Broad substrate specificity of snake venom fibrinolytic enzymes: Possible role in haemorrhage

Maruyama, Masugi; Sugiki, Masahiko; Yoshida, Etsuo; Shimaya, Kazuhiro; Mihara, Hisashi

doi:10.1016/0041-0101(92)90514-6

Cited by 76 publications

(38 citation statements)

References 16 publications

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“…Enzymatic hydrolysis of basement membrane (BM) components has been proposed as a key event in the onset of capillary vessel disruption [5,8]. In vitro, SVMPs are able to digest the BM preparation Matrigel [9], as well as puriWed BM components [9][10][11][12][13][14]. Most of these analyses have been performed by observing the patterns of hydrolysis in SDS-PAGE.…”

mentioning

confidence: 99%

Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: A biochemical and immunohistochemical study

Escalante

Shannon

Moura-da-Silva

et al. 2006

Archives of Biochemistry and Biophysics

103

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The hemorrhagic activity characteristic of viperid snake envenomations is due to the action of venom metalloproteinases (SVMPs) on the capillary vessel basement membrane (BM). This study compared the action of two SVMPs on BM in vitro (degradation of Matrigel) and in vivo (immunohistochemical assessment of BM markers in mouse gastrocnemius muscle). SVMPs BaP1 (belonging to the P-I class) and jararhagin (of the P-III class) had a similar proteolytic activity on azocasein and degraded Matrigel with a slightly diVerent cleavage pattern, since BaP1 exerted a limited proteolysis of both laminin and nidogen, whereas jararhagin predominantly degraded nidogen. In contrast with this pattern of limited proteolysis of BM proteins observed in vitro, immunohistochemical analysis of laminin, nidogen and type IV collagen, as well as of the endothelial cell marker VEGFR-2, in the hemorrhagic areas in the muscle, revealed a pronounced reduction in the immunostaining of these three BM components, associated with a loss of the endothelial cell marker. BM of muscle Wbers was aVected to a lesser extent. In conclusion, in vitro results demonstrated that SVMPs induce a pattern of limited proteolysis on BM components. The drastic loss of these antigens in aVected capillaries in vivo is likely to depend on the combination of limited proteolysis of BM and the action of hemodynamic biophysical forces, previously shown to play a role in SVMP-induced capillary damage, which may cause a mechanical disruption of BM structure.

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mentioning

confidence: 99%

Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: A biochemical and immunohistochemical study

Escalante

Shannon

Moura-da-Silva

et al. 2006

Archives of Biochemistry and Biophysics

103

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“…The specific type-IV collagenase activity of jarara fibrases III and IV was 7.6 ±0.3 and 16.5 ±1.2 units/mg, respectively. The num bers of type-IV collagenase units of jararafi brases III and IV which gave the MHD calcu lated from the average specific type-IV colla genase activities of the two enzymes (0.24 and 0.56, respectively) were much lower than that of bacterial specific collagenase (191.4) [3]. It is strongly suggested that jararafibrases III and IV can degrade the vascular basement membrane as single enzymes and induce local hemorrhage.…”

mentioning

confidence: 94%

“…It is known that local hemorrhage and necrosis are major complications following snake envenomation by the Bothrops species, and the efficacy of antivenom therapy is very limited towards such complications. Local hemorrhage after snake envenomation is con ceivably induced by disruption of the vascular basement membrane in the bitten area, occur ring simultaneously with abnormal hemostat ic systems [1][2][3]. Kamiguti et al [2] reported that systemic hemorrhage could also be in duced by a hemorrhagic enzyme through an identical mechanism.…”

Section: Introductionmentioning

confidence: 99%

“…Many purified hemorrhagic fac tors possess proteinase activities, especially fibrinolytic activity [5][6][7][8][9]. It was suggested that the broad substrate specificities of the enzymes might enable them to degrade the vascular basement membrane directly and to induce hemorrhage as single enzymes [3]. Although B. jararaca venom appears to contain other fibrinolytic enzymes, we have been unable to purify them because of the large amount of contamination.…”

