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1.
Phytomedicine ; 78: 153314, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32882582

RESUMO

BACKGROUND: Sarsasapogenin (Sar) shows good effects on diabetic nephropathy (DN) through inhibition of the NLRP3 inflammasome, yet the potential mechanism is not well known. PURPOSE: This study was designed to explore the regulation of thrombin and/or its receptor protease-activated receptor 1 (PAR-1) on the NLRP3 inflammasome and NF-κB signaling in DN condition, and further expounded the molecular mechanism of Sar on DN. METHODS: Streptozotocin-induced diabetic rats were treated by gavage with Sar (0, 20 and 60 mg/kg) for consecutive 10 weeks. Then urine and serum were collected for protein excretion, creatinine, urea nitrogen, and uric acid assay reflecting renal functions, renal tissue sections for periodic acid-Schiff staining and ki67 expression reflecting cell proliferation, and renal cortex for the NLRP3 inflammasome and NF-κB signaling as well as thrombin/PAR-1 signaling. High glucose-cultured human mesangial cells (HMCs) were used to further investigate the effects and mechanisms of Sar. RESULTS: Sar markedly ameliorated the renal functions and mesangial cell proliferation in diabetic rats, and suppressed activation of the NLRP3 inflammasome and NF-κB in renal cortex. Moreover, Sar remarkably down-regulated PAR-1 in protein and mRNA levels but didn't affect thrombin activity in kidney, although thrombin activity was significantly decreased in the renal cortex of diabetic rats. Meanwhile, high glucose induced activation of the NLRP3 inflammasome and NF-κB, and increased PAR-1 expression while didn't change thrombin activity in HMCs; however, Sar co-treatment ameliorated all the above indices. Further studies demonstrated that PAR-1 knockdown attenuated activation of the NLRP3 inflammasome and NF-κB, and Sar addition strengthened these effects in high glucose-cultured HMCs. CONCLUSION: Sar relieved DN in rat through inhibition of the NLRP3 inflammasome and NF-κB by down-regulating PAR-1 in kidney.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Células Mesangiais/efeitos dos fármacos , Receptor PAR-1/metabolismo , Espirostanos/farmacologia , Animais , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamassomos/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Células Mesangiais/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nefrite/tratamento farmacológico , Nefrite/metabolismo , Ratos Sprague-Dawley , Receptor PAR-1/genética , Trombina/metabolismo
2.
Bratisl Lek Listy ; 121(8): 600-604, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726124

RESUMO

AIM: To investigate the changes of water content in brain tissue, the expression of AQP4mRNA after cerebral hemorrhage in rats, and the intervention effect of Protease activated receptor 1 inhibitor (PAR1 inhibitor) on both. METHODS: Establish sham operation group (Sham group), ICH group, ICH+PAR1 inhibitor high-dose group (PI(H)group), ICH+PAR1 inhibitor low-dose group (PI(L)group), 25 in each group. Neural dysfunction scores were performed at 1d, 3d, 7d, 14d, and 21d after surgery, and brain water content and AQP4mRNA content were measured. RESULTS: Results: The neurological dysfunction and cerebral edema of rats with cerebral hemorrhage reached the peak at 3 days after operation. With the increase of time, the water content and AQP4mRNA content in the PL(H)group were higher than those in the PI(L)group. The differences were statistically significant. CONCLUSIONS: Appropriate inhibition of PAR1 can alleviate cerebral edema around the hematoma and play a role in improving the function of nerve defects. The mechanism may be realized by down-regulating the expression of AQP4mRNA in brain tissue (Tab. 3, Fig. 3, Ref. 25).


