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1.
Cardiovasc Hematol Agents Med Chem ; 17(2): 115-124, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31622211

RESUMO

AIM: This study aims to find out the components responsible for the antithrombotic activity of Nelumbo nucifera. MATERIAL AND METHODS: Petroleum ether, chloroform and hydroalcoholic extracts of whole plant of Nelumbo nucifera (Lotus) were prepared and assessed for its thrombolytic, anti-platelet aggregation activity and bleeding time. The extracts were further analyzed through HPTLC and GC-MS. Statistical analysis was conducted through ANOVA trailed by Tukey's multiple comparison test test. RESULTS: Hydroalcoholic extract showed the highest activity at the concentration of 400µg/ml in thrombolytic assay (42.03 ± 5.76), anti-platelet aggregation assay (57.93 ± 1.68) and bleeding time (70.17 ± 2.16) in comparison to clopodigrel (33.76 ± 3.43), aspirin (66.55 ± 1.86) and aspirin (93.85 ± 2.75) at the concentration of 100 µg/ml respectively. 25 peaks were identified through GC-MS, out of which, ferulic acid (14.2µ/g) and quercetin (5.4 µ/g) are active chemical compounds. HPTLC showed different chromatograms in hydroalcoholic extracts like (1) chlorogenic, (2) quercetin, (3) benzoic acid, (4) caffeic acid, (5) ferulic acid, (6) kaempferol, and (7) gallic acid. CONCLUSION: Based on these findings, flavonoids present in hydroalcoholic extract may be developed into a drug for clinical application for the treatment of thrombosis in patients.


Assuntos
Fibrinolíticos/uso terapêutico , Flavonoides/uso terapêutico , Nelumbo/química , Extratos Vegetais/uso terapêutico , Inibidores da Agregação de Plaquetas/uso terapêutico , Trombose/tratamento farmacológico , Animais , Tempo de Lise do Coágulo de Fibrina , Fibrinolíticos/química , Flavonoides/química , Masculino , Extratos Vegetais/química , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/química , Ratos Wistar , Terapia Trombolítica
2.
Eur J Med Chem ; 183: 111722, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31563807

RESUMO

Thrombosis is a pathological coagulation process and can lead to many serious thrombotic diseases. Here, we report a novel potent antithrombotic compound (6k) based on isosteviol with anticoagulant and antiplatelet activities. 6k selectively inhibited FXa (Ki = 0.015 µM) against a panel of serine proteases and showed excellent anticoagulant activity (significant prolongation of ex vivo PT and aPTT over the vehicle, p < 0.01). 6k also significantly inhibited ADP-induced platelet aggregation in rats relative to the vehicle (p < 0.01). Furthermore, 6k exhibited potent ex vivo and in vivo antithrombotic activity in rats relative to the vehicle (p < 0.01 and p < 0.0001, respectively). Novel structure 6k, with potent antithrombotic activity, is expected to lead a promising approach for the development of antithrombotic agents.


Assuntos
Diterpenos de Caurano/química , Diterpenos de Caurano/farmacologia , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Inibidores da Agregação de Plaquetas/química , Inibidores da Agregação de Plaquetas/farmacologia , Trombose/tratamento farmacológico , Animais , Descoberta de Drogas , Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Feminino , Humanos , Masculino , Tempo de Tromboplastina Parcial , Agregação Plaquetária/efeitos dos fármacos , Ratos , Ratos Wistar , Trombina/metabolismo
3.
ACS Appl Mater Interfaces ; 11(35): 31627-31637, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31412200

RESUMO

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray's law and comprises a network of channels ranging in size from capillaries (∼50 µm) to large arterioles and venules (∼400 µm). Micro-PIV (micro-particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.


