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1.
Phytochemistry ; 165: 112025, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207449

RESUMO

Thrombin inhibitor therapy is one of the most effective therapeutic strategies for the prevention and treatment of cardiovascular and thrombotic diseases. Although several marketed direct thrombin inhibitors (DTIs) have been widely used in clinic, the potentially serious complications of these DTIs prompted the researchers to find more DTIs with improved safety profiles. Herein, we report that natural anthraquinones in Cassiae semen (the seed of Cassia obtusifolia L. or C. tora L.), including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin, display strong to moderate inhibition on human thrombin, with the IC50 values ranging from 9.08 µM to 27.88 µM. Further investigation on the inhibition kinetics demonstrates that these anthraquinones are mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, while obtusifolin and aurantio-obtusin show strong thrombin inhibition capacity, with the Ki values of 9.63 µM and 10.30 µM, respectively. Docking simulations demonstrate that both obtusifolin and aurantio-obtusin can simultaneously bind on the catalytic cavity and the two anion binding exosites (ABE1 and ABE2), while the hydroxyl group at the C-7 site and the methoxyl group at the C-8 site can create key interactions with the amino acids surrounding the catalytic cavity via hydrogen bonding. All these findings suggest that obtusifolin and aurantio-obtusin are strong thrombin inhibitors possessing a unique anthraquinone skeleton, and could be used as lead compounds for the development of new thrombin inhibitors with improved properties.


Assuntos
Antraquinonas/farmacologia , Cassia/química , Simulação de Acoplamento Molecular , Inibidores de Serino Proteinase/farmacologia , Trombina/antagonistas & inibidores , Antraquinonas/química , Antraquinonas/isolamento & purificação , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/isolamento & purificação , Relação Estrutura-Atividade , Trombina/metabolismo
2.
Int J Nanomedicine ; 14: 4059-4069, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31213815

RESUMO

Background and purpose: Traumatic brain injury (TBI) is a major disease without effective treatment. Recently, Tat-NR2B9c peptide emerged as a promising neuroprotective agent, but limited in clinical translation by it low brain penetrability. We synthesized Tat-NR2B9c loaded self-assembled activatable protein nanoparticles, termed TN-APNPs, and demonstrated that TN-APNPs enhanced the delivery of Tat-NR2B9c to the brain lesion in stroke. Herein we developed a novel approach to further engineering TN-APNPs for targeted delivery of Tat-NR2B9c to the injured brain with enhanced efficiency through conjugation of CAQK or CCAQK, a short peptide. Methods: Short peptide-conjugated TN-APNPs were synthesized by conjugated with CAQK or CCAQK via a click condensation reaction with CBT, then analyzed by dynamic light scattering, transmission electron microscopy and thrombin responsive assay. Characterization of short peptide-conjugated TN-APNPs were investigated by using cell excitotoxicity assay and transwell blood-brain-barrier model in vitro, and pharmacokinetics, IVIS imaging system and confocal analysis in TBI-bearing mice. Evaluation of therapeutic effects were analyzed by H&E staining, Elevated Plus Maze analysis and Rotarod test. Results: CAQK-conjugated TN-APNPs (C-TN-APNPs) and CCAQK-conjugated TN-APNPs (CC-TN-APNPs) were spherical in morphology and 30 nm in diameter. In vitro studies revealed that TN-APNPs, C-TN-APNPs and CC-TN-APNPs were responsive to thrombin cleavage, reduced the cytotoxicity of Tat-NR2B9c, and increased BBB permeability of Tat-NR2B9c. CC-TN-APNPs demonstrated the better circulation time, better targeting ability and penetrating efficiency to the injured brain, and better therapeutic benefits in vivo studies. Conclusion: This study demonstrated CC-TN-APNPs as a promising therapeutic for clinical management of TBI.


Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Nanopartículas/química , Peptídeos/uso terapêutico , Animais , Lesões Encefálicas Traumáticas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/metabolismo , Portadores de Fármacos/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Peptídeos/síntese química , Peptídeos/farmacologia , Teste de Desempenho do Rota-Rod , Trombina/uso terapêutico
3.
Biochimie ; 163: 12-20, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31059753

RESUMO

In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ±â€¯11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.


Assuntos
Catepsina L/metabolismo , Rhipicephalus/enzimologia , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Catepsina L/química , Catepsina L/isolamento & purificação , Bovinos , Cinética , Modelos Moleculares , Proteólise
4.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 5-11, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102351

RESUMO

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-ß-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -ß-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS: Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.


Assuntos
Pirofosfatases/química , Canais de Cátion TRPM/química , Canais de Cátion TRPM/isolamento & purificação , Escherichia coli/genética , Glucosídeos/química , Humanos , Domínios Proteicos , Estabilidade Proteica , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/metabolismo
5.
Eur J Med Chem ; 176: 187-194, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31103899

RESUMO

Inhibitors of the flaviviral serine proteases, which are crucial for the replication of dengue and West-Nile virus, have attracted much attention over the last years. A dibasic 4-guanidinobenzoate was previously reported as inhibitor of the dengue protease with potency in the low-micromolar range. In the present study, this lead structure was modified with the intent to explore structure-activity relationships and obtain compounds with increased drug-likeness. Substitutions of the guanidine moieties, the aromatic rings, and the ester with other functionalities were evaluated. All changes were accompanied by a loss of inhibition, indicating that the 4-guanidinobenzoate scaffold is an essential element of this compound class. Further experiments indicate that the target recognition of the compounds involves the reversible formation of a covalent adduct.


Assuntos
Amidas/química , Antivirais/química , Carbamatos/química , Ésteres/química , Inibidores da Tripsina/química , Amidas/síntese química , Antivirais/síntese química , Carbamatos/síntese química , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Estabilidade de Medicamentos , Ésteres/síntese química , Estrutura Molecular , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Inibidores da Tripsina/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/enzimologia
6.
Anal Chim Acta ; 1070: 112-122, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31103164

RESUMO

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4-177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2-199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Sistemas On-Line , Técnica de Seleção de Aptâmeros/métodos , Animais , Calorimetria , Bovinos , Eletroforese Capilar , Humanos , Imunoglobulina G/análise , Lactoferrina/análise , Ligantes , Muramidase/análise , Muramidase/metabolismo , Trombina/análise
7.
J Extra Corpor Technol ; 51(1): 29-37, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30936586

RESUMO

Direct thrombin inhibitors (DTIs), such as bivalirudin and dabigatran, have maintained steady inpatient and outpatient use as substitutes for heparin and warfarin, respectively, because of their high bioavailability and relatively safe "on-therapy" range. Current clinical methods lack the capacity to directly quantify plasma DTI concentrations across wide ranges. At present, the gold standard is the ecarin clotting time (ECT), where ecarin maximizes thrombin activity and clotting time is evaluated to assess DTIs' anticoagulation capability. This work focused on the development of a microfluidic paper analytic device (µPAD) that can quantify the extent of anticoagulation as well as DTI concentration within a patient's whole blood sample. Capillary action propels a small blood sample to flow through the nitrocellulose paper channels. Digital images of whole blood migration are then captured by our self-coded Raspberry Pi and/or the Samsung Galaxy S8 smartphone camera. Both the flow length and the blue absorbance from the plasma front on the µPAD were measured, allowing simultaneous, dual assays: ecarin clotting test (ECT) and ecarin chromogenic assay (ECA). Statistically significant (p < .05) changes in flow and absorbance were observed within our translational research study. Currently, there are no quantitative, commercially available point-of-care tests for the ECT and ECA within the United States. Both the ECT and ECA assays could be instrumental to differentiate between supratherapeutic and subtherapeutic incidents during bridging anticoagulant therapy and limit the unwarranted use of reversal agents.


