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Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms
~To~xlrnn, Vol. 27, No. 12, pp. 1397-1399, 1989 . C/I~IOd 1D Grcat BIIIBIO.
0041-0IO1J89 ß.00 + .00 ® 1989 Pennon Pew plc
SHORT COMMiJNICATION CROSS-NEUTRALIZATION OF THROMBIN-LIKE ENZYMES IN SNAKE VENOMS BY POLYVALENT ANTIVENOMS Ixe Ct,nus and D. MESs
Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G . (Acceptedjor publication 14 June 1989)
I. CLnus and D. MESS . Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms. Toxicon 27, 1397-1399, 1989 .-Five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the thrombin-like activity of snake venoms
(Bitis gabonica, Agkistrodon acutus, Bothrops riper, B. atrox, Crotalus adamanteus) . Considerable cross-neutralization was observed . Anti-coagulane anti-
bodies were isolated from an antivenom by affinity chromatography using a purified enzyme from Bibs gabonica venom. These antibodies neutralized the activity of most snake venom coagulant enzymes. DISTURBANCE of hemostasis is one of the most serious symptoms in snake bite . Specific venom constituents activate blood coagulation factors such as factor V or X, or act as convertases of prothrombin or as thrombin-like enzymes (KoxxAI,nc, 1985). This may cause complete and long-lasting incoagulability of blood. Besides substitution therapy with plasma or plasma derivatives the use of antivenoms is the only effective treatment. But for most antivenoms information about their capacity to neutralize these venom activities are not available. Moreover, cross-reactivity of antivenoms to venoms which have not been used in the immunization schedule is a common and most interesting phenomenon (KoxxAI,Ix and TASORSxA, 1983 ; MESS, 1986; MESS et al., 1988). In the present paper we describe the neutralization of the fibrinogen coagulant activity of snake venoms by commercial antivenoms. Venom from Bibs gabonica was purchased from Latoxan, Rosans, France . The venoms from Crotalus adamanteus and Bothrops riper (Costa Rica) were a gift from Dr F. Kornalik, Prague, CSSR; Bothrops atrox venom (Brazil) was kindly provided by Dr K. Stocker, Pentapharm AG, Basel, Switzerland, and Agkistrodon acutus venom by Dr Wang, Fuzhou, China . The following antivenoms were used for neutralization studies: polyvalent North, Central Africa and Orient (Near and Middle East) antivenom from Behringwerke AG, Marburg, F.R .G .; polyvalent antivenom from South African Institute for Medical Research (SAIMR), Johannesburg, South Africa ; polyvalent Crotalidaeantivenom from Wyeth Laboratories, Philadelphia, PA, U.S.A. Thrombin-like activity was tested by adding 0.1 ml of venom solution (25 hg) to 0.2 ml of bovine fibrinogen solution (2 mg/ml saline). The mixture was incubated at 37°C and the time until clot formation was measured. For neutralization testing 50 hg of venom (in 0.1 ml saline) was incubated with 0.1 ml of antivenom (0.1 ml saline as control) for 30 min at 37°C; 0.1 ml of the reaction mixture (containing 25 hg venom) was used for testing residual enzyme activity . Complete inhibition was assumed, when no clot formation occurred within 3 min; 1397
1398
Short Communication
zero inhibition, when the clotting time was identical to that of the control sample . Enzyme-antibodies from Behringwerke-0rient antivenom were isolated by affinity chromatography as described previously (MEHS et al., 1988) using a semi-pure thrombinlike enzyme obtained from Bitis gabonica venom by gel filtration and DEAE-cellulose chromatography (PIRKLE et al., 1986) . An enzyme from Agkistrodon acutus venom was purified by gel filtration and affinity chromatography on benzamidine-Sepharose (KOMORI et al., 198 .
