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1.
Med Hypotheses ; 143: 110150, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763660

RESUMO

COVID-19 due to the SARS-CoV-2 infection is a multi-systemic immune syndrome affecting mainly the lungs, oropharyngeal region, and other vascular endothelial beds. There are tremendous ongoing efforts for the aim of developing drugs against the COVID-19 syndrome-associated inflammation. However, currently no specific medicine is present for the absolute pharmacological cure of COVID-19 mucositis. The re-purposing/re-positioning of already existing drugs is a very important strategy for the management of ongoing pandemy since the development of a new drug needs decades. Apart from altering angiotensin signaling pathways, novel drug candidates for re-purposing comprise medications shall target COVID-19 pathobiology, including pharmaceutical formulations that antagonize proteinase-activated receptors (PARs), mainly PAR-1. Activation of the PAR-1, mediators and hormones impact on the hemostasis, endothelial activation, alveolar epithelial cells and mucosal inflammatory responses which are the essentials of the COVID-19 pathophysiology. In this context, Ankaferd hemostat (Ankaferd Blood Stopper, ABS) which is an already approved hemostatic agent affecting via vital erythroid aggregation and fibrinogen gamma could be a potential topical remedy for the mucosal management of COVID-19. ABS is a clinically safe and effective topical hemostatic agent of plant origin capable of exerting pleiotropic effects on the endothelial cells, angiogenesis, cell proliferation and vascular dynamics. ABS had been approved as a topically applied hemostatic agent for the management of post-surgical/dental bleedings and healing of infected inflammatory mucosal wounds. The anti-inflammatory and proteinase-activated receptor axis properties of ABS with a considerable amount of oestrogenic hormone presence highlight this unique topical hemostatic drug regarding the clinical re-positioning for COVID-19-associated mucositis. Topical ABS as a biological response modifier may lessen SARS-CoV-2 associated microthrombosis, endothelial dysfunction, oropharyngeal inflammation and mucosal lung damage. Moreover, PAR-1 inhibition ability of ABS might be helpful for reducing the initial virus propagation and mocasal spread of COVID-19.


Assuntos
Anti-Inflamatórios/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/complicações , Estrogênios/fisiologia , Hemostáticos/uso terapêutico , Mucosite/tratamento farmacológico , Pandemias , Fitoestrógenos/uso terapêutico , Fitoterapia , Extratos Vegetais/uso terapêutico , Pneumonia Viral/complicações , Receptor PAR-1/antagonistas & inibidores , Administração Tópica , Distribuição por Idade , Anti-Inflamatórios/administração & dosagem , Infecções por Coronavirus/sangue , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/fisiopatologia , Reposicionamento de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Estrogênios/agonistas , Hemostáticos/administração & dosagem , Humanos , Mucosite/etiologia , Fitoestrógenos/administração & dosagem , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Pneumonia Viral/sangue , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , Receptor PAR-1/fisiologia , Estomatite/tratamento farmacológico , Estomatite/etiologia , Trombofilia/sangue , Trombofilia/etiologia
2.
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
3.
Arterioscler Thromb Vasc Biol ; 40(8): 1891-1904, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32493172

