- Email: [email protected]
Some clinical snakebite problems
Report and Abstracts
587
technique in - 1 set pulses. Whole cell currents elicited by acetylcholine (ACh) were up to 4 nA at -70 mV membrane potential (V,), and were rapidly desensitixing. Recovery from dmensitization was 95% complete within 2 set and complete within 20 sec. The ncso for ACh was 10 FM. PhTX-343 was applied in the presence of lo@! ACh in the range 0.01 @v+ to 1OOpM. The IC, at -75 mV was 1 PM, but was voltage dependent, i.e. at more negative V, the toxin was more potent and less so at less negative V,. The responses recovered from toxin, to control levels within 1min when ACh was applied at 20 set intervals (three pulses). We are grateful to Professor K. Nakanishi for PhTX-343, and to Dr P. Sells for TE671 cells. Brackley er al. (1993) J. Phurn~uc.exp. Ther. 266, 1573-1580. McAllister et al. (1977) Int. J. Cancer 24, 206-212. Roxental et al. (1989) J. Pharmuc. exp. Ther 249, 123-130. Schoepfer et al. (1988) FEBS Len. 226, 235-240. Bioinformatic approaches to toxin structure-activity. M. Nyegaard, L. Cardle and M. J. Dufton (Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow Gl IXL, U.K.). The archetypal ‘three-loop* or ‘three-finger’ neurotoxins and cytotoxins, examples of which occur throughout elapid snake venoms, provide an excellent opportunity to investigate toxin structure-activity relationships because so many distinct pharmacological effects are associated with one basic molecular shape. In an attempt to define the key functional areas in a more systematic way than previous studies, a novel theoretical methodology which considers residue evolution in three dimensions was applied to the major subtypes. The methodology ranks the importance of residue positions according to the evolutionary conservation of their surroundings, not just their individual variation, and produces ‘conservation maps’ for each toxin family upon a representative chain fold. Control analyses of carboxypeptidases A, cytochromes c, phospholipases A, and proteinase inhibitors have confirmed the predictive accuracy of the method. The short and long alpha-neurotoxin families have two very similar foci of 3D conservation in their respective structures. One of these sites is adjacent to the disulphidebridged ‘core’ while the other encompasses the exposed end of the central loop. However, the cytotoxin and muscarinic toxin families differ by showing high conservation in the vicinity of their N-termini while their central loop area is variable. Thereby, some areas conserved in the nicotinic neurotoxins have varied in the cytotoxins/muscarinic toxins and vice versa. One possibility, therefore, is that opposite ends of the molecular configuration are prominent in nicotinic versus muscarinic/cytotoxic actions (in accord with some experimental evidence). All toxin groups showed the highest levels of local conservation at a point near the central disulphide-bridged area. However, this was a surface-accessible region to one side of the molecular core (i.e. exposed to the ‘concave face’ of the molecule), and not the core itself. There are consistent structural differences between the toxin types when this locality is compared. Interestingly, the character of this locality appears important for determining the disposition of the third major chain loop in such molecules. Much of the outcome of the evolutionary analysis is compatible with the available experimental evidence on the functional regions, but the consistent and extreme conservation near the molecular core seems at odds with the expectation that the analysis primarily highlights features which are directly fimctionai rather than merely structural. However, if there is a common theme running through these molecules in terms of mode of action or type of target molecule sought, it is anticipated that this area will be both involved in and responsible for the underlying differentiation in specificity. Some cfinicui’snakebite problems. D. A. Warrell (Centre for Tropical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.). Snakebite is an important medical problem in many tropical countries and is responsible for SO,ooO-100,000 deaths each year. However, clinical studies of snakebite patienti are made dithcult by the uneven geographical distribution of cases, their heterogeneity, and by the problem of creating research facilities in the rural locations where snakebite is most common. Having identified a clinical snakebite problem which demands investigation, the researcher must establish which hospital(s) admits sufficient cases for a study, discover the season(s) of peak incidence, decide which species are most commonly responsible, develop the research protocol, and assemble the necessary team of investigators. Therapeutic intervention with antivenom, and in some cases ancillary treatments, should be included in most clinical studies of snakebite as very few clinical trials of these treatments have been carried out. During the last 20 years, the Oxford-Liverpool Venom Research Group has carried out clinical studies in Nigeria, Thailand, Burma, Sri Lanka, Papua New Guinea, Brazil, and Ecuador. In Acre, Western Brazil, and the El Oriente