Toxicon 93 (2015) 41e50
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The lethality test used for estimating the potency of antivenoms against Bothrops asper snake venom: Pathophysiological mechanisms, prophylactic analgesia, and a surrogate in vitro assay n, Andrea Oviedo, Teresa Escalante, Gabriela Solano, Alexandra Rucavado, Francisco Chaco María Gutie rrez* 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 23 September 2014 Received in revised form 31 October 2014 Accepted 5 November 2014 Available online 6 November 2014
The potency of antivenoms is assessed by analyzing the neutralization of venom-induced lethality, and is expressed as the Median Effective Dose (ED50). The present study was designed to investigate the pathophysiological mechanisms responsible for lethality induced by the venom of Bothrops asper, in the experimental conditions used for the evaluation of the neutralizing potency of antivenoms. Mice injected with 4 LD50s of venom by the intraperitoneal route died within ~25 min with drastic alterations in the abdominal organs, characterized by hemorrhage, increment in plasma extravasation, and hemoconcentration, thus leading to hypovolemia and cardiovascular collapse. Snake venom metalloproteinases (SVMPs) play a predominat role in lethality, as judged by partial inhibition by the chelating agent CaNa2EDTA. When venom was mixed with antivenom, there was a venom/antivenom ratio at which hemorrhage was significantly reduced, but mice died at later time intervals with evident hemoconcentration, indicating that other components in addition to SVMPs also contribute to plasma extravasation and lethality. Pretreatment with the analgesic tramadol did not affect the outcome of the neutralization test, thus suggesting that prophylactic (precautionary) analgesia can be introduced in this assay. Neutralization of lethality in mice correlated with neutralization of in vitro coagulant activity in human plasma. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Antivenom Bothrops asper Lethality Neutralization Hemorrhage Hemoconcentraton
1. Introduction The most traditional way to assess the overall toxicity of venoms or toxins is based on the study of their capacity to induce death in experimental animals, which is performed by the estimation of the Median Lethal Dose (LD50). For this, various doses of venom, or toxin, are injected in animals, usually mice, by intravenous (i.v.), intraperitoneal (i.p.), or subcutaneous (s.c.) routes. Deaths occurring during a defined time span, usually 24 or 48 h, are recorded, and LD50 is estimated by using appropriate statistical methods such as probits, Spearman-Karber or non-parametric tests (WHO, 1981, 2010). On the other hand, the gold standard in the assessment of the preclinical efficacy of antivenoms or antitoxins is the analysis of the neutralization of lethality. In this case, a fixed amount of venom, or toxin, is incubated with various dilutions of antivenom. Then, aliquots of the mixtures, containing a ‘challenge dose’ of venom, i.e. a number of
* Corresponding author. rrez). E-mail address: [emailprotected] (J.M. Gutie http://dx.doi.org/10.1016/j.toxicon.2014.11.223 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
LD50s (usually 3e6), are injected in experimental animals and lethality is recorded. Neutralization is generally expressed as the Median Effective Dose (ED50), defined as the venom/antivenom ratio at which 50% of the injected animals survive (WHO, 2010). Despite the widespread use of these experimental protocols in the analysis of venom toxicity and in the assessment of the neutralizing potency of antivenoms, in many cases the lethality test remains a ‘black box’, since the main pathological and pathophysiological mechanisms involved in lethality in these assays have not been explored in detail. In the case of predominantly neurotoxic venoms, such as those of the majority of elapid snake species, neuromuscular paralysis leading to respiratory arrest is the mechanism of death. In contrast, in the case of viperid snake venoms, which induce a complex pattern of systemic pathophysiological effects, the study of the mechanisms of death in experimental rrez et al., 2013). This is models has received little attention (Gutie relevant for understanding the test itself and the neutralizing ability of antivenoms, i.e. which systemic venom effects are being abrogated by antivenoms in the experimental conditions in which these tests are performed.
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In Central America, a polyspecific antivenom (‘suero antiofídico polivalente’) is used in the treatment of envenomings caused by ~ os and Cerdas, 1980; Gutie rrez, 2010). It is viperid snakes (Bolan produced by immunizing horses with a mixture of the venoms of ~ os and Bothrops asper, Crotalus simus and Lachesis stenophrys (Bolan Cerdas, 1980; Angulo et al., 1997). The quality control of this antivenom includes a test for the neutralization of lethality induced by these venoms in mice, using the i.p. route and a challenge dose of venom corresponding to 4 LD50s. The design and analytical properties of this test have been previously characterized (Solano et al., 2010). The present investigation was designed to study the pathological and pathophysiological alterations occurring in mice by this dose of B. asper venom, in order to understand the mechanisms of lethality induced by venom alone and by mixtures of venom and antivenom, as applied to the lethality neutralization assay. In addition, an in vitro test for the study of neutralization of venom by this antivenom was investigated, with the aim of finding a surrogate laboratory alternative that could replace the mouse lethality test and, consequently, reduce the number of animals used in the quality control of this antivenom. Finally, the introduction of precautionary (prophylactic) analgesia in this test, based on the administration of tramadol, was analyzed, as an attempt to decrease the suffering and distress induced in mice in this acute toxicity assay. 2. Materials and methods 2.1. Venom and antivenom A pool of venom was prepared from more than 40 adult specimens of B. asper collected in the Pacific versant of Costa Rica and maintained at the serpentarium of Instituto Clodomiro Picado. Once obtained, venom was freeze-dried and stored at 20 C. The polyspecific (‘polyvalent’) antivenom (‘suero antiofìdico polivalente’, batch 5361213POLQ), manufactured at Instituto Clodomiro Picado, was used. In addition, the following batches were utilized for the study of the correlation between neutralization of lethality and coagulant effect: 4030906POLQ, 4051106POLQ, 4061106POLQ, 4070107POLQ, 4120507POLQ, 4180707POLQ, 4190807POLQ, and 4250208POLQ. In some cases, in-process samples of two batches of this antivenom were also used. Polyvalent antivenom is prepared by fractionating the plasma of horses immunized with a mixture of venoms of B. asper, C. simus and L. stenophrys. It is composed of whole IgG molecules purified by caprylic acid precipitation of nonimmunoglobulin plasma proteins (Rojas et al., 1994). 2.2. Animals CD-1 mice of 16e18 g were used throughout the study. All experiments involving mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of Universidad de Costa Rica. 2.3. Pathological and pathophysiological alterations in mice receiving a challenge dose of 4 LD50s of venom The LD50 of B. asper venom in mice, using the i.p. route as routinely performed in the Quality Control Laboratory of Instituto Clodomiro Picado, is 62.5 mg/16e18 g mice. For the study on the mechanisms of lethality, groups of mice (16e18 g) were injected i.p. with 250 mg venom (corresponding to 4 LD50s), dissolved in 0.5 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS). The time of death of mice was recorded, and the pathological and pathophysiological alterations at the time of death were analyzed, as described below.
