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Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis
International Journal of Biological Macromolecules 95 (2017) 263–267
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis Anna L. Jacob-Ferreira ∗ , Danilo L. Menaldo, Marco A. Sartim, Thalita B. Riul, Marcelo Dias-Baruffi, Suely V. Sampaio ∗ Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil
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Article history: Received 20 September 2016 Received in revised form 16 November 2016 Accepted 17 November 2016 Available online 19 November 2016 Keywords: Batroxase Snake venom Metalloproteases Antithrombotic agents Venous thrombosis
a b s t r a c t Background: Snake venoms are great sources of bioactive molecules, which may be used as models for new drugs. Toxins that interfere in hemostasis have received considerable attention over the years. Objectives: This study aimed at the evaluation of the antithrombotic activity of Batroxase, a P-I metalloprotease from Bothrops atrox venom, in an animal model of venous thrombosis. Methods: The antithrombotic activity of Batroxase was tested in vivo in a model based on two factors of the Virchow’s Triad: blood flow alterations (partial stenosis of the inferior vena cava), and vessel wall injury (10% ferric chloride for 5 min), in comparison with sodium heparin (positive control) and saline (negative control). Bleeding/clotting time was assessed by a tail bleeding assay. The immunogenicity of Batroxase was also analyzed. Results: Batroxase (12 mg/kg) reduced thrombus formation in 81%, similarly to heparin (100 U/kg), which reduced it in 85% in comparison with the saline group. Both Batroxase and heparin increased bleeding/clotting time in approximately 3 fold. Immunizations of rabbits with Batroxase do not result in detectable levels of antibodies against this metalloprotease. Conclusion: Batroxase presents antithrombotic activity in vivo. Moreover, its lack of immunogenicity increases the interest on its possible therapeutic potential over thrombogenic disorders. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The approach on toxinology research involves the study on toxins from plants, animals and microbial sources, in order to understand their characteristics, metabolism, and functions [1]. These investigation efforts intend not only to prevent and treat their effects on envenomation, but also their actions on different scenarios, e.g. the activation or inhibition of the hemostatic system [2–5]. Bothrops snake venom metalloproteases (SVMPs) are the main class of toxins responsible for triggering hemostasis events, inducing blood coagulation disorders, and leading to hemorrhage, as they are capable of degrading proteins of vessel membranes
∗ Corresponding authors at: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universio dade de São Paulo, FCFRP-USP, Av. do Café, s/n , CEP 14040-903, Ribeirão Preto-SP, Brazil. E-mail addresses: jacob [email protected] (A.L. Jacob-Ferreira), [email protected] (S.V. Sampaio). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.063 0141-8130/© 2016 Elsevier B.V. All rights reserved.
allowing blood extravasation, or acting on coagulation factors activators [6–10]. Another feature involving some SVMPs concerns the capacity to act directly on fibrin, degrading clots and preventing the formation of new clots. The therapeutic potential of fibrin(ogen)olytic SVMPs is recently being explored for the treatment of patients with cardio- and cerebrovascular disorders, as described for Fibrolase and its recombinant analog Alfimeprase [11,12]. Our research group purified and biochemically characterized a neutral (pI 7.5) P-I class (∼25 kDa) metalloprotease from Bothrops atrox snake venom: Batroxase, which is capable of degrading components of the extracellular matrix, such as type IV collagen and fibronectin, as well as components of the coagulation cascade, as fibrinogen and fibrin [13]. This enzyme had its fibrin(ogen)olytic activity inhibited by ␣2-macroglobulin [14]. Most importantly, Batroxase presented a dose-dependent in vivo thrombolytic activity, similar to the clinically relevant drug Alteplase (tissue-type plasminogen activator), without, however, affecting bleeding/clotting time of the studied animals [14]. The inactivation of the metalloprotease by ␣2-macroglobulin may reduce its
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activity, but also its potential side effects, as seen for bleeding time [14]. In order to continue the exploitation of Batroxase’s therapeutic potential for the future development and production of new drugs with effects over hemostasis, the present study aimed at assessing its antithrombotic activity and also its immunogenicity. 2. Methods The equipment and other materials were described in the course of the methodology, and unspecified reagents used were of analytical grade. 2.1. Isolation of Batroxase from Bothrops atrox venom Batroxase was obtained through chromatographic fractionation of Bothrops atrox venom, acquired from Centre of Extraction of Animal Toxins (Morungaba-SP, Brazil), as previously described [14,15]. Briefly, two consecutive chromatographic steps on resins obtained from GE Healthcare (Chicago, IL, USA) were performed. First a size exclusion chromatography on Sephacryl S-200, followed by anion exchange chromatography on DEAE Sepharose. Latter, the fraction containing the metalloprotease was ultrafiltered in concentrator tube with polyethersulfone membrane with cut-off of 3000MWCO, Vivaspin® 20 (Sartorius, Goettingen, Germany), and the isolated protein was quantified using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer instructions, separated in aliquots of 1 mg/tube, lyophilized and stored at −20 ◦ C until its use in the experiments. 