RESUMO

OBJECTIVE: Platelets are critical to the formation of a hemostatic plug and the pathogenesis of atherothrombosis. Preclinical animal models, especially the mouse, provide an important platform to assess the efficacy and safety of antiplatelet drugs. However, these studies are limited by inherent differences between human and mouse platelets and the species-selectivity of many drugs. To circumvent these limitations, we developed a new protocol for the adoptive transfer of human platelets into thrombocytopenic nonobese diabetic/severe combined immune deficiency mice, that is, a model where all endogenous platelets are replaced by human platelets in mice accepting xenogeneic tissues. Approach and Results: To demonstrate the power of this new model, we visualized and quantified hemostatic plug formation and stability by intravital spinning disk confocal microscopy following laser ablation injury to the saphenous vein. Integrin αIIbß3-dependent hemostatic platelet plug formation was achieved within ≈30 seconds after laser ablation injury in humanized platelet mice. Pretreatment of mice with standard dual antiplatelet therapy (Aspirin+Ticagrelor) or PAR1 inhibitor, L-003959712 (an analog of vorapaxar), mildly prolonged the bleeding time and significantly reduced platelet adhesion to the site of injury. Consistent with findings from clinical trials, inhibition of PAR1 in combination with dual antiplatelet therapy markedly prolonged bleeding time in humanized platelet mice. CONCLUSIONS: We propose that this novel mouse model will provide a robust platform to test and predict the safety and efficacy of experimental antiplatelet drugs and to characterize the hemostatic function of synthetic, stored and patient platelets.


Assuntos
Plaquetas/fisiologia , Hemostasia/efeitos dos fármacos , Transferência Adotiva , Animais , Benzofuranos/farmacologia , Carbamatos/farmacologia , Terapia Antiplaquetária Dupla/efeitos adversos , Humanos , Masculino , Camundongos , Modelos Animais , Receptor PAR-1/antagonistas & inibidores
4.
Arterioscler Thromb Vasc Biol ; 40(8): 1905-1917, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32580633

RESUMO

OBJECTIVE: Remodeling of the extracellular matrix plays a vital role in cardiovascular diseases. Using a mouse model of postnatal ascending aortic aneurysms (termed Fbln4SMKO), we have reported that abnormal mechanosensing led to aneurysm formation in Fbln4SMKO with an upregulation of the mechanosensitive transcription factor, Egr1 (Early growth response 1). However, the role of Egr1 and its upstream regulator(s) in the initiation of aneurysm development and their relationship to an aneurysmal microenvironment are unknown. Approach and Results: To investigate the contribution of Egr1 in the aneurysm development, we deleted Egr1 in Fbln4SMKO mice and generated double knockout mice (DKO, Fbln4SMKO; Egr1-/-). Aneurysms were prevented in DKO mice (42.8%) and Fbln4SMKO; Egr1+/- mice (26%). Ingenuity Pathway Analysis identified PAR1 (protease-activated receptor 1) as a potential Egr1 upstream gene. Protein and transcript levels of PAR1 were highly increased in Fbln4SMKO aortas at postnatal day 1 before aneurysm formed, together with active thrombin and MMP (matrix metalloproteinase)-9, both of which serve as a PAR1 activator. Concordantly, protein levels of PAR1, Egr1, and thrombin were significantly increased in human thoracic aortic aneurysms. In vitro cyclic stretch assays (1.0 Hz, 20% strain, 8 hours) using mouse primary vascular smooth muscle cells induced marked expression of PAR1 and secretion of prothrombin in response to mechanical stretch. Thrombin was sufficient to induce Egr1 expression in a PAR1-dependent manner. CONCLUSIONS: We propose that thrombin, MMP-9, and mechanical stimuli in the Fbln4SMKO aorta activate PAR1, leading to the upregulation of Egr1 and initiation of ascending aortic aneurysms.


Assuntos
Aneurisma da Aorta Torácica/etiologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Receptor PAR-1/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas da Matriz Extracelular/deficiência , Feminino , Humanos , Masculino , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Pessoa de Meia-Idade , Receptor PAR-1/antagonistas & inibidores , Estresse Mecânico , Trombina/farmacologia
5.
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
6.
Breast Cancer Res Treat ; 180(2): 379-384, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32034579

