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.     

Hydrolysis of L-cystine-di-beta-naphthylamide and neurohypophyseal peptides by the plasma of the snake Bothrops jararaca.

1. Bothrops jararaca plasma or serum hydrolysed L-cystine-di-beta-naphthylamide (CNAse activity) at a degree comparable to that of plasma or serum in pregnant women. 2. In adult snakes, activity was less in males. It was not altered in pregnancy but increased after delivery, being higher at pH 6.4 (unspecific enzymes) than at pH 7.9 (true pregnant woman plasma oxytocinase). 3. Its optimum pH was 5.9, different from that of other known enzymes that hydrolyse the same substrate. 4. Bothrops jararaca plasma also hydrolysed vasopressin, oxytocin and vasotocin. 5. These hydrolysing activities were unexpected for an ovoviviparous reptile.     

Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes

Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life.     

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.     

The defensive strike of five species of lanceheads of the genus Bothrops (Viperidae).

We studied the defensive strike of one species of each of five recognized lineages within the genus Bothrops, namely, B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. pauloensis. The defensive strike of the studied species was in general similar to that of Crotalus viridis and C. atrox, but some important differences were observed. Bothrops alternatus and B. pauloensis struck preferentially from a tight body posture, whereas B. jararaca and B. moojeni from a loose body posture. Defensive strikes were either true or false (during the latter, the mouth remains closed or partially open). Almost all strikes were successful; only on a few occasions snakes missed their target (flawed strikes). Strike variables were very conservative among the five species, especially strike distance and height, and one possible explanation may be related to constraints imposed on strike variables as a way of increasing strike accuracy.     

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.     

Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.     

The kininases of Bothrops jararaca plasma

Evidence is presented to suggest that kininase activity of Bothrops jararaca plasma is due to the presence of at least three distinct enzymes: a carboxypeptidase B type enzyme, similar to that found in human plasma in that its activity is enhanced by Co2+ (1 X 10(-4) M); a carboxypeptidase B type enzyme whose activity is unaffected by Co2+, and an enzyme which cleaves bradykinin to liberate Phe-Arg as the major peptide fragment formed. The latter enzyme is responsible for the major kininase activity of this snake plasma and is identified as a dipeptide hydrolase.     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.     

Inhibition of prothrombin activation by bothrojaracin, a C-type lectin from Bothrops jararaca venom

Bothrojaracin is a potent and specific alpha-thrombin inhibitor (Kd approximately 0.6 nM) isolated from Bothrops jararaca venom. It binds to both of thrombin's anion-binding exosites (1 and 2), thus inhibiting the ability of the enzyme to act upon several natural macromolecular substrates, such as fibrinogen, platelet receptor, protein C, and factor V. Additionally, bothrojaracin interacts with prothrombin (Kd approximately 30 nM), as previously determined by a solid-phase assay. However, there is no information concerning the effect of this interaction on prothrombin activation and whether the binding of bothrojaracin can occur in plasma. Here, we show that bothrojaracin specifically interacts with prothrombin in human plasma. It is an effective anticoagulant after activation of the intrinsic pathway of blood coagulation, and analysis of prothrombin conversion in plasma shows that bothrojaracin strongly reduces alpha-thrombin formation. To determine whether this effect is due exclusively to inhibition of feedback reactions involving the thrombin-induced activation of factors V and VIII, we analyzed the effect of bothrojaracin on the activation of purified prothrombin by Oxyuranus scutellatus venom. As with plasma, bothrojaracin greatly inhibited thrombin formation, suggesting a direct interference in the prothrombin activation by the enzyme found in this venom (scuterin, a prothrombin activator described as a factor Xa/factor Va-like complex). Altogether, we suggest that bothrojaracin exerts its anticoagulant effect in plasma by two distinct mechanisms: (1) it binds generated thrombin and inhibits exosite 1 dependent activities such as fibrinogen clotting and factor V activation, and (2) it interacts with prothrombin and decreases its proteolytic activation. Thus, bothrojaracin may be useful in the search for thrombin inhibitors that bind both the zymogen and the active enzyme.     

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.     

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.     

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.     

Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom

Snake venoms of the Viperidae family contain a numberof proteins that cause hemostatic disturbances. Enveno-mation of this family is characterized by hemorrhage,edema, local tissue damage, myonecrosis, fibrinolytic andkinin releasing activities [1]. In southeastern Brazil, theviper Bothrops jararaca (Viperidae) is responsible for 90%of snakebite accidents [2]. The enzymes that have proteolytic, coagulate andhemorraghic activities can activate or interfere withthe process of coagulation, and…     

Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest

The Brazilian Atlantic Forest is one of the world's major biodiversity hotspots and is threatened by a severe habitat loss. Yet little is known about the processes that originated its remarkable richness of endemic species. Here we present results of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene of the pitviper, jararaca lancehead (Bothrops jararaca), and two closely related insular species (Bothrops insularis and Bothrops alcatraz), endemic of this region. Phylogenetic and network analyses revealed the existence of two well-supported clades, exhibiting a southern and a northern distribution. The divergence time of these two phylogroups was estimated at 3.8 million years ago, in the Pliocene, a period of intense climatic changes and frequent fragmentation of the tropical rainforest. Our data also suggest that the two groups underwent a large size expansion between 50,000 and 100,000 years ago. However, the southern group showed a more marked signal of population size fluctuation than the northern group, corroborating evidences that southern forests may have suffered a more pronounced reduction in area in the late Pleistocene. The insular species B. alcatraz and B. insularis presented very low diversity, each one sharing haplotypes with mainland individuals placed in different subclades. Despite their marked morphological and behavioural uniqueness, these two insular species seem to have originated very recently and most likely from distinct costal B. jararaca populations, possibly associated with late Pleistocene or Holocene sea level fluctuations.     

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.     

Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro

This protocol details the optimal conditions to establish a long-term primary culture of secretory cells from the venom gland of the Bothrops jararaca snake. Furthermore, these conditions allow the production and secretion of venom into the culture medium. Snake venom is a rich source of active molecules and has been used for bioprospection studies. However, obtaining enough venom from snakes is a major obstacle. Secretory cells of venom glands are capable of producing active toxins. Therefore, a culture of secretory cells is a good in vitro system to acquire the venom of snakes without capturing the animal from the wild. The protocol described here provides a rapid (approximately 4 h) and reproducible means of producing sufficient amounts of snake venom for biological investigations.     

Predatory Behavior of the Opossum Didelphis albiventris on the Pitviper Bothrops jararaca

The predatory behavior of Didelphis albiventris (Didelphidae) on Bothrops jararaca (Viperidae) was described from encounters provoked in captivity. One adult and four young opossums presented the same behavior when killing and ingesting the snakes. Opossums subjugated the snakes by biting them in the head or neck region and began ingestion by eating this part first. The total time spent in subjugation plus ingestion was 24:23 min (SD = 8:08 min). Although D. albiventris is resistant to snake venom, the behavioral sequence observed is, probably, not a specialization for snake predation. It probably consists of a general and primitive pattern of prey manipulation observed in carnivorous mammals.