Induction of microvascular leakage and histamine release by promutoxin, an Arg49 phospholipase A2

It has been recognized that phospholipase A₂ (PLA₂) is a crucial factor of snake venom induced inflammation. Recently, promutoxin, a novel member of minor subgroup of snake venom PLA₂ (R49 PLA₂) has been characterized in our laboratory, but its roles in induction of inflammation remain uninvestigated. Using highly purified promutoxin, we found this enzymatically inactive PLA₂ provoked a dose-dependent increase in microvascular leakage in the skin of rats. Pretreatment of rats with compound 48/80 diminished promutoxin-induced skin reaction and reduced mast cell numbers in rats. Cyproheptadine, terfenadine, Ginkgolide B and heparin inhibited promutoxin elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, promutoxin was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting promutoxin elicited histamine release. Provocation of microvascular leakage and mast cell activation by promutoxin suggests further that snake venom induced inflammation is related to mast cell activation and certain anti-inflammatory drugs could be therapeutic effective in treating snake wound.     

Inflammatory effects of snake venom myotoxic phospholipases A2

Snake venom phospholipases A₂ (PLA₂) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA₂s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA₂s. Since the discovery that mammalian PLA₂ is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA₂s are being actively investigated, and part of the knowledge on secretory PLA₂ effects has been gained by using snake venom PLA₂s as tools, due to their high structural homology with human secretory PLA₂s. The inflammatory events evoked by PLA₂s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA₂s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA₂s with cell membranes potentially involved in the PLA₂-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA₂s and their mechanisms of action.     

PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom,Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

A monomeric basic PLA₂ (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA₂ enzyme class and displays conserved domains as the catalytic network, Ca²⁺-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA₂ showed an allosteric behavior and its enzymatic activity was dependent on Ca²⁺. Examination of PhTX-II PLA₂ by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA₂ causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA₂ that contributes with toxic actions caused by P. hyoprora venom.     

Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake

We report the purification and biochemical/pharmacological characterization of two myotoxic PLA₂ (BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M_r 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂, had a sequence of 121 amino acid residues. BbTX-II: SLFELGKMILQETGKNPAKSYGAYGCYCGVLGRGKPKDATDRCCYVHKCCYKLTGCDNKKKDRYSYSWKDKTIVCGENNPCLKELCECDKAVAICLRENLNTYNKKYRYHLKPLCKKADAC with pI value 8.73. BbTX-III: SLWEWGQMILKETGKNPFPYYGAYGCYCGWGGRRKPKDATDRCCFVHDCCRYKKLTGCPKTNDRYSYSRLDYTIVCGEDDPCKEICECDKAAAVCFRENLRTYNKKYMAHLRVLCKKDKPC with a pI value of 8.46. BbTX-III presented PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45 °C. Maximum PLA₂ activity required Ca²⁺. In vitro, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA₂ homologue, occur in the absence of a measurable PLA₂ activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.     

A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A2s

Venom phospholipase A₂s (PLA₂s) display a wide spectrum of pharmacological activities and, based on the wealth of biochemical and structural data currently available for PLA₂s, mechanistic models can now be inferred to account for some of these activities. A structural model is presented for the role played by the distribution of surface electrostatic potential in the ability of myotoxic D49/K49 PLA₂s to disrupt multilamellar vesicles containing negatively charged natural and non-hydrolyzable phospholipids. Structural evidence is provided for the ability of K49 PLA₂s to bind phospholipid analogues and for the existence of catalytic activity in K49 PLA₂s. The importance of the existence of catalytic activity of D49 and K49 PLA₂s in myotoxicity is presented.     

The role of phospholipases A2 in the stimulation of neutrophil motility by cobra venoms.