Assuntos
Edema Encefálico , Hemorragia Cerebral , Receptor PAR-1 , Animais , Aquaporina 4/metabolismo , Encéfalo , Modelos Animais de Doenças , Hematoma , Ratos , Receptor PAR-1/metabolismo
3.
Br J Pharmacol ; 177(21): 4971-4974, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32639031

RESUMO

In the search to rapidly identify effective therapies that will mitigate the morbidity and mortality of COVID-19, attention has been directed towards the repurposing of existing drugs. Candidates for repurposing include drugs that target COVID-19 pathobiology, including agents that alter angiotensin signalling. Recent data indicate that key findings in COVID-19 patients include thrombosis and endotheliitis. Activation of proteinase-activated receptor 1 (PAR1), in particular by the serine protease thrombin, is a critical element in platelet aggregation and coagulation. PAR1 activation also impacts on the actions of other cell types involved in COVID-19 pathobiology, including endothelial cells, fibroblasts and pulmonary alveolar epithelial cells. Vorapaxar is an approved inhibitor of PAR1, used for treatment of patients with myocardial infarction or peripheral arterial disease. We discuss evidence for a possible beneficial role for vorapaxar in the treatment of COVID-19 patients and other as-yet non-approved antagonists of PAR1 and proteinase-activated receptor 4 (PAR4). LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Lactonas/administração & dosagem , Pneumonia Viral/tratamento farmacológico , Piridinas/administração & dosagem , Receptor PAR-1/antagonistas & inibidores , Animais , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Humanos , Lactonas/farmacologia , Pandemias , Inibidores da Agregação de Plaquetas/administração & dosagem , Inibidores da Agregação de Plaquetas/farmacologia , Pneumonia Viral/virologia , Piridinas/farmacologia , Receptor PAR-1/metabolismo , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/metabolismo
4.
PLoS One ; 15(5): e0231944, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365105

RESUMO

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1ß, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.


Assuntos
Miométrio/efeitos dos fármacos , Trombina/farmacologia , Contração Uterina/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Feminino , Humanos , Contração Muscular/efeitos dos fármacos , Miométrio/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Imagem com Lapso de Tempo , Contração Uterina/fisiologia
5.
Lab Invest ; 100(8): 1057-1067, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32341518

RESUMO

Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell-cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1-TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR-TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.


Assuntos
Inflamação/genética , Receptor PAR-1/genética , Receptor PAR-2/genética , Transdução de Sinais/genética , Canais de Cátion TRPV/genética , Animais , Cálcio/metabolismo , Permeabilidade Capilar/genética , Edema/genética , Edema/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Canais de Cátion TRPV/metabolismo
6.
Eur J Histochem ; 64(2)2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32236088

RESUMO

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.


Assuntos
Neurogênese/efeitos dos fármacos , Nós Neurofibrosos/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Trombina/farmacologia , Animais , Cálcio/metabolismo , Feminino , Masculino , Neuritos/efeitos dos fármacos , Células PC12 , Pirróis/farmacologia , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Receptor PAR-1/metabolismo , Nervo Isquiático/efeitos dos fármacos , Tapsigargina/farmacologia
7.
Am J Physiol Renal Physiol ; 318(5): F1067-F1073, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200667

RESUMO

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg-1·day-1), PAR2 antagonist (FSLLRY, 3 mg·kg-1·day-1), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Iminas/farmacologia , Rim/efeitos dos fármacos , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-2/antagonistas & inibidores , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibrose , Humanos , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais
8.
Arterioscler Thromb Vasc Biol ; 40(5): 1275-1288, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212848

RESUMO

OBJECTIVE: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on ß-arrestin1. CONCLUSIONS: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.


Assuntos
Anti-Inflamatórios/administração & dosagem , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fator VIIa/administração & dosagem , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Receptor PAR-1/metabolismo , Animais , Modelos Animais de Doenças , Endocitose , Células Endoteliais/metabolismo , Endotoxinas , Feminino , Homozigoto , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Transgênicos , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/metabolismo , Mutação Puntual , Receptor PAR-1/genética , Transdução de Sinais
9.
Proc Natl Acad Sci U S A ; 117(9): 5039-5048, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071217

RESUMO

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.


