Clinical and Evolutionary Implications of Dynamic Coagulotoxicity Divergences in Bothrops (Lancehead Pit Viper) Venoms.

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes.

BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms.

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

Snake venom color and L-amino acid oxidase: An evidence of long-term captive Crotalus durissus terrificus venom plasticity.

The venom color variation of Crotalus durissus terrificus (Cdt) is attributed to the presence of the toxin L-amino acid oxidase (LAAO). During the venom milking routine of Instituto Butantan, we have noticed that most venoms of captive Cdt specimens show a yellowish color, while most venoms of wild specimens are white. Here we describe a comparative analysis of long-term captive (LTC) and recently wild-caught (RWC) Cdt, focusing on LAAO variation. For the identification of LAAO in individual venoms, four different approaches were employed: evaluation of the enzymatic activity, SDS-PAGE, Western blotting, and ELISA. In addition, mass spectrometry analysis was performed using pooled samples. Although some variation among these methodologies was observed, it was possible to notice that the presence of LAAO was significantly higher in the venom of LTC individuals. LAAO was identified in 60-80% LTC specimens and in only 10-12% of RWC specimens. Furthermore, this enzyme accounts for 5.6% of total venom proteins of LTC Cdt pooled venom, while it corresponds to only 0.7% of RWC Cdt pooled venom. These findings strongly suggest that captive maintenance increases the expression of LAAO in Cdt venom.

Venom complexity of Bothrops atrox (common lancehead) siblings.

BACKGROUND: Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. METHODS: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. RESULTS: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. CONCLUSION: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.

Geographic variation of individual venom profile of Crotalus durissus snakes.

BACKGROUND: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. METHODS: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. RESULTS: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. CONCLUSIONS: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Venom complexity of Bothrops atrox (common lancehead) siblings

Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. Methods: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. Results: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. Conclusion: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Evaluation of the Inhibitory Potential of Casuarictin, an Ellagitannin Isolated from White Mangrove (Laguncularia racemosa) Leaves, on Snake Venom Secretory Phospholipase A2.

Ellagitannins constitute the largest group of hydrolyzable tannins of plants, and, from this group, casuarictin (Casu) was identified in some plant species. However, to our knowledge, no investigation of secretory phospholipase A2 (sPLA2) inhibition by Casu has been performed yet. Casuarictin was isolated by chromatography n-butanol (n-BuOH) partition of Laguncularia racemosa leaves. The pharmacological and biological effects of Casu were evaluated on isolated sPLA2 from the rattlesnake (Crotalus durissus terrificus) and using a plant bacterial strain. The compound was able to form a protein complex consisting of a stable sPLA2 + Casu complex. Analyses carried out with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) revealed that the molecular mass of sPLA2 increased from 14,425.62 to 15,362.74 Da. The enzymatic activity of the sPLA2 + Casu complex was significantly lower than that of native sPLA2. Besides, molecular interactions of Casu with sPLA2 were able to virtually abolish the native edematogenic effect as well as myonecrosis induced by the protein when injected 10 min after sPLA2. Therefore, Casu may be considered a potential anti-inflammatory that can be used to treat edema and myonecrosis induced by serine-secreting phospholipase A2. In addition, the compound also showed great antimicrobial potential.

LC-ESI-IT-MS/MS and MALDI-TOF Approach: Identification of Natural Polymers from Rhizophora mangle Barks and Determination of Their Analgesic and Anti-inflammatory Properties.

We recognize the chemical composition of the acetonic extract of Rhizophora mangle barks (AERM) using mass spectrometry analysis [liquid chromatography (LC)-ESI-IT-MS/MS and matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF)]. Analgesic activity was evaluated by formalin and tail-flick experimental assays. Anti-inflammatory activity was performed by paw edema test induced by carrageenan and 48/80 compounds. The first series of experiments involved [LC]-FIA-IT-MS/MS with 11 separated catechins derivatives until degree of polymerization 3 (DP3). The spectra obtained by MALDI-TOF analysis of the AERM presented two homologous series: one based on polymers of m/z 288 Da increments (up to DP12) and another series based on polymers of m/z [288 + 162] Da increments (up to DP11). In addition to these series of flavan-3-ol, each DP had a subset of masses with a variation of - 16 Da (homologous series of afzelechins-m/z 873-3465 Da) and + 16 Da (homologous series of gallocatechins-m/z 905-3497 Da). A similar pattern with homologous series of gallocatechins and afzelechins could also be observed for a fifth and a sixth monohexoside series: glucogallocatechins (m/z 779-3371) and glucoafzelechins (m/z 747-3339). The intraperitoneal administration of different doses of AERM (50, 150 and 300 µg mL-1) have a morphine-like effect and intense anti-inflammatory activity.

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.

Evaluation of Potential Thrombin Inhibitors from the White Mangrove (Laguncularia racemosa (L.) C.F. Gaertn.).

The aim of this work was to verify the effects of methanol (MeOH) and hydroalcoholic (HA) extracts and their respective partition phases obtained from white mangrove (Laguncularia racemosa (L.) C.F. Gaertn.) leaves on human thrombin activity. Among the extracts and phases tested, only the ethyl acetate and butanolic partitions significantly inhibited human thrombin activity and the coagulation of plasma in the presence of this enzyme. Chromatographic analyses of the thrombin samples incubated with these phases revealed that different compounds were able to interact with thrombin. The butanolic phase of the MeOH extract had the most potent inhibitory effects, reducing enzymatic activity and thrombin-induced plasma coagulation. Two glycosylated flavonoids in this partition were identified as the most potent inhibitors of human thrombin activity, namely quercetin-3-O-arabinoside (QAra) and quercetin-3-O-rhamnoside (Qn). Chromatographic analyses of thrombin samples incubated with these flavonoids demonstrated the chemical modification of this enzyme, suggesting that the MeOH extract contained other compounds that both induced structural changes in thrombin and diminished its activity. In this article, we show that despite the near absence of the medical use of mangrove compounds, this plant contains natural compounds with potential therapeutic applications.