Proteolítica

Januario AH, Santos SL, Marcussi S, Mazzi MV, Pietro RC, Sato DN, Ellena J, Sampaio SV, Franca SC, Soares AM.

Neo-clerodane diterpenoid, a new metalloprotease snake venom inhibitor from Baccharis trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic properties.

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plant natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja, has been popularly used to treat liver diseases, rheumatism, diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7alpha-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C₂₀H₂₈O₅, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic properties against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metalloproteases, basic and acidic phospholipases A₂. To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future studies.

Marcussi, S(2); Fernandes, VC(1); Mazzi, MV(3); Cambraia, RS(1); Sant'Ana, CD(3); Silveira, LB(1); França, SC(1); Giglio, JR(2); Soares, AM(1)

Isolamento e caracterização bioquímica de uma Metaloprotease não-hemorrágica do veneno da serpente B. jararacussu

As metaloproteases de venenos de serpentes são responsáveis por diversos efeitos tóxicos e farmacológicos induzidos por diferentes serpentes. Estudos de mecanismo de ação dessas proteínas tem sido intensamente realizados buscando entender sua participação nos envenenamentos ofídicos. Este trabalho tem como objetivo isolar e caracterizar enzimaticamente uma nova metaloprotease do veneno de B. jararacussu, buscando o entendimento de seus mecanismos de ação. A protease de baixo peso molecular denominada BjussuMP-II foi isolada do veneno de B. jararacussu em duas etapas cromatográficas, uma troca iônica em CM-Sepharose pH 8,0, seguida por uma coluna hidrofóbica, Phenyl-Sepharose. BjussuMP-II apresentou uma única cadeia polipeptídica de PM ~ 22.000 em SDS-PAGE. Esta protease não induziu atividade hemorrágica in vivo e/ou coagulante in vitro, no entanto mostrou-se altamente proteolítica sobre o fibrinogênio e a caseína. Realizou-se uma caracterização enzimática da BjussuMP-II variando sua concentração, tempo de incubação, estabilidade em pH e temperatura, e incubação com diferentes íons. Os aspectos abordados neste trabalho poderão trazer informações complementares sobre mecanismos de ação, e o melhor entendimento dos efeitos induzidos pelas metaloproteases de venenos de serpentes e da participação, direta ou sinérgica destas proteínas nos envenenamentos causados pela serpente Bothrops jararacussu.

Bucaretchi F, Herrera SR, Hyslop S, Baracat EC, Vieira RJ

From January, 1984 to March, 1999, 31 children under 15 y old (ages 1-14 y, median 8 y) were admitted after being bitten by rattlesnakes (Crotalus durissus ssp). One patient was classified as "dry-bite", 3 as mild envenoming, 9 as moderate envenoming and 18 as severe envenoming. Most patients had neuromuscular manifestations, such as palpebral ptosis (27/31), myalgia (23/31) and weakness (20/31). Laboratory tests suggesting rhabdomyolysis included an increase in total blood creatine kinase (CK, 28/29) and lactate dehydrogenase (LDH, 25/25) levels and myoglobinuria (14/15). The main local signs and symptoms were slight edema (20/31) and erythema (19/31). Before antivenom (AV) administration, blood coagulation disorders were observed in 20/25 children that received AV only at our hospital (incoagulable blood in 17/25). AV early reactions were observed in 20 of these 25 cases (9/9 patients not pretreated and 11/16 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). There were no significant differences in the frequency of patients with AV early reactions between the groups that were and were not pretreated (Fisher's exact test, p = 0.12). Patients admitted less than and more than 6 h after the bite showed the same risk of developing severe envenoming (Fisher's exact test, p = 1). No children of the first group (< 6 h) showed severe complications whereas 3/6 children admitted more than 6 h post-bite developed acute renal failure. Patients bitten in the legs had a higher risk of developing severe envenoming (Fisher's exact test, p = 0.04). There was a significant association between both total CK and LDH blood enzyme levels and severity (p < 0.001 for CK and p < 0.001 for LDH; Mann-Whitney U test). No deaths were recorded.

Otero R^a, Nunez V^a, Jimenez SL^a, Fonnegra Ra, Osorio RG^a, Garcia ME^a, Diaz A^a

Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18–20 g). After preincubation of sublethal doses of every extract (0.5–4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD₅₀) (99.3 μg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia ( Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45–80%; 10–30% survival rate in the control group receiving the venom alone; P<0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 μg=1.5 i.m. LD₅₀), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A₂ activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 μg) in agarose-erythrocyte-egg yolk gels.