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1.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498156

RESUMO

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aminoácido Oxirredutases/metabolismo , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Derme/citologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transglutaminases/metabolismo
2.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440782

RESUMO

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga-/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19-56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga-/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19-56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.


Assuntos
Biomarcadores , Plaquetas/metabolismo , Fibrinogênio/metabolismo , Trombose Venosa/sangue , Trombose Venosa/etiologia , Animais , Sequência de Bases , Coagulação Sanguínea , Modelos Animais de Doenças , Éxons , Fibrinogênio/química , Fibrinogênio/genética , Edição de Genes , Expressão Gênica , Plasmídeos/genética , Ativação Plaquetária , RNA Guia , Deleção de Sequência , Trombose Venosa/diagnóstico , Peixe-Zebra
3.
Arterioscler Thromb Vasc Biol ; 41(3): 1092-1104, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472402

RESUMO

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Fibrinogênio/metabolismo , Fragmentos de Peptídeos/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de Superfície
4.
Methods Mol Biol ; 2147: 45-54, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840809

RESUMO

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication-in one step-of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition.To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers-namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen-that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications.


Assuntos
Biopolímeros/química , Bioimpressão/métodos , Ácidos Polimetacrílicos/síntese química , Impressão Tridimensional , Tecidos Suporte/química , Biopolímeros/efeitos da radiação , Bioimpressão/instrumentação , Sulfatos de Condroitina/química , Fibrinogênio/química , Gelatina/química , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Tinta , Luz , Processos Fotoquímicos , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Propriedades de Superfície/efeitos da radiação , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
5.
J Vis Exp ; (166)2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33346198

RESUMO

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (>100 µm) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel's 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user's needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.


Assuntos
Hidrogéis/química , Imageamento Tridimensional , Microscopia , Módulo de Elasticidade , Fibrina/química , Fibrinogênio/química , Análise de Elementos Finitos , Polimerização , Silicones/química , Software , Trombina/química , Interface Usuário-Computador
6.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33322044

RESUMO

We identified a novel heterozygous variant, Bßp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bßp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bßp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bßγ complex in Bßp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bßp.P234 residue is located in the contact region between the Bß and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bßp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bß and γ chains impact the assembly and secretion of fibrinogen.


Assuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Multimerização Proteica , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Fibrinogênio/química , Fibrinogênio/metabolismo , Humanos , Transporte Proteico
7.
Nat Commun ; 11(1): 4907, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999289

RESUMO

Global alterations in the metabolic network provide substances and energy to support tumor progression. To fuel these metabolic processes, extracellular matrix (ECM) plays a dominant role in supporting the mass transport and providing essential nutrients. Here, we report a fibrinogen and thrombin based coagulation system to construct an artificial ECM (aECM) for selectively cutting-off the tumor metabolic flux. Once a micro-wound is induced, a cascaded gelation of aECM can be triggered to besiege the tumor. Studies on cell behaviors and metabolomics reveal that aECM cuts off the mass transport and leads to a tumor specific starvation to inhibit tumor growth. In orthotopic and spontaneous murine tumor models, this physical barrier also hinders cancer cells from distant metastasis. The in vivo gelation provides an efficient approach to selectively alter the tumor mass transport. This strategy results in a 77% suppression of tumor growth. Most importantly, the gelation of aECM can be induced by clinical operations such as ultrasonic treatment, surgery or radiotherapy, implying this strategy is potential to be translated into a clinical combination regimen.


Assuntos
Materiais Biomiméticos/administração & dosagem , Matriz Extracelular/química , Neoplasias/terapia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Materiais Biomiméticos/química , Materiais Biomiméticos/efeitos da radiação , Linhagem Celular Tumoral/transplante , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quimiorradioterapia/métodos , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Feminino , Fibrinogênio/administração & dosagem , Fibrinogênio/química , Fibrinogênio/efeitos da radiação , Géis , Humanos , Injeções Intravenosas , Metabolômica , Camundongos , Neoplasias/metabolismo , Trombina/administração & dosagem , Trombina/química , Trombina/efeitos da radiação , Terapia por Ultrassom/métodos , Ondas Ultrassônicas
8.
Nat Commun ; 11(1): 5468, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122656

RESUMO

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10-50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.


