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1.
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
2.
PLoS One ; 15(1): e0227543, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31995579

RESUMO

Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.


Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
3.
Soft Matter ; 15(44): 9076-9084, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31651923

RESUMO

Phospholipids are found throughout the natural world, including the lung surfactant (LS) layer that reduces pulmonary surface tension and enables breathing. Fibrinogen, a protein involved in the blood clotting process, is implicated in LS inactivation and the progression of disorders such as acute respiratory distress syndrome. However, the interaction between fibrinogen and LS at the air-water interface is poorly understood. Through a combined microrheological, confocal and epifluorescence microscopy approach we quantify the interfacial shear response and directly image the morphological evolution when a model LS monolayer is penetrated by fibrinogen. When injected into the subphase beneath a monolayer of the phospholipid dipalmitoylphosphatidylcholine (DPPC, the majority component of LS), fibrinogen preferentially penetrates disordered liquid expanded (LE) regions and accumulates on the boundaries between LE DPPC and liquid condensed (LC) DPPC domains. Thus, fibrinogen is line active. Aggregates grow from the LC domain boundaries, ultimately forming a percolating network. This network stiffens the interface compared to pure DPPC and imparts the penetrated monolayer with a viscoelastic character reminiscent of a weak gel. When the DPPC monolayer is initially compressed beyond LE-LC coexistence, stiffening is significantly more modest and the penetrated monolayer retains a viscous-dominated, DPPC-like character.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Fibrinogênio/química , Surfactantes Pulmonares/química , Adsorção , Elasticidade , Imãs , Reologia , Tensão Superficial , Viscosidade
4.
Analyst ; 144(16): 4848-4857, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31294736

RESUMO

Fibrinogen is a blood protein that is essential for clotting. It is converted into the polymer fibrin by the blood enzymes thrombin and factor XIIIa. Fibrinogen is one of the first proteins to be depleted in heavily bleeding patients. Patients with early hypofibrinogenemia need urgent fibrinogen replenishment to prevent the onset of haemorrhage and death. However, currently there is no rapid, sensitive, cheap and easy-to-use fibrinogen assay that can detect fibrinogen concentrations. In this study, we have developed a new paper-based diagnostic to quantify the fibrinogen concentration in blood at room temperature. This diagnostic is a 2-step process: first, plasma is added onto thrombin-treated paper strips where fibrinogen is converted to fibrin; then the strips are placed into an aqueous dye bath where elution occurs. The test operates by measuring the change in hydrophobicity, which increases with fibrinogen concentration under otherwise constant conditions. The diagnostic can precisely measure fibrinogen concentration within the range of 0-2 g L-1, which is ideal for the clinical diagnosis of hypofibrinogenemia. Furthermore, testing needs only 12 µL of plasma, 60 mU of thrombin and 7.5 minutes of testing. This diagnostic has the potential to revolutionise point of care testing and save many lives.


Assuntos
Técnicas de Química Analítica/métodos , Fibrinogênio/análise , Papel , Afibrinogenemia/diagnóstico , Animais , Compostos Azo/química , Bovinos , Técnicas de Química Analítica/instrumentação , Corantes/química , Fator XIIIa/química , Fibrina/química , Fibrinogênio/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Testes Imediatos , Soroalbumina Bovina/química , Trombina/química , Viscosidade
5.
Int J Biol Macromol ; 137: 405-419, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31265849

RESUMO

We present a computational analysis coupled with experimental studies, focusing on the binding-interaction between beta-adrenoreceptor blocking agents (acebutolol and propranolol) with fibrinogen protein (E-region). Herein, computational modeling on structural validation and flexibility properties of fibrinogen E-region showed that the E-region interacting residues, which form the funnel-shaped hydrophobic cavity for ligand-binding, can be efficiently modeled. The obtained free energy of binding (FEB) values for the docking complexes, namely acebutolol/fibrinogen E-region and propranolol/fibrinogen E-region, were very close and amounted to - 6.9 kcal/mol and - 6.8 kcal/mol, respectively. They were supported by a high binding-accuracy (R.M.S.D < 2 Å) for the best crystallographic binding-poses in both cases. In this regard, we identify a docking-mechanism of interaction for the propranolol and acebutolol mainly based on non-covalent hydrophobic contacts with the fibrinogen E-region binding-site. Besides, the beta-adrenoreceptor blocking agents are able to induce local perturbations affecting particularly the fibrinogen E-region allosteric residues linked to significant changes in the inter-residue communication and flexibility properties of residue network. In this sense, we show that the key biophysical parameters like frequency and collectivity degree may be compromised in different ways by the interaction with acebutolol and propranolol. Isothermal titration calorimetry, zeta potential and small angle X-ray scattering (SAXS) measurements were performed to complete and corroborate computational analysis. The combined experimental results point out that acebutolol acts to a lesser extent to fibrinogen structure than propranolol.


