RESUMO
This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.
Assuntos
Fibrinolíticos/química , Nanopartículas de Magnetita/química , Mucor/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Cromatografia por Troca Iônica , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacologia , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mucor/química , Mucor/genética , Peptídeo Hidrolases/farmacologia , TemperaturaRESUMO
Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson's disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.
Assuntos
Afibrinogenemia/genética , Fibrinogênio/química , Doença de Parkinson/genética , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , alfa-Sinucleína/química , Afibrinogenemia/tratamento farmacológico , Afibrinogenemia/metabolismo , Afibrinogenemia/patologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Coagulantes/uso terapêutico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Fibrinogênio/genética , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Inibidores de Proteases/uso terapêutico , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/tratamento farmacológico , Deficiência de alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease that causes joint destruction. Although its etiology remains unknown, citrullinated proteins have been considered as an auto-antigen able to trigger an inflammatory response in RA. Herein, we modified the classical antigen-induced arthritis (AIA) model by using citrullinated human plasma fibrinogen (hFIB) as an immunogen to investigate the mechanism of inflammation-driven joint damage by citrullinated hFIB in C57BL/6 mice. We found that hFIB-immunized mice showed high serum levels of anti-citrullinated peptides antibodies (ACPAs). Moreover, hFIB immunized mice showed increased mechanical hyperalgesia, massive leukocyte infiltration, high levels of inflammatory mediators, and progressive joint damage after the intra-articular challenge with citrullinated hFIB. Interestingly, hFIB-induced arthritis was dependent on IL-23/IL-17 immune axis-mediated inflammatory responses since leukocyte infiltration and mechanical hyperalgesia were abrogated in Il17ra-/- and Il23a-/- mice. Thus, we have characterized a novel model of experimental arthritis suitable to investigate the contribution of ACPAs and Th17 cell-mediated immune response in the pathogenesis of RA.
Assuntos
Artrite/induzido quimicamente , Fibrinogênio/toxicidade , Inflamação/induzido quimicamente , Interleucina-23/metabolismo , Animais , Citrulinação , Fibrinogênio/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina G , Inflamação/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-23/genética , Masculino , Camundongos , Camundongos Knockout , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismoRESUMO
Fibrinogen plays a vital role in normal homeostasis by promoting platelet aggregation, clot formation and fibrinolysis. It is quantified in finished pharmaceutical products using different methods described in pharmacopoeia, but these are inaccurate, difficult to validate and do not allow for identification of aggregates or protein products of the same formulation. The aim of this study was to develop and validate a method for quantification of the content of fibrinogen and other proteins present in pharmaceutical formulations by comparing it with current pharmacopeial methods. Fibrinogen was quantified in two commercial products and compared to a pharmacopeial method using a validated method for size-exclusion high-pressure liquid chromatography (SEC-HPLC). The fibrinogen level was in accordance with both products' specifications. The SEC-HPLC method showed that the percentage of fibrinogen was 94.88 for one product and 50.68 for the other, and detected high molecular weight aggregates in the second product. The SEC-HPLC method that we developed is an improvement to the current pharmacopeial method, because it allows for quantification of fibrinogen and determination of product purity. This is important because greater purity can reduce potential adverse effects of pharmaceutical products in patients.
