NEUTRALIZATION OF THE CYTOLYTIC AND MYOTOXIC ACTIVITIES OF PHOSPHOLIPASES A(2) FROM BOTHROPS-ASPER SNAKE-VENOM BY GLYCOSAMINOGLYCANS OF THE HEPARIN/HEPARAN SULFATE FAMILY

Basic phospholipases A(2) from the venom of Bothrops asper exhibit skeletal muscle damaging activity in vivo, and cytolytic activity to a variety of cell types in culture. Glycosaminoglycans of the heparin/heparan sulfate family were found to be potent blockers of the cytolytic action in vitro, and, as well, to be able to neutralize the muscle damaging activity of purified myotoxins and crude venom in vivo. However, the neutralizing effect of heparins was more potent in vitro than in vivo. The cytolytic activity of myotoxin II (a lysine-49 phospholipase A(2) isoform) and its inhibition by heparin was characterized. The neutralizing effect of heparin did not depend on its anticoagulant activity, since both standard heparin and heparin with low affinity for antithrombin (LA-heparin) had a similar efficiency. Heparan sulfate and low molecular mass heparin (5 kDa) also neutralized myotoxin II. In contrast, different heparin-derived disaccharides were unable to block cytolysis, implying a requirement for a longer carbohydrate chain structure for the interaction with the protein. By affinity chromatogaphy and gel diffusion, it was demonstrated that heparins form a complex with all isoforms of basic venom myotoxins, held at least in part by electrostatic interactions. The phospholipase A(2) activity of myotoxin III, a related aspartate-49 isoform from the same venom, was unaffected by heparins, despite the fact that its myotoxic activity was inhibited, indicating a dissociation of the two actions.