Asunto(s)
Mutación , Humanos , Chile , Glicoproteínas de Membrana Plaquetaria/genética , Efecto FundadorRESUMEN
Despite abundant evidence demonstrating that platelets foster metastasis, anti-platelet agents have low therapeutic potential due to the risk of hemorrhages. In addition, whether platelets can regulate metastasis at the late stages of the disease remains unknown. In this study, we subject syngeneic models of metastasis to various thrombocytopenic regimes to show that platelets provide a biphasic contribution to metastasis. While potent intravascular binding of platelets to tumor cells efficiently promotes metastasis, platelets further support the outgrowth of established metastases via immune suppression. Genetic depletion and pharmacological targeting of the glycoprotein VI (GPVI) platelet-specific receptor in humanized mouse models efficiently reduce the growth of established metastases, independently of active platelet binding to tumor cells in the bloodstream. Our study demonstrates therapeutic efficacy when targeting animals bearing growing metastases. It further identifies GPVI as a molecular target whose inhibition can impair metastasis without inducing collateral hemostatic perturbations.
Asunto(s)
Plaquetas , Metástasis de la Neoplasia , Glicoproteínas de Membrana Plaquetaria , Animales , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Humanos , Ratones , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Línea Celular Tumoral , Femenino , Ratones Endogámicos C57BLRESUMEN
Platelet-activating factor (PAF) plays a significant role in several leucocyte functions, including platelet aggregation and inflammation. Additionally, PAF has a role in the behavioral and physiological changes in mammals. However, the effect of PAF has not been well studied in birds. Therefore, the study aimed to determine if PAF affects feeding behavior, voluntary activity, cloacal temperature, and feed passage through the digestive tract in chicks (Gallus gallus). We also studied the involvement of PAF in the innate immune system induced by lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Both intraperitoneal (IP) and intracerebroventricular (ICV) injections of PAF significantly decreased food intake. IP injection of PAF significantly decreased voluntary activity and slowed the feed passage from the crop, whereas ICV injection had no effect. Conversely, ICV injection of PAF significantly increased the cloacal temperature, but IP injection had no effect. The IP injection of LPS significantly reduced the mRNA expression of lysophosphatidylcholine acyltransferase 2, an enzyme responsible for PAF production in the heart and pancreas. On the other hand, LPS significantly increased the mRNA expression of the PAF receptor in the peripheral organs. The present study shows that PAF influences behavioral and physiological responses and is related to the response against bacterial infections in chicks.
Asunto(s)
Temperatura Corporal , Pollos , Cloaca , Buche de las Aves , Ingestión de Alimentos , Factor de Activación Plaquetaria , Animales , Masculino , Temperatura Corporal/efectos de los fármacos , Cloaca/efectos de los fármacos , Cloaca/fisiología , Buche de las Aves/efectos de los fármacos , Buche de las Aves/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Lipopolisacáridos/farmacología , Factor de Activación Plaquetaria/farmacología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.
Asunto(s)
Plaquetas , Agregación Plaquetaria , Quinasa Syk , Animales , Ratones , Fosforilación , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Quinasa Syk/genética , Quinasa Syk/metabolismo , TirosinaRESUMEN
Introduction: Platelets play an important role in cardiovascular diseases. After acute myocardial infarction, platelets display enhanced activation and migrate into the infarct zone. Furthermore, platelets trigger acute inflammation and cardiac remodeling leading to alterations in scar formation and cardiac function as observed in thrombocytopenic mice. GPVI is the major collagen receptor in platelets and important for platelet activation and thrombus formation and stability. Antibody induced deletion of GPVI at the platelet surface or treatment of mice with recombinant GPVI-Fc results in reduced inflammation and decreased infarct size in a mouse model of AMI. However, the role of GPVI has not been fully clarified to date. Methods/Results: In this study, we found that GPVI is not involved in the inflammatory response in experimental AMI using GPVI deficient mice that were analyzed in a closed-chest model. However, reduced platelet activation in response to GPVI and PAR4 receptor stimulation resulted in reduced pro-coagulant activity leading to improved cardiac remodeling. In detail, GPVI deficiency in mice led to reduced TGF-ß plasma levels and decreased expression of genes involved in cardiac remodeling such as Col1a1, Col3a1, periostin and Cthrc1 7 days post AMI. Consequently, collagen quality of the scar shifted to more tight and less fine collagen leading to improved scar formation and cardiac function in GPVI deficient mice at 21d post AMI. Conclusion: Taken together, this study identifies GPVI as a major regulator of platelet-induced cardiac remodeling and supports the potential relevance of GPVI as therapeutic target to reduce ischemia reperfusion injury and to improve cardiac healing.
