RESUMO
Connexins oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular trafficking of molecules. In this study, we report the expression and function of an orphan connexin, connexin-62 (Cx62), in human and mouse (Cx57, mouse homolog) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62, and 3-dimensional structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using fluorescence recovery after photobleaching analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and hemostasis. This was associated with elevated protein kinase A-dependent signaling in a cyclic adenosine monophosphate-independent manner and was not observed in Cx57-deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterized connexin in regulating the function of circulating cells.
Assuntos
Plaquetas/metabolismo , Conexinas/fisiologia , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Conexinas/sangue , Conexinas/química , Conexinas/deficiência , Conexinas/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Junções Comunicantes/fisiologia , Hemostasia/fisiologia , Humanos , Integrinas/sangue , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Adesividade Plaquetária , Agregação Plaquetária , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Trombose/sangueRESUMO
Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.
Assuntos
Anti-Infecciosos , Venenos de Cnidários , Colubridae , Humanos , Animais , Camundongos , Staphylococcus aureus , Austrália , Cicatrização , Anti-Infecciosos/farmacologia , Venenos de Cnidários/farmacologia , Colágeno/farmacologia , Peptídeos/farmacologia , Citocinas/farmacologia , MamíferosRESUMO
Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.
Assuntos
Anexina A1 , Animais , Humanos , Camundongos , Anexina A1/metabolismo , Plaquetas/metabolismo , Inflamação/metabolismo , Neutrófilos/metabolismo , Peptídeos/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismoRESUMO
Cardiovascular diseases (CVDs) are a primary cause of deaths worldwide. Thrombotic diseases, specifically stroke and coronary heart diseases, account for around 85% of CVDs-induced deaths. Platelets (small circulating blood cells) are responsible for the prevention of excessive bleeding upon vascular injury, through blood clotting (haemostasis). However, unnecessary activation of platelets under pathological conditions, such as upon the rupture of atherosclerotic plaques, results in thrombus formation (thrombosis), which can cause life threatening conditions such as stroke or heart attack. Therefore, antiplatelet medications are usually prescribed for people who are at a high risk of thrombotic diseases. The currently used antiplatelet drugs are associated with major side effects such as excessive bleeding, and some patients are resistant to these drugs. Therefore, numerous studies have been conducted to develop new antiplatelet agents and notably, to establish the relationship between edible plants, specifically fruits, vegetables and spices, and cardiovascular health. Indeed, healthy and balanced diets have proven to be effective for the prevention of CVDs in diverse settings. A high intake of fruits and vegetables in regular diet is associated with lower risks for stroke and coronary heart diseases because of their plethora of phytochemical constituents. In this review, we discuss the impacts of commonly used selected edible plants (specifically vegetables, fruits and spices) and/or their isolated compounds on the modulation of platelet function, haemostasis and thrombosis.
Assuntos
Plaquetas/metabolismo , Plantas Comestíveis/química , Animais , Ensaios Clínicos como Assunto , Fungos/química , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Testes de Função PlaquetáriaRESUMO
Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.
Assuntos
Cobras Corais , Mordeduras de Serpentes , Animais , Cobras Corais/metabolismo , Venenos Elapídicos/química , Antivenenos , Fosfolipases A2/metabolismo , Venenos de Serpentes/metabolismo , Elapidae/metabolismoRESUMO
Aberrant activation of signalling pathways has been postulated to promote age related changes in skeletal muscle. Cell signalling activation requires not only the expression of ligands and receptors but also an appropriate environment that facilitates their interaction. Here we first examined the expression of SULF1/SULF2 and members of RTK (receptor tyrosine kinase) and the Wnt family in skeletal muscle of normal and a mouse model of accelerated ageing. We show that SULF1/SULF2 and these signalling components, a feature of early muscle development are barely detectable in early postnatal muscle. Real time qPCR and immunocytochemical analysis showed gradual but progressive up-regulation of SULF1/SULF2 and RTK/Wnt proteins not only in the activated satellite cells but also on muscle fibres that gradually increased with age. Satellite cells on isolated muscle fibres showed spontaneous in vivo satellite cell activation and progressive reduction in proliferative potential and responsiveness to HGF (hepatocyte growth factor) and dysregulated myogenic differentiation with age. Finally, we show that SULF1/SULF2 and RTK/Wnt signalling components are expressed in progeric mouse muscles at earlier stage but their expression is attenuated by an intervention that promotes muscle repair and growth.