Assuntos
Antagonistas Adrenérgicos beta/metabolismo , Fibrinogênio/química , Fibrinogênio/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Simulação de Acoplamento Molecular , Propranolol/metabolismo , Ligação Proteica , Domínios Proteicos , Termodinâmica
6.
Biomater Sci ; 7(9): 3764-3778, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31342016

RESUMO

Fluorinated polymers are strong candidates for development of new cardiovascular medical devices, due to their lower thrombogenicity as compared to other polymers used for cardiovascular implants. Few studies have reported the development of fluorinated polyesters and their potential in blood contact applications has never been examined. In this study, we developed a versatile method for preparing trifluoromethyl-functionalized poly(lactic acid) that can be potentially extended to prepare a new class of polyesters with various halogen or halocarbon substitutions. The resulting fluorinated polymer was hydrophobic relative to poly(lactic acid) and extracts from this polymer showed no in vitro cytotoxicity to NIH-3T3 mouse fibroblast cells. A preliminary consideration of the blood interactions of the CF3-functionalized polyester was evaluated by measuring the amount of the adsorbed albumin and fibrinogen from human blood plasma. The fluorinated polyester adsorbed and retained higher amounts of albumin and fibrinogen with a higher albumin/fibrinogen ratio as compared to poly(lactic acid), suggesting enhanced hemocompatibility. Plasma protein adsorption is the first event that occurs seconds after device implantation and controlling the adsorbed proteins will dictate the performance of medical implants.


Assuntos
Materiais Biocompatíveis/química , Hidrocarbonetos Fluorados/sangue , Hidrocarbonetos Fluorados/química , Poliésteres/química , Adsorção , Animais , Materiais Biocompatíveis/síntese química , Células Cultivadas , Fibrinogênio/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Estrutura Molecular , Células NIH 3T3 , Poliésteres/síntese química , Albumina Sérica Humana/química , Propriedades de Superfície
7.
Nanoscale ; 11(20): 10023-10033, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31086875

RESUMO

Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.


Assuntos
Plaquetas/metabolismo , Microscopia de Fluorescência , Plaquetas/citologia , Plaquetas/ultraestrutura , Linhagem Celular Tumoral , Técnicas de Cocultura , Fibrinogênio/química , Fibrinogênio/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Nanoestruturas/química , Selectina-P/química , Selectina-P/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Toxins (Basel) ; 11(5)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067768

RESUMO

The functional activities of Anguimorpha lizard venoms have received less attention compared to serpent lineages. Bite victims of varanid lizards often report persistent bleeding exceeding that expected for the mechanical damage of the bite. Research to date has identified the blockage of platelet aggregation as one bleeding-inducing activity, and destructive cleavage of fibrinogen as another. However, the ability of the venoms to prevent clot formation has not been directly investigated. Using a thromboelastograph (TEG5000), clot strength was measured after incubating human fibrinogen with Heloderma and Varanus lizard venoms. Clot strengths were found to be highly variable, with the most potent effects produced by incubation with Varanus venoms from the Odatria and Euprepriosaurus clades. The most fibrinogenolytically active venoms belonged to arboreal species and therefore prey escape potential is likely a strong evolutionary selection pressure. The results are also consistent with reports of profusive bleeding from bites from other notably fibrinogenolytic species, such as V. giganteus. Our results provide evidence in favour of the predatory role of venom in varanid lizards, thus shedding light on the evolution of venom in reptiles and revealing potential new sources of bioactive molecules useful as lead compounds in drug design and development.


Assuntos
Fibrinogênio/química , Lagartos , Peçonhas/química , Animais , Coagulação Sanguínea , Humanos , Tromboelastografia
9.
SAR QSAR Environ Res ; 30(5): 363-382, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31112078

RESUMO

In the current study, we have developed predictive quantitative structure-activity relationship (QSAR) models for cellular response (foetal rate lung fibroblast proliferation) and protein adsorption (fibrinogen adsorption (FA)) on the surface of tyrosine-derived biodegradable polymers designed for tissue engineering purpose using a dataset of 66 and 40 biodegradable polymers, respectively, employing two-dimensional molecular descriptors. Best four individual models have been selected for each of the endpoints. These models are developed using partial least squares regression with a unique combination of six and four descriptors for cellular response and protein adsorption, respectively. The generated models were strictly validated using internal and external metrics to determine the predictive ability and robustness of proposed models. Subsequently, the validated individual models for each response endpoints were used for the generation of 'intelligent' consensus models ( http://teqip.jdvu.ac.in/QSAR_Tools/DTCLab/ ) to improve the quality of predictions for the external data set. These models may help in prediction of virtual polymer libraries for rational design/optimization for properties relevant to biomedical applications prior to their synthesis.


