Toxicon 90 (2014) 26e35
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Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2 María Gutie rrez* Ruth Arce-Bejarano, Bruno Lomonte, Jose Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, 1000 San Jos e, Costa Rica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2014 Received in revised form 14 July 2014 Accepted 23 July 2014 Available online 1 August 2014
Intravascular hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of intravascular hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing intravascular hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of intravascular hemolysis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Coral snake venom Micrurus fulvius Phospholipase A2 Intravascular hemolysis
1. Introduction Intravascular hemolysis is an infrequent clinical finding in snakebite envenomings, although it has been observed after bites by several species, such as Daboia russelli * Corresponding author. Tel.: þ506 2511 7865; fax: þ506 2292 0485. E-mail addresses: [email protected], [email protected] rrez). (J.M. Gutie http://dx.doi.org/10.1016/j.toxicon.2014.07.010 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
(Phillips et al., 1988; Mukherje et al., 2000). In some cases, microangiopathic hemolysis occurs, as a consequence of the mechanical damage generated in erythrocyte membrane by microthrombi deposited in the microvasculature (Warrell, 1996; Joseph et al., 2007). Few direct hemolytic components have been identified in snake venoms, such as cobra venom ‘cardiotoxins’ (also called ‘direct lytic factors’), which are membrane-damaging proteins of the threefinger toxin family (Slotta and Vick, 1969). On the other
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hand, few phospholipases A2 (PLA2) are directly hemolytic, i.e. able to disrupt the integrity of erythrocyte plasma membrane in the absence of exogenously added phospholipids, despite the fact that many PLA2s exert cytotoxicity in a variety of cell types (Chwetzoff et al., 1989; Lomonte et al., 1994, 1999). An example of direct hemolytic agent is the hemoglobinuria-inducing PLA2 from the venom of the elapid Micropechis ikaheka (Gao et al., 1999). Hence, the search for novel directly hemolytic toxins in snake venoms is important to understand the pathophysiology of some envenomings and to develop experimental models of intravascular hemolysis. Envenomings by American elapid species of the genus Micrurus are characterized by a predominant neurotoxic effect, resulting in flaccid paralysis which may lead to respiratory arrest and death (Vital-Brazil, 1987). Moreover, at least at the experimental level, some Micrurus venoms rrez et al., 1992; Barros induce myotoxicity in mice (Gutie n et al., 1999; de Roodt et al., 2012) et al., 1994; Alape-Giro and in ectothermic prey (Urdaneta et al., 2004), and clinical reports have described pain and other alterations in envenomings by coral snakes (Manock et al., 2008; de Roodt et al., 2013; Wood et al., 2013). In addition to the characteristic neurotoxic manifestations, a unique clinical presentation described in dogs envenomed by the North American Eastern coral snake, Micrurus fulvius, is intravascular hemolysis, associated with anemia and hemoglorez et al., binuria (Marks et al., 1990; Peterson, 2006; Pe 2012). Although this effect has not been described in human cases (Kitchens and van Mierop, 1987; Wood et al., 2013), we have observed intravascular hemolysis in our experimental studies in mice injected with M. fulvius venom, thus opening the possibility of developing an experimental model of this pathophysiological alteration. In the present report we describe a model of intravascular hemolysis induced by the venom of M. fulvius in mice, and assess the direct hemolytic potential of this venom on erythrocytes of various species in vitro. In addition, we identify two major PLA2 fractions which reproduce the in vivo hemolytic effect of the whole venom, and provide the complete primary structure of one of these hemolytic enzymes. 2. Materials and methods 2.1. Venom and antivenom Venom of M. fulvius was purchased from SigmaeAldrich (Missouri, USA). The monospecific anti-Micrurus nigrocinctus antivenom manufactured at Instituto Clodomiro Picado (Universidad de Costa Rica; batch 5200313ACLQ) was used in some experiments. It is produced by fractionation of hyperimmune horse plasma with caprylic acid (Rojas et al., 1994), and is composed of whole IgG molecules. 2.2. Experiments in vivo Groups of four CD-1 mice (16e18 g) received an intravenous (i.v.) injection of various doses of M. fulvius venom, dissolved in 100 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS). Control mice received 100 mL of PBS alone. After
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1 h, mice were bled from the tail and blood was collected into heparinized capillary tubes. After centrifugation, the hematocrit was determined. In addition, a sample of urine was collected and observed for the presence of red pigmentation. The Hemolytic Dose 50% (HD50) was estimated as the dose of venom that reduced the hematocrit by 50%, as compared to the hematocrit of mice injected with PBS. In another set of experiments, a dose of 5 mg venom, dissolved in 100 mL PBS was injected into groups of mice. One hr after injection, mice were bled as described, blood was collected into heparinized capillary tubes and centrifuged. Some mice showing evident hemoglobinuria with reduction in hematocrit were euthanized by CO2 inhalation 1 h after injection, and kidneys were dissected out, cut into small pieces and added to 10% formalin. After routine processing for embedding in paraffin, sections approximately 8 mm thick were obtained and placed on glass slides, followed by staining with hematoxylin-eosin for microscopic observation. In addition, blood smears were prepared from samples collected 1 h after venom injection, and were stained with Wright for microscopic observation of erythrocyte morphology. In order to assess the role of PLA2 activity in the hemolytic effect, 300 mL of a solution of 3 mg/mL venom, dissolved in 0.1 M Tris, 0.7 mM EDTA, pH 8.0, was incubated with 20 mL of 0.03 mM p-bromophenacyl bromide (pBPB) for 24 h at room temperature (22e25 C). Inactivated venom was assessed for in vivo hemolytic activity as described above. The protocols of the experiments involving the use of mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. 