Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Prothrombin and Factor X activating properties of Bothrops erythromelas venom Masugi Maruyama, Aura S. Kamiguti, Sandra C. Tomy, Luci C. Antonio, Masahiko Sugiki & Hisashi Mihara To cite this article: Masugi Maruyama, Aura S. Kamiguti, Sandra C. Tomy, Luci C. Antonio, Masahiko Sugiki & Hisashi Mihara (1992) Prothrombin and Factor X activating properties of Bothrops erythromelas venom, Annals of Tropical Medicine & Parasitology, 86:5, 549-556, DOI: 10.1080/00034983.1992.11812706 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812706
Published online: 15 Nov 2016.
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Citing articles: 1 View citing articles
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Date: 03 April 2017, At: 19:27
Annals of Tropical Medicine and Parasitology, Vol. 86, No.5, 549-556 (1992)
Prothrombin and Factor X activating properties of Bothrops erythromelas venom BY MASUGI MARUYAMA Department ofPhysiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-ken, 889-16, Japan
AURAS. KAMIGUTI, SANDRA C. TOMY, LUCI C. ANTONIO Laboratory of Hematology, Instituto But ant an, Sao Paulo, Brazil MASAHIKO SUGIKI
AND
HISASHI MIHARA
Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-ken, 889-16, Japan Received 23 June 1992, Accepted 29 June 1992
The enzymatic properties of Factor II (FII) and Factor X (FX) activators from Bothrops erythromelas venom were investigated. Both activators were inhibited by ethylenediaminetetraacetate (EDTA) and 1,10-phenanthroline, and are thought to be metalloproteinases with molecular weights of 90 kDa and 70-90 kDa, respectively. The activity of the FII activator in the crude venom was about 30 times greater than that in Oxyuranus scutellatus venom and the level ofFX activator activity, which was eN+ ion dependent, was similar to that in Daboia russelli venom. The venom also had two haemorrhagic factors (58 and 105 kDa) and two fibrinolytic enzymes (18 and 58 kDa).
In South America, about 90% of snakebites on man are caused by Bothrops spp. (WHO, 1981). The venoms of all but one of these species contain thrombin-like enzymes and envenomed patients develop pseudo-disseminated intravascular coagulation syndrome (pseudo-DIC), with decreased fibrinogen levels and unclottable blood (Rosenfeld, 1971; Kamiguti et al., 1986). Several of these thrombin-like enzymes have been isolated and characterized (Fung et al., 1971; Aronson, 1976; Ortiz and Gubenseck, 1976; Stocker and Barlow, 1976; Stocker et al., 1982). Nahas et al. (1979) demonstrated procoagulant activities in various Bothrops venoms and Factor X- and Factor 11-activating enzymes were isolated from Bothrops atrox venom 0003-4983/92/050549 + 08 $08.00/0
(Hofman and Bon, 1987a,b). The procoagulant/ thrombin-like enzyme ratio was found to be higher in young Bothrops than in adults (Furtado et al., 1991 ); patients bitten by young B. jararaca had increased cross-linked fibrin degradation products, and decreased levels of Factors V and VIII because of thrombin formation (Maruyama et al., 1990). The cross-linked fibrin deposits are thought to produce severe microangiopathy; fibrin deposits caused by the action of the thrombin-like enzymes are very prone to fibrinolytic activity and are readily eliminated from the circulation. Bothrops erythromelas is commonly found in north-eastern Brazil (Hoge and Hoge, 1978/ 1979) and is thought to be the most common © 1992 Liverpool School of Tropical Medicine
550
MARUYAMA ET AL.