region of Ecuador, indigenous Indian tribes, such as the Cashinawa, Shuar, and Waorani, and immigrant ‘seringueiros’ are exposed to bites by Borhrops atrox. Hospital treatment with antivenom may be inaccessible or require many days travel; a high incidence of mortality and morbidity has been reported by anthropologists. In Burma, Sri Lanka, and other rice-growing countries of south-east Asia, fanners face the dangers of Russell’s viper bites during planting and harvesting of,the paddy. Rubber, coffee, and other plantation workers are frequently bitten by Malayan pit vipers, in Thailand, peninsular Malaysia, Indo-China and Java. In the southern coastal area of New Guinea, Papuan taipans are responsible for many cases of severe neurotoxic evenoming. In the Guinea savanna of West Africa, saw-scaled or carpet vipers (Echis ocellutus) cause many
588
Report and Abstracts
deaths of farmers and herdsmen. In all these locations, there is a need for early administration of safe and effective antivenoms at the most peripheral level of the health service or even as a first-aid measure. Screening ofuntivenoms. R. D. G. Theakston (Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, U.K.). Initial assessment of antivenoms is normally carried out by screening in experimental animals, using the median effective dose (ED& assay combined with a range of other in vivo tests to evaluate neutralization of venom haemorrhagic, necrotizing and defibrinogenating activities. fn vitro tests currently recommended include those for procoagulant and neurotoxic activities. Once these tests have been performed and satisfactory results obtained, the antivenom, providing it meets all the standard requirements pertaining to stability, lack of pyrogenicity, and sterility, and has achieved ethical committee clearance, can be assessed in human patients, preferably on a comparative basis, using clinical criteria such as restoration of blood coagulability, resolution of neurotoxic signs and/or abolition of venom-induced spontaneous bleeding. An additional more objective assessment is the evaluation of the pharmacological picture of clearence of venom antigen in relation to the circulating levels of antivenom using enzyme immunoassay (EIA). Using these techniques, we have shown that in Brazil the three main Borhrops antivenoms are highly effective in neutralizing Bothrops venoms and are possibly being administered in excessive doses to patients, resulting in an unacceptably high incidence of early anaphylactic reactions. In Thailand, a comparative experimental and clinical trial of three antivenoms active against the venom of the Malayan pit viper (Cullosefasmn rhodostoma) showed good correlation between experimental and clinical results and in Sri Lanka the relative inefficacy of Indian HatBcine antivenom against the venom of Sri Lankan Russell’s viper (Duboia russe/lipulchellu) is about to result in the production of a new antivenom produced using Sri Lankan, as opposed to Indian, venom. Recently these procedures have also been used to assess a new Fab sheep .&his ocellutus antivenom in Nigeria where currently no antivenom is available. In conclusion, using a combination of experimental, clinical and immunological procedures, it is possible to obtain a highly accurate assessment of the efficacy of a particular antivenom. Inhibition ofplureler
function by snake venom. A. S. Kamiguti (Department Liverpool University Hospital, Liverpool, U.K.).
of Haematology of the Royal
The most well-studied snake venom components capable of inhibiting platelet function are the disintegrins and the phospholipase A2 (PLA,) enzymes. Recently this activity appears also to be shared by venom haemorrhagic factors. The mechanisms by which these different components affect platelet function were reviewed. Briefly, disintegrins inhibit platelets by the high-affinity binding to platelet glycoprotein IIb/IIIa (gp IIb/IIIa), the fibrinogen binding site to platelet surface. These cysteine-rich polypeptides are more effective than the synthetic linear RGDS peptides in the inhibitory capacity. Venom PLA, enzymes are less selective in the inhibition, as it appears that they are capable to inhibit platelet function independent of the activation mechanism involved in platelet response. A PLA, from Pseudechis pupuunus venom, similarly to the whole venom of Mcropechis ikuheku, has the inhibitory activity dependent on a yet unidentified plasma component. The major haemorrhagic factor from the venom of Borhrops jarurucu, jararhagin, has inhibitory effects in the platelet responses evoked by collagen and ristocetin. The inhibition appears to be dependent on proteolysis of ligands involved in platelet response together with alterations of platelet surface glycoproteins, in particular gp Ia/IIa, the collagen receptor. Jararhagin, an a-tibrinogenase, cleaves the C-terminal region of fibrinogen Aa chains; however, the loss of the 23,000mol. wt fibrinogen fragment still allows the molecule to interact with platelets. The role of these components in bleeding disorders frequently seen in envenoming will be discussed. Studies on phospholipuse A, isolated from Pupuun black snake (Pseudechis papunaus) venom. G. D. Laing,’ A.