Assuntos
Materiais Biocompatíveis/química , Fibrinogênio/química , Modelos Moleculares , Polímeros/química , Adsorção/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Análise dos Mínimos Quadrados , Estrutura Molecular , Polímeros/farmacologia , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes
10.
PLoS One ; 14(5): e0217601, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120999

RESUMO

A cell therapy product of transforming growth factor (TGF)-ß1-transduced chondrocytes has been commercialized to treat osteoarthritis of the knee via intra-articular injection. The need for arthroscopic application of the cells to simultaneously treat intra-articular pathologies of knee osteoarthritis is increasingly urgent. The purpose of this study was to optimize TGF-ß1-transduced chondrocytes for arthroscopic application. The optimal composition of chondrocytes and thrombin was initially determined by measuring the consolidation time of a diverse ratio of chondrocytes and thrombin mixed with 1 ml of fibrinogen. The consolidation time of the diverse ratio of fibrinogen and atelocollagen mixed with the determined optimal ratio of chondrocytes and thrombin was evaluated. The mixture of the determined optimal ratio of TGF-ß1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin was applied to the cartilage defect of the 3D printed knee joint model arthroscopically. The status of the mixture in the defect was then evaluated. Chondrogenic activities of TGF-ß1-transduced chondrocytes mixed with atelocollagen were evaluated. The determined ratio of TGF-ß1-transduced chondrocytes to thrombin was 8:2 and that of fibrin to atelocollagen was also 8:2. Excellent maintenance of conformation of the mixture of TGF-ß1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin in the cartilage defect of the 3D printed knee joint model was observed arthroscopically. Increased chondrogenic activities were observed in the group of TGF-ß1-transduced chondrocytes mixed with atelocollagen. TGF-ß1-transduced chondrocytes can be applied arthroscopically to treat cartilage defects of the knee at an optimized mixing ratio of atelocollagen, fibrinogen, and thrombin.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Osteoartrite/tratamento farmacológico , Fator de Crescimento Transformador beta1/genética , Artroscopia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Colágeno/química , Colágeno/farmacologia , Fibrina/química , Fibrina/farmacologia , Fibrinogênio/química , Fibrinogênio/farmacologia , Humanos , Injeções Intra-Articulares , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Osteoartrite/genética , Osteoartrite/patologia , Impressão Tridimensional , Regeneração/efeitos dos fármacos , Regeneração/genética , Trombina/química , Trombina/farmacologia
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 220: 117143, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31136867

RESUMO

This study describes synthesis of N­acetyl­l­cysteine-capped CdTe quantum dots (QDs) and investigates their interaction with plasma protein fibrinogen (FIB) and the structural changes of FIB. It is shown that the interaction of QDs with FIB is a spontaneous process and the major driving forces are van der Waals forces and hydrogen bonds. Multi-spectroscopic measurements show that the intrinsic fluorescence of FIB was quenched and secondary and tertiary structures were altered due to the interaction with QDs. In addition, the aggregation state of FIB was altered in the presence of QDs. Furthermore, the formed complexes of FIB with QDs reduced the cytotoxicity of QDs. The coating of FIB on QDs could lower intracellular QDs uptake and therefore result in less released cadmium ions and ROS productions. This study, therefore, might be helpful to the comprehensive understanding of QDs toxicity and provide evidence for assessing the safe application of nanoparticles.