Assuntos
Materiais Biomiméticos , Fibrinolíticos/química , Hedera/anatomia & histologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Dispositivos Lab-On-A-Chip , Folhas de Planta/anatomia & histologia , Trombose/metabolismo , Ativador de Plasminogênio Tecidual/química , Transporte Biológico , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Trombose/patologia
4.
Carbohydr Polym ; 222: 115025, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320079

RESUMO

Fucosylated chondroitin sulfate (FCS) oligosaccharides of specific molecular weight have shown potent anticoagulant activities with selectivity towards intrinsic factor Xase complex. However, the preparation of FCS oligosaccharides by traditional methods requires multiple purification steps consuming large amounts of time and significant resources. The current study focuses on developing a method for the rapid preparation of FCS oligomers from sea cucumber Pearsonothuria graeffei having 6-18 saccharide residues. The key steps controlling molecular weight (Mw) and purity of these FCS oligomers were evaluated. Structural analysis showed the resulting FCS oligomers were primarily l-Fuc3,4diS-α1,3-d-GlcA-ß1,3-(d-GalNAc4,6diS-ß1,4-[l-Fuc3,4diS-α1,3-]d-GlcA-ß1,3-)nd-anTal-ol4,6diS (n = 1˜5) accompanied by partial de-fucosylation and/or de-sulfation. In vitro and in vivo experiments demonstrate that these FCS oligomers selectively inhibit intrinsic factor Xase complex and exhibit remarkable antithrombotic activity without hemorrhagic and hypotension side effects. This method is suitable for large-scale preparation of FCS oligosaccharides as clinical anticoagulants.


Assuntos
Anticoagulantes/uso terapêutico , Sulfatos de Condroitina/uso terapêutico , Fator IXa/antagonistas & inibidores , Fator VIIIa/antagonistas & inibidores , Fibrinolíticos/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Sequência de Carboidratos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Cisteína Endopeptidases , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Masculino , Camundongos , Coelhos , Ratos Sprague-Dawley , Pepinos-do-Mar/química , Trombose Venosa/tratamento farmacológico
5.
Int J Biol Macromol ; 134: 622-630, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31047931

RESUMO

Thrombin, a multifunctional serine protease responsible for the proteolytic hydrolysis of soluble fibrinogen, plays a pivotal role in the blood coagulation cascade. Currently, thrombin inhibitor therapy has been recognized as an effective therapeutic strategy for the prevention and treatment of thrombotic diseases. In this study, the inhibitory effects of natural constituents in St. John's Wort against human thrombin are carefully investigated by a fluorescence-based biochemical assay. The results clearly demonstrate that most of naphthodianthrones, flavonoids and biflavones exhibit strong to moderate inhibition on human thrombin. Among all tested compounds, hypericin shows the most potent inhibitory capability against thrombin, with the IC50 value of 3.00 µM. Further investigation on inhibition kinetics demonstrates that hypericin is a potent and reversible inhibitor against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki value of 2.58 µM. Inhibition kinetic analyses demonstrate that hypericin inhibits thrombin-mediated Z-GGRAMC acetate hydrolysis in a mixed manner, which agrees well with the results from docking simulations that hypericin can bind on both catalytic cavity and anion binding exosites. All these findings suggest that hypericin is a natural thrombin inhibitor with a unique dianthrone skeleton, which can be used as a good candidate to develop novel thrombin inhibitors with improved properties.


Assuntos
Fibrinolíticos/química , Fibrinolíticos/farmacologia , Hypericum/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Relação Dose-Resposta a Droga , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Perileno/análogos & derivados , Perileno/química , Perileno/farmacologia , Proteólise , Relação Estrutura-Atividade
6.
Mar Drugs ; 17(3)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884850

RESUMO

Marine organisms exhibit some advantages as a renewable source of potential drugs, far beyond chemotherapics. Particularly, the number of marine natural products with antithrombotic activity has increased in the last few years, and reports show a wide diversity in scaffolds, beyond the polysaccharide framework. While there are several reviews highlighting the anticoagulant and antithrombotic activities of marine-derived sulfated polysaccharides, reports including other molecules are sparse. Therefore, the present paper provides an update of the recent progress in marine-derived sulfated polysaccharides and quotes other scaffolds that are being considered for investigation due to their antithrombotic effect.


Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Organismos Aquáticos/química , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Relação Estrutura-Atividade
7.
Mar Drugs ; 17(3)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871149

RESUMO

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.


