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1.
Physiol Rev ; 103(1): 717-785, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-35901239

RESUMO

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.


Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Humanos , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G , Peptídeo Hidrolases/metabolismo , Homeostase
2.
Biomed Res Int ; 2022: 3865844, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246974

RESUMO

Matriptases are cell surface proteolytic enzymes belonging to the type II transmembrane serine protease family that mediate inflammatory skin disorders and cancer progression. Matriptases may affect the development of periodontitis via protease-activated receptor-2 activity. However, the cellular mechanism by which matriptases are involved in periodontitis is unknown. In this study, we examined the antiperiodontitis effects of matriptase on Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS)-stimulated human gingival fibroblasts (HGFs). Matriptase small interfering RNA-transfected HGFs were treated with PG-LPS. The mRNA and protein levels of proinflammatory cytokines and matrix metalloproteinase 1 (MMP-1) were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA), respectively. Western blot analyses were performed to measure the levels of Toll-like receptor 4 (TLR4)/interleukin-1 (IL-1) receptor-associated kinase (IRAK)/transforming growth factor ß-activated kinase 1 (TAK1), p65, and p50 in PG-LPS-stimulated HGFs. Matriptase downregulation inhibited LPS-induced proinflammatory cytokine expression, including the expression of IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-Iß. Moreover, matriptase downregulation inhibited PG-LPS-stimulated MMP-1 expression. Additionally, we confirmed that the mechanism underlying the effects of matriptase downregulation involves the suppression of PG-LPS-induced IRAK1/TAK1 and NF-κB. These results suggest that downregulation of matriptase PG-LPS-induced MMP-1 and proinflammatory cytokine expression via TLR4-mediated IRAK1/TAK1 and NF-κB signaling pathways in HGFs.


Assuntos
Fibroblastos , Metaloproteinase 1 da Matriz , Periodontite , Serina Endopeptidases , Citocinas/metabolismo , Regulação para Baixo , Fibroblastos/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/toxicidade , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , NF-kappa B/metabolismo , Periodontite/genética , Periodontite/metabolismo , Porphyromonas gingivalis , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Ativados por Proteinase/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
3.
FASEB J ; 36(10): e22564, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36165219

RESUMO

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.


Assuntos
Coagulantes , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Modelos Animais de Doenças , Fator VII , Fibrinogênio , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Histonas , Camundongos , Peptídeo Hidrolases , Receptores Ativados por Proteinase , Serina Endopeptidases/genética , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/genética
4.
Cell Mol Biol Lett ; 27(1): 77, 2022 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088291

RESUMO

Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.


Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais/fisiologia
5.
Front Immunol ; 13: 912748, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35844627

RESUMO

Candida albicans Sap6, a secreted aspartyl protease (Sap), contributes to fungal virulence in oral candidiasis. Beside its protease activity, Sap6 contains RGD (RGDRGD) motif required for its binding to host integrins. Sap6 activates immune cells to induce proinflammatory cytokines, although its ability to interact and activate human oral epithelial cells (OECs) remain unknown. Addition of purified recombinant Sap6 (rSap6) to OECs resulted in production of IL-1ß and IL-8 cytokines similar to live hyphal C. albicans. OECs exposed to rSap6 showed phosphorylation of p38 and MKP1 and expression of c-Fos not found with C. albicans Δsap6, heat-inactivated Sap6, or rSap6ΔRGD . Heat inactivated rSap6 was able to induce IL-1ß but not IL-8 in OECs, while rSap6ΔRGD induced IL-8 but not IL-1ß suggesting parallel signaling pathways. C. albicans hyphae increased surface expression of Protease Activated Receptors PAR1, PAR2 and PAR3, while rSap6 increased PAR2 expression exclusively. Pretreatment of OECs with a PAR2 antagonist blocked rSap6-induced p38 MAPK signaling and IL-8 release, while rSap6ΔRGD had reduced MKP1 signaling and IL-1ß release independent from PAR2. OECs exposed to rSap6 exhibited loss of barrier function as measured by TEER and reduction in levels of E-cadherin and occludin junctional proteins that was prevented by pretreating OECs with a PAR2 antagonist. OECs treated with PAR2 antagonist also showed reduced rSap6-mediated invasion by C. albicans cells. Thus, Sap6 may initiate OEC responses mediated both through protease activation of PAR2 and by its RGD domain. This novel role of PAR2 suggests new drug targets to block C. albicans oral infection.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Candida albicans , Proteínas Fúngicas/metabolismo , Receptor PAR-2/metabolismo , Estomatite/microbiologia , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Receptores Ativados por Proteinase/metabolismo
6.
Diabet Med ; 39(8): e14868, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35514270

