A basic phospholipase A2 (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery® Bio Wide column. From liquid chromatography-electrospray 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 LmrTX from L. muta rhombeata and other PLA2 from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA2 activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering 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 ± 7.8 min. LmrTX, the PLA2 from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/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.
Bibliographical noteFunding Information:
We acknowledge the Mass spectrometry Laboratory at Brazilian Biosciences National laboratory, CNPEM-ABTLUS, Campinas, Brazil, for their support with the mass spectrometric analyses. The authors thank Mr. Paulo A. Baldasso for general technical assistance. This work was supported by the São Paulo Research Foundation – FAPESP (Process 09/02299-8 ) and is part of Post-Doctoral project by Daniela Carla da Silva Damico and FAPESP ( 2010/19916-7 ) to Claudio Chrysostomo Werneck.
- Anticoagulant activity
- Arterial thrombosis
- Lachesis muta rhombeata venom
- Phospholipase A
- Photochemical injury
- Platelet aggregation