Assuntos
Proteínas de Bactérias/química , Endopeptidases/biossíntese , Fibrinolíticos/química , Peptídeo Hidrolases/química , Poríferos/microbiologia , Streptomyces/química , Streptomyces/enzimologia , Animais , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/farmacologia , Filogenia , Espectroscopia de Infravermelho com Transformada de Fourier , Trombose/tratamento farmacológico
8.
ACS Appl Mater Interfaces ; 11(10): 9777-9785, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30785265

RESUMO

Surface wetting occurring in daily life causes undesired contaminations, which are critical issues in various fields. To solve these problems, the nonwetting property of a superhydrophobic (SH) surface has proven its utility by preventing contaminant infiltration, serious infections, or malfunction. However, the application of SH surfaces in the biomedical field has been limited due to the weak durability and toxicity of the related components. To overcome these limitations, we developed a robust and biocompatible SH surface through combinational biomimicking of three natural organisms, lotus leaf, mussel, and sandcastle worm, for the first time. Using the water-immiscible and polycationic characteristics of mussel adhesive protein (iMglue), an SH iMglue-SiO2(TiO2/SiO2)2 coating was fabricated by solution-based electrical charge-controlled layer-by-layer growth of nanoparticles (NPs). The fabricated iMglue-SiO2(TiO2/SiO2)2 SH surface showed excellent durable nonwetting properties and was applied to an intracatheter tube coating to develop antithrombotic catheters under blood flow. Furthermore, we developed a iMglue-employed SH patch for a tissue closure bandage by spraying hydrophobic SiO2 NPs on the iMglue-covered cotton pads. The prepared iMglue-employing SH patch showed perfect bifunctionality with excellent antibiofouling and tissue closure capabilities. Our work presents a novel, useful strategy for fabricating a biomedically multifunctional, robust SH surface through combinational mimicking of natural organisms.


Assuntos
Materiais Biomiméticos/farmacologia , Fibrinolíticos/farmacologia , Nanopartículas/química , Agentes Molhantes/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Incrustação Biológica , Materiais Biomiméticos/química , Bivalves/química , Fibrinolíticos/química , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lotus/química , Folhas de Planta/química , Proteínas/química , Dióxido de Silício/química , Propriedades de Superfície , Titânio/química , Água/química , Agentes Molhantes/química
9.
Food Funct ; 10(2): 886-892, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30693925

RESUMO

Various bioactive peptides are identified from casein hydrolysates. YQEPVLGPVR (PICA), a novel antithrombotic peptide derived from beta-casein (fragment 193-202), was identified by high-performance liquid chromatography - liquid chromatography-mass spectrometry/mass spectrometry. The anticoagulation activity assay showed that this peptide has a strong anticoagulant activity. It was proved that the peptide did not interact with the active site of thrombin to inhibit thrombin, and that it inhibited thrombin activity by binding the exosite-1 of thrombin, which was also confirmed by the fibrinogen clotting time assay. It was shown that PICA prolonged fibrinogen clotting time in a dose-dependent manner. Secondary structures of the thrombin-PICA complex were also measured by circular dichroism to prove that PICA can combine with thrombin. Moreover, Discovery Studio 2017 R2 software was used for molecular docking to provide the potential mechanism for the antithrombotic activity of the peptide. These results suggested that PICA probably can be used as an antithrombotic ingredient in the functional food industry.


Assuntos
Caseínas/química , Fibrinolíticos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fibrinogênio/antagonistas & inibidores , Fibrinogênio/química , Fibrinolíticos/química , Modelos Moleculares , Conformação Proteica , Trombina/química
10.
J Ethnopharmacol ; 231: 394-402, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30359761