Assuntos
Proteômica , Receptor PAR-1/metabolismo , Transdução de Sinais , Trombina/metabolismo , Proteínas de Ligação a Calmodulina , Proteínas de Transporte , Células Endoteliais/metabolismo , Humanos , Proteínas dos Microfilamentos , Fosforilação , Inibidor da Proteína C/metabolismo
10.
Biochem Pharmacol ; 175: 113849, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32059841

RESUMO

Gestational diabetes mellitus (GDM) is associated with an increased risk of progressing to type 2 DM and cardiovascular disease; however, the pathogenesis is still poorly understood. This study was to investigate roles of thrombin and its receptor protease-activated receptor 1 (PAR-1) and NLRP1 inflammasome in endothelial injury in GDM condition. Umbilical cord and plasma of GDM patients and high glucose (HG) cultured human umbilical vein endothelial cells (HUVECs) were used to examine the pathological changes of these pathways. Meanwhile, ameliorative effects and potential mechanisms of a natural product sarsasapogenin (Sar) were investigated in HUVECs. Thrombin/PAR-1 pathway, advanced glycation endproducts (AGEs) and their receptor (RAGE) axis, and the nucleotide-binding domain and leucine-rich repeat containing protein 1 (NLRP1) inflammasome were activated in GDM condition and HG-cultured HUVECs, accompanied by endothelial injury (decreased cell viability and increased lactate dehydrogenase release). Nevertheless, thrombin inhibition or PAR-1 antagonism caused decreases in AGEs formation and RAGE expression in HG-cultured HUVECs, while AGEs inhibition or RAGE antagonism declined PAR-1 expression not thrombin activity. Furthermore, thrombin inhibition or PAR-1 antagonism restrained NLRP1 inflammasome activation in HG-cultured HUVECs; meanwhile, NLRP1 expression and interleukin 18 levels were remarkably reduced in HG-cultured HUVECs after PAR-1 knockdown. Interestingly, Sar co-treatment could suppress thrombin/PAR-1 pathway, NLRP1 inflammasome, and AGEs/RAGE axis. Together, endothelial damages in GDM were likely due to enhanced interaction between AGEs/RAGE axis and thrombin/PAR-1 pathway, followed by NLRP1 inflammasome activation. Moreover, Sar may act as a protective agent against endothelial injury in chronic HG condition.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Gestacional/patologia , Endotélio Vascular/patologia , Inflamassomos/metabolismo , Receptor PAR-1/metabolismo , Trombina/metabolismo , Glicemia/análise , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura/química , Diabetes Gestacional/sangue , Diabetes Gestacional/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Gravidez , Receptor PAR-1/genética , Transdução de Sinais , Espirostanos/farmacologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismo
11.
PLoS One ; 15(2): e0222685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078628

RESUMO

Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that there is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.


Assuntos
Sinais Direcionadores de Proteínas/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endopeptidases/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Inibidores de Proteases/farmacologia , Receptor PAR-2/genética , Receptores de Superfície Celular , Tripsina/metabolismo
12.
Nat Mater ; 19(2): 218-226, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959953

RESUMO

Haematopoietic cells and platelets employ G-protein-coupled receptors (GPCRs) to sense extracellular information and respond by initiating integrin-mediated adhesion. So far, such processes have not been demonstrated in non-haematopoietic cells. Here, we report that the activation of protease-activated receptors PAR1 and PAR2 induce multiple signalling pathways to establish α5ß1-integrin-mediated adhesion. First, PARs signal via Gßγ and PI3K to α5ß1-integrins to adopt a talin- and kindlin-dependent high-affinity conformation, which triggers fibronectin binding and initiates cell adhesion. Then, within 60 s, PARs signal via Gα13, Gαi, ROCK and Src to strengthen the α5ß1-integrin-mediated adhesion. Furthermore, PAR signalling changes the abundance of numerous proteins in the adhesome assembled by α5ß1-integrins, including Gα13, vacuolar protein-sorting-associated protein 36, and band 4.1-like protein 4B or 5, and accelerates cell adhesion maturation, spreading and migration. The mechanistic insights describe how agonist binding to PAR employs GPCR and integrin-signalling pathways to initiate and regulate adhesion and to guide physiological responses of non-haematopoietic cells.