2.3.1. Macroscopic observations and histological analysis Immediately after death, the peritoneal cavity of mice was opened and observed for macroscopic alterations. Then, samples of the following organs were collected: mesentery, small intestine, large intestine, liver, kidneys, diaphragm, lungs, heart and brain. Tissue samples were immediately placed in 10% formalin fixative solution, and were processed routinely for embedding in paraffin. Then, 4 mm sections were collected, placed in slides and stained with hematoxylin and eosin for microscopic observation. Control mice injected with 0.5 mL of PBS were sacrificed by an overdose of ketamine and xylazine at approximately 20 min after injection, which corresponds to the time of death of mice receiving venom. Upon sacrifice, samples of the various organs were obtained and processed as described. 2.3.2. Quantification of peritoneal hemorrhage and changes in hematocrit, coagulation time and plasma creatine kinase (CK) activity Groups of mice were injected with 4 LD50s of venom, as described. Twenty min after injection, animals were sacrificed by CO2 inhalation. Immediately afterwards, 1.0 mL of PBS was injected in the peritoneal cavity and, after a mild massage, an incision was made in the abdominal wall and a sample of peritoneal fluid was collected from this cavity. Samples were diluted 1:2 with PBS-1% Triton X-100 to induce erythrocyte lysis. After centrifugation for 5 min at 1000 g, the absorbance of the supernatant at 540 nm was recorded as a quantitative estimation of the amount of hemoglobin present in the lavage fluid, which correlates with the extent of hemorrhage. In another group of mice injected with 4 LD50s of venom, a blood sample was collected by cardiac puncture, under anesthesia, 20 min after injection. Blood was immediately placed in heparinized microcapillary tubes and centrifuged for estimating the hematocrit. Another blood sample was placed in dry glass tubes and allowed to stand at room temperature, and clotting times were recorded. In parallel, the creatine kinase (CK) activity of plasma, obtained by centrifugation of blood collected in heparinized microcapillaries, was quantified as an index of myonecrosis rrez et al., 1980), using a commercial kit (CK LIQUI-UV, (Gutie Stanbio Lab., Texas, USA). In all cases, a control group of mice was injected with 0.5 mL of PBS, and analyses were performed as described. In order to identify venom components responsible for hemoconcentration, similar experiments were performed by injecting i.p. a purified PI metalloproteinase (SVMP) (BaP1) rrez et al., 1995) and a fraction containing a mixture of (Gutie myotoxic Asp49 phospholipase A2 (PLA2) and Lys49 PLA2 homologue, isolated from the venom of B. asper by ion-exchange chromatography on CM-Sepharose, using a KCl gradient (Lomonte and rrez, 1989). The amounts injected were 75 mg of BaP1 and Gutie 100 mg of PLA2 myotoxin fraction, which roughly correspond to the amounts of PI SVMPs and myotoxic Asp49 and Lys49 PLA2s present in 250 mg of B. asper venom, on the basis of the proteomic analysis of the venom of adult specimens from the Pacific population of n et al., 2008). Costa Rica (Alape-Giro 2.3.3. Quantification of increase in vascular permeability in the peritoneal cavity Groups of mice were injected with 250 mg venom dissolved in 0.5 mL PBS, as described. Immediately before venom injection, mice received an i.v. injection of 200 mL of a solution of 2 mg/mL Evans Blue (SigmaeAldrich, Missouri, USA), dissolved in PBS. Twenty min after venom administration, mice were sacrificed by CO2 inhalation, and 1.0 mL of PBS was injected in the peritoneal cavity. After a mild massage, an incision was performed in the abdominal wall and a sample of peritoneal fluid was collected. Samples were diluted 1:4 with PBS and, after 5 min centrifugation at 1000 g, the absorbance
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of the supernatant was recorded at 620 nm as a quantitative index of the amount of Evans Blue extravasated in the peritoneal cavity. Control mice received a similar i.v. injection of Evans Blue solution, followed by 0.5 mL of PBS i.p. Twenty min after injection, mice were sacrificed by CO2 inhalation and Evans Blue in the peritoneal cavity was quantified as described. 2.4. Effect of inhibitors on the lethal activity and associated alterations induced by venom in mice In order to assess the role of SVMPs, PLA2, serine proteinases and basic myotoxic PLA2s and PLA2 homologues in lethality, the following inhibitors were used: 300 mM CaNa2EDTA, a chelating agent that inhibits SVMPs (SigmaeAldrich; Rucavado et al., 2000), 1.4 mM p-bromophenacyl bromide (pBPB), an inhibitor of PLA2s rrez, 1997), 5 mM 4-(2(SigmaeAldrich; Díaz-Oreiro and Gutie aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), a hydrosoluble irreversible inhibitor of serine proteinases (SigmaeAldrich, Powers et al., 2002), and 2 mg/mL fucoidan, a carbohydrate that inhibits basic myotoxic PLA2s and PLA2 homologues (Sigma Aldrich; Angulo and Lomonte, 2003). In the cases of CaNa2EDTA, AEBSF, and fucoidan, venom solutions were prepared in PBS and incubated with the inhibitors at room temperature (22e25 C) for 30 min. In the case of pBPB, venom dissolved in 1.0 mL PBS was mixed with 150 mL of pBPB dissolved in ethanol and incubated for 3 h at room temperature. The inhibition of hemorrhagic activity of venom by EDTA, of PLA2 activity by pBPB, of thrombin-like activity by AEBSF, and of myotoxic activity by fucoidan was confirmed by performing the corresponding tests, as previously described (Rucavado et al., 2000, 2005; Díaz-Oreiro and rrez, 1997; Angulo and Lomonte, 2003). Gutie To assess the role of basic myotoxic PLA2s and PLA2 homologues by an alternative approach, anti-myotoxic PLA2 antibodies were isolated from polyvalent antivenom by affinity chromatography on a CNBr-activated Sepharose CL-4B (GE Healthcare) column, containing immobilized myotoxic Asp49 PLA2 and Lys49 PLA2 homologue isolated from the venom of B. asper, as described above. Aliquots of antivenom, diluted 1:2 with PBS, were passed several times through the column for 20 min and then washed with PBS. Bound antibodies were eluted with 0.1 M glycine, pH 3.0, and collected in 0.5 M Tris, pH 8.8, buffer. The purified antibodies were dialyzed against water, lyophilized and dissolved in PBS for neutralization studies. The ability of these antibodies to neutralize myotoxicity of crude venom was assessed by quantifying the plasma CK activity in mice injected with either 50 mg B. asper venom or mixtures of venom and affinity-purified antibodies. For assessing the action of the various inhibitors or antibodies on venom lethality, mixtures containing venom and either PBS, inhibitors or antibodies were prepared and incubated for the times described above; in the case of antibodies, incubations were carried out at room temperature (22e25 C) for 30 min. Then, aliquots of the mixtures, containing 250 mg venom, were injected i.p. in mice, and lethality, as well as some pathological and pathophysiological alterations, were analyzed as described above. In some experiments, mixtures of two inhibitors or inhibitors and antibodies were tested for their ability to abrogate venom toxicity. In these cases, the time of death was also recorded.