2.3. Identification of hemoglobin in urine by mass spectrometry The presence of hemoglobinuria was assessed by obtaining urine samples from mice injected i.v. with 5 mg venom, PLA2-17, or PLA2-18, 1 h after injection. Control mice received PBS instead of venom. Urine proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 12% acrylamide concentration under reducing conditions (Laemmli, 1970). Gels were stained with Coomassie Blue R250 and the bands migrating at ~15 kDa were excised, subjected to automated in-gel reduction with DTT, alkylation with iodoacetamide, and digestion with sequencing grade trypsin on a ProGest (Digilab) processor. The resulting peptides were analyzed by MALDI-TOF-TOF on a Proteomics Analyzer 4800 Plus mass spectrometer (Applied Biosystems) as described (Lomonte et al., 2014). Fragmentation spectra were searched against the UniProt-SwissProt database for Mus musculus (20120527) using the Paragon® algorithm of ProteinPilot v.4 (ABSciex) at 95% confidence. 2.4. Neutralization of in vivo hemolysis by antivenom A solution of M. fulvius venom was incubated with M. nigrocinctus antivenom, at a ratio of 200 mg venom/mL antivenom. Controls included venom incubated without antivenom, and antivenom incubated without venom. After 30 min of incubation at 37 C, aliquots of 100 mL of the mixtures, containing 10 mg venom, were injected i.v. in
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mice, and hemolytic activity was assessed 1 h after injection, as described above. 2.5. Experiments in vitro Hemolysis of erythrocytes from different species was assessed in a fluid phase system in tubes. CD-1 mice were bled by cardiac puncture, under anesthesia with ketamine and xylazine. Blood was placed in Eppendorf tubes containing 3.8% (w:v) sodium citrate as anticoagulant. Likewise, human, horse, rabbit and dog blood samples were collected by venipuncture using sodium citrate as anticoagulant. After centrifugation, red blood cell package was washed five times with 0.14 M NaCl, 0.01 M Tris, pH 7.7 (TBS). Then, a 5% erythrocyte suspension was prepared in TBS. Fifty mL of erythrocyte suspension were mixed with 250 mL containing various amounts of M. fulvius venom dissolved in TBS containing 10 mM CaCl2 (TBS-Ca2þ), pH 7.7. Controls included erythrocytes incubated with TBS-Ca2þ alone (0% hemolysis) and erythrocytes incubated with 0.1% Triton X-100 in water (100% hemolysis). In some experiments with mouse erythrocytes, 25 mL of homologous serum was added to the mixture in order to assess the potential role of complement, or other serum components, in this phenomenon. Tubes were incubated for 60 min at 37 C with mild stirring every 20 min. Then, they were centrifuged at 3000 rpm for 5 min, and the absorbances of the supernatants were recorded at 540 nm using a microwell plate reader, as a quantitative index of hemolysis. Results were expressed as percentage hemolysis, taking as 100% the absorbance of samples incubated with Triton. Experiments were performed in triplicates. 2.6. Effect of physical strain on venom-induced hemolysis In order to assess whether rheological factors associated with blood flow operating in blood vessels may contribute to the intravascular hemolysis induced by M. fulvius venom, an in vitro model of physical strain was developed. Blood was collected from mice by cardiac puncture under anesthesia, using sodium citrate as anticoagulant, as described above. After several washings in TBS, the erythrocyte pellet was resuspended in TBS-Ca2þ to attain a hematocrit of 40%. Aliquots of 200 mL of erythrocyte suspensions were incubated, for 30 min at 37 C, with either 10 mL TBS or 10 mg M. fulvius venom, dissolved in 10 mL TBS. Then, samples of either control or venom-treated erythrocyte suspensions were passed five times through a 0.5 mL syringe with a needle (31G 8 mm), in order to simulate the physical stress that blood undergoes in the vasculature in vivo. Other control and venom-treated samples were not submitted to this physical stress. Afterwards, samples were centrifuged at 3000 g for 5 min and the supernatants were collected. Absorbances at 540 nm were recorded as a quantitative index of hemoglobin in the supernatant. 2.7. Fractionation of venom by HPLC M. fulvius venom (2 mg) was dissolved in 200 mL of water containing 0.1% trifluoroacetic acid (TFA), and fractionated by reverse-phase HPLC on a C18 column (4.6 250 mm, 5 mM
particle; Teknochroma) using an Agilent 1200 chromatograph. Proteins were eluted at 1 mL/min by applying a gradient toward acetonitrile with 0.1% TFA, as previously ndez et al., 2011), and monitored at 215 nm. described (Ferna Fractions were collected manually, dried by vacuum centrifugation at 45 C, and redissolved in PBS for further characterization. Protein concentration of the fractions was adjusted by measuring their absorbance at 280 nm on a NanoDrop 2000c reader (Thermo Scientific). Molecular masses of the fractions of interest were determined by MALDI-TOF in positive linear mode, using sinapinic acid as matrix. In addition, venom fractions were analyzed by SDSPAGE and the resulting protein bands were digested with trypsin for identification by MALDI-TOF-TOF as described above. All fragmentation spectra were inspected manually in order to confirm the peptide sequences obtained automatically by the Paragon algorithm of ProteinPilot. 2.8. Characterization of venom fractions showing hemolytic activity Several HPLC fractions were tested for in vivo and in vitro hemolytic activity, as described above for the whole venom. The PLA2 activity of the hemolytic fractions was determined on the monodisperse synthetic substrate 4-nitro-3octanoyl-benzoic acid (NOBA) (Holzer and Mackessy, 1996). Varying amounts of enzymes, dissolved in 25 mL of water, were added to 200 mL of 10 mM Tris, 10 mM CaCl2, 0.1 M NaCl, pH 8.0, in triplicate wells of a microplate. After mixing, 25 mL of NOBA (1 mg/mL in acetonitrile) were added, to achieve a final substrate concentration of 0.32 mM. The mixtures were incubated for 60 min at 37 C, and absorbances were recorded at 405 nm. PLA2 activity was expressed as the change in absorbance. In order to assess whether these hemolytic components were also myotoxic, mice received an intramuscular (i.m.) injection of 10 mg, dissolved in 100 mL PBS. Two hr after injection, mice were bled from the tail and blood was collected in heparinized capillary tubes and centrifuged. The plasma creatine kinase (CK) activity was quantified by using a commercial kit (CK rrez et al., 1980). LIQUI-UV, Stanbio Lab., Texas, USA) (Gutie 2.9. Statistical analyses The significance of the differences between the means of various experimental groups was assessed by Analysis of Variance, followed by Tukey test. A p value of 12 different PLA2 isoform transcripts.
