Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring.

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 µg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Unveiling the peptidome diversity of Lachesismuta snake venom: Discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides.

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesismuta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

What's in a mass?

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

Danger in the Canopy. Comparative Proteomics and Bioactivities of the Venoms of the South American Palm Pit Viper Bothrops bilineatus Subspecies bilineatus and smaragdinus and Antivenomics of B. b. bilineatus (Rondônia) Venom against the Brazilian Pentabothropic Antivenom.

We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

Comparative proteomic profiling and functional characterization of venom pooled from captive Crotalus durissus terrificus specimens and the Brazilian crotalic reference venom.

The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom.

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature.

BACKGROUND: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. METHODS: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. RESULTS: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. CONCLUSION: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Functional and proteomic comparison of Bothrops jararaca venom from captive specimens and the Brazilian Bothropic Reference Venom.

Snake venom is a variable phenotypic trait, whose plasticity and evolution are critical for effective antivenom production. A significant reduction of the number of snake donations to Butantan Institute (São Paulo, Brazil) occurred in recent years, and this fact may impair the production of the Brazilian Bothropic Reference Venom (BBRV). Nevertheless, in the last decades a high number of Bothrops jararaca specimens have been raised in captivity in the Laboratory of Herpetology of Butantan Institute. Considering these facts, we compared the biochemical and biological profiles of B. jararaca venom from captive specimens and BBRV in order to understand the potential effects of snake captivity upon the venom composition. Electrophoretic analysis and proteomic profiling revealed few differences in venom protein bands and some differentially abundant toxins. Comparison of enzymatic activities showed minor differences between the two venoms. Similar cross-reactivity recognition pattern of both venoms by the antibothropic antivenom produced by Butantan Institute was observed. Lethality and neutralization of lethality for B. jararaca venom from captive specimens and BBRV showed similar values. Considering these results we suggest that the inclusion of B. jararaca venom from captive specimens in the composition of BBRV would not interfere with the quality of this reference venom. BIOLOGICAL SIGNIFICANCE: Snakebite envenomation is a neglected tropical pathology whose treatment is based on the use of specific antivenoms. Bothrops jararaca is responsible for the majority of snakebites in South and Southeastern Brazil. Its venom shows individual, sexual, and ontogenetic variability, however, the effect of animal captivity upon venom composition is unknown. Considering the reduced number of wild-caught snakes donated to Butantan Institute in the last decades, and the increased life expectancy of the snakes raised in captivity in the Laboratory of Herpetology, this work focused on the comparative profiling of B. jararaca venom from captive snakes and the Brazilian Bothropic Reference Venom (BBRV). BBRV is composed of venom obtained upon the first milking of wild-caught B. jararaca specimens, and used to assess the potency of all bothropic antivenoms produced by Brazilian suppliers. The use of proteomic strategies, added to biochemical and neutralization tests, allowed to conclude that, despite some subtle differences detected between these two venoms, venom from captive specimens could be used in the BBRV composition without affecting its quality in antivenom potency assays.

Examination of biochemical and biological activities of Bothrops jararaca (Serpentes: Viperidae; Wied-Neuwied 1824) snake venom after up to 54 years of storage.

The number of snakes donated to the Brazilian Instituto Butantan has been decreasing in the past 10 years. This circumstance motivated us to compare the properties of five venom pools of Bothrops jararaca snake stored for up to 54 years. Results showed differences among venom pools regarding enzymatic and other biological activities, such as caseinolytic, phospholipase A2, hemorrhagic and coagulant activities, as well as antigenicity. Protein content, reverse-phase chromatographic profile, and immunorecognition by commercial Bothrops antivenom were comparable for all venom pools, although lethality of the most recent preparations was higher. Since the lowest functional activities did not always correspond to older venoms, differences among venom pools used for antivenom production during the period 1963-2008 may correlate with the different proportions of venoms from different localities used in their generation, rather than to long-term storage. We conclude that B. jararaca venoms properly stored for long periods of time retain their structural and pharmacological activities, thus representing useful materials for scientific research and antivenom production.

Cloning, Characterization and Anti-Inflammatory Properties of Bothrops jararaca Snake Antithrombin.

Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was ~30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development.

Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity.

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. In this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. In contrast, AaTIΔ (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. In the competition assay, rAaTI, AaTIΔ or C-terminal AaTI acidic tail-thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. In conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTIΔ form also prolongs thrombin time.

Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatoryand immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilatorsand platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegyptishowed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands,carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor).Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. In this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able toprolong prothrombin time, activated partial thromboplastin time and thrombin time. In contrast, AaTID(rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. In the competition assay, rAaTI, AaTID or C-terminal AaTI acidic tailethrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibitionassay of rAaTI showed an uncompetitive inhibition mechanism. In conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTID form also prolongs thrombin time.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity.

