Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

Toxicon 60 (2012) 70–82

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Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity A.C.O. Cintra a, L.G.B. De Toni a, M.A. Sartim a, J.J. Franco a, R.C. Caetano a, M.T. Murakami b, S.V. Sampaio a, * a

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, 14040-903 Ribeirão Preto, São Paulo, Brasil b Laboratório Nacional de Biociências (LNBio), Campinas, São Paulo, Brasil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 December 2011 Received in revised form 8 February 2012 Accepted 20 March 2012 Available online 30 March 2012

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPa-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and b-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and b-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

Keywords: Snake venom metalloproteinase Substrate specificity Bothrops atrox venom Fibrinolytic and thrombolytic activity

1. Introduction Bothrops snake related ophidic accidents are characterized by local effects, such as vessel basement membrane proteolysis, hemorrhage, necrosis, edema and leukocyte infiltration (Fox and Serrano, 2009; Teixeira et al., 2009), and systemic effects, such as coagulopathies, nephrotoxicity, hemodynamic dysfunction and cardiotoxicity (Rosenfeld and Kalen, 1971; Gutiérrez et al., 1995, 2005; * Corresponding author. Tel.: þ55 16 3602 4287; fax: þ55 16 3602 4725. E-mail address: [email protected] (S.V. Sampaio). 0041-0101/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.toxicon.2012.03.018

Fernandes et al., 2006). Venom metalloproteinases play an important role in envenomation physiopathology because of their proteolytic activity toward several biological substrates. Snake venom metalloproteinases (SVMPs) are classified into four groups (PI to PIV) based on their molecular mass, domain structure and hemorrhagic intensity. PI-group SVMPs consist of metalloproteinases that contain only the proteinase domain, have molecular masses ranging from 20–30 kDa and weak hemorrhagic activity. The PII group is comprised of 30–60 kDa proteins that contain both proteinase and disintegrin-like domains. PIII group proteins include a cysteine-rich domain, and PIV proteins

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contain an additional lectin-like domain (Fox and Serrano, 2005; Du et al., 2006; Fox and Bjarnason, 1995). Several PI-group SVMPs from different snake venoms have been isolated and characterized, including Neuwiedase from Bothrops neuwiedi (Rodrigues et al., 2000), BaPI (Gutiérrez et al., 2005) and BH2 (Borkow et al., 1993) from Bothrops asper, BlaH1 from Bothrops lanceolatus (Stroka et al., 2005), CcH1 from Cerastes cerastes (Boukhalfa-Abib et al., 2009), BjussuMPII from Bothrops jararacussu (Marcussi et al., 2007) and Agkislysin from Agkistrodon acutus (Wang et al., 2004), Bothrojaractivase from Bothrops jararaca (Berguer et al., 2008) and metalloproteinases HT-a, -c, -d and -e from the Crotalus genera (Bjarnason and Fox, 1994). These proteinases have several common hemostasis-disturbing activities, such as fibrin(ogen)olysis, coagulation factor activation (factor X and II), induction or inhibition of platelet aggregation and activation of the coagulation process via proteolytic activity (Fox and Serrano, 2009; Kamigutti, 2005; Jia et al., 1996; Bjarnason and Fox, 1994). Therefore, the study of the effects of this class of toxins on thrombosis and clotting is important for understanding the physiopathology of snake envenomation. Furthermore, potential clinical or pharmacological applications of these proteins as thrombolytic and fibrinolytic agents have been discussed (Fujimura et al., 1996; Rodrigues et al., 2004; Gutiérrez and Rucavado, 2000; Jia et al., 2009; Toombs, 2001; Swenson et al., 2004). In the present study, we describe the purification and biochemical and functional characterization of Batroxase, which is a new PI-class metalloproteinase from Bothrops atrox snake venom that has fibrinolytic and thrombolytic activities. 2. Materials and methods 2.1. Venom Crude desiccated B. atrox venom (Pará state) was purchased from SANMARU serpentarium (Taquaral, São Paulo, Brazil). 2.2. Animals Four- to six-week-old male Swiss mice, weighing 18–20 g each, were obtained from the Biotery of Isogenic Experimental Animals at the Pharmaceutical Science School of Ribeirão Preto (USP). The procedures used during the experiments were approved by the Animal Ethical Use Committee of the USP-Ribeirão Preto campus (protocol number 02.09.2009). 2.3. Blood The blood and plasma used in the experiments were donated by healthy volunteers who were not using any medications, in accordance with the authorization of the Ethics and Human Research Committee of the USP (protocol number 148). 2.4. Reagents

b-mercaptoethanol, sodium dodecyl sulfate (SDS) and Coomasie Brilliant Blue G 250 were obtained from GE Life

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Sciences, USA. Phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetra acetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), dithiothreitol (DTT), iodoacetoamide, substrates (type IV collagen, plasmin, fibrinogen, fibronectin, laminin and human plasminogen), enzymes (tripsin, chymotrypsin, streptococcus aureus V8 protease, urokinase and thrombin) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Adenosine diphosphate (ADP) was from Helena Laboratories (Beaumont – TX). All other chemical were of analytical or sequencing grade. 2.5. Batroxase purification Crude venom from Bothrops atrox (500 mg) was dissolved in 50 mM ammonium bicarbonate (ambic) buffer, pH 8, and clarified by centrifugation at 10,000  g for 10 min. The supernatant solution was fractionated on a Sephadex G-75 chromatography column (100 cm  4 cm, GE Life Sciences, USA), which was equilibrated and eluted with the same buffer. Elution was performed at 30 mL/h and monitored by spectrometry at 280 nm. The eluted fractions were assayed for hemorrhagic activity and evaluated by SDS-PAGE. A 20 mg sample of the hemorrhagic fraction Ba III was diluted in 50 mM ammonium bicarbonate buffer, pH 7.4, and applied on a Shodex ES-502N 7C ion exchange column (7.6 mm  10 cm–Shimadzu, Japan). The solution was also analyzed via high-performance liquid chromatography (HPLC) (Shimadzu, Japan) using 50 mM ambic pH 7.4 as buffer A and 500 mM ambic pH 7.4 as buffer B. The material was eluted using a linear gradient of buffer B from 0 to 100% with a flow rate of 0.6 mL/min. The eluted material was monitored at 280 nm. The resulting fractions (ES I and ES II) were assayed for hemorrhagic activity, and fraction ES I was found to induce hemorrhage. The homogeneity of the purified metalloproteinase was evaluated by reverse-phase chromatography using a C-18 ODS column (25 cm  46 mm – Shimadzu, Japan) which was previously equilibrated with 0.1% TFA (solvent A) and them submitted to a linear gradient of acetonitrile 70% (solvent B) from 0 to 100% over 75 min. The eluted material was monitored at 280 nm. 2.6. Biochemical characterization Protein quantification was performed using the microbiuret method, according to Itzhaki and Gill (1964). A calibration curve was determined using different concentrations of bovine serum albumin (from 0.1 to 2.0 mg/mL). The protein contents of crude B. atrox and each chromatographic fraction were assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) using a 13.5% gel containing Tris– glycine pH 8.3 and 0.01% SDS, some samples being treated with ß-mercaptoethanol (Laemmli, 1970). After the electrophoretic procedure, the gel was stained with 0.2% Coomassie Brilliant Blue G 250. The molecular mass standards (GE Life Sciences, USA) consisted of the following: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovoalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.1 kDa) and a-lactoalbumin (14.4 kDa).