Assuntos
Antitrombinas , Sistemas Automatizados de Assistência Junto ao Leito , Anticoagulantes , Testes de Coagulação Sanguínea , Endopeptidases , Humanos , Microfluídica , Trombina
8.
Angiol Sosud Khir ; 25(1): 131-142, 2019.
Artigo em Russo | MEDLINE | ID: mdl-30994619

RESUMO

The problem of haemostasis in cardiovascular surgery is of current concern. Recent trends are towards increased use of topical haemostatic agents. Tachocomb has been used for more than 30 years in abdominal surgery and oncology. The purpose of the present publication is to analyse the literature data and formulate the indications for the use of Tachocomb in cardiovascular surgery. Multicenter randomized and local studies have demonstrated efficacy of Tachocomb in treatment of surgical haemorrhage in operations on the heart, thoracic aorta, carotid arteries and lower-limb arteries, resulting in significantly decreased time to achieve haemostasis, decreased volume of blood loss and haemotrasfusion, as well as reduced frequency of complications. Also shown was economic efficacy, including a shortened length of patients' hospital stay. This is followed by describing a wide spectrum of examples of alternative use of Tachocomb, including its use for seamless closure of defects of cardiac chamber walls, aero- and lymphostasis, prevention of formation of commissures. Convincing experimental and clinical results make it possible to formulate a series of indications for the use of Tachocomb in cardiovascular surgery.


Assuntos
Procedimentos Cirúrgicos Cardiovasculares , Hemostáticos , Trombina , Perda Sanguínea Cirúrgica , Hemostasia , Hemostasia Cirúrgica , Hemostáticos/uso terapêutico , Humanos
9.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979095

RESUMO

Many nucleic acid-protein structures have been resolved, though quantitative structure-activity relationship remains unclear in many cases. Thrombin complexes with G-quadruplex aptamers are striking examples of a lack of any correlation between affinity, interface organization, and other common parameters. Here, we tested the hypothesis that affinity of the aptamer-protein complex is determined with the capacity of the interface to dissipate energy of binding. Description and detailed analysis of 63 nucleic acid-protein structures discriminated peculiarities of high-affinity nucleic acid-protein complexes. The size of the amino acid sidechain in the interface was demonstrated to be the most significant parameter that correlates with affinity of aptamers. This observation could be explained in terms of need of efficient energy transfer from interacting residues. Application of energy dissipation theory provided an illustrative tool for estimation of efficiency of aptamer-protein complexes. These results are of great importance for a design of efficient aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Ácidos Nucleicos/química , Proteínas/química , Fenômenos Biofísicos , Transferência de Energia , Fenômenos Mecânicos , Complexos Multiproteicos/química , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Trombina/química
10.
Biosens Bioelectron ; 133: 177-182, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928736

RESUMO

In this paper, a three-line LFB was successfully developed by adding a thrombin line to a conventional two-line LFB for the detection of thrombin in a wide range of human serum. We introduced a thrombin line between the test line and the control line. The concentration of thrombin in the sample was quantitatively related to the signal formation on the three lines of the LFB. We can make use of signal on three lines to quantitative determinate the thrombin by data processing. The detection range of thrombin concentrations measured in 10 min was 1 nM to 100 µM and the LOD was 0.85 nM. Our approach paves way for rapid and sensitive thrombin detection and a superior device for testing in a wide range of physiological concentrations, which also can be used in other hook-effect-limited aptamers or antibodies based sandwich LFBs, and has a high accuracy even within the range of the hook-effect.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Nanopartículas Metálicas/química , Trombina/isolamento & purificação , Ouro/química , Humanos , Limite de Detecção , Trombina/química
12.
Ann Hematol ; 98(8): 1855-1865, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30997536

