Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

André L Fuly; Olga L T Machado; Elias W Alves; Célia R Carlinis

doi:10.1055/s-0038-1665414

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1997; 78(05): 1372-1380
DOI: 10.1055/s-0038-1665414

Review Article

Schattauer GmbH Stuttgart

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A₂ Isolated from Lachesis muta (Bushmaster) Snake Venom

André L Fuly

¹The Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;

,

Olga L T Machado

²Laboratório de Químíca e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil

,

Elias W Alves

¹The Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;

²Laboratório de Químíca e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil

,

Célia R Carlinis

¹The Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;

› Author Affiliations

Abstract

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Summary

Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A₂ activities. A phospholipase A₂, denoted LM-PLA₂ was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C₂/C₁₈ column. LM-PLA₂ presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA₂ with other PLA₂ from different sources.

LM-PLA₂ displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA₂ displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA₂ as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA₂ is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA₂ was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA₂ from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA₂s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

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References

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