuntos
Autoimunidade/imunologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Haplótipos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Quinases da Família src/genética , Adulto , Alelos , Desequilíbrio Alélico , Especificidade de Anticorpos/imunologia , Linhagem Celular , Sangue Fetal/citologia , Homozigoto , Humanos , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Linfócitos T/enzimologia , Quinases da Família src/sangueRESUMO
BACKGROUND AND OBJECTIVES: Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbß3, and in vivo. METHODS AND RESULTS: Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. CONCLUSION: These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbß3, which requires prior signaling through Src.
Assuntos
Plaquetas/enzimologia , Ativação Plaquetária , Quinases da Família src/sangue , Animais , Proteínas de Transporte/metabolismo , Forma Celular , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Camundongos , Camundongos Knockout , Mutação , Peptídeos/metabolismo , Ativação Plaquetária/genética , Adesividade Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas c-fyn , Transdução de Sinais , Trombose/sangue , Trombose/enzimologia , Trombose/genética , Fatores de Tempo , Quinases da Família src/deficiência , Quinases da Família src/genéticaRESUMO
OBJECTIVE: Our aim in this study was to provide novel information on the molecular mechanisms playing a major role in the unwanted platelet activation associated with ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS: We compared the platelet proteome of 11 STEMI patients to a matched control group of 15 stable chronic ischemic cardiopathy patients. In addition, we did a prospective study to follow the STEMI patients over time. Proteins were separated by high-resolution 2D gel electrophoresis, identified by mass spectrometry, and validated by Western blotting. Platelets from STEMI patients on admission displayed 56 protein spot differences (corresponding to 42 unique genes) compared with the control group. The number of differences decreased with time during the patients' follow-up. Interestingly, the adapter protein CrkL and the active form of Src (phosphorylated in Tyr418) were found to be upregulated in platelets from STEMI patients. Major signaling pathways related to the proteins identified include integrin, integrin-linked kinase, and glycoprotein VI (GPVI) signaling. Interestingly, a study on an independent cohort of patients showed a higher degree of activation of GPVI signaling in STEMI patients. CONCLUSIONS: CrkL, the active form of Src, and GPVI signaling are upregulated in platelets from STEMI patients.
Assuntos
Síndrome Coronariana Aguda/fisiopatologia , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Eletrocardiografia , Infarto do Miocárdio/sangue , Infarto do Miocárdio/fisiopatologia , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Síndrome Coronariana Aguda/sangue , Proteínas Adaptadoras de Transdução de Sinal/sangue , Idoso , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Integrinas/sangue , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Estudos Prospectivos , Proteínas Serina-Treonina Quinases/sangue , Proteômica , Regulação para Cima , Quinases da Família src/sangueRESUMO
In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbß3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.
Assuntos
Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Venenos de Escorpião/farmacologia , Quinases da Família src/sangue , Animais , Plaquetas/metabolismo , Células Cultivadas , Galinhas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteínas da Membrana de Plaquetas/deficiência , Glicoproteínas da Membrana de Plaquetas/genética , Transdução de Sinais , TransfecçãoRESUMO
Platelet activation is critically regulated by an increase in intracellular calcium concentration ([Ca2+](i)). Although Ca2+ release from intracellular Ca2+ stores and subsequent store-operated Ca2+ entry are often thought to be the major contributors to increases in [Ca2+](i) evoked by most agonists, high concentrations of thrombin activate a Ca2+ entry pathway that is independent of Ca2+ store depletion (known as 'non-capacitative cation entry'-NCCE). The channel that conducts NCCE has not previously been clearly identified, and the mechanisms that regulate its activation are also unknown. Here we have investigated NCCE using fura-2-loaded human platelets. To investigate NCCE independently of other Ca2+ signaling pathways, the intracellular Ca2+ stores were first rapidly depleted in the absence of extracellular Ca2+. Sr2+ was then added to monitor maximal store-operated cation influx. Thrombin was then added to stimulate NCCE. Flufenamic acid, which inhibits Ca2+ entry through most TRPC isoforms, but potentiates entry through TRPC6, was found to block store-operated cation entry. In contrast, thrombin-induced NCCE was increased, suggesting the possible involvement of TRPC6. Since TRPC6 is regulated by Src family tyrosine kinases in some cells, we investigated the possible role of this kinase family in NCCE. PP2, a Src family tyrosine kinase inhibitor, completely abolished thrombin-induced NCCE. Furthermore, NCCE was enhanced by phenylarsine oxide and could be directly induced by vanadyl hydroperoxide, both tyrosine phosphatase inhibitors. These data indicate that Src family tyrosine kinase activation is a required step in NCCE activation. In conclusion NCCE may be an important regulator of platelet activation when local thrombin concentrations are high.
Assuntos
Plaquetas/metabolismo , Cálcio/sangue , Trombina/metabolismo , Quinases da Família src/sangue , Plaquetas/enzimologia , Canais de Cálcio/sangue , Cátions Bivalentes/metabolismo , Ativação Enzimática , Humanos , Ativação Plaquetária/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6RESUMO
BACKGROUND: The integrin alpha(IIb)beta(3) is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of alpha(IIb)beta(3), for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD-ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. OBJECTIVES: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. METHODS: We assessed the effect of lotrafiban, and other alpha(IIb)beta(3) antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro-coagulant activity) and dense granule release. RESULTS: In all cases, no effect of alpha(IIb)beta(3) antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)-associated FcR gamma-chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y(12) antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. CONCLUSIONS: These data provide a mechanism for enhancement of platelet activity by alpha(IIb)beta(3) inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.
