Jack bean (Canavalia ensiformis) urease induces eicosanoid-modulated hemocyte aggregation in the Chagas' disease vector Rhodnius prolixus.

Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein.

A phospholipase A2 gene is linked to Jack bean urease toxicity in the Chagas' disease vector Rhodnius prolixus.

BACKGROUND: Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including exocytosis induction and insecticidal effects. The hemipteran Rhodnius prolixus is one of the known susceptible models for this toxicity. It has been shown that Jack bean urease (JBU) has deleterious effects on R. prolixus, and these effects are modulated by eicosanoids, which are synthesized in a cascade involving phospholipase A2 (PLA2) enzymes. METHODS: R. prolixus genome was screened for putative PLA2s and matching transcripts were cloned. Predicted amino acid sequences were analyzed and transcript distribution among tissues was determined by qPCR. RNAi techniques were used and subsequent JBU toxicity assays were performed. RESULTS: Two PLA2 genes were identified, Rhopr-PLA2III and Rhopr-PLA2XII. The transcripts are widely distributed in the tissues but at different levels. The analyses fit the putative proteins into groups III and XII of secretory PLA2s. After 70% of Rhopr-PLA2XII expression was knocked down, JBU's toxicity was decreased by more than 50% on 5th instars R. prolixus. CONCLUSIONS: Rhopr-PLA2XII gene is linked to JBU's toxic effect in R. prolixus and our findings support previous studies demonstrating that eicosanoids modulate this toxicity. GENERAL SIGNIFICANCE: Besides identifying and characterizing two PLA2 genes in the major Chagas' disease vector R. prolixus, we have shown that the potent toxicity of JBU is linked to one of these genes. Our results contribute to the general comprehension of urease's mechanisms of action in insects, and, potentially, to studies on the control of the Chagas' disease parasite transmission.

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut.

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E(2) (PGE(2)) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE(2) increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth.

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD(50) 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1cm)(1%) of 6.3, and fluorescence emission in the 290-450 nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 microg/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens.

Jaburetox-2Ec: an insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis.

Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.

Antifungal activity of plant and bacterial ureases.

Ureases (EC 3.5.1.5) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Produced by plants, fungi and bacteria, but not by animals, ureases share significant homology and similar mechanisms of catalysis, although differing in quaternary structures. While fungal and plant ureases are homo-oligomeric proteins of 90 kDa subunits, bacterial ureases are multimers of two (e.g. Helicobacter pylori) or three subunit complexes. It has been proposed that in plants these enzymes are involved in nitrogen bioavailability and in protection against pathogens. Previous studies by our group have shown that plant ureases, but not a bacterial (Bacillus pasteurii) urease, display insecticidal activity. Herein we demonstrate that (Glycine max) embryo-specific soybean urease, jackbean (Canavalia ensiformis) major urease and a recombinant H. pylori urease impair growth of selected phytopathogenic fungi at sub-micromolar concentrations. This antifungal property of ureases is not affected by treatment of the proteins with an irreversible inhibitor of the ureolytic activity. Scanning electron microscopy of urease-treated fungi suggests plasmolysis and cell wall injuries. Altogether, our data indicate that ureases probably contribute to the plant arsenal of defense compounds against predators and phytopathogens and that the urease defense mechanism is independent of ammonia release from urea.

Ureases display biological effects independent of enzymatic activity: is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Bacillus pasteurii urease shares with plant ureases the ability to induce aggregation of blood platelets.

Ureases (EC 3.5.1.5) are highly homologous enzymes found in plants, bacteria and fungi. Canatoxin, an isoform Canavalia ensiformis urease, has several biological properties unrelated to its ureolytic activity, like platelet-aggregating and pro-inflammatory effects. Here, we describe that Bacillus pasteurii urease (BPU) also induces aggregation of rabbit platelets, similar to the canatoxin-induced effect (ED(50) 0.4 and 0.015 mg/mL, respectively). BPU induced-aggregation was blocked in platelets pretreated with dexamethasone and esculetin, a phospholipase A(2) and a lipoxygenase inhibitor, respectively, while platelets treated with indomethacin, a cyclooxygenase inhibitor, showed increased response to BPU. Methoxyverapamil (Ca(2+) channel blocker) and AMP (ADP antagonist) abrogated urease-induced aggregation, whereas the PAF-acether antagonist Web2170 had no effect. We concluded that platelet aggregation induced by BPU is mediated by lipoxygenase-derived eicosanoids and secretion of ADP from the platelets through a calcium-dependent mechanism. Potential relevance of these findings for bacterium-plant interactions and pathogenesis of bacterial infections are discussed.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10 percent of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Insecticidal effects of canatoxin on the cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae).