2.2. Animals Male Wistar rats (250–270 g) and adult female New Zealand White rabbits were obtained from the Central Animal Facility of USP (Ribeirão Preto-SP), and maintained under controlled conditions of temperature (24 ◦ C) and brightness (12 h light/dark cycles), with free access to food and water. In the previous day from the venous thrombosis experiments, rats had their food removed overnight, to allow the emptying of their intestine and facilitate its handling during the surgical procedure. All experiments involving animals were performed according to the Brazilian College of Animal Experimentation (COBEA) guidelines and experimental protocols were approved by the Ethics Committee on Animal Use of Ribeirão Preto campus, University of São Paulo, protocol number 12.1.1809.53.2. 2.3. Assessment of the antithrombotic activity of Batroxase on venous thrombosis In order to analyze the antithrombotic activity of Batroxase in comparison to the antithrombotic drug of clinical use (sodium heparin, Hepamax-S® , Blausiegel Ltd, Brazil), a model of partial blood stasis and vascular damage of the inferior vena cava was used [16]. Wistar rats (250–270 g, N = 3–5/group) were anesthetized with an association of ketamine (80 mg/kg) and xylazine (10 mg/kg) IP, and laid down in supine position. The abdomen was opened by incision along the linea alba toward the sternum, followed by the inferior vena cava exposure. The partial stasis was induced by tying a cotton thread just below the junction of the vena cava with the left renal vein. A blind 21G needle was placed between the node and the inferior vena cava, which was then removed to allow partial blood flow and patterned formation of partial stasis. The tested drug was injected through the left femoral vein of the animal over 2 min before the induction of the thrombus, which was done by a filter paper (5 × 5 mm) saturated with 10% ferric chloride solution (10 L), which remained in contact with the vein for 5 min, causing vascular damage and enabling the induction of the thrombus
Fig. 1. Schematic protocols. Panel A: Antithrombotic test in vivo. It was first performed the surgery for cannulation of the left femoral vein, isolation and partial stenosis of the inferior vena cava (approximately 30 min). Then, stimuli (saline, heparin or Batroxase) were injected through the left femoral vein, during the 2 min that precede the red thrombus induction by a piece of filter paper saturated with ferric chloride solution at 10% placed in contact with the vascular wall for 5 min. After, a period of 30 min was waited in order to allow the formation/stability of thrombus. During this period of wait, the bleeding/clotting time of the animals was accompanied for up to 30 min. Panel B: Evaluation of Batroxase effects over biochemical analysis of rats’ blood. For the biochemical analysis of the rats’ blood, animals were anesthetized, and stimuli (saline, heparin or Batroxase) were injected through their tail vein over a 2 min period, as for the antithrombotic study protocol. Another 30 min was waited before cardiac puncture, in order to mimic the bleeding time evaluation period. Panel C: Rabbits immunization protocol: Immunogenicity of Batroxase was tested in rabbits. Blood samples were collected before the immunization protocol starts (pre). In the first day, Batroxase (BTX) emulsified with complete Freund adjuvant (CFA) was subcutaneously injected in the neck region of the rabbits. The immunization was repeated every 15 days for 3 more times, using incomplete Freund’s adjuvant (IFA) instead of CFA. After 5 days of the fourth immunization, another blood sample was collected through the ear vein for ELISA analyses, and 5 days later, animals were anesthetized for complete blood collection by cardiac puncture.
(Fig. 1A). After the 5 min of induction with ferric chloride, a 30 min period was waited in order to allow the formation of the red thrombus. During this period, the bleeding/clotting time of the animals was assessed. Different doses of sodium heparin (40, 90, 100 and 120 U/kg) and a dose of Batroxase (12 mg/kg) were evaluated and compared with a saline group. The Batroxase dose was chosen based on the dose that had previously shown thrombolytic activity in this venous thrombosis model [14]. 2.4. Evaluation of Batroxase changes over bleeding/clotting time of animals For the analysis of the bleeding/coagulation time [17], after the drug administration, the tail of anesthetized rats was heated in water (40 ◦ C) for 1 min, and after drying, a small cut at the tip of the tail was performed with the use of a razor (Laser® , Laser Shaving Ltd, United Kingdom). Bleeding time starts when the first blood drop touches the filter paper, and it was recorded at 30 s intervals, accompanied until bleeding stopped or to a maximum of 30 min. After the end of the experiments, the inferior vena cava was excised for immediately weighing of the thrombus formed (wet thrombus). A thrombus of the saline group, fixed in 10% formol, was prepared and stained using hematoxylin and eosin for the histological analyses of the venous thrombus characteristics. 2.5. Evaluation of Batroxase effects over biochemical analysis of rats’ blood For the biochemical analysis of the rats’ blood, animals (N = 4/group) were anesthetized and drugs (saline, heparin 100 U/kg and Batroxase 12 mg/kg) were injected through their tail vein over a 2 min period, as for the antithrombotic study protocol. Another 30 min period was waited before cardiac puncture, to mimic the thrombus formation/bleeding time evaluation period.
