RESUMO
Arterial thrombosis contributes to some of the most frequent causes of mortality globally, such as myocardial infarction and stroke. Platelets are essential mediators of physiological haemostasis and pathological thrombosis. Platelet activation is controlled by a multitude of signalling pathways. Upon activation, platelets shed platelet-derived extracellular vesicles (pEVs). In this Special Issue: Extracellular Vesicles, Moon et al. investigate the impact of various platelet agonists (thrombin, ADP, collagen) on the proteome of pEVs. The study demonstrates that pEVs exhibit an agonist-dependent altered proteome compared to their parent cells, with significant variations in proteins related to coagulation, complement, and platelet activation. The study observes the rapid generation of pEVs following agonist stimulation with specific proteome alterations that underscore an active packaging process. This commentary highlights the implications of their findings and discusses the role of pEV cargo in cardiovascular disease with potential novel therapeutic and diagnostic opportunities.
Assuntos
Plaquetas , Vesículas Extracelulares , Ativação Plaquetária , Proteoma , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Proteoma/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteômica/métodos , Trombina/metabolismo , Trombina/farmacologiaRESUMO
BACKGROUND: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown. METHODS: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury. RESULTS: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors. CONCLUSIONS: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.
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Armadilhas Extracelulares , Fígado , Trombose , Armadilhas Extracelulares/metabolismo , Animais , Camundongos , Fígado/patologia , Fígado/metabolismo , Trombose/etiologia , Trombose/patologia , Colestase/patologia , Colestase/complicações , Modelos Animais de Doenças , Masculino , Tromboplastina/metabolismo , Trombofilia/etiologia , Trombofilia/sangue , Fibrina/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Humanos , Infiltração de Neutrófilos , Fator Xa/metabolismo , Trombina/metabolismoRESUMO
BACKGROUND: Acute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model. METHODS: In vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically. RESULTS: In vitro, IL-1ß, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group. CONCLUSION: Intratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.
Assuntos
Lesão Pulmonar Aguda , Vesículas Extracelulares , Células-Tronco Mesenquimais , Camundongos Endogâmicos ICR , Trombina , Animais , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/transplante , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/microbiologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Camundongos , Células-Tronco Mesenquimais/metabolismo , Células RAW 264.7 , Trombina/metabolismo , Escherichia coli , Masculino , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/terapia , Resultado do Tratamento , Modelos Animais de Doenças , HumanosAssuntos
Células Endoteliais , Etanol , Pulmão , Receptor PAR-1 , Trombina , Humanos , Trombina/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-1/genética , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Etanol/farmacologia , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Multimerização Proteica/efeitos dos fármacosRESUMO
Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.
Assuntos
Sistema de Sinalização das MAP Quinases , Músculo Liso Vascular , Trombina , Trombina/farmacologia , Trombina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Rigidez Vascular/efeitos dos fármacos , Células Cultivadas , Ratos , Humanos , Ratos Sprague-Dawley , Hormônios Peptídicos/metabolismo , Hormônios Peptídicos/farmacologia , Hormônios Peptídicos/genéticaRESUMO
INTRODUCTION: There are important pharmacological differences between direct oral anticoagulants (DOAC) and a deeper knowledge of how they influence different aspects of hemostasis in patients on treatment is desirable. MATERIALS AND METHODS: Blood samples from patients on dabigatran (n = 23), rivaroxaban (n = 26), or apixaban (n = 20) were analyzed with a fibrin network permeability assay, a turbidimetric clotting and lysis assay, the calibrated automated thrombogram (CAT), plasma levels of thrombin-antithrombin complex (TAT) and D-dimer, as well as DOAC concentrations, PT-INR and aPTT. As a comparison, we also analyzed samples from 27 patients on treatment with warfarin. RESULTS: Patients on dabigatran had a more permeable fibrin network, longer lag time (CAT and turbidimetric assay), and lower levels of D-dimer in plasma, compared with patients on rivaroxaban- and apixaban treatment, and a more permeable fibrin network than patients on warfarin. Clot lysis time was slightly longer in patients on dabigatran than in patients on rivaroxaban. Warfarin patients formed a more permeable fibrin network than patients on apixaban, had longer lag time than patients on rivaroxaban (CAT assay), and lower peak thrombin and ETP compared to patients on treatment with both FXa-inhibitors. CONCLUSIONS: Results from this study indicate dabigatran treatment is a more potent anticoagulant than apixaban and rivaroxaban. However, as these results are not supported by clinical data, they are probably more related to the assays used and highlight the difficulty of measuring and comparing the effect of anticoagulants.