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The isoelectric focusing of the purified metalloproteinase was performed according to Vesterberg (1972). Ampholytes with pH values ranging from 3.5 to 10.0 (GE Life Sciences, USA) were used to form the pH gradient on the gel. The molecular weight of Batroxase was determined by mass spectrometry analysis on an Axima Performance MALDI-TOF mass spectrometer (Shimadzu, Japan) in linear mode. The sample was diluted in 100 mL of water and added to the matrix (alpha-cyano-4-hydroxycinnamic acid) at a proportion of 1:3. 2.7. Hemorrhagic activity The hemorrhagic activity was assessed according to Nikai et al. (1984). Samples containing 2.5, 5.0, 10, 25 and 50 mg of Batroxase in 50 mL of phosphate-buffered saline (PBS) were injected intradermally into the dorsal skin of mice. Three hours after the injection, the animals were sacrificed in a CO2 chamber, and the dorsal skin was removed. The MHD was defined as the protein dose that produced hemorrhages with a mean diameter of 10 mm, as calculated using the perpendicular major diameters of the hemorrhagic spot. Groups of 5 animals were tested, and control group animals were injected with PBS only. All chromatographic fractions were assayed for hemorrhagic activity.

2.11. Fibrinogenolytic activity The ability of Batroxase to digest fibrinogen was evaluated using the method published by Edgar and Prentice (1973), with some modifications. A 25 ml aliquot of fibrinogen solution (2.0 mg/mL in 25 mM Tris–HCl pH 7.4) was incubated with several concentrations of Batroxase (0.25, 0.5, 1, 2, 6, 8 and 10 mg in 5 mL 25 mM Tris–HCl pH 7.4) at 37  C for 90 min. The reaction was stopped with 20 mL of 50 mM Tris–HCl pH 6.8 containing 10% glycerol (v/v), 4% SDS (w/v), 0.05% bromophenol blue (v/v) and 4% b-mercaptoethanol (v/v), followed by heating at 100  C for 5 min. After reduction and denaturation, the samples were assayed for fibrinogen hydrolysis by 13.5% SDS-PAGE. The fibrinogen digestion kinetics were evaluated by incubating a fixed concentration of Batroxase with fibrinogen for different time intervals (0, 5, 10, 15, 30, 60 and 120 min) at 37  C. The fibrinogenolytic activity was also tested under different pH values (2.5; 3.0; 4.0; 5.0; 6.0; 7.0; 9.0 and 10.0) and temperature conditions (80, 20, 5, 37, 50 and 100  C). Protease inhibitors (EDTA, EGTA, PMSF) and bmercaptoethanol were assayed for inhibition of fibrinogen hidrolysis by Batroxase. The GE Life Sciences molecular mass standards were used.

2.8. Thrombolytic activity

2.12. Proteolytic activity of Batroxase on different substrates

The thrombolytic activity of different concentrations of Batroxase (25, 50 and 100 mg/500 mL of PBS) was evaluated by incubation for 24 h at 37  C with clots induced “in vitro” in 500 mL of human whole blood using 24-well plates (Gremski et al., 2007). The control group consisted of 500 mL of whole blood incubated with 500 mL of PBS. After incubation, the plates were photographed, the clot was removed and both perpendicular major diameters of the clot were measured. The clot digestion value was expressed as the mean clot diameter (cm).

2.12.1. Type IV collagen Type IV collagen solution (4 mg/mL) was prepared in 10 mM Tris–HCl pH 7.4 containing 10 mM NaCl and incubated with different concentrations of Batroxase. The reaction was stopped by adding 20 mL of 50 mM Tris–HCl pH 6.8 containing 10% glycerol (v/v), 4% SDS (w/v), 0.05% bromophenol blue (v/v) and 4% b-mercaptoethanol (v/v), followed by heating at 100  C for 5 min. The substrate digestion was analyzed by 7.5% SDS-PAGE.

2.9. Coagulant activity The effect of Batroxase on coagulation was evaluated using human plasma (200 mL) incubated with different concentrations of the metalloproteinase (0.1, 0.2, 0.4, 0.8, 1.6 and 2.0 mg/25 mL) at 37  C. As a control, human plasma (200 mL) was added to 25 mL of CaCl2 at 0.25 mM, which induced clot formation within 3 min (Selistre et al., 1990). The minimum coagulant dose (MCD) was calculated as the minimum amount of protein that was able to induce plasma clotting in 60 seconds. 2.10. Fibrinolytic activity The fibrinolytic activity was assessed in Petri plates containing fibrin according to Leitão et al. (2000). Aliquots of 30 mL containing different concentrations of Batroxase (0.5, 1.0, 4.0, 6.0, 8.0, 10, 20 and 40 mg) were added to cavities on the fibrin gel and incubated at 37  C for 24 h. The fibrinolytic activity was evaluated visually and quantified according to the halo diameter, which was compared to a positive control (plasmin 10 mg) and a negative control (PBS only).