S. Kamiguti,2 M. Wilkinson,’ G. Lowe* and R. D. G. Theakston’ (‘Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.; *Department of Haematology, Royal Liverpool Hospital, Liverpool, U.K.; and ‘Department of Biochemistry, University of Liverpool, Liverpool, U.K.). A phospholipase A, of mol. wt 17,200 ~16.7, was purified from the venom of the Papuan black snake (Pseudechis As well as exhibiting strong anticoagulant activity, the enzyme inhibited collagen-induced platelet aggregation in a dose-dependent manner. Electron microscopy of platelets incubated with the enzyme revealed loss of discoid shape and dramatic changes in the cell cytoskeleton. The PLA, enzyme was subjected to amino acid sequencing which revealed close homology (up to 91%) with PLA, enzymes isolated from Pseudechis uusrrulis (king brown snake) venom.
pupuunus) by a two-stage procedure using ion-exchange and hydrophobic chromatography.
Molecular studies of haemorrhugins from Kenyun Echis (Echis pyramidum leakeyi) venom. A. M. Moura-Da-
Silva,‘.2,3M. J. I. Paine,‘,* R. D. G. Theakston’ and J. M. Crampton* (‘Snake Venom Research Unit, and ‘Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.; and ‘Instituto Butantan, Slo Paulo, Brazil). Local and systemic haemorrhage caused by metalloproteases are important clinical symptoms in envenoming by vipers. Haemorrhagic metalloproteases from pit vipers have been cloned and characterized; however, little
587
technique in - 1 set pulses. Whole cell currents elicited by acetylcholine (ACh) were up to 4 nA at -70 mV membrane potential (V,), and were rapidly desensitixing. Recovery from dmensitization was 95% complete within 2 set and complete within 20 sec. The ncso for ACh was 10 FM. PhTX-343 was applied in the presence of lo@! ACh in the range 0.01 @v+ to 1OOpM. The IC, at -75 mV was 1 PM, but was voltage dependent, i.e. at more negative V, the toxin was more potent and less so at less negative V,. The responses recovered from toxin, to control levels within 1min when ACh was applied at 20 set intervals (three pulses). We are grateful to Professor K. Nakanishi for PhTX-343, and to Dr P. Sells for TE671 cells. Brackley er al. (1993) J. Phurn~uc.exp. Ther. 266, 1573-1580. McAllister et al. (1977) Int. J. Cancer 24, 206-212. Roxental et al. (1989) J. Pharmuc. exp. Ther 249, 123-130. Schoepfer et al. (1988) FEBS Len. 226, 235-240. Bioinformatic approaches to toxin structure-activity. M. Nyegaard, L. Cardle and M. J. Dufton (Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow Gl IXL, U.K.). The archetypal ‘three-loop* or ‘three-finger’ neurotoxins and cytotoxins, examples of which occur throughout elapid snake venoms, provide an excellent opportunity to investigate toxin structure-activity relationships because so many distinct pharmacological effects are associated with one basic molecular shape. In an attempt to define the key functional areas in a more systematic way than previous studies, a novel theoretical methodology which considers residue evolution in three dimensions was applied to the major subtypes. The methodology ranks the importance of residue positions according to the evolutionary conservation of their surroundings, not just their individual variation, and produces ‘conservation maps’ for each toxin family upon a representative chain fold. Control analyses of carboxypeptidases A, cytochromes c, phospholipases A, and proteinase inhibitors have confirmed the predictive accuracy of the method. The short and long alpha-neurotoxin families have two very similar foci of 3D conservation in their respective structures. One of these sites is adjacent to the disulphidebridged ‘core’ while the other encompasses the exposed end of the central loop. However, the cytotoxin and muscarinic toxin families differ by showing high conservation in the vicinity of their N-termini while their central loop area is variable. Thereby, some areas conserved in the nicotinic neurotoxins have varied in the cytotoxins/muscarinic toxins and vice versa. One possibility, therefore, is that opposite ends of the molecular configuration are prominent in nicotinic versus muscarinic/cytotoxic actions (in accord with some experimental evidence). All toxin groups showed the highest levels of local conservation at a point near the central disulphide-bridged area. However, this was a surface-accessible region to one side of the molecular core (i.e. exposed to the ‘concave face’ of the molecule), and not the core itself. There are consistent structural differences between the toxin types when this locality is compared. Interestingly, the character of this locality appears important for determining the disposition of the third major chain loop in such molecules. Much of the outcome of the evolutionary analysis is compatible with the available experimental evidence on the functional regions, but the consistent and extreme conservation near the molecular core seems at odds with the expectation that the analysis primarily highlights features which are directly