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We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase\u2010<jats:sc>D<\/jats:sc>, causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose\u2010dependent and time\u2010dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP\u2010phospholipase\u2010<jats:sc>D<\/jats:sc> protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin\u2010treated erythrocytes reacted with annexin\u2010V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase\u2010<jats:sc>D<\/jats:sc>, which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine\u2010protease inhibitor) that had no effect on hemolysis. By site\u2010directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that <jats:italic>L. intermedia<\/jats:italic> venom phospholipase\u2010<jats:sc>D<\/jats:sc> triggers direct human blood cell hemolysis in a catalytic\u2010dependent manner. J. Cell. Biochem. 107: 655\u2013666, 2009. \u00a9 2009 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/jcb.22148","type":"journal-article","created":{"date-parts":[[2009,5,19]],"date-time":"2009-05-19T14:49:09Z","timestamp":1242744549000},"page":"655-666","source":"Crossref","is-referenced-by-count":54,"title":["Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase\u2010<scp>D<\/scp> (dermonecrotic toxin) from brown spider venom"],"prefix":"10.1002","volume":"107","author":[{"given":"Daniele","family":"Chaves\u2010Moreira","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga M.","family":"Chaim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Youssef B.","family":"Sade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K\u00e1tia 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