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antivenom, all mice survived, whereas when using a ratio of 6.75 mg venom/mL antivenom all mice died. To compare these findings with those of mice injected with venom alone, tissue samples were collected for histological examination, and evaluation of hematocrit and hemorrhagic activity was carried out 20 min after injection of the mixtures, as described. Neutralization of hemorrhagic activity of venom by antivenom was also assessed by rrez et al., 1985). Briefly, venom was the mouse skin test (Gutie incubated with either PBS or antivenom, at venom/antivenom ratios of 1.33 or 6.75 mg venom/mL antivenom. Incubations were carried out at 37 C for 30 min. Then, 100 mL of the mixtures, containing 20 mg venom, were injected intradermally in mice (18e20 g). After 2 h animals were sacrificed by CO2 inhalation, and the hemorrhagic area in the inner side of the skin was measured. 2.6. Effect of tramadol in the estimation of the Median Effective Dose (ED50) of antivenom To evaluate whether the use of precautionary analgesia by tramadol affects the estimation of the ED50, groups of mice (16e18 g) were pretreated subcutaneously with either 100 mL of distilled water or of tramadol chlorhydrate (50 mg/kg; Laboratorio Sanderson S.A., Chile), dissolved in water; this dose was selected on the basis of previous studies in mice (Raffa et al., 1992). Fifteen min after these treatments, groups of five mice were injected i.p. with mixtures containing 250 mg venom and various dilutions of antivenom in a total volume of 0.5 mL; mixtures of venom and antivenom were prepared and incubated for 30 min at 37 C before injection. Deaths occurring during 48 h were recorded, and ED50 was estimated by probits. 2.7. Correlation between neutralization of lethality and neutralization of coagulant activity in vitro In order to search for surrogate in vitro methods that correlate with the lethality test used in the assessment of antivenom potency, samples of eight different batches of polyvalent antivenom were obtained and diluted to a different extent in order to have a broad spectrum of neutralizing potencies. For neutralization of lethality, mixtures of a fixed dose of venom and various dilutions of antivenom were prepared and incubated at 37 C for 30 min. Then, aliquots of 0.5 mL of the mixtures, containing 4 LD50s of venom, were injected into groups of six mice (16e18 g). Deaths were recorded during 48 h and the ED50 was estimated by probits. In parallel, neutralization by these antivenoms of in vitro coagulant effect of B. asper venom was studied. Mixtures of a fixed dose of venom and variable dilutions of antivenom were prepared and incubated as described. Then, aliquots of 100 mL of the mixtures, containing two Minimum Coagulant Doses of venom, were added to 200 mL of citrated plasma obtained from the blood of healthy et al., 1989). human donors, and clotting times were recorded (Gene The Minimum Coagulant Dose of venom is the dose that induces et al., coagulation of plasma in 60 s (Theakston and Reid, 1983; Gene 1989). Controls included venom incubated without antivenom. Neutralizing efficacy of antivenom was expressed as Effective Dose (ED), corresponding to the ratio mg venom/mL antivenom at which the clotting time was prolonged three times as compared to clot et al., 1989). ting time of plasma incubated with venom alone (Gene
2.5. Pathological and pathophysiological alterations in mice injected with mixtures of venom and antivenom
2.8. Statistical analyses
In order to discern whether the cause of death is similar in mice injected with venom alone or in mice receiving mixtures of venom and antivenom, solutions of two different venom: antivenom ratios were prepared. In one case, using a ratio of 1.33 mg venom/mL
For determining the significance of the difference between the mean values of two experimental groups, a Student's t test was used. When more than two groups were analyzed, mean values were compared by ANOVA, followed by Tukey test for comparing
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pairs of means. For the analysis of correlation between neutralization tests, the value of r was calculated. A p value < 0.05 was considered significant. 3. Results 3.1. Pathology and pathophysiology induced by i.p. injection of 4 LD50s of venom The potency test used to evaluate the neutralization of lethal activity in the quality control of antivenoms at Instituto Clodomiro Picado involves the injection, in 16e18 g mice, of a challenge dose of venom consisting of 4 LD50s, which corresponds to 250 mg B. asper venom when using the i.p. route. Hence, an analysis of pathological and pathophysiological effects was performed when injecting this dose in mice. The time of death was 25 ± 3 min. Macroscopic examination of the peritoneal cavity of mice showed a conspicuous hemorrhage, especially in the mesentery and in the intestines, with the presence of a hemorrhagic fluid in the cavity. Extensive hemorrhage was observed histologically in the mesentery, with abundant masses of erythrocytes in areas of adipocytes, as well as inside lymphatic vessels (Fig. 1). Abundant extravasated erythrocytes were also observed surrounding skeletal muscle fibers in the diaphragm, with the presence of scattered necrotic muscle fibers (Fig. 1). Hemorrhagic foci were observed in the walls of both small and large intestines. In contrast, no evident histological alterations were noticed in kidneys and liver, with the exception of hyperemia. Moreover, macroscopic and microscopic examination of the thoracic organs did not reveal hemorrhage, with the exception of isolated foci in the myocardium. Neither hemorrhage nor edema was observed in lungs and brain. Hyperemia was the most consistent microscopic finding in heart and lungs. In agreement with histological observations, a significant increment in the amount of hemoglobin was detected in peritoneal