Assuntos
Dabigatrana , Fibrina , Fibrinólise , Pirazóis , Piridonas , Rivaroxabana , Trombina , Humanos , Dabigatrana/farmacologia , Rivaroxabana/farmacologia , Pirazóis/farmacologia , Piridonas/farmacologia , Piridonas/uso terapêutico , Trombina/metabolismo , Masculino , Fibrina/metabolismo , Feminino , Idoso , Fibrinólise/efeitos dos fármacos , Pessoa de Meia-Idade , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Permeabilidade/efeitos dos fármacos , Varfarina/farmacologia , Idoso de 80 Anos ou mais , Anticoagulantes/farmacologiaRESUMO
BACKGROUND/AIM: How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. MATERIALS AND METHODS: Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. RESULTS: Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. CONCLUSION: Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.
Assuntos
Fibrinólise , Glucocorticoides , Inibidor 1 de Ativador de Plasminogênio , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Fibrinólise/efeitos dos fármacos , Glucocorticoides/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Trombina/metabolismo , Trombina/farmacologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Ativação Transcricional/efeitos dos fármacos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Coagulação Sanguínea/efeitos dos fármacosRESUMO
OBJECTIVES: To explore the evidence, urinary biomarkers, and partial mechanisms of hypercoagulability in the pathogenesis of IgA vasculitis (IgAV). METHODS: Differential expression of proteins in the urine of 10 healthy children and 10 children with IgAV was screened using high-performance liquid chromatography-tandem mass spectrometry, followed by Reactome pathway analysis. Protein-protein interaction (PPI) network analysis was conducted using STRING and Cytoscape software. In the validation cohort, 15 healthy children and 25 children with IgAV were included, and the expression levels of differential urinary proteins were verified using enzyme-linked immunosorbent assay. RESULTS: A total of 772 differential proteins were identified between the IgAV group and the control group, with 768 upregulated and 4 downregulated. Reactome pathway enrichment results showed that neutrophil degranulation, platelet activation, and hemostasis pathways were involved in the pathogenesis of IgAV. Among the differential proteins, macrophage migration inhibitory factor (MIF) played a significant role in neutrophil degranulation and hemostasis, while thrombin was a key protein in platelet activation and hemostasis pathways. PPI analysis indicated that thrombin directly interacted with several proteins involved in inflammatory responses, and these interactions involved MIF. Validation results showed that compared to healthy children, children with IgAV had significantly higher urine thrombin/creatinine and urine MIF/creatinine levels (P<0.05). CONCLUSIONS: Thrombin contributes to the pathogenesis of IgAV through interactions with inflammatory factors. Urinary thrombin and MIF can serve as biomarkers reflecting the hypercoagulable and inflammatory states in children with IgAV.
Assuntos
Vasculite por IgA , Proteômica , Trombina , Humanos , Criança , Masculino , Proteômica/métodos , Feminino , Vasculite por IgA/urina , Trombina/metabolismo , Fatores Inibidores da Migração de Macrófagos/urina , Mapas de Interação de Proteínas , Pré-Escolar , Oxirredutases IntramolecularesAssuntos
Cirrose Hepática , Protrombina , Trombina , Humanos , Protrombina/metabolismo , Cirrose Hepática/sangue , Trombina/metabolismo , Coagulação Sanguínea , Deficiência de Antitrombina III/sangue , Deficiência de Antitrombina III/complicações , Deficiência de Antitrombina III/diagnóstico , Deficiência de Antitrombina III/genéticaRESUMO
Peptides and molecular residues sourced from the fragmentation of the extracellular matrix (ECM) can exacerbate a plethora of cellular functions. We selected a natural ECM-derived complex peptide mixture to functionalize sodium alginate. Three alginate derivatives (sodium alginate conjugated with ECM) SALE-1, SALE-2, and SALE-3 were synthesized using the lowest (10 % w/w), moderate (50 % w/w), and highest (100 % w/w) concentrations of ECM. Thereafter, they were used to fabricate three groups of mat scaffolds EMAT-1 (ECM derivatized alginate thrombin-mat), EMAT-2, and EMAT-3, respectively by the freeze-drying process. To enhance the hemostatic activity, thrombin was loaded onto the scaffolds. Another group, AT, without any derivatized alginate was additionally included in order to comparative analysis. Physical characteristics revealed that the porous mat scaffold showed enhancement in degradation and swelling ability with the increase in ECM content. The higher cell proliferation, migration, and cell viability were noticed in the higher ECM-containing samples EMAT-2 and EMAT-3. In vivo studies using rodent hepatic and rabbit ear models were carried out to ensure the hemostatic ability of the scaffolds. EMAT-2 and EMAT-3 demonstrate excellent liver regeneration ability in rat models. Moreover, the rat cutaneous wound model depicted that EMAT-3 dramatically elevated the skin's healing ability, thereby rendering it an excellent candidate for future clinical application in wound healing.