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito's development.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombincoagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator andantimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. Inthis paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity withinsect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptaseinhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized therecombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (Ki) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito’s development.

Bothrops jararaca fibrinogen and its resistance to hydrolysis evoked by snake venoms.

Fibrinogen is an essential protein involved in several steps of hemostasis, being associated with the final steps of the blood coagulation mechanism. Herein, we describe the purification and characterization of a reptile fibrinogen, obtained from Bothrops jararaca plasma. Native B. jararaca fibrinogen showed a molecular mass of 372 kDa, and the reduced and alkylated fibrinogen molecule showed three chains of 71, 60 and 55 kDa, which are similar to the molecular masses of human and bovine Aalpha, Bbeta and gamma fibrinogen chains. Remarkably, B. jararaca fibrinogen was clotted by bovine thrombin, but B. jararaca, Crotalus durissus terrificus and Lachesis muta rhombeata venoms could not induce its clotting or hydrolysis. Thus, despite the similarities between B. jararaca and mammalian fibrinogens, the former shows distinctive features, which protect B. jararaca snakes from accidental envenomation.

Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera: Reduviidae).

Recently, we have cloned several Kazal-type serine protease inhibitors from the midgut of the Triatoma infestans bug. A single gene composed of multi Kazal-type domains, in tandem, encodes these inhibitors. In this work, we describe the purification and characterization of recombinant infestins 3-4 and 4, which are potent factor XIIa inhibitors (KI=67 pM and 128 pM, respectively). We also identified the first native factor XIIa inhibitor from a hematophagous insect. The factor XIIa inhibitory activity of infestin 4 demonstrates extremely efficient anticoagulant activity, prolonging activated partial thromboplastin time by approximately 3 times. Our results suggest that infestins perform a very important role in the T. infestans midgut during meal acquisition and digestion by controlling blood coagulation by means of inhibiting thrombin and factor XIIa.

Characterization of Bothrops jararaca coagulation inhibitor (BjI) and presence of similar protein in plasma of other animals.

BjI, a protein isolated from Bothrops jararaca snake blood, inhibits the coagulant activity of thrombin. This protein presents two bands of 109 and 138 kDa by SDS-PAGE under reducing conditions. In order to verify the presence of BjI-like proteins in plasma of other animals (reptiles and non-reptiles), we raised a specific polyclonal antibody in mice to it, and we verified immunological cross-reaction by western blotting, considering as positive reactions the development of bands with either 109 or 138 kDa. Similar proteins were identified in Bothrops neuwiedi and Crotalus durissus terrificus snakes. In contrast, no BjI-like protein in other classes of animals was noticeable, nor in other snakes tested. Interestingly, a prolonged thrombin time was found only in snake plasmas that showed similar BjI proteins. BjI bound to two proteins of B. jararaca venom, identified by western blotting. The N-terminal of the B. jararaca venom proteins showed similarity with thrombin-like proteins isolated from other snake venoms. In conclusion, there are similar proteins to BjI in plasmas of B. neuwiedi and Crotalus durissus terrificus, and these proteins also prolong thrombin time. Moreover, these results evidence the presence of target enzymes in snake venom for plasma BjI.

A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization.

A novel thrombin inhibitor, Bothrops jararaca inhibitor (BjI), has been identified and purified from B. jararaca snake blood by two anionic chromatographic steps. Purified BjI showed two polypeptide chains with molecular masses of 109 and 138 kDa, by SDS-PAGE in reducing conditions. On the other hand, in nonreducing conditions the molecular masses were 150 and 219 kDa, suggesting that the polypeptide chain 109 kDa can be a dimer form linked by disulfide bond. However, the native BjI shows a molecular mass higher than 1000 kDa by gel filtration chromatography, indicating the need of a quaternary structure formation for the blood coagulation inhibition. BjI is a specific thrombin coagulant activity inhibitor that does not affect other thrombin functions, such as: amidolytic and platelet aggregation activities. BjI is not an antithrombin-like inhibitor. Fibrinogen and heparin competition ELISA assays with BjI and thrombin showed that fibrinogen does not interfere in the BjI and thrombin binding, however, heparin interferes in BjI and thrombin interaction, suggesting that BjI binds to heparin site or other sites close to it. Our findings indicate that BjI is an exosite binding thrombin inhibitor, specific upon coagulant activity thrombin inhibitor, without any anti-platelet aggregation activity.