Toxicon 60 (2012) 773–781 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity Daniela C.S. Damico a,*, T. Vassequi-Silva a, F.D. Torres-Huaco a, A.C.C. Nery-Diez a, R.C.G. de Souza b, S.L. Da Silva c, C.P. Vicente d, C.B. Mendes e, E. Antunes e, C.C. Werneck a, Sérgio Marangoni a a Department of Biochemistry, Institute of Biology, University of Campinas (UNICAMP), PO Box 6109, CEP 13083-970, Campinas, SP, Brazil b Hospitalar Foundation of Itacaré, Itacaré, BA, Brazil c Federal University of São João Del Rei – UFSJ, Chemistry, Biotecnology and Bioprocess Department, Ouro Branco, MG, Brazil d Department of Anatomy, Cell Biology and Physiology and Biophysics, Institute of Biology, University of Campinas (UNICAMP), Brazil e Department of Pharmacology, State University of Campinas, SP, Brazil article info abstract Article history: A basic phospholipase A2 (LmrTX) isoform was isolated from Lachesis muta rhombeata Received 4 January 2012 snake venom and partially characterized. The venom was fractionated by molecular Received in revised form 13 June 2012 exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase Accepted 19 June 2012 Ò HPLC on a C-5 Discovery Bio Wide column. From liquid chromatography–electrospray Available online 29 June 2012 ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA2 Keywords: LmrTX from L. muta rhombeata and other PLA from snake venoms, like CB1 and CB2 from Phospholipase A 2 2 fi Lachesis muta rhombeata venom Crotalus durissus terri cus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA2 Arterial thrombosis activity in the presence of a synthetic substrate and alkylation of histidine residues Photochemical injury significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and Anticoagulant activity antithrombotic activity. In this study, we examined the ability of the LmrTX in altering Platelet aggregation thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 Æ 7.8 min. LmrTX, the PLA2 from L. muta rhombeata venom, caused a change in the occlusion time to 99 Æ 10 min with doses of 7.5 mg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction anthropized areas, such as the Atlantic Forest of southern Bahia (Souza et al., 2007). Much is said about the great The bushmaster is the largest venomous snake in the capacity of adult inoculation of the Lachesis, but the Americas and the second largest in the world, reaching severity of the accident is independent of the size of the 3.40 m. Individuals exceeding 2.80 m in length are rare in animal (França and Cardoso, 1989), since, unlike what can Brazil (Souza et al., 2007). The Lachesis accidents statistic be seen in Bothrops, where the size of the animal is the misleads without a context: the 1.6–2.4% of total snakebites main prognostic factor of evolution accidents (França and at the national level become 17% in the Amazon (Ramza, Cardoso, 1989), even in surface scratches, inoculation 1994) or more than that in highly fragmented and with a single prey and accidents with young animals, characterized by low volume of the Lachesis venom inoc- * Corresponding author. Tel.: þ55 19 3521 6132; fax: þ55 19 3521 6129. ulated, can still cause early serious and systemic effect E-mail address: [email protected] (D.C.S. Damico). (Souza et al., 2007). This is probably due to the cascade of 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.06.010 774 D.C.S. Damico et al. / Toxicon 60 (2012) 773–781 effects triggered by the self pharmacological post inocula- acid (solvent A). The resulting solution was clarified by tion, as well as the synergy between the various actions of centrifugation and the supernatant was further submitted Ò the poison, namely: intense local pain, swelling, profuse to a reversed phase chromatography on a C5 Discovery Bio bleeding at the site of the bite, diarrhea and abdominal Wide Pore 10 mm (25 cm  4.6 mm). Fractions were eluted pain, vomiting, bradycardia, hypotension/profuse sweating, using a linear gradient (0–100%, v/v) of acetonitrile (solvent inability to swallow or painful attempt to do so, dysphagia B) at a constant flow rate of 1.0 ml/min over 50 min, and the and shock (Souza et al., 2007). resulting fractions were manually collected. The elution Proteins found in snake venom that affect the clotting profile of both analyses was monitored at 280 nm, and the factors can be classified as those who work in coagulation collected