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PubMed1993

Model for the interaction of crotoxin, a phospholipase A2 neurotoxin, with presynaptic membranes.

Crotoxin is a phospholipase A2 neurotoxin that impairs the release of acetylcholine at neuromuscular junctions, primarily at the presynaptic level. It associates a phospholipase A2 subunit, CB, with a chaperon subunit, CA. We have shown elsewhere that the purely cholinergic synaptosomes from the Torpedo electric organ provided a convenient model to study the pharmacology of crotoxin and other related neurotoxins [Délot, E., & Bon, C. (1992) J. Neurochem. 58, 311-319]. In the present experiments, we labeled crotoxin with 125I and demonstrated saturable binding to Torpedo presynaptic membranes. In the range of concentrations that was effective on synaptosomes, crotoxin bound to a single class of sites without cooperativity. The binding was affected by divalent cations, and its kinetics was rather complex. We observed a competition between crotoxin and related neurotoxins, but not CB. Although CA could not bind, it could compete efficiently with crotoxin. We compare our results with those previously obtained by others on guinea pig brain membranes. On Torpedo membranes, as on all models tested, CB was the major species bound to the membrane, while CA remained in solution. However, the mechanism underlying this phenomenon had never been clarified. The major question is the time scale of the events, i.e., does CB bind before or after dissociating from CA? Our results indicate that the predominant pathway involves the formation of a ternary complex between crotoxin's subunits and the acceptor site preceding the release of CA.

Acetylcholine↗

PubMed2001

Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney.

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g% of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport (%TNa+). The infusion of Crotalus durissus terrificus venom (10 microg/ml) and crotoxin (10 microg/ml) increased GFR (control80 = 0.78 +/- 0.07, venom80 = 1.1 +/- 0.07, crotoxin80 = 2.0 +/- 0.05 ml g(-1) min(-1), P<0.05) and UF (control80 = 0.20 +/- 0.02, venom80 = 0.32 +/- 0.03, crotoxin80 = 0.70 +/- 0.05 ml g(-1) min(-1), P<0.05), and decreased %TNa+ (control100 = 75.0 +/- 2.3, venom100 = 62.9 +/- 1.0, crotoxin80 = 69.0 +/- 1.0 ml g(-1) min(-1), P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney.

Animals↗

PubMed1993

Effects of crotoxin on the action potential kinetics of frog skeletal muscle.

1. The effect of crotoxin on the action potential kinetics of frog (Rana catesbeiana; 60-80 g) skeletal muscle was studied using a modified Ringer solution containing 1.6 mM KCl. 2. Crotoxin affected the kinetics of the action potential in a dose-dependent manner (90 to 460 nM). At 230 nM, crotoxin prolonged the duration of the action potential (from 1.1 +/- 0.1 to 1.6 +/- 0.1 ms) and slowed the rates of depolarization (from 282.0 +/- 7.3 to 196.0 +/- 13.2 V/s) and repolarization (from -81.4 +/- 6.9 to -55.6 +/- 3.8 V/s), in a dose-dependent manner. Its phospholipase subunit (component B) was five times less effective. 3. No effect of crotoxin was observed in the presence of 2.5 mM KCl or when SrCl2 was substituted for CaCl2. Lowering the muscle temperature to 12 degrees C did not reduce the effect of crotoxin. 4. No effect on the passive membrane response to hyperpolarizing current pulses was observed, which implies no major effect on the membrane resistance and capacitance. 5. It is concluded that crotoxin reduces the Na+ current and slows down the repolarization mechanism. This effect is probably not dependent on the phospholipase A2 activity of crotoxin and is inhibited by the substitution of Sr2+ for Ca2+.

Action Potentials↗

PubMed1985

Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vas deferens.