Assuntos
Compostos de Cádmio/química , Fibrinogênio/química , Coroa de Proteína/química , Pontos Quânticos/química , Telúrio/química , Animais , Compostos de Cádmio/metabolismo , Compostos de Cádmio/toxicidade , Calorimetria/métodos , Células Cultivadas , Dicroísmo Circular , Fibrinogênio/metabolismo , Hepatócitos/efeitos dos fármacos , Ligação de Hidrogênio , Camundongos Endogâmicos C57BL , Conformação Proteica , Pontos Quânticos/metabolismo , Pontos Quânticos/toxicidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Telúrio/metabolismo , Telúrio/toxicidade , Termodinâmica
13.
Int J Biol Macromol ; 134: 262-268, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31078590

RESUMO

Herein we present a tuned cell attachment on self-healable double network hydrogel bearing dynamic covalent bonds and hydrogen bonds. Agar formed first network, while glycol chitosan and oxidized carboxylmethyl cellulose formed second network in the resultant double network hydrogel. Because of the simple one-pot preparation, the hydrogel can be injected by using syringes. The moduli of the hydrogel were improved compared to that of the parent single-network. The hydrogel exhibited self-healing ability without need for heating or cut surface treatment. The incorporation of agar in the double network induced the enhanced protein adsorption, and the following cell attachments were governed by the adsorbed protein states. Therefore, the double network hydrogel holds great potential for applications in various biomedical applications.


Assuntos
Ágar/química , Carboximetilcelulose Sódica/química , Adesão Celular/efeitos dos fármacos , Quitosana/química , Hidrogéis/química , Hidrogéis/farmacologia , Fenômenos Mecânicos , Adsorção , Albuminas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Fibrinogênio/química , Camundongos , Células NIH 3T3
14.
Dokl Biochem Biophys ; 484(1): 37-41, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012009

RESUMO

Oxidation of fibrinogen with hypochlorite inhibited the fibrin network self-assembly even at the lowest concentration of the oxidant. The analysis of the results of protein electrophoresis at this hypochlorite concentration showed the absence of fragmentation of the protein and covalent cross-linking of its chains. The study of the areas responsible for the conversion of fibrinogen into fibrin by mass spectrometry showed that they are not subject to oxidative damage. However, we identified oxidized amino acid residues, which could affect the protofibril aggregation.


Assuntos
Fibrina/química , Fibrinogênio/química , Hipoclorito de Sódio/química , Feminino , Humanos , Masculino , Oxirredução
15.
Biophys Chem ; 249: 106149, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30981137

RESUMO

Proteins in solution affect the structural and dynamic properties of the bulk water at the protein-water interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step toward the determination of the size of the water ordering contributions originate in proteins systems.


Assuntos
Fibrinogênio/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ribonuclease Pancreático/química , Albumina Sérica Humana/química , Água/química , Humanos , Propriedades de Superfície , Termodinâmica
16.
Vox Sang ; 114(4): 374-385, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937927

RESUMO

BACKGROUND/OBJECTIVES: Prothrombin complex concentrates (PCC) are increasingly administered off-label in the United States to treat bleeding in cardiovascular surgical patients and carry the potential risk for acquired thromboembolic side-effects after surgery. Therefore, we hypothesized that the use of low-dose 3-factor (3F) PCC (20-30 IU/kg), as part of a transfusion algorithm, reduces bleeding without increasing postoperative thrombotic/thromboembolic complications. MATERIALS/METHODS: After IRB approval, we retrospectively analysed 114 consecutive, complex cardiovascular surgical patients (age > 18 years), between February 2014 and June 2015, that received low-dose 3F-PCC (Profilnine® ), of which seven patients met established exclusion criteria. PCC was dosed according to an institutional perioperative algorithm. Allogeneic transfusions were recorded before and after PCC administration (n = 107). The incidence of postoperative thromboembolic events was determined within 30 days of surgery, and Factor II levels were measured in a subset of patients (n = 20) as a quality control measure to avoid excessive PCC dosing. RESULTS: Total allogeneic blood product transfusion reached a mean of 12·4 ± 9·9 units before PCC and 5·0 ± 6·3 units after PCC administration (P < 0·001). The mean PCC dose was 15·8 ± 7·1 IU/kg. Four patients (3·8%) each experienced an ischaemic stroke on postoperative day 1, 2, 4 and 27. Seven patients (6·5%) had acquired venous thromboembolic disease within 10 days of surgery. Median factor II level after transfusion algorithm adherence and PCC administration was 87%. CONCLUSIONS: 3F-PCC use for refractory bleeding after cardiovascular surgery resulted in reduced transfusion of allogeneic blood and blood products. Adherence to this algorithmic approach was associated with an acceptable incidence of postoperative thrombotic/thromboembolic complications.