Assuntos
Adesão Celular , Integrina alfa5beta1/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais , Células HEK293 , Humanos , Talina/metabolismo
13.
FASEB J ; 34(1): 1079-1090, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914657

RESUMO

Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)-1, -2, -3, and -4 on FSAP-mediated signaling in HEK293T and A549 cells. Cleavage of PAR-reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR-2 and to a lesser degree PAR-1, but not PAR-3 or PAR-4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR-2, but not PAR-1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N-terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR-1, but not PAR-2, prevented the apoptosis-inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR-1 and -2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.


Assuntos
Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Apoptose , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Peptídeos/química , Fosforilação , Isoformas de Proteínas , Transdução de Sinais , Trombose
14.
Circ Res ; 126(4): 471-485, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31910739

RESUMO

RATIONALE: BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. OBJECTIVE: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. METHODS AND RESULTS: The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis. CONCLUSIONS: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.


Assuntos
Endotélio Vascular/fisiopatologia , Proteínas Tirosina Quinases/deficiência , Receptor PAR-1/metabolismo , Sepse/metabolismo , Trombina/metabolismo , Animais , Permeabilidade Capilar/genética , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/genética , Sepse/genética , Sepse/fisiopatologia , Transdução de Sinais/efeitos dos fármacos
15.
Am J Physiol Lung Cell Mol Physiol ; 318(1): L192-L199, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31664854

RESUMO

We evaluated the mechanisms underlying protease-activated receptor 1 (PAR1)-mediated activation of nodose C-fibers in mouse lungs. The PAR1-induced action potential discharge at the terminals was strongly inhibited in phospholipase C-ß3 (PLCß3)-deficient animals. At the level of the cell soma, PAR1 activation led to an increase in cytosolic calcium that was largely inhibited by transient receptor potential (TRP) A1 antagonism. Patch-clamp recordings, however, revealed that neither TRPA1 nor TRPV1 or any other ruthenium red-sensitive ion channels are required for the PAR1-mediated inward current or membrane depolarization in isolated nodose neurons. Consistent with these findings, PAR1-mediated action potential discharge in mouse lung nodose C-fiber terminals was unaltered in Trpa1/Trpv1 double-knockout animals and Trpc3/Trpc6 double-knockout animals. The activation of the C-fibers was also not inhibited by ruthenium red at concentrations that blocked TRPV1- and TRPA1-dependent responses. The biophysical data show that PAR1/Gq-mediated activation of nodose C-fibers may involve multiple ion channels downstream from PLCß3 activation. TRPA1 is an ion channel that participates in PAR1/Gq-mediated elevation in intracellular calcium. There is little evidence, however, that TRPA1, TRPV1, TRPC3, TRPC6, or other ruthenium red-sensitive TRP channels are required for PAR1/Gq-PLCß3-mediated membrane depolarization and action potential discharge in bronchopulmonary nodose C-fibers in the mouse.


Assuntos
Pulmão/metabolismo , Fibras Nervosas Amielínicas/metabolismo , Receptor PAR-1/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Potenciais de Ação/fisiologia , Animais , Brônquios/metabolismo , Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gânglio Nodoso/metabolismo , Fosfolipase C beta/metabolismo , Células Receptoras Sensoriais/metabolismo
16.
Am J Sports Med ; 48(1): 197-209, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31765237

