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

Abstract

SummaryCrude 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.

Anti-snake venom effect of secodolastane diterpenes isolated from Brazilian marine brown alga Canistrocarpus cervicornis against Lachesis muta venom

“…muta snake lyophilized venom was provided from Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil, and LM-PLA 2 -I was isolated accordingly to Fuly et al (1997;2002). Balb/c mice (18-20 g) were obtained from the Núcleo de Animais de Laboratório of the Universidade Federal Fluminense (CEPA: 200/10).…”

“…The objective of this work is to evaluate the effect of diterpenes isolated from Brazilian C. cervicornis marine alga against in vivo and in vitro activities of L. muta snake venom, as well as against the phospholipase A 2 enzyme (denoted LM-PLA2-I) previously isolated from its venom (Fuly et al, 1997).…”

Inhibitory effect of a Brazilian marine brown alga Spatoglossum schröederi on biological activities of Lachesis muta snake venom

“…Moreover, the inhibitory effect of seaweed was also evaluated against a phospholipase A 2 enzyme (denoted LM-PLA2-I) previously isolated from this venom (Fuly et al, 1997).…”

Caracterização funcional e estrutural de uma nova fosfolipase A2 ácida de Bothrops moojeni

“…Entretanto, algumas exceções foram descritas como: uma PLA 2 de Bothrops alternatus que induz hemorragia pulmonar e necrose em células do músculo cardíaco de murinos (NISENBOM et al, 1986; e outra recentemente isolada de Bothrops pauloensis, denominada Bp-PLA 2 que induz mionecrose . Fuly e colaboradores demonstraram que as PLA 2 s ácidas da peçonha de Lachesis muta também eram capazes de induzir mionecrose e inibir a agregação plaquetária (FULY et al, 1997;. Estas enzimas de caráter ácido ainda merecem maiores estudos na tentativa de melhor entender a correlação entre estrutura, função catalítica e tóxica, além de elucidar suas possíveis aplicações farmacológicas.…”

“…Esta composição pode explicar a capacidade desta região de interagir e penetrar nas camadas lipídicas das membranas celulares, uma propriedade relacionada com a atividade miotóxica dessas toxinas. Já nas PLA 2 s Asp49, a região C-terminal é composta predominantemente por resíduos hidrofílicos e aniônicos VALENTIN e LAMBEAU 2000;LOMONTE et al, 2003;OHNO et al, 2003;DOS SANTOS et al, 2009;TEIXEIRA et al ,2011 (FULY et al, 1997;SERRANO et al, 1999;ANDRIÃO-ESCARSO et al, 2002;KETELHUT et al, 2003;ROBERTO et al, 2004a, b ;MAGRO et al, 2003;COGO et al, 2006;MODESTO et al, 2006;RODRIGUES et al, 2007TEIXEIRA et al, 2011). A aquisição de informações adicionais irá contribuir para a melhor compreensão dos mecanismos de ação destas enzimas individualmente e da sua participação em envenenamentos ofídicos.…”