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This new protocol (Fig. 1B) was performed in order to reduce the possible alterations caused by the venous thrombosis surgery, e.g. inflammatory reactions, which could disturb the results of the blood alterations caused by drugs only. Blood was collected in appropriate anticoagulants (EDTA or sodium citrate), and taken to the Clinical Analysis Service Facility of the School of Pharmaceutical Sciences of Ribeirão Preto, USP, for the analysis of prothrombin time, activated partial thromboplastin time, fibrinogen concentration and hemogram (complete blood count). 2.6. Batroxase’s immunogenicity The immunogenicity of the isolated Batroxase was tested in 2 adult female New Zealand White rabbits (Fig. 1C). Blood samples were collected through the ear vein to obtain pre-immune serum (negative control). Batroxase (200 g) diluted in 1 mL of sterile PBS and emulsified with 1 mL of complete Freund adjuvant (CFA, Difco Laboratories Inc., Detroit, MI, USA) was injected into 4 sites subcutaneously in the neck region of the rabbits. The immunization was repeated every 15 days for 3 more times, using incomplete Freund’s adjuvant (IFA) instead of CFA. After 5 days of the fourth immunization, another blood sample was collected through the ear vein for analyses, and 5 days later, animals were anesthetized for complete blood collection by cardiac puncture. Serum was obtained by centrifugation at 1500 g for 10 min, and assayed for the presence of Batroxase’s antibodies by enzyme-linked immunosorbent assay (ELISA). The immunogenicity of Batroxase was monitored by ELISA. Initially, 50 L of Batroxase dissolved in PBS (20 g/mL) was loaded into wells (1 g/well) of a 96 wells plate, and incubated overnight at 4 ◦ C for antigen immobilization. Sequentially, each well was loaded with blocking buffer (3% gelatin, 0.5% Tween 20 in PBS, 200 L/well) and incubated for 2 h at 37 ◦ C. Then, samples of preimmune- or immune-sera (50 L/well), diluted 1:1000 in PBS, were loaded to the plate and incubated for 1 h at 37 ◦ C. After, wells were loaded with 50 L of peroxidase-conjugated Affinipure goat anti-rabbit IgG antibody (Jackson ImmunoReseach, West Grove, PA, USA) diluted 1:5000 in PBS containing 1% gelatin and 0.05% Tween 20, followed by incubation for 1 h at 37 ◦ C. All of the above steps were followed by washing each well with 200 L PBS containing 0.1% Tween. Sequentially, TMB solution (tetramethylbenzidine, Organon Teknika, Boxtel, Netherlands) in the presence of hydrogen peroxide was loaded to the plate. Finally, after 10 min, this reaction was stopped by adding 2 N sulfuric acid solution (50 L/well), and the absorbance at 450 nm was read in a spectrophotometer (Spectramax Plus – Molecular Devices). 2.7. Statistical analyses The results were expressed as means ± SEM. The comparisons between groups were assessed by one-way analysis of variance followed by Dunnett’s multiple comparison tests. A probability value <0.05 was considered significant.
Fig. 2. Antithrombotic activity of Batroxase on venous thrombosis. Stimuli (saline, heparin or Batroxase) were injected during 2 min, before the induction of the formation of thrombus by a 10% solution of ferric chloride, and it was waited a 30 min period for the formation/stabilization of the thrombus. At the end of the experiment, the inferior vena cava was excised and the thrombus present was isolated and weighed for analysis of antithrombotic activity of Batroxase. Saline (negative control), heparin in different doses from 10 to 120 U/kg (positive control), Batroxase (BTX) at 12 mg/kg n = 3–5/group, * p < 0.05 vs. saline.
Fig. 3. Bleeding/clotting time of Batroxase-treated animals. The analysis of the bleeding/clotting time was performed in the same anesthetized animals used for the analysis of antithrombotic activity in vivo. After administration of saline, heparin or Batroxase, a small cut at the tip of the tail was made using a razor. Bleeding time starts when the first blood drop touches the filter paper, and was recorded at 30 s intervals, until bleeding stopped or to a maximum of 30 min. Saline (negative control), Heparin in different doses from 10 to 120 U/kg (positive control), Batroxase (BTX) at 12 mg/kg n = 3–5/group, * p < 0.05 vs. saline.
there was no thrombus formation (higher doses are not shown in figures). 3.2. Batroxase changes over bleeding/clotting time of animals Both Batroxase and heparin in different doses caused an increase of approximately 3 times in the bleeding/clotting time of rats, in comparison with the saline group (Fig. 3).
3. Results 3.3. Batroxase effects over biochemical analysis of rats’ blood 3.1. Antithrombotic activity of Batroxase on venous thrombosis The antithrombotic activity of Batroxase (12 mg/kg) was similar to the result obtained with heparin (100 U/kg), a clinically relevant anticoagulant, with a reduction of 81 and 85% of the weight of the thrombus formed, respectively (Fig. 2). Heparin antithrombotic activity was dose-dependent, and with doses higher than 120 U/kg
Biochemical analyses such as hemogram (complete blood count), prothrombin time, activated partial thromboplastin time and fibrinogen concentration were done on the blood of rats treated with saline, Batroxase (12 mg/kg) and heparin (100 U/kg) (N = 4/group), and there were no significant differences between groups (data not shown).
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Fig. 4. Histological analyses of the venous (red) thrombus. A thrombus of the saline group stained with hematoxylin and eosin. E: erythrocytes; F: fibrin; V: Vena Cava’s wall; I: vessel injury; A: Aorta’s wall.