RESUMO

Haemophilia A (HA) is caused by a lack or reduced amount of factor VIII protein (FVIII). About one-third of patients with non-severe HA carrying specific missense mutations show discrepant results between FVIII activity (FVIII:C), measured by one-stage or chromogenic two-stage assays. The aim of this study was to elucidate the mechanism underlying the assay discrepancy in vitro and in silico. Thirteen missense mutations in the Factor 8-gene associated with discrepant results in patients were transiently expressed. FVIII:C of the mutations was determined using two one-stage assays (FVIII:C1st, FVIII:CBonn) and a two-stage chromogenic assay (FVIII:Cchr). Furthermore, thrombin generation test (TGT) and in silico analysis were performed to investigate the haemostatic potential as well as the structural impact of the variants, respectively. For the majority (9/13) of the analysed mutations, the discrepancy was confirmed. Moreover, we established a modified TGT protocol for in vitro characterization of FVIII. Hence, TGT parameters were significantly impaired in the group of variants associated with higher chromogenic values. Additionally, in silico analysis revealed the impact of the mutations on FVIII protein structure leading to assay discrepancy. Moreover, the data shows that also among one-stage clotting assays, assay discrepancy is observed. Our results show that for the majority of mutations, application of a global assay like TGT method could help to improve diagnosis or correct assessment of the severity of HA.


Assuntos
Bioensaio/normas , Fator VIII/química , Hemofilia A/diagnóstico , Hemofilia A/genética , Mutação de Sentido Incorreto , Testes de Coagulação Sanguínea , Simulação por Computador , Fator VIII/genética , Fator VIII/metabolismo , Expressão Gênica , Hemofilia A/sangue , Hemofilia A/patologia , Humanos , Masculino , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Índice de Gravidade de Doença , Trombina/química , Trombina/metabolismo
13.
Immunity ; 50(4): 1024-1026, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995493

RESUMO

Hemostasis and immunity were long considered entirely separate entities. In this issue of Immunity, Burzynski et al. (2019) find that thrombin, the key enzyme within the coagulation cascade, activates IL-1α, a central pleiotropic pro-inflammatory cytokine, to promote wound healing and platelet production following ectoderm injury.


Assuntos
Interleucina-1alfa , Trombina , Coagulação Sanguínea , Sistema Imunitário , Imunidade Inata
14.
Transfusion ; 59(S2): 1560-1567, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30980741

RESUMO

BACKGROUND: Cryoprecipitate's shelf life is limited due to concerns over decreased clotting factor activity and contamination with extended storage. Hemostatic characteristics of thawed cryoprecipitate stored up to 35 days at refrigerated and room temperatures were assessed. STUDY DESIGN AND METHODS: Pooled cryoprecipitate was thawed and aliquoted for storage at 1-6°C or 21-24°C. Samples were tested immediately after thawing and at 4 h, 24 h, 72 h, and weekly for 35 days. At each time point fibrinogen, factor VIII (FVIII), and von Willebrand factor (vWF) were assessed. Thrombin generation and rotational thromboelastometry (ROTEM) were also performed. Further, packed red cells, platelet concentrates, frozen plasma, and stored cryoprecipitate were combined (1:1:1:1) to simulate massive transfusion and analyzed by ROTEM. Day 35 samples were cultured for bacterial contamination. RESULTS: Precipitation was observed in refrigerated samples; however, these aggregates were easily resuspended upon warming in a 37°C water bath. No significant changes were observed in fibrinogen concentration or ROTEM at either temperature. FVIII and vWF declined significantly during storage. vWF, clot time, and thrombin generation were significantly better preserved with refrigeration. With simulated massive transfusion, fibrinogen function remained at or above the established range for whole blood at both storage temperatures. Bacterial contamination was not observed in cold stored or room temperature cryoprecipitate. CONCLUSION: The fibrinogen concentration and function of cryoprecipitate at extended storage durations are adequate for fibrinogen replacement in critical bleeding. These results support extension of the shelf life of cryoprecipitate when used for fibrinogen replacement.


