Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.823
Filtrar
1.
Tohoku J Exp Med ; 255(1): 1-8, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34511578

RESUMO

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.


Assuntos
Nefropatias Diabéticas/etiologia , Óxido Nítrico Sintase Tipo III/deficiência , Receptores Ativados por Proteinase/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Coagulação Sanguínea , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Inibidores do Fator Xa/farmacologia , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Receptores Ativados por Proteinase/deficiência , Receptores Ativados por Proteinase/genética , Transdução de Sinais , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502228

RESUMO

Extracellular vesicles (EVs) compose a heterogenous group of membrane-derived particles, including exosomes, microvesicles and apoptotic bodies, which are released into the extracellular environment in response to proinflammatory or proapoptotic stimuli. From earlier studies suggesting that EV shedding constitutes a cellular clearance mechanism, it has become evident that EV formation, secretion and uptake represent important mechanisms of intercellular communication and exchange of a wide variety of molecules, with relevance in both physiological and pathological situations. The putative role of EVs in hemostasis and thrombosis is supported by clinical and experimental studies unraveling how these cell-derived structures affect clot formation (and resolution). From those studies, it has become clear that the prothrombotic effects of EVs are not restricted to the exposure of tissue factor (TF) and phosphatidylserines (PS), but also involve multiplication of procoagulant surfaces, cross-linking of different cellular players at the site of injury and transfer of activation signals to other cell types. Here, we summarize the existing and novel clinical and experimental evidence on the role and function of EVs during arterial and venous thrombus formation and how they may be used as biomarkers as well as therapeutic vectors.


Assuntos
Biomarcadores/metabolismo , Comunicação Celular , Vesículas Extracelulares/metabolismo , Tromboplastina/metabolismo , Trombose/patologia , Animais , Humanos , Trombose/metabolismo
3.
Biomed Pharmacother ; 139: 111569, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243622

RESUMO

BACKGROUND: Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PURPOSE: The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. METHODS: 50 µl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 µg/kg, 10 µg/kg, 50 µg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 µg/kg, 10 µg/kg, 50 µg/kg) were intraperitoneally injected or 10 µg/50 µl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. RESULTS: LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/ß and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. CONCLUSIONS: TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.


Assuntos
Diterpenos/farmacologia , Fibrinólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , NF-kappa B/metabolismo , Fenantrenos/farmacologia , Trombofilia/induzido quimicamente , Trombofilia/tratamento farmacológico , Animais , Modelos Animais de Doenças , Compostos de Epóxi/farmacologia , Pulmão/metabolismo , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Síndrome do Desconforto Respiratório , Transdução de Sinais/efeitos dos fármacos , Trombofilia/metabolismo , Tromboplastina/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205482

RESUMO

Tissue factor (TF) plays an important role in the progression and angiogenesis of tumor cells. The present study investigated the mechanism of interleukin-1ß (IL-1ß)-induced TF expression in A549 lung cancer cells. Based on mRNA and protein analyses, including appropriate inhibitor experiments, IL-1ß was shown to induce TF expression in a time-dependent manner, mediated by IL-1 receptor-dependent phosphorylation of the mitogen-activated protein kinases (MAPK) p38, p42/44 and c-jun N-terminal kinase (JNK), as well as the Src kinase and the epidermal growth factor receptor (EGFR). Thereby, inhibition of EGFR transactivation by the Src inhibitor PP1 or direct EGFR inhibition by the EGFR tyrosine kinase inhibitor (TKI) erlotinib led to a reduction of IL-1ß-induced TF expression and to a suppression of p42/44 MAPK and EGFR activation, while IL-1ß-induced p38 MAPK and JNK activation remained unchanged. A knockdown of EGFR by siRNA was associated with decreased IL-1ß-mediated p42/44 MAPK activation, which was no longer inhibitable by erlotinib. Concentration-dependent inhibition of IL-1ß-induced TF expression was also observed in the presence of gefitinib and afatinib, two other EGFR TKIs. In summary, our results suggest that IL-1ß leads to increased TF formation in lung cancer cells via both Src/EGFR/p42/44 MAPK-dependent and EGFR-independent signaling pathways, with the latter mediated via p38 MAPK and JNK.


