Protective effect of schizolobium parahyba flavonoids against snake venoms and isolated toxins

Four compounds (isoquercitrin, myricetin-3-O-glucoside, catechin and gallocatechin) were isolated from lyophilized aqueous extract of Schizolobium parahyba leaves by chromatography on Sephadex LH-20, followed by semipreparative HPLC using a C-18 column, and identified by 1H and 13C NMR. The compounds were then, tested against hemorrhagic and fibrinogenolytic activities of Bothrops crude venoms and isolated metalloproteinases. The inhibitors neutralized the biological and enzymatic activities of Bothrops venoms and toxins isolated from B. jararacussu and B. neuwiedi venoms. The results showed that gallocatechin and myricetin-3-O-glucoside are good inhibitors of hemorrhagic and fibrinogenolytic activities of metalloproteinases, respectively. Gallocatechin also inhibited the myotoxic activity of both B. alternatus venom and BnSP-6 (Lys49 PhospholipaseA2 from B. neuwiedi). Circular dichroism and docking simulation studies were performed in order to investigate the possible interaction between BnSP-6 and gallocatechin. This is the first time these compounds and their anti-ophidian properties are reported for S. parahyba species. Forthcoming studies involving X-ray co-crystallization, will be of great importance for the development of new therapeutic agents for the treatment of ophidian accidents and for the better understanding of the structure/function relationship of venom toxins.     

Triterpenoid saponins, new metalloprotease snake venom inhibitors isolated from Pentaclethra macroloba.

We report here the antiproteolytic and antihemorrhagic properties of triterpenoid saponin inhibitors, named macrolobin-A and B, from Pentaclethra macroloba, against Bothrops snake venoms. The inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. Clotting and fibrinogenolytic activities induced by snake venoms and isolated thrombin-like enzymes were partially inhibited. Furthermore, the potential use of these inhibitors to complement antivenom therapy as an alternative treatment and/or used as molecular models for development of new therapeutical agents in the treatment of snake bite envenomations needs to be evaluated in future studies.     

Purification and characterization of a hemorrhagic metalloproteinase from Bothrops lanceolatus (Fer-de-lance) snake venom.

Bothrops snake venoms contain metalloproteinases that contribute to the local effects seen after envenoming. In this work, a hemorrhagic metalloproteinase (BlaH1) was purified from the venom of the snake Bothrops lanceolatus by a combination of gel filtration, affinity (metal chelating) and hydrophobic interaction chromatographies. The hemorrhagin was homogeneous by SDS-PAGE and had a molecular mass of 28 kDa that was unaltered by treatment with beta-mercaptoethanol. BlaH1 gave a single band in immunoelectrophoresis and immunoblotting using commercial bothropic antivenom. BlaH1 had hemorrhagic, caseinolytic, fibrinogenolytic, collagenolytic and elastinolytic activities, but no phospholipase A(2) activity. The hemorrhagic and caseinolytic activities were inhibited by EDTA, indicating that they were metal ion-dependent. In contrast, aprotinin, benzamidine and PMSF did not affect these activities. The caseinolytic activity of BlaH1 had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at > or =70 degrees C. The hemorrhagic activity was neutralized by commercial bothropic antivenom. These properties suggest that this new hemorrhagin belongs to class P-I snake venom metalloproteinases.     

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon15, 161–167, 1977.—In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon acutus venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did ε-aminocaproic acid. Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due-to a direct action on fibrin. Both EDTA (5 × 10^-4 M) and cysteine (5 × 10^-3 M) completely inhibited the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the fibrinolytic principle.     

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.     

Neutralization of pharmacological and toxic activities of bothrops snake venoms by Schizolobium parahyba (Fabaceae) aqueous extract and its fractions

The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Atlantic Forest (Brazil), was tested to analyse its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus (BaltCV) and Bothrops moojeni (BmooCV) snake venoms. Sp inhibited 100% of lethality, blood incoagulability, haemorrhagic and indirect haemolytic activities at a 1:10 ratio (venom/extract, w/w), as well as coagulant activity at a 1:5 ratio (venom/extract, w/w) induced by both venoms. BaltCV fibrinogenolytic activity was also neutralized by Sp at a 1:10 ratio, resulting in total protection of fibrinogen Bbeta chain and partial protection of Aalpha chain. Interaction tests have demonstrated that, at certain extract/proteins ratios, Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analysed ophidian activities. Sp aqueous extract chromatography on Sephadex LH-20 was carried out aiming at the separation of these compounds that mask the obtained results. Thus, the fractionation of Sp resulted in three fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analysed for their ability to inhibit the BaltCV fibrinogenolytic activity. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 showed only partial inhibition of this venom activity. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp aqueous extract, together with tannins, also contains other compounds that can display specific inhibitory activity against snake venom toxins.     