Asunto(s)
Infarto del Miocardio , Glicoproteínas de Membrana Plaquetaria , Animales , Ratones , Cicatriz , Colágeno/metabolismo , Inflamación , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores de Colágeno , Remodelación VentricularRESUMEN
Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria , Quinasa Syk/metabolismo , Tirosina , Animales , Plaquetas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Fosforilación , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Inmunológicos/metabolismo , Quinasa Syk/genética , Tirosina/metabolismoRESUMEN
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Tetraspaninas/genética , Tetraspaninas/metabolismo , Plaquetas/metabolismo , Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Dominios Proteicos , Proteolisis , Especificidad por Sustrato , Tetraspaninas/químicaRESUMEN
Ethanol has been demonstrated to exert profound effects upon cells and tissues via multiple mechanisms. One recently appreciated means by which cells can communicate with other cells is via the production and release of extracellular vesicles. Though smaller exosomes have been demonstrated to be released in response to ethanol exposure, the ability of ethanol to modulate the generation and release of larger microvesicle particles (MVP) is lesser studied. The present studies examined the ability of exogenous ethanol to generate MVP with a focus on skin cells. Acute ethanol exposure resulted in augmented MVP release in keratinocytes and in the skin and blood of mice. Unlike other stimuli such as ultraviolet B radiation or thermal burn injury, ethanol-mediated MVP release was independent of the Platelet-activating Factor receptor (PAFR). However, ethanol pretreatment was found to augment thermal burn injury-induced MVP in a PAFR-dependent manner. These studies provide a novel mechanism for ethanol-mediated effects, that could be relevant in the significant toxicity associated with thermal burn injury in the setting of alcohol intoxication.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Etanol/toxicidad , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Animales , Línea Celular , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Ischemic stroke is an inflammation-related disease, during which process activation of NLRP3 inflammasome and subsequent pyroptosis play crucial roles. Platelet-activating factor (PAF) is a potent phospholipid regulator of inflammation which exerts its effect via binding specific PAF receptor (PAFR). However, whether PAFR contributes to pyroptosis during ischemia/reperfusion (I/R) injury remains to be elucidated. To explore the underlying effect of PAFR on ischemic stroke from the perspective of pyroptosis, mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) injury and primary cultures of mice cerebral cortical neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) injury to mimic I/R in vivo and in vitro, after which indexes associated with pyroptosis were analyzed. Intriguingly, our results indicated that inhibition of PAFR with its inhibitor XQ-1H or PAFR siRNA exerted a neuroprotective effect against I/R injury both in vivo and in vitro. Furthermore, inflammasome activation and pyroptosis after ischemic challenge were attenuated by XQ-1H or PAFR siRNA. Besides, the protection of XQ-1H was abolished by PAF stimulaiton to some extent. Moreover, XQ-1H or PAFR siRNA alleviated the neuronal pyroptosis induced by LPS and nigericin (an NLRP3 activator) in cortical neurons. Taken together, this study firstly demonstrates that PAFR is involved in neuronal pyroptosis after I/R injury, and XQ-1H, a specific PAFR inhibitor, has a promising prospect in attenuating I/R injury from the perspective of anti-pyroptosis.
Asunto(s)
Ginkgólidos/farmacología , Lactonas/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Daño por Reperfusión/metabolismo , Animales , Modelos Animales de Enfermedad , Inflamasomas/metabolismo , Masculino , Ratones , Neuronas/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2-depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2-depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.
Asunto(s)
Lesión Renal Aguda , Rabdomiólisis , Lesión Renal Aguda/etiología , Animales , Plaquetas , Femenino , Hemo , Lectinas Tipo C/genética , Ratones , Glicoproteínas de Membrana Plaquetaria/genética , EmbarazoRESUMEN
A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.