Fig. 7. Amino acid sequence of the phospholipase A2 from fraction 17 of M. fulvius venom (numbering from Fig. 5). The protein was in-gel digested with trypsin and the indicated peptides were de novo sequenced by MALDI-TOF-TOF mass spectrometry. The sequence shows 100% identity with the RNA transcript “PLA2 2b” (UniProtKB accession U3EPF0) reported in the venom gland transcriptome of this species (Margres et al., 2013). The final dipeptide at the C-terminus (indicated with **) could not be detected in the tryptic digest, but its identity is inferred from the conservation of these two amino acids in the reported venom gland PLA2 transcripts of M. fulvius, and the concordance between the observed (Fig. 6F) and the expected mass values.
membrane was analyzed, particularly regarding the relative amounts of phosphatidylcholine (PC) and sphingomyelin (SM), the two most abundant phospholipids in the outer monolayer of this membrane. As depicted in Fig. 9, the highest PC/SM ratios occur in dog and mouse erythrocytes, whereas the lowest ratios occur in human and rabbit erythrocytes. Interestingly, dog and mouse erythrocytes present the highest susceptibility to the hemolytic action of venom, whereas human and rabbit cells are largely resistant at the venom concentrations tested. This agrees with clinical findings since envenomings by M. fulvius in humans
are not associated with hemolysis (Kitchens and van rez et al., 2012). Hence, there seems to Mierop, 1987; Pe be a general trend of higher susceptibility to hemolysis by M. fulvius venom in erythrocytes with higher PC/SM ratios. Since snake venom PLA2s are able to hydrolyze PC, but not SM, this correlation suggests that the higher susceptibility of dog and mouse erythrocytes to hemolytic PLA2s depends on the higher extent of hydrolysis of PC of the outer monolayer of the membrane, thus weakening membrane stability and rendering it more susceptible to disruption and cell lysis.
Fig. 8. (A) Phospholipase A2 activity of hemolytic fractions (17 and 18) from Micrurus fulvius venom in vitro, determined on 4-nitro-3-octanoyl-benzoic acid (NOBA), as described in Materials and Methods. Each point represents mean ± SD of three replicates. (B) SDS-PAGE of the fractions (10 mg) under reducing conditions, showing a main band at ~14 kDa, and minor dimeric aggregates at ~23 kDa. M: Molecular mass standards.
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Fig. 9. Relative abundances (%) of phosphatidylcholine and sphingomyelin in the external leaflet of the membrane of erythrocytes from different species of mammals. Values were obtained from the following references: Nelson (1967), Virtanen et al. (1998), and Shevchenko and Shishkina (2011).