RESUMO

BACKGROUND: Meniscal injury is very common, and injured meniscal tissue has a limited healing ability because of poor vascularity. Platelets contain both pro- and anti-angiogenic factors, which can be released by platelet selective activation. HYPOTHESIS: Platelets release a high level of vascular endothelial growth factor (VEGF) when they are activated by protease-activated receptor 1 (PAR1), whereas the platelets release endostatin when they are activated by protease-activated receptor 4 (PAR4). The PAR1-treated platelets enhance the proliferation of meniscal cells in vitro and promote in vivo healing of wounded meniscal tissue. STUDY DESIGN: Controlled laboratory study. METHOD: Platelets were isolated from human blood and activated with different reagents. The released growth factors from the activated platelets were determined by immunostaining and enzyme-linked immunosorbent assay. The effects of the platelets with different treatments on meniscal cells were tested by an in vitro model of cell culture and an in vivo model of wounded meniscal healing. RESULTS: The results indicated that platelets contained both pro- and antiangiogenic factors including VEGF and endostatin. In unactivated platelets, VEGF and endostatin were contained inside of the platelets. Both VEGF and endostatin were released from the platelets when they were activated by thrombin. However, only VEGF was released from the platelets when they were activated by PAR1, and only endostatin was released from the platelets when they were activated by PAR4. The rat meniscal cells grew much faster in the medium that contained PAR1-activated platelets than in the medium that contained either PAR4-activated platelets or unactivated platelets. The wounds treated with PAR1-activated platelets healed faster than those treated with either PAR4-activated platelets or unactivated platelets. Many blood vessel-like structures were found in the wounded menisci treated with PAR1-activated platelets. CONCLUSION: The PAR1-activated platelets released high levels of VEGF, which increased the proliferation of rat meniscal cells in vitro, enhanced the vascularization of menisci in vivo, and promoted healing of wounded menisci. CLINICAL RELEVANCE: Our results suggested that selective activated platelets can be used clinically to enhance healing of wounded meniscal tissue.


Assuntos
Plaquetas/metabolismo , Menisco/lesões , Neovascularização Fisiológica/fisiologia , Ativação Plaquetária/fisiologia , Animais , Endostatinas/metabolismo , Feminino , Humanos , Ratos , Ratos Nus , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Circ Res ; 126(4): 486-500, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31859592

RESUMO

RATIONALE: A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown. OBJECTIVE: In this study, we hypothesized that rivaroxaban's antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis. METHODS AND RESULTS: In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban's antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban's anticoagulatory capacity. CONCLUSIONS: Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.


Assuntos
Artérias/metabolismo , Plaquetas/efeitos dos fármacos , Fator Xa/farmacologia , Receptor PAR-1/agonistas , Rivaroxabana/farmacologia , Trombose/prevenção & controle , Animais , Artérias/patologia , Plaquetas/metabolismo , Inibidores do Fator Xa/farmacologia , Fibrinolíticos/administração & dosagem , Fibrinolíticos/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Receptor PAR-1/metabolismo , Rivaroxabana/administração & dosagem , Trombose/metabolismo
18.
J Surg Res ; 246: 568-583, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31653415