RESUMO

BACKGROUND: Antiplatelet therapy is a cornerstone in the secondary prevention of ischemic events following percutaneous coronary intervention (PCI). The new P2Y12 receptor inhibitors prasugrel and ticagrelor have been shown to improve patients' outcomes. Whether or not these drugs have equal efficacy in individuals with or without diabetes is disputed. Furthermore, platelets can be activated by thrombin, which is, at least in part, independent of P2Y12 -mediated platelet activation. Protease-activated receptor (PAR)-1 and -4 are thrombin receptors on human platelets. We sought to compare the in vitro efficacy of prasugrel (n = 121) and ticagrelor (n = 99) to inhibit PAR-mediated platelet aggregation in individuals with type 2 diabetes (prasugrel n = 26, ticagrelor n = 29). MATERIALS AND METHODS: We compared P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation as assessed by multiple electrode platelet aggregometry between prasugrel- and ticagrelor-treated patients without and with type 2 diabetes who underwent acute PCI. RESULTS: Overall, there were no differences of P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation between prasugrel- and ticagrelor-treated patients. However, both drugs inhibited P2Y12 -mediated platelet aggregation stronger, and thereby to a similar extent in patients with type 2 diabetes than in those without diabetes. There was no correlation between either P2Y12 -, or PAR-1- or PAR-4-mediated platelet aggregation and levels of HbA1c or the body mass index (BMI). However, we observed patients with high residual platelet reactivity in response to PAR-1 and PAR-4 stimulation in all cohorts. CONCLUSION: Prasugrel and ticagrelor inhibit P2Y12 - and PAR-mediated platelet aggregation in individuals with diabetes to a similar extent, irrespective of HbA1c levels and BMI.


Assuntos
Síndrome Coronariana Aguda , Diabetes Mellitus Tipo 2 , Intervenção Coronária Percutânea , Síndrome Coronariana Aguda/terapia , Adenosina/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Cloridrato de Prasugrel/farmacologia , Cloridrato de Prasugrel/uso terapêutico , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Receptores Ativados por Proteinase , Ticagrelor/farmacologia , Ticagrelor/uso terapêutico
7.
J Med Chem ; 65(7): 5575-5592, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35349275

RESUMO

Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.


Assuntos
Infarto do Miocárdio , Trombose , Animais , Humanos , Lactonas , Infarto do Miocárdio/tratamento farmacológico , Inibidores da Agregação Plaquetária , Ratos , Receptor PAR-1 , Receptores Ativados por Proteinase , Trombose/induzido quimicamente , Trombose/tratamento farmacológico
8.
Blood Adv ; 6(11): 3494-3506, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35359002

RESUMO

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.


Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Trombose , ChAdOx1 nCoV-19 , Citometria de Fluxo , Heparina/uso terapêutico , Humanos , Imunoglobulinas Intravenosas/efeitos adversos , Fator Plaquetário 4 , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores Ativados por Proteinase/uso terapêutico , Trombocitopenia/diagnóstico , Trombose/tratamento farmacológico
9.
Am J Pathol ; 192(2): 361-378, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35144762

RESUMO

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.