fimctionai rather than merely structural. However, if there is a common theme running through these molecules in terms of mode of action or type of target molecule sought, it is anticipated that this area will be both involved in and responsible for the underlying differentiation in specificity. Some cfinicui’snakebite problems. D. A. Warrell (Centre for Tropical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.). Snakebite is an important medical problem in many tropical countries and is responsible for SO,ooO-100,000 deaths each year. However, clinical studies of snakebite patienti are made dithcult by the uneven geographical distribution of cases, their heterogeneity, and by the problem of creating research facilities in the rural locations where snakebite is most common. Having identified a clinical snakebite problem which demands investigation, the researcher must establish which hospital(s) admits sufficient cases for a study, discover the season(s) of peak incidence, decide which species are most commonly responsible, develop the research protocol, and assemble the necessary team of investigators. Therapeutic intervention with antivenom, and in some cases ancillary treatments, should be included in most clinical studies of snakebite as very few clinical trials of these treatments have been carried out. During the last 20 years, the Oxford-Liverpool Venom Research Group has carried out clinical studies in Nigeria, Thailand, Burma, Sri Lanka, Papua New Guinea, Brazil, and Ecuador. In Acre, Western Brazil, and the El Oriente region of Ecuador, indigenous Indian tribes, such as the Cashinawa, Shuar, and Waorani, and immigrant ‘seringueiros’ are exposed to bites by Borhrops atrox. Hospital treatment with antivenom may be inaccessible or require many days travel; a high incidence of mortality and morbidity has been reported by anthropologists. In Burma, Sri Lanka, and other rice-growing countries of south-east Asia, fanners face the dangers of Russell’s viper bites during planting and harvesting of,the paddy. Rubber, coffee, and other plantation workers are frequently bitten by Malayan pit vipers, in Thailand, peninsular Malaysia, Indo-China and Java. In the southern coastal area of New Guinea, Papuan taipans are responsible for many cases of severe neurotoxic evenoming. In the Guinea savanna of West Africa, saw-scaled or carpet vipers (Echis ocellutus) cause many
588
Report and Abstracts
deaths of farmers and herdsmen. In all these locations, there is a need for early administration of safe and effective antivenoms at the most peripheral level of the health service or even as a first-aid measure. Screening ofuntivenoms. R. D. G. Theakston (Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, U.K.). Initial assessment of antivenoms is normally carried out by screening in experimental animals, using the median effective dose (ED& assay combined with a range of other in vivo tests to evaluate neutralization of venom haemorrhagic, necrotizing and defibrinogenating activities. fn vitro tests currently recommended include those for procoagulant and neurotoxic activities. Once these tests have been performed and satisfactory results obtained, the antivenom, providing it meets all the standard requirements pertaining to stability, lack of pyrogenicity, and sterility, and has achieved ethical committee clearance, can be assessed in human patients, preferably on a comparative basis, using clinical criteria such as restoration of blood coagulability, resolution of neurotoxic signs and/or abolition of venom-induced spontaneous bleeding. An additional more objective assessment is the evaluation of the pharmacological picture of clearence of venom antigen in relation to the circulating levels of antivenom using enzyme immunoassay (EIA). Using these techniques, we have shown that in Brazil the three main Borhrops antivenoms are highly effective in neutralizing Bothrops venoms and are possibly being administered in excessive doses to patients, resulting in an unacceptably high incidence of early anaphylactic reactions. In Thailand, a comparative experimental and clinical trial of three antivenoms active against the venom of the Malayan pit viper (Cullosefasmn rhodostoma) showed good correlation between experimental and clinical results and in Sri Lanka the relative inefficacy of Indian HatBcine antivenom against the venom of Sri Lankan Russell’s viper (Duboia russe/lipulchellu) is about to result in the production of a new antivenom produced using Sri Lankan, as opposed to Indian, venom. Recently these procedures have also been used to assess a new Fab sheep .&his ocellutus antivenom in Nigeria where currently no antivenom is available. In conclusion, using a combination of experimental, clinical and immunological procedures, it is possible to obtain a highly accurate assessment of the efficacy of a particular antivenom. Inhibition ofplureler
function by snake venom. A. S. Kamiguti (Department Liverpool University Hospital, Liverpool, U.K.).