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), Rehmanniae Radix (RR, derived from the root of Rehmannia glutinosa (Gaertn.) DC.) is commonly used as natural medicine for thousands of years, two types including the dried and rice-wine processed RR were used for different clinical purposes respectively, which were the typical case that pharmaceutical effect changed by processing in TCM. AIM OF STUDY: The goal of this study was to investigate the differences in the antithrombosis and hematopoietic effects of extracts of dried and processed RR (DRR and PRR) in vivo, and to explore the chemical basis underlying changes of medicinal properties caused by processing. MATERIALS AND METHODS: The aqueous extracts of DRR and PRR were prepared. Protective effect of varying doses of different extracts were investigated in type-I carrageenan induced mice tail thrombosis and cyclophosphamide induced myelosuppression model. The chemical composition of DRR and PRR extracts were determined by High Performance Liquid Chromatography coupled with tandem quadrupole time-of-flight Mass Spectrometry (HPLC/Q-TOF-MS). RESULTS: In antithrombosis activity tests, PRR possessed less ameliorated effects than DRR in the model mouse on body temperature, tail thrombus length and blood flow. Both DRR and PRR had no significant influence on prothrombin time (PT) and activated partial thromboplastin time (APTT), only high dose DRR could decrease the content of fibrinogen (FIB) in plasma. Histological examination of lung tissue suggested that thrombosis was significantly improved in DRR-H group. For myelosuppression model, only PRR could improve peripheral hemogram, both DRR and PRR had hematopoietic effects as demonstrated by their abilities to ameliorate the bone marrow nucleated cells (BMNC) and pathology of bone marrow tissue. The hematopoietic effects of PRR were significantly more potent than that of DRR at the concentration of 9 g/kg. By comparing the chemical composition, we found that iridoid glycosides were decreased and furfural derivatives increased in DRR after processing which may be the chemical mechanism contribute to the differences in efficacy. CONCLUSIONS: According to the results of this research, processing with rice wine for nine cycles significantly reduced antithrombotic effects and enhanced the hematopoietic effects of DRR as demonstrated in model mice. It can scientifically explain the different effect among two types of RR in clinical through the diverse method of processing and usage. Meanwhile, the predicted activity compounds from two types of RR can be potential candidates for the treatment of thrombosis and anemia.


Assuntos
Fibrinolíticos/farmacologia , Hematínicos/farmacologia , Extratos Vegetais/farmacologia , Rehmannia , Animais , Dessecação , Fibrinolíticos/química , Hematínicos/química , Hematopoese/efeitos dos fármacos , Masculino , Camundongos Endogâmicos ICR , Oryza , Extratos Vegetais/química , Raízes de Plantas/química , Rehmannia/química , Trombose/tratamento farmacológico , Vinho
11.
ACS Appl Mater Interfaces ; 11(2): 1951-1956, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30571091

RESUMO

Thrombus diseases, induced by blood stasis or vascular embolization normally, frequently occur with high disability and mortalities worldwide. At present, drug thrombolysis, a primary clinical therapy for blood clot lysis, could increase the lethal risk for hemorrhage when thrombolysis agents are overused in the whole body. Therefore, a novel and advanced therapy for blood clot lysis, based on remote physical signals, is helpful for assisting clinical therapy. Here, we used the localized light-Au-hyperthermia (LAH) treatment, induced by gold nanorods (Au NRs) irradiated with near-infrared light (808 nm), for precise, rapid, and drug-free blood clot lysis. The LAH technology was first introduced in the murine hematoma model and the murine myocardial infarction model for blood clot lysis. Compared with traditional therapy, LAH was assured to shorten the time of detumescence in the murine hematoma model owing to their precise and localized hyperthermia. Meanwhile, we also discovered that LAH was a benefit to vascular recanalization in the murine myocardial infarction model. In addition, the Au NRs used in LAH present ideal biocompatibility in the murine model, which endows it to be suitable for blood clot lysis in vivo.