In addition to the hydrolysis of plasma membrane phospholipids in mouse erythrocytes, the strong hemolysis observed in vivo might be also mediated by biophysical rheological factors operating in the circulation, i.e. by the effect that blood flow-dependent physical stress has on the stability of erythrocytes. It is noteworthy that a much lower dose of M. fulvius venom is necessary to induce hemolysis in vivo than on washed erythrocytes in vitro. To explore the hypothesis that rheological factors operating in vivo may be playing a role, we designed an experiment in which, after incubation of washed mouse erythrocytes with the venom in vitro, the cell suspension was subjected to a mechanical strain generated by the forced passage of cells through a needle, as an experimental model of blood flow through the vasculature. Results clearly show that erythrocytes treated with venom and submitted to this physical strain were lysed to a much higher extent than erythrocytes incubated with venom without physical strain and control erythrocytes incubated with saline solution and subjected to physical strain. Thus, the intense hemolysis observed in vivo is likely to be the consequence of two independent events, i.e. an initial hydrolysis of membrane phospholipids by the PLA2s, which weakens the stability of the membrane, and the subsequent mechanical strain generated by rheological factors operating in the circulation. Intravascular hemolysis has been reported in canine cases of envenoming by M. fulvius (Marks et al., 1990; rez et al., 2012) and, therefore, is Peterson, 2006; Pe clinically relevant in Veterinary Medicine. An antivenom raised against the venom of the Central American coral snake M. nigrocinctus proved effective in the neutralization of intravascular hemolysis induced by M. fulvius, even when M. nigrocinctus venom is devoid of hemolytic effect in mice (our observations). Since M. nigrocinctus venom contains abundant PLA2s, it is suggested that antibodies against non-hemolytic PLA2s from M. nigrocinctus cross-react with the hemolytic PLA2s of M. fulvius venom. Previous observations have demonstrated
the effectiveness of this antivenom to neutralize lethal and myotoxic activities of M. fulvius venom (Arce et al., 2003). Our results now show that the antivenom is also effective in the abrogation of intravascular hemolysis induced by M. fulvius, further evidencing the extensive immunological cross-reactivity between the venoms of both species. In conclusion, the venom of M. fulvius induces a potent intravascular hemolytic effect in mice due to the direct action of PLA2s on erythrocyte membrane. Venom-induced direct hemolysis in vitro was observed with mouse and dog erythrocytes, but not in human, rabbit and horse red blood cells. Such differences in susceptibility to venom might be related to the relative amounts of PC and SM in the erythrocyte membrane. The potent in vivo hemolytic effect observed in mice might be due to the combination of biochemical events, i.e. phospholipid hydrolysis by venom PLA2s, and rheological events, i.e. the physical strain imposed on erythrocytes by blood flow through the microvasculature. The hemolytic effect is neutralized by a monospecific antivenom raised against the venom of M. nigrocinctus. The experimental model described can be useful to explore pathophysiological alterations associated with acute intravascular hemolysis and to investigate the structural determinants that allow some PLA2s to disrupt the integrity of erythrocyte plasma membrane, an effect that only few snake venom PLA2s are able to induce. Ethical statement The protocols used in this study followed national and international ethical guidelines in the use of laboratory animals and in the performance of scientific research. Acknowledgements This study was supported in part by grants CRP/COS1301 from the International Centre for Genetic Engineering and Biotechnology (ICGEB, Italy) and from Vicerrectoría de n, University of Costa Rica. The valuable supInvestigacio port of Diana Mora-Obando, Daniela Solano, Edwin Mosnchez, Paola Rey-Sua rez, and coso, Arturo Vargas, Melvin Sa enz is greatly acknowledged, as well as the Ar anzazu Sa collaboration of Drs Gustavo Rojas, Jenaro Murillo, Diego rez in various aspects of this study. Valerio, and Carlos Sua Thanks are also due to Dr Michael Schaer (College of Veterinary Medicine, University of Florida) for sharing his valuable clinical experience on cases of canine envenomings by M. fulvius in the USA. Conflict of interest The authors declare that there are no conflicts of interest regarding this work. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.07. 010.
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(Micrurus lemniscatus helleri): case report from eastern Ecuador and review. Trans. R. Soc. Trop. Med. Hyg. 102, 1127e1132. Margres, M.J., Aronow, K., Loyacano, J., Rokyta, D.R., 2013. The venomgland transcriptome of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the intragenomic evolution of venoms. BMC Genomics 14, 531. Marks, S.L., Mannella, C., Schaer, M., 1990. Coral snake envenomation in the dog: report of four cases and review of the literature. J. Am. Anim. Hosp. Assoc. 26, 629e634. rrez, J.M., Lomonte, B., 2008. Cellular pathology Montecucco, C., Gutie induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell. Mol. Life Sci. 65, 2897e2912. Mukherje, A.K., Ghosal, S.K., Maity, C.R., 2000. Some biochemical properties of Russell's viper (Daboia russelli) venom from Eastern India: correlation with clinic-pathological manifestation in Russell’s viper bite. Toxicon 38, 163e175. Nelson, G.J., 1967. Lipid composition of erythrocytes in various mammalian species. Biochim. Biophys. Acta 144, 221e232. Olamendi-Portugal, T., Batista, C., Restano-Cassulini, R., Pando, V., VillaHernandez, O., Zavaleta-Martínez-Vargas, A., Salas-Arruz, M.C., Rodríguez de la Vega, R.C., Becerril, B., Possani, L., 2008. Proteomic analysis of the venom from the fish eating coral snake Micrurus surinamensis: novel toxins, their function and phylogeny. Proteomics 8, 1919e1932. rez, M.L., Fox, K., Schaer, M., 2012. A retrospective evaluation of coral Pe snake envenomation in dogs and cats (20 cases) (1996e2011). J. Vet. Med. Crit. Care 22, 682e689. Peterson, M.E., 2006. Snake bite: coral snakes. Clin. Tech. Small Anim. Pract. 