Assuntos
Interleucina-1beta/metabolismo , Tromboplastina/metabolismo , Células A549 , Receptores ErbB/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases
5.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299151

RESUMO

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Túbulos Renais/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais/efeitos dos fármacos , Receptor PAR-2/genética , Edulcorantes/farmacologia
6.
J Intern Med ; 290(3): 677-692, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34080738

RESUMO

BACKGROUND: Prognostic markers for disease severity and identification of therapeutic targets in COVID-19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID-19 patients with different disease severity at admission and longitudinally during hospitalization. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS-CoV-2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS-CoV-2-negative bacterial sepsis (n = 5) and healthy controls (n = 10). RESULTS: COVID-19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA-DR) and exhaustion (PD-1) markers on T cells were increased compared with controls, but comparable between COVID-19 severity groups. Non-classical monocytes and monocytic HLA-DR expression decreased whereas monocytic PD-L1 and CD142 expression increased with COVID-19 severity. RNA sequencing exhibited increased plasma B-cell activity in critical COVID-19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease. CONCLUSION: Critical COVID-19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T-cell immune response. Plasma B-cell activity and calprotectin were higher in critical COVID-19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID-19 should be further explored.


Assuntos
COVID-19/genética , COVID-19/imunologia , Leucócitos Mononucleares/imunologia , Transcriptoma , Imunidade Adaptativa , Adulto , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Antígenos HLA-DR/imunologia , Humanos , Imunidade Inata , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-7/imunologia , Complexo Antígeno L1 Leucocitário/sangue , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Fenótipo , Receptor de Morte Celular Programada 1/imunologia , SARS-CoV-2 , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologia , Tromboplastina/imunologia , Tromboplastina/metabolismo
7.
Lab Invest ; 101(10): 1394-1402, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34145381

RESUMO

Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.


Assuntos
Ácidos e Sais Biliares/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Tromboplastina/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Desoxicólico/farmacologia , Células Hep G2 , Humanos , Isoxazóis/farmacologia , Fígado/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo
8.
Eur Rev Med Pharmacol Sci ; 25(10): 3886-3897, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34109597

RESUMO

OBJECTIVE: Platelets, blood coagulation along with fibrinolysis are greatly involved in the pathophysiology of infectious diseases induced by bacteria, parasites and virus. This phenomenon is not surprising since both the innate immunity and the hemostatic systems are two ancestral mechanisms which closely cooperate favoring host's defense against foreign invaders. However, the excessive response of these systems may be dangerous for the host itself. MATERIALS AND METHODS: We searched and retrieved the articles, using the following electronic database: MedLine and Embase. We limited our search to articles published in English, but no restrictions in terms of article type, publication year, and geography were adopted. RESULTS: The hemostatic phenotype of the infectious diseases is variable depending on the points of attack of the different involved pathogens. Infectious diseases which show a prothrombotic phenotype are bacterial sepsis, SARS-CoV-2 and malaria. However, among the bacterial sepsis, Yersinia Pestis is characterized by a profibrinolytic behavior. On the contrary, the hemorrhagic fevers, due to Dengue and Ebola virus, mainly exploit the activation of fibrinolysis secondary to a huge endothelial damage which can release a large amount of t-PA in the early phase of the diseases. CONCLUSIONS: Blood coagulation and fibrinolysis are greatly activated based on the strategy of the different infectious agents which exploit the excess of response of both systems to achieve the greatest possible virulence.


Assuntos
Coagulação Sanguínea , COVID-19/patologia , Fibrinólise , COVID-19/complicações , COVID-19/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Monócitos/citologia , Monócitos/metabolismo , Monócitos/virologia , SARS-CoV-2/isolamento & purificação , Tromboplastina/metabolismo , Vírus/patogenicidade
9.
EBioMedicine ; 67: 103369, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33971404

RESUMO

BACKGROUND: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. METHODS: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA. FINDINGS: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels. INTERPRETATION: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity. FUNDING: Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.


Assuntos
COVID-19/complicações , Vesículas Extracelulares/imunologia , Tromboplastina/metabolismo , Trombose/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Superfície/análise , Biomarcadores/análise , COVID-19/sangue , COVID-19/imunologia , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Suíça , Trombose/etiologia , Trombose/imunologia , Fator de Necrose Tumoral alfa/sangue
10.
Front Immunol ; 12: 664209, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054832