The effects of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus on mammalian autonomic neuromuscular transmission, have been investigated. In the longitudinal muscle of the guinea-pig ileum, crotoxin induced a dose-dependent contraction which was followed by relaxation, in spite of the continued presence of the toxin. The contractile response was inhibited by indomethacin, tetrodotoxin, verapamil or nifedipine, but was unaffected by atropine, propranolol, mepyramine or methysergide. In addition, crotoxin caused a presynaptic inhibition of the electrically-evoked twitch of the longitudinal muscle of the guinea-pig ileum. In the guinea-pig vas deferens crotoxin also caused an inhibition of the response to field stimulation. The inhibition was reversible after washing and the preparation remained insensitive to further doses of the toxin. The inhibitory effects of crotoxin were not mediated by noradrenaline and were not due to a non-specific smooth muscle depression, because it was not associated with any reduction in motor responses to acetylcholine, ATP, bradykinin or substance P. Pre-incubation of the guinea-pig vas deferens with indomethacin blocked the inhibitory effects of the toxin. This suggests that the presynaptic activity of crotoxin in the vas deferens might be mediated by prostaglandins.

Animals↗

PubMed1991

Immunochemical cross-reactivity of two phospholipase A2 neurotoxins, agkistrodotoxin and crotoxin.

Polyclonal rabbit antisera were raised against the phospholipase A2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A2 from Vipera berus venom, but not other phospholipases A2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.

Animals↗

PubMed1994

Crotoxin induces aggregation of human washed platelets.

Crotoxin, the main toxic component isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, is a reversible protein complex composed of a non-toxic non-enzymatic acidic polypeptide (crotapotin) and a toxic basic phospholipase A2 (PLA2). In this study, we have evaluated the ability of crotoxin to induced aggregation in human washed platelets. Human washed platelet aggregation was monitored in a Payton aggregometer and thromboxane B2 (TXB2) release measured by direct radioimmunoassay (RIA). Crotoxin (15-50 micrograms/ml) produced dose-dependent and irreversible human washed platelet aggregation, which was inhibited by pre-incubation of the platelets with sodium nitroprusside (50-500 microM) or iloprost (8-80 nM). Crotoxin also induced TXB2 release (207 +/- 8 ng/ml, n = 6), and although indomethacin significantly reduced the release of TXB2 (to 23.5 +/- 5 ng/ml, P < 0.001, n = 6), it did not inhibit crotoxin-induced aggregation. Our results clearly demonstrate that crotoxin induces human washed platelet aggregation and that this phenomenon is independent of the formation of pro-aggregatory arachidonic acid metabolites.

Amino Acid Sequence↗

PubMed1994

Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA2 subunit of crotoxin and neutralize its pharmacological activity.

Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A2. Polyclonal antibodies against three synthetic peptides selected from the C-terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin.

Amino Acid Sequence↗

PubMed2004

A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus.

In Brazil, the Crotalus durissus terrificus subspecie is the most studied, particularly concerning its crotoxin. Crotoxin is the major toxic component of the South American rattlesnake Crotalus durissus venom. It is composed of two different subunits, CA called crotapotin and CB weakly toxic phospholipase A2 with high enzymatic activity. In this paper, we decided to make a study of the main toxic characteristics of crotoxin (CTX) and CB fraction from the other subspecies, Crotalus durissus cascavella and of Crotalus durissus collilineatus, in comparison with those of C. d. terrificus. Ours results have shown that the venoms presented similar chromatographic profiles and the purified fractions were free of contaminants. Regarding the toxic activities, the DL50 of the crotoxins showed no significant differences between the subspecies. The smaller toxicity of CB indicated that the toxicity of the crotoxin complex depends on the interaction between CA and CB. CTX and fraction CB of the three species of Crotalus showed negligible proteolytic activity. C. d. terrificus CTX presented higher PLA2 activity when compared with the others two subspecies. The oedema induced by CB developed later than the CTX and reached its peak 3 h after the injection. The myotoxic activity was determined by assaying serum CK levels. Mice injected with CTX of C. d. terrificus presented greater myotoxic activity compared to the others. The myotoxic activity of CB from the three subspecies was lower than the activity of the crotoxin, reinforcing the idea that the fraction CA increases the toxicity of CB.