All antivenoms tested were able to neutralize (i.e. inhibit) the coagulant activity of the venoms to varying degrees (Table 1): the activity of Bibs gabonica venom was best neutralized by Behringwerke and SAIMR antivenoms, that of Bothrops atrox, B. riper and Crotalus adamanteus venom by Wyeth antivenom. Remarkable cross-reactivity was observed in the case of the Chinese Agkistrodon acutus venom. Behringwerke-0rient as well as SAIMR antivenom exhibited high potency in neutralizing the coagulant activity of this venom. Moreover, SAIMR antivenom neutralized the activity of Bothrops atrox and of Crotalus adamanteus venom to the same extent as Wyeth antivenom. TABLE I . NEUTRALIZATION OF FIBRINOGEN COAGULANT ACTIVITY BY POLYVALENT ANTIVENOM3
Venom
Antivenom
Antivenom dilutions Undil.
Agkistrodon acutes
Bitfis gabonica
Bothrops asper
Bothrops atrox
Crotales adamanteur
Behr . NO-Afr . Behr . Orient Behr . Cent. Afr. SAIMR Wyeth Hehr . NO-Afr. Behr . Orient Behr . Cent . Afr. SAIMR Wyeth
1/2
1/4
--81-_
1/8
1/l6
1/32
1/64 --27--
--8~--
_ßg_____27--
-,80--~2-_
-18--~7-- -31---31--
Behr . NO-Afr. Behr .Orient Behr . Cent . Afr. SAIMR Wyeth
--22--~1---55--~8--
Hehr . NO-Afr. Hehr .Orient Hehr . Cent . Afr. SAIMR Wyeth
--77---88---72--
Behr . NO-Afr. Behr .Orient Behr . Cent . Afr. SAIMR Wyeth
0 --58---~5--
-5~---~7----~-
-~~____2¢_
-70--~~-
--71--78-_
Fifty micrograms of venom in 0.1 ml of saline were mined with 0.1 ml of antivenom or antivenom dilutions and incubated at 37°C for 30 min and then 0.1 ml was tested for thrombin-like activity. Behr., Behringwerke antivenom; NO-Afr ., North Africa; Orient, Near and Middle East; Cent . Afr., Central Africa; SAIMR, South African Institute for Medical Research; Wyeth, Wyeth anti-Crotalidae antivenom. Solid lines indicate complete inhibition, numbers % inhibition (mean value of four experiments) .
1399
Short Communication TAHLE 2 . NFUTR~ il!~ATION OF FIBRINOGEN-OOAGULANT ACtIVITY OF SNAKE VENOMS BY AN7~ODII~ L90LA7~ FROM BEFfltINGWERKE ORIENT AN7iVENOb1 BY AFFINTfY CHROi~tATOGRAPHY USING Bit7.4 gabonica ENZYAlE AS LIGAND Venoms
% Neutralization (i.e . enzyme inhibition) Fraction I Fraction II (14 mg/ml) (1 .2 mg/ml)
Agkistrodon ocular Bitis gabonica Bothrops riper Bothrops atrox Crotales adammtteus Same conditions as in Table
0 0 0 0 0 1.
Mean values
58 f 7 35 f, 5 31 f 3 28 f 3 0 of four experiments f S .D.