Assuntos
Alginatos , Matriz Extracelular , Liofilização , Fígado , Pele , Alicerces Teciduais , Cicatrização , Animais , Alginatos/química , Matriz Extracelular/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Coelhos , Cicatrização/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Ratos , Alicerces Teciduais/química , Masculino , Humanos , Ratos Sprague-Dawley , Proliferação de Células/efeitos dos fármacos , Trombina/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
Pulmonary embolism (PE) is a heterogenous condition with variable clinical presentations. Thrombin generation potential (TGP) and biomarkers, and blood cellular indices can reflect the underlying pathophysiology and risk stratification of PE. This case-control study analyzed TGP in 209 PE patients from Loyola University, Pulmonary Embolism Response Team program compared to normal human plasma (NHP) controls. The present study evaluates TGP and biomarkers, and cellular indices in relation to PE severity, according to the European Society of Cardiology (ESC) guidelines. Statistical analysis including median with interquartile range (IQR), 2-tailed Wilcoxon Mann-Whitney test, Chi-square test, and Spearman Correlational analysis were performed. There were 209 patients with PE, with an almost equal distribution between sex, and a median age of 63 years. Significant downregulation in peak thrombin and endogenous thrombin potential (ETP), as well as upregulation in lag time, were observed in PE patients versus controls. Biomarker analysis revealed pronounced elevations, with D-dimer demonstrating the most significant increase. Blood cellular indices also rose in PE patients, correlating with disease severity. PE severity was associated with higher TGP and biomarker levels. Mortality rates differed significantly across risk categories and were highest in patients with elevated cellular indices. TGP and biomarkers are intricately linked to PE severity and can aid in risk stratification. Elevated cellular indices are associated with increased mortality, highlighting their potential as prognostic markers. These findings could enhance the precision of PE management strategies.
Assuntos
Biomarcadores , Embolia Pulmonar , Trombina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores/sangue , Estudos de Casos e Controles , Embolia Pulmonar/sangue , Trombina/metabolismo , Trombina/biossíntese , Trombina/análiseRESUMO
Among biosensing systems for sensitive diagnoses fluorescence enhancement techniques have attracted considerable attention. This study constructed a simple multilayered structure comprising a plane metal mirror coated with a plasma-polymerized film (PPF) as an optical interference layer on a glass slide for fluorescence enhancement. Plasma polymerization enables the easy deposition of organic thin films containing functional groups, such as amino groups. This study prepared PPFs using acetonitrile as a monomer, and the influences of washing and the output powers of plasma polymerization on PPF thickness were examined by Fourier transform infrared spectroscopy. This is because controlling the PPF thickness is vital in fluorescence enhancement. Multilayered glass slides prepared using a silver layer with 84 nm-thick acetonitrile PPFs exhibited 11- and 281-fold fluorescence enhancements compared with those obtained from the substrates with a bare surface and only modified by the silver layer, respectively. Oligonucleotides labeled with a thiol group and cyanine5 were successfully immobilized on the multilayered substrates, and the fluorescence of the acetonitrile PPFs was superior to that of the allylamine and cyclopropylamine PPFs. Furthermore, an aptamer-based sandwich assay targeting thrombin was performed on the multilayered glass slides, resulting in an approximately 5.1-fold fluorescence enhancement compared with that obtained from the substrate with a bare surface. Calibration curves revealed the relationship between fluorescence intensity and thrombin concentration of 10-1000 nM. This study demonstrates that PPFs can function as materials for fluorescence enhancement, immobilization for biomaterials, and aptamer-based sandwich assays.