3.4. Batroxase’s immunogenicity Batroxase was considered non immunogenic, as after the scheme of 4 immunization cycles, there were no differences in the pre- and post-immune serum of the rabbits tested, analyzed by ELISA (data not shown). 4. Discussion The present study shows the in vivo antithrombotic activity of Batroxase, a metalloprotease isolated from Bothrops atrox snake venom, in a model of venous thrombosis. Thrombosis is a failure of hemostasis, a process regulated by the vessel wall, platelets and coagulation factors, keeping the blood in a liquid state, free of clots, and inducing rapid formation of a plug when there is a vascular injury [18]. Venous thromboembolism, which includes deep vein thrombosis and pulmonary embolism, is a major cause of morbidity and mortality worldwide, being the third most common cardiovascular disorder after coronary artery disease and cerebrovascular disease [19]. The experimental model of venous thrombosis used in this study is characterized by two of the three factors of Virchow’s triad: endothelial injury induced by ferric chloride, and disturbance of the blood flow due to the partial stasis [16]. This model is most similar to clinical conditions, since it is believed that the rupture of the vascular wall is essential for triggering the hemostasis process [16]. Also, it is more reproducible inter/intra laboratory than the use of hypercoagulability, as the activity of thromboplastin from different sources can considerably vary, reflecting in differences of the necessary dose for this experimental model of venous thrombosis [16]. As observed in our results (Fig. 4), venous thrombi are rich in fibrin and poor in platelets, and are also called red thrombi, due to the high incorporation of red blood cells; and they may be prevented by the use of antithrombotic/anticoagulant drugs [20]. Snake venom metalloproteases have received considerable attention in recent years as they may exert various biological activities, which can interfere in cardiovascular diseases, such as proteolytic, fibrin(ogen)olytic, apoptotic, prothrombin activation, factor X activation, alterations on platelets aggregation, and coagulant activity [18,21–24]. Batroxase, a metalloprotease isolated from Bothrops atrox venom, was recently purified and biochemically characterized in our laboratory, and presented antithrombotic and thrombolytic activities in vitro, as it was capable of degrading components of the coagulation cascade, such as fibrinogen and fibrin [13]. In this work, we demonstrated that this toxin also prevents thrombi formation in vivo, in an experimental animal model of venous thrombosis. Batroxase (12 mg/kg) presented antithrombotic activity similar to the clinically relevant drug, heparin (100 U/kg), with reduction of 81 and 85% of the thrombus weight, respectively. This antithrombotic activity may be due to Batroxase’s capacity of cleaving fibrinogen in a form that would not allow fibrin to polymerize
[13]. The dose of Batroxase tested in this work (12 mg/kg) was based in our previous study, which showed that Batroxase presented thrombolytic activity in vivo, in this same venous thrombosis model [14]. In this study, we observed an increase in the tail bleeding/clotting time of rats, both with heparin and Batroxase, during the antithrombotic study protocol. This increase was not observed for Batroxase in our previous work. We have previously shown that Batroxase was inhibited by ␣2-macroglobulin, a plasmatic molecule capable of inhibiting different proteases by the venous fly trap hypothesis, as it forms a complex with the protease and reduces its access to macromolecular substrates [14,25]. The bleeding time alterations observed in this work may be due to the higher plasmatic concentration of Batroxase resulting of the antithrombotic protocol, as the drug was intravenously administrated during only 2 min, in comparison to the 30 min administration of the thrombolytic protocol. Thus, the elevated concentration of Batroxase may have exceeded the inhibition capacity of ␣2-macroglobulin, which was not able to reduce its possible hemorrhagic collateral effect. Interestingly, no significant differences were found on biochemical analyses of the rats’ blood. Venous thromboembolism is associated with high risk of recurrence after a first event, and on cessation of anticoagulant therapy [19]. As our goal is to explore the therapeutic potential of the toxin for its use as a model for future development and production of new drugs, we evaluated Batroxase’s immunogenicity. Remarkably, Batroxase did not cause immunogenicity in the studied animals. This characteristic increases the interest in the clinical use of Batroxase. 5. Conclusion This study shows the in vivo antithrombotic activity of Batroxase in a model of venous thrombosis, which increases our interest in its potential as a biological model for drugs that may interfere in hemostasis. Its lack of immunogenicity increases the interest on its possible therapeutic potential over thrombogenic disorders. Competing interests The authors declare that there are no competing interests. Ethical statement The authors declare they have followed the ethical requirements for this publication. Authors’ contributions ALJF conceived and accomplishment this study, and wrote the manuscript. DLM, MAS and TBR assisted in the biochemical and functional experiments, while MDB and SVS supervised and critically discussed the study. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank the financial support provided by the São Paulo Research Foundation (FAPESP, grants 2012/215699 and 2011/23236-4) and the National Council for Scientific and Technological Development (CNPq, grant 476932/2012-2 and 487351/2012-6). The funding agencies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. We are also grateful to Clinical Analysis Service facil-