Assuntos
Fatores de Coagulação Sanguínea/biossíntese , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptor da Anafilatoxina C5a/metabolismo , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais , Animais , Fatores de Coagulação Sanguínea/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Receptores Ativados por Proteinase/genética
10.
Int J Mol Sci ; 23(3)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35163205

RESUMO

Acute Respiratory Distress Syndrome is the most common cause of respiratory failure among critically ill patients, and its importance has been heightened during the COVID-19 pandemic. Even with the best supportive care, the mortality rate in the most severe cases is 40-50%, and the only pharmacological agent shown to be of possible benefit has been steroids. Mesenchymal stromal cells (MSCs) have been tested in several pre-clinical models of lung injury and been found to have significant therapeutic benefit related to: (a) potent immunomodulation; (b) secretion of epithelial and endothelial growth factors; and (c) augmentation of host defense to infection. Initial translational efforts have shown signs of promise, but the results have not yielded the anticipated outcomes. One potential reason is the relatively low survival of MSCs in inflammatory conditions as shown in several studies. Therefore, strategies to boost the survival of MSCs are needed to enhance their therapeutic effect. Protease-activated receptors (PARs) may represent one such possibility as they are G-protein coupled receptors expressed by MSCs and control several facets of cell behavior. This review summarizes some of the existing literature about PARs and MSCs and presents possible future areas of investigation in order to develop potential, PAR-modified MSCs with enhanced therapeutic efficiency.


Assuntos
Sobrevivência de Enxerto/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Receptores Ativados por Proteinase/fisiologia , Síndrome do Desconforto Respiratório/terapia , Animais , COVID-19/genética , COVID-19/patologia , COVID-19/terapia , Sobrevivência Celular/genética , Estado Terminal/terapia , Humanos , Células-Tronco Mesenquimais/fisiologia , Receptores Ativados por Proteinase/genética , Receptores Ativados por Proteinase/metabolismo , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2/fisiologia , Transdução de Sinais/fisiologia , Transfecção , Resultado do Tratamento
11.
Prostate ; 82(6): 723-739, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35167724

RESUMO

BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.


Assuntos
Neoplasias Ósseas , Neoplasias da Próstata , Receptor PAR-2/metabolismo , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Animais , Neoplasias Ósseas/secundário , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Neoplasias da Próstata/patologia , Receptores Ativados por Proteinase/metabolismo
12.
FEBS J ; 289(14): 4000-4020, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35175692

RESUMO

Inflammatory diseases have become increasingly prevalent throughout the world. Coronavirus disease 2019 (COVID-19), which has recently become pandemic, also exhibits hyperinflammation and cytokine release syndrome. To address inflammation-related diseases, numerous molecular targets have been explored in preclinical studies and clinical trials. Among them, the protease-activated receptors (PARs) that belong to G protein-coupled receptors are one of the most popular classes of drug targets, participating in inflammatory signalling and diseases. PARs activation can trigger downstream intracellular signalling to modulate a variety of inflammatory responses in multiple systems, including nervous, respiratory, digestive, circulatory, urinary and immune systems. Importantly, there are the Yin-Yang effects, comprising anti- and pro-inflammatory roles, of PARs activation in different types of inflammations, and these are context-dependent. Alternatively, it was recently revealed that PARs not only modulate inflammatory-related tumour progression, but also participate in inflammatory cytokine release related to COVID-19 via direct interaction with severe acute respiratory syndrome coronavirus 2 protein, suggesting that PARs also participate in other diseases via inflammatory responses. In this review, we renew and discuss the findings of PARs as molecular targets for treating inflammatory diseases, highlighting the novel roles of PARs and facilitating a better understanding of their designated values in the specific inflammatory environment.


Assuntos
Receptores Ativados por Proteinase , Humanos , Inflamação , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais/fisiologia
13.
Br J Pharmacol ; 179(10): 2086-2099, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34235728