TABLE 1 Biological activities ofBothrops erythromelas venom
Minimum coagulating dose On plasma (llg ml- 1) On fibrinogen (1-!g ml- 1) Fibrinolytic activity (mm 2)* Haemorrhagic activity (mm 2)t FII activator activity (units mg- 1) FX activator activity (units mg- 1)
4·8
> 1000 116± 16·1 421±38-4 1729 5·6
*Thirty J.ll of venom (I mg ml- 1) were applied to a standard fibrin plate (0·6%). Mean ± standard deviation (S.D.) of three replicates. tFiftyJ.!lofvenom(l mgml 1)wasinjectedintradermally. Mean ± S.D. of six replicates.
cause of snakebite on man m the area. The venom of this snake is unique among Bothrops venoms in having powerful procoagulant activity while lacking thrombin-like activity (Nahas et a!., 1979; Furtado eta!., 1991). In the present study, the biochemical and physiochemical properties of the procoagulant components of this venom were investigated. MATERIALS AND METHODS Materials The test material was a pool of lyophilized venom collected from 50 adult B. erythromelas maintained in the Herpetology Laboratory, Instituto Butantan, and this was compared with the lyophilized venoms (both from Sigma) of Daboia russelli and Oxyuranus scutellatus. Each venom was reconstituted in physiological saline and then kept frozen at - 20°C until used. Dithiothreitol and 1,10-phenanthroline were from Nacalai Tesque, Osaka, Japan, and elastatinal was from the Peptide Institute, Osaka, Japan. The chromogenic substrates, benzoyi-Ile-Giu-(y-OCH3)-GiyArg-benzoyi-Ile-Giu-Giy-Arg-p-nitroanilide hydrochloride (s-2222) and H-D-Phe-Pip-Argp-nitroanilide hydrochloride (s-2238) were purchased from Daiichi Pure Chemicals, Tokyo, Japan. Minimum Coagulant Dose The minimum coagulant doses on plasma and fibrinogen were estimated using the method of Theakston and Reid (1983).
Molecular Sieve Chromatography Two ml of B. erythromelas venom (5 mg ml- 1 in saline) were applied to a 1· 5 x 9 5 em Sephacryl S-300 column, equilibrated with Tris-buffer at a flow rate of 0· 5 ml/minute. Individual fractions were assayed for procoagulant, fibrinolytic and haemorrhagic activities. Factor II- and Factor X-activator Assays Factor II- and Factor X-activating activities were assayed by two- and one-step methods. TWO-STEP METHOD
FII activating activity was measured using a specific synthetic substrate for Fila. Fifty !J.I of various concentrations of venom solution and 50 !J.l of purified human FII (one unit ml- 1; Sigma) were mixed with 300 !J.I of 50 mM NaCI in 50 mM Tris-HCI buffer, pH 8·0 (Tris-buffer). The mixture was incubated for two minutes at 37°C and then the reaction was stopped by the addition of 200 !J.I of 50 mM ethylenediaminetetraacetate (EDT A) in Tris-buffer. One hundred !J.I of 3 mM s-2238, a specific synthetic substrate for thrombin (Fila), was then added and incubated for one minute at 37°C, before 100 !J.I of 50% acetic acid was added to stop the reaction. The optical density (o.D.) of the mixture was then measured at 405 nm. To estimate FX activating activity, 50 !J.I of varying concentrations of venom, 50 !J.I of purified human FX (one unit ml- \Sigma) and 50 !J.I of 100 mM CaCI 2 were mixed with 250 !J.I Tris-buffer. The subsequent procedures were identical to those for the FII assay except the substrate, for activated FX (FXa), was s-2222 and the first and second incubation steps were for 15 and 30 minutes, respectively. One unit of activating activity was defined as the amount of activator which activated one unit of Factors II or X (from Sigma) per minute at 37°C. Factors II and X, fully activated by the venoms of 0. scutellatus and D. russelli, respectively, were used as standards. ONE-STEP METHOD