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ity of the School of Pharmaceutical Sciences of Ribeirão Preto, USP, for the biochemical analysis of the animals’ blood; Prof. Dr. Karina Fittipaldi Bombonato Prado and Mr. Dimitrius Leonardo Pitol of the School of Dentistry of Ribeirão Preto, USP, for the histological analyses of the venous thrombus; and Mr. Rubens Eduardo da Silva of the School of Pharmaceutical Sciences of Ribeirão Preto, USP, for their support in the immunogenicity assay. References [1] M.E. Lima, A.M.C. Pimenta, M.F. Martin-Eauclaire, R.B. Zingali, H. Rochat, Animal Toxins: State of the Art –perspectives in Health and Biotechnology, UFMG, Belo Horizonte, 2009 (p. 750). [2] S. Braud, C. Bon, A. Wisner, Snake venom acting on hemostasis, Biochimie 82 (2000) 851–859. [3] C.T. Cologna, S. Marcussi, J.R. Giglio, A.M. Soares, E.C. Arantes, Tityus serrulatus scorpion venom and toxins: an overview, Protein Pept. Lett. 16 (2009) 920–932. [4] R.S. Rodrigues, L.F. Izidoro, S.V. de Oliveira Jr, A.M. Sampaio, V.M. Soares, Snake venom phospholipases A2: a new class of antitumor agents, Protein Pept. Lett. 16 (2009) 894–898. [5] L.R. Ayres, A.R. Récio, S.M. Burin, J.C. Pereira, A.C. Martins, S.V. Sampaio, F.A. Castro, L.S. Pereira-Crott, Bothrops snake venoms and their isolated toxins, and L-amino acid oxidase and a serine protease, modulate human complement system pathways, J. Venom. Anim. Toxins Incl. Trop. Dis. 21 (2015) 29. [6] J.W. Fox, S.M. Serrano, Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases, Toxicon 45 (2005) 969–985. [7] J.O. Costa, C.B. Petric, A. Hamaguchi, M.I. Homsi-Brandeburgo, C.Z. Oliveira, A.M. Soares, F. Oliveira, Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom, J. Venom. Anim. Toxins Incl. Trop. Dis. 13 (2007) 640–654. [8] M. Suntravat, M. Yusuksawad, A. Sereemaspun, J.C. Pérez, I. Nuchprayoon, Effect of purified Russell’s viper venom-factor X activator (RVV-X) on renal hemodynamics, renal functions, and coagulopathy in rats, Toxicon 8 (2011) 230–238. [9] K.M. Yamashita, A.F. Alves, K.C. Barbaro, M.L. Santoro, Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation, PLoS Negl. Trop. Dis. 8 (2014) e2814. [10] J.M. Gutiérrez, T. Escalante, A. Rucavado, C. Herrera, Hemorrhage caused by snake venom metalloproteinases: a journey of discovery and understanding, Toxins (Basel) 8 (2016) 93.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis Anna L. Jacob-Ferreira ∗ , Danilo L. Menaldo, Marco A. Sartim, Thalita B. Riul, Marcelo Dias-Baruffi, Suely V. Sampaio ∗ Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 20 September 2016 Received in revised form 16 November 2016 Accepted 17 November 2016 Available online 19 November 2016 Keywords: Batroxase Snake venom Metalloproteases Antithrombotic agents Venous thrombosis
a b s t r a c t Background: Snake venoms are great sources of bioactive molecules, which may be used as models for new drugs. Toxins that interfere in hemostasis have received considerable attention over the years. Objectives: This study aimed at the evaluation of the antithrombotic activity of Batroxase, a P-I metalloprotease from Bothrops atrox venom, in an animal model of venous thrombosis. Methods: The antithrombotic activity of Batroxase was tested in vivo in a model based on two factors of the Virchow’s Triad: blood flow alterations (partial stenosis of the inferior vena cava), and vessel wall injury (10% ferric chloride for 5 min), in comparison with sodium heparin (positive control) and saline (negative control). Bleeding/clotting time was assessed by a tail bleeding assay. The immunogenicity of Batroxase was also analyzed. Results: Batroxase (12 mg/kg) reduced thrombus formation in 81%, similarly to heparin (100 U/kg), which reduced it in 85% in comparison with the saline group. Both Batroxase and heparin increased bleeding/clotting time in approximately 3 fold. Immunizations of rabbits with Batroxase do not result in detectable levels of antibodies against this metalloprotease. Conclusion: Batroxase presents antithrombotic activity in vivo. Moreover, its lack of immunogenicity increases the interest on its possible therapeutic potential over thrombogenic disorders. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The approach on toxinology research involves the study on toxins from plants, animals and microbial sources, in order to understand their characteristics, metabolism, and functions [1]. These investigation efforts intend not only to prevent and treat their effects on envenomation, but also their actions on different scenarios, e.g. the activation or inhibition of the hemostatic system [2–5]. Bothrops snake venom metalloproteases (SVMPs) are the main class of toxins responsible for triggering hemostasis events, inducing blood coagulation disorders, and leading to hemorrhage, as they are capable of degrading proteins of vessel membranes
∗ Corresponding authors at: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universio dade de São Paulo, FCFRP-USP, Av. do Café, s/n , CEP 14040-903, Ribeirão Preto-SP, Brazil. E-mail addresses: jacob [email protected] (A.L. Jacob-Ferreira), [email protected] (S.V. Sampaio). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.063 0141-8130/© 2016 Elsevier B.V. All rights reserved.