Assuntos
Fibrinolíticos , Ouro , Hipertermia Induzida/métodos , Nanopartículas Metálicas , Nanotubos/química , Terapia Trombolítica/métodos , Animais , Modelos Animais de Doenças , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacologia , Ouro/química , Ouro/farmacocinética , Ouro/farmacologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Ratos
12.
Int J Nanomedicine ; 13: 7835-7844, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538462

RESUMO

Background: Due to the discovery that deep venous thrombosis (DVT) inhibitor is of chemotherapeutic importance, the nano-property of dimethyl 2,2'-[2,2'-(ethane-1,1-diyl) bis(1H-indole-3,2-diyl)]-diacetate (DEBIC), a recently reported antitumor agent, is worthy of characterization. Materials and methods: One-pot reaction was used to prepare DEBIC. Electrospray Ionization (+/-)-Fourier Transform-Ion Cyclotron Resonance-Mass Spectrometer (ESI(+/-)-FT-ICR-MS), quadrupole Collision Induced Dissociation (qCID) and nuclear overhauser effect spectroscopy spectra were used to present the assembly of DEBIC. Transmission electron microscopy, scanning electron microscopy, atomic force microscopy and Faraday-Tyndall effect were used to visualize the nano-property of DEBIC. Rat models were used to evaluate DVT inhibition and the bleeding reaction of DEBIC. Results: One-pot reaction can provide DEBIC in acceptable yield and high purity. In water, rat plasma and lyophilized powders of DEBIC existed as particles of small nano-size. In vivo DEBIC inhibited DVT in a dose-dependent manner. The minimal effective dose of DEBIC was 1.7 µmol/kg. Even the dose of 36 µmol/kg/day DEBIC did not induce bleeding side effect in DVT rats like in warfarin (0.82 µmol/kg/day). Conclusion: DEBIC is a small molecule capable of nano-scale assembly, inhibiting venous thrombosis and inducing no bleeding side effect.


Assuntos
Fibrinolíticos/uso terapêutico , Hemorragia/complicações , Indóis/uso terapêutico , Nanopartículas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Trombose Venosa/tratamento farmacológico , Animais , Dimerização , Fibrinolíticos/sangue , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Liofilização , Indóis/sangue , Indóis/química , Indóis/farmacologia , Masculino , Nanopartículas/ultraestrutura , Tamanho da Partícula , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray
13.
Mar Drugs ; 16(11)2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30424528

RESUMO

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1⁻MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1⁻MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1⁻MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1⁻MSP-F4 with molecular weights of 24⁻240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.


Assuntos
Anticoagulantes/farmacologia , Clorófitas/química , Desoxiaçúcares/farmacologia , Fibrinolíticos/farmacologia , Mananas/farmacologia , Alga Marinha/química , Ácidos/química , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Testes de Coagulação Sanguínea , Desoxiaçúcares/química , Desoxiaçúcares/isolamento & purificação , Suplementos Nutricionais , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Humanos , Masculino , Mananas/química , Mananas/isolamento & purificação , Peso Molecular , Ratos , Ratos Sprague-Dawley , Análise Espectral/métodos , Sulfatos/química
14.
Int J Mol Sci ; 19(11)2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445728

RESUMO

A new series of 1,3,4-oxadiazoles derivatives was synthesized, characterized, and evaluated for their in vitro and in vivo anti-thrombotic activity. Compounds (3a⁻3i) exhibited significant clot lysis with respect to reference drug streptokinase (30,000 IU), and enhanced clotting time (CT) values (130⁻342 s) than heparin (110 s). High affinity towards 1NFY with greater docking score was observed for the compounds (3a, 3i, 3e, 3d, and 3h) than the control ligand RPR200095. In addition, impressive inhibitory potential against factor Xa (F-Xa) was observed with higher docking scores (5612⁻6270) with Atomic Contact Energy (ACE) values (-189.68 to -352.28 kcal/mol) than the control ligand RPR200095 (Docking score 5192; ACE -197.81 kcal/mol). In vitro, in vivo, and in silico results proposed that these newly synthesized compounds might be used as anticoagulant agents.