Analysis of Variance↗

PubMed2006

Crotoxin induces actin reorganization and inhibits tyrosine phosphorylation and activity of small GTPases in rat macrophages.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom. Previous work of our group demonstrated that this toxin or its phospholipase A(2) subunit inhibits macrophage spreading and phagocytosis. The phagocytic activity of macrophages is controlled by the rearrangement of actin cytoskeleton and activity of the small Rho GTPases. The effect of crotoxin and its subunit on actin reorganization and tyrosine phosphorylation in rat peritoneal macrophages, during phagocytosis of opsonized zymosan, was presently investigated. The crude venom was used as positive control. In addition, the effect of crotoxin on the activity of Rho and Rac1 small GTPases was examined. Transmission electron studies showed that the venom or crotoxin decreased the extent of spread cells and increased microprojections often extended from macrophage surface. Immunocytochemical assays demosntrated that the venom or toxins increased F-actin content in the cytoplasm of these cells, but induced a marked decrease of phosphotyrosine. These effects were abolished by treatment with zileuton, a 5-lipoxygenase inhibitor. Furthermore, crotoxin decreased membrane-associated RhoA and Rac1 in translocation assays. The present results indicate that the crotalid venom and crotoxin are able to induce cytoskeleton rearrangement in macrophages. This effect is associated with inhibition of tyrosine phosphorylation and of the activity of proteins involved in intracellular signalling pathways important for the complete phagocytic activity of these cells.

Actins↗

PubMed1997

Time factor in the detection of circulating whole venom and crotoxin and efficacy of antivenom therapy in patients envenomed by Crotalus durissus.

Thirty-seven patients envenomed by Crotalus durissus were classified into three groups according to the interval between the bite and hospital admission (delta T): group 1 (n = 14, delta T < 4 hr), group 2 (n = 14, delta T > 4 hr < 8 hr) and group 3 (n = 9, delta T > 8 hr). Venous blood from these patients was sampled for biochemical and hematological analysis and for whole venom, crotoxin and antivenom enzyme-linked immunosorbent assays before antivenom treatment (T0) and at 1 hr (T1), 6 hr (T6), 12 hr (T12) and 24 hr (T24) after the start of antivenom therapy. The patients were treated with 100-200 ml (10-20 ampules) of C. durissus antivenom. Whole venom and crotoxin were detected in 13 (92.8%) and 11 (78.6%) of 14 group 1 patients, respectively, in 11 (78.6%) and six (42.9%) of 14 group 2 patients, respectively, and in two (22.2%) and one (11.1%) of nine group 3 patients, respectively, before antivenom treatment. Data from this study show that whole venom and crotoxin were not detected in most of patients when the time elapsed between the bite and hospital admission was greater than 8 hr, and crotoxin was not detected in most of the patients who were admitted to the hospital at times ranging from 4 to 8 hr after the snakebite. Plasma whole venom, crotoxin and antivenom levels measured over time in these patients show the efficacy of antivenom treatment, since circulating venom and crotoxin were no longer detected 1 hr after antivenom therapy and high antivenom titers persisted for at least 24 hr after serotherapy.

Adolescent↗

PubMed1988

Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities.

Crotoxin, the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus is a mixture of several isoforms that differ slightly in their molecular structure. The toxin consists of two nonidentical subunits: a basic and weakly toxic phospholipase A2, component B, and an acidic and nontoxic subunit, component A. In the present investigation, we have used fast-performance liquid chromatography (FPLC) on anionic and cationic exchange columns to purify isoforms of both crotoxin subunits. Two component A isoforms and four component B isoforms were obtained in a homogeneous state, and their purity was verified by isoelectric focusing in polyacrylamide gels. The amino acid composition of the purified component A and component B isoforms was in good agreement with the protein sequences determined previously with mixtures of isoforms. The amino acid compositions indicated that for both crotoxin components the isoforms differed only by the replacement of few amino acid residues. Eight crotoxin complexes have been prepared in a homogeneous state by reassociation of pure component A and component B isoforms. The quantitative comparison of enzymatic and pharmacological properties of the reconstituted crotoxins indicated that the two component A isoforms had identical properties, whereas the four component B isoforms fell in two classes: crotoxin complexes formed with component B isoforms of the first class were enzymatically less active and pharmacologically more potent than those obtained with component B isoforms of the second class.

