Hemolytic and Anticoagulant Study of the Neurotoxin Vipoxin and Its Components—Basic Phospholipase A₂ and an Acidic Inhibitor

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V. N. Atanasov¹, D. Danchev², M. Mitewa¹, and S. Petrova^3*

¹Laboratory of Biocoordination and Bioanalytical Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Sofia University, J. Bourchier Ave. 1, 1164 Sofia, Bulgaria; fax: +359-2-962-5438; E-mail: vatanasov@chem.uni-sofia.bg; mmitewa@chem.uni-sofia.bg

²Unit of Hemostasis, Central Clinical Laboratory, Military Medical Academy, G. Sofiiski Str. 3, 1603 Sofia, Bulgaria; fax: +359-2-952-6536; E-mail: dobridanchev@yahoo.com

³Laboratory of Enzymology, Department of Biochemistry, Faculty of Biology, Sofia University, Dr. Tsankov Ave. 8, 1164 Sofia, Bulgaria; fax: +359-2-865-6641; E-mail: spetrova@biofac.uni-sofia.bg

^* To whom correspondence should be addressed.

Received June 2, 2008; Revision received September 5, 2008

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In the present study, we demonstrate for the first time that the potent neurotoxin vipoxin from the venom of Vipera ammodytes meridionalis exhibits hemolytic and anticoagulant properties. By investigating the effects of phospholipids and calcium ions on hemolysis, we established that the phospholipase A₂ (PLA₂) enzyme activity is responsible for the hemolytic properties. This was confirmed by chemical modification of the PLA₂ active-site histidine residue with p-bromophenacylbromide. Applying different clotting assays, we show that the PLA₂ is a weakly anticoagulant enzyme, which affects intrinsic tenase complex by the hydrolysis of procoagulant phospholipids, rather than by nonenzymatic mechanisms (binding to specific coagulation factors). The whole complex—vipoxin—does not affect the coagulation system.

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KEY WORDS: vipoxin, phospholipase A₂, hemolysis, anticoagulant activity

DOI: 10.1134/S0006297909030055

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