Assuntos
Fibrinolíticos/química , Fibrinolíticos/farmacologia , Simulação de Acoplamento Molecular , Oxidiazóis/química , Oxidiazóis/farmacologia , Adulto , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Humanos , Imagem Tridimensional , Masculino , Ratos Sprague-Dawley , Eletricidade Estática , Fatores de Tempo , Adulto Jovem
15.
Toxins (Basel) ; 10(11)2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30373118

RESUMO

Antistasin, first identified as a potent inhibitor of the blood coagulation factor Xa, is a novel family of serine protease inhibitors. In this study, we purified a novel antistasin-type inhibitor from leech Poecilobdella manillensis called poecistasin. Amino acid sequencing of this 48-amino-acid protein revealed that poecistasin was an antistasin-type inhibitor known to consist of only one domain. Poecistasin inhibited factor XIIa, kallikrein, trypsin, and elastase, but had no inhibitory effect on factor Xa and thrombin. Poecistasin showed anticoagulant activities. It prolonged the activated partial thromboplastin time and inhibited FeCl3-induced carotid artery thrombus formation, implying its potent function in helping Poecilobdella manillensis to take a blood meal from the host by inhibiting coagulation. Poecistasin also suppressed ischemic stroke symptoms in transient middle cerebral artery occlusion mice model. Our results suggest that poecistasin from the leech Poecilobdella manillensis plays a crucial role in blood-sucking and may be an excellent candidate for the development of clinical anti-thrombosis and anti-ischemic stroke medicines.


Assuntos
Fibrinolíticos , Sanguessugas , Inibidores de Serino Proteinase , Inibidores da Tripsina , Animais , Lesões das Artérias Carótidas/tratamento farmacológico , Fator XIIa/antagonistas & inibidores , Fator Xa/metabolismo , Feminino , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Hormônios de Invertebrado , Camundongos Endogâmicos C57BL , Elastase Pancreática/antagonistas & inibidores , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Inibidores de Serino Proteinase/uso terapêutico , Trombina/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/uso terapêutico
16.
Nucleic Acids Res ; 46(22): 12177-12185, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30357392

RESUMO

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties.


Assuntos
Aptâmeros de Nucleotídeos/química , Fibrinolíticos/química , Trombina/química , Motivos de Aminoácidos , Anticoagulantes/química , Dicroísmo Circular , Cristalografia por Raios X , Fibrinogênio/química , Quadruplex G , Humanos , Ligantes , Modelos Moleculares , Mutação , Oligonucleotídeos/química , Ligação Proteica , Conformação Proteica
17.
Carbohydr Polym ; 202: 554-562, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30287035

RESUMO

Glycoconjugates extracted from Genipa americana leaves (PE-Ga) were separated into two fractions, denominated as PFI and PFII (total carbohydrate: 23-36%/uronic acid: 9-30%; protein:4-5%; polyphenols:0.776-0.812 mg/g), mainly composed by arabinose, galactose and uronic acid and presenting high (PFI) and low (PFII) molecular weight (based on polyacrylamide electrophoresis gel and gel permeation chromatography). Uronic acid was also detected by FT-IR (wavenumbers: 1410 and 1333 cm-1) and NMR (α-GalpA). Deproteinization of glycoconjugates showed reduced protein and polyphenol levels with loss of its biological effects. PE-Ga and PFII prolonged clotting time-aPTT (3.6 and 1.8x), while PE-Ga and PFI inhibited by 48% (100 µg/µL) the ADP-induced platelet aggregation. In vivo, these glycoconjugates at 1 mg/kg inhibited (37-53%) venous thrombus formation (4.7 ± 0.1 mg) and increased bleeding time (PE-Ga and PFI:3.0x; PFII:1.7x vs. PBS:906 ± 16.7 s). In conclusion, the arabinogalactan-rich glycoconjugate of G. americana leaves, containing uronic acid, present antiplatelet, anticoagulant (intrinsic/common pathway) and antithrombotic effects, with low hemorrhagic risk.