Asunto(s)
Micropartículas Derivadas de Células/inmunología , Tolerancia Inmunológica/efectos de la radiación , Queratinocitos/inmunología , Rayos Ultravioleta , Animales , Línea Celular , Micropartículas Derivadas de Células/genética , Femenino , Humanos , Ratones , Ratones Noqueados , Factor de Activación Plaquetaria/genética , Factor de Activación Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/inmunologíaRESUMEN
ABSTRACT: Myocardial infarction (MI) is a leading cause of heart failure all over the world. Long noncoding RNAs have been reported to be associated with the development of MI. In this article, we aimed to explore the effects of long noncoding RNA small nuclear RNA host gene 7 (SNHG7) on MI and the possible mechanism. In this study, an MI model was established by ligating the left anterior descending coronary artery of mice. Cardiac fibroblasts (CFs) derived from neonatal mice were activated by angiotensin II (Ang-II) treatment. The expression of SNHG7 and miR-455-3p was examined by quantitative real-time polymerase chain reaction, and protein levels of platelet-activating factor receptor (PTAFR) and fibrosis-related proteins were analyzed by western blot assay. Cell apoptosis of CFs was monitored by flow cytometry. Enzyme-linked immunosorbent assay was performed to evaluate inflammatory responses in CFs. Moreover, dual-luciferase reporter assay was used to confirm the target relationship between miR-455-3p and SNHG7 or PTAFR. LncRNA SNHG7 and PTAFR were upregulated, whereas miR-455-3p was downregulated in cardiac tissues of mice with MI and Ang-II-induced CFs. SNHG7 depletion or miR-455-3p overexpression attenuated Ang-II-induced apoptosis, fibrosis, and inflammation in CFs, which was severally weakened by miR-455-3p inhibition or PTAFR upregulation. LncRNA SNHG7 targeted miR-455-3p, and PTAFR was a target of miR-455-3p. LncRNA SNHG7 depletion exerted protective roles in apoptosis, fibrosis, and inflammation in Ang-II-induced CFs by regulating miR-455-3p/PTAFR axis, providing a potential molecular target for MI therapy.
Asunto(s)
MicroARNs/genética , Infarto del Miocardio/genética , Glicoproteínas de Membrana Plaquetaria/genética , ARN Largo no Codificante/genética , Receptores Acoplados a Proteínas G/genética , Animales , Apoptosis/genética , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fibroblastos/citología , Fibrosis/genética , Técnicas de Silenciamiento del Gen , Inflamación/genética , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/fisiopatología , ARN Nuclear Pequeño/genética , Regulación hacia ArribaRESUMEN
Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.
Asunto(s)
Cisteína-Endopeptidasas Gingipaínas/farmacología , Glicoproteínas de Membrana Plaquetaria/genética , Porphyromonas gingivalis/metabolismo , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Factores de Virulencia/farmacología , Células A549 , Adhesión Bacteriana/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Fimbrias Bacterianas/química , Regulación de la Expresión Génica , Cisteína-Endopeptidasas Gingipaínas/deficiencia , Cisteína-Endopeptidasas Gingipaínas/genética , Interacciones Huésped-Patógeno/genética , Humanos , Lipopolisacáridos/farmacología , Modelos Biológicos , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidad , Alveolos Pulmonares/microbiología , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Factores de Virulencia/deficiencia , Factores de Virulencia/genéticaRESUMEN
The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5'-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.
Asunto(s)
Activación Plaquetaria , Trombosis , Animales , Plaquetas , Hemostasis , Ratones , Glicoproteínas de Membrana Plaquetaria/genética , Trombosis/genéticaRESUMEN
Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia , Células HEK293 , Humanos , Ligandos , Microscopía Fluorescente , Mutación , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Imagen Individual de Molécula , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
Asunto(s)
Complejo Antígeno-Anticuerpo , Plaquetas , Glicoproteínas de Membrana Plaquetaria , Multimerización de Proteína , Anticuerpos de Dominio Único , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Humanos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacologíaRESUMEN
OBJECTIVE: Coronary artery disease (CAD) is the main cause of mortality worldwide. How stable coronary artery disease (SCAD) progresses to acute myocardial infarction (AMI) is not known. This study was aimed to explore the differentially expressed genes (DEGs) and pathways involved in the progression of SCAD to AMI. MATERIALS AND METHODS: Publicly available gene-expression profiles (GSE71226, GSE97320, GSE66360) were downloaded from the Gene Expression Omnibus (GEO) database and integrated to identify DEGs. The GSE59867 dataset was further used to verify the result of screened DEGs. Functional-enrichment analyses, protein-protein interaction network, microRNA-transcription factor (TF)-mRNA regulatory network, and drug-gene network were visualized. RESULTS: Sixty common DEGs (CDEGs) were screened between the SCAD-Control group and AMI-Control group in the integrated dataset. Four upregulated DEGs were selected from GSE59867. Twenty hub genes were discovered, and three significant modules were constructed in the PPI network. The intersection of functional and pathway-enrichment analyses of 60 CDEGs and the module DEGs indicated that they were mainly involved in "inflammatory response", "immune response", and "cytokine-cytokine receptor interaction". A miRNA-TF-mRNA regulatory network comprised 87 miRNAs, 16 upregulated target DEGs and 7 TFs. CONCLUSIONS: We identified several important genes and miRNAs involved in the progression of SCAD to AMI: platelet activating factor receptor (PTAFR), aquaoporin-9 (AQP9), toll-like receptor-4 (TLR4), human constitutive androstane receptor-3 (HCAR3), leucine-rich-α2 glycoprotein-1 (LRG1), mothers Against Decapentaplegic Homolog 4 (SMAD4) and miRNA-149-5p, miRNA-6778-3p, and miRNA-520a-3p. Inflammation and the immune response had important roles in the progression from SCAD to AMI.