Assuntos
Diferenciação Celular , Músculo Esquelético/crescimento & desenvolvimento , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células Satélites de Músculo Esquelético/citologia , Sulfatases/genética , Sulfatases/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , Proteínas Wnt/metabolismoRESUMO
Cardiovascular diseases represent a major cause of mortality and morbidity in the world, and specifically, thrombotic conditions such as heart attacks and strokes are caused by unwarranted activation of platelets and subsequent formation of blood clots (thrombi) within the blood vessels during pathological circumstances. Therefore, platelets act as a primary therapeutic target to treat and prevent thrombotic conditions. Current treatments are limited due to intolerance, and they are associated with severe side effects such as bleeding complications. Hence, the development of novel therapeutic strategies for thrombotic diseases is an urgent priority. Flavonoids are naturally occurring plant-derived molecules that exert numerous beneficial effects in humans through modulating the functions of distinct cell types. However, naturally occurring flavonoids suffer from several issues such as poor solubility, lipophilicity, and bioavailability, which hinder their efficacy and potency. Despite these, flavonoids act as versatile templates for the design and synthesis of novel molecules for various therapeutic targets. Indeed, several synthetic flavonoids have recently been developed to improve their stability, bioavailability, and efficacy, including for the modulation of platelet function. Here, we provide insight into the actions of certain natural flavonoids along with the advantages of synthetic flavonoids in the modulation of platelet function, haemostasis, and thrombosis.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Trombose/tratamento farmacológico , Trombose/metabolismo , Animais , Disponibilidade Biológica , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Flavonoides/síntese química , Flavonoides/química , Humanos , Trombose/sangue , Resultado do TratamentoRESUMO
The Eph kinases, EphA4 and EphB1, and their ligand, ephrinB1, have been previously reported to be present in platelets where they contribute to thrombus stability. Although thrombus formation allows for Eph-ephrin engagement and bidirectional signaling, the importance specifically of Eph kinase or ephrin signaling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signaling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signaling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together, these data demonstrate that EphB2 signaling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/enzimologia , Ativação Plaquetária/fisiologia , Receptor EphB2/metabolismo , Transdução de Sinais/fisiologia , Animais , Plaquetas/citologia , Camundongos , Camundongos Transgênicos , Receptor EphB2/genéticaRESUMO
OBJECTIVE: Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6α-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. APPROACH AND RESULTS: FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin αIIbß3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. CONCLUSIONS: This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.
Assuntos
Plaquetas/efeitos dos fármacos , Ácido Quenodesoxicólico/análogos & derivados , Hemostasia/efeitos dos fármacos , Isoxazóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Trombose/prevenção & controle , Animais , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácido Quenodesoxicólico/farmacologia , GMP Cíclico/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Genótipo , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Citoplasmáticos e Nucleares/sangue , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Trombose/sangue , Fatores de TempoRESUMO
OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.
Assuntos
Plaquetas/enzimologia , Membrana Celular/enzimologia , Megacariócitos/enzimologia , Ativação Plaquetária , Isomerases de Dissulfetos de Proteínas/sangue , Actinas/sangue , Animais , Plaquetas/efeitos dos fármacos , Proteínas Sanguíneas/deficiência , Proteínas Sanguíneas/genética , Calnexina/sangue , Membrana Celular/efeitos dos fármacos , Genótipo , Humanos , Megacariócitos/efeitos dos fármacos , Fusão de Membrana , Proteínas de Membrana/sangue , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Ativação Plaquetária/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/biossíntese , Transporte Proteico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangueRESUMO
Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer's disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-ß peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.
Assuntos
Inflamação/metabolismo , NF-kappa B/metabolismo , Benzamidas/farmacologia , Linhagem Celular , Escherichia coli/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Nitrilas/farmacologia , Salmonella/imunologia , Sulfonas/farmacologiaRESUMO
Platelets are anucleated blood cells that participate in a wide range of physiological and pathological functions. Their major role is mediating haemostasis and thrombosis. In addition to these classic functions, platelets have emerged as important players in the innate immune system. In particular, they interact with leukocytes, secrete pro- and anti-inflammatory factors, and express a wide range of inflammatory receptors including Toll-like receptors (TLRs), for example, Toll-like receptor 4 (TLR4). TLR4, which is the most extensively studied TLR in nucleated cells, recognises lipopolysaccharides (LPS) that are compounds of the outer surface of Gram-negative bacteria. Unlike other TLRs, TLR4 is able to signal through both the MyD88-dependent and MyD88-independent signalling pathways. Notably, despite both pathways culminating in the activation of transcription factors, TLR4 has a prominent functional impact on platelet activity, haemostasis, and thrombosis. In this review, we summarise the current knowledge on TLR4 signalling in platelets, critically discuss its impact on platelet function, and highlight the open questions in this area.
Assuntos
Plaquetas/metabolismo , Trombose/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Hemostasia/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbß3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbß3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.
Assuntos
Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Colágeno/metabolismo , Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Inibidores de Proteínas Quinases/toxicidade , Pirazóis/toxicidade , Pirimidinas/toxicidade , Adenina/análogos & derivados , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Tirosina Quinase da Agamaglobulinemia , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Hemorragia/sangue , Humanos , Piperidinas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/sangue , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Fatores de Risco , Fatores de TempoRESUMO
Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.