RESUMO

BACKGROUND: Coagulation disturbances in several liver diseases lead to thrombin generation, which triggers intracellular injury via activation of protease-activated receptor-1 (PAR-1). Little is known about the thrombin/PAR-1 pathway in hepatic ischemia-reperfusion injury (IRI). The present study aimed to clarify whether a newly selective PAR-1 antagonist, vorapaxar, can attenuate liver damage caused by hepatic IRI, with a focus on apoptosis and the survival-signaling pathway. METHODS: A 60-min hepatic partial-warm IRI model was used to evaluate PAR-1 expression in vivo. Subsequently, IRI mice were treated with or without vorapaxar (with vehicle). In addition, hepatic sinusoidal endothelial cells (SECs) pretreated with or without vorapaxar (with vehicle) were incubated during hypoxia-reoxygenation in vitro. RESULTS: In naïve livers, PAR-1 was confirmed by immunohistochemistry and immunofluorescence analysis to be located on hepatic SECs, and IRI strongly enhanced PAR-1 expression. In IRI mice models, vorapaxar treatment significantly decreased serum transaminase levels, improved liver histological damage, reduced the number of apoptotic cells as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining (median: 135 versus 25, P = 0.004), and induced extracellular signal-regulated kinase 1/2 (ERK 1/2) cell survival signaling (phospho-ERK/total ERK 1/2: 0.96 versus 5.34, P = 0.004). Pretreatment of SECs with vorapaxar significantly attenuated apoptosis and induced phosphorylation of ERK 1/2 in vitro (phospho-ERK/total ERK 1/2: 0.66 versus 3.04, P = 0.009). These changes were abolished by the addition of PD98059, the ERK 1/2 pathway inhibitor, before treatment with vorapaxar. CONCLUSIONS: The results of the present study revealed that hepatic IRI induces significant enhancement of PAR-1 expression on SECs, which may be associated with suppression of survival signaling pathways such as ERK 1/2, resulting in severe apoptosis-induced hepatic damage. Thus, the selective PAR-1 antagonist attenuates hepatic IRI through an antiapoptotic effect by the activation of survival-signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Lactonas/administração & dosagem , Fígado/irrigação sanguínea , Piridinas/administração & dosagem , Receptor PAR-1/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Receptor PAR-1/metabolismo , Traumatismo por Reperfusão/etiologia , Trombina/metabolismo
19.
Mediators Inflamm ; 2019: 4952131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31814803

RESUMO

Background: Thrombin could elicit degranulation of mast cells involved in numerous physiologic and pathologic processes; however, the detailed scrutiny of this procedure and further research of possible cell signaling pathways are lacking. Methods: P815 mouse mast cells were exposed to various concentrations of thrombin for 16 h. Expression of protease-activated receptor (PAR)1, PAR2, PAR3, and PAR4 mRNA in P815 was analyzed by quantitative real-time PCR (qRT-PCR) and the fittest concentration of thrombin was decided. Then, secretions of mediators from P815 stimulated by thrombin 0.2 U/ml were determined using enzyme-linked immunosorbent assay (ELISA) and Luminex liquichip; the possible cell signaling pathways were measured by immunoblotting. Furthermore, inhibition of thrombin inhibitor (hirudin), PAR1 inhibitor (SCH79797), and MAPK inhibitors (SB203580, PD98059, and SP600125) on the mediator section was evaluated by ELISA and Luminex liquichip. Results: Thrombin 0.2 U/ml induced the elevated expression of PAR1, PAR2, PAR3, and PAR4, as well as the increasing level of phospho-IκBα, phospho-SAPK/JNK MAPK, phospho-P38 MAPK (Thr180/Tyr182), and phospho-ERK1/2 MAPK (p44/42) in P815. Secretion of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), interleukin- (IL-) 2, IL-6, chemokine ligand- (CCL-) 2, chemokine (C-X-C motif) ligand- (CXCL-) 1, and CXCL-5 from P815 increased apparently; this effect could be diminished by hirudin, whereas SCH79797 and MAPK inhibitors (SB203580, PD98059, and SP600125) diminish the secretions with weaker effect. Conclusion: We found the expression of PAR mRNA in P815, activation of signaling pathways of nuclear factor-kappaB (NF-κB), and mitogen-activated protein kinases (MAPKs) including C-Jun NH2-terminal kinase (JNK), P38, and extracellular signal-regulated kinase 1/2 (ERK1/2), and the release of multiple inflammatory mediators stimulated by thrombin, as well as the inhibition of the inflammatory releases by hirudin, SCH79797, and MAPK inhibitors including SB203580, PD98059, and SP600125.


Assuntos
Citocinas/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Mol Vis ; 25: 546-558, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31673221

RESUMO

Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.


Assuntos
Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/citologia , Trocador de Sódio e Cálcio/metabolismo , Trombina/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Forma Celular/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Fragmentos de Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos Long-Evans , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trítio/metabolismo
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