Asunto(s)
Biología Computacional , Enfermedad de la Arteria Coronaria/metabolismo , Infarto del Miocardio/metabolismo , Enfermedad Aguda , Acuaporinas/genética , Acuaporinas/inmunología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , MicroARNs/genética , MicroARNs/inmunología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Mapas de Interacción de Proteínas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/inmunología , Proteína Smad4/genética , Proteína Smad4/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Platelets are increasingly being recognized for playing roles beyond thrombosis and hemostasis. Today we know that they mediate inflammation by direct interactions with innate immune cells or secretion of cytokines/chemokines. Here we review their interactions with neutrophils and monocytes/macrophages in infection and sepsis, stroke, myocardial infarction and venous thromboembolism. We discuss new roles for platelet surface receptors like GPVI or GPIb and also look at platelet contributions to the formation of neutrophil extracellular traps (NETs) as well as to deep vein thrombosis during infection, e.g. in COVID-19 patients.
Asunto(s)
Plaquetas/inmunología , COVID-19/inmunología , Infarto del Miocardio/inmunología , Neutrófilos/inmunología , Sepsis/inmunología , Accidente Cerebrovascular/inmunología , Tromboembolia Venosa/inmunología , Plaquetas/patología , COVID-19/genética , COVID-19/patología , COVID-19/virología , Comunicación Celular/genética , Comunicación Celular/inmunología , Citocinas/genética , Citocinas/inmunología , Trampas Extracelulares/genética , Trampas Extracelulares/inmunología , Regulación de la Expresión Génica , Humanos , Inflamación , Macrófagos/inmunología , Macrófagos/patología , Monocitos/inmunología , Monocitos/patología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Neutrófilos/patología , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/inmunología , Sepsis/genética , Sepsis/patología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Tromboembolia Venosa/genética , Tromboembolia Venosa/patologíaAsunto(s)
Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombosis/patología , Animales , Plaquetas/metabolismo , Plaquetas/patología , Progresión de la Enfermedad , Eliminación de Gen , Humanos , Ratones , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/genética , Trombosis/genética , Trombosis/metabolismoRESUMEN
BACKGROUND: Previous studies had shown that glycosaminoglycan (GAG) from Urechis unicinctus exerts an obvious antiplatelet aggregation effect. OBJECTIVE: This study aims to investigate the effects of GAG from Urechis unicinctus on ADP-induced platelet calcium and membrane glycoprotein expressions in rats. METHODS: Fura-2/AM fluorescence probe was used to measure intracytosolic free-calcium concentration ([Ca2+]i) of platelets and calculate platelet calcium influx and release concentrations. Flow cytometry was used to detect the expressions of platelet membrane glycoproteins GPIIb/IIIa (PAC-1) and P-selectin (CD62P) in rats. RESULTS: The results showed that the GAG from U. unicinctus significantly inhibited the release of platelet calcium (p < 0.01) and the expressions of platelet GPII b/IIIa and P-selectin (p < 0.01) induced by ADP in rats but had no significant effect on the influx of platelet calcium (p > 0.01). CONCLUSIONS: This study indicated that GAG may inhibit platelet activation and aggregation by reducing the release of Ca2+ and ADP-induced expression of platelet membrane glycoprotein in rats.