Assuntos
Fatores de Coagulação Sanguínea/química , Coagulação Sanguínea , Hemorragia/terapia , Hemostasia , Tromboembolia/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Testes de Coagulação Sanguínea , Plaquetas/citologia , Transfusão de Sangue , Ponte Cardiopulmonar , Feminino , Fibrinogênio/química , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Estudos Retrospectivos , Estados Unidos , Adulto Jovem
17.
Free Radic Res ; 53(4): 430-455, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30935261

RESUMO

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bß, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole "a" and hole "b" to knob "A" and knob "B", respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046. Highlights Various oxidative modifications were detected in fibrinogen by mass spectrometry αC-connector has been shown to be most susceptible to oxidation E region proved to be least vulnerable to the action of the oxidising agent Some of the Met residues in the fibrinogen structure could operate as ROS scavengers.


Assuntos
Fibrinogênio/química , Espectrometria de Massas/métodos , Ozônio/farmacologia , Fragmentos de Peptídeos/química , Fibrinogênio/efeitos dos fármacos , Humanos , Oxirredução , Fragmentos de Peptídeos/efeitos dos fármacos
18.
J Biol Chem ; 294(22): 8834-8847, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30992366

RESUMO

Proteinases are essential drivers of allergic airway disease and innate antifungal immunity in part through their ability cleave the clotting factor fibrinogen (FBG) into fibrinogen cleavage products (FCPs) that signal through Toll-like receptor 4 (TLR4). However, the mechanism by which FCPs engage TLR4 remains unknown. Here, we show that the proteinases from Aspergillus melleus (PAM) and other allergenic organisms rapidly hydrolyze FBG to yield relatively few FCPs that drive distinct antifungal mechanisms through TLR4. Functional FCPs, termed cryptokines, were characterized by rapid loss of the FBG α chain with substantial preservation of the ß and γ chains, including a γ chain sequence (Fibγ390-396) that binds the integrin Mac-1 (CD11b/CD18). PAM-derived cryptokines could be generated from multiple FBG domains, and the ability of cryptokines to induce fungistasis in vitro and innate allergic airway disease in vivo strongly depended on both Mac-1 and the Mac-1-binding domain of FBG (Fibγ390-396). Our findings illustrate the essential concept of proteinase-activated immune responses and for the first time link Mac-1, cryptokines, and TLR4 to innate antifungal immunity and allergic airway disease.


Assuntos
Aspergillus/imunologia , Antígeno CD11b/metabolismo , Fibrinogênio/metabolismo , Proteínas Fúngicas/metabolismo , Imunidade Inata , Peptídeo Hidrolases/metabolismo , Animais , Aspergillus/enzimologia , Antígeno CD11b/deficiência , Antígeno CD11b/genética , Modelos Animais de Doenças , Fibrinogênio/química , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptor 4 Toll-Like/metabolismo
19.
J Chem Phys ; 150(15): 155103, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31005110

RESUMO

In this paper, we studied the influence of nonmagnetic iron oxide nanoparticles on fibrin gel formation and its structure using dynamic light scattering. The surface of nanoparticles produced by a new method in acoustoplasma discharge with cavitation has specific morphology and accelerates the rate of fibrin gel formation, i.e., activates the enzyme thrombin. We studied changes in the form of autocorrelation functions of the scattered light intensity for fibrinogen-thrombin samples with different thrombin concentrations as well as the nanoparticles addition. Appearance of the power-law term in the function was an indicator of gel formation in the sample. Application of Martin's theory allows estimating the exponent φ of power-law function and the contribution of the diffusive mode of protofibrils. We found that an increase in thrombin concentration or its activation with iron oxide nanoparticles leads to decreasing contribution of the diffusive mode, and increasing contribution of the exponent of power-law function. The values of fractal dimension Df calculated using Muthukumar's theory are 1.61 ± 0.13 and 1.69 ± 1.11 for samples with low and high concentrations of thrombin respectively and 1.77 ± 0.08 for the sample with thrombin activated by nanoparticles. Such an increase in fractal dimension shows an increase in the complexity of the fibrin gel structure (or density).


Assuntos
Compostos Férricos/química , Fibrina/química , Fractais , Géis/química , Nanopartículas Metálicas/química , Fibrinogênio/química , Humanos , Luz , Espalhamento de Radiação , Trombina/química
20.
J Biol Chem ; 294(22): 8773-8778, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31028172

RESUMO

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.


Assuntos
Fator XIIIa/química , Fibrina/química , Mapeamento de Peptídeos/métodos , Peptídeos/análise , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Fator XIIIa/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Fibrinogênio/química , Humanos , Lisina/química , Espectrometria de Massas , Trombose/metabolismo , Trombose/patologia
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