2.12.2. Fibronectin Fibronectin (4 mg/mL) in 10 mM Tris–HCl pH 7.4 and 10 mM NaCl was incubated with Batroxase at a molar ratio of 1:50 enzyme:substrate at 37  C for 2, 6, 12 and 24 h. The hydrolysis was interrupted by adding 20 mL of 50 mM Tris– HCl pH 6.8 containing 10% glycerol (v/v), 4% SDS, 0.05% bromophenol blue (v/v) and 4% b-mercaptoethanol (v/v), followed by heating at 100  C for 5 min. The fibronectin hydrolysis was analyzed by 7.5% SDS-PAGE. The Spectra multicolor broad range protein ladder (260–10 kDa) was used as a molecular mass standard. 2.12.3. Laminin A stock solution of laminin (4 mg/mL) was prepared in 50 mM Tris–HCl pH 7.4, 10 mM NaCl and 2 mM CaCl2. The substrate was incubated with Batroxase at a molar ratio of 1:50 at 37  C for 2, 6, 12 and 24 h. After incubation, 20 mL of stop solution containing 1 M urea, 4% ß-mercaptoethanol (v/v) and 4% SDS (w/v) was added, and the material was heated for 15 min at 100  C. The extracellular matrix component digestion was analyzed by 7.5% SDS-PAGE. The Spectra multicolor broad range protein ladder (260– 10 kDa) was used as the molecular mass standard.

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2.12.4. Fibrin To evaluate the proteolytic activity of Batroxase on fibrin, a clot was induced by incubating a fibrinogen solution (10 mg/mL in HEPES) with thrombin at 37  C for 1 h. The clot was then dissolved and transferred in 100 mL aliquots to glass tubes and incubated with 5 mg of Batroxase at 37  C. The reaction was interrupted at different time points (0, 15, 30, 60 and 120 min and 12 h) by adding 20 mL of a solution containing 1 M urea, 4% ß-mercaptoethanol (v/v) and 4% SDS (w/v), and it was left to incubate overnight. The digestion products were analyzed by 7.5% SDS-PAGE. The Page ruler pre-stained protein ladder (170–35 kDa, Fermentas, USA) was used as the molecular mass standards. 2.12.5. Plasminogen Human plasminogen (30 mg) was incubated with Batroxase (5 mg) in 10 mM Tris–HCl buffer containing 10 mM CaCl2, pH 8.5, for different time intervals at 37  C. The reaction was stopped by adding sample buffer containing a reducing agent. The digestion was analyzed by 10% SDS-PAGE. As a positive control, urokinase (625 U/mL) was used as a known plasminogen activator. 2.12.6. Matrigel digestion A 100 mL aliquot of Matrigel (BD Bioscience) in 50 mM Tris–HCl buffer containing 20 mM CaCl2, pH 7.6, was incubated with 10 mg Batroxase at 37  C, for different time intervals. The reaction was stopped by adding sample buffer containing a reducing agent, and the digestion was analyzed by SDS-PAGE in a 4–15% gradient gel under reducing conditions. As a negative control, Matrigel was incubated with the sample buffer only for 180 min. As a positive control, the Matrigel was incubated with 10 mg B. atrox crude venom for 180 min. 2.13. Platelet aggregation assay Platelet-rich plasma (PRP) was prepared from freshly collected human plasma by centrifugation of whole blood at 1000  g for 10 min. Plasma-poor platelets (PPP) were obtained from PRP by centrifugation at 1000  g for 15 min. Platelet aggregation was monitored turbidimetrically using an aggregometer (Chrono-Log Corporation). The PRP presented a platelet count of 3  105 cells/mL. For each assay, 10 or 20 mg Batroxase was added to 500 ml of PRP, and the aggregation was monitored for 2 min at 37  C with stirring. Subsequently, ADP (final concentration 10 mM) was added, and the solution was monitored for an additional 6 min. As a positive control for maximal aggregation, ADP only was added to PRP and monitored for 6 min. The values obtained for Batroxase were compared with those obtained for ADP only. 2.14. Determination of Batroxase amino acid sequence A 150 mg sample of the purified protein was diluted in 50 mM ambic buffer, pH 8.0, and reduced with dithiothreitol (DTT) at a molar ratio of 50:1 (w/w) for 1 hour at 56  C. The material was then alkylated with 10 mL iodoacetamide (1 mg/mL) for 30 min in the dark. A 50 mg sample of this reduced and alkylated Batroxase (RA-Batroxase) was submitted to trypsin proteolytic digestion at a molar ratio

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of 2:100 (w/w, enzyme:protein) for 4 h at 37  C. For chymotrypsin hydrolysis, 50 mg of RA-Batroxase was suspended in 100 mM Tris–HCl containing 10 mM CaCl2, pH 7.8, at a molar ratio of 1:60 (w/w, enzyme:protein) and incubated for 3 h at 37  C. Streptococcus aureus V8 protease (in 10 mM ambic pH 8.0) was then added at a molar ratio of 3:100 (w/w, enzyme:protein), and the reaction was incubated at 37  C for 18 h. The hydrolyzed material was subjected to electrospray ionization mass spectrometry (ESI) using a quadrupole-time-of-flight mass spectrometer (QTof Ultima, Waters/Micromass) coupled to an ultraperformance liquid chromatography (UPLC) system (NanoAcquity, Waters). The peptides generated by digestion were desalted on-line using a Waters Symmetry C18 trap column (5 mm  180 mm  20 mm). Elution was performed in a BEH 130 C18 (1.7 mm  75 mm  100 mm) column using a 0–60% (v/v) acetonitrile gradient for 1 h. The spectra were acquired using data-directed analysis by selecting the doubly and triply charged peptides for MS/MS experiments. All of the MS/MS spectra were processed using the Mascot Distiller software and the MASCOT search engine (Matrix Science, Boston). The N-terminal amino acid sequence of Batroxase was determined using the native protein obtained from reverse-phase chromatography using a C-18 column (as described previously). The sequencing procedure was performed using a PPSQ-33A automated protein microsequencer (Shimadzu, Japan). Both the N-terminal protein sequence obtained by automatic sequencer and the internal peptide digested material obtained from the mass spectrometry were used to search for related protein sequences in the SWISS-PROT/TREMBL database with the BLAST FASTA program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The homology between Batroxase and other proteinases was evaluated using the NCBI protein data bank. Alignments were refined using the program CLUSTAL 2.0.11. 2.15. Molecular modeling The atomic coordinates of class I snake venom metalloproteinase from Bothrops moojeni (BmooMPa-I, PDB ID: 3GBO, Akao et al., 2010) were used as a 3D template for restraint-based modeling and implemented in the MODELLER program (Fiser and Sali, 2003a). The overall model was improved by enforcing the proper stereochemistry using spatial restraints and CHARMM energy terms, followed by conjugate gradient simulation based on the variable target function method (Fiser and Sali, 2003a). Loops were optimized using MODLOOP (Fiser and Sali, 2003b) based on the satisfaction of spatial restraints, without relying on a database of known protein structures. The DOPE potential was evaluated for all models, and the model with the lowest global score was selected for explicit solvent molecular dynamics simulation using the GROMACS package (Lindahl et al., 2001) and the GROMOS-96 (43a1) force field to check its stability and consistency. The overall and local quality of the final model was assessed by VERIFY3D (Eisenberg et al., 1997), PROSA (Wiederstein and Sippl, 2007) and VADAR (Willard et al., 2003). Three-dimensional structures were analyzed and compared using the program PyMoL (www.pymol.org).