Assuntos
Acetonitrilas , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Acetonitrilas/química , Polimerização , Espectrometria de Fluorescência/métodos , Prata/química , Trombina/análise , Humanos , Carbocianinas/química , Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Corantes Fluorescentes/químicaRESUMO
Rapid and safe hemostasis is crucial for the survival of bleeding patients in prehospital care. It is urgent to develop high performance hemostatic material to control the massive hemorrhage in the military field and accidental trauma. In this work, an efficient protein hemostat of thrombin was immobilized onto commercial gauze, which was mediated by self-polymerization and anchoring of tannic acid (TA). Through TA treatment, the efficient immobilization of thrombin was achieved, preserving both the biological activity of thrombin and the physical properties of the dressing, including absorbency, breathability, and mechanical performance. Moreover, in the presence of TA coating and thrombin, Gau@TA/Thr could obviously shortened clotting time and enriched blood components such as plasma proteins, platelets, and red blood cells, thereby exhibiting an enhanced in vitro coagulation effect. In SD rat liver volume defect and artery transection hemorrhage models, Gau@TA/Thr still had outstanding hemostatic performance. Besides, the Gau@TA/Thr gauze had inherent antibacterial property and demonstrated excellent biocompatibility. All results suggested that Gau@TA/Thr would be a potential candidate for treating uncontrollable hemorrhage in prehospital care.
Assuntos
Bandagens , Coagulação Sanguínea , Hemorragia , Hemostáticos , Taninos , Trombina , Taninos/química , Taninos/farmacologia , Animais , Hemorragia/tratamento farmacológico , Trombina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Ratos , Hemostáticos/farmacologia , Hemostáticos/química , Ratos Sprague-Dawley , Masculino , Anti-Infecciosos/farmacologia , Humanos , Proteínas Imobilizadas/farmacologia , Proteínas Imobilizadas/química , Modelos Animais de Doenças , PolifenóisRESUMO
BACKGROUND: During pituitary surgery, CSF leaks are often treated by intrasellar packing, using muscle or fat grafts. However, this strategy may interfere with the interpretation of postoperative MRI and may impact the quality of resection in cases of second surgery, due to the existence of additional fibrous tissue. We present an alternative technique, using a diaphragm reconstruction with a heterologous sponge combining fibrinogen and thrombin (TachoSil), applied in selected patients with low-flow CSF leaks. This study investigates the surgical outcome of patients treated with this strategy. METHODS: From a cohort of 2231 patients treated from June 2011 to June 2023 by endoscopic endonasal approach for pituitary surgery, the surgical technique of diaphragm repair with TachoSil patch performed in 55 patients (2.6%) was detailed, and the rate of closure failure was analyzed at 6 months postoperatively. No intrasellar packing was used and sellar floor reconstruction was performed whenever possible. The rate of postoperative CSF leak was compared with that reported in three previous publications that also used the TachoSil patch technique. RESULTS: Patients were mostly women (F/M ratio: 1.2) with a median age of 53.6 years. Surgery was indicated for non-functioning adenomas, Cushing's disease, acromegaly, and Rathke's cleft cysts in 38/55 (69.1%), 6/55 (10.9%), 5/55 (9.1%) and 6/55 (10.9%) patients respectively. The rate of postoperative CSF leak was 1.8% (n = 1/55), which was not significantly different from that reported in the three cohorts from the literature (2.8%, p > 0.05). No postoperative meningitis was recorded. CONCLUSIONS: In highly selected patients with low-flow CSF leaks related to small focal diaphragm defects, diaphragm reconstruction using a TachoSil patch can be a safe and valuable alternative to intrasellar packing.