RESUMO

PURPOSE: Protease-activated receptor 1 (PAR1) is a signaling protein ubiquitously present on the surface of tumor cells, and its homologous protein fragment, PAR1-activating peptide (P1AP), can inhibit protein signal transduction of PAR1/G in tumor cells. pH (Low) insertion peptide (pHLIP) can target the acidic tumor microenvironment (TME) and can be used as an excellent carrier to deliver P1AP to tumor cells for therapeutic purposes. METHODS: PAR1 expression on the surface of MDA-MB-231 cells and human MCF10A mammary epithelial cells was observed. The binding between fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells under different pH values was analyzed. The effect of pHLIP(Var7)-P1AP on the proliferation of MDA-MB-231 cells was analyzed under the conditions of pH 7.4 and 6.0. RESULTS: PAR1 was highly expressed on the surface of MDA-MB-231 cells. In an acidic environment (pH 6.0 and 5.0), fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells had a high binding ability, and the binding ability increased with the decrease in pH. In an acidic environment (pH 6.0), pHLIP(Var7)-P1AP significantly inhibited MDA-MB-231 cell proliferation. With 0.5 µg, 1 µg, 2 µg, 4 µg, and 8 µg of pHLIP(Var7)-P1AP, the cell proliferation inhibition rates were 3.39%, 5.27%, 14.29%, 22.14%, and 35.69%, respectively. CONCLUSION: PAR1 was highly expressed on the surface of MDA-MB-231 cells. pHLIP(Var7)-P1AP can effectively target MDA-MB-231 cells in an acidic environment and inhibit the growth of MDA-MB-231 cells by inhibiting the signal transduction of PAR1/G protein.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas de Membrana/farmacologia , Oligopeptídeos/farmacologia , Receptor PAR-1/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Concentração de Íons de Hidrogênio , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/metabolismo , Microambiente Tumoral
7.
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
8.
J Neurosci ; 40(7): 1483-1500, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31911460

RESUMO

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knock-out of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knock-out in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knock-out mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.SIGNIFICANCE STATEMENT Replacement of oligodendroglia and myelin regeneration holds tremendous potential to improve function across neurological conditions. Here we demonstrate Protease Activated Receptor 1 (PAR1) is an important regulator of the capacity for myelin regeneration across two experimental murine models of myelin injury. PAR1 is a G-protein-coupled receptor densely expressed in the CNS, however there is limited information regarding its physiological roles in health and disease. Using a combination of PAR1 knock-out mice, oligodendrocyte monocultures and oligodendrocyte-astrocyte cocultures, we demonstrate blocking PAR1 improves myelin production by a mechanism related to effects across glial compartments and linked in part to regulatory actions toward growth factors such as BDNF. These findings set the stage for development of new clinically relevant myelin regeneration strategies.


Assuntos
Doenças Desmielinizantes/fisiopatologia , Regeneração Nervosa/efeitos dos fármacos , Receptor PAR-1/antagonistas & inibidores , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Axônios/patologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Quelantes/toxicidade , Técnicas de Cocultura , Cobre , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/patologia , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Perfilação da Expressão Gênica , Lisofosfatidilcolinas/toxicidade , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/fisiologia , Regeneração Nervosa/fisiologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Receptor PAR-1/deficiência , Receptor PAR-1/fisiologia , Teste de Desempenho do Rota-Rod , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Substância Branca/efeitos dos fármacos , Substância Branca/patologia
9.
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
10.
J Vasc Access ; 21(4): 467-474, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31774037

RESUMO

BACKGROUND: Protease-activated receptor-1 antagonism by vorapaxar could facilitate arteriovenous fistula maturation but may increase bleeding risk. OBJECTIVE: The primary objective of the Vorapaxar Study for Maturation of arteriovenous fistula for Hemodialysis Access (VorapAccess) was to determine if vorapaxar improves arteriovenous fistula functional maturation in patients with end-stage renal disease. METHODS: VorapAccess was a randomized, placebo-controlled, double-blind pilot trial comparing 2.5 mg vorapaxar per day with placebo for twelve weeks starting on day two after arteriovenous fistula creation. The primary outcome was time to functional maturation defined as successful cannulation for six hemodialysis sessions within three weeks. The planned sample size was 50 participants. The study was terminated early after withdrawal of planned financial support. Given the small number of randomized patients, we performed descriptive analyses without inference testing. RESULTS: A total of 13 participants were randomly allocated study drug (six vorapaxar and seven placebo). The median age was 56 years and seven participants (54%) were female. The median (minimum-maximum) days to functional maturation were 169 (77-287) days in the vorapaxar group and 145 (48-198) days in the placebo group. Six of the 13 (46%) participants had arteriovenous fistula functional maturation within 180 days; two of six (33%) in the vorapaxar group and four of seven (57%) in the placebo group. There was one bleeding event in the placebo group. CONCLUSION: Fewer than half of participants had functional maturation within 180 days after surgery, suggesting a major need for agents or strategies that enhance arteriovenous fistula maturation.