of Haematology of the Royal
The most well-studied snake venom components capable of inhibiting platelet function are the disintegrins and the phospholipase A2 (PLA,) enzymes. Recently this activity appears also to be shared by venom haemorrhagic factors. The mechanisms by which these different components affect platelet function were reviewed. Briefly, disintegrins inhibit platelets by the high-affinity binding to platelet glycoprotein IIb/IIIa (gp IIb/IIIa), the fibrinogen binding site to platelet surface. These cysteine-rich polypeptides are more effective than the synthetic linear RGDS peptides in the inhibitory capacity. Venom PLA, enzymes are less selective in the inhibition, as it appears that they are capable to inhibit platelet function independent of the activation mechanism involved in platelet response. A PLA, from Pseudechis pupuunus venom, similarly to the whole venom of Mcropechis ikuheku, has the inhibitory activity dependent on a yet unidentified plasma component. The major haemorrhagic factor from the venom of Borhrops jarurucu, jararhagin, has inhibitory effects in the platelet responses evoked by collagen and ristocetin. The inhibition appears to be dependent on proteolysis of ligands involved in platelet response together with alterations of platelet surface glycoproteins, in particular gp Ia/IIa, the collagen receptor. Jararhagin, an a-tibrinogenase, cleaves the C-terminal region of fibrinogen Aa chains; however, the loss of the 23,000mol. wt fibrinogen fragment still allows the molecule to interact with platelets. The role of these components in bleeding disorders frequently seen in envenoming will be discussed. Studies on phospholipuse A, isolated from Pupuun black snake (Pseudechis papunaus) venom. G. D. Laing,’ A.
S. Kamiguti,2 M. Wilkinson,’ G. Lowe* and R. D. G. Theakston’ (‘Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.; *Department of Haematology, Royal Liverpool Hospital, Liverpool, U.K.; and ‘Department of Biochemistry, University of Liverpool, Liverpool, U.K.). A phospholipase A, of mol. wt 17,200 ~16.7, was purified from the venom of the Papuan black snake (Pseudechis As well as exhibiting strong anticoagulant activity, the enzyme inhibited collagen-induced platelet aggregation in a dose-dependent manner. Electron microscopy of platelets incubated with the enzyme revealed loss of discoid shape and dramatic changes in the cell cytoskeleton. The PLA, enzyme was subjected to amino acid sequencing which revealed close homology (up to 91%) with PLA, enzymes isolated from Pseudechis uusrrulis (king brown snake) venom.
pupuunus) by a two-stage procedure using ion-exchange and hydrophobic chromatography.
Molecular studies of haemorrhugins from Kenyun Echis (Echis pyramidum leakeyi) venom. A. M. Moura-Da-
Silva,‘.2,3M. J. I. Paine,‘,* R. D. G. Theakston’ and J. M. Crampton* (‘Snake Venom Research Unit, and ‘Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.; and ‘Instituto Butantan, Slo Paulo, Brazil). Local and systemic haemorrhage caused by metalloproteases are important clinical symptoms in envenoming by vipers. Haemorrhagic metalloproteases from pit vipers have been cloned and characterized; however, little