Amino Acids↗

PubMed1989

Binding of crotoxin, a presynaptic phospholipase A2 neurotoxin, to negatively charged phospholipid vesicles.

Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.

Chemical Phenomena↗

PubMed1988

Early induction by crotoxin of biphasic frequency changes and giant miniature endplate potentials in frog muscle.

1. Following the addition of crotoxin (250 nM) at the frog neuromuscular junction, there was an initial fall in frequency of miniature endplate potentials (m.e.p.ps), followed by a secondary rise which was characterized by the appearance of large spontaneous potentials (giants, g.m.e.p.ps) and an occasional large potential of the burst type. 2. In the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesicamol), an inhibitor of vesicular acetylcholine uptake, the frequency of g.m.e.p.ps induced by crotoxin was reduced. 3. The characteristic changes in m.e.p.p. frequency and amplitude distribution were absent with crotoxin in Sr-EGTA Ringer. In the presence of high concentrations of Mn (3.6 or 5.4 mM with 0.9 mM Ca), the crotoxin-induced initial fall and the onset of the secondary rise in m.e.p.p. and g.m.e.p.p. frequencies were slower. The timing of these phases was unaffected by Ca concentrations ranging from 6.3 to 0.9 mM. 4. High concentrations of Mn ions partially inhibited the phospholipase A2 activity of crotoxin on artificial phospholipid membranes. This also supports the involvement of the Ca-dependent phospholipase A2 subunit in both phases of the physiological action of the toxin. 5. G.m.e.p.ps were associated with a moderate increase in m.e.p.p. frequency (2-3 s-1) and were of a time-course similar to that of m.e.p.ps. They persisted after washing with medium lacking Ca ions and in the presence of Ca-Mn Ringer that blocked evoked responses. 6. It is concluded that crotoxin, acting through its phospholipase A2 subunit, produces specific disturbances of synaptic exocytosis and vesicle formation in the axolemma of the motor nerve terminal which lead to biphasic changes in m.e.p.p. frequency and the onset of large spontaneous potentials.

Animals↗

PubMed2000

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum.

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0. 03-1 microM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 microM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Animals↗

PubMed2000

Nature of the postsynaptic action of crotoxin at guinea-pig diaphragm end-plates.

Crotoxin is known to desensitize the nicotinic receptor of Torpedo marmorata and Electrophorus electricus electroplaques. The aim of the present study was to elucidate whether the postsynaptic effect of crotoxin at a mammalian muscle end-plate is also caused by receptor desensitization or results from a curaremimetic action. For this purpose, we investigated the action of 4-aminopyridine (4-AP) on crotoxin-induced blockade of miniature end-plate potentials (m.e.p.p.s) and of the depolarization of end-plates produced by carbachol. The experiments were carried out in guinea-pig diaphragms bathed in Tyrode solution at 37 degrees C and gassed with 95% O2, 5% CO2. The potentials were measured with conventional techniques using glass microelectrodes. Even at low concentrations, crotoxin blocked the m.e.p.p.s and this blockade was antagonized by 4-AP. Neostigmine was without effect. 4-AP did not restore the m.e.p.p.s blocked by either d-tubocurarine (dTc) or beta-bungarotoxin (beta-BTX). 4-AP also antagonized the crotoxin-induced blockade of the end-plate depolarization produced by carbachol. These results show that the postsynaptic effect of crotoxin at the guinea-pig muscle end-plate also results from nicotinic receptor desensitization.

4-Aminopyridine↗

PubMed1998

Differential biodistribution of native and 2 kGy 60Co irradiated crotoxin in tissues of CBA/J mice.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Animals↗

PubMed1977

Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of beta-bungarotoxin, crotoxin and taipoxin.

1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.

Animals↗

PubMed2006

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom.

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Amino Acid Sequence↗