Assuntos
Conexinas/genética , Infarto do Miocárdio/genética , Acidente Vascular Cerebral/genética , Trombose/genética , Plaquetas/metabolismo , Plaquetas/patologia , Comunicação Celular/genética , Conexinas/metabolismo , Junções Comunicantes/genética , Junções Comunicantes/fisiologia , Hemostasia/genética , Humanos , Infarto do Miocárdio/fisiopatologia , Transdução de Sinais , Acidente Vascular Cerebral/fisiopatologia , Trombose/fisiopatologiaRESUMO
Statins are widely prescribed cholesterol-lowering drugs that are a first-line treatment of coronary artery disease and atherosclerosis, reducing the incidence of thrombotic events such as myocardial infarction and stroke. Statins have been shown to reduce platelet activation, although the mechanism(s) through which this occurs is unclear. Because several of the characteristic effects of statins on platelets are shared with those elicited by the inhibitory platelet adhesion receptor PECAM-1 (platelet endothelial cell adhesion molecule-1), we investigated a potential connection between the influence of statins on platelet function and PECAM-1 signaling. Statins were found to inhibit a range of platelet functional responses and thrombus formation in vitro and in vivo. Notably, these effects of statins on platelet function in vitro and in vivo were diminished in PECAM-1(-/-) platelets. Activation of PECAM-1 signaling results in its tyrosine phosphorylation, the recruitment and activation of tyrosine phosphatase SHP-2, the subsequent binding of phosphoinositol 3-kinase (PI3K), and diminished PI3K signaling. Statins resulted in the stimulation of these events, leading to the inhibition of Akt activation. Together, these data provide evidence for a fundamental role of PECAM-1 in the inhibitory effects of statins on platelet activation, which may explain some of the pleiotropic actions of these drugs.
Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Fibrinolíticos/farmacologia , Indóis/farmacologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Relação Dose-Resposta a Droga , Fluvastatina , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbß3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of ß3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3ß Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbß3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.
Assuntos
Dinaminas/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Ativação Plaquetária/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Fator de von Willebrand/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Humanos , Camundongos , Camundongos Knockout , Ativação Plaquetária/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/farmacologia , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais/fisiologia , Tiazolidinas/farmacologiaRESUMO
OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbß3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.
Assuntos
Plaquetas/efeitos dos fármacos , Flavonas/farmacologia , Hemostasia/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Trombose/prevenção & controle , Animais , Plaquetas/enzimologia , Sinalização do Cálcio/efeitos dos fármacos , Moléculas de Adesão Celular/sangue , GMP Cíclico/sangue , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/sangue , Fosfatidilinositol 3-Quinase/sangue , Fosfoproteínas/sangue , Fosforilação , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/sangue , Trombose/sangue , Fatores de TempoRESUMO
We thank the author for showing interest in our article [...].
Assuntos
Neurite Óptica , Toxinas Biológicas , Animais , Naja naja , BungarusRESUMO
Snakebite envenoming and its resulting complications are serious threats to the health of vulnerable people living in rural areas of developing countries. The knowledge of the heterogeneity of symptoms associated with snakebite envenoming and their management strategies is vital to treat such life-threatening complications to save lives. Russell's viper envenomation induces a diverse range of clinical manifestations from commonly recognised haemotoxic and local effects to several rare conditions that are often not reported. The lack of awareness about these unusual manifestations can affect prompt diagnosis, appropriate therapeutic approaches, and positive outcomes for patients. Here, we report pulmonary thromboembolism that developed in three patients following Russell's viper envenomation and demonstrate their common clinical features and diagnostic and therapeutic approaches used. All patients showed clinical signs of local (oedema) and systemic (blood coagulation disturbances) envenomation, which were treated using polyvalent antivenom. They exhibited elevated heart rates, breathlessness, and reduced oxygen saturation, which are non-specific but core parameters in the diagnosis of pulmonary embolism. The recognition of pulmonary embolism was also achieved by an electrocardiogram, which showed sinus tachycardia and computed tomography and echocardiogram scans further confirmed this condition. Anti-coagulant treatment using low-molecular-weight heparin offered clinical benefits in these patients. In summary, this report reinforces the broad spectrum of previously unreported consequences of Russell's viper envenomation. The constant updating of healthcare professionals and the dissemination of major lessons learned in the clinical management of snakebite envenoming through scientific documentation and educational programs are necessary to mitigate the adverse impacts of venomous snakebites in vulnerable communities.
Assuntos
Antivenenos , Daboia , Embolia Pulmonar , Mordeduras de Serpentes , Mordeduras de Serpentes/complicações , Mordeduras de Serpentes/tratamento farmacológico , Embolia Pulmonar/etiologia , Embolia Pulmonar/tratamento farmacológico , Humanos , Animais , Masculino , Antivenenos/uso terapêutico , Venenos de Víboras/toxicidade , Adulto , Feminino , Pessoa de Meia-Idade , Anticoagulantes/uso terapêuticoRESUMO
Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell's viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian 'Big Four' snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell's viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell's viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.