Assuntos
Biomimética , Plaquetas/efeitos dos fármacos , Oligopeptídeos/sangue , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Benzodiazepinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ativação Enzimática , Humanos , Cadeias gama de Imunoglobulina/sangue , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Ligantes , Fosfatidilserinas/sangue , Fosforilação , Piperidinas/farmacologia , Inibidores da Agregação Plaquetária/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/sangue , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2Y12 , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Quinase Syk , Fatores de Tempo , Tirofibana , Tirosina/análogos & derivados , Tirosina/sangue , Tirosina/farmacologia , Quinases da Família src/sangueRESUMO
Prostate and breast cancer cells preferentially metastasize to bone, whereupon a complex interaction between metastatic tumor cells, osteoclasts, and osteoblasts results in the development of bone lesions that cause significant pain and patient morbidity. For patients with bone lesions, the goals of treatment are to decrease tumor growth, prevent further metastases, and inhibit tumor-associated bone pathology. Preclinical data suggest that SRC, a nonreceptor tyrosine kinase, is an important signaling molecule during the processes of osteoclast-mediated bone resorption, tumor growth, and metastasis, and that SRC has a role in hormone receptor signaling and resistance. As such, SRC represents a logical target for the treatment of advanced metastatic prostate or breast cancer. SRC-targeting agents, including dasatinib, saracatinib, and bosutinib, are currently in clinical development for patients with solid tumors. Preliminary data from phase 1/2 trials, including tumor responses and bone-specific activity in patients with prostate or breast cancer, demonstrate that SRC inhibitors have potential in the clinical setting. Data arising from ongoing and future clinical trials will confirm whether SRC inhibitors provide clinical benefits for patients with advanced disease.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases da Família src/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/prevenção & controle , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Quinases da Família src/sangueRESUMO
OBJECTIVES: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. however, data concerning expression, regulation and function of eNOS AND iNOS in platelets remain controversial. METHODS AND RESULTS: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate (L-arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC beta(1)-subunit-Tyr(192). CONCLUSIONS: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.
Assuntos
Plaquetas/enzimologia , Guanilato Ciclase/sangue , Óxido Nítrico Sintase/sangue , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , GMP Cíclico/sangue , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/química , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II/sangue , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , RNA Mensageiro/sangue , RNA Mensageiro/genética , Ristocetina/farmacologia , Solubilidade , ômega-N-Metilarginina/farmacologia , Quinases da Família src/sangue , Fator de von Willebrand/farmacologiaRESUMO
PURPOSE: Dasatinib (BMS-354825) is a potent, oral multi-targeted kinase inhibitor. It is an effective therapy for patients with imatinib-resistant or -intolerant Ph+ leukemias,. It has demonstrated promising preclinical anti-tumor activity, and is under clinical evaluation in solid tumors. To support the clinical development of dasatinib, we identified a pharmacodynamic biomarker to assess in vivo SRC kinase inhibition, with subsequent evaluation in cancer patients. METHODS: The biomarker, phosphorylated SRC (phospho-SRC), was first identified in human prostate PC-3 tumor cells and peripheral blood mononuclear cells (PBMCs) in vitro. It was further assessed in nude mice bearing PC-3 xenografts. Phospho-SRC[pY418] in tumors and PBMC were measured by western blot analysis, and were quantified by ELISA assays. Dasatinib plasma concentrations were determined using LC/MS/MS. RESULTS: In PC-3 cells, dasatinib showed dose-dependent anti-proliferative effect, which correlated with the inhibition of phospho-SRC[pY418] and of SRC kinase activity. With a single oral dose of 50 or 15 mg/kg, tumoral phospho-SRC[pY418] was maximally inhibited at 3 h, partially reversed between 7 and 17 h, and completely recovered after 24 h post dose. At 5 mg/kg, tumoral phospho-SRC[pY418] inhibition was less pronounced and recovered more rapidly to baseline level within 24h. Dasatinib (1 mg/kg) resulted in little inhibition. In PBMCs, a similar time course and extent of phospho-SRC[pY418] inhibition was observed. Inhibition of phospho-SRC[pY418] in vivo appeared to correlate with the preclinical in vivo efficacy and PK profiles of dasatinib in mice. CONCLUSIONS: Phospho-SRC[pY418] may potentially be used as a biomarker to enable assessment of target inhibition in clinical studies exploring dasatinib antitumor activity.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Monitoramento de Medicamentos/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Quinases da Família src/análise , Adenocarcinoma/química , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Biomarcadores , Divisão Celular/efeitos dos fármacos , Dasatinibe , Feminino , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/sangue , Fosfoproteínas/antagonistas & inibidores , Fosforilação , Neoplasias da Próstata/química , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Organismos Livres de Patógenos Específicos , Especificidade por Substrato , Tiazóis/administração & dosagem , Tiazóis/farmacocinética , Tiazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/sangueRESUMO
In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.