allowing blood extravasation, or acting on coagulation factors activators [6–10]. Another feature involving some SVMPs concerns the capacity to act directly on fibrin, degrading clots and preventing the formation of new clots. The therapeutic potential of fibrin(ogen)olytic SVMPs is recently being explored for the treatment of patients with cardio- and cerebrovascular disorders, as described for Fibrolase and its recombinant analog Alfimeprase [11,12]. Our research group purified and biochemically characterized a neutral (pI 7.5) P-I class (∼25 kDa) metalloprotease from Bothrops atrox snake venom: Batroxase, which is capable of degrading components of the extracellular matrix, such as type IV collagen and fibronectin, as well as components of the coagulation cascade, as fibrinogen and fibrin [13]. This enzyme had its fibrin(ogen)olytic activity inhibited by ␣2-macroglobulin [14]. Most importantly, Batroxase presented a dose-dependent in vivo thrombolytic activity, similar to the clinically relevant drug Alteplase (tissue-type plasminogen activator), without, however, affecting bleeding/clotting time of the studied animals [14]. The inactivation of the metalloprotease by ␣2-macroglobulin may reduce its
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activity, but also its potential side effects, as seen for bleeding time [14]. In order to continue the exploitation of Batroxase’s therapeutic potential for the future development and production of new drugs with effects over hemostasis, the present study aimed at assessing its antithrombotic activity and also its immunogenicity. 2. Methods The equipment and other materials were described in the course of the methodology, and unspecified reagents used were of analytical grade. 2.1. Isolation of Batroxase from Bothrops atrox venom Batroxase was obtained through chromatographic fractionation of Bothrops atrox venom, acquired from Centre of Extraction of Animal Toxins (Morungaba-SP, Brazil), as previously described [14,15]. Briefly, two consecutive chromatographic steps on resins obtained from GE Healthcare (Chicago, IL, USA) were performed. First a size exclusion chromatography on Sephacryl S-200, followed by anion exchange chromatography on DEAE Sepharose. Latter, the fraction containing the metalloprotease was ultrafiltered in concentrator tube with polyethersulfone membrane with cut-off of 3000MWCO, Vivaspin® 20 (Sartorius, Goettingen, Germany), and the isolated protein was quantified using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer instructions, separated in aliquots of 1 mg/tube, lyophilized and stored at −20 ◦ C until its use in the experiments. 2.2. Animals Male Wistar rats (250–270 g) and adult female New Zealand White rabbits were obtained from the Central Animal Facility of USP (Ribeirão Preto-SP), and maintained under controlled conditions of temperature (24 ◦ C) and brightness (12 h light/dark cycles), with free access to food and water. In the previous day from the venous thrombosis experiments, rats had their food removed overnight, to allow the emptying of their intestine and facilitate its handling during the surgical procedure. All experiments involving animals were performed according to the Brazilian College of Animal Experimentation (COBEA) guidelines and experimental protocols were approved by the Ethics Committee on Animal Use of Ribeirão Preto campus, University of São Paulo, protocol number 12.1.1809.53.2. 2.3. Assessment of the antithrombotic activity of Batroxase on venous thrombosis In order to analyze the antithrombotic activity of Batroxase in comparison to the antithrombotic drug of clinical use (sodium heparin, Hepamax-S® , Blausiegel Ltd, Brazil), a model of partial blood stasis and vascular damage of the inferior vena cava was used [16]. Wistar rats (250–270 g, N = 3–5/group) were anesthetized with an association of ketamine (80 mg/kg) and xylazine (10 mg/kg) IP, and laid down in supine position. The abdomen was opened by incision along the linea alba toward the sternum, followed by the inferior vena cava exposure. The partial stasis was induced by tying a cotton thread just below the junction of the vena cava with the left renal vein. A blind 21G needle was placed between the node and the inferior vena cava, which was then removed to allow partial blood flow and patterned formation of partial stasis. The tested drug was injected through the left femoral vein of the animal over 2 min before the induction of the thrombus, which was done by a filter paper (5 × 5 mm) saturated with 10% ferric chloride solution (10 L), which remained in contact with the vein for 5 min, causing vascular damage and enabling the induction of the thrombus
Fig. 1. Schematic protocols. Panel A: Antithrombotic test in vivo. It was first performed the surgery for cannulation of the left femoral vein, isolation and partial stenosis of the inferior vena cava (approximately 30 min). Then, stimuli (saline, heparin or Batroxase) were injected through the left femoral vein, during the 2 min that precede the red thrombus induction by a piece of filter paper saturated with ferric chloride solution at 10% placed in contact with the vascular wall for 5 min. After, a period of 30 min was waited in order to allow the formation/stability of thrombus. During this period of wait, the bleeding/clotting time of the animals was accompanied for up to 30 min. Panel B: Evaluation of Batroxase effects over biochemical analysis of rats’ blood. For the biochemical analysis of the rats’ blood, animals were anesthetized, and stimuli (saline, heparin or Batroxase) were injected through their tail vein over a 2 min period, as for the antithrombotic study protocol. Another 30 min was waited before cardiac puncture, in order to mimic the bleeding time evaluation period. Panel C: Rabbits immunization protocol: Immunogenicity of Batroxase was tested in rabbits. Blood samples were collected before the immunization protocol starts (pre). In the first day, Batroxase (BTX) emulsified with complete Freund adjuvant (CFA) was subcutaneously injected in the neck region of the rabbits. The immunization was repeated every 15 days for 3 more times, using incomplete Freund’s adjuvant (IFA) instead of CFA. After 5 days of the fourth immunization, another blood sample was collected through the ear vein for ELISA analyses, and 5 days later, animals were anesthetized for complete blood collection by cardiac puncture.
(Fig. 1A). After the 5 min of induction with ferric chloride, a 30 min period was waited in order to allow the formation of the red thrombus. During this period, the bleeding/clotting time of the animals was assessed. Different doses of sodium heparin (40, 90, 100 and 120 U/kg) and a dose of Batroxase (12 mg/kg) were evaluated and compared with a saline group. The Batroxase dose was chosen based on the dose that had previously shown thrombolytic activity in this venous thrombosis model [14]. 2.4. Evaluation of Batroxase changes over bleeding/clotting time of animals For the analysis of the bleeding/coagulation time [17], after the drug administration, the tail of anesthetized rats was heated in water (40 ◦ C) for 1 min, and after drying, a small cut at the tip of the tail was performed with the use of a razor (Laser® , Laser Shaving Ltd, United Kingdom). Bleeding time starts when the first blood drop touches the filter paper, and it was recorded at 30 s intervals, accompanied until bleeding stopped or to a maximum of 30 min. After the end of the experiments, the inferior vena cava was excised for immediately weighing of the thrombus formed (wet thrombus). A thrombus of the saline group, fixed in 10% formol, was prepared and stained using hematoxylin and eosin for the histological analyses of the venous thrombus characteristics. 2.5. Evaluation of Batroxase effects over biochemical analysis of rats’ blood For the biochemical analysis of the rats’ blood, animals (N = 4/group) were anesthetized and drugs (saline, heparin 100 U/kg and Batroxase 12 mg/kg) were injected through their tail vein over a 2 min period, as for the antithrombotic study protocol. Another 30 min period was waited before cardiac puncture, to mimic the thrombus formation/bleeding time evaluation period.