21, 183e186. Phillips, R.E., Theakston, R.D.G., Warrell, D.A., Galigedara, Y., Abeysekera, D.T., Dissanayaka, P., Hutton, R.A., Aloysius, D.J., 1988. Paralysis, rhabdomyolysis and haemolysis caused by bites of Russell's viper (Vipera russelli pulchela) in Sri Lanka: failure of Indian (Haffkine) antivenom. Quat. J. Med. 68, 691e715. Ramsey, H.W., Taylor, W.J., Boruchow, I.B., Snyder, G.K., 1972. Mechanism of shock produced by an elapid snake venom (Micrurus f. fulvius) venom in dogs. Am. J. Physiol. 222, 782e786. rez, P., Nún ~ ez, V., Gutie rrez, J.M., Lomonte, B., 2011. Proteomic and Rey-Sua biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica. J. Proteomics 75, 655e667. nez, J.M., Gutie rrez, J.M., 1994. Caprylic acid fractionation of Rojas, G., Jime hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351e363. Shevchenko, O.G., Shishkina, L.N., 2011. Comparative analysis of phospholipid composition in blood erythrocytes of various species of mouse-like rodents. J. Evol. Biochem. Physiol. 47, 179e186. Slotta, K.H., Vick, J.A., 1969. Identification of the direct lytic factor from cobra venom as cardiotoxin. Toxicon 6, 167e173. ~ os, F., Gutie rrez, J.M., 2004. Feeding behavior and Urdaneta, A.H., Bolan venom toxicity of coral snake Micrurus nigrocinctus (Serpentes:Elapidae) on its natural prey in captivity. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 138, 485e492. Vergara, I., Pedraza-Escalona, M., Paniagua, D., Restano-Cassulini, R., n, A., 2014. Eastern coral Zamudio, F., Batista, C.V., Possani, L.D., Alago snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2. J. Proteomics 105, 295e306. Virtanen, J.A., et al., 1998. Phospholipid composition of mammalian red cell membrane can be rationalized by superlattice model. Proc. Natl. Acad. Sci. U. S. A. 95, 4964e4969. Vital-Brazil, O., 1987. Coral snake venoms: mode of action and pathophysiology of experimental envenomation. Rev. Inst. Med. Trop. S~ ao Paulo 29, 119e126. Warrell, D.A., 1996. Clinical features of envenoming from snake bites. In: Bon, C., Goyffon, M. (Eds.), Envenomings and Their Treatment. Fonrieux, Lyon, pp. 63e76. dation Marcel Me Weiss, R., McIsaac, R.J., 1971. Cardiovascular and muscular effects of venom from coral snake, Micrurus fulvius. Toxicon 9, 219e228. Wood, A., Schauben, J., Thundiyil, J., Kunisaki, T., Sollee, D., LewisYounger, C., Bernstein, J., Weisman, R., 2013. Review of Eastern coral snake (Micrurus fulvius fulvius) exposures managed by the Florida Poison Information Center Network: 1998e2010. Clin. Toxicol. 51, 783e788.
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Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2 María Gutie rrez* Ruth Arce-Bejarano, Bruno Lomonte, Jose Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, 1000 San Jos e, Costa Rica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2014 Received in revised form 14 July 2014 Accepted 23 July 2014 Available online 1 August 2014
Intravascular hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of intravascular hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing intravascular hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of intravascular hemolysis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Coral snake venom Micrurus fulvius Phospholipase A2 Intravascular hemolysis
1. Introduction Intravascular hemolysis is an infrequent clinical finding in snakebite envenomings, although it has been observed after bites by several species, such as Daboia russelli * Corresponding author. Tel.: þ506 2511 7865; fax: þ506 2292 0485. E-mail addresses: [email protected], [email protected] rrez). (J.M. Gutie http://dx.doi.org/10.1016/j.toxicon.2014.07.010 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
(Phillips et al., 1988; Mukherje et al., 2000). In some cases, microangiopathic hemolysis occurs, as a consequence of the mechanical damage generated in erythrocyte membrane by microthrombi deposited in the microvasculature (Warrell, 1996; Joseph et al., 2007). Few direct hemolytic components have been identified in snake venoms, such as cobra venom ‘cardiotoxins’ (also called ‘direct lytic factors’), which are membrane-damaging proteins of the threefinger toxin family (Slotta and Vick, 1969). On the other
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hand, few phospholipases A2 (PLA2) are directly hemolytic, i.e. able to disrupt the integrity of erythrocyte plasma membrane in the absence of exogenously added phospholipids, despite the fact that many PLA2s exert cytotoxicity in a variety of cell types (Chwetzoff et al., 1989; Lomonte et al., 1994, 1999). An example of direct hemolytic agent is the hemoglobinuria-inducing PLA2 from the venom of the elapid Micropechis ikaheka (Gao et al., 1999). Hence, the search for novel directly hemolytic toxins in snake venoms is important to understand the pathophysiology of some envenomings and to develop experimental models of intravascular hemolysis. Envenomings by American elapid species of the genus Micrurus are characterized by a predominant neurotoxic effect, resulting in flaccid paralysis which may lead to respiratory arrest and death (Vital-Brazil, 1987). Moreover, at least at the experimental level, some Micrurus venoms rrez et al., 1992; Barros induce myotoxicity in mice (Gutie n et al., 1999; de Roodt et al., 2012) et al., 1994; Alape-Giro and in ectothermic prey (Urdaneta et al., 2004), and clinical reports have described pain and other alterations in envenomings by coral snakes (Manock et al., 2008; de Roodt et al., 2013; Wood et al., 2013). In addition to the characteristic neurotoxic manifestations, a unique clinical presentation described in dogs