lavage fluid from envenomed mice, in contrast to mice injected with PBS (Fig. 2A). In addition, envenomed mice developed a process of hemoconcentration, as revealed by an increment in the hematocrit from 39 ± 2% (control mice) to 54 ± 2% (envenomed mice) (p < 0.05) (Fig. 2B), hence indicating that, in addition to overt hemorrhage, there is loss of plasma from the circulation, which leads to hemoconcentration. In agreement, a prominent increment in vascular permeability was detected in the peritoneal cavity, as shown by the increase in the amount of Evans Blue in the peritoneal lavage fluid (Fig. 2C). Regarding the effect of venom in clotting time, blood from mice receiving PBS had clotting times of 194 ± 80 s, whereas blood collected from envenomed mice remained unclotted after 20 min of incubation, thus revealing a state of defibrinogenation. In addition, a prominent increment in plasma CK activity occurred after 20 min in mice injected with 250 mg venom, as a consequence of myonecrosis (Fig. 2D). Neither the PI SVMP BaP1 nor a fraction containing a mixture of myotoxic Asp49 PLA2 and Lys49 PLA2 homologue reproduced the increment in hematocrit, i.e. hemoconcentration, observed with the crude venom (results not shown). 3.2. Effect of inhibitors of venom components in lethality and other parameters In agreement with previous observations (Rucavado et al., 2000), incubation of B. asper venom with 300 mM CaNa2EDTA abrogated hemorrhagic activity, as judged by the intradermal mouse test. The serine proteinase inhibitor AEBSF significantly prolonged the clotting time of a fibrinogen solution to which venom was added, evidencing the inhibition of a thrombin-like rez et al., 2008). Likeserine proteinase present in this venom (Pe wise, after three hours of incubation, pBPB reduced by 80% the PLA2 activity of the venom, in agreement with inhibition of a myotoxic rrez, PLA2 isolated from B. asper venom (Díaz-Oreiro and Gutie
Fig. 1. Light micrographs of sections of mesentery (A, B) and diaphragm (C, D) from mice injected by the i.p. route with either 500 mL PBS (A, C) or 4 DL50s (250 mg) B. asper venom, dissolved in 500 mL PBS (B, D). Upon death of envenomed mice, which occurred approximately 20 min after injection, the abdominal cavity was opened and tissues were dissected out, fixed in formalin and processed for histological evaluation. Mice injected with PBS were sacrificed by an overdose of anesthetic (xylazine and ketamine) 20 min after injection. Notice prominent hemorrhage, i.e. abundant erythrocytes, in tissues from envenomed mice, whereas tissues from PBS-injected mice show a normal histological appearance. Hematoxylin and eosin staining. Bar represents 200 mm in A, and 100 mm in B, C, and D.
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Fig. 2. Effects induced by the i.p. injection of 4 LD50s of B. asper venom (250 mg in 500 mL PBS) in mice. All analyses were performed 20 min after injection of either venom or PBS. (A) Hemorrhage, quantified on the basis of absorbance at 540 nm of peritoneal lavage fluid. (B) Hematocrit. (C) Plasma extravasation, quantified on the basis of extravasation of Evans Blue in the peritoneal cavity. (D) Myonecrosis: 20 min after either venom or PBS injection, a blood sample was collected by cardiac puncture, under anesthesia, and plasma CK activity was quantified. The details of the experimental protocols are described in Materials and Methods. Results are expressed as mean ± S.D. (n ¼ 5). *p < 0.05 (Student's t test) when compared with values of PBS-injected controls.
1997). Moreover, fucoidan abrogated venom myotoxicity, as assessed by quantification of plasma CK activity (Angulo and Lomonte, 2003). Affinity-purified antibodies against a myotoxic fraction, composed of Asp49 and Lys49 myotoxic PLA2s from B. asper venom, reduced by 80% the increment of plasma CK activity induced by this venom. These inhibitors and antibodies were then used to assess the role of SVMPs, serine proteinases, PLA2s, and basic myotoxins in venom-induced lethality. Incubation of venom with CaNa2EDTA prevented death in two out of five envenomed animals; a similar result was observed when using the peptidomimetic hydroxamate metalloproteinase inhibitor Batimastat (Table 1); this inhibitor has been shown to abrogate hemorrhagic activity of B. asper venom (Rucavado et al., 2000). Moreover, in the mice that died, the time of death was significantly prolonged by CaNa2EDTA treatment, as compared to mice injected with venom alone (Fig. 3). Incubation of venom with either AEBSF, fucoidan or pBPB did not protect from lethality and the times of death did not differ significantly from mice receiving venom alone (not shown). When CaNa2EDTA was combined with either pBPB or AEBSF there was not an increment in the protection conferred by CaNa2EDTA alone (Table 1; Fig. 3). On the other hand, all mice injected with venom incubated with CaNa2EDTA and fucoidan died, showing profuse hemorrhage in the peritoneal cavity; this is likely to be the consequence of the anticoagulant activity of fucoidan, and
hampered the further use of this inhibitor in the study. When CaNa2EDTA was combined with anti-myotoxin antibodies, no further protection, in terms of survival percentage, was observed as compared to mice receiving venom and CaNa2EDTA alone (Table 1); however, the time of death was significantly prolonged when antibodies were combined with CaNa2EDTA (Fig. 3). Injections of the inhibitors or antibodies alone did not induce any evidence of toxicity in mice. On the basis of these results, it was decided to study in further detail the inhibition of venom by CaNa2EDTA. Histological examination of mesentery, small intestine, large intestine and diaphragm from samples collected 20 min after injection of venom inhibited with CaNa2EDTA showed a drastic reduction in the extent of hemorrhage, as compared to mice injected with venom alone. Only few isolated hemorrhagic foci
Table 1 Lethality induced in mice by the injection of 4 DL50s of B. asper venom either alone or preincubated with various inhibitors or antibodies. Treatment
Dead mice/injected mice
PBS Venom Venom Venom Venom Venom Venom Venom
0/5 5/5 3/5 3/5 3/5 6/6 5/6 4/5
þ þ þ þ þ þ
EDTA Batimastat EDTA þ pBPB EDTA þ fucoidan EDTA þ AEBSF EDTA þ anti-myotoxin antibodies
Fig. 3. Time of death of mice injected with either 250 mg B. asper venom or with mixtures of this dose of venom and various inhibitors or anti-myotoxin antibodies. Venom was mixed with either PBS, CaNa2EDTA or mixtures of CaNa2EDTA and other inhibitors or antibodies, as described in Materials and Methods. After injections, animals were observed and the time of death was recorded. All mice injected with venom alone died, whereas only 60e80% of mice receiving venom and inhibitors died (see Table 1). Results are expressed as mean ± S.D. (n ¼ 3e5). *p < 0.05 when compared to time of death of mice injected with venom alone.**p < 0.05 when compared to time of death of mice from the other experimental groups (ANOVA followed by Tukey test).