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This new protocol (Fig. 1B) was performed in order to reduce the possible alterations caused by the venous thrombosis surgery, e.g. inflammatory reactions, which could disturb the results of the blood alterations caused by drugs only. Blood was collected in appropriate anticoagulants (EDTA or sodium citrate), and taken to the Clinical Analysis Service Facility of the School of Pharmaceutical Sciences of Ribeirão Preto, USP, for the analysis of prothrombin time, activated partial thromboplastin time, fibrinogen concentration and hemogram (complete blood count). 2.6. Batroxase’s immunogenicity The immunogenicity of the isolated Batroxase was tested in 2 adult female New Zealand White rabbits (Fig. 1C). Blood samples were collected through the ear vein to obtain pre-immune serum (negative control). Batroxase (200 g) diluted in 1 mL of sterile PBS and emulsified with 1 mL of complete Freund adjuvant (CFA, Difco Laboratories Inc., Detroit, MI, USA) was injected into 4 sites subcutaneously in the neck region of the rabbits. The immunization was repeated every 15 days for 3 more times, using incomplete Freund’s adjuvant (IFA) instead of CFA. After 5 days of the fourth immunization, another blood sample was collected through the ear vein for analyses, and 5 days later, animals were anesthetized for complete blood collection by cardiac puncture. Serum was obtained by centrifugation at 1500 g for 10 min, and assayed for the presence of Batroxase’s antibodies by enzyme-linked immunosorbent assay (ELISA). The immunogenicity of Batroxase was monitored by ELISA. Initially, 50 L of Batroxase dissolved in PBS (20 g/mL) was loaded into wells (1 g/well) of a 96 wells plate, and incubated overnight at 4 ◦ C for antigen immobilization. Sequentially, each well was loaded with blocking buffer (3% gelatin, 0.5% Tween 20 in PBS, 200 L/well) and incubated for 2 h at 37 ◦ C. Then, samples of preimmune- or immune-sera (50 L/well), diluted 1:1000 in PBS, were loaded to the plate and incubated for 1 h at 37 ◦ C. After, wells were loaded with 50 L of peroxidase-conjugated Affinipure goat anti-rabbit IgG antibody (Jackson ImmunoReseach, West Grove, PA, USA) diluted 1:5000 in PBS containing 1% gelatin and 0.05% Tween 20, followed by incubation for 1 h at 37 ◦ C. All of the above steps were followed by washing each well with 200 L PBS containing 0.1% Tween. Sequentially, TMB solution (tetramethylbenzidine, Organon Teknika, Boxtel, Netherlands) in the presence of hydrogen peroxide was loaded to the plate. Finally, after 10 min, this reaction was stopped by adding 2 N sulfuric acid solution (50 L/well), and the absorbance at 450 nm was read in a spectrophotometer (Spectramax Plus – Molecular Devices). 2.7. Statistical analyses The results were expressed as means ± SEM. The comparisons between groups were assessed by one-way analysis of variance followed by Dunnett’s multiple comparison tests. A probability value <0.05 was considered significant.
Fig. 2. Antithrombotic activity of Batroxase on venous thrombosis. Stimuli (saline, heparin or Batroxase) were injected during 2 min, before the induction of the formation of thrombus by a 10% solution of ferric chloride, and it was waited a 30 min period for the formation/stabilization of the thrombus. At the end of the experiment, the inferior vena cava was excised and the thrombus present was isolated and weighed for analysis of antithrombotic activity of Batroxase. Saline (negative control), heparin in different doses from 10 to 120 U/kg (positive control), Batroxase (BTX) at 12 mg/kg n = 3–5/group, * p < 0.05 vs. saline.
Fig. 3. Bleeding/clotting time of Batroxase-treated animals. The analysis of the bleeding/clotting time was performed in the same anesthetized animals used for the analysis of antithrombotic activity in vivo. After administration of saline, heparin or Batroxase, a small cut at the tip of the tail was made using a razor. Bleeding time starts when the first blood drop touches the filter paper, and was recorded at 30 s intervals, until bleeding stopped or to a maximum of 30 min. Saline (negative control), Heparin in different doses from 10 to 120 U/kg (positive control), Batroxase (BTX) at 12 mg/kg n = 3–5/group, * p < 0.05 vs. saline.
there was no thrombus formation (higher doses are not shown in figures). 3.2. Batroxase changes over bleeding/clotting time of animals Both Batroxase and heparin in different doses caused an increase of approximately 3 times in the bleeding/clotting time of rats, in comparison with the saline group (Fig. 3).
3. Results 3.3. Batroxase effects over biochemical analysis of rats’ blood 3.1. Antithrombotic activity of Batroxase on venous thrombosis The antithrombotic activity of Batroxase (12 mg/kg) was similar to the result obtained with heparin (100 U/kg), a clinically relevant anticoagulant, with a reduction of 81 and 85% of the weight of the thrombus formed, respectively (Fig. 2). Heparin antithrombotic activity was dose-dependent, and with doses higher than 120 U/kg
Biochemical analyses such as hemogram (complete blood count), prothrombin time, activated partial thromboplastin time and fibrinogen concentration were done on the blood of rats treated with saline, Batroxase (12 mg/kg) and heparin (100 U/kg) (N = 4/group), and there were no significant differences between groups (data not shown).