Assuntos
Criopreservação , Fator VIII/metabolismo , Fibrinogênio/metabolismo , Hemostáticos/metabolismo , Transfusão de Sangue , Humanos , Tromboelastografia , Trombina/metabolismo , Fatores de Tempo , Fator de von Willebrand/metabolismo
15.
Ann Vasc Surg ; 59: 173-183, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31028852

RESUMO

BACKGROUND: Ultrasound-guided thrombin injection (UGTI) has become the method of choice in the treatment of pseudoaneurysm caused by endovascular procedures because it is minimally invasive, costs less, and effective, with short hospitalization time. The objective was identify the morphological aspects of femoral pseudoaneurysms and clinical aspects of patients that may lead to the failure of UGTI in femoral pseudoaneurysms after cardiac catheterization. POPULATION AND METHOD: From December 2012 to December 2016, 60 patients with pseudoaneurysms caused by cardiac catheterization were referred to the interventional radiology unit to be treated with UGTI. Medical charts were retrospectively reviewed for comorbidities, use of antiplatelet agents, anticoagulation, indication of cardiac catheterization, and so forth. Morphological aspects of the pseudoaneurysms such as volume, diameter (anteroposterior, laterolateral, and longitudinal), length, and diameter of the neck were analyzed. RESULTS: Technical success of UGTI was achieved in 100%. No clinical aspects of the patients were statistically significant for UGTI failure in occlusion of the pseudoaneurysms. For morphological aspects of pseudoaneurysm: anteroposterior (P = 0.029), longitudinal (P = 0.020), and neck diameters (P = 0.004) were statistically significant for UGTI failure. Logistic regression analysis for longitudinal diameter showed that for each centimeter, there was a 2.66 chance of failure of pseudoaneurysm thrombosis in a single thrombin injection session (95% confidence interval: 1.33-5.30). For longitudinal and neck diameters greater than 1.8 cm and 0.55 cm, respectively, there is a greater probability of needing more than one UGTI session for complete thrombosis. CONCLUSIONS: Among variables, the longitudinal dimension was more significant, and in a larger diameter, the treatment with thrombin injection presented greater complexity.


Assuntos
Falso Aneurisma/tratamento farmacológico , Cateterismo Cardíaco/efeitos adversos , Cateterismo Periférico/efeitos adversos , Artéria Femoral , Trombina/administração & dosagem , Lesões do Sistema Vascular/tratamento farmacológico , Falso Aneurisma/diagnóstico por imagem , Falso Aneurisma/etiologia , Cateterismo Cardíaco/métodos , Cateterismo Periférico/métodos , Feminino , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/lesões , Humanos , Injeções Intra-Arteriais , Masculino , Punções , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Trombina/efeitos adversos , Falha de Tratamento , Ultrassonografia de Intervenção , Lesões do Sistema Vascular/diagnóstico por imagem , Lesões do Sistema Vascular/etiologia
16.
Cardiovasc Diabetol ; 18(1): 49, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992036