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were observed in the mesentery, whereas hemorrhage was largely absent in the intestines and diaphragm (Fig. 4). In agreement, the amount of hemoglobin present in peritoneal lavage fluid was significantly reduced as compared to mice receiving venom alone (Fig. 5A). Incubation with CaNa2EDTA partially reduced the increment in vascular permeability induced by venom in the peritoneal cavity (Fig. 5B) and the extent of hemoconcentration, i.e. hematocrit increment (Fig. 5C). Regarding coagulation, mice injected with venom incubated with CaNa2EDTA were partially defibrinogenated since one sample did not clot at 20 min and three additional samples developed a weak clot after 20e25 min of incubation at room temperature. Hence, CaNa2EDTA inhibited defibrinogenating activity only to a partial extent.
3.3. Pathological and pathophysiological alterations in mice injected with mixtures of venom and antivenom In order to explore the alterations occurring in mice injected with venom and antivenom, thus reproducing the circumstances of an antivenom potency assay, two venom/antivenom ratios were selected. At the ratio of 1.33 mg venom/mL antivenom, all mice survived, whereas at the ratio of 6.75 mg venom/mL antivenom all mice died, although the time of death was prolonged more than 120 min, as compared to ~20 min in mice injected with venom alone. Hemorrhagic activity, as assessed by the mouse intradermal test, revealed that hemorrhage was abrogated at the ratio 1.33 mg venom/mL antivenom, whereas the hemorrhagic area in the skin was reduced by 85% at a ratio of 6.75 mg venom/mL antivenom, although the values of skin hemorrhage in the two groups receiving
Fig. 5. Effect of CaNa2EDTA in the effects induced by B. asper venom. Venom was mixed with either PBS or 300 mM CaNa2EDTA. Then, aliquots of 500 mL of the mixtures, containing 250 mg venom, were injected i.p. in mice. Control mice received 500 mL of PBS alone. Twenty min after injection, hemorrhage, Evans Blue extravasation and hematocrit were determined as described in Materials and Methods. Results are presented as mean ± S.D. (n ¼ 5). *p < 0.05 when compared to venom alone; **p < 0.05 when compared to PBS controls (ANOVA followed by Tukey test).
Fig. 4. Light micrographs of sections of mesentery (A) and diaphragm (B) from mice injected by the i.p. route with 250 mg B. asper venom previously incubated with 300 mM CaNa2EDTA (see Materials and Methods for details), in a total volume of injection of 500 mL. Mice were sacrificed 20 min after injections, the abdominal cavity was opened and tissues were dissected out, placed in formalin solution, and routinely processed for histological analysis. Notice the absence of hemorrhagic lesions in both tissues. Hematoxylin and eosin staining. Bar represents 200 mm.
venom and antivenom were not significantly different (p > 0.05) (Fig. 6A). Histological analysis performed in samples collected 20 min after injection of venom/antivenom mixtures showed clear differences between the two groups. Mice injected with a mixture having a ratio of 1.33 mg venom/mL antivenom showed a normal histological pattern in mesentery, large intestine, small intestine and diaphragm, whereas sections of tissues from mice receiving a mixture with a ratio of 6.75 mg venom/mL antivenom revealed moderate hemorrhage in the mesentery and scant hemorrhagic foci in the intestine, together with areas of myonecrosis in the diaphragm (not shown). Likewise, the hematocrit in mice injected with the mixture at a ratio of 6.75 mg venom/mL antivenom did not differ significantly from the values of mice injected with venom alone, evidencing lack of neutralization of this effect. In contrast, hematocrit in mice receiving venom and antivenom at a ratio of 1.33 mg venom/mL antivenom was significantly different from that of mice injected with venom alone and with venom and antivenom
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Fig. 6. Neutralization of toxic effects of B. asper venom by polyvalent antivenom. Mixtures of venom and antivenom were prepared as to attain venom/antivenom ratios of 1.33 and 6.75 mg venom/mL antivenom. Controls included venom incubated with PBS instead of antivenom. After incubation, the hemorrhagic activity of the mixtures was assessed by the mouse skin test (A) (see Materials and Methods for details). Groups of mice were injected i.p. with 500 mL of the mixtures, containing 250 mg venom. After 20 min, mice were bled by cardiac puncture, under anesthesia, and the hematocrit was determined (B). Results are expressed as mean ± S.D. (n ¼ 5). *p < 0.05 when compared with the group receiving venom alone. **p < 0.05 when comparing the two antivenom-treated groups (ANOVA followed by Tukey test).