envenomed by the North American Eastern coral snake, Micrurus fulvius, is intravascular hemolysis, associated with anemia and hemoglorez et al., binuria (Marks et al., 1990; Peterson, 2006; Pe 2012). Although this effect has not been described in human cases (Kitchens and van Mierop, 1987; Wood et al., 2013), we have observed intravascular hemolysis in our experimental studies in mice injected with M. fulvius venom, thus opening the possibility of developing an experimental model of this pathophysiological alteration. In the present report we describe a model of intravascular hemolysis induced by the venom of M. fulvius in mice, and assess the direct hemolytic potential of this venom on erythrocytes of various species in vitro. In addition, we identify two major PLA2 fractions which reproduce the in vivo hemolytic effect of the whole venom, and provide the complete primary structure of one of these hemolytic enzymes. 2. Materials and methods 2.1. Venom and antivenom Venom of M. fulvius was purchased from SigmaeAldrich (Missouri, USA). The monospecific anti-Micrurus nigrocinctus antivenom manufactured at Instituto Clodomiro Picado (Universidad de Costa Rica; batch 5200313ACLQ) was used in some experiments. It is produced by fractionation of hyperimmune horse plasma with caprylic acid (Rojas et al., 1994), and is composed of whole IgG molecules. 2.2. Experiments in vivo Groups of four CD-1 mice (16e18 g) received an intravenous (i.v.) injection of various doses of M. fulvius venom, dissolved in 100 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS). Control mice received 100 mL of PBS alone. After
27
1 h, mice were bled from the tail and blood was collected into heparinized capillary tubes. After centrifugation, the hematocrit was determined. In addition, a sample of urine was collected and observed for the presence of red pigmentation. The Hemolytic Dose 50% (HD50) was estimated as the dose of venom that reduced the hematocrit by 50%, as compared to the hematocrit of mice injected with PBS. In another set of experiments, a dose of 5 mg venom, dissolved in 100 mL PBS was injected into groups of mice. One hr after injection, mice were bled as described, blood was collected into heparinized capillary tubes and centrifuged. Some mice showing evident hemoglobinuria with reduction in hematocrit were euthanized by CO2 inhalation 1 h after injection, and kidneys were dissected out, cut into small pieces and added to 10% formalin. After routine processing for embedding in paraffin, sections approximately 8 mm thick were obtained and placed on glass slides, followed by staining with hematoxylin-eosin for microscopic observation. In addition, blood smears were prepared from samples collected 1 h after venom injection, and were stained with Wright for microscopic observation of erythrocyte morphology. In order to assess the role of PLA2 activity in the hemolytic effect, 300 mL of a solution of 3 mg/mL venom, dissolved in 0.1 M Tris, 0.7 mM EDTA, pH 8.0, was incubated with 20 mL of 0.03 mM p-bromophenacyl bromide (pBPB) for 24 h at room temperature (22e25 C). Inactivated venom was assessed for in vivo hemolytic activity as described above. The protocols of the experiments involving the use of mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. 2.3. Identification of hemoglobin in urine by mass spectrometry The presence of hemoglobinuria was assessed by obtaining urine samples from mice injected i.v. with 5 mg venom, PLA2-17, or PLA2-18, 1 h after injection. Control mice received PBS instead of venom. Urine proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 12% acrylamide concentration under reducing conditions (Laemmli, 1970). Gels were stained with Coomassie Blue R250 and the bands migrating at ~15 kDa were excised, subjected to automated in-gel reduction with DTT, alkylation with iodoacetamide, and digestion with sequencing grade trypsin on a ProGest (Digilab) processor. The resulting peptides were analyzed by MALDI-TOF-TOF on a Proteomics Analyzer 4800 Plus mass spectrometer (Applied Biosystems) as described (Lomonte et al., 2014). Fragmentation spectra were searched against the UniProt-SwissProt database for Mus musculus (20120527) using the Paragon® algorithm of ProteinPilot v.4 (ABSciex) at 95% confidence. 2.4. Neutralization of in vivo hemolysis by antivenom A solution of M. fulvius venom was incubated with M. nigrocinctus antivenom, at a ratio of 200 mg venom/mL antivenom. Controls included venom incubated without antivenom, and antivenom incubated without venom. After 30 min of incubation at 37 C, aliquots of 100 mL of the mixtures, containing 10 mg venom, were injected i.v. in
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mice, and hemolytic activity was assessed 1 h after injection, as described above. 2.5. Experiments in vitro Hemolysis of erythrocytes from different species was assessed in a fluid phase system in tubes. CD-1 mice were bled by cardiac puncture, under anesthesia with ketamine and xylazine. Blood was placed in Eppendorf tubes containing 3.8% (w:v) sodium citrate as anticoagulant. Likewise, human, horse, rabbit and dog blood samples were collected by venipuncture using sodium citrate as anticoagulant. After centrifugation, red blood cell package was washed five times with 0.14 M NaCl, 0.01 M Tris, pH 7.7 (TBS). Then, a 5% erythrocyte suspension was prepared in TBS. Fifty mL of erythrocyte suspension were mixed with 250 mL containing various amounts of M. fulvius venom dissolved in TBS containing 10 mM CaCl2 (TBS-Ca2þ), pH 7.7. Controls included erythrocytes incubated with TBS-Ca2þ alone (0% hemolysis) and erythrocytes incubated with 0.1% Triton X-100 in water (100% hemolysis). In some experiments with mouse erythrocytes, 25 mL of homologous serum was added to the mixture in order to assess the potential role of complement, or other serum components, in this phenomenon. Tubes were incubated for 60 min at 37 C with mild stirring every 20 min. Then, they were centrifuged at 3000 rpm for 5 min, and the absorbances of the supernatants were recorded at 540 nm using a microwell plate reader, as a quantitative index of hemolysis. Results were expressed as percentage hemolysis, taking as 100% the absorbance of samples incubated with Triton. Experiments were performed in triplicates. 