Specific antibodies were isolated from liehringwerke-0rient antivenom by affinity chromatography using a semi-pure coagulant enzyme from Bitis gabonica venom as ligand . The unadsorbed material which passed through the column (fraction I; Table 2) had no neutralization capacity towards the coagulant activity of the five venoms; but the adsorbed antibodies (fraction In which were eluted from the column by lowering the pH of the buffer to 3.0 proved to be effective in neutralizing most venom coagulants except that of Crotales adamantees venom. On the other hand, antibodies were not retained when the pure coagulant enzyme from Agkistrodon ocular venom was used as ligand . The antivenom passed the affinity column unadsorbed, which contrasts with the observation that the antivenom effectively neutralizes the venom's coagulant activity . However, this may demonstrate that binding kinetics of the antibodies may vary considerably . Neutralizing antibodies are firmly bound by the enzyme from Bitis gabonica venom, but no such high affinity to antibodies is observed in the case of Agkistrodon acutes venom enzyme . Our experiments confirm that commercial antivenoms indeed neutralize thrombin-like activities of snake venoms and that cross-neutralization capacities of the antivenoms exist to a large extent (KORNA1.Ix and TABORSKA, 1983). This suggests common antigenic properties of the venom thrombin-like enzymes. According to the recommendations of the World Health Organization (WHO, 1981) antivenom testing should include other venom activities such as hemorrhage, necrotizing, coagulanes and in vivo-defibrination beside LDP. Information about these neutralizing capacities of antivenoms and their potential cross-reactivities are crucial for a rational and effective treatment of snake bite . REFERENCES Ko~1oR1, Y ., Nllw, T ., YAMAUCHI, T. and $UGIHARA, H . (198 A comparative study of clotting factors from the venom of Agkistrodort acetes collected in China and Taiwan . Comp . Biochem. Physiol. 88B, 64349. KORNALIK, F. (1985) The influence of snake venom enzymes on blood coagulation . Pharmac. Ther . 29, 353--005 . KORNALIK, F. and TABORSKA, E . (1983) Neutralization of lethal, local and systemic effects of snake venoms by monovalent and polyvalent antivenoms . In : Proceedings 5th European Symposium on Animal . Plant and Microbial Toxins, pp .9-15 (MESS, D . and HABERI~Ia, G., Eds) . Hannover: European Section of the international Society of Toxinology . MEas, D. (1986) Myotoxic activity of phospholipases A, isolated from cobra venoms : neutralization by polyvalent antivenoms. Toxicon 24, 1001-1008. MESS, D., POtn,u1ANN, S . and VoN TENSFOLDE, W. (1988) Snake venom hemorrhagins : neutralization by commercial antivenoms . Toxicon 26, 45358. PmICI .E, H ., TFD :ODOR, L, MIYADA, D . and $ISOdON3, G . (1986) Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions . 1. biol. Chem. 261, 8830-8835 . WHO (1981) Progress in the characterization of venoms and standardization of antivenoms . WHO Offset Publication No. 58, Geneva: World Health Organization .
0041-0IO1J89 ß.00 + .00 ® 1989 Pennon Pew plc
SHORT COMMiJNICATION CROSS-NEUTRALIZATION OF THROMBIN-LIKE ENZYMES IN SNAKE VENOMS BY POLYVALENT ANTIVENOMS Ixe Ct,nus and D. MESs
Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G . (Acceptedjor publication 14 June 1989)
I. CLnus and D. MESS . Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms. Toxicon 27, 1397-1399, 1989 .-Five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the thrombin-like activity of snake venoms
(Bitis gabonica, Agkistrodon acutus, Bothrops riper, B. atrox, Crotalus adamanteus) . Considerable cross-neutralization was observed . Anti-coagulane anti-
bodies were isolated from an antivenom by affinity chromatography using a purified enzyme from Bibs gabonica venom. These antibodies neutralized the activity of most snake venom coagulant enzymes. DISTURBANCE of hemostasis is one of the most serious symptoms in snake bite . Specific venom constituents activate blood coagulation factors such as factor V or X, or act as convertases of prothrombin or as thrombin-like enzymes (KoxxAI,nc, 1985). This may cause complete and long-lasting incoagulability of blood. Besides substitution therapy with plasma or plasma derivatives the use of antivenoms is the only effective treatment. But for most antivenoms information about their capacity to neutralize these venom activities are not available. Moreover, cross-reactivity of antivenoms to venoms which have not been used in the immunization schedule is a common and most interesting phenomenon (KoxxAI,Ix and TASORSxA, 1983 ; MESS, 1986; MESS et al., 1988). In the present paper we describe the neutralization of the fibrinogen coagulant activity of snake venoms by commercial antivenoms. Venom from Bibs gabonica was purchased from Latoxan, Rosans, France . The venoms from Crotalus adamanteus and Bothrops riper (Costa Rica) were a gift from Dr F. Kornalik, Prague, CSSR; Bothrops atrox venom (Brazil) was kindly provided by Dr K. Stocker, Pentapharm AG, Basel, Switzerland, and Agkistrodon acutus venom by Dr Wang, Fuzhou, China . The following antivenoms were used for neutralization studies: polyvalent North, Central Africa and Orient (Near and Middle East) antivenom from Behringwerke AG, Marburg, F.R .G .; polyvalent antivenom from South African Institute for Medical Research (SAIMR), Johannesburg, South Africa ; polyvalent Crotalidaeantivenom from Wyeth Laboratories, Philadelphia, PA, U.S.A. Thrombin-like activity was tested by adding 0.1 ml of venom solution (25 hg) to 0.2 ml of bovine fibrinogen solution (2 mg/ml saline). The mixture was incubated at 37°C and the time until clot formation was measured. For neutralization testing 50 hg of venom (in 0.1 ml saline) was incubated with 0.1 ml of antivenom (0.1 ml saline as control) for 30 min at 37°C; 0.1 ml of the reaction mixture (containing 25 hg venom) was used for testing residual enzyme activity . Complete inhibition was assumed, when no clot formation occurred within 3 min; 1397
1398
Short Communication
zero inhibition, when the clotting time was identical to that of the control sample . Enzyme-antibodies from Behringwerke-0rient antivenom were isolated by affinity chromatography as described previously (MEHS et al., 1988) using a semi-pure thrombinlike enzyme obtained from Bitis gabonica venom by gel filtration and DEAE-cellulose chromatography (PIRKLE et al., 1986) . An enzyme from Agkistrodon acutus venom was purified by gel filtration and affinity chromatography on benzamidine-Sepharose (KOMORI et al., 198 .
All antivenoms tested were able to neutralize (i.e. inhibit) the coagulant activity of the venoms to varying degrees (Table 1): the activity of Bibs gabonica venom was best neutralized by Behringwerke and SAIMR antivenoms, that of Bothrops atrox, B. riper and Crotalus adamanteus venom by Wyeth antivenom. Remarkable cross-reactivity was observed in the case of the Chinese Agkistrodon acutus venom. Behringwerke-0rient as well as SAIMR antivenom exhibited high potency in neutralizing the coagulant activity of this venom. Moreover, SAIMR antivenom neutralized the activity of Bothrops atrox and of Crotalus adamanteus venom to the same extent as Wyeth antivenom. TABLE I . NEUTRALIZATION OF FIBRINOGEN COAGULANT ACTIVITY BY POLYVALENT ANTIVENOM3
Venom
Antivenom
Antivenom dilutions Undil.
Agkistrodon acutes
Bitfis gabonica
Bothrops asper
Bothrops atrox
Crotales adamanteur
Behr . NO-Afr . Behr . Orient Behr . Cent. Afr. SAIMR Wyeth Hehr . NO-Afr. Behr . Orient Behr . Cent . Afr. SAIMR Wyeth
1/2
1/4
--81-_
1/8
1/l6
1/32
1/64 --27--
--8~--
_ßg_____27--
-,80--~2-_
-18--~7-- -31---31--
Behr . NO-Afr. Behr .Orient Behr . Cent . Afr. SAIMR Wyeth
--22--~1---55--~8--
Hehr . NO-Afr. Hehr .Orient Hehr . Cent . Afr. SAIMR Wyeth
--77---88---72--
Behr . NO-Afr. Behr .Orient Behr . Cent . Afr. SAIMR Wyeth
0 --58---~5--
-5~---~7----~-
-~~____2¢_
-70--~~-
--71--78-_
Fifty micrograms of venom in 0.1 ml of saline were mined with 0.1 ml of antivenom or antivenom dilutions and incubated at 37°C for 30 min and then 0.1 ml was tested for thrombin-like activity. Behr., Behringwerke antivenom; NO-Afr ., North Africa; Orient, Near and Middle East; Cent . Afr., Central Africa; SAIMR, South African Institute for Medical Research; Wyeth, Wyeth anti-Crotalidae antivenom. Solid lines indicate complete inhibition, numbers % inhibition (mean value of four experiments) .