Assuntos
Anticoagulantes/farmacologia , Fibrinolíticos/farmacologia , Galactanos/farmacologia , Glicoconjugados/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Rubiaceae/química , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Galactanos/química , Galactanos/isolamento & purificação , Glicoconjugados/química , Glicoconjugados/isolamento & purificação , Voluntários Saudáveis , Humanos , Folhas de Planta/química , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/química , Inibidores da Agregação de Plaquetas/isolamento & purificação , Ratos , Ratos Wistar , Trombose Venosa/tratamento farmacológico
18.
Int J Mol Sci ; 19(10)2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287737

RESUMO

Sipunculus nudus, an old marine species, has great potential for use as functional seafood due to its various bioactivities. Its potential antithrombotic activity pushed us to isolate the bio-active components bio-guided by tracking fibrinolytic activity. As a result, a novel protease named as SK (the kinase obtained from S. nudus) was obtained, which possessed a molecular weight of 28,003.67 Da and 15 N-terminal amino acid sequences of PFPVPDPFVWDTSFQ. SK exerted inhibitory effects on thrombus formation through improving the coagulation system with dose-effect relationship within a certain range. Furthermore, in most cases SK got obviously better effect than that of urokinase. With the help of untargeted mass spectrometry-based metabolomics profiling, arachidonic acid, sphingolipid, and nicotinate and nicotinamide mechanism pathways were found to be important pathways. They revealed that the effect mechanism of SK on common carotid arterial thrombosis induced by FeCl3 was achieved by inhibiting vessel contraction, platelet aggregation, adhesion, and release, correcting endothelial cell dysfunction and retarding process of thrombus formation. This study demonstrated SK was a promising thrombolytic agent on the basis of its comprehensive activities on thrombosis, and it should get further exploitation and utilization.


Assuntos
Trombose das Artérias Carótidas/tratamento farmacológico , Fibrinolíticos/metabolismo , Proteínas de Helminto/metabolismo , Nematoides/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Fibrinolíticos/química , Fibrinolíticos/uso terapêutico , Proteínas de Helminto/química , Proteínas de Helminto/uso terapêutico , Masculino , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Ratos , Ratos Sprague-Dawley
19.
Int J Biol Macromol ; 120(Pt B): 1817-1822, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30223052

RESUMO

The sulfated polysaccharide NP2 was isolated and purified from Nemacystus decipiens, the structure and antithrombotic activity of NP2 was further studied. NP2 was composed of fucose, glucuronic acid, galactose and xylose at molar ratios of 76.3:20.5:1.5:1.7. ES-CID-MS/MS results showed that NP2 had a backbone of α (1 → 3)-linked fucose and a branch was composed of Fuc-(2 → 1)-GlcA, which was agree with the results of NMR and methylation analysis. The results also show that the sulfate groups were substituted at the C2 or C4 positions of the fucose residues. In addition, analysis of the antithrombotic activity results indicated that NP2 can increase the percentage of t-PA/PAI-1, thereby suggesting that NP2 has high fibrinolytic activity and should be explored as a novel antithrombotic agent.


Assuntos
Fibrinolíticos/química , Fibrinolíticos/farmacologia , Feófitas/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Metilação , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo
20.
Bioorg Chem ; 81: 468-480, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30243238

RESUMO

In the present study, novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinolines (4a-l) were recognized and evaluated as G-Protein Coupled Receptor (GPCR) ligands through molecular evaluations. Thrombin mediates adhesion of mast cell, a type of cell abundantly found in connective tissue and releasing histamine and other substances during inflammatory and allergic reactions, through phosphoinositol 3-kinase pathway. With this background, as preliminary, 4a-l are resolute to be potential leads, designated from their effective phosphoinositol 3-kinase (PI3-Kinase) inhibition potentials, best-docked scores, comparative ligand efficiency, and significant structural attributes evaluated by ab initio simulations. Since thrombin is one of the main reason for various cancer invasion in association with PI3Kinase, a thrombolytic potential of the compounds also analyzed. The experimental in vitro studies confirmed the significant enhancement as PI3Kinase inhibitors and appreciable enhancement in MTT assay of breast and skin cancer cell lines. Significantly, acetophenone substituent in the quinoline scaffold could be coherent to note the significant binding affinity to all the evaluated drug targets.


Assuntos
Antineoplásicos/farmacologia , Fibrinolíticos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Quinolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Descoberta de Drogas , Fibrinolíticos/química , Halogenação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/química , Receptor PAR-1/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
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