2.6. Effect of physical strain on venom-induced hemolysis In order to assess whether rheological factors associated with blood flow operating in blood vessels may contribute to the intravascular hemolysis induced by M. fulvius venom, an in vitro model of physical strain was developed. Blood was collected from mice by cardiac puncture under anesthesia, using sodium citrate as anticoagulant, as described above. After several washings in TBS, the erythrocyte pellet was resuspended in TBS-Ca2þ to attain a hematocrit of 40%. Aliquots of 200 mL of erythrocyte suspensions were incubated, for 30 min at 37 C, with either 10 mL TBS or 10 mg M. fulvius venom, dissolved in 10 mL TBS. Then, samples of either control or venom-treated erythrocyte suspensions were passed five times through a 0.5 mL syringe with a needle (31G 8 mm), in order to simulate the physical stress that blood undergoes in the vasculature in vivo. Other control and venom-treated samples were not submitted to this physical stress. Afterwards, samples were centrifuged at 3000 g for 5 min and the supernatants were collected. Absorbances at 540 nm were recorded as a quantitative index of hemoglobin in the supernatant. 2.7. Fractionation of venom by HPLC M. fulvius venom (2 mg) was dissolved in 200 mL of water containing 0.1% trifluoroacetic acid (TFA), and fractionated by reverse-phase HPLC on a C18 column (4.6 250 mm, 5 mM
particle; Teknochroma) using an Agilent 1200 chromatograph. Proteins were eluted at 1 mL/min by applying a gradient toward acetonitrile with 0.1% TFA, as previously ndez et al., 2011), and monitored at 215 nm. described (Ferna Fractions were collected manually, dried by vacuum centrifugation at 45 C, and redissolved in PBS for further characterization. Protein concentration of the fractions was adjusted by measuring their absorbance at 280 nm on a NanoDrop 2000c reader (Thermo Scientific). Molecular masses of the fractions of interest were determined by MALDI-TOF in positive linear mode, using sinapinic acid as matrix. In addition, venom fractions were analyzed by SDSPAGE and the resulting protein bands were digested with trypsin for identification by MALDI-TOF-TOF as described above. All fragmentation spectra were inspected manually in order to confirm the peptide sequences obtained automatically by the Paragon algorithm of ProteinPilot. 2.8. Characterization of venom fractions showing hemolytic activity Several HPLC fractions were tested for in vivo and in vitro hemolytic activity, as described above for the whole venom. The PLA2 activity of the hemolytic fractions was determined on the monodisperse synthetic substrate 4-nitro-3octanoyl-benzoic acid (NOBA) (Holzer and Mackessy, 1996). Varying amounts of enzymes, dissolved in 25 mL of water, were added to 200 mL of 10 mM Tris, 10 mM CaCl2, 0.1 M NaCl, pH 8.0, in triplicate wells of a microplate. After mixing, 25 mL of NOBA (1 mg/mL in acetonitrile) were added, to achieve a final substrate concentration of 0.32 mM. The mixtures were incubated for 60 min at 37 C, and absorbances were recorded at 405 nm. PLA2 activity was expressed as the change in absorbance. In order to assess whether these hemolytic components were also myotoxic, mice received an intramuscular (i.m.) injection of 10 mg, dissolved in 100 mL PBS. Two hr after injection, mice were bled from the tail and blood was collected in heparinized capillary tubes and centrifuged. The plasma creatine kinase (CK) activity was quantified by using a commercial kit (CK rrez et al., 1980). LIQUI-UV, Stanbio Lab., Texas, USA) (Gutie 2.9. Statistical analyses The significance of the differences between the means of various experimental groups was assessed by Analysis of Variance, followed by Tukey test. A p value of 12 different PLA2 isoform transcripts.
Fig. 7. Amino acid sequence of the phospholipase A2 from fraction 17 of M. fulvius venom (numbering from Fig. 5). The protein was in-gel digested with trypsin and the indicated peptides were de novo sequenced by MALDI-TOF-TOF mass spectrometry. The sequence shows 100% identity with the RNA transcript “PLA2 2b” (UniProtKB accession U3EPF0) reported in the venom gland transcriptome of this species (Margres et al., 2013). The final dipeptide at the C-terminus (indicated with **) could not be detected in the tryptic digest, but its identity is inferred from the conservation of these two amino acids in the reported venom gland PLA2 transcripts of M. fulvius, and the concordance between the observed (Fig. 6F) and the expected mass values.
membrane was analyzed, particularly regarding the relative amounts of phosphatidylcholine (PC) and sphingomyelin (SM), the two most abundant phospholipids in the outer monolayer of this membrane. As depicted in Fig. 9, the highest PC/SM ratios occur in dog and mouse erythrocytes, whereas the lowest ratios occur in human and rabbit erythrocytes. Interestingly, dog and mouse erythrocytes present the highest susceptibility to the hemolytic action of venom, whereas human and rabbit cells are largely resistant at the venom concentrations tested. This agrees with clinical findings since envenomings by M. fulvius in humans
are not associated with hemolysis (Kitchens and van rez et al., 2012). Hence, there seems to Mierop, 1987; Pe be a general trend of higher susceptibility to hemolysis by M. fulvius venom in erythrocytes with higher PC/SM ratios. Since snake venom PLA2s are able to hydrolyze PC, but not SM, this correlation suggests that the higher susceptibility of dog and mouse erythrocytes to hemolytic PLA2s depends on the higher extent of hydrolysis of PC of the outer monolayer of the membrane, thus weakening membrane stability and rendering it more susceptible to disruption and cell lysis.