Assuntos
Derivação Arteriovenosa Cirúrgica , Falência Renal Crônica/terapia , Lactonas/uso terapêutico , Inibidores da Agregação de Plaquetas/uso terapêutico , Piridinas/uso terapêutico , Diálise Renal , Extremidade Superior/irrigação sanguínea , Idoso , Derivação Arteriovenosa Cirúrgica/efeitos adversos , California , Método Duplo-Cego , Término Precoce de Ensaios Clínicos , Feminino , Hemorragia/induzido quimicamente , Humanos , Falência Renal Crônica/diagnóstico , Lactonas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Inibidores da Agregação de Plaquetas/efeitos adversos , Piridinas/efeitos adversos , Receptor PAR-1/antagonistas & inibidores , Diálise Renal/efeitos adversos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
11.
Chin J Nat Med ; 17(8): 591-599, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472896

RESUMO

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.


Assuntos
Sanguessugas/química , Receptor PAR-1/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Perfilação da Expressão Gênica , Sanguessugas/genética , Sanguessugas/metabolismo , Camundongos Endogâmicos ICR , Modelos Moleculares , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/química , Inibidores da Agregação de Plaquetas/isolamento & purificação , Inibidores da Agregação de Plaquetas/farmacologia , Inibidores da Agregação de Plaquetas/uso terapêutico , Proteômica , Ratos Sprague-Dawley , Glândulas Salivares/química , Glândulas Salivares/metabolismo , Trombose/prevenção & controle
12.
Biomed Res Int ; 2019: 2187306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467874

RESUMO

Thrombin plays a pivotal role in the pathogenesis of atherosclerosis. Baicalin, an active flavonoid compound, was shown to attenuate the development of atherosclerosis, but the mechanism remains elusive. In the present study, the role and mechanism of baicalin in thrombin-induced cell injury was investigated in human umbilical vein endothelial cells (HUVECs). Our results showed that baicalin significantly reduced thrombin-induced apoptosis of HUVECs. Additional experiments showed that baicalin inhibited thrombin-induced NF-κB activation and PAR-1 expression. In addition, baicalin decreased thrombin-induced PAR-1 expression by inhibiting ERK pathway. These results indicated that baicalin has protective effects on thrombin-induced cell injury in HUVECs possibly through inhibition of PAR-1 expression and its downstream NF-κB activation, which was mediated by ERK1/2 activation.


Assuntos
Aterosclerose/tratamento farmacológico , Flavonoides/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Receptor PAR-1/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/genética , Substâncias Protetoras/farmacologia , Receptor PAR-1/genética , Trombina/toxicidade
13.
Structure ; 27(10): 1517-1526.e3, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31422910

RESUMO

G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states.