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2.16. Statistical analysis

content under reduced conditions (Fig. 1A insert) shows that Ba III contained two proteins, with one main band presenting a molecular mass of approximately 27 kDa and the second band presenting a molecular mass of approximately 17 kDa. Ba III was submitted to a second purification procedure using anion exchange chromatography (Fig. 1B). Unbound material was eluted in 50 mM ambic pH 7.4, whereas the bound proteins were eluted with a linear gradient of increasing concentrations of ambic pH 7.4, up to 500 mM. The resulting fractions (ES I and ES II) were assayed for hemorrhagic activity, and fraction ES I was able to induce dorsal skin hemorrhage in mice. SDS-PAGE (Fig. 1B insert) shows that ES I produced a single protein band of approximately 27 kDa under reducing conditions. To confirm the purity of the fraction, ES I was submitted to reverse phase chromatography on HPLC, which revealed a single homogenous peak (Fig. 1C). In addition, isoelectric focusing produced a single protein band with a pI of 7.5 (Fig. 1D). The MALDI-TOF mass spectrometry analysis, based on a single charged molecule, identified a protein

The results obtained were expressed as the mean  standard deviation (SD) and statistically analyzed by applying a one-way ANOVA, followed by the Tukey method. Differences with p < 0.05 were considered statistically significant. 3. Results 3.1. Purification and biochemical characterization A new proteinase isolated from the venom of Bothrops atrox, which is a snake native to the state of Pará in Brazil, was obtained by two chromatographic procedures. The first step consisted of gel filtration on a Sephadex G-75 column under alkaline conditions (pH 8.0). The chromatogram shown in Fig. 1A illustrates the five major fractions obtained (Ba I to Ba V). Fraction Ba III presented hemorrhagic activity. The SDS-PAGE analysis of the fraction

B

1,2

2,5 100 2,0

0,8 0,6 0,4

80 1,5 60 1,0

40

0,5

20

0,2 0,0

50

100

150

200

250

0

100

0 200

150

Time (min)

0,20 100 0,15

D 11.5

80

10.0 8.5

60

0,10

40 0,05

20 0

0,00

% (B)

Absorbance 280 nm

50

Fraction number

pH

C

0,0

0

% (B)

1,0

Absorbance 280 nm

Absorbance 280 nm

A

7.0

pI 7.5

5.5 4.0 2.5 1.0

0

20

40

Time (min)

60

80

0

2

4

6

8

10

Migration (cm)

Fig. 1. Purification of Batroxase from B. atrox. (A) Gel filtration chromatography of B. atrox crude venom using a Sephadex G-75 column. Fractions were eluted in 50 mM ambic buffer, pH 8.0, and the Ba III fraction presented hemorrhagic activity. (A insert) SDS-PAGE (12% gel) analysis of gel filtration fractions under reduced conditions: Lane 1, molecular weight standard; Lane 2, Ba III; Lane 3, Ba II; Lane 4, Ba I; Lane 5, B. atrox crude venom. (B) Anion exchange chromatography of fraction Ba III using an ES-502 N 7 C column. The unbound material was washed out with 50 mM ambic pH 7.4 (buffer A), and the bound material was eluted with 500 mM ambic pH 7.4 (buffer B) in a linear gradient from 0% to 100% buffer B. The procedure was performed over 200 min, with a flow rate of 0.6 mL/min. The material was monitored at 280 nm. (B insert) SDS-PAGE (12% gel) analysis of Batroxase, purified by anion exchange chromatography, under reducing conditions. Lane 1, Batroxase (ES I fraction); Lane 2, molecular mass standard. (C) The hemorrhagic fraction ES I was submitted to reverse phase chromatography using a C-18 column to evaluate sample homogeneity. The column was equilibrated with 0.1% TFA (solvent A) and eluted with a linear gradient from 0% to 100% of 70% acetonitrile (solvent B) over 75 min, with a flow rate of 1 mL/min. The eluted material was monitored at 280 nm. (D) Isoelectric focusing – pH gradient migration pattern of isoelectric focusing. (D insert) Electrofocusing in a 7% polyacrylamide gel.

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with a molecular mass of 22.9 kDa (data not shown). Taken together, these results confirm the isolation of Batroxase, a new protein from Bothrops atrox snake venom.

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The amount of 100 mg Batroxase completely dissolved the clot (Fig. 2B). 3.4. Fibrinolytic activity

3.2. Hemorrhagic activity Batroxase was able to induce hemorrhaging after intradermal injection in the dorsal skin of mice, with a DMH of 10 mg (Fig. 2A). 3.3. Thrombolytic activity The thrombolytic activity was evaluated via “in vitro” clot degradation using different concentrations of Batroxase, which induced clot lysis in a concentration-dependent manner, with doses of 50 mg (p <0.05) and 100 mg (p < 0.001) presenting greater activity than the PBS control.