Fifty !J.I of venom solution, 50 !J.I of purified FII or FX (one unit ml-\ Sigma) and 100111 of 3 mM substrate (s-2238 or s-2222) were mixed
B. ERYTHROMELAS VENOM FII AND FX ACTIVATORS
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ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Prothrombin and Factor X activating properties of Bothrops erythromelas venom Masugi Maruyama, Aura S. Kamiguti, Sandra C. Tomy, Luci C. Antonio, Masahiko Sugiki & Hisashi Mihara To cite this article: Masugi Maruyama, Aura S. Kamiguti, Sandra C. Tomy, Luci C. Antonio, Masahiko Sugiki & Hisashi Mihara (1992) Prothrombin and Factor X activating properties of Bothrops erythromelas venom, Annals of Tropical Medicine & Parasitology, 86:5, 549-556, DOI: 10.1080/00034983.1992.11812706 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812706
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [NUS National University of Singapore]
Date: 03 April 2017, At: 19:27
Annals of Tropical Medicine and Parasitology, Vol. 86, No.5, 549-556 (1992)
Prothrombin and Factor X activating properties of Bothrops erythromelas venom BY MASUGI MARUYAMA Department ofPhysiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-ken, 889-16, Japan
AURAS. KAMIGUTI, SANDRA C. TOMY, LUCI C. ANTONIO Laboratory of Hematology, Instituto But ant an, Sao Paulo, Brazil MASAHIKO SUGIKI
AND
HISASHI MIHARA
Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-ken, 889-16, Japan Received 23 June 1992, Accepted 29 June 1992
The enzymatic properties of Factor II (FII) and Factor X (FX) activators from Bothrops erythromelas venom were investigated. Both activators were inhibited by ethylenediaminetetraacetate (EDTA) and 1,10-phenanthroline, and are thought to be metalloproteinases with molecular weights of 90 kDa and 70-90 kDa, respectively. The activity of the FII activator in the crude venom was about 30 times greater than that in Oxyuranus scutellatus venom and the level ofFX activator activity, which was eN+ ion dependent, was similar to that in Daboia russelli venom. The venom also had two haemorrhagic factors (58 and 105 kDa) and two fibrinolytic enzymes (18 and 58 kDa).
In South America, about 90% of snakebites on man are caused by Bothrops spp. (WHO, 1981). The venoms of all but one of these species contain thrombin-like enzymes and envenomed patients develop pseudo-disseminated intravascular coagulation syndrome (pseudo-DIC), with decreased fibrinogen levels and unclottable blood (Rosenfeld, 1971; Kamiguti et al., 1986). Several of these thrombin-like enzymes have been isolated and characterized (Fung et al., 1971; Aronson, 1976; Ortiz and Gubenseck, 1976; Stocker and Barlow, 1976; Stocker et al., 1982). Nahas et al. (1979) demonstrated procoagulant activities in various Bothrops venoms and Factor X- and Factor 11-activating enzymes were isolated from Bothrops atrox venom 0003-4983/92/050549 + 08 $08.00/0
(Hofman and Bon, 1987a,b). The procoagulant/ thrombin-like enzyme ratio was found to be higher in young Bothrops than in adults (Furtado et al., 1991 ); patients bitten by young B. jararaca had increased cross-linked fibrin degradation products, and decreased levels of Factors V and VIII because of thrombin formation (Maruyama et al., 1990). The cross-linked fibrin deposits are thought to produce severe microangiopathy; fibrin deposits caused by the action of the thrombin-like enzymes are very prone to fibrinolytic activity and are readily eliminated from the circulation. Bothrops erythromelas is commonly found in north-eastern Brazil (Hoge and Hoge, 1978/ 1979) and is thought to be the most common © 1992 Liverpool School of Tropical Medicine
550
MARUYAMA ET AL.