Assuntos
Dissulfetos/química , Fibrinogênio/química , Trombose/metabolismo , Alquilação , Testes de Coagulação Sanguínea , Fibrina/biossíntese , Fibrinólise , Hepatócitos , Humanos , Espectrometria de Massas , Oxirredução , Polimerização , Trombina/metabolismo
9.
Nat Commun ; 11(1): 5431, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110079

RESUMO

Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.


Assuntos
Adesinas Bacterianas/química , Infecções Estafilocócicas/microbiologia , Staphylococcus/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Fibrinogênio/química , Fibrinogênio/metabolismo , Humanos , Ligação Proteica , Análise Espectral , Infecções Estafilocócicas/metabolismo , Staphylococcus/química , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimento
10.
Biomolecules ; 10(8)2020 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-32784866

RESUMO

Glycosylation may strongly affect protein structure and functions. A high risk of cardiovascular complications seen in patients with end-stage renal disease (ESRD) is, at least partly associated with delayed clot formation, increased clot strength, and delayed cloth lysis. Taking into consideration that fibrinogen mediates these processes, we isolated fibrinogen from the plasma from patients with ESRD on peritoneal dialysis (ESRD-PD), and examined glycosylation of native fibrinogen and its subunits by lectin-based microarray and lectin blotting. Compared to healthy controls, fibrinogen from patients had increased levels of A2BG2 and decreased levels of FA2 glycan. The distribution of glycans on individual chains was also affected, with the γ chain, responsible for physiological functions of fibrinogen (such as coagulation and platelet aggregation), being most prone to these alterations. Increased levels of multi-antennary N-glycans in ESRD-PD patients were also associated with the type of dialysis solutions, whereas an increase in the fucosylation levels was strongly related to the peritoneal membrane damage. Consequently, investigation of fibrinogen glycans can offer better insight into fibrinogen-related complications observed in ESRD-PD patients and, additionally, contribute to prognosis, choice of personalised therapy, determination of peritoneal membrane damage, and the length of utilization of peritoneum for dialysis.


Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Fucose/metabolismo , Falência Renal Crônica/sangue , Diálise Peritoneal , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Glicosilação , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Lectinas/sangue , Lectinas/química , Masculino , Pessoa de Meia-Idade , Polissacarídeos/sangue , Polissacarídeos/química , Polissacarídeos/metabolismo , Prognóstico , Análise Serial de Proteínas
11.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32566134

RESUMO

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbß3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.


Assuntos
Plaquetas/citologia , Colágeno Tipo IV/química , Fibrinogênio/química , Adesividade Plaquetária , Células Cultivadas , Humanos , Microtúbulos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Pseudópodes
12.
Int J Hematol ; 112(3): 331-340, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32562089

RESUMO

We identified two heterozygous dysfibrinogenemias, Bßp.Gly45Cys (Kyoto VII; K-VII) and Bßp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bßp.G45C has been well analyzed; however, that of Bßp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bßp.G45C and Bßp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bßp.G45C was impaired more than Bßp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bßp.G45C and thrombosis for patients with Bßp.R74C.


Assuntos
Afibrinogenemia/genética , Afibrinogenemia/metabolismo , Fibrina/genética , Fibrina/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Adulto , Criança , Feminino , Fibrinogênio/química , Variação Genética , Hemorragia/etiologia , Hemorragia/genética , Heterozigoto , Humanos , Masculino , Estrutura Molecular , Polimerização , Risco , Trombose/etiologia , Trombose/genética
13.
J Vis Exp ; (160)2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32568238

RESUMO

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients' hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.


Assuntos
Testes de Coagulação Sanguínea/métodos , Fibrinogênio/química , Tromboelastografia/métodos , Trombose/sangue , Adulto , Animais , Bovinos , Humanos
14.
PLoS Negl Trop Dis ; 14(6): e0008379, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479494

RESUMO

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/genética
15.
PLoS One ; 15(4): e0232010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324839

RESUMO

Rheumatoid arthritis (RA), caused by the abnormal recognition of human joint cells by autoimmune antibodies, remains the world's most prevalent autoimmune disease, with over five million people affected and as much as 4% of the population at risk of RA. To prevent rapid disease development, hormonal and anti-inflammatory therapies require fast and reliable RA diagnosis. However, difficulty in detecting early specific biomarkers for RA means that it is unclear when treatment needs to begin. Here, we combined synthesis of citrullinated peptide epitopes with molecular diagnostics to verify a new specific biomarker for early RA diagnosis and flare prediction. A fibrinogen-derived 21-amino-acid-long citrullinated peptide showed high reactivity toward autoantibodies in RA samples. Additionally, the level of antibodies to this epitope was elevated prior to flares. In contrast, other citrullinated protein variants had lower reactivity and poorer sensitivity to disease activity. In conclusion, fibrinogen-derived epitope E2 subjected to citrullination facilitated a reliable RA diagnosis with a strong correlation to disease activity. This is of a high value for the diagnosis and management of RA patients who respond poorly to treatment.


Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/metabolismo , Epitopos/imunologia , Peptídeos Cíclicos/imunologia , Adolescente , Adulto , Artrite Reumatoide/imunologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Citrulina/metabolismo , Diagnóstico Precoce , Epitopos/química , Feminino , Fibrinogênio/química , Humanos , Masculino , Peptídeos Cíclicos/química , Índice de Gravidade de Doença , Exacerbação dos Sintomas , Adulto Jovem
16.
Mar Drugs ; 18(4)2020 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-32290502

RESUMO

: Enzymatic hydrolysis of native collagen and fibrinogen was carried out under comparable conditions at room temperature. The molecular weight parameters of proteins before and after hydrolysis by thrombin were monitored by gel-penetrating chromatography (GPC). An analysis of the experiment results shows that the molecular weight parameters of the initial fibrinogen (Fn) and cod collagen (CC) are very similar. High molecular CC decays within the first minute, forming two low molecular fractions. The main part (~80%) falls on the fraction with a value of Mw less than 10 kDa. The initial high molecular fraction of Fn with Mw ~320-340 kDa is not completely hydrolyzed even after three days of control. The presence of low molecular fractions with Mw ~17 and Mw ~10 kDa in the solution slightly increases within an hour and noticeably increases for three days. The destruction of macromolecules of high molecular collagen to hydrolysis products appears almost completely within the first minute mainly to the polymer with Mw ~10 kDa, and enzymatic hydrolysis of fibrinogen proceeds slower than that of collagen, but also mainly to the polymer with Mw ~10 kDa. Comparative photos of the surfaces of native collagen, fibrinogen and the scaffold based on them were obtained.


Assuntos
Colágeno/química , Fibrinogênio/química , Peixes , Hemostáticos/química , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Hidrólise , Trombina/química
17.
Sci Rep ; 10(1): 5111, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198419

RESUMO

Fibrin is an optimal scaffold for tissue-engineering applications because it mimics the extracellular matrix. Despite this interesting feature, fibrin gel owns only poor mechanical properties that limit its applications. Different approaches have been used for fibrin electrospinning, however all the methods investigated required washing steps, cross-linking agent treatment or immersion. The aim of this work was to produce a bilayered fibrin/polyurethane scaffold by combination of the electrospun method and the spray, phase-inversion method for the preparation of a fibrin nanostructured layer to be attached onto a poly(ether)urethane microporous support layer. The synthetic layer was obtained by the spray, phase-inversion technique onto a rotating metallic collector, while fibrinogen was processed to obtain a nanofibrous structure by electrospinning. Finally, fibrin polymerization was obtained by thrombin solution spraying onto the electrospun nanofibers. SEM analysis showed the formation of filamentous structure with diameter in the range of µm attached onto the synthetic layer. This scaffold could be applied in soft tissue regeneration such as wound healing or as drug delivery system.


Assuntos
Fibrina/metabolismo , Regeneração Tecidual Guiada/métodos , Poliuretanos/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Sistemas de Liberação de Medicamentos/métodos , Matriz Extracelular , Fibrinogênio/química , Teste de Materiais , Nanofibras/química , Trombina/química , Cicatrização/fisiologia
18.
Anal Chim Acta ; 1102: 72-83, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32043998

RESUMO

Critical bleeding causes over 2 million deaths a year. Early hypofibrinogenemia is a strong predictor of mortality in critically bleeding patients. The early replenishment of fibrinogen can significantly improve outcomes. However, over replenishment can also be dangerous. Furthermore, there is no rapid, cheap, hand-held diagnostic that can aid critically bleeding patients in fibrinogen replacement therapy. In this study, we have developed a hand-held paper diagnostic that measures plasma fibrinogen concentrations. The diagnostic has the potential to be used as a point of care device both inside and outside of hospital settings. It can vastly reduce the time to treatment for fibrinogen replacement therapy. The diagnostic is a two-step process. First, thrombin and plasma are added onto horizontially-orientated paper strips where the fibrinogen is converted into fibrin, drastically increasing the plasma's hydrophobicity. Second, an aqueous blue dye is pipetted onto the strips and allowed to wick through the fibrin. The distance the blue dye wicks through the strip correlates precisely to the fibrinogen concentration. The diagnostic can provide results within a minute. It can distinguish low fibrinogen concentrations (ie. <2 g/L) from normal fibrinogen concentrations. It shows remarkable reproducibility between healthy individuals. It is unaffected by common blood conditions such as acidosis, blood alcohol, severe hypertriglyceridemia, severe haemolysis and warfarin administration. Finally, it is unaffected by humidity and can withstand cold temperatures.