at a ratio of 6.75 mg venom/mL antivenom, being similar to that of mice injected with PBS alone (Fig. 6B). Thus, animals receiving the mixture at a ratio of 6.75 mg venom/mL antivenom, all of which died, showed a significant reduction in the hemorrhagic activity of the venom but not in hemoconcentration, hence dissociating these two pathophysiological events. 3.4. Effect of pretreatment with tramadol in the estimation of antivenom potency Two independent experiments were performed with two different samples of polyvalent antivenom obtained along the process of plasma fractionation. For each experiment, the ED50s of antivenoms against the venom of B. asper were estimated in parallel in two groups of mice, one pretreated by the s.c. route with the analgesic tramadol and another pretreated with water. ED50 values for these two groups of mice were: Antivenom sample (1): 2.5 mg venom/mL antivenom (95% confidence limits: 1.8e3.3) for mice pretreated with tramadol, and 2.2 mg venom/mL antivenom (95% confidence limits: 1.5e2.8) for mice pretreated with water. Antivenom sample (2): 1.9 mg venom/mL antivenom (95% confidence limits: 0.5e3.0) for mice pretreated with tramadol, and 2.2 mg venom/mL antivenom (95% confidence limits: 1.8e3.2) for mice
Fig. 7. Correlation between the ability of polyvalent antivenom to neutralize lethal and in vitro coagulant activities. Eight batches of antivenom were diluted to a variable extent as to have a spectrum of neutralizing potencies. Then, mixtures of a fixed dose of venom and variable dilutions of antivenoms were prepared and incubated at 37 C for 30 min. The neutralization of lethality was assessed in mice and the neutralization of coagulant activity was determined in citrated human plasma, as described in Materials and Methods. Neutralization was expressed as Median Effective Dose (ED50) for lethality and Effective Dose (ED) for coagulant effect. A significant correlation was observed between the two determinations (r ¼ 0.883, p < 0.005).
pretreated with water. Hence, there was not a significant variation in the estimation of ED50 of these samples of antivenom in mice receiving a prophylactic injection of tramadol as compared to mice pretreated only with water. 3.5. Correlation between the neutralization of lethal and in vitro coagulant activities Eight different batches of antivenom were diluted to a variable extent as to have a wide spectrum of neutralizing potencies. Then, these antivenoms were tested in parallel for the neutralization of lethal and in vitro coagulant activities. Since experiments with inhibitors evidenced a key role of SVMPs in lethality, and since in vitro coagulant activity of B. asper venom is induced predominantly by SVMPs (Loría et al., 2003; Rucavado et al., 2004), the possibility of a correlation between neutralization of lethality and neutralization of coagulant activity was considered. As shown in Fig. 7, there was a significant correlation between the neutralization of these two activities (r ¼ 0.883, p < 0.005). 4. Discussion Despite the relevance of the antivenom potency assay, which is the gold standard in the quality control of antivenoms (WHO, 2010), the pathological and pathophysiological alterations occurring in mice in the conditions in which this test is performed remain poorly known for many medically-relevant venoms. We have studied the alterations induced by the venom of B. asper in mice, using the venom dose routinely employed for the antivenom potency test in the Quality Control Laboratory of Instituto Clodomiro Picado, i.e. 250 mg in 16e18 g mice, corresponding to four LD50s. The rationale of this study is that deciphering the mechanisms underlying lethality in this model is important to understand the neutralizing profile of the polyspecific antivenom used for the treatment of envenomings by pit vipers in Central America. Results show that (a) the vast majority of pathological alterations occur in the organs of the abdomen, in agreement with the route of venom injection (i.p.) and with the rapid onset of death (~20 min); (b) the most conspicuous pathological alteration is hemorrhage in the peritoneal cavity, and especially in the mesentery, with extravasation also observed in the intestinal wall and in diaphragm; (c) mice develop a state of incoagulability which is likely to contribute to extravasation, hypovolemia and lethality; (d) there is a significant increment in vascular permeability, evidenced by the increase in the amount of Evans Blue in the peritoneal fluid; (e) there is a higher extent of plasma extravasation than of whole
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blood extravasation, thus resulting in hemoconcentration, i.e. increment in hematocrit; and (f) there are limited pathological alterations in organs outside the abdomen, such as heart, lungs and brain. These observations strongly suggest that the cause of death in this experimental model is massive extravasation in the abdominal cavity and organs, generating a fulminant cardiovascular collapse within ~20 min. These phenomena of hemoconcentration and hemorrhage, associated with increments in lactic acid secondary to ischemia, were previously described in a rat model of systemic envenoming by Crotalus atrox venom (Carlson et al., 1975). Systemic hemorrhage and plasma extravasation leading to cardiovascular shock are some of the main clinical manifestations in severe envenomings by B. asper and other viperid species (Warrell, ~ o, 2009). 2004; Otero-Patin The identity of the venom components responsible for the observed effects and lethality was assessed using inhibitors of specific venom enzymes and antibodies against basic myotoxins. The most abundant components of B. asper venom are SVMPs, PLA2s, including Asp49 PLA2s and Lys49 PLA2 homologues, and serine n et al., 2008). In proteinases, as analyzed by proteomics (Alape-Giro addition, studies on the experimental pathology and pathophysiology of envenomings by B. asper have shown that SVMPs are responsible for local and systemic hemorrhage, coagulopathy and inflammation, whereas PLA2s and PLA2 homologues induce myotoxicity, damage to lymphatic vessels and inflammation, and serine proteinases provoke clotting disturbances and increments in rrez et al., 2009a, 2009b; Teixeira et al., vascular permeability (Gutie 2003, 2009). Hence, there is agreement between the predominant effects in the pathophysiology of envenoming and the relative abundance of SVMPs, PLA2s and serine proteinases in the venom. Incubation of venom with the chelating agent CaNa2EDTA, or with the synthetic peptidomimetic hydroxamate metalloproteinase inhibitor Batimastat, resulted in the survival of approximately 40% of mice injected, and in the prolongation of the time of death of mice that did not survive, thus underscoring the relevance of SVMPs in venom-induced lethality. A previous study demonstrated