TABLE 1 Biological activities ofBothrops erythromelas venom
Minimum coagulating dose On plasma (llg ml- 1) On fibrinogen (1-!g ml- 1) Fibrinolytic activity (mm 2)* Haemorrhagic activity (mm 2)t FII activator activity (units mg- 1) FX activator activity (units mg- 1)
4·8
> 1000 116± 16·1 421±38-4 1729 5·6
*Thirty J.ll of venom (I mg ml- 1) were applied to a standard fibrin plate (0·6%). Mean ± standard deviation (S.D.) of three replicates. tFiftyJ.!lofvenom(l mgml 1)wasinjectedintradermally. Mean ± S.D. of six replicates.
cause of snakebite on man m the area. The venom of this snake is unique among Bothrops venoms in having powerful procoagulant activity while lacking thrombin-like activity (Nahas et a!., 1979; Furtado eta!., 1991). In the present study, the biochemical and physiochemical properties of the procoagulant components of this venom were investigated. MATERIALS AND METHODS Materials The test material was a pool of lyophilized venom collected from 50 adult B. erythromelas maintained in the Herpetology Laboratory, Instituto Butantan, and this was compared with the lyophilized venoms (both from Sigma) of Daboia russelli and Oxyuranus scutellatus. Each venom was reconstituted in physiological saline and then kept frozen at - 20°C until used. Dithiothreitol and 1,10-phenanthroline were from Nacalai Tesque, Osaka, Japan, and elastatinal was from the Peptide Institute, Osaka, Japan. The chromogenic substrates, benzoyi-Ile-Giu-(y-OCH3)-GiyArg-benzoyi-Ile-Giu-Giy-Arg-p-nitroanilide hydrochloride (s-2222) and H-D-Phe-Pip-Argp-nitroanilide hydrochloride (s-2238) were purchased from Daiichi Pure Chemicals, Tokyo, Japan. Minimum Coagulant Dose The minimum coagulant doses on plasma and fibrinogen were estimated using the method of Theakston and Reid (1983).
Molecular Sieve Chromatography Two ml of B. erythromelas venom (5 mg ml- 1 in saline) were applied to a 1· 5 x 9 5 em Sephacryl S-300 column, equilibrated with Tris-buffer at a flow rate of 0· 5 ml/minute. Individual fractions were assayed for procoagulant, fibrinolytic and haemorrhagic activities. Factor II- and Factor X-activator Assays Factor II- and Factor X-activating activities were assayed by two- and one-step methods. TWO-STEP METHOD
FII activating activity was measured using a specific synthetic substrate for Fila. Fifty !J.I of various concentrations of venom solution and 50 !J.l of purified human FII (one unit ml- 1; Sigma) were mixed with 300 !J.I of 50 mM NaCI in 50 mM Tris-HCI buffer, pH 8·0 (Tris-buffer). The mixture was incubated for two minutes at 37°C and then the reaction was stopped by the addition of 200 !J.I of 50 mM ethylenediaminetetraacetate (EDT A) in Tris-buffer. One hundred !J.I of 3 mM s-2238, a specific synthetic substrate for thrombin (Fila), was then added and incubated for one minute at 37°C, before 100 !J.I of 50% acetic acid was added to stop the reaction. The optical density (o.D.) of the mixture was then measured at 405 nm. To estimate FX activating activity, 50 !J.I of varying concentrations of venom, 50 !J.I of purified human FX (one unit ml- \Sigma) and 50 !J.I of 100 mM CaCI 2 were mixed with 250 !J.I Tris-buffer. The subsequent procedures were identical to those for the FII assay except the substrate, for activated FX (FXa), was s-2222 and the first and second incubation steps were for 15 and 30 minutes, respectively. One unit of activating activity was defined as the amount of activator which activated one unit of Factors II or X (from Sigma) per minute at 37°C. Factors II and X, fully activated by the venoms of 0. scutellatus and D. russelli, respectively, were used as standards. ONE-STEP METHOD
Fifty !J.I of venom solution, 50 !J.I of purified FII or FX (one unit ml-\ Sigma) and 100111 of 3 mM substrate (s-2238 or s-2222) were mixed
B. ERYTHROMELAS VENOM FII AND FX ACTIVATORS
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