Canatoxin (CNTX) is a variant form of urease isolated from Canavalia ensiformis (Leguminosaea) seeds. A possible role in the plant defense was proposed for CNTX, due to its toxicity upon feeding to the beetle Callosobruchus maculatus, and the hematophagous bug, Rhodnius prolixus. The toxic effect is caused by a canatoxin-derived peptide ( approximately 10kDa) formed by insect cathepsin-like digestive enzymes. In order to evaluate their potential as bioinsecticides, the effects of CNTX and its peptide were evaluated on a phytophagous hemipteran insect Dysdercus peruvianus, a pest of cotton culture. For the bioassays, the insects fed on gelatin capsules containing powdered cotton seeds, mixed with the freeze-dried protein and other test materials and were observed for survival rate, weight gain and molting. Ingestion of canatoxin, or a recombinant 10kDa peptide derived from it, severely affected young forms of the insects, delaying their development or leading to their death. In contrast, adults were insensitive to diets containing higher concentrations of canatoxin. Cathepsin-like proteinases predominated and showed distinct pattern of enzymatic activities in midguts homogenates according to the developmental stage of the insect, a fact which may explain the different susceptibility of nymphs as compared to adult D. peruvianus. The data presented confirm the potential use of canatoxin-like proteins and derived peptides as bioinsecticides.

Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity.

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.

Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional and toxic proteins.

The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars.

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus).

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes.

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.

Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern.

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1-34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding alpha and beta chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.

cAMP does not inhibit convulxin-induced tyrosyl-phosphorylation of human platelet proteins, including PLCgamma2, but completely blocks the integrin alphaIIb beta3-dependent dephosphorylation step: comparisons with RGDS peptide, cytochalasin D, and phenylarsine oxide.

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom, induces platelet aggregation, phospholipase C (PLC) activation, and tyrosyl-phosphorylation (PTP) of multiple proteins, including PLCgamma2 by a mechanism independent of integrin alphaIIb beta3. However, PTP induced by Cvx is followed by a dephosphorylation step in a platelet aggregation-dependent manner. Here we show that increasing intraplatelet content of cAMP with forskolin is associated with the inhibition of Cvx-induced platelet aggregation, ATP secretion, and inositol-phosphates production. However, the early onset of Cvx-induced PTP is not sensitive to cAMP (including PLCgamma2), and it also occurs in the presence of integrin alphaIIb beta3-antagonist (RGDS peptide, RGDS) or inhibitors of actin polymerization (cytochalasin D, CD) and tyrosine-phosphatases (phenylarsine oxide, PAO). However, forskolin, RGDS, and CD prevented the dephosphorylation step together with inhibition of platelet aggregation, whereas in the presence of phenylarsine oxide (PAO) the dephosphorylation step was replaced by an increase in the number and intensity of tyrosyl-phosphorylated proteins. Our data provide evidence to conclude that (i) cAMP inhibits platelet aggregation at a downstream site to PLCgamma2 tyrosyl-phosphorylation; (ii) Cvx-induced PTP is independent on integrin alphaIIb beta3 engagement, actin polymerization, and tyrosine-phosphatases activation; (iii) integrin alphaIIb beta3 mediates the dephosphorylation step in a platelet aggregation-dependent manner; and (iv) Cvx and collagen stimulate platelets by a similar signal transduction pathway.

Convulxin induces platelet activation by a tyrosine-kinase-dependent pathway and stimulates tyrosine phosphorylation of platelet proteins, including PLC gamma 2, independently of integrin alpha IIb beta 3.

1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+ concentration ([Ca2+]in) due to both Ca2+ entry and internal Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]in in Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLC gamma 2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin alpha IIb beta 3 is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin alpha IIb beta 3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLC gamma 2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin alpha IIb beta 3.

Variability of bothrojaracin isoforms and other venom principles in individual jararaca (Bothrops jararaca) snakes maintained under seasonally invariant conditions.

Bothrojaracin (BJC) is a potent thrombin inhibitor isolated from the venom of Bothrops jararaca. Venoms from individual snakes have been shown to vary in BJC content, and more than one molecular variant (isoform) has been identified in the same venom. In order to determine whether the production of this protein and its isoforms varies under seasonally invariant conditions, an analysis was made of BJC isolated from venoms collected individually once a month for 10 months from two female B. jararaca snakes kept under conditions of constant temperature and photoperiod. The crude venom from each individual snake exhibited a characteristic pattern of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with no noticeable variation throughout the collecting period. BJC from individual venoms was purified by gel filtration on Sephacryl S-200 followed by an affinity column (PPACK-thrombin Sepharose). BJC content and other activities such as phospholipase A2, azocaseinolytic activity and inhibition of thrombin-induced platelet aggregation varied considerably among the samples. Purified BJC from both snakes inhibited fibrin coagulation and migrated as a single band of 27,000 mol. wt on SDS-PAGE. However, the BJC pattern on non-denaturing PAGE differed between the two snakes, with four to six bands per sample each month, which were all recognized by polyclonal anti-BJC antibodies. The isoelectric focusing pattern of BJC was also characteristic for each snake, with only minor differences throughout the collecting period. These results indicate that under seasonally invariant conditions: (1) there was a considerable variation over the 10-month period in the production of BJC and other important venom activities such as phospholipase A2 and proteinases; (2) individual B. jararaca snakes produced a distinctive array of BJC isoforms; and (3) despite quantitative differences, there were essentially no qualitative differences in the production of BJC isoforms by individual snakes during the 10-month period.