RESUMO

Rationale: Systemic activation of procoagulant and inflammatory mechanisms has been implicated in the pathogenesis of COVID-19. Knowledge of activation of these host response pathways in the lung compartment of COVID-19 patients is limited. Objectives: To evaluate local and systemic activation of coagulation and interconnected inflammatory responses in critically ill COVID-19 patients with persistent acute respiratory distress syndrome. Methods: Paired bronchoalveolar lavage fluid and plasma samples were obtained from 17 patients with COVID-19 related persistent acute respiratory distress syndrome (mechanical ventilation > 7 days) 1 and 2 weeks after start mechanical ventilation and compared with 8 healthy controls. Thirty-four host response biomarkers stratified into five functional domains (coagulation, complement system, cytokines, chemokines and growth factors) were measured. Measurements and Main Results: In all patients, all functional domains were activated, especially in the bronchoalveolar compartment, with significantly increased levels of D-dimers, thrombin-antithrombin complexes, soluble tissue factor, C1-inhibitor antigen and activity levels, tissue type plasminogen activator, plasminogen activator inhibitor type I, soluble CD40 ligand and soluble P-selectin (coagulation), next to activation of C3bc and C4bc (complement) and multiple interrelated cytokines, chemokines and growth factors. In 10 patients in whom follow-up samples were obtained between 3 and 4 weeks after start mechanical ventilation many bronchoalveolar and plasma host response biomarkers had declined. Conclusions: Critically ill, ventilated patients with COVID-19 show strong responses relating to coagulation, the complement system, cytokines, chemokines and growth factors in the bronchoalveolar compartment. These results suggest a local pulmonary rather than a systemic procoagulant and inflammatory "storm" in severe COVID-19.


Assuntos
COVID-19/imunologia , Estado Terminal , Pulmão/metabolismo , Síndrome do Desconforto Respiratório/imunologia , SARS-CoV-2/fisiologia , Tromboplastina/metabolismo , Idoso , Coagulação Sanguínea , Estudos de Coortes , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Seguimentos , Humanos , Imunidade Inata , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Respiração Artificial
11.
J Toxicol Sci ; 46(4): 187-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33814512

RESUMO

Tissue factor (TF) is the initiator of the coagulation cascade, constitutively expressed in subendothelial cells such as vascular smooth muscle cells and initiating rapid coagulation when the vascular vessel is damaged. TF has been shown to be involved in the development and progression of atherosclerosis. Arsenic, an environmental pollutant, is related to the progression of atherosclerosis, although the pathogenic mechanisms are not fully elucidated. In the present study, we investigated the effect of arsenite on the expression of TF in human aortic smooth muscle cells (HASMCs) and the underlying molecular mechanisms. We found that (1) arsenite stimulated TF synthesis and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in HASMCs, (2) sulforaphane, an Nrf2 activator, also stimulated TF synthesis in HASMCs, and (3) arsenite-induced upregulation of TF synthesis was prevented by Nrf2 knockdown in HASMCs. These results suggest that arsenite promotes TF synthesis by activating the Nrf2 pathway in HASMCs and that the induction of TF expression by arsenite may be related to the progression of atherosclerosis.


Assuntos
Aorta/citologia , Arsenitos/toxicidade , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Tromboplastina/metabolismo , Aterosclerose/etiologia , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos/toxicidade , Fator 2 Relacionado a NF-E2/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sulfóxidos/toxicidade , Tromboplastina/genética
12.
Biomolecules ; 11(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917935

RESUMO

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.


Assuntos
Fator VII/química , Fator VIIa/química , Precursores de Proteínas/química , Regulação Alostérica , Domínio Catalítico , Análise por Conglomerados , Fator VII/metabolismo , Fator VIIa/metabolismo , Humanos , Simulação de Dinâmica Molecular , Análise de Componente Principal , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Tromboplastina/química , Tromboplastina/metabolismo , Tripsina/metabolismo
13.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805059

RESUMO

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.


Assuntos
Selectina-P/química , Neoplasias Pancreáticas/metabolismo , Ativação Plaquetária , Trombina/química , Trombose Venosa/metabolismo , Plaquetas/metabolismo , Linhagem Celular Tumoral , Humanos , Ligantes , Adesividade Plaquetária , Agregação Plaquetária , Fatores de Risco , Trombofilia , Tromboplastina/metabolismo , Trombose Venosa/complicações
14.
Eur J Pharmacol ; 901: 174059, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33794215