Neutrophil (PMN) accumulation frequently occurs at the site of snakebite as part of the inflammatory response to envenoming. We demonstrate here that the venoms of the cobras, Naja naja and N. mossambica, and two purified venom phospholipase A(2)s (PLA(2)s) isolated from the latter venom, stimulate CD11b translocation from the PMN granule store to the plasma membrane and enhance neutrophil motility on collagen-coated surfaces. These effects were partially attenuated by the PLA(2) inhibitor, aristolochic acid, and almost completely abolished by the specific cytosolic PLA(2) inhibitor, methylarachidonylfluorophosphonate (MAFP). Annexin V and inhibitors of collagenase, cyclo-oxygenase and lipo-oxygenase, all inhibited PMN motility to a variable extent. FACS analysis and confocal microscopy showed that Annexin V interfered with binding and rapid endocytosis of the venom PLA(2). These results indicate that venom and venom PLA(2) preparations first caused a non-enzymatic stimulation of PMN leading to the activation of cytosolic PMN PLA(2) and production of arachidonate metabolites involved in stimulation of PMN degranulation and motility. The evidence suggests that venom PLA(2) then interacts with anionic phospholipids exposed on stimulated PMN, becomes endocytosed, and then contributes itself to the production of chemoattractants responsible for PMN accumulation at the site of the snakebite.     

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA₂ from Agkistrodon contortrix laticinctus snake venom and Lys49PLA₂-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA₂-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.     

cDNA cloning, structural, and functional analyses of venom phospholipases A2 and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi

Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.     

Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A₂ (PLA₂) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA₂. They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA₂s from venoms of Bothrops genus. The two PLA₂ variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA₂s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.     

Isolation and Characterization of A2-EPTX-Nsm1a,a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom

Phospholipase A₂ (PLA₂) toxins are one of the main toxin families found in snake venom. PLA₂ toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA₂ components_, there is limited information on the function and activities of PLA₂ toxins from the venom. In this study, a secretory PLA₂ from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA₂ from venoms of other Naja species. The PLA₂ activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA₂ isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.     

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2

Bothrops marajoensis is found in the savannah of Marajó Island in the State of Pará and regions of Amapá State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A₂ (PLA₂). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA₂ showed amino acid identity with other lysine K49 sPLA₂s of snake venom. B. marajoensis venom (30 μg/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA₂ did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA₂ decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA₂ induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system.     

Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

We report the detailed molecular characterization of two PLA₂s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA₂s, named BlK-PLA₂ and BlD-PLA₂, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA₂s, and show a high degree of sequence similarity to homologous snake venom PLA₂s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA₂s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA₂s were assessed by quantitative Real-Time PCR. Bl-PLA₂s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA₂s as tools of research.     

Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora

Two distinct phospholipase A₂ (PLA₂) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA₂ purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits, A and B, inhibited both the acidic and basic PLA₂s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLIβ and PLIγ, respectively. A PLIα homolog (PLIα-like protein; PLIα-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLIα, but lacked in the inhibitory activity toward the acidic and basic PLA₂s. The cDNAs encoding PLIα-LP, PLIβ, PLIγ-A, and PLIγ-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.     

Inhibitory effect of phospholipase A2 isolated from Crotalus durissus terrificus venom on macrophage function

Recent work demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, inhibits macrophage spreading and phagocytic activities. The crotoxin molecule is composed of two subunits, an acidic non-toxic and non-enzymatic polypeptide named crotapotin and a weakly toxic basic phospholipase A₂ (PLA₂). In the present work, the active subunit responsible for the inhibitory effect of crotoxin on macrophage function was investigated. Peritoneal macrophages harvested from naive rats were used. Crotapotin (2.12, 3.75, or 8.37 nM/ml), added for 2 h to the medium of peritoneal cell incubation, did not modify the spreading and phagocytic activities of these cells. On the other hand, the PLA₂ (1.43, 2.86, or 6.43 nM/ml) subunit caused a significant reduction (30, 33, and 35%, respectively) of the spreading activity. The PLA₂ also inhibited the phagocytosis of opsonised zymosan, opsonised sheep erythrocytes, and Candida albicans, indicating that this inhibitory effect is not dependent on the type of receptor involved in the phagocytosis process. The inhibitory effect of PLA₂ was not due to loss of cell membrane integrity, since macrophage viability was higher than 95%. These findings indicate that the inhibitory effect of crotoxin on macrophage spreading and phagocytic activities is caused by the phospholipase A₂ subunit.     