Bothrops sp. snake venoms: comparison of some biochemical and physicochemical properties and interference in platelet functions.

Procoagulant, proteolytic, phospholipase and platelet pro-aggregating and inhibiting activities were screened for pooled venoms of seven Bothrops species as well as Crotalus durissus terrificus and Lachesis muta snakes typical of the Brazilian territory. As reported by other authors, we also found that examination of the electrophoretic and gel filtration patterns of Bothrops snakes venoms could not be used for identification of the species of a given venom because of the lack of marked interspecific differences within the same genus. Our data indicated that B. cotiara, B. alternatus and B. atrox possess no platelet pro-aggregating activity, low inhibitory effect on platelet aggregation and very low or intermediate levels for the other activities. B. moojeni, B. neuwiedi and B. jararacussu whose venoms possess high procoagulant, platelet pro-aggregating and phospholipase activities are low in both proteolytic and platelet inhibitory activities. B. jararaca venom showed the highest inhibitory effect on platelet aggregation and very low platelet pro-aggregating activity. Compared with the Bothrops venoms studied, L. muta venom showed that highest proteolytic activity while C. d. terrificus venom presented remarkable high platelet pro-aggregating and phospholipase activities. In all venoms, proteolytic activity could be completely inhibited by EDTA (2 mM) alone. In contrast, the presence of phenylmethylsulfonyl fluoride (5 mM) inhibited partially the caseinolytic activity of all venoms, except that L. muta venom, which was almost completely blocked by this reagent. Altogether, these data confirm the presence of high levels of metalloproteinases in the venoms of Crotalinae snakes. Most of these enzymes are dependent of the availability of Ca2+, being much less the same concerning the presence of serine residues in their active sites. The data indicated that the presence and levels of procoagulant, azocaseinolytic and phospholipase A2 activities alone could not differentiated the species of the Bothrops venoms studied, particularly in the cases of B. jararaca, B. moojeni and B. atrox. However, the platelet inhibiting property of low doses of B. jararaca venom can be useful to differentiate it from B. moojeni venom. In the same way, the platelet pro-aggregating activity of high doses of B. jararaca venom may be used to distinguish it from B. atrox crude venom, otherwise very similar but incapable to activate platelets. In conclusion, our comparative screening of biological properties has indicated that platelet studies may serve as a tool to distinguish among venoms that otherwise behave biochemically in a very similar way. Although promising, the general applicability of platelet activation studies by snake venoms for classification or taxanomical purposes has yet to be extended to other family of snakes to be proven useful.

Human platelets activation by convulxin is accompanied by tyrosyl-phosphorylation of PLCgamma2 and occurs independently of integrin alphaIIbbeta3.

In the present report we show that convulxin (Cvx), a C-type lectin from Crotalus durissus terrificus venom, induces platelet agregation and phospholipase C (PLC) activation by a protein tyrosine kinase (PTK)-dependent pathway. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelet proteins with molecular masses of 40, 72/74, 78/80 and 120 kDa, followed by dephosphorylation of some proteins. However, platelet aggregation was accompanied by the phosphorylation of a 105-kDa molecular mass protein. Furthermore, Cvx stimulates a rapid-tyrosyl phosphorylation of a 145-kDa protein that was identified as PLC gamma 2. Protein tyrose phosphatase (PTP) induced by Cvx was not blocked when platelets were stimulated in the presence of indomethacin, apyrase, EDTA or RGDS peptide, but inhibited by staurosporine and genistein. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and it is independent of platelet aggregation or fibrinogen binding to integrin alphaIIbbeta3. On the other hand, the phosphorylation step, and the phosphorylation of the 105-kDa protein, were both inhibited by RGDS and EDTA, which suggests that the integrin alphaIIbbeta3 beta is involved in these steps. Our results, taken together, show that Cvx induces platelet IIb 3 aggregation in a similar manner as collagen and collagen-related peptides that also trigger platelet aggregation by a PTK-dependent pathway, and stimulate tyrosyl-phosphorylation of PLC gamma 2. However, Cvx is unique among platelet receptor agonists, because under test-tube stirring conditions it induces a PTP profile independently of integrin alphaIIbbeta3.