Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and
hemolysis. The molecular mechanism by which these toxins cause
hemolysis is under investigation; literature data have suggested that
enzyme catalysis is necessary for the
biological activities triggered by the toxin. However, the way by which
phospholipase-D activity is directly related with human
hemolysis has not been determined. To evaluate how
brown spider venom phospholipase-D activity causes
hemolysis, we examined the impact of recombinant
phospholipase-D on human red blood cells. Using six different purified recombinant
phospholipase-D molecules obtained from a
cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and
phospholipase-D activity. Studying recombinant
phospholipase-D, a potent hemolytic and
phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic
sphingomyelin (SM),
lysophosphatidylcholine (LPC), and
lyso-platelet-activating factor. Additionally, we determined that the toxin degrades
phospholipids in a
detergent extract of human erythrocytes, as well as
phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant
phospholipase-D treatments caused
hemolysis of human erythrocytes. This
hemolysis, dependent on products of metabolism of
phospholipids, is also dependent on
calcium ion concentration because the percentage of
hemolysis increased with an increase in the dose of
calcium in the medium. Recombinant
phospholipase-D treatment of human erythrocytes stimulated an influx of
calcium into the cells that was detected by a
calcium-sensitive
fluorescent probe (Fluo-4). This
calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by
L-type calcium channel inhibitors but not by a
T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of
calcium activated potassium channels. Finally, this inhibition of
hemolysis following recombinant
phospholipase-D treatment occurred in a concentration-dependent manner in the presence of
L-type calcium channel blockers such as
nifedipine and
verapamil. The data provided herein, suggest that the
brown spider venom phospholipase-D-induced
hemolysis of human erythrocytes is dependent on the metabolism of membrane
phospholipids, such as SM and LPC, generating bioactive products that stimulate a
calcium influx into red blood cells mediated by the L-type channel.