Assuntos
Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Fibrinogênio/análise , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Soluções Tampão , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Fibrinogênio/química , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Few studies have addressed gene expression of hemostasis-related factors during acute thrombo-hemorrhagic diseases. Bites by the lanced-headed viper Bothrops jaracaca induce rapid hemostatic disturbances in victims, leading to systemic bleedings, thrombocytopenia and consumption coagulopathy. Although circulating levels of coagulation factors recover rapidly after administration of specific antivenom therapy, it is unclear if B. jararaca venom (BjV) upregulates the mRNA synthesis of hepatic hemostasis-related factors, or if the recovery occurs under basal conditions after the neutralization of venom components by antivenom. Thus, we aimed to investigate if BjV regulates gene expression of important hemostasis-related factors synthetized by the liver. On that account, Swiss mice were injected with saline or BjV (1.6 mg/kg b.w, s.c.), and after 3, 6 and 24 h blood samples and liver fragments were collected to analyze mRNA expression by real-time qPCR. Increased gene expression of fibrinogen chains, haptoglobin and STAT3 was observed during envenomation, particularly at 3 and 6 h. At 24h, mRNA levels of F10 were raised, while those of Serpinc1, Proc and Adamts13 were diminished. Surprisingly, F3 mRNA levels were steadily decreased at 3 h. Gene expression of Thpo, F7, F5 Tfpi, Mug1 was unaltered. mRNA levels of Vwf, P4hb, F8, F2, Plg, and Serpinf2 were minimally altered, but showed important associations with Nfkb1 gene expression. In conclusion, snakebite envenomation upregulates hepatic mRNA synthesis particularly of fibrinogen chains, and acute-phase markers. This response explains the fast recovery of fibrinogen levels after antivenom administration to patients bitten by B. jararaca snakes.
Assuntos
Proteínas Sanguíneas/genética , Regulação da Expressão Gênica , Hemostasia/genética , Fígado/metabolismo , Mordeduras de Serpentes/metabolismo , Animais , Antivenenos/uso terapêutico , Transtornos da Coagulação Sanguínea , Bothrops/metabolismo , Modelos Animais de Doenças , Fibrinogênio/química , Fibrinogênio/genética , Fibrinogênio/metabolismo , Haptoglobinas/metabolismo , Hemorragia , Hemostáticos , Masculino , Camundongos , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Mordeduras de Serpentes/sangue , Trombocitopenia , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
OBJECTIVES: The aim of this study was to describe the histological and clinical outcome of "dentin block" (a mixture of autologous particulate dentin, leukocyte- and platelet-rich fibrin (L-PRF), and liquid fibrinogen) in alveolar ridge preservation. MATERIAL AND METHODS: Ten extraction sockets were grafted with "dentin block," a mixture of particulate autologous dentin with chopped leukocyte-platelet-rich fibrin (L-PRF) membranes at a 1:1 ratio, and liquid fibrinogen as a binder. Two grafted sites were followed at 4 and 5 months, and 6 sites at 6 months. Biopsies were taken from the core of the grafted site for histologic and histo-morphometric analysis. RESULTS: All patients completed the study without any adverse event. The vertical and horizontal dimensions of the alveolar ridge were preserved or even increased after 4, 5, or 6 months and remained stable after 6 months of the implant placement. The histological examination revealed a median relative percentage of bone, dentin, and connective tissue of 57.0, 0.9, and 39.3%, respectively. A comparison of samples at different time points (4, 5, and 6 months) showed a progressive increase in the proportion of bone with a decrease in the proportion of dentin. The bone was compact with normal osteocytes and moderate osteoblastic activity. In 4 out of 10 samples, no dentin was observed; in the other samples, it represented 1-5% (with geometric fragments). CONCLUSIONS: Dentin block showed to be a suitable bone substitute in an alveolar ridges preservation model. CLINICAL RELEVANCE: The promising results of dentin block as a bone substitute in alveolar ridge preservation could have an important clinical impact considering this biomaterial brings together the regenerative potential of three autologous products with excellent biological and clinical behavior, low risk of adverse effects, and feasible acquisition.
Assuntos
Aumento do Rebordo Alveolar , Substitutos Ósseos/uso terapêutico , Dentina/química , Fibrinogênio/química , Fibrina Rica em Plaquetas/química , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Extração Dentária , Alvéolo DentalRESUMO
BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.