1399
Short Communication TAHLE 2 . NFUTR~ il!~ATION OF FIBRINOGEN-OOAGULANT ACtIVITY OF SNAKE VENOMS BY AN7~ODII~ L90LA7~ FROM BEFfltINGWERKE ORIENT AN7iVENOb1 BY AFFINTfY CHROi~tATOGRAPHY USING Bit7.4 gabonica ENZYAlE AS LIGAND Venoms
% Neutralization (i.e . enzyme inhibition) Fraction I Fraction II (14 mg/ml) (1 .2 mg/ml)
Agkistrodon ocular Bitis gabonica Bothrops riper Bothrops atrox Crotales adammtteus Same conditions as in Table
0 0 0 0 0 1.
Mean values
58 f 7 35 f, 5 31 f 3 28 f 3 0 of four experiments f S .D.
Specific antibodies were isolated from liehringwerke-0rient antivenom by affinity chromatography using a semi-pure coagulant enzyme from Bitis gabonica venom as ligand . The unadsorbed material which passed through the column (fraction I; Table 2) had no neutralization capacity towards the coagulant activity of the five venoms; but the adsorbed antibodies (fraction In which were eluted from the column by lowering the pH of the buffer to 3.0 proved to be effective in neutralizing most venom coagulants except that of Crotales adamantees venom. On the other hand, antibodies were not retained when the pure coagulant enzyme from Agkistrodon ocular venom was used as ligand . The antivenom passed the affinity column unadsorbed, which contrasts with the observation that the antivenom effectively neutralizes the venom's coagulant activity . However, this may demonstrate that binding kinetics of the antibodies may vary considerably . Neutralizing antibodies are firmly bound by the enzyme from Bitis gabonica venom, but no such high affinity to antibodies is observed in the case of Agkistrodon acutes venom enzyme . Our experiments confirm that commercial antivenoms indeed neutralize thrombin-like activities of snake venoms and that cross-neutralization capacities of the antivenoms exist to a large extent (KORNA1.Ix and TABORSKA, 1983). This suggests common antigenic properties of the venom thrombin-like enzymes. According to the recommendations of the World Health Organization (WHO, 1981) antivenom testing should include other venom activities such as hemorrhage, necrotizing, coagulanes and in vivo-defibrination beside LDP. Information about these neutralizing capacities of antivenoms and their potential cross-reactivities are crucial for a rational and effective treatment of snake bite . REFERENCES Ko~1oR1, Y ., Nllw, T ., YAMAUCHI, T. and $UGIHARA, H . (198 A comparative study of clotting factors from the venom of Agkistrodort acetes collected in China and Taiwan . Comp . Biochem. Physiol. 88B, 64349. KORNALIK, F. (1985) The influence of snake venom enzymes on blood coagulation . Pharmac. Ther . 29, 353--005 . KORNALIK, F. and TABORSKA, E . (1983) Neutralization of lethal, local and systemic effects of snake venoms by monovalent and polyvalent antivenoms . In : Proceedings 5th European Symposium on Animal . Plant and Microbial Toxins, pp .9-15 (MESS, D . and HABERI~Ia, G., Eds) . Hannover: European Section of the international Society of Toxinology . MEas, D. (1986) Myotoxic activity of phospholipases A, isolated from cobra venoms : neutralization by polyvalent antivenoms. Toxicon 24, 1001-1008. MESS, D., POtn,u1ANN, S . and VoN TENSFOLDE, W. (1988) Snake venom hemorrhagins : neutralization by commercial antivenoms . Toxicon 26, 45358. PmICI .E, H ., TFD :ODOR, L, MIYADA, D . and $ISOdON3, G . (1986) Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions . 1. biol. Chem. 261, 8830-8835 . WHO (1981) Progress in the characterization of venoms and standardization of antivenoms . WHO Offset Publication No. 58, Geneva: World Health Organization .