MECHANISM OF INDUCTION OF COMPLEMENT SUSCEPTIBILITY OF ERYTHROCYTES BY SPIDER AND BACTERIAL SPHINGOMYELINASES

Tambourgi D. V.(1), Silva, M.S.(1), Billington, S. J., (2)Andrade, R.M.G.(1), Magnol, F.C.i(1), Glenn Songer, J.(2), Van den Berg, C.W.(3)

(1)Laboratório de Imunoquímica, Instituto Butantan, Brazil; (2)Department of Veterinary Science and Microbiology, University of Arizona, USA, (3)Department of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, UK.

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induces Complement (C) dependent lysis of autologous erythrocytes by induction of cleavage of cell surface glycophorins (GPs) through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway (AP) of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to that observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human E was C dependent and if cleavage of GPs occurred. We show here that haemolysis of both PLD and P1 treated human E was C dependent, but while PLD mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathway. P1 but not PLD induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases Annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidyl serine (PS) to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is due to exposure of PS, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Supported by the Wellcome Trust as a Collaborative Research Initiative Grant to DVT and CWB

CORRESPONDENCE TO:

Tambourgi D. V., Laboratório de Imunoquímica, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil, Email: dvtambourgi@yahoo.com