fractions were lyophilized and conserved at factors, including activating factor V, factor X activator, À20 C. The homogeneity of the final material was assessed prothrombin activators and enzymes Thrombin-like, anti- by mass spectrometry. coagulant factors, including proteins that bind to factors IX/ X, protein C activators, inhibitors of thrombin and phos- 2.4. Phospholipase A2 activity pholipase A2, and those that act in fibrinolysis, including fibrinolytic enzymes and plasminogen activators (Kini, PLA2 activity was measured using the assay described by 2005). Cho and Kezdy (1991) and Holzer and Mackessy (1996) So far, five PLA2 isoenzymes have been isolated from modified for 96-well plates (Beghini et al., 2000). The Lachesis spp. venoms: two acidic (LmPLA2I and LmPLA2II) standard assay mixture contained 200 ml of buffer (10 mM from L. muta (Fuly et al., 2003); two basic (LmTX-I and Tris–HCl, 10 mM CaCl2 and 100 mM NaCl, pH 8.0), 20 mlof LmTX-II) from L. muta muta (Damico et al., 2005) and one synthetic chromogenic substrate 4-nitro-3-(octanoyloxy) (LsPA-1) from Lachesis stenophrys (de Assis et al., 2008). benzoic acid 3 mM, 20 ml of water, and 20 ml of PLA2 in However, none have been purified from L. muta rhombeata a final volume of 260 ml. After adding PLA2 isoforms (20 mg), and studied in relation to the anticoagulant activity. the mixture was incubated for up to 40 min at 37 C, In this study, we report for the first time, the purifica- absorbance reading at intervals of 10 min. The enzyme tion, prediction of primary structure, anticoagulant and activity, expressed as the initial velocity of the reaction (V0), antithrombotic activity of the PLA2 from L. muta rhombeata was calculated based on the increase of absorbance after venom and its relation with its enzymatic activity. 20 min. 2. Materials and methods 2.5. Determination of the molecular mass of the purified protein by mass spectrometry 2.1. Venom and reagents An aliquot (4.5 ml) of the protein was inject by C18 Venom was collected in Serra Grande Center (IBAMA (100 mm  100 mm) RP-UPLC (nanoAcquity UPLC, Waters) authorization number 24945-1), Bahia State Brazil, the only coupled with nano-electrospray tandem mass spectrom- facility in the country totally dedicated to study and pres- etry on a Q-Tof Ultima API mass spectrometer (MicroMass/ ervation of the Atlantic Bushmaster, L. muta rhombeata Waters) at a flow rate of 600 nl/min. The gradient was 0– (www.lachesisbrasil.com.br). All chemicals and reagents 50% acetonitrile in 0.1% formic acid over 45 min. The were of analytical or sequencing grade. instrument was operated in MS continuum mode and the data acquisition was from m/z 100–3000 at a scan rate of 1 s 2.2. Animals and an interscan delay of 0.1 s. The spectra were accumu- lated over about 300 scans, and the multiple charged data 7–8 weeks C57BL6 mice were supplied by the Animal produced by the mass spectrometer on the m/z scale were Services Unit of the State University of Campinas (UNI- converted to the mass (molecular weight) scale using CAMP). Mice were housed at room temperature on a 12 h Maximum Entropy-based software (1) supplied with the light/dark cycle and had free access to food and water. All Masslynx 4.1 software package. The processing parameters procedures were performed according to the general were: output mass range 6000–20,000 Da at a ‘resolution’ guidelines proposed by the Brazilian Council for Animal of 0.1 Da/channel; the simulated isotope pattern model was Experimentation (COBEA) and approved by the university’s used with the spectrum blur width parameter set to 0.2 Da, Committee for Ethics in Animal Experimentation (CEEA/ the minimum intensity ratios between successive peaks UNICAMP) number 1790-1. were 20% (left and right). The deconvoluted spectrum was then smoothed (2  3 channels, Savitzky Golay smooth) 2.3. Venom fractionation and the centroid mass values were obtained using 80% of the peak top and a minimum peak width at half height of 4 One hundred mg of crude venom of L. muta rhombeata channels. was dissolved in 1 ml of 0.2 M Ammonium bicarbonate buffer, pH 8.0. After centrifugation at 5.000 g for 5 min, 2.6. Analysis of tryptic digests the supernatant was loaded onto a Sephadex G75 column (1.5 cm  90 cm), previously equilibrated with the same The protein was reduced (DTT 5 mM for 25 min at 56 C) solution, under a flow rate of 12 ml/h.