The fibrinolysis assay consisted of incubation of Batroxase in a gel containing fibrin. Batroxase was able to induce fibrin hydrolysis at all concentrations tested, and there was no significant difference from the hydrolysis induced by plasmin. This activity was concentrationdependent up to 8 mg of Batroxase; higher concentrations did not induce additional fibrin hydrolysis (Fig. 2C). 3.5. Fibrinogenolytic activity The fibrinogen digestion by Batroxase was monitored by SDS-PAGE under reducing conditions. The concentrations used for the experiment induced substrate digestion

Fig. 2. Functional characterization of Batroxase. (A) Determination of the minimal hemorrhage dose (MHD) of Batroxase. Different concentrations of Batroxase (2.5; 5.0; 10.0; 25.0 and 50.0 mg in 50 mL of PBS) were injected intradermally into the dorsal skin of mice, and the hemorrhage halo formation was measured after three hours. The results were expressed as the mean halo diameter (cm)  standard deviation (SD). (A insert) Hemorrhage visualized in dorsal skin (ex situ). Each concentration was tested in a group of 5 mice (n ¼ 5). (B) Thrombolytic activity. Different amounts of Batroxase (25, 50 and 100 mg) were diluted in 500 mL PBS and added to 500 mL of human whole blood clots, and the mixture was incubated at 37  C for 24 h. The results obtained were expressed as the mean thrombus diameter (cm)  SD (n ¼ 3). The Tukey test was used for statistical analysis. *p < 0.05 and **p < 0.001 compared with the control (PBS). (B insert) Side and upper views of a 24-well plate showing thrombus clot reduction. Upper panel, side view; Lower panel, upper view after supernatant removal for thrombus measurement. (C) Different concentrations of Batroxase were incubated in pools formed in a fibrin gel to evaluate the fibrinolytic activity. Control groups were incubated in the presence of 10 mg plasmin (PC) or PBS only (NC). The results were expressed as the mean diameter of the halo formed (cm) by fibrin digestion. The Tukey test was used for statistical analysis; *p < 0.05 compared with the control (PBS). (C insert) Photo of the fibrin gel activity plate. Pool 1, PBS; Pool 2, 0.5; Pool 3, 1.0; Pool 4, 4.0; Pool 5, 6.0; Pool 6, 8.0; Pool 7, 10.0; Pool 8, 20.0; Pool 9, 40.0 mg Batroxase; Pool 10, 5 mg plasmin; Pool 11, 10 mg plasmin (PC).

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(Fig. 3A). From 0.5 mg of the proteinase, hydrolysis of the a and b chain of fibrinogen, but not the g chain, was observed (Fig. 3A). After the critical concentration of Batroxase was determined, digestions performed for different periods of incubation showed that fibrinogen was digested at all time periods tested, and 30 min of incubation was determined as optimal for this activity (Fig. 3B, lane 7). The optimal temperature and pH for fibrinogen proteolysis by Batroxase were 37  C and pH 5.0 (data not shown). Ion-chelating agents such as EDTA and EGTA, as well as the reducing agent b-mercaptoethanol, were able to completely inhibit the substrate digestion (data not shown). These results confirm that Batroxase is able to digest the fibrinogen molecule as a metalloproteinase. 3.6. Proteolytic effects on different substrates 3.6.1. Extracellular matrix components At amounts of 8 mg and higher, Batroxase was able to induce the partial digestion of the a 1 and a 2 chains of type IV collagen, and the substrate was completely degraded with 10 mg of Batroxase (Fig. 4A, lane 6 and 7, respectively). Batroxase was able to cleave fibronectin subunits A and B after 60 min of incubation, presenting a complete substrate digestion at 240 min (Fig. 4B, lane 3–6) and was not able to digest laminin, even with long periods of incubation (data not shown). 3.6.2. Coagulation components As illustrated in Fig. 4C, Batroxase was able to digest the fibrin, preferentially the b chain. After 15 min of incubation, a decrease of the b chain could be noted, with complete hydrolysis occurring after 60 min. The a and g chains of fibrin remained intact, but the g-g dimer was gradually digested (Fig. 4C, lane 5). Fig. 4D shows the SDS-PAGE analysis of the proteolytic fragmentation of plasminogen by Batroxase. The band with a molecular mass of 83 kDa is represents plasminogen

(Fig. 4D, lane 1). The incubation of plasminogen with urokinase generated proteolytic fragments with an apparent molecular mass of 66 kDa, which corresponded to the heavy chain of plasmin (compared with the plasmin control band: Fig. 4D, lanes 2 and 3). This pattern was not observed when the substrate was incubated with Batroxase, which generated fragments ranging from 20 to 38 kDa, independent of the time of incubation (Fig. 4D, lanes 5–8). These results suggest that the proteolysis of plasminogen by Batroxase is different from that by urokinase. 3.6.3. Matrigel digestion After 15 min of incubation of Matrigel with Batroxase, the a 1, a and g laminin chains were digested, and no nidogen proteolysis was observed (Fig. 4E, lanes 7–10). A similar response was observed upon the incubation of Matrigel with B. atrox crude venom (Fig. 4E, lane 6). 3.7. Platelet aggregation Neither 10 nor 20 mg of metalloproteinase was able to induce platelet aggregation after two minutes of incubation. Subsequently, to evaluate whether Batroxase could inhibit human platelet aggregation, 10 mM ADP was added to medium containing Batroxase and PRP. The incubation was monitored for six minutes, and there was no significant effect on the platelet aggregation response compared with treatment with ADP only (Fig. 5). 3.8. Batroxase primary sequence The amino acid sequence of Batroxase was determined for the 45 initial (N-terminal) residues by automatic Edman degradation. The remaining primary sequence of the proteinase was determined by mass spectrometry by overlapping the amino acid sequences of the digested peptides (T4, Ch5, Ch6, SV8-1, Ch7, Ch8, SV8-3 and Ch 10)

Fig. 3. Fibrinogenolytic activity. (A) Concentration-response curve generated by fibrinogen digestion: Lane 1, fibrinogen 2 mg/mL (Fib); Lane 2, 10 mg Batroxase (BTX); Lane 3, Fib þ 0.25 mg BTX; Lane 4, Fib þ 0.5 mg BTX; Lane 5, Fib þ 1.0 mg BTX; Lane 6, Fib þ 2.0 mg BTX; Lane 7, Fib þ 6.0 mg BTX; Lane 8, Fib þ 8.0 mg BTX; Lane 9, Fib þ 10.0 mg BTX; Lane 10, molecular weight standard. (B) Time-dependent fibrinogen digestion. Lane 1, 2 mg/mL Fib; Lane 2, 1 mg BTX. Lanes 3 to 9 each contain 2 mg/mL Fib and 1 mg BTX, incubated for different durations: Lane 3, 0 min; Lane 4, 5 min; Lane 5, 10 min; Lane 6, 15 min; Lane 7, 30 min; Lane 8, 60 min; Lane 9, 90 min. Lane 10, molecular mass standard.