Simplified procedures for the isolation of HF3, bothropasin, disintegrin-like/cysteine-rich protein and a novel P-I metalloproteinase from Bothrops jararaca venom

HF3 and bothropasin are P-III hemorrhagic snake venom metalloproteinases (SVMPs) of Bothrops jararaca. The DC protein is composed of the disintegrin-like/cysteine-rich domains derived from the autolysis of P-III SVMPs. Here we describe simplified procedures for the isolation of HF3, bothropasin, the DC protein, and BJ-PI, a novel P-I SVMP. The isolated proteins were identified by mass spectrometry. BJ-PI is a potent caseinolytic enzyme devoid of hemorrhagic activity. HF3, bothropasin and BJ-PI show distinct fibrinogenolytic activities.     

α-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon rhodostoma (Malayan pit viper) snake venom was separated into eleven fractions. Fraction II had fibrinogenolytic activity, and when further purified by gel filtration was homogeneous, as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. It had a single peptide chain with a molecular weight of 25,360 and an isoelectric point greater than 10. The fibrinogenolytic activity was completely destroyed after heating for 30 min at 60°C at pH 5.6, 7.4 or 8.8. This enzyme cleaved specifically the α(A) chain of monomeric fibrinogen, without cleaving the β(B) chain or γ chain. The specific fibrinogenolytic activity was 51 mg fibrinogen/min per mg protein. This enzyme showed proteolytic activities toward fibrinogen, fibrin and casein, but was devoid of phospholipase A and tosyl-l-arginine methylester esterase activities which are found in the crude venom. The fibrinogenolytic activity was inhibited by EDTA and cysteine, but not by ε-aminocaproic acid.     

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom.

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A(2) activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.     

Characterization of hemorrhagic principles from Trimeresurus gramineus snake venom

In addition to alpha-fibrinogenase (hemorrhagin I, HR1), a potent hemorrhagic principle (hemorrhagin II, HR2) was purified from Trimeresurus gramineus venom. It was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. HR2 was a single peptide chain containing 10% carbohydrate with a molecular weight of 81,500. It possessed 669 amino acid residues per molecule, while HR1 contained only 203 amino acid residues per molecule with a molecular weight of 23,500. Both hemorrhagins possessed proteolytic activities toward fibrinogen, casein and azocoll. However, the proteolytic activities of HR1 were much more potent than those of HR2. They were devoid of TAME-esterase and phospholipase A2 activities which were found in crude venom. beta-Mercaptoethanol and antivenin completely inhibited the hemorrhagic activities of HR1 and HR2, while epsilon-aminocaproic acid, trasylol, p-bromophenacyl bromide, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor did not. EDTA completely inhibited the hemorrhagic, fibrinogenolytic and caseinolytic activities of HR1. EDTA also completely inhibited the caseinolytic and fibrinogenolytic activities of HR2, but only partially inhibited its hemorrhagic activity. Subsequent addition of Zn2+ (5 mM) reversed the EDTA-induced inhibitory effect on the hemorrhagic activity of HR1. However, ZN2+ did not reverse the EDTA-induced inhibitory effect on the HR2-induced hemorrhagic activity. These hemorrhagins were found to be ZN2+-containing metalloproteinases. Therefore, the hemorrhagic activity of HR1 seems to be related to its proteolytic activity while that of HR2 seems to be unrelated to its proteolytic activity.     

Species difference in the fibrinogenolytic effects of alpha- and beta-fibrinogenases from Trimeresurus mucrosquamatus snake venom

Alpha- and beta-fibrinogenases prepared from Trimeresurus mucrosquamatus venom digested specifically the alpha(A) and beta(B) chains of the fibrinogen molecule, respectively. alpha-Fibrinogenase digested bovine fibrinogen more markedly than human fibrinogen, while beta-fibrinogenase digested human fibrinogen more markedly than bovine fibrinogen. Human fibrin was also digested by both enzymes. Plasma fibrinogens of 4 animal species were digested by alpha-fibrinogenase to the same degree, while those by beta-fibrinogenase in the following order: human greater than dog greater than guinea-pig greater than rabbit. The fibrinogenolytic effects of alpha-fibrinogenase on human fibrinogen were strongly inhibited by sera of the 4 animal species, while those of beta-fibrinogenase were inhibited in the following order: rabbit greater than guinea-pig greater than dog greater than human. It was concluded that the different activities of the protease inhibitors in the plasma of animal species are mainly responsible for the sensitivity differences.     

Factor X converting and thrombin-like activities of Bothrops Jararaca snake venom

Factor X converting and thrombin-like activities of Bothrops jararaca venom. Toxicon15, 107–114, 1977.—Bothrops jararaca venom is able to activate factor X and to convert fibrinogen to fibrin by a thrombin-like activity. The two activities behaved as if they were due to distinct enzyme proteins as determined by chromatography on Sephadex G-100, isoelectric focusing, disc gel electrophoresis and heat-treatment.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom

The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten species of Costa Rican crotaline snakes were studied, together with the neutralization of these effects by a polyvalent antivenom. The venoms of Bothrops asper, B. schlegelii, B. nummifer, B. godmani, Lachesis muta and Crotalus durissus induced a coagulant effect in vitro, and all of them, with the exception of B. nummifer, also induced defibrination in vivo. The four non-coagulant venoms (B. lateralis, B. ophryomegas, B. nasuta and B. picadoi) induced a degradation of the alpha (A) chain of fibrinogen, thereby inhibiting coagulation. However, they did not induce defibrination upon i.v. injection. All of the venoms showed fibrinolytic activity in vitro. Polyvalent antivenom was effective in the neutralization of coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of these venoms, with the exception of coagulant effect induced by C. durissus venom. Since only three venoms are used in the immunization of horses, these results demonstrate the high degree of immunological cross reactivity between components affecting coagulation in Costa Rican crotaline snake venoms.     

Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom

A hemorrhagic metalloproteinase, named Batx-I, was isolated from the venom of Bothrops atrox specimens (from Southeastern Colombian region) by a combination of CM-Sephadex C25 ion-exchange and Affi-gel Blue affinity chromatographies. This enzyme accounts for about 45% of venom proteins, and it has an ESI-MS isotope-averaged molecular mass of 23296.2 Da and a blocked N-terminus. Two internal fragments sequenced by mass spectrometric analysis showed similarity to other SVMPs from Bothrops venoms. To investigate the possible participation of Batx-I in the envenomation pathophysiology, proteolytic, fibrinogenolytic, hemorrhagic, and other biological activities were evaluated. The minimal hemorrhagic dose obtained was 17 μg/20 g body weight. The enzyme showed proteolytic activity on azocasein, comparable with activity of BaP1. This activity was inhibited by EDTA and 1, 10 o-phenanthroline but not by aprotinin, pepstatin A or PMSF. Fibrinogenolytic activity was analyzed by SDS-PAGE, revealing a preference for degrading the Aα- and Bβ-chains, although partial degradation of the γ-chain was also detected. The protein lacks coagulant and defibrinating activity. The CK levels obtained, clearly reflects a myotoxic activity induced by Batx-I. The hemorrhagic and fibrinogenolytic activities exhibited by the isolated PI-SVMP may play a role in the hemorrhagic and blood-clotting disorders observed in patients bitten by B. atrox in Colombia.     

The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper)

An inhibitory effect of Bothrops castelnaudi venom was observed on the following systems: prothrombin time, activated partial thromboplastin time, thrombin time, thromboplastin generation time, activation of factor X by Russell's viper venom and Russell's viper venom activated factor X (factor Xa). This effect did not require previous incubation and was prevented by the addition of Bothrops-antivenom. The prolonged activated partial thromboplastin time was not shortened by increased phospholipid concentration (0.5-10 mg/ml), suggesting that the inhibitory effect is not due to an anti-phospholipid activity. No significant fibrinogenolytic activity was detected upon incubation of human fibrinogen with the venom, since physiological levels of thrombin-clottable material were still present. Compared to Bothrops jararaca venom, the proteolytic activity on casein and on azocoll was very low. Thrombin-induced clots of human plasma and fibrinogen were not lysed by the venom within 24 hr. The results indicate that the anticoagulant effect of Bothrops castelnaudi venom is exerted at least at two levels of the blood coagulation mechanism: (1) before prothrombin activation, by inhibiting factor X-activation and factor Xa activity; (2) by direct action on thrombin.     

Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom

A hemorrhagic toxin was isolated from Mojave rattlesnake venom. The isoelectric point of the toxin was 4.7 and its mol. wt was 27,000. Concentrations as low as 2 micrograms injected s.c. in mice caused hemorrhage greater than 5 mm in diameter. The toxin was fibrinogenolytic and hydrolyzed hide powder azure, casein and collagen. The toxin also partially inactivated complement. It had no activity against elastin, fibrin, and the chromogenic substrates S-2805, S-2302 and S-2238. Its esterolytic activity was 3% of the activity of the unfractionated venom. The enzymatic and hemorrhagic activities were inhibited by EDTA. The hemorrhagic toxin was absent or in low quantities in Mojave rattlesnake venoms containing Mojave toxin. Chromatography by HPLC easily distinguishes Mojave rattlesnake venoms into two types by the presence or absence of the hemorrhagic toxin.     

Pathological and biochemical changes induced in mice after intramuscular injection of venom from newborn specimens of the snake Bothrops asper (Terciopelo).

Venom from newborn Bothrops asper snakes has higher lethal, hemorrhagic, edema-forming, proteolytic and defibrinating activities than venom from adult B. asper specimens. Electrophoretic analysis confirmed the variation between these venoms. Intramuscular injection of 100 micrograms of venom from newborn specimens in mice induced defibrination, together with moderate increments of serum levels of lactate dehydrogenase, creatine kinase, hemoglobin and total proteins. A conspicuous hemorrhage developed in injected muscle rapidly after envenomation, probably due to a drastic alteration in capillaries and larger blood vessels. Other histological alterations included moderate myonecrosis, lung collapse and prominent renal damage, characterized by tubular necrosis and hyalinization. Polyvalent antivenom effectively neutralized lethal, hemorrhagic and indirect hemolytic activities of newborn B. asper venom, although requiring higher antivenom doses than neutralization of venom from adult B. asper.     

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C₁₆₄I₁₆₅M₁₆₆ motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.