the role of SVMPs in the systemic alterations, including lethality, induced by B. asper venom (Rucavado et al., 2004). Likewise, SVMPs play a key role in the systemic effects induced by Bothrops jararaca venom (Yamashita et al., 2014). Our observations indicate that CaNa2EDTA drastically reduced peritoneal hemorrhage and partially, but not completely, inhibited the increase in vascular permeability and hemoconcentration. In contrast, neither PLA2 nor serine proteinase inhibitors had an effect on venom lethality, indicating that these enzymes do not play a key role in this effect. This was further confirmed by experiments in which these inhibitors were combined with CaNa2EDTA; in these circumstances, no improvement of the inhibitory effect of CaNa2EDTA alone was observed in terms of survival and time of death. The role of basic myotoxic PLA2s and PLA2 homologues, which n et al., comprise approximately 40% of the venom (Alape-Giro 2008) and exert a significant pro-inflammatory activity in addition to myonecrosis (Teixeira et al., 2003), was initially assessed with the use of fucoidan, an inhibitor of these myotoxins. However, owing to the relatively high dose of fucoidan needed to inhibit the amount of myotoxins present in 250 mg of venom, the anticoagulant effect of this carbohydrate provoked widespread hemorrhage which contributed to venom toxicity. To circumvent this problem, we purified antibodies from the antivenom against a mixture of myotoxic Asp49 PLA2s and Lys49 PLAs homologues from this venom. Despite being effective in the neutralization of myotoxicity induced by the venom, the combination of these antibodies and CaNa2EDTA did not increase the protection conferred by CaNa2EDTA in terms of percentage of surviving mice. However, addition of anti-myotoxin antibodies to venom-EDTA mixtures prolonged
the time of death as compared to mice injected with venom and EDTA alone, hence suggesting that basic myotoxic components contribute, albeit to a minor extent, to the pathophysiological alterations that precipitate death in this model. If SVMPs are not the only culprit of B. asper venom-induced lethality, and if PLA2s, PLA2 homologues and serine proteinases do not seem to be responsible for the SVMP-independent toxicity leading to death, which components are then involved in this ‘residual’ lethal activity once these major venom toxins are inhibited? Minor components in this venom, identified by proteomic analysis, include L-amino acid oxidase, cysteine-rich secretory proteins n (CRISPs), C-type lectin-like proteins and disintegrins (Alape-Giro et al., 2008). However, on the basis of the known pharmacological activities of these proteins, as well as their low concentration in the venom, it is highly unlikely that they play a significant role in lethality. Two possible explanations for our findings are: (a) Even in conditions where SVMPs, PLA2s and serine proteinases enzymatic activities are largely inhibited, these abundant components may still stimulate resident cells, especially macrophages, of the peritoneal cavity, by catalytically-independent mechanisms, to generate inflammatory mediators which induce increment in vascular permeability and other systemic alterations; (b) the toxicity that remains after SVMPs and myotoxic PLA2 inhibition might be due to a synergistic effect of various venom components which, by themselves, are not sufficiently toxic but, when combined in the venom, increase their toxicity. The validity of these hypotheses and the nature of these components remain to be investigated. The observation that venom inhibited with CaNa2EDTA is devoid of hemorrhagic activity, but is still able to induce an increment in extravasation of Evans Blue, strongly suggests that the remaining effect responsible for lethality, once SVMPs are inhibited, is associated with increment in vascular permeability resulting in plasma exudation, which might lead to cardiovascular collapse. Experiments with antivenom support this view. When a high dose of antivenom was used (1.33 mg venom/mL antivenom), not only hemorrhage, but also hemoconcentration were abrogated, and all mice survived. In contrast, when using a ratio of 6.75 mg venom/mL antivenom, in which all mice died, hemorrhage was largely reduced, but plasma extravasation leading to hemoconcentration remained, hence explaining lethality secondary to plasma loss. In these mice, the time of death was prolonged to more than 120 min, as compared with venom alone (~20 min), probably reflecting the time span required to generate enough plasma extravasation to cause a significant hypovolemia. Hence, the combination of overt hemorrhage and plasma exudation due to increment in vascular permeability are likely to be the main culprits of mouse lethality in this assay. Our study also aimed at finding alternatives to the use of mice in this test and to reduce the animal suffering involved. We first explored whether the use of the analgesic tramadol can be introduced in the antivenom potency test. Observations indicate that a dose of tramadol of 50 mg/kg, administered 15 min before injection of venom and antivenom mixtures, does not affect the outcome of the antivenom potency test. Although the analgesic effect of this drug lasts for only 2e3 h (Ide et al., 2006), this intervention would contribute to reduce the acute distress and pain generated during the first hours of this test. On the other hand, in the search for surrogate in vitro assays for testing neutralization of antivenom, we took into consideration the fact that high molecular mass P-III SVMPs are the most potent hemorrhagic components in viperid rrez venoms, including B. asper venom (Borkow et al., 1993; Gutie et al., 1995, 2005; Franceschi et al., 2000), and that another P-III SVMP, the prothrombin activator basparin A, is the most important procoagulant component of this venom (Loría et al., 2003; Rucavado et al., 2004). Since SVMP-induced extravasation seems
n et al. / Toxicon 93 (2015) 41e50 F. Chaco