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Fig. 4. Proteolytic digestion of different substrates. (A) SDS-PAGE (7.5%) analysis of type IV collagen fragment digestion by different concentrations of Batroxase. Lane 1, type IV collagen (2 mg/mL). Lanes 2–7 contain 2 mg/mL collagen incubated at 37  C for 2 hours with different amounts of Batroxase: Lane 2, 1 mg; Lane 3, 2 mg; Lane 4, 4 mg; Lane 5, 6 mg: Lane 6, 8 mg; Lane 7, 10 mg. (B) SDS-PAGE (7.5%) analysis of fibronectin fragments digested by Batroxase after different periods of incubation. Lane 1, fibronectin (1 mg/mL). Lanes 2–6 contain 1 mg/mL fibronectin and 5 mg Batroxase incubated at 37  C for different time periods: Lane 2, 0 min; Lane 3, 60 min; Lane 4, 240 min; Lane 5, 360 min; Lane 6, 480 mins. Lane 7, molecular weight standard. (C) Fibrin digestion fragment analysis by SDS-PAGE (12.0% gel). Lane 1, fibrin (2.5 mg/250 mL). Lanes 2–7 contain 5 mg/5 mL Batroxase incubated with 2.5 mg/250 mL fibrin (obtained from thrombin clots formed from human plasma and subsequently dissolved) at 37  C for different time periods. Lane 2, 0 min; Lane 3, 15 min; Lane 4, 30 min; Lane 5, 60 min; Lane 6, 120 min; Lane 7, 12 h. Lane 8, molecular mass standard. (D) Plasminogen digestion fragment analysis by SDS-PAGE (10.0% gel). Lane 1, plasminogen (30 mg); Lane 2, plasminogen (30 mg) incubated with urokinase (625 U/mL) at 37  C for 1 h. Lanes 3–9 contain 30 mg plasminogen incubated with 5 mg Batroxase at 37  C for different time periods: Lane 4, 0 min; Lane 5, 15 min; Lane 6, 30 min; Lane 7, 60 min; Lane 8, 120 min. Lane 10, molecular mass standard. (E) Matrigel component digestion fragment analysis by PAGE (gel gradient 4–15%). Matrigel was incubated with B. atrox crude venom or Batroxase at a molar ratio of 1:4 at 37  C: Lane 2, B. atrox crude venom (10 mg); Lane 3, Batroxase (5 mg); Lane 4, molecular weight standard; Lane 5, Matrigel (1, laminin chain a 1; 2, laminin chain b and g; 3, nidogen chain); Lane 6, Matrigel þ crude venom. Lanes 7–10 contain Batroxase (5 mg) incubated with Matrigel at a molar ratio of 1:40 for different time periods: Lane 7, 15 min; Lane 8, 30 min; Lane 9, 60 min; Lane 10, 180 min.

obtained by trypsin, chymotrypsin and S. aureus V8 protease hydrolysis. As illustrated in Fig. 6, Batroxase contains 202 amino acid residues, with a high content of lysine, arginine, glutamic acid and aspartic acid (glutamic acid and aspartic acid were identified as glutamine and asparagine). The multiple amino acid sequence alignment of Batroxase with other PI-class SVMPs identified by protein data bank BLAST (PubMed – Medline) was created using Clustal 2.0.11 software (Fig. 7). Batroxase has a high structural identity with other Bothrops spp. metalloproteinases, and a multiple alignment analysis revealed a strong identity to other SVMPs: B. atrox atrolysin, 89%; B. insularis insularinase A precursor, 84%; B. jararaca jararafibrase 2 precursor, 80%; Agkistrodon contortrix contortrix fibrolase and alfimeprase, 58% and 58%, respectively;

Bothrops moojeni BmooMPa-I, 54%; and Vipera lebetina lebetase, 53%. 3.9. Tertiary structure of Batroxase The modeled atomic structure of Batroxase showed good local and global stereochemical properties with a Z-score of 6.8, which was compatible with the values obtained for experimentally determined structures. Analyses of the Ramachandran plot indicate that 94% of the Batroxase residues are in the most favorable regions, and 6% are in additional allowed regions. In addition, the local quality assessed by plotting the energies as a function of the amino acid positions shows no positive values, which indicates the good stereochemical quality of the model and its suitability for structural analyses and comparisons (Fig. 8).

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Fig. 5. Platelet aggregation assay. Different concentrations of Batroxase (BTX) were added to PRP (platelet-rich plasma). After two minutes, the platelet aggregation agonist ADP (10 mM) was added. Platelet aggregation was monitored throughout the assay for a total duration of 8 min. ADP 10 mM, BTX 10 mg and BTX 20 mg.

4. Discussion According to Araújo et al. (2007), ophidic accidents are an important public health issue. Bothrops snakes (family Viperidae) are responsible for most envenomation cases in Brazil. In 2005, approximately 29,000 cases of envenomation were reported, 88% of which were caused by Bothrops spp. snakes. Bothrops snake venoms contain a great variety of biologically active proteic, including metalloproteinases, that play an important role in the pathophysiological envenomation process.

