Snake venom phospholipase A2s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2.

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell-cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on ß-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Jabuticaba (Plinia jaboticaba) skin extracts as inhibitors of phospholipases A2 and proteases.

The phenolic extracts of jabuticaba skin flour (JSF) were characterized by HPLC, and evaluated for their modulating action upon phospholipases A2 and proteases of snake venom, aiming at their possible use in the treatment of the various diseases associated with the action of venom toxins. Two types of extracts were prepared from JSF: aqueous and methanolic. These extracts, evaluated at different ratios, (venom: extract, m/m), significantly inhibited the phospholipase activity induced by the venom of Bothrops moojeni and Crotalus durissus terrificus, except for Bothrops atrox venom. The greatest hemolysis inhibitory action was observed for the methanolic extract, when incubated with venoms of B. moojeni and C. durissus terrificus, with inhibitions between 21 and 100%. Thrombolysis induced by venoms of B. moojeni and C. durissus terrificus was inhibited by both extracts, ranging from 32 to 83% and 51 to 83% for the aqueous and methanolic extracts, respectively. Both extracts extended coagulation time, induced by the venoms of B. moojeni and Lachesis muta muta. Inhibitory actions are related to phenolic compounds, such as gallic, syringic and p-coumaric acids, besides catechin, epigallocatechin gallate, epicatechin; resveratrol and quercetin, present in the extracts of jabuticaba skin flour, confirming their potential for nutraceutical use.

Proteomic Analysis and Immuno-Profiling of Eastern India Russell's Viper ( Daboia russelii) Venom: Correlation between RVV Composition and Clinical Manifestations Post RV Bite.

The proteomes of Russell's viper venom (RVV) from Burdwan (RVV B) and Nadia (RVV N), the two districts of West Bengal, eastern India (EI), were investigated by gel-filtration chromatography (GFC) followed by tandem mass spectrometry of tryptic fragments of the fractions. A total of 73 and 69 proteins belonging to 15 snake venom protein families were identified in RVV B and RVV N, respectively, by MS/MS search against Viperidae (taxid 8689) protein entries of the nonredundant NCBI database. The minor differences in venom composition of both the EI RV were established unequivocally by their biochemical and pharmacological properties and by SDS-PAGE, gel filtration chromatography, and LC-MS/MS analyses. The composition of EI RVVs was well correlated with published reports on the pathophysiology of RV-envenomed patients from this part of the country. Venom-antivenom cross-reactivity determined by ELISA, Western blotting, and antivenomics approaches demonstrated poor recognition of low molecular mass (<20 kDa) RVV proteins by commercial polyvalent antivenoms, which was substantiated by neutralization of RVV enzymes by antivenom.

Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

Inhibitory effects of ascorbic acid toward snake venom metalloproteinase (SVMP) from Indian Echis carinatus venom: Insights from molecular modeling and binding studies.

Classical antivenom therapy is unable to shield complications of viper bite and has limitations such as anaphylaxis and serum sickness. Snake venom metalloproteinases are responsible for local tissue damage and hemorrhage at the bitten site in viper envenomation, and this has led to a persistent search for metalloproteinase inhibitors. Here, we report the inhibitory effects of ascorbic acid against metalloproteinase from Echis carinatus venom both in-silico and in-vitro. Ascorbic acid effectively inhibited the proteolytic activity of E. carinatus venom in a dose-dependent manner. Interaction studies of ascorbic acid with purified ecarin using isothermal titration calorimetry showed favorable binding energy and energetics. The molecular docking of ascorbic acid with ecarin revealed important interactions with residues at the active site pocket of ecarin. It was observed that the ligand behaves as a chelating inhibitor. Thus, the backbone structural scaffold of ascorbic acid can find potential use as building blocks in designing drug-like molecules for viper bite management.

Antiplatelet and anticoagulant activities of two phospholipase A2s purified from Cerastes cerastes venom: Structure-function relationship.

