Intravascular hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms.

A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe intravascular hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause intravascular hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mN m-1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.

Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: identification of directly hemolytic phospholipases A2.

Intravascular hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of intravascular hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing intravascular hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of intravascular hemolysis.