Assuntos
Guanidinas/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptor PAR-1/química , Receptor PAR-1/metabolismo , Sítios de Ligação , Guanidinas/química , Humanos , Ligantes , Modelos Moleculares , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inibidores , Imagem Individual de Molécula
14.
Thromb Haemost ; 119(9): 1394-1402, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31291665

RESUMO

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16- monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16- monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16- monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16- monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endotoxemia/metabolismo , Inflamação/metabolismo , Monócitos/imunologia , Receptor PAR-1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Voluntários Saudáveis , Humanos , Lactonas/administração & dosagem , Lactonas/farmacologia , Lipopolissacarídeos/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/genética , Receptores de IgG/metabolismo , Tromboplastina/metabolismo , Transcriptoma , Regulação para Cima
15.
FASEB J ; 33(10): 10966-10972, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31287960

RESUMO

Vorapaxar-dependent protease-activated receptor (PAR)-1 inhibition diminishes diabetic nephropathy in experimental type 1 diabetes. As most patients with diabetic nephropathy suffer from type 2 diabetes, the aim of this study was to investigate whether PAR-1 inhibition also limits diabetic nephropathy in experimental type 2 diabetes. Consequently, leptin-deficient black and tan brachyuric (BTBRob/ob) mice were randomly assigned to vorapaxar (1.75 mg/kg; twice weekly via oral gavage) or vehicle treatment, whereas matched wild-type (WT) BTBR (BTBRWT) mice served as nondiabetic controls. Weight and (nonfasting) blood glucose levels were monitored for up to 18 wk, after which kidney function and histologic damage was evaluated postmortem. We show that blood glucose levels and body weight increased in diabetic BTBRob/ob mice compared with nondiabetic BTBRWT controls. Vorapaxar-dependent PAR-1 inhibition reduced but did not normalize blood glucose levels in BTBRob/ob mice, whereas it potentiated the increase in body weight. Vorapaxar did not, however, preserve kidney function, whereas it only minimally reduced histopathological signs of kidney injury. Overall, we thus show that PAR-1 inhibition reduces blood glucose levels during the progression of diabetic nephropathy in experimental type 2 diabetes but does not improve renal function. This is in contrast to the therapeutic potential of vorapaxar in type 1 diabetes-induced nephropathy, highlighting the importance of disease-dependent treatment modalities.-Waasdorp, M., Florquin, S., Duitman, J., Spek, C. A. Pharmacological PAR-1 inhibition reduces blood glucose levels but does not improve kidney function in experimental type 2 diabetic nephropathy.


Assuntos
Glicemia/metabolismo , Nefropatias Diabéticas/tratamento farmacológico , Rim/efeitos dos fármacos , Lactonas/uso terapêutico , Piridinas/uso terapêutico , Receptor PAR-1/antagonistas & inibidores , Animais , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/metabolismo , Feminino , Rim/fisiopatologia , Lactonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/farmacologia , Receptores para Leptina/deficiência , Receptores para Leptina/genética
16.
Bioorg Med Chem ; 27(17): 3788-3796, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31320211

RESUMO

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Oxazóis/farmacologia , Receptor PAR-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/química , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Humanos , Ligantes , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Agregação Plaquetária/efeitos dos fármacos , Receptor PAR-1/metabolismo , Relação Estrutura-Atividade
17.
Eur J Oral Sci ; 127(4): 287-293, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175838

RESUMO

Arginine-specific cysteine proteinases, such as Arg-gingipain B (RgpB), mediate inflammation by activating protease-activated receptors (PARs). Arg-gingipain B is produced by Porphyromonas gingivalis, and is implicated in the causation of periodontal disease. The purpose of the present study was to observe the influence of recombinant RgpB protein (rRgpB) on PAR activation by monitoring intracellular Ca2+ ion concentration ([Ca2+]i) and inositol-1,4,5-triphosphate (IP3) levels in human gingival fibroblasts (HGFs). Our findings showed that rRgpB could cause a transient increase in [Ca2+]i. This increase in [Ca2+]i was completely suppressed by vorapaxar, a PAR-1 antagonist. Recombinant Arg-gingipain B increased the concentration of IP3, reaching a maximum at 60 s after treatment; this was completely inhibited by vorapaxar. We therefore conclude that rRgpB-induced calcium signaling in HGFs is mainly caused by PAR-1 activation. This suggests that PAR-1 activation plays a significant role in chronic inflammatory periodontal disease induced by P. gingivalis RgpB.


