RESUMO
Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Bothrops/metabolismo , Coagulantes/farmacologia , Venenos de Crotalídeos/enzimologia , Fator Xa/metabolismo , Leucócitos/efeitos dos fármacos , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Protrombina/metabolismo , Tromboplastina/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Estabilidade Enzimática , Feminino , Humanos , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/metabolismo , Cinética , Leucócitos/metabolismo , Masculino , Metaloendopeptidases/isolamento & purificação , Metaloproteases/isolamento & purificação , Pessoa de Meia-Idade , Temperatura , Adulto JovemRESUMO
The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.
Assuntos
Calotropis/enzimologia , Coagulantes/química , Cisteína Endopeptidases/química , Látex/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Coagulação Sanguínea , Cromatografia por Troca Iônica , Coagulantes/isolamento & purificação , Coagulantes/farmacologia , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Estrutura Secundária de Proteína , Proteólise , Tempo de Protrombina , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.
Assuntos
Animais , Camundongos , Coagulação Sanguínea , Bothrops , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia em Agarose , Cromatografia por Troca Iônica , Costa Rica , Coagulantes/administração & dosagem , Coagulantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Fibrinogênio/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Mordeduras de Serpentes/fisiopatologia , Trombina/químicaRESUMO
A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.
Assuntos
Coagulação Sanguínea , Bothrops , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia em Agarose , Cromatografia por Troca Iônica , Coagulantes/administração & dosagem , Coagulantes/farmacologia , Costa Rica , Avaliação Pré-Clínica de Medicamentos , Fibrinogênio/metabolismo , Camundongos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Mordeduras de Serpentes/fisiopatologia , Trombina/químicaRESUMO
A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).
Assuntos
Coagulantes/isolamento & purificação , Coagulantes/farmacologia , Venenos de Crotalídeos/enzimologia , Trombina/isolamento & purificação , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Coagulação Sanguínea , Bothrops , Bovinos , Coagulantes/metabolismo , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Venenos de Crotalídeos/toxicidade , Fator XIII/efeitos dos fármacos , Fator XIII/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Hidrólise , Cinética , Camundongos , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Trombina/antagonistas & inibidoresRESUMO
A thrombin-like enzyme, balterobin, was purified from the venom of Bothrops alternatus. The purification steps included Sephadex G-75, heparin-sepharose and reverse phase HPLC C-18 column. Balterobin showed an apparent molecular weight of 30,000 in non-reduced conditions and displays a specific coagulant activity of 32.8 NIH units/mg over bovine fibrinogen. It also exhibits arginine amidase activity on DL-BAPNA. Like thrombin-like enzymes from other snakes, balterobin possesses valine as N-terminal residue, and is inhibited by PMSF.