In the present study, we describe the purification and biochemical characterization of a new hemorrhagic metalloproteinase from Bothrops atrox snake venom. The proteinase was isolated by consecutive gel filtration and anion exchange chromatography, which provided a high level of homogeneity as confirmed by reverse phase chromatography, SDS-PAGE, isoelectric focusing and N-terminal amino acid sequencing. The purification of PI-class SVMPs is commonly performed using two to three chromatographic steps that predominantly consist of gel filtration and ionic exchange techniques (Mandelbaum et al., 1982; Muniz et al., 2008). The purified SVMPs include leucurolysin-A from Bothrops leucurus (Gremski et al., 2007), bothropasin from Bothrops jararaca (Muniz et al., 2008), BaH4 from Bothrops asper (Franceschi et al., 2000) and ammodytagin from Vipera ammodytes ammodytes (Kurtovi c et al., 2011). BaH1 was isolated from Bothrops asper venom in three chromatographic steps using gel filtration, ion exchange and hydrophobic interaction methods (Borkow et al., 1993). Other PI SVMPs were obtained by different procedures: atroxlysin-I from Bothrops atrox (Sanchez et al., 2010) was isolated by two gel filtration steps, and B-mooMPa-I was isolated from Bothrops moojeni using a combination of gel filtration, ionic exchange and affinity chromatography techniques (Bernardes et al., 2008). Batroxase comprises approximately 1.2% (w/w) of the crude B. atrox snake venom, with a pI of 7.5 and a molecular mass of 22.9 kDa, as determined by mass spectrometry (data not shown), or w27 kDa, as determined by SDS-PAGE under reduced conditions (Fig. 1B insert). PI-class SVMPs, which display a single proteolytic domain, have molecular masses from w20 to 30 kDa (Lopes et al., 2009), as represented by BnP1 from Bothrops neuwiedi (Baldo et al., 2008) at 24 kDa, BlaH1 from Bothrops

Fig. 6. The complete amino acid sequence of Batroxase. Peptides were obtained from the digestion of Batroxase by chymotrypsin (Ch), trypsin (T), glu-c and asp-c (SV 8). The complete amino acid sequence was deduced from the sequences of the following overlapping peptides: T4, Ch5, Ch6, SV8-1, Ch7, Ch8, SV8 -3 and Ch 10.

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Fig. 7. Amino acid sequence multiple alignment of Batroxase with other PI-class SVMPs. The alignment was performed using Clustal software, version 2.0.11, with the primary sequences of other PI-class snake venom metalloproteinases obtained by BLAST FASTA program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). (NCBI – Medline). Gaps are indicated by “–”; “*” indicates positions that are fully conserved; “:” indicates positions that are strongly conserved and “.” indicates positions that are weakly conserved in atroxlysin from Bothrops atrox (Peru) (gi/353526296); the insularinase A precursor from Bothrops insularis (gi/82197476); jararafibrase 2 from Bothrops jararaca (VMJAR-BOTJA); fibrolase from Agkistrodon contortrix contortrix (gi/26393688); alfimeprase from Agkistrodon contortrix contortrix (gi/462095); BmooMPa-I from Bothrops moojeni (229462813); and lebetase from Vipera lebetina (gi/123901597).

lanceolatus (Stroka et al., 2005) at 28 kDa, leucurolysin-A from Bothrops leucurus (Gremski et al., 2007) at 23 kDa and atroxlysin-I from Bothrops atrox (Sanchez et al., 2010) at 23 kDa. Envenomation by Bothrops spp. venoms is characterized by local and systemic hemorrhage caused by the proteolytic digestion of extracellular matrix components (Escalante et al., 2011). The contribution of Batroxase to the hemorrhagic process was initially evaluated in the dorsal skin of mice. Batroxase was found to have an MHD of 10 mg, which was similar to that of other SVMPs; for example, atrolysin C

and D from Crotalus atrox have MHDs of 8 and 11 mg, respectively (Bjarnason and Fox, 1994), BaP1 has an MHD of 20 mg (Gutiérrez et al., 2005) and atroxlysin-I has an MHD of 19.9 mg (Sanchez et al., 2010). These doses are relatively high compared with those of PII and PIII SVMPs, which have MHDs from 0.2 to 4 mg; for example, BaH1 from Bothrops asper (Borkow et al., 1993) has an MHD of 0.2 mg, B-JussuMP-I from Bothrops jararacussu has an MHD of 4 mg (Mazzi et al., 2006) and BaH4 from Bothrops asper has an MHD of 2 mg (Franceschi et al., 2000). Based on these results, we consider Batroxase to be a weakly hemorrhagic

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Fig. 8. Schematic illustration of the modeled structure of Batroxase. The three catalytic histidine residues are shown in stick representation with carbon atoms in yellow. The zinc ion is drawn as a green sphere. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

metalloproteinase. To determine the mechanism underlying the induction of hemorrhage by Batroxase, its capacity to digest extracellular matrix components was assessed. Batroxase was able to hydrolyze type IV collagen and fibronectin molecules, and it also degraded the a 1, a and g chains of laminin in Matrigel, but it was not able to digest isolated laminin. No nidogen proteolysis was detected. According to Bou-Gharios et al. (2004), the basement membranes of blood vessels consist mainly of laminin, collagen and fibronectin. Therefore, the ability of Batroxase to hydrolyze these components is consistent with its ability to induce hemorrhage by degrading extracellular matrix components of the blood vessel basement membranes. Batroxase was able to digest fibrinogen by cleaving the a and b chains. Furthermore, the fibrinogen hydrolysis occurred in a concentration-dependent manner and was inhibited by EDTA and EGTA, which indicates that its metalloproteinase character was important for inducing proteolysis. According to Mosesson (2005), under physiological conditions, fibrin is formed by the cleavage of the fibrinogen a chain by thrombin. However, the results obtained showed that a and b chain cleavage by Batroxase suggests that the fibrin formed might not be able to polymerize. Thus, the activity of Batroxase on the fibrinogen molecule likely indicates a consumption of this substrate and an inhibition of clot and thrombus formation. Several PI SVMPs are able to preferentially digest the a chain of the fibrinogen molecule, e.g., BnPI from Bothrops neuwiedi (Baldo et al., 2008), BlaH1 from Bothrops lanceolatus (Stroka et al., 2005), Atroxlysin-I from Bothrops atrox (Sanchez et al., 2010), BmooMPa-I from Bothrops moojeni (Bernardes et al., 2008) and Neuwiedase from Bothrops neuwiedi (Rodrigues et al., 2001). Fibrinolytic activity has been reported for several PIclass SVMPs, such as Neuwiedase (Rodrigues et al., 2001) and BnP1 from Bothrops neuwiedi (Baldo et al., 2008), Bothrojaractivase from Bothrops jararaca (Berguer et al., 2008), Berythractivase from Bothrops erythromelas (Silva et al., 2003), BthMP from Bothrops moojeni (Lopes et al.,

