Haity Moussatché

Natural anti-snake venom proteins☆

The resistance of several animals to snake venom has been reviewed. Some general concepts are introduced to allow the comparative evaluation of the resistance of different animals studied by different investigators. The purification and properties of several factors isolated from the serum of different animals by some researchers are described: Trimeresurus flavoviridis (Omori-Satoh et al., 1972); Vipera palaestinae (Ovadia et al., 1975, 1977); Sigmodon hispidus (Pichyangkul and Perez, 1981); Didelphis virginiana and Didelphis marsupialis (Menchaga and Perez, 1981; Moussatché et al., 1979, 1980, 1981; Perales et al., 1986, 1989a,b); Neotoma micropus (Garcia and Perez, 1984); Erinaceus europaeus (de Witt and Weströmm, 1987); Herpestes edwardsii (Tomihara et al., 1987); Dinodon semicarinatus (Tomihara et al., 1988); and Philander opossum (Domont et al., 1989). The protective antihemorrhagic and antineurotoxic factors have some common characteristics: they are acid proteins with isoelectric points ranging between 4.0 and 5.4; their molecular masses vary from 52 to 90 kDa, with one exception of 780 kDa; none has proteolytic activity; their pH and thermostabilities are high and they seem to be glycoproteins. No precipitation lines are formed between the neutralizing proteins and the venoms upon immunodiffusion, indicating that the serum protective factors are not immunoglobulins. The possible mode of action of the antineurotoxic factor isolated from Vipera palaestinae by Ovadia et al. (1977) is shortly discussed as well as the possibility that the antihemorrhagic factors may act by a similar mechanism.

Neutralization of the oedematogenic activity of Bothrops Jararaca venom on the mouse paw by an antibothropic fraction isolated from Opossum (Didelphis Marsupialis) serum

The pharmacological modulation of mice paw oedema produced byBothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1–30 μg/paw) produced a dose-and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48h. BJV heated at 100°C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25–200 μg/paw) isolated fromDidelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited.

Isolation and partial characterization of an anti-bothropic complex from the serum of South American Didelphidae

An anti-bothropic fraction (ABF) with anti-Bothrops jararaca venom activity tested in mice was isolated from the serum of some South American Didelphidae (Didelphis marsupialis, Philander opossum and Lutreolina crassicaudata) by DEAE-Sephacel chromatography. ABF from D. marsupialis was shown to be 12 times more active in protection assays on a weight basis than the serum proteins. A similar fraction obtained from Metachirus nudicaudatum serum was shown to be inactive. An anti-bothropic complex (ABC) was isolated from D. marsupialis ABF. HPLC gel permeation chromatography of ABC from D. marsupialis indicated the presence of a main peak with mol. wt of 84,000. SDS-PAGE of this ABC showed the presence of two subunits of 48,000 and 43,000. The active ABF isolated from P. opossum and L. crassicaudata also showed the presence of these subunits by SDS-PAGE. Isolation of the 48,000 mol. wt D. marsupialis subunit by HPLC-hydrophobic interaction chromatography demonstrated that the 43,000 subunit was essential for the protective action of the complex. Both subunits from D. marsupialis, P. opossum and L. crassicaudata were Western-blotted and N-terminal sequenced. No N-terminal amino acid was found for the 43,000 subunit, whereas for the 48,000 subunit a high degree of homology was found: D. marsupialis: H2N-L K A M D P T P P L W I K T E X P . ; L. crassicaudata: H2N-L K A M D P T P P L W I Q T E . . . ; P. opossum: H2N-L K A M D T T P E . . . No significant homology with known proteins was detected.

Inhibitory properties of the antibothropic complex from the South American opossum (Didelphis marsupialis) serum

The South American opossum Didelphis marsupialis is known to be highly resistant to snake envenomation. In this paper it is shown that the opossum serum inhibits haemorrhage induced by both Crotalinae and Viperinae venoms. Tested against Bothrops jararaca (jararaca) venom, the antibothropic complex (ABC) isolated from the opossum serum was at least six times more antihaemorrhagic than the commercial antivenom. ABC showed no proteolytic activity by itself and was not hydrolysed by the venom. It inhibited the hydrolysis of casein by B. jararaca venom, but did not inhibit its hydrolytic activities upon N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA). The inhibitor did not interfere with trypsin and bacterial collagenase activities on BAPNA and N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA), respectively. It reduced chymotrypsin hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) because ABC is also a substrate for this enzyme. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B. jararaca venom preferentially degraded fibrinogen A alpha-chain and fibrin alpha-chain. Tested on extracellular matrix proteins, the venom hydrolysed collagen IV, gelatins I and V, laminin and fibronectin, besides depolimerizing collagen I alpha-chain dimers. Fibrillar collagen V was not digested. These hydrolyses were inhibited by ABC and by EDTA. Our results show that the antibothropic complex is a venom metalloproteinase inhibitor, which could, at least partially, account for its antihaemorrhagic activity. Electrophoretic evidence indicated non-covalent complex formation between the antihaemorrhagic factor and component(s) of B. jararaca venom.

Inhibition of the hyperalgesic activity of Bothrops jararaca venom by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

The antibothropic fraction (ABF) already isolated from Didelphis marsupialis serum, inhibits the haemorrhagic, oedematogenic, myonecrotic and lethal activities of Bothrops jararaca venom (Bjv). The aim of this work was to verify the capability of ABF to inhibit the hyperalgesic activity of Bjv. Intraplantar injection of Bjv induced hyperalgesia in a time- and dose-dependent manner and ABF administered in situ concomitantly with Bjv or i.v. 30 min before venom injection reduced the induced hyperalgesia. This same effect was observed when ABF was intravenously injected at 5 and 15 min after Bjv. Our results show that ABF inhibits also the hyperalgesia induced by Bjv.

Detection of an antibothropic fraction in opossum (Didelphis marsupialis) milk that neutralizes Bothrops jararaca venom

An antibothropic fraction (ABF) from Didelphis marsupialis (opossum) serum, which is responsible for the neutralization of Bothrops jararaca venom was isolated by Perales et al. [Perales, J., Moussatché, H., Marangoni, S., Oliveira, B. and Domont, G. B. (1994). Isolation and partial characterization of an antibothropic complex from the serum of South American Didelphidae. Toxicon 32, 1237-1249]. The aim of this work was to verify the presence of this factor in opossums milk, which could represent an additional protection for the neonatal opossum against bothropic venoms. An active milk fraction was isolated and showed similar physicochemical, structural, antigenic and biological properties when compared to ABF, indicating that they are probably the same protein.

New methodology for the obtainment of antibothropic factors from the South American opossum (Didelphis marsupialis) and jararaca snake (Bothrops jararaca).

The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice.