RESUMO
Artrhospira (Spirulina) platensis produced fibrinolytic enzyme under mixotrophic conditions using corn steep liquor (CSL). The enzyme was extracted, purified by combination of two chromatographic techniques and biochemically characterized. Maximum fibrinolytic production (268.14 U mg-1) was obtained using liquid medium culture composed by 0.2% CLS after 10th day of cultivation. Fibrinolytic activity was higher when extracted by homogenization methods and was purified 32.72-fold with specific activity of 7988 U mg-1. Fibrin zymography showed an active band, indicated acts as a plasmin-like protein with molecular weight of 72 kDa. Fibrinolytic enzyme have optimum pH of 6.0, stable in the range of 6.0 to 10.0 during 24 h and optimum temperature at 40 °C with a stability below 50 °C. Fibrinolytic enzyme is a serine metalloprotease by to be enhanced by Fe2+ and inhibited by PMSF. The enzyme has higher enzymatic activity than most other fibrinolytic enzymes and is stable at temperature and pH human physiological. Overall, the fibrinolytic enzyme from A. platensis has attractive biochemical properties to potential applications in the treatment of thrombosis.
Assuntos
Meios de Cultura/química , Fibrinolíticos/metabolismo , Spirulina/enzimologia , Biomassa , Precipitação Química , Estabilidade Enzimática , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Fotossíntese , TemperaturaRESUMO
Acetylsalicylic acid (ASA) intoxication is potentially lethal. After ingestion, AAS is rapidly transformed into salicylic acid that dissociates into an hydrogen ion plus salicylate. Salicylate is the main form of AAS in the body and produces multiple alterations. Initially, the stimulation of the ventilatory center promotes a respiratory alkalosis. Then, the mitochondrial dysfunction induced by salicylate, will generate a progressive metabolic acidosis due to the accumulation of ketoacids, lactic acid and dicarboxylic acids among others. Another alterations include hydro electrolytic disorders, gastrointestinal lesions, neurological involvement, ototoxicity and coagulopathy. The correct handling of acetylsalicylic acid intoxication requires an thorough knowledge of its pharmacokinetics and pharmacodynamics. Treatment consists in life support measures, gastric lavage, activated charcoal and urinary alkalization to promote the excretion of salicylates. In some occasions, it will be necessary to start renal replacement therapy as soon as possible.
Assuntos
Humanos , Aspirina/intoxicação , Aspirina/metabolismo , Fibrinolíticos/intoxicação , Fibrinolíticos/metabolismo , Overdose de Drogas/fisiopatologia , Overdose de Drogas/terapia , Acidose/induzido quimicamente , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Aspirina/administração & dosagem , Overdose de Drogas/metabolismo , Hipoglicemia/induzido quimicamente , Hipotensão/induzido quimicamente , Mitocôndrias/efeitos dos fármacosRESUMO
Antithrombin (AT) is a serpin that inhibits mainly thrombin and fXa after being activated by binding to glycosaminoglycans as heparin and heparan sulfate. Upon binding, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. Recently, a new compound, named TMI, was discovered in our group with nanomolar affinity to antithrombin, and shown to be able to induce a partial activation of antithrombin. As TMI represents an original scaffold for structural optimizations aiming the development of new antithrombotic drugs, the present work demonstrated, through a series of molecular dynamics simulations, that TMI is able to modulate AT reactive center loop flexibility similarly to what is observed to heparin, as well as exposing AT P1 residue, Arg393. These results represent the first atomic level indication of AT conformational activation by TMI, and may offer a predictive basis for future studies aiming TMI structural optimization.
Assuntos
Antitrombinas/química , Ativadores de Enzimas/química , Fibrinolíticos/química , Heparina/química , Heparitina Sulfato/química , Fosfatos de Inositol/química , Regulação Alostérica , Antitrombinas/metabolismo , Sítios de Ligação , Desenho de Fármacos , Ativadores de Enzimas/metabolismo , Fator Xa/química , Fibrinolíticos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Trombina/químicaAssuntos
Aspirina/metabolismo , Aspirina/intoxicação , Overdose de Drogas/fisiopatologia , Overdose de Drogas/terapia , Fibrinolíticos/metabolismo , Fibrinolíticos/intoxicação , Acidose/induzido quimicamente , Aspirina/administração & dosagem , Overdose de Drogas/metabolismo , Humanos , Hipoglicemia/induzido quimicamente , Hipotensão/induzido quimicamente , Mitocôndrias/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
OBJECTIVES: In this work, we further investigated the effect of the compound LASSBio-752 in thrombosis models in rats. METHODS: Arterial and venous thrombosis model, ex-vivo recalcification time and aPTT and PT. KEY FINDINGS: In the venous thrombosis model, oral administration of LASSBio-752 [48.2 mg (100 µmol)/kg] one hour before the thrombus induction decreased thrombus weight by 37 ± 0.2%. Interestingly, the antithrombotic action of this compound [48.2 mg (100 µmol)/kg] occurred at 87.5 ± 2.1% of inhibition after 24 h of administration and showed a lasting activity. When tested on the arterial thrombosis model, after a 1-h interval, there was already an increase in time to total occlusion of 34 ± 2.4 min, but the greatest effect was observed at intervals between 6 and 15 h of administration, when no occlusion of the artery was observed. The antithrombotic effect was reduced after 24 h when the occlusion time was 23.8 ± 2.3 min, close to that of the control, 17.6 ± 2.0 min. We also observed that bleeding was not excessive in any of the intervals tested. CONCLUSIONS: Our results indicate that compound LASSBio-752 is a potential candidate for utilization in the treatment of thromboembolic diseases.
