RESUMO
Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.