Assuntos
Células Endoteliais/metabolismo , Fibrinogênios Anormais/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Trombose , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Coagulação Sanguínea , Linhagem Celular , Fibrina/química , Fibrinogênio/química , Fibrinólise/fisiologia , Humanos , Trombina/metabolismo , Trombose/metabolismoRESUMO
Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28â¯kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bß chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bß chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bß fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Fibrina/química , Fibrinogênio/metabolismo , Serina Proteases/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Fibrinogênio/química , Humanos , Concentração de Íons de Hidrogênio , Lorazepam , Serina Proteases/química , Adulto JovemRESUMO
Bothrops venezuelensis snake venoms, from five localities in the North-Central Venezuelan regions, showed biochemical and haemostatic differences. In this study, bioactivities of B. venezuelensis venoms from different regions (Aragua state; Waraira Repano (Capital District); Baruta, La Boyera and Lagunetica (Miranda state)) were compared using both natural and synthetic substrates. The protein contents of these venoms were Lagunetica 89%, La Boyera 79%, Baruta 71%, Waraira Repano 68% and Aragua 64%. Toxic activities effects were: Intraperitoneal LD50s: Aragua-14â¯mg/kg; Waraira Repano-6.4â¯mg/kg; Baruta: 8.3â¯mg/kg; La Boyera-4.4â¯mg/kg; Lagunetica-16.2â¯mg/kg. The MHD results: Aragua-21.4⯵g/mouse; Waraira Repano-2.5⯵g/mouse; Baruta-1.2⯵g/mouse; La Boyera-1.4⯵g/mouse and Lagunetica-12⯵g/mouse. The hide powder azure results: Aragua-1.24â¯U/mg; La Boyera-2.26â¯U/mg; Baruta-2.83â¯U/mg; Lagunetica-3.28â¯U/mg and Waraira Repano-5.77â¯U/mg. Esterase specific activity on BAEE results: Waraira Repano-666.66â¯U/mg; La Boyera-805.5â¯U/mg; Baruta-900.00â¯U/mg; Lagunetica-922.19â¯U/mg and Aragua-1960.67â¯U/mg. Casein zymography showed digestion bands in the molecular weight above 100 and at 66.2 and 21.5â¯kDa. Analysis of casein degradation by SDS-PAGE showed two different degradation patterns. Fibrinolytic activity (mm2/µg) on fibrin plates results: Aragua-6.07; Lagunetica-27.6; Waraira Repano-35.7; La Boyera-44.27 and Baruta-45.63. In the fibrinogenolytic assay, the five venoms completely degraded the α chain after 1â¯min of incubation. None of the venoms completely degraded the ß and γ chains after 24â¯h incubation. The research indicated that venoms of B. venezuelensis of different geographic areas in Venezuela exhibit variances in composition and component concentrations; except the Aragua venom, all of them had high proteolytic activities.
Assuntos
Bothrops , Venenos de Crotalídeos/toxicidade , Animais , Coagulação Sanguínea/efeitos dos fármacos , Caseínas/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Fibrinogênio/química , Fibrinólise/efeitos dos fármacos , Geografia , Hemorragia/induzido quimicamente , Dose Letal Mediana , Camundongos , Proteólise/efeitos dos fármacos , VenezuelaRESUMO
Homocysteine and its cyclic ester homocysteine thiolactone (HTL) have been involved in the detrimental consequences associated to hyperhomocysteinemia, an independent risk factor for vascular diseases. HTL reacts with protein lysine residues in a process named N-homocysteinylation. The aim of our study was to evaluate the in vitro effects of HTL on the fibrinogen through electrophoretic methods. Fibrinogen was incubated with HTL at different molar ratios and structural changes of the protein were assessed by polyacrylamide gel electrophoresis (PAGE), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF). Ellman´s reaction, CZE and proton nuclear magnetic resonance (1H NMR) were used to evaluate HTL hydrolyisis. On denaturing PAGE numerous bands were observed, being the three lower bands identical to those obtained by treatment with 2-mercaptoethanol. This effect was also detected by CZE. The results show a reducing action of HTL on the fibrinogen molecule, probably attributed to the sulfhydryl groups generated by N-homocysteinylation and/or by the ones present in the homocysteine molecule yielded by HTL hydrolysis. In order to distinguish between these two options, HTL stability was evaluated at different pH and incubation times. The results showed minimum HTL hydrolysis in our experimental conditions. We postulate that the reducing effect observed would be mainly associated to the new sulfhydryl groups generated by the N-homocysteinylation process. Moreover, a displacement of the HTL-treated fibrinogen isoforms towards more acidic pH values was detected. The structural changes of N-homocysteinylated fibrinogen could be involved in the pathological consequences of hyperhomocysteinemia.