to be the main factor behind the lethal effect of this venom, it was hypothesized that antibodies against hemorrhagic P-III SVMP would be also effective against this procoagulant enzyme, and that a parallel immune response against these two types of P-III SVMPs develops in immunized horses. On this basis it was tested whether neutralization of the coagulant activity of this venom in vitro may be a useful surrogate test for the lethality assay in vivo. Our observations support this hypothesis, since there was a significant correlation between neutralization of lethality and neutralization of coagulant effect on human plasma in vitro. This opens the possibility of using this assay for following the development of antivenom immune response in horses immunized with B. asper venom, and for assessing antivenom potency during the fractionation of hyperimmune plasma, leaving the mouse test only for the last stages of antivenom production and for the quality control of the final product. This would represent a significant reduction in the number of mice used in the manufacture of antivenom. Our findings agree with those of Pommuttakun and Ranatabanangkoon (2014) and Oguiura et al. (2014) who described the usefulness of the neutralization of coagulant effect to assess potency for antivenoms against the venoms of the viperids Calloselasma rhodostoma and B. jararaca, respectively. However, the observed correlation between neutralization of in vitro coagulant and lethal activities described herein may not occur in the cases of other venoms and antivenoms, and such correlation has to be assessed on a case by case basis. In conclusion, the main effects which determine lethality in mice injected with four LD50s of B. asper venom by the i.p. route are peritoneal hemorrhage and increment in vascular permeability, together with coagulopathy. These effects provoke a rapid hypovolemia and cardiovascular collapse. SVMPs are the main toxins responsible for lethality; in addition, other unidentified venom components also contribute to plasma extravasation and hypovolemia, and are responsible for lethality in conditions where SVMPs are inhibited. Thus, lethality in this assay is the consequence of the combined action of SVMPs and other as yet unidentified components, probably acting in a synergistic fashion. The prophylactic use of the analgesic tramadol does not affect the outcome of the antivenom potency assay when using B. asper venom and polyspecific antivenom, hence constituting an alternative to reduce the pain and distress of animals in this test. Finally, there is a significant correlation between the neutralization of lethality and of coagulant activity of B. asper venom in vitro. This opens the possibility of using this alternative test to assess the development of antivenom antibodies in horses and to follow antivenom titers during the fractionation of horse plasma, thus contributing to a significant reduction in the number of mice used for in-process antivenom quality control. Ethical statement The protocols used in this study followed ethical guidelines in the use of laboratory animals and in the performance of scientific research. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors thank Bruno Lomonte for providing fucoidan, and Daniela Solano for collaboration in laboratory work. The provision of several batches of antivenom by the Industrial Division of Instituto Clodomiro Picado is greatly acknowledged. This study was
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n, supported by FEES-CONARE and Vicerrectoría de Investigacio n received Universidad de Costa Rica (project 741-B2-652). F. Chaco n for this investigation. support from Vicerrectoría de Investigacio Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.11.223. References n, A., Sanz, L., Escolano, J., Flores-Díaz, M., Madrigal, M., Sasa, M., Alape-Giro Calvete, J.J., 2008. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J. Proteome Res. 7, 3556e3571. rrez, J.M., 1997. Clinical and laboratory alterations in Angulo, Y., Estrada, R., Gutie horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom. Toxicon 35, 81e90. Angulo, Y., Lomonte, B., 2003. Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A2. Biochem. Pharmacol. 66, 1993e2000. ~ os, R., Cerdas, L., 1980. Produccio n y control de sueros antiofídicos en Costa Bolan Rica. Bol. Oficina Sanit. Panam. 88, 189e196. rrez, J.M., Ovadia, M., 1993. Isolation and characterization of Borkow, G., Gutie synergistic hemorrhagins from the venom of the snake Bothrops asper. Toxicon 31, 1137e1150. Carlson, R.W., Schaeffer, R.C., Whigham, H., Michaels, S., Russell, F.E., Weil, M.H., 1975. Rattlesnake venom shock in the rat: development of a method. Am. J. Physiol. 229, 1668e1674. rrez, J.M., 1997. Chemical modification of histidine and lysine Díaz-Oreiro, C., Gutie residues of myotoxic phospholipases A2 isolated from Bothrops asper and Bothrops godmani snake venoms: effects on enzymatic and pharmacological properties. Toxicon 35, 241e252. rrez, J.M., 2000. Purification and charFranceschi, A., Rucavado, A., Mora, N., Gutie acterization of BaH4, a hemorrhagic metalloproteinase from the venom of the snake Bothrops asper. Toxicon 38, 63e77. , J.A., Roy, A., Rojas, G., Gutie rrez, J.M., Cerdas, L., 1989. Comparative study on Gene coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841e848. rrez, J.M., 2010. Snakebite envenomation in Central America. In: Mackessy, S.P. Gutie (Ed.), Handbook of Venoms and Toxins of Reptiles. CRC Press, Florida, pp. 491e507. rrez, J.M., Arroyo, O., Bolan ~ os, R., 1980. Mionecrosis, hemorragia y edema Gutie n blanco. Toxicon 18, inducidos por el veneno de Bothrops asper en rato 603e610. rrez, J.M., Escalante, T., Rucavado, A., 2009b. Experimental pathophysiology of Gutie systemic alterations induced by Bothrops asper snake venom. Toxicon 54, 976e987. rrez, J.M., Gene , J.A., Rojas, G., Cerdas, L., 1985. Neutralization of proteolytic Gutie and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23, 887e892. rrez, J.M., Romero, M., Díaz, C., Borkow, G., Ovadia, M., 1995. Isolation and Gutie characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo). Toxicon 33, 19e29. rrez, J.M., Rucavado, A., Chaves, F., Díaz, C., Escalante, T., 2009a. Experimental Gutie pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 54, 958e975. rrez, J.M., Rucavado, A., Escalante, T., Díaz, C., 2005. Hemorrhage induced by Gutie snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45, 997e1011. Villalta, M., Vargas, M., rrez, J.M., Solano, G., Pla, D., Herrera, M., Segura, A., Gutie n, G., 2013. Assessing the preclinical effiSanz, L., Lomonte, B., Calvete, J.J., Leo cacy of antivenoms: from the lethality neutralization assay to antivenomics. Toxicon 69, 168e179. Ide, S., Minami, M., Ishihara, K., Uhl, G.R., Sora, I., Ikeda, K., 2006. m opioid receptordependent and independent components in effects of tramadol. Neuropharmacology 51, 651e658. rrez, J.M., 1989. A new muscle damaging toxin, myotoxin II, from Lomonte, B., Gutie the venom of the snake Bothrops asper (terciopelo). Toxicon 27, 725e733. Loría, G.D., Rucavado, A., Kamiguti, A.S., Theakston, R.D.G., Fox, J.W., Alape, A., rrez, J.M., 2003. Characterization of ‘basparin A’, a prothrombin-activating Gutie metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis. Arch. Biochem. Biophys. 418, 13e24. Oguiura, N., Kapronezai, J., Ribeiro, T., Rocha, M.M., Medeiros, C.R., Marcelino, J.R., Prezoto, B.C., 2014. An alternative micromethod to assess the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom. Toxicon 90, 148e154. ~ o, R., 2009. Epidemiological, clinical and therapeutic aspects of Bothrops Otero-Patin asper bites. Toxicon 54, 998e1011.
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