RESUMO

The role of CXC chemokine ligand 16 (CXCL16), oxidized LDL (ox-LDL), tissue factor (TF) and autophagy-induced beta cell death in type 1 diabetes mellitus (T1DM) pathogenesis is still unclear. We examined the therapeutic potential and mechanism of resveratrol (RES) against T1DM. Diabetes was induced in Balb/c mice by i. p. injection of 55 mg/kg streptozotocin (STZ) for five consecutive days. The control group received vehicles. RES or (RES + STZ) groups received RES (50 mg/kg, i. p.) daily for 12 days starting from the fourth day of buffer or STZ injections, respectively. Blood glucose, serum insulin, beta cell mass, serum lipid profiles, histological changes, oxidative stress biomarkers were determined. Moreover, CXCL16, TF, ox-LDL, P62 and LC3 tissue expression were also analyzed. Diabetic mice showed a marked deterioration in biochemical, physical and oxidative stress parameters. Interestingly, immunofluorescence analysis showed a remarkable elevation in CXCL16 (12 folds), ox-LDL (9 folds), TF (8.3 folds) in pancreatic B-cells. Moreover, western blotting revealed a profound increase in ox-LDL (2.6 folds), TF (3.2 folds), while a significant decline in P62 (0.34) and LC3 (0.25) when compared to control. RES mitigated biochemical, physical, oxidative imbalance and distorted pancreatic architecture in T1DM mice. Intriguingly, CXCL16, ox-LDL, TF and autophagic markers were also restored after RES treatment. Our data give the first direct evidence that beta cell-specific CXCL16/ox-LDL pathway activation is a potential trigger of TF activation and autophagic beta cell death in T1DM. Moreover, RES may have potential therapeutic applications for prevention of T1DM mainly via ameliorating this pathway.


Assuntos
Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Quimiocina CXCL16/efeitos dos fármacos , Diabetes Mellitus Tipo 1/prevenção & controle , Lipoproteínas LDL/efeitos dos fármacos , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tromboplastina/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/prevenção & controle , Teste de Tolerância a Glucose , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico
15.
Front Immunol ; 12: 625957, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767697

RESUMO

Endotoxin-induced lung injury is one of the major causes of death induced by endotoxemia, however, few effective therapeutic options exist. Hydrogen inhalation has recently been shown to be an effective treatment for inflammatory lung injury, but the underlying mechanism is unknown. In the current study we aim to investigate how hydrogen attenuates endotoxin-induced lung injury and provide reference values for the clinical application of hydrogen. LPS was used to establish an endotoxin-induced lung injury mouse model. The survival rate and pulmonary pathologic changes were evaluated. THP-1 and HUVECC cells were cultured in vitro. The thioredoxin 1 (Trx1) inhibitor was used to evaluate the anti-inflammatory effects of hydrogen. Hydrogen significantly improved the survival rate of mice, reduced pulmonary edema and hemorrhage, infiltration of neutrophils, and IL-6 secretion. Inhalation of hydrogen decreased tissue factor (TF) expression and MMP-9 activity, while Trx1 expression was increased in the lungs and serum of endotoxemia mice. LPS-stimulated THP-1 and HUVEC-C cells in vitro and showed that hydrogen decreases TF expression and MMP-9 activity, which were abolished by the Trx1 inhibitor, PX12. Hydrogen attenuates endotoxin-induced lung injury by decreasing TF expression and MMP-9 activity via activating Trx1. Targeting Trx1 by hydrogen may be a potential treatment for endotoxin-induced lung injury.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Anti-Inflamatórios/farmacologia , Hidrogênio/farmacologia , Pulmão/efeitos dos fármacos , Tiorredoxinas/metabolismo , Tromboplastina/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos ICR , Infiltração de Neutrófilos/efeitos dos fármacos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Edema Pulmonar/prevenção & controle , Transdução de Sinais , Células THP-1
16.
Clin Appl Thromb Hemost ; 27: 10760296211003983, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33784877