Assuntos
Sinalização do Cálcio , Fibroblastos/metabolismo , Cisteína Endopeptidases Gingipaínas/farmacologia , Porphyromonas gingivalis/enzimologia , Receptor PAR-1/metabolismo , Proteínas de Bactérias/farmacologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Inositol 1,4,5-Trifosfato , Lactonas/farmacologia , Piridinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Proteínas Recombinantes/farmacologia
18.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31197089

RESUMO

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.


Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptor PAR-1/metabolismo , Transdução de Sinais , Trombina/farmacologia , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
19.
Neurosci Lett ; 692: 64-68, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30391321

RESUMO

The blood-brain barrier (BBB) is a unique structure that controls substances exchange between the systemic circulation and the brain. Disruption of its integrity contributes to the development and progression of a variety of brain disorders including stroke, epilepsy and neurodegenerative diseases. It was shown that intracerebral thrombin level substantially increases following status epilepticus (SE). Inhibition of protease-activated receptor 1 (PAR1), the major thrombin receptor in the brain, produces an anti-epileptogenic and neuroprotective effects in an experimental model of temporal lobe epilepsy (TLE). Since serine proteases and PAR1 are implicated in the synaptic plasticity and memory formation, the aim of the present study was to elucidate the involvement of PAR1 in synaptic plasticity and behavior deficits following SE. Using lithium-pilocarpine model of TLE, we demonstrate that inhibition of PAR1 rescues SE-induced synaptic plasticity deficits in CA1 region of hippocampus. Although treatment with PAR1 antagonist does not ameliorate spatial learning deficits, it attenuates anxiolytic-like behavior in experimental rats after SE. Taken together; our data suggest an important role of PAR1 in SE-induced synaptic and behavioral alterations and provide a new insight into cellular mechanisms underlying behavioral impairments associated with epilepsy.


Assuntos
Região CA1 Hipocampal/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Potenciação de Longa Duração , Receptor PAR-1/antagonistas & inibidores , Estado Epiléptico/fisiopatologia , Animais , Comportamento Animal/efeitos dos fármacos , Região CA1 Hipocampal/efeitos dos fármacos , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Lítio/administração & dosagem , Masculino , Pilocarpina/administração & dosagem , Pirróis/administração & dosagem , Quinazolinas/administração & dosagem , Ratos Wistar , Estado Epiléptico/induzido quimicamente
20.
Apoptosis ; 23(11-12): 679-694, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30196356

RESUMO

A novel activating peptide was designed and synthesized from V. cholerae hemagglutinine protease (HAP) mediated cleavage site of mouse PAR1. The peptide "PFISED" interacts with PAR1 in a new site which is different from its thrombin mediated conventional activation site and induced a series of new downstream signaling pathways. The peptide showed apoptosis in human and mouse breast (MCF-7 and EAC) and colon (HT29 and CT26) cancer cells where as in the same peptide concentration in normal human breast epithelial cells (MCF-10A), normal human fibroblast cells (MRC-5), normal mouse peritoneal macrophage cells and normal mouse breast and colon tissues did not show any effect. Treatment with this peptide enhanced the survival kinetics of EAC induced mice. The peptide mediated apoptosis was inhibited in presence of PAR1 inhibitor and was significantly reduced in si-PAR1 treated cells that indicate the activating peptide "PFISED" induced PAR1 mediated apoptosis of colon and breast cancer cells. This peptide induced over expression and activation of PAR1 and its downstream MAP kinase and NFκB signaling pathways. These signaling pathways enhanced the cellular ROS level to kill malignant cells. We report a novel pro-apoptotic peptide which can selectively kill malignant cells via its specific target receptor PAR1 which is over expressed in the malignant cells and can be used as a molecular target therapy for cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Peptídeos/farmacologia , Receptor PAR-1/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Células MCF-7 , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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