RESUMO

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with a hypercoagulable state and increased neutrophil extracellular traps formation (NETosis). We investigated predictors of NETosis and cell death markers in circulating blood and their association with a prothrombotic state in T2DM. METHODS: In a cross-sectional study involving 113 T2DM patients aged 63.7 ± 8.2 years, we investigated citrullinated histone H3 (H3Cit), cell-free deoxyribonucleic acid (cfDNA), myeloperoxidase, neutrophil elastase, and inflammation markers, along with thrombin generation (TG), plasma clot lysis time (CLT), clot permeability (Ks) and fibrinolysis inhibitors. RESULTS: On multivariate logistic regression analysis adjusted for age and gender, predictors of high H3Cit (≥ 7.36 ng/mL, upper quartile) were: glycated hemoglobin (HbA1c) ≥ 7.0% and interleukin-6. Interleukin-6 was also found to be a predictor of high cfDNA (≥ 2.84 µg/mL, upper quartile) along with glucose. Citrullinated histone H3 and cfDNA correlated positively with CLT and inversely with Ks, while TG associated solely with cfDNA. These associations were not seen with myeloperoxidase and neutrophil elastase. Patients with previous myocardial infarction (n = 21, 18.6%) had higher H3Cit (+108%, p < 0.001) and cfDNA (+45%, p = 0.022). On multivariable analysis adjusted for potential confounders, H3Cit and cfDNA, along with plasminogen activator inhibitor-1 and concomitant cardiovascular disease, were predictors of CLT. Citrullinated histone H3 alone was a predictor of Ks and only cfDNA was a predictor of peak thrombin generated. CONCLUSIONS: In T2DM, NETosis detectable in circulating blood is associated with inflammatory state and a prothrombotic state, especially hypofibrinolysis.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Armadilhas Extracelulares/metabolismo , Fibrinólise , Trombose/sangue , Idoso , Biomarcadores/sangue , Glicemia/metabolismo , Ácidos Nucleicos Livres/sangue , Citrulinação , Estudos Transversais , DNA/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Hemoglobina A Glicada/metabolismo , Histonas/sangue , Humanos , Mediadores da Inflamação/sangue , Elastase de Leucócito/sangue , Masculino , Pessoa de Meia-Idade , Peroxidase/sangue , Fatores de Risco , Trombina/metabolismo , Trombose/diagnóstico , Trombose/etiologia
17.
Medicine (Baltimore) ; 98(15): e15223, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30985725

RESUMO

OBJECTIVES: The purpose of this study was to compare transurethral resection of the prostate (TURP) versus holmium laser enucleation of the prostate (HoLEP) in patients with benign prostatic hyperplasia (BPH) focusing on perioperative thrombin generation and fibrinolysis. METHODS: Sixty-five BPH patients were prospectively randomly assigned to undergo TURP (n = 32) or HoLEP (n = 33). The prothrombin fragment (PF) 1+2, thrombin-antithrombin complex (TAT), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) were measured preoperatively, at the 1st day and 3rd day after surgery. RESULTS: PF1+2, TAT, t-PA, and PAI-1 significantly elevated at day 1 and day 3 after surgery (P < .05) and remarkedly decreased from the 1st day to the 3rd day (P < .05). The levels of PF1+2 and TAT were higher in TURP group postoperatively than that in HoLEP group (P < .05). There is no significant difference between 2 groups in regard of t-PA and PAI-1 (P > .05). CONCLUSION: The activation of thrombin generation and fibrinolysis system were noticed in BPH patients after TURP or HoLEP. TURP may associate with a higher hypercoagulable thrombotic risk than HoLEP.


Assuntos
Hólmio/uso terapêutico , Terapia a Laser , Hiperplasia Prostática/sangue , Hiperplasia Prostática/cirurgia , Ressecção Transuretral da Próstata , Idoso , Fibrinólise , Humanos , Masculino , Período Perioperatório , Próstata/cirurgia , Trombina/metabolismo , Resultado do Tratamento
18.
Chem Commun (Camb) ; 55(39): 5627-5630, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31025680

RESUMO

The reversible photocontrol of an enzyme governing blood coagulation is demonstrated. The thrombin binding aptamer (TBA), was rendered photochromic by modification with two anthracene groups. Light-triggered anthracene photodimerisation distorts its structure, inhibiting binding of the enzyme thrombin, which in turn triggers catalysis and the resulting clotting process.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Quadruplex G , Trombina/metabolismo , Raios Ultravioleta , Antracenos/química , Aptâmeros de Nucleotídeos/química , Biocatálise , Coagulação Sanguínea , Dicroísmo Circular , Dimerização , Humanos , Ligação Proteica , Temperatura Ambiente , Trombina/química
19.
Adv Mater ; 31(21): e1808262, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30972819

RESUMO

Nucleic acid aptamers selected for thrombin binding have been previously shown to possess anticoagulant activity; however, problems with rapid renal clearance and short circulation half-life have prevented translation to clinical usefulness. Here, a family of self-folding, functional RNA origami molecules bearing multiple thrombin-binding RNA aptamers and showing significantly improved anticoagulant activity is described. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures are designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'-fluoro-modified C- and U-nucleotides is shown to increase nuclease resistance and stability during long-term storage. Specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA is demonstrated. The RNA origami constructs show anticoagulant activity sevenfold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity is maintained after at least 3 months of storage in buffer at 4 °C. Additionally, inhibition of thrombin is shown to be reversed by addition of single-stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.