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Fig. 4. Histological analyses of the venous (red) thrombus. A thrombus of the saline group stained with hematoxylin and eosin. E: erythrocytes; F: fibrin; V: Vena Cava’s wall; I: vessel injury; A: Aorta’s wall.
3.4. Batroxase’s immunogenicity Batroxase was considered non immunogenic, as after the scheme of 4 immunization cycles, there were no differences in the pre- and post-immune serum of the rabbits tested, analyzed by ELISA (data not shown). 4. Discussion The present study shows the in vivo antithrombotic activity of Batroxase, a metalloprotease isolated from Bothrops atrox snake venom, in a model of venous thrombosis. Thrombosis is a failure of hemostasis, a process regulated by the vessel wall, platelets and coagulation factors, keeping the blood in a liquid state, free of clots, and inducing rapid formation of a plug when there is a vascular injury [18]. Venous thromboembolism, which includes deep vein thrombosis and pulmonary embolism, is a major cause of morbidity and mortality worldwide, being the third most common cardiovascular disorder after coronary artery disease and cerebrovascular disease [19]. The experimental model of venous thrombosis used in this study is characterized by two of the three factors of Virchow’s triad: endothelial injury induced by ferric chloride, and disturbance of the blood flow due to the partial stasis [16]. This model is most similar to clinical conditions, since it is believed that the rupture of the vascular wall is essential for triggering the hemostasis process [16]. Also, it is more reproducible inter/intra laboratory than the use of hypercoagulability, as the activity of thromboplastin from different sources can considerably vary, reflecting in differences of the necessary dose for this experimental model of venous thrombosis [16]. As observed in our results (Fig. 4), venous thrombi are rich in fibrin and poor in platelets, and are also called red thrombi, due to the high incorporation of red blood cells; and they may be prevented by the use of antithrombotic/anticoagulant drugs [20]. Snake venom metalloproteases have received considerable attention in recent years as they may exert various biological activities, which can interfere in cardiovascular diseases, such as proteolytic, fibrin(ogen)olytic, apoptotic, prothrombin activation, factor X activation, alterations on platelets aggregation, and coagulant activity [18,21–24]. Batroxase, a metalloprotease isolated from Bothrops atrox venom, was recently purified and biochemically characterized in our laboratory, and presented antithrombotic and thrombolytic activities in vitro, as it was capable of degrading components of the coagulation cascade, such as fibrinogen and fibrin [13]. In this work, we demonstrated that this toxin also prevents thrombi formation in vivo, in an experimental animal model of venous thrombosis. Batroxase (12 mg/kg) presented antithrombotic activity similar to the clinically relevant drug, heparin (100 U/kg), with reduction of 81 and 85% of the thrombus weight, respectively. This antithrombotic activity may be due to Batroxase’s capacity of cleaving fibrinogen in a form that would not allow fibrin to polymerize
[13]. The dose of Batroxase tested in this work (12 mg/kg) was based in our previous study, which showed that Batroxase presented thrombolytic activity in vivo, in this same venous thrombosis model [14]. In this study, we observed an increase in the tail bleeding/clotting time of rats, both with heparin and Batroxase, during the antithrombotic study protocol. This increase was not observed for Batroxase in our previous work. We have previously shown that Batroxase was inhibited by ␣2-macroglobulin, a plasmatic molecule capable of inhibiting different proteases by the venous fly trap hypothesis, as it forms a complex with the protease and reduces its access to macromolecular substrates [14,25]. The bleeding time alterations observed in this work may be due to the higher plasmatic concentration of Batroxase resulting of the antithrombotic protocol, as the drug was intravenously administrated during only 2 min, in comparison to the 30 min administration of the thrombolytic protocol. Thus, the elevated concentration of Batroxase may have exceeded the inhibition capacity of ␣2-macroglobulin, which was not able to reduce its possible hemorrhagic collateral effect. Interestingly, no significant differences were found on biochemical analyses of the rats’ blood. Venous thromboembolism is associated with high risk of recurrence after a first event, and on cessation of anticoagulant therapy [19]. As our goal is to explore the therapeutic potential of the toxin for its use as a model for future development and production of new drugs, we evaluated Batroxase’s immunogenicity. Remarkably, Batroxase did not cause immunogenicity in the studied animals. This characteristic increases the interest in the clinical use of Batroxase. 5. Conclusion This study shows the in vivo antithrombotic activity of Batroxase in a model of venous thrombosis, which increases our interest in its potential as a biological model for drugs that may interfere in hemostasis. Its lack of immunogenicity increases the interest on its possible therapeutic potential over thrombogenic disorders. Competing interests The authors declare that there are no competing interests. Ethical statement The authors declare they have followed the ethical requirements for this publication. Authors’ contributions ALJF conceived and accomplishment this study, and wrote the manuscript. DLM, MAS and TBR assisted in the biochemical and functional experiments, while MDB and SVS supervised and critically discussed the study. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank the financial support provided by the São Paulo Research Foundation (FAPESP, grants 2012/215699 and 2011/23236-4) and the National Council for Scientific and Technological Development (CNPq, grant 476932/2012-2 and 487351/2012-6). The funding agencies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. We are also grateful to Clinical Analysis Service facil-
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