portions from glycophorins molecules, rendering the cells sensitive to the lytic action of the autologous complement system.. This study aimed to identify possible membrane proteases involved in this process. The toxin was expressed, purified and showed to be functionally active, being able to render erythrocytes susceptible to the autologous complement-dependent lysis, in a dose dependent manner, and to hydrolyze sphingomyelin.. Treatment of human erythrocytes with the toxin caused the removal of the membrane glycophorins, but did not act on Kell, CD59, DAF and CR1 and induced deposition of C1q, C3, C4, C5b-9, factor B and properdin. Complement-dependent hemolysis, induced by the toxin, was prevented by pretreatment with inhibitors of metalloproteinases, galardin, phenanthroline and bestatin, but not with PMSF, a serine proteinase inhibitor, and simvastatin, an inhibitor of elastase and MMP-9. The proteolytic activity of erythrocyte membranes was explored by using fluorescent substrates. In these assays, it was found that the membrane preparations have basal activity upon the peptides used. However, after treatment with SMase D, the proteolytic activity of the membranes was higher than the control using the substrate Abz-FRSSR-EDDnp. The preincubation with PMSF, simvastatin and, to a lesser degree, with phenanthroline, prevented the increase of this activity. In conclusion, the results suggest that SMase D from L. intermedia spider venom is capable of activating proteases in red blood cells membrane involved in the transformation of human erythrocytes in autologous cell complement activator and which are sensitive to the action of metalloproteinases inhibitors, and others, sensitive to serine proteinases inhibitors, which appear not to be directly involved in the phenomenon of hemolysis and whose biological role has yet to be clarified.