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Vazamento de Líquido Cefalorraquidiano , Combinação de Medicamentos , Fibrinogênio , Procedimentos de Cirurgia Plástica , Trombina , Humanos , Feminino , Pessoa de Meia-Idade , Trombina/uso terapêutico , Masculino , Fibrinogênio/uso terapêutico , Adulto , Vazamento de Líquido Cefalorraquidiano/cirurgia , Idoso , Procedimentos de Cirurgia Plástica/métodos , Estudos de Coortes , Diafragma/cirurgia , Complicações Pós-Operatórias , Neoplasias Hipofisárias/cirurgia , Resultado do Tratamento , Rinorreia de Líquido Cefalorraquidiano/cirurgia , Hipófise/cirurgia , Tampões de Gaze CirúrgicosRESUMO
BACKGROUND: The convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of complex samples by solid-phase extraction (SPE). Compared to the in-tube solid-phase microextraction in fused-silica capillaries, micro SPE in plastic pipette tips have fascinating merits for the easily operated enrichment of trace target analytes from biological samples. However, the poor compatibility of organic-inorganic hybrid monoliths with plastics leads to the rare appearance of commercial hybrid monolithic pipette tips (HMPTs). Therefore, how to synthesize the organic-inorganic hybrid monolithic materials with better extraction performance in plastic pipette tips becomes a challenge. RESULTS: We develop a facile and cheap strategy to immobilize organic-inorganic hybrid monoliths in pipette tips. Melamine sponge was employed as the supporting skeleton to in situ assemble amine- and thiol-bifunctionalized hybrid monolithic material via "one pot" in a pipette tip, and gold nanoparticles (GNPs) and thiol-modified aptamer against human α-thrombin were sequentially attached to the hybrid monolith within the HMPTs. The average coverage density of the aptamer with GNPs as an intermediary reached as high as 818.5 pmol µL-1. The enriched thrombin concentration was determined by a sensitive enzymatic chromogenic assay with the limit of detection of 2 nM. The extraction recovery of thrombin at 10 nM in human serum was 86.1 % with a relative standard deviation of 6.1 %. This proposed protocol has been applied to the enrichment and determination of thrombin in real serum sample with strong anti-interference ability, low limit of detection and high recovery. SIGNIFICANCE: The amine- and thiol-bifunctionalized HMPTs prepared with sponge as the skeleton frame provided a novel substrate material to decorate aptamers for efficient enrichment of proteins. This enlightens us that we can take advantage of the tunability of sponge assisted HMPTs to produce and tailor a variety of micro SPE pipette tips for broader applications on the analysis of trace targets in complex biological, clinic and environmental samples.
Assuntos
Aptâmeros de Nucleotídeos , Trombina , Triazinas , Triazinas/química , Triazinas/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Humanos , Trombina/análise , Trombina/isolamento & purificação , Ouro/química , Nanopartículas Metálicas/química , Extração em Fase Sólida/métodosRESUMO
The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological rolesâsubstrate, receptor, and inhibitorâand were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.
Assuntos
Peptídeos , Pontos Quânticos , Trombina , Pontos Quânticos/química , Peptídeos/química , Peptídeos/metabolismo , Trombina/metabolismo , Trombina/análise , Trombina/química , Fator Xa/metabolismo , Fator Xa/química , Proteólise , Humanos , Propriedades de Superfície , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/químicaRESUMO
Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Bicamadas Lipídicas , Técnicas de Microbalança de Cristal de Quartzo , Trombina , Trombina/análise , Bicamadas Lipídicas/química , Aptâmeros de Nucleotídeos/química , Humanos , Fosfatidilcolinas/químicaRESUMO
A mid-infrared label-free immunoassay-based biosensor is an effective device to help identify and quantify biomolecules. This biosensor employs a surface-enhanced infrared absorption spectroscopy, which is a highly potent sensing technique for detecting minute quantities of analytes. In this study, a biosensor was constructed using a metamaterial absorber, which facilitated strong coupling effects. For maximum coupling effect, it is necessary to enhance the near-field intensity and the spatial and spectral overlap between the optical cavity resonance and the vibrational mode of the analyte. Due to significant peak splitting, conventional baseline correction methods fail to adequately analyze such a coupling system. Therefore, we employed a coupled harmonic oscillation model to analyze the spectral distortion resulting from the peak splitting induced by the strong coupling effect. The proposed biosensor with a thrombin-binding aptamer-based immunoassay could achieve a limit of detection of 267.4 pM, paving the way for more efficient protein detection in clinical practice.