Fig. 8. (A) Phospholipase A2 activity of hemolytic fractions (17 and 18) from Micrurus fulvius venom in vitro, determined on 4-nitro-3-octanoyl-benzoic acid (NOBA), as described in Materials and Methods. Each point represents mean ± SD of three replicates. (B) SDS-PAGE of the fractions (10 mg) under reducing conditions, showing a main band at ~14 kDa, and minor dimeric aggregates at ~23 kDa. M: Molecular mass standards.
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Fig. 9. Relative abundances (%) of phosphatidylcholine and sphingomyelin in the external leaflet of the membrane of erythrocytes from different species of mammals. Values were obtained from the following references: Nelson (1967), Virtanen et al. (1998), and Shevchenko and Shishkina (2011).
In addition to the hydrolysis of plasma membrane phospholipids in mouse erythrocytes, the strong hemolysis observed in vivo might be also mediated by biophysical rheological factors operating in the circulation, i.e. by the effect that blood flow-dependent physical stress has on the stability of erythrocytes. It is noteworthy that a much lower dose of M. fulvius venom is necessary to induce hemolysis in vivo than on washed erythrocytes in vitro. To explore the hypothesis that rheological factors operating in vivo may be playing a role, we designed an experiment in which, after incubation of washed mouse erythrocytes with the venom in vitro, the cell suspension was subjected to a mechanical strain generated by the forced passage of cells through a needle, as an experimental model of blood flow through the vasculature. Results clearly show that erythrocytes treated with venom and submitted to this physical strain were lysed to a much higher extent than erythrocytes incubated with venom without physical strain and control erythrocytes incubated with saline solution and subjected to physical strain. Thus, the intense hemolysis observed in vivo is likely to be the consequence of two independent events, i.e. an initial hydrolysis of membrane phospholipids by the PLA2s, which weakens the stability of the membrane, and the subsequent mechanical strain generated by rheological factors operating in the circulation. Intravascular hemolysis has been reported in canine cases of envenoming by M. fulvius (Marks et al., 1990; rez et al., 2012) and, therefore, is Peterson, 2006; Pe clinically relevant in Veterinary Medicine. An antivenom raised against the venom of the Central American coral snake M. nigrocinctus proved effective in the neutralization of intravascular hemolysis induced by M. fulvius, even when M. nigrocinctus venom is devoid of hemolytic effect in mice (our observations). Since M. nigrocinctus venom contains abundant PLA2s, it is suggested that antibodies against non-hemolytic PLA2s from M. nigrocinctus cross-react with the hemolytic PLA2s of M. fulvius venom. Previous observations have demonstrated
the effectiveness of this antivenom to neutralize lethal and myotoxic activities of M. fulvius venom (Arce et al., 2003). Our results now show that the antivenom is also effective in the abrogation of intravascular hemolysis induced by M. fulvius, further evidencing the extensive immunological cross-reactivity between the venoms of both species. In conclusion, the venom of M. fulvius induces a potent intravascular hemolytic effect in mice due to the direct action of PLA2s on erythrocyte membrane. Venom-induced direct hemolysis in vitro was observed with mouse and dog erythrocytes, but not in human, rabbit and horse red blood cells. Such differences in susceptibility to venom might be related to the relative amounts of PC and SM in the erythrocyte membrane. The potent in vivo hemolytic effect observed in mice might be due to the combination of biochemical events, i.e. phospholipid hydrolysis by venom PLA2s, and rheological events, i.e. the physical strain imposed on erythrocytes by blood flow through the microvasculature. The hemolytic effect is neutralized by a monospecific antivenom raised against the venom of M. nigrocinctus. The experimental model described can be useful to explore pathophysiological alterations associated with acute intravascular hemolysis and to investigate the structural determinants that allow some PLA2s to disrupt the integrity of erythrocyte plasma membrane, an effect that only few snake venom PLA2s are able to induce. Ethical statement The protocols used in this study followed national and international ethical guidelines in the use of laboratory animals and in the performance of scientific research. Acknowledgements This study was supported in part by grants CRP/COS1301 from the International Centre for Genetic Engineering and Biotechnology (ICGEB, Italy) and from Vicerrectoría de n, University of Costa Rica. The valuable supInvestigacio port of Diana Mora-Obando, Daniela Solano, Edwin Mosnchez, Paola Rey-Sua rez, and coso, Arturo Vargas, Melvin Sa enz is greatly acknowledged, as well as the Ar anzazu Sa collaboration of Drs Gustavo Rojas, Jenaro Murillo, Diego rez in various aspects of this study. Valerio, and Carlos Sua Thanks are also due to Dr Michael Schaer (College of Veterinary Medicine, University of Florida) for sharing his valuable clinical experience on cases of canine envenomings by M. fulvius in the USA. Conflict of interest The authors declare that there are no conflicts of interest regarding this work. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.07. 010.
R. Arce-Bejarano et al. / Toxicon 90 (2014) 26e35
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