This study aimed to elucidate anticoagulant/antiplatelet mechanisms of two previously purified PLA2 s from Cerastes cerastes venom, here, termed Cc1 -PLA2 and Cc2 -PLA2 . Both PLA2 s present close molecular weights of 13,534 and 13,430 Da and Isoectric pH (pI) 7.38 and 7.86 respectively, for Cc1 -PLA2 and Cc2 -PLA2 . These Ca2+ -dependent enzymes showed a high catalytic activity upon phospholipids, inducing indirect hemolysis, since they conserve the catalytic domain of PLA2 s 26 CYCGWGGKG34 . They exhibited dual inhibition of platelet aggregation by targeting P2 Y12 and TPα receptors preventing Adenosine diphosphate/arachidonate binding and blood clotting. These effects are due to the interaction of Cc1 -PLA2 s/Cc2 -PLA2 s with factor FXa through a noncatalytic PL-independent mechanism leading to nonreleased thrombin. Both proteins consist of 120 amino acid residues and share similar three-dimensional structures close to other SV-PLA2 s. Structural data of PLA2 s allowed the relevant residues involved in binding to FXa and platelet receptors. These findings may lead to the design of novel noncompetitive FXa inhibitors.

Isolation and characterization of an anti-leishmanial disintegrin from Cerastes cerastes venom.

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.

Dacin, one metalloproteinase from Deinagkistrodon acutus venom inhibiting contraction of mouse ileum muscle.

BACKGROUND: Mice were bitten by five-pace vipers (Deinagkistrodon acutus), and then envenomed. It was well-known that the snake venom mainly disturbed the blood homeostasis of the envenomed victims. Ocassionally, we found that the venom of D. acutus could inhibit the contraction tension of mouse ileum, so in this study we aimed to identify the active component inhibiting the contraction tension of mouse ileum in the snake venom. RESULTS: The active component inhibiting the contraction tension of mouse ileum, designated as Dacin, was isolated from D. acutus venom, purified to protein homogeneity and composed of a single peptide chain, about 23 kDa analyzed by SDS-PAGE, and 22, 947. 9 Da measured by MALDI-TOF-MS. Not only the results of its PMF blasted by Mascot indicated that Dacin may be one snake venom metalloproteinase (SVMP), but also the results of the biochemical and in-vivo assays as follow demonstrated that it was one SVMP: it cleaved Aα and Bß chains, not Cγ of bovine fibrinogen within 1 h, and also hydrolyzed fibrin polymer; besides its fibrino(geno)lytic activities were strongly inhibited by ß- mercaptoethanol, EDTA and EGTA; and it could induce a hemorrhagic reaction under the dorsal skin of mouse. In the isolated tissue assays, Dacin caused the concentration-dependent and time-dependent inhibitory actions on the spontaneous contraction tension of the ileum smooth muscle of mouse, and the inhibitory effects were irreversible. CONCLUSIONS: Taken together, for the first time one active component (Dacin, a SVMP) that irreversibly inhibited the spontaneous contraction tension of mouse ileum has been isolated and identified from D. acutus venom. The findings may provide not only a new insight for toxicological researches on SVMPs and venoms of the vipers, but also a reference for clinicians to treat the snake-bitten victims. However, Dacin's inhibitory molecular mechanism will be further studied in the future.

Biochemical and biological characterization of a dermonecrotic metalloproteinase isolated from Cerastes cerastes snake venom.

A dermonecrotic metalloproteinase (CcD-II) was isolated from C. cerastes venom. Venom fractionation was performed using three chromatographic steps (molecular exclusion on Sephadex G-75, ion-exchange on DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography on C8 column). CcD-II presented an apparent molecular mass of 39.9 kDa and displayed a dermonecrotic activity with a minimal necrotic dose of 0.2 mg/kg body weight. CcD-II showed proteolytic ability on casein chains and on α and ß fibrinogen chains that was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline while remained unaffected by phenylmethylsulphonyl fluoride and heparin. CcD-II displayed gelatinase activity and degraded extracellular matrix compounds (type-IV collagen and laminin). These results correlated with histopathological analysis showing a complete disorganization of collagenous skin fibers. These data suggested that CcD-II belongs to P-II class of snake venom metalloproteinase. The characterization of venom compounds involved in tissue damage may contribute in the development of new therapeutic strategies in envenomation.

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators.

The present study describes the purification and partial characterization of a basic anticoagulant PLA2 enzyme named as Rv(i) PLA2 from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA2 family. The peptide sequence showed similarity to uncharacterized basic PLA2 enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA2 exhibited strong phospholipase A2 and anticoagulant activity. It also induced expression of COX-2 and TNF-α mRNA in a dose-dependent manner in phorbol 12-myristate 13-acetate differentiated THP-1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA2 with p-bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA2 might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.