Assuntos
Benzenossulfonatos/química , Corantes/química , Fibrinogênio/análise , Papel , Afibrinogenemia/diagnóstico , Temperatura Baixa , Fibrinogênio/química , Humanos , Umidade , Interações Hidrofóbicas e Hidrofílicas , Testes Imediatos , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Trombina/química
19.
J Agric Food Chem ; 68(10): 3132-3139, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32064873

RESUMO

Thrombin is currently one of the important targets for the treatment and prevention of thrombosis. At present, there are few reports on the application of lactoferrin peptides in anticoagulation. In this study, a peptide with the amino acid sequence of LRPVAAEIY (LF-LR) derived from lactoferrin was shown to possess antithrombotic activity. LF-LR (5 mM) significantly prolonged activated partial thromboplastin time, prothrombin time, and thrombin time for 13.4, 1.7, and 5.1 s, respectively. It prolonged the coagulation time of fibrinogen from 15.3 ± 0.4 to 20.2 ± 0.5 s by affecting the conformation of thrombin. Using circular dichroism analysis, LF-LR can increase the α-helix content of thrombin from 25.6 to 56.7% and made the ß-sheet disappear. In addition, LF-LR also quenched fluorescence of thrombin at about 346 nm (λEx = 280 nm). By means of molecular docking, it was found that LF-LR could bind to both the active site and the exosite-I of thrombin, and the combined LYS60F, TRP60D, ASP189, LYS36, and ARG77A are typical amino acids in the two domains, respectively.


Assuntos
Anticoagulantes/química , Lactoferrina/química , Peptídeos/química , Trombina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Bovinos , Fibrinogênio/química , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Tempo de Trombina
20.
Clin Appl Thromb Hemost ; 26: 1076029619894057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32013532

RESUMO

Hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome is a serious complication of pregnancy. Postpartum hemorrhage indicates poor prognosis of pregnant women with HELLP syndrome. The aim of our study is to investigate the predictive value of coagulation markers for postpartum hemorrhage of pregnant women with HELLP syndrome. In a retrospective cohort study, 106 patients who were diagnosed as pregnant women with HELLP syndrome in Peking University Third Hospital from August 2010 to January 2017 were analyzed. The demographic characters of maternal and fetus, days of hospital stay, postpartum complications, and the laboratory tests of coagulation markers within 3 days before delivery were collected. In addition, 100 healthy pregnant women were collected as a control group. The result showed that the incidence of preeclampsia in pregnant women with postpartum hemorrhage was higher than that in pregnant women without hemorrhage (P = .011). The level of fibrinogen (FIB) in postpartum hemorrhage pregnant women with HELLP syndrome was lower than that in nonpostpartum hemorrhage pregnant women with HELLP syndrome and healthy pregnant women (2.3 [1.68-2.81] vs 3.64 ± 0.95, P = .000; 2.3 [1.68-2.81] vs 4.48 ± 0.62, P = .000). Multivariate analysis showed that decreased FIB levels independently predicted the postpartum hemorrhage of pregnant women with HELLP syndrome (odds ratio = 7.374, 95% confidence interval [CI], 1.551-35.05, P = .012). The receiver operating characteristic curve showed that the area under the curve of FIB level when predicting postpartum hemorrhage is 0.841 (95% CI, 0.708-0.976). When the cutoff value of FIB was 3.04 g/L, the sensitivity was 90.90% and the specificity was75.80%. Therefore, the low level of prenatal FIB is a reliable biomarker to predict postpartum hemorrhage of pregnant women with HELLP syndrome, which make it useful for pregnant women with HELLP syndrome in guiding surveillance therapy and prognosis assessment.


Assuntos
Fibrinogênio/química , Síndrome HELLP/sangue , Hemorragia Pós-Parto/sangue , Adulto , Feminino , Humanos , Gravidez , Estudos Retrospectivos
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