Assuntos
Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , RNA/química , Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Humanos , Conformação de Ácido Nucleico , Tempo de Tromboplastina Parcial , RNA/farmacologia , Trombina/antagonistas & inibidores , Trombina/genética , Trombina/metabolismo
20.
Br J Radiol ; 92(1098): 20180893, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982331

RESUMO

OBJECTIVE: Evaluate the factors associated with pseudoaneurysm development and morphology. Measure ultrasound-guided thrombin injection (UGTI) and surgical treatment outcomes for pseudoaneurysms, and the necessity for post-primary intervention (reintervention). METHODS: Retrospective analysis of ultrasound scans and CT angiography of peripheral pseudoaneurysm interventions from February 2011 to April 2017. 99 patients (61.6% female) were identified; median age 72 years (range 12-89). Multivariate analysis of patient demographics, including pre-intervention anticoagulant and antiplatelet medication was performed. RESULTS: Primary intervention for peripheral pseudoaneurysms (62.6% common femoral artery) included 93 UGTI and 6 surgical repairs; with 12 reinterventions (9 UGTI and 3 surgical). Pseudoaneurysm incidence for vascular interventional radiology (VIR) was 0.48% (31/6451) and cardiology was 0.24% (60/25,229). Rates for primary success, immediate complications, 30-day mortality and reintervention were measured respectively for UGTI (98.9%, 3.0%, 2.0%, and 11.8%) and surgical repair (100%, 0.0%, 22.2% and 16.7%). Reintervention risk factors included pre-intervention thrombocytopaenia (<150 × 109/L) ( p = 0.025) and pseudoaneurysms following vascular surgery (p = 0.033). Other positive associations for reintervention (non-significant) included use of a sheath size > 6 Fr ( p = 0.108) or arterial closure device ( p = 0.111) during the pseudoaneurysm causative procedure. The pre-intervention warfarin subgroup developed a larger mean pseudoaneurysm sac size (4.21 cm, range 0.9-7.6), compared to no treatment (p = 0.003), aspirin (p = 0.005) and clopidogrel (p = 0.026) subgroups. UGTI dosage for thrombosis had a positive correlation with incremental sac size increase (p < 0.001). CONCLUSION: The main reintervention risk factor was pre-intervention thrombocytopaenia, with additional positive associations including pseudoaneurysms caused by surgery, increased sheath size and arterial closure devices. Warfarinized patients developed larger sac-sized pseudoaneurysms compared with other pre-intervention regimens, with positive correlation of higher UGTI dosage required for thrombosis. ADVANCES IN KNOWLEDGE: Patients with pre-intervention thrombocytopaenia and pseudoaneurysms attributed to vascular surgery are subgroups that may benefit from post-intervention imaging surveillance due to significant reintervention risk.


Assuntos
Falso Aneurisma/etiologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Doença Arterial Periférica/etiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Falso Aneurisma/diagnóstico por imagem , Falso Aneurisma/cirurgia , Anticoagulantes/uso terapêutico , Criança , Angiografia por Tomografia Computadorizada , Feminino , Hemostáticos/administração & dosagem , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/diagnóstico por imagem , Doença Arterial Periférica/cirurgia , Inibidores da Agregação de Plaquetas/uso terapêutico , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Reoperação/estatística & dados numéricos , Estudos Retrospectivos , Trombina/administração & dosagem , Ultrassonografia , Ultrassonografia de Intervenção , Adulto Jovem
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