RESUMO

Identifying drug targets mitigating vascular dysfunction, thrombo-inflammation and thromboembolic complications in COVID-19 is essential. COVID-19 coagulopathy differs from sepsis coagulopathy. Factors that drive severe lung pathology and coagulation abnormalities in COVID-19 are not understood. Protein-protein interaction studies indicate that the tagged viral bait protein ORF9c directly interacts with PAR2, which modulates host cell IFN and inflammatory cytokines. In addition to direct interaction of SARS-CoV-2 viral protein with PARs, we speculate that activation of PAR by proteases plays a role in COVID-19-induced hyperinflammation. In COVID-19-associated coagulopathy elevated levels of activated coagulation proteases may cleave PARs in association with TMPRSS2. PARs activation enhances the release of cytokines, chemokines and tissue factor expression to propagate IFN-dependent inflammation, leukocyte-endothelial interaction, vascular permeability and coagulation responses. This hypothesis, corroborated by in vitro findings and emerging clinical evidence, will focus targeted studies of PAR1/2 blockers as adjuvant drugs against cytokine release syndrome and COVID-19-associated coagulopathy. LINKED ARTICLES: This article is part of a themed issue on The second wave: are we any closer to efficacious pharmacotherapy for COVID 19? (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.10/issuetoc.


Assuntos
Síndrome da Liberação de Citocina/tratamento farmacológico , Humanos , Inflamação , Receptores Ativados por Proteinase , SARS-CoV-2
14.
Thromb Haemost ; 122(6): 961-973, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34619794

RESUMO

The Ig-ITIM bearing receptors, PECAM-1 and CEACAM1, have been shown net negative regulators of platelet-collagen interactions and hemiITAM signaling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2, protease activated receptor (PAR4), ADP purinergic receptors, and thromboxane receptor (TP) A2 pathways. In addition, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and the TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared with hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen-related peptide or rhodocytin. In contrast, DKO platelets showed increased amounts of P-selectin exposure upon stimulation with PAR4 agonist peptide or thrombin but not Pecam-1-/- , Ceacam1-/- , or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced α-granule release via PAR4/Gαq/PLC signaling without crosstalk with Src/Syk or G12/13 signaling pathways. Severely delayed clot retraction in vitro was observed in DKO phenotype. The DKO model revealed a significant increase in thrombus formation compared with the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared with Pecam-1-/- , Ceacam1-/- , and WT platelets. This study demonstrates that PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signaling, platelet-collagen interactions, and PAR4 Gαq protein- coupled signaling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.


Assuntos
Selectina-P , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Trombose , Difosfato de Adenosina/metabolismo , Animais , Antígenos CD , Plaquetas/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular , Colágeno/metabolismo , Camundongos , Selectina-P/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Receptores Ativados por Proteinase/metabolismo , Receptores Purinérgicos/metabolismo , Trombose/genética , Trombose/metabolismo
15.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639054

RESUMO

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.


Assuntos
Colite/metabolismo , Peptídeos/metabolismo , Receptores Ativados por Proteinase/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores , Colite/etiologia , Colite/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Masculino , Peptídeos/química , Proteólise , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Cells ; 10(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34359930

RESUMO

Chronic spontaneous urticaria (CSU) is a common skin disorder characterized by an almost daily recurrence of wheal and flare with itch for more than 6 weeks, in association with the release of stored inflammatory mediators, such as histamine, from skin mast cells and/or peripheral basophils. The involvement of the extrinsic coagulation cascade triggered by tissue factor (TF) and complement factors, such as C3a and C5a, has been implied in the pathogenesis of CSU. However, it has been unclear how the TF-triggered coagulation pathway and complement factors induce the activation of skin mast cells and peripheral basophils in patients with CSU. In this review, we focus on the role of vascular endothelial cells, leukocytes, extrinsic coagulation factors and complement components on TF-induced activation of skin mast cells and peripheral basophils followed by the edema formation clinically recognized as urticaria. These findings suggest that medications targeting activated coagulation factors and/or complement components may represent new and effective treatments for patients with severe and refractory CSU.