Inhibition of phospholipase A2 activities and some inflammatory responses by the marine product ircinin

The marine product ircinin has been tested for its effects on secretory and cytosolic phospholipase A₂ (PLA₂) activities in vitro as well as for inhibition of cellular functions in human neutrophils and inflammatory responses in mice. Ircinin inhibited Naja naja venom, human synovial recombinant, bee venom and zymosan-injected rat air pouch PLA₂ with IC₅₀ values in the M range, similar to those of the known inhibitor scalaradial. On the other hand, ircinin was less active on cytosolic PLA₂ from human monocytes and decreased potently the release of LTB₄ in human neutrophils. This marine product affected weakly human neutrophil functions like superoxide generation and degranulation. In the zymosaninjected rat air pouch ircinin inhibited in vivo the activity of PLA₂ present in exudates and reduced dose-dependently myeloperoxidase levels, whereas cell migration was inhibited only at the highest dose tested. This compound exerted a potent anti-oedematous effect after topical application in the mouse ear oedema test. Ircinin is a new inhibitor of PLA₂ activity and our results suggest a potential role for this marine product as an inhibitor of inflammatory processes.     

Anti-Neurotoxins from Micrurus mipartitus in the Development of Coral Snake Antivenoms

In Colombia, the genus Micrurus includes 30 species, of which M. mipartitus and M. dumerilii are the most widely distributed. Micrurus causes less than 3% of the approximately 5000 cases of snakebite per year. The elapid envenomation caused by the snakes from the Micrurus genus, are characterized by the severity of their clinical manifestations, due to the venom neurotoxic components such as three-finger toxins (3FTx) and phospholipases (PLA₂). The treatment for snakebites is the administration of specific antivenoms, however, some of them have limitations in their neutralizing ability. A strategy proposed to improve antivenoms is to produce antibodies against the main components of the venom. The aim of this work was to produce an antivenom, using an immunization protocol including the main 3FTx and PLA₂ responsible for M. mipartitus lethality. The antibody titers were determined by ELISA in rabbits’ serum. The immunized animals elicited a response against toxins and whole venom. The Immunoglobulin G (IgGs) obtained were able to neutralize the lethal effect of their homologous toxins. A combination of antivenom from M. mipartitus with antitoxins improved their neutralizing ability. In the same way, a mixture of anti 3FTx and PLA₂ protected the mice from a 1.5 median lethal dose (LD₅₀) of M. mipartitus venom. The results showed that this might be a way to improve antibody titers specificity against the relevant toxins in M. mipartitus venom and indicated that there is a possibility to develop and use recombinant 3FTx and PLA₂ toxins as immunogens to produce antivenoms. Additionally, this represents an alternative to reduce the amount of venom used in anti-coral antivenom production.     

Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A2 from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A₂ (PLA₂) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA₂ edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K_I) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA₂ in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA₂ (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA₂.     

Composition and Acute Inflammatory Response from Tetraponera rufonigra Venom on RAW 264.7 Macrophage Cells

Tetraponera rufonigra (Arboreal Bicoloured Ant) venom induces pain, inflammation, and anaphylaxis in people and has an increased incident in Southeast Asia regions. The bioactive components and mechanism of action of the ant venom are still limited. The aim of this research was to identify the protein composition and inflammatory process of the ant venom by using RAW 264.7 macrophage cells. The major venom proteins are composed of 5’ nucleotidase, prolyl endopeptidase-like, aminopeptidase N, trypsin-3, venom protein, and phospholipase A₂ (PLA₂). The venom showed PLA₂ activity and represented 0.46 μg of PLA₂ bee venom equivalent/μg crude venom protein. The venom induced cytotoxic in a dose- and time-dependent manner with IC₂₀ approximately at 4.01 µg/mL. The increased levels of COX-2 and PGE₂ were observed after 1 h of treatment correlating with an upregulation of COX-2 expression. Moreover, the level of mPGES-1 expression was obviously increased after 12 h of venom induction. Hence, our results suggested that the induction of COX-2/mPGEs-1 pathway could be a direct pathway for the ant venom-induced inflammation.     

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A₂ (PLA₂) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA₂-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA₂-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA₂-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA₂-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA₂s. The phylogenetic analyses showed that BpPLA₂-TXI forms a group with other acidic D49 PLA₂s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.     

In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper

The antimicrobial and antiparasite activity of phospholipase A₂ (PLA₂) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A₂ (PLA₂) (fraction V) and another containing a PLA₂ homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA₂ and its homologue have antiplasmodial potential.