Section: Introductionmentioning

confidence: 99%

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Purification and Characterization of Low Molecular Weight Fibrinolytic/Hemorrhagic Enzymes from Snake (Bothrops jararaca) Venom

Maruyama

Tanigawa²,

Sugiki³

et al. 1993

Enzyme Protein

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Two low molecular weight fibrinolytic/hemorrhagic enzymes, jararafibrase III and jararafîbrase IV, were purified from Bothrops jararaca venom using a fast protein liquid chromatography system. The purified jararafibrase III and jararafibrase IV were single chain proteins with molecular weights of 20,400 ± 500 and 21,200 ± 400, respectively, by SDS-PAGE. The isoelectric points of jararafibrase III and jararafibrase IV were 9.4 and 6.9, respectively. The activity of the enzyme was inhibited by 1,10-phenanthroline and EDTA, suggesting that both enzymes were metalloproteinases. The specific fibrinolytic activities of jararafibrase III and jararafibrase IV were 7.5 ±0.4 and 6.5 ±1.6 units/mg protein, respectively. The enzymes induced local hemorrhage in the skin of rats. The minimal hemorrhagic doses of jararafibrase III and IV were 31.0 and 34.0 μg/rat, respectively. The enzymes displayed broad substrate specificities like the previously purified jararafibrases I and II. Jararafibrases III and IV degraded type-IV collagen, gelatin, laminin and fibronectin into smaller fragments. The specific activities of jararafibrase III for type-IV collagen and gelatin were 7.6 ± 0.3 and 43 ±11 units/mg protein, respectively. The specific activities of jararafibrase IV foi type IV-collagen and gelatin were 16.5 ± 1.2 and 112 ± 9 units/ mg protein, respectively.

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“…Following a 1 h at 37 C, two aliquots were taken for analysis. One was subjected to electrophoresis on a nondenaturing gel (20% acrylamide) to observe the ecotin-HP complex formation, as described by Maruyama and coworkers 30 . Urokinase-type plasminogen activator (u-PA)(1 IU) was added to the other aliquot, and the reaction was started after 2 min at 37 C by the addition of SPECTROZYME Õ UK (chromogenic substrate for the amidolytic assay of urokinase -American Diagnostica Inc., Stamford, CT) (0.2 mM, final concentration), as described by Castro and coworkers 20 ).…”

Section: Native Page and Western-blot Analysismentioning

confidence: 99%

Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure–activity relationship study and analysis of interactions

Sathler¹,

Lourenço²,

Miceli

et al. 2013

Journal of Enzyme Inhibition and Medicinal Chemistry

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Recently the literature described the binding of Haptoglobin (HP) with ecotin, a fold-specific serine-proteases inhibitor with an anticoagulant profile and produced by Escherichia coli. In this work, we used some in silico and in vitro techniques to evaluate HP 3D-fold and its interaction with wild-type ecotin and two variants. Our data showed HP models conserved trypsin fold, in agreement to the in vitro immunological recognition of HP by trypsin antibodies. The analysis of the three ecotin-HP complexes using the mutants RR and TSRR/R besides the wild type revealed several hydrogen bonds between HP and ecotin secondary site. These data are in agreement with the in vitro PAGE assays that showed the HP-RR complex in native gel conditions. Interestingly, the ternary complex interactions varied depending on the inhibitor structure and site-directed mutation. The interaction of HP with TSRR/R involved new residues compared to wild type, which infers a binding energy increase caused by the mutation.

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Broad substrate specificity of snake venom fibrinolytic enzymes: Possible role in haemorrhage

Cited by 76 publications

References 16 publications

Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: A biochemical and immunohistochemical study

Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: A biochemical and immunohistochemical study

Purification and Characterization of Low Molecular Weight Fibrinolytic/Hemorrhagic Enzymes from Snake (Bothrops jararaca) Venom

Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure–activity relationship study and analysis of interactions

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