2009) and Atroxlysin-1 from Bothrops asper (Sanchez et al., 2010). Batroxase was able to induce fibrin digestion in a concentration-dependent manner up to 8 mg. The lack of further digestion at higher concentrations was probably the result of the total consumption of the fibrin in the gel. To confirm that the fibrinolytic hydrolysis mediated by Batroxase was not the result of the activation of plasminogen to generate plasmin, Batroxase was incubated with plasminogen, and the resulting fragments were analyzed. Batroxase did not digest plasminogen to generate plasmin, which confirms that it induces fibrinolysis independently of other agents. A thrombus is formed by the aggregation of platelets on the fibrin clot mesh. Because of its ability to induce fibrinolysis, Batroxase reduced the size of an “in vitro” induced thrombus in a 50 mg treatment after 24 hours of incubation, and it completely degraded the thrombus in a 100 mg. With the same amounts, Leucurolysin-a from Bothrops leucurus (Gremski et al., 2007) was also able to dissolve a thrombus “in vitro”, with the maximal activity observed for a 100 mg treatment. Batroxase did not affect human platelet aggregation by the agonist ADP. This characteristic capacity has been reported for other PI-class SVMPs, such as Neuwiedase from Bothrops neuwiedi (Rodrigues et al., 2001) because this class contains only a proteolytic domain. PII class SVMPs possess the proteolytic domain and a disintegrin domain that contains an RGD site that enables interactions with other integrins on platelet surface, thereby preventing platelet aggregation by agonists (Calvette et al., 1991). In the PIII class SVMPs, such as in Basparin A from Bothrops asper (Loría et al., 2003), an additional cysteine-rich domain further facilitates platelet aggregation. Several snake venom metalloproteinases are capable of inducing an incoagulable plasma condition because of their ability to consume plasma coagulation factors (Kamigutti, 2005). Similar to other PI-class SVMPs, Batroxase did not induce plasma coagulation, which facilitates the hemorrhagic process. The primary sequence of Batroxase was determined by N-terminal amino acid sequencing by automatic Edman degradation, and the digested peptides obtained by trypsin proteolysis were sequenced by mass spectrometry. These analyses indicated that Batroxase is composed of 202 amino acids. Additionally, a primary structure analysis showed that Batroxase lacks N-glycosylation sites (N-X-S/ T); its zinc-binding motif (HELGHNLGISH) is fully conserved when compared with that of other SVMPs; and it contains a C164I165 M166 motif associated with a “Met-turn”. PI-class SVMPs may be sub-characterized according to disulfide bridge content (Fox and Serrano, 2005); PIa proteins such as HT-2 from Crotalus ruber ruber, have two disulfide bridges, whereas PIb proteins such as Fibrolase from Agkistrodon contortrix contortrix and Lebetase from Vipera lebetina (Bello et al., 2006) have three disulfide bridges. Batroxase presented seven cysteine residues that are fully conserved with those in the other metalloproteinases, with matching such as Cys117–Cys197, Cys157– Cys181 and Cys159–Cys164. According to our tertiary structure analyses, Batroxase forms an a-b-a fold that is stabilized by three disulfide

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bridges (above) similar to those of other class PI SVMPs (Gomis-Rüth et al., 1994; Gong et al., 1998; Akao et al., 2010) (Fig. 8). The active site is nested in the interface between the upper (approximately 150 N-terminal residues) and lower domain (approximately 50C-terminal residues), with all catalysis-relevant residues and the zincbinding motif fully conserved. In class PI SVMPs, the zinc ion is coordinated by the N32 atoms of the three catalytic histidines (His142, His146 and His152) and up to three solvent molecules. Typically, one solvent molecule coordinating the zinc ion is polarized by the residue Glu143, which permits a nucleophilic attack on the scissile peptide bond of a polypeptide chain substrate. In astacin, this typical interaction is replaced by one involving the hydroxyl group of Tyr169 side chain (Bode et al., 1992). Similar to BmooMPa-I and other class PI SVMPs, the calcium-binding site at the crossover region of the N- and C-termini is also conserved. The calcium ion is considered to play a structural role in PI-class SVMPs (Gomis-Rüth et al., 1994; Akao et al., 2010). The present study thus characterizes Batroxase as a PIb class SVMP with weak hemorrhagic activity that is possibly mediated by the proteolysis of blood vessel basement membrane components such as laminin, type IV collagen and fibronectin. Because of its capacity to promote fibrinolytic and thrombolytic activity independently of plasminogen activation, Batroxase may be an interesting tool for novel therapeutic approaches for the treatment of coagulation disorders, as was recently reported for alfimeprase, which is a recombinant protein obtained from snake venom fibrolase (Toombs, 2001). Acknowledgements Dra. Eliane C. Arantes from FCFRP-USP, Ribeirão Preto, for her cooperation to determine the N-terminal sequence. Dr. José Cesar Rosa from Faculty of Medicine of Ribeirão Preto – USP, for his cooperation to determine molecular mass. This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). Conflict of interest statement No conflict of interest to declare. References Akao, P.K., Tonoli, C.C., Navarro, M.S., Cintra, A.C., Neto, J.R., Arni, R.K., Murakami, M.T., 2010. Structural studies of BmooMPalpha-I, a nonhemorrhagic metalloproteinase from Bothrops moojeni venom. Toxicon 55, 361–368. Araújo, S.D., De Souza, A., Nunes, F.P., Gonçalves, L.R., 2007. Effect of dexamethasone associated with serum therapy on treatment of Bothrops jararaca venom-induced paw edema in mice. Inflamm. Res. 56, 409–413. Baldo, C., Tanjoni, I., León, I.R., Batista, I.F., Della-Casa, M.S., Clissa, P.B., Weinlich, R., Lopes-Ferreira, M., Lebrun, I., Amarante-Mendes, G.P., Rodrigues, V.M., Perales, J., Valente, R.H., Moura-Da-silva, A.M., 2008. BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP. Toxicon 51, 54–65. Bello, C.A., Hermogenes, A.L., Magalhaes, A., Veiga, S.S., Gremski, L.H., Richardson, M., Sanchez, E.F., 2006. Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom. Biochimie 88, 189–200.

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