LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

Daniela C.S. Damico; T. Vassequi-Silva; F. D. Torres-Huaco; A. C.C. Nery-Diez; R. C.G. de Souza; S. L. Da Silva; C. P. Vicente; C. B. Mendes; E. Antunes; C. C. Werneck; Sérgio Marangoni

doi:10.1016/j.toxicon.2012.06.010

LmrTX, a basic PLA₂ (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

Daniela C.S. Damico, T. Vassequi-Silva, F. D. Torres-Huaco, A. C.C. Nery-Diez, R. C.G. de Souza, S. L. Da Silva, C. P. Vicente, C. B. Mendes, E. Antunes, C. C. Werneck, Sérgio Marangoni

Research output: Contribution to journal › Original Article › peer-review

24 Scopus citations

Abstract

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.

Original language	American English
Pages (from-to)	773-781
Number of pages	9
Journal	Toxicon
Volume	60
Issue number	5
DOIs	https://doi.org/10.1016/j.toxicon.2012.06.010
State	Indexed - Oct 2012
Externally published	Yes

Bibliographical note

Funding 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.

Keywords

Anticoagulant activity
Arterial thrombosis
Lachesis muta rhombeata venom
Phospholipase A
Photochemical injury
Platelet aggregation

Access to Document

10.1016/j.toxicon.2012.06.010

Cite this

Damico, D. C. S., Vassequi-Silva, T., Torres-Huaco, F. D., Nery-Diez, A. C. C., de Souza, R. C. G., Da Silva, S. L., Vicente, C. P., Mendes, C. B., Antunes, E., Werneck, C. C., & Marangoni, S. (2012). LmrTX, a basic PLA₂ (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity. Toxicon, 60(5), 773-781. https://doi.org/10.1016/j.toxicon.2012.06.010

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title = "LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity",

abstract = "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{\textregistered} 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.",

keywords = "Anticoagulant activity, Arterial thrombosis, Lachesis muta rhombeata venom, Phospholipase A, Photochemical injury, Platelet aggregation",

author = "Damico, {Daniela C.S.} and T. Vassequi-Silva and Torres-Huaco, {F. D.} and Nery-Diez, {A. C.C.} and {de Souza}, {R. C.G.} and {Da Silva}, {S. L.} and Vicente, {C. P.} and Mendes, {C. B.} and E. Antunes and Werneck, {C. C.} and S{\'e}rgio Marangoni",

note = "Funding 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 S{\~a}o 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.",

year = "2012",

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doi = "10.1016/j.toxicon.2012.06.010",

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pages = "773--781",

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Damico, DCS, Vassequi-Silva, T, Torres-Huaco, FD, Nery-Diez, ACC, de Souza, RCG, Da Silva, SL, Vicente, CP, Mendes, CB, Antunes, E, Werneck, CC & Marangoni, S 2012, 'LmrTX, a basic PLA₂ (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity', Toxicon, vol. 60, no. 5, pp. 773-781. https://doi.org/10.1016/j.toxicon.2012.06.010

TY - JOUR

T1 - LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

AU - Damico, Daniela C.S.

AU - Vassequi-Silva, T.

AU - Torres-Huaco, F. D.

AU - Nery-Diez, A. C.C.

AU - de Souza, R. C.G.

AU - Da Silva, S. L.

AU - Vicente, C. P.

AU - Mendes, C. B.

AU - Antunes, E.

AU - Werneck, C. C.

AU - Marangoni, Sérgio

N1 - Funding 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.

PY - 2012/10

Y1 - 2012/10

N2 - 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.

AB - 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.

KW - Anticoagulant activity

KW - Arterial thrombosis

KW - Lachesis muta rhombeata venom

KW - Phospholipase A

KW - Photochemical injury

KW - Platelet aggregation

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U2 - 10.1016/j.toxicon.2012.06.010

DO - 10.1016/j.toxicon.2012.06.010

M3 - Original Article

C2 - 22750534

AN - SCOPUS:84863842064

SN - 0041-0101

VL - 60

SP - 773

EP - 781

JO - Toxicon

JF - Toxicon

IS - 5

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