Comparative assay of Vipera ammodytes antivenom potency.

The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bß chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential.

BACKGROUND: Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates. METHODS: A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice. RESULTS: Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α2-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α2MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity. CONCLUSIONS: Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy. GENERAL SIGNIFICANCE: Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.

Purification, characterization and antibacterial activity of L-amino acid oxidase from Cerastes cerastes.

Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram-positive and Gram-negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An L-amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS-PAGE. LC-MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin-resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 µg/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on L-leucine at pH 8.0 and 20°C were Km = 0.06 mmol and Vmax = 164 mmol/min.

Antibacterial potential of a basic phospholipase A2 (VRV-PL-V) of Daboia russellii pulchella (Russell's Viper) venom.

Microbial/bacterial resistance against antibiotics is considered as a potentially serious threat to public health. Further, as these antibiotics elicit side effects, there is interest in developing new molecules with novel modes of action from diverse organisms. Along these lines, in this study the antibacterial potential of the basic protein VRV-PL-V (Vipera russellii venom phospholipase A2 fraction V) of Daboia russellii pulchella venom was evaluated. VRV-PL-V demonstrated a potent antibacterial activity against all the human pathogenic strains tested. It inhibited more effectively Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis when compared to Gram-negative bacteria like Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, and Salmonella paratyphi. It inhibited bacterial growth with MIC values ranging from 13 to 24 µg/ml. The antibacterial potential of VRV-PL-V was comparable to the standards used like gentamycin, chloramphenicol, and streptomycin. There was a strong correlation between PLA2 activities and hemolytic and antibacterial activity. It was found that even in the presence of p-bromophenacyl bromide (an inhibitor of PLA2 enzymatic activity), there was marked antibacterial activity, suggesting dissociation or partial overlapping of the bactericidal/antimicrobial domains. Therefore, this study shows that although there is a strong correlation between enzymatic and antimicrobial activities of VRV-PL-V, it may also possess other properties that mimic bactericidal/membrane permeability-increasing protein.

Aptamer RA36 inhibits of human, rabbit, and rat plasma coagulation activated with thrombin or snake venom coagulases.

RA36 DNA aptamer is a direct anticoagulant prolonging clotting time of human, rabbit, and rat plasma in the thrombin time test. Anticoagulant activity of RA36 is lower than that of recombinant hirudin. During inhibition of human plasma clotting activated with echitox (coagulase from Echis multisquamatus venom), the aptamer presumably binds to meisothrombin exosite I. The sensitivity of human plasma to the aptamer 5-fold surpasses that of rat plasma. Analysis of RA36 binding to coagulase of Agkistrodon halys venom (ancistron) is required for proving the effect of aptamer on polymerization of human fibrinogen.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bß and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Analysis of phospholipase A2, L-amino acid oxidase, and proteinase enzymatic activities of the Lachesis muta rhombeata venom.

The study of venom components is an important step toward understanding the mechanism of action of such venoms and is indispensable for the development of new therapies. This work aimed to investigate the venom of Lachesis muta rhombeata and evaluate enzymes related to its toxicity. Phospholipase A2 (PLA(2)), L-amino acid oxidase (LAAO), and proteinase activities were measured, and the molecular weights were estimated. We found the venom to contain one PLA(2) (17 kDa), one LAAO (132 kDa), and three serine proteinases (40, 31, and 20 kDa). Although only serine proteinases were observed in the zymogram, metalloproteinases were found to contribute more to the total proteolytic activity than did serine proteinases. The work confirmed the presence of highly active enzymes; and, moreover, we proposed a novel method for confirming the presence of LAAOs by zymography. We also suggested a simple step to increase the sensitivity of proteinase assays.

A [Lys49]phospholipase A2 from Protobothrops flavoviridis venom induces caspase-independent apoptotic cell death accompanied by rapid plasma-membrane rupture in human leukemia cells.

Protobothrops flavoviridis venom contains plural phospholipase A(2) (PLA(2)) isozymes. A [Lys(49)]PLA(2) called BPII induced cell death in human leukemia cells. PLA2, an [Asp(49)]PLA(2) that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA(2) lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.