RESUMO

COVID-19 (Coronavirus Disease 2019) is a highly contagious infection and associated with high mortality rates, primarily in elderly; patients with heart failure; high blood pressure; diabetes mellitus; and those who are smokers. These conditions are associated to increase in the level of the pulmonary epithelium expression of angiotensin-converting enzyme 2 (ACE-2), which is a recognized receptor of the S protein of the causative agent SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Severe cases are manifested by parenchymal lung involvement with a significant inflammatory response and the development of microvascular thrombosis. Several factors have been involved in developing this prothrombotic state, including the inflammatory reaction itself with the participation of proinflammatory cytokines, endothelial dysfunction/endotheliitis, the presence of antiphospholipid antibodies, and possibly the tissue factor (TF) overexpression. ARS-Cov-19 ACE-2 down-regulation has been associated with an increase in angiotensin 2 (AT2). The action of proinflammatory cytokines, the increase in AT2 and the presence of antiphospholipid antibodies are known factors for TF activation and overexpression. It is very likely that the overexpression of TF in COVID-19 may be related to the pathogenesis of the disease, hence the importance of knowing the aspects related to this protein and the therapeutic strategies that can be derived. Different therapeutic strategies are being built to curb the expression of TF as a therapeutic target for various prothrombotic events; therefore, analyzing this treatment strategy for COVID-19-associated coagulopathy is rational. Medications such as celecoxib, cyclosporine or colchicine can impact on COVID-19, in addition to its anti-inflammatory effect, through inhibition of TF.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/metabolismo , Celecoxib/uso terapêutico , Colchicina/uso terapêutico , Ciclosporina/uso terapêutico , SARS-CoV-2/metabolismo , Tromboplastina/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/epidemiologia , Citocinas/metabolismo , Humanos
17.
Nat Commun ; 12(1): 1693, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727531

RESUMO

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.


Assuntos
Entropia , Ensaios de Triagem em Larga Escala , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Coagulação Sanguínea , Fluorescência , Células HEK293 , Humanos , Metaloproteinases da Matriz/metabolismo , Peptídeos/metabolismo , Especificidade por Substrato , Tromboplastina/metabolismo
18.
Blood Adv ; 5(3): 628-634, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33560376

RESUMO

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease.


Assuntos
COVID-19/patologia , Vesículas Extracelulares/metabolismo , Tromboplastina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , COVID-19/complicações , COVID-19/virologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Modelos de Riscos Proporcionais , Curva ROC , Risco , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Trombose/diagnóstico , Trombose/etiologia
19.
Clin Appl Thromb Hemost ; 27: 1076029620943300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33586482

RESUMO

Sepsis-associated disseminated intravascular coagulation (DIC) is related to marked hemostatic changes such as transient thrombocytopenia secondary to the endogenous activation and consumption of platelets. This study measured markers of platelet function in 103 adult ICU patients with clinically established sepsis-associated DIC to determine the biomarker association with disease severity. Patients were categorized as having no DIC, nonovert DIC, or overt DIC using the International Society of Thrombosis and Hemostasis scoring system. Plasma levels of CD40L, platelet factor 4 (PF4), platelet-derived microparticles, and microparticle-associated tissue factor were quantified. Markers of platelet activation were significantly elevated in patients with DIC compared to healthy individuals. This increase was independent of platelet count. Levels of PF4 differed based on the severity of DIC and differentiated nonsurvivors and survivors. These findings suggest that the markers of platelet activation in DIC may not be regulated by the number of circulating platelets and may be independent of the factors leading to their consumption.


Assuntos
Plaquetas/metabolismo , Coagulação Intravascular Disseminada/diagnóstico , Ativação Plaquetária , Fator Plaquetário 4/sangue , Sepse/complicações , Adulto , Idoso , Biomarcadores/sangue , Ligante de CD40/sangue , Estudos de Casos e Controles , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/etiologia , Coagulação Intravascular Disseminada/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Sepse/sangue , Sepse/diagnóstico , Sepse/mortalidade , Índice de Gravidade de Doença , Tromboplastina/metabolismo , Adulto Jovem
20.
Infect Genet Evol ; 90: 104751, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33540085

RESUMO

COVID-19 is the currently evolving viral disease worldwide. It mainly targets the respiratory organs, tissues and causes illness. A plethora of studies has been performing to bring proper treatment and prevent people from the infection. Likewise, susceptibility to some infectious diseases has been associated with blood group phenotypes. The co-relationship of blood group with the occurrence of SARS-CoV-2 infection and death has been examined in numerous studies. This review explained the described studies regarding the correlation of blood group and the other essential factors with COVID-19.


Assuntos
Sistema ABO de Grupos Sanguíneos/genética , COVID-19/epidemiologia , COVID-19/etiologia , Suscetibilidade a Doenças , Fenótipo , SARS-CoV-2 , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/imunologia , Sistema ABO de Grupos Sanguíneos/metabolismo , Coronavirus/classificação , Coronavirus/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ligação Proteica , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Relação Estrutura-Atividade , Tromboplastina/metabolismo , Fator de von Willebrand/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...