Assuntos
Coagulação Sanguínea , Urticária Crônica/sangue , Urticária Crônica/patologia , Proteínas do Sistema Complemento/metabolismo , Mastócitos/patologia , Animais , Humanos , Leucócitos/metabolismo , Receptores Ativados por Proteinase/metabolismo
17.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445691

RESUMO

The essential role of G-protein coupled receptors (GPCRs) in tumor growth is recognized, yet a GPCR based drug in cancer is rare. Understanding the molecular path of a tumor driver gene may lead to the design and development of an effective drug. For example, in members of protease-activated receptor (PAR) family (e.g., PAR1 and PAR2), a novel PH-binding motif is allocated as critical for tumor growth. Animal models have indicated the generation of large tumors in the presence of PAR1 or PAR2 oncogenes. These tumors showed effective inhibition when the PH-binding motif was either modified or were inhibited by a specific inhibitor targeted to the PH-binding motif. In the second part of the review we discuss several aspects of some cardinal GPCRs in tumor angiogenesis.


Assuntos
Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Ativados por Proteinase/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Domínios de Homologia à Plecstrina/genética , Domínios de Homologia à Plecstrina/fisiologia , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores Ativados por Proteinase/genética , Transdução de Sinais/fisiologia
18.
Sci Rep ; 11(1): 14264, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253819

RESUMO

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.


Assuntos
Quimiocina CXCL10/metabolismo , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/metabolismo , Fibroblastos/metabolismo , Miocardite/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Ativados por Proteinase/metabolismo , Animais , Autofagia , Linhagem Celular , Deleção de Genes , Humanos , Imunidade Inata , Inflamação , Mediadores da Inflamação , Macrófagos/imunologia , Masculino , Camundongos , Miocárdio/imunologia , Ratos , Trombina/metabolismo , Replicação Viral
19.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063076

RESUMO

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11-18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.


Assuntos
Envelhecimento/fisiologia , Plaquetas/metabolismo , Colágeno/farmacologia , Trombospondina 1/farmacologia , Difosfato de Adenosina/farmacologia , Adolescente , Adulto , Plaquetas/efeitos dos fármacos , Criança , Venenos de Crotalídeos/farmacologia , Exocitose/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Lectinas Tipo C , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Receptores Ativados por Proteinase/metabolismo , Trombospondina 1/química
20.
J Diabetes Investig ; 12(12): 2172-2182, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34043882

RESUMO

AIMS/INTRODUCTION: Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs). Recent data have shown that PARs influence the development of glomerular diseases including diabetic kidney disease (DKD) by regulating inflammation. Heparin cofactor II (HCII) specifically inactivates thrombin; thus, we hypothesized that low plasma HCII activity correlates with DKD development, as represented by albuminuria. MATERIALS AND METHODS: Plasma HCII activity and spot urine biomarkers, including albumin and liver-type fatty acid-binding protein (L-FABP), were determined as the urine albumin-to-creatinine ratio (uACR) and the urine L-FABP-to-creatinine ratio (uL-FABPCR) in 310 Japanese patients with diabetes mellitus (176 males and 134 females). The relationships between plasma HCII activities and those DKD urine biomarkers were statistically evaluated. In addition, the relationship between plasma HCII activities and annual uACR changes was statistically evaluated for 201/310 patients (115 males and 86 females). RESULTS: The mean plasma HCII activity of all participants was 93.8 ± 17.7%. Multivariate-regression analysis including confounding factors showed that plasma HCII activity independently contributed to the suppression of the uACR and log-transformed uACR values (P = 0.036 and P = 0.006, respectively) but not uL-FABPCR (P = 0.541). In addition, plasma HCII activity significantly and inversely correlated with annual uACR and log-transformed uACR increments after adjusting for confounding factors (P = 0.001 and P = 0.014, respectively). CONCLUSIONS: The plasma HCII activity was inversely and specifically associated with glomerular injury in patients with diabetes. The results suggest that HCII can serve as a novel predictive factor for early-stage DKD development, as represented by albuminuria.


Assuntos
Albuminúria/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/urina , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/urina , Cofator II da Heparina/análise , Adulto , Idoso , Albuminas/metabolismo , Albuminúria/urina , Biomarcadores/sangue , Biomarcadores/urina , Creatinina/urina , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etiologia , Proteínas de Ligação a Ácido Graxo/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Ativados por Proteinase/sangue , Análise de Regressão , Trombina/metabolismo
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