Assuntos
Doenças das Artérias Carótidas/tratamento farmacológico , Fibrinolíticos/administração & dosagem , Hemostáticos/administração & dosagem , Trombose Venosa/tratamento farmacológico , Administração Oral , Animais , Doenças das Artérias Carótidas/metabolismo , Feminino , Fibrinolíticos/metabolismo , Hemostáticos/metabolismo , Masculino , Ratos , Ratos Wistar , Tromboembolia/tratamento farmacológico , Tromboembolia/metabolismo , Trombose/tratamento farmacológico , Trombose/metabolismo , Resultado do Tratamento , Trombose Venosa/metabolismoRESUMO
Thrombin is a multifunctional enzyme with a key role in the coagulation cascade. Its functional modulation can culminate into normal blood coagulation or thrombosis. Thus, the identification of novel potent inhibitors of thrombin are of immense importance. Sculptin is the first specific thrombin inhibitor identified in the transcriptomics analysis of tick's salivary glands. It consists of 168 residues having four similar repeats and evolutionary diverged from hirudin. Sculptin is a competitive, specific and reversible inhibitor of thrombin with a Ki of 18.3 ± 1.9 pM (k on 4.04 ± 0.03 × 107 M-1 s-1 and k off 0.65 ± 0.04 × 10-3 s-1). It is slowly consumed by thrombin eventually losing its activity. Contrary, sculptin is hydrolyzed by factor Xa and each polypeptide fragment is able to inhibit thrombin independently. A single domain of sculptin alone retains ~45% of inhibitory activity, which could bind thrombin in a bivalent fashion. The formation of a small turn/helical-like structure by active site binding residues of sculptin might have made it a more potent thrombin inhibitor. In addition, sculptin prolongs global coagulation parameters. In conclusion, sculptin and its independent domain(s) have strong potential to become novel antithrombotic therapeutics.
Assuntos
Fibrinolíticos/química , Hirudinas/química , Fragmentos de Peptídeos/química , Peptídeos/química , Trombose/prevenção & controle , Animais , Ligação Competitiva , Coagulação Sanguínea/fisiologia , Domínio Catalítico , Cristalografia por Raios X , Fator Xa/química , Fator Xa/metabolismo , Fibrinolíticos/metabolismo , Expressão Gênica , Hirudinas/genética , Hirudinas/metabolismo , Humanos , Hidrólise , Ixodidae/química , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Trombose/sangue , Trombose/patologiaRESUMO
Low levels of high-density lipoprotein-cholesterol (HDL-C) constitute an independent biomarker of cardiovascular morbi-mortality. However, recent advances have drastically modified the classical and limited view of HDL as a carrier of 'good cholesterol', and have revealed unexpected levels of complexity in the circulating HDL particle pool. HDL particles are indeed highly heterogeneous in structure, intravascular metabolism and biological activity. This review describes recent progress in our understanding of HDL subpopulations and their biological activities, and focuses on relationships between the structural, compositional and functional heterogeneity of HDL particles.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Antioxidantes/metabolismo , Doenças Cardiovasculares/metabolismo , HDL-Colesterol/metabolismo , Fibrinolíticos/metabolismo , Vasodilatadores/metabolismo , Animais , Anti-Inflamatórios não Esteroides/classificação , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/classificação , Antioxidantes/farmacologia , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Biomarcadores/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , HDL-Colesterol/classificação , HDL-Colesterol/farmacologia , Citoproteção , Fibrinolíticos/classificação , Fibrinolíticos/farmacologia , Regulação da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasodilatadores/classificação , Vasodilatadores/farmacologiaRESUMO
Background Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 2² factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm² was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.
Assuntos
Peptídeo Hidrolases/biossíntese , Streptomyces/enzimologia , Fibrinolíticos/metabolismo , Glycine max , Modelos Estatísticos , Actinobacteria , Farinha , Glucose/análise , LíquensRESUMO
Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide â4-α-IdoA2S-1â4-α-GlcNS6S-1â. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1â4-α-GlcA and α-IdoA2S-1â4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fibrinolíticos/farmacologia , Heparina/farmacologia , Mucosa Intestinal/química , Sulfatos/farmacologia , Animais , Resinas de Troca Aniônica , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Anticoagulantes/toxicidade , Proteínas Antitrombina/metabolismo , Bovinos , Cromatografia por Troca Iônica , Dissacarídeos/metabolismo , Modelos Animais de Doenças , Fator Xa/metabolismo , Inibidores do Fator Xa , Feminino , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/metabolismo , Fibrinolíticos/toxicidade , Glicosilação , Hemorragia/induzido quimicamente , Heparina/química , Heparina/isolamento & purificação , Heparina/metabolismo , Heparina/toxicidade , Antagonistas de Heparina/farmacologia , Heparina Liase/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Estrutura Molecular , Tempo de Tromboplastina Parcial , Protaminas/farmacologia , Protrombina/antagonistas & inibidores , Protrombina/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Sulfatos/química , Sulfatos/isolamento & purificação , Sulfatos/metabolismo , Sulfatos/toxicidade , Suínos , Tromboplastina , Trombose Venosa/sangue , Trombose Venosa/induzido quimicamente , Trombose Venosa/prevenção & controleRESUMO
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.