THE ANTICOAGULANT PHOSPHOLIPASE A2 FROM VIPER A BERUS VENOM

Abstract

An anticoagulant protein has been isolated by several Chromatographie steps from the venom of vipera berus orientale. The inhibitor appeared as a protein with a molecular weight of 13,400 formed by 119 amino acid residues, with six disulfide bridges without free SH groups. Its isoelectric point was 9.2. Deprived of any proteolytic activity, it displayed a high phospholipase A2 activity in the presence of calcium. Its enzymatic activity was studied on isolated phospholipids, on whole platelet phospholipid and on brain cephalin. The anticoagulant activity on plasma was explained by antagonism to phospholipid procoagulant activity. One μg impaired the clotting of 1 ml of citrated recalcified human plasma. The unit of berus anticoagulant activity was similar to that of the heparin anticoagulant unit. Both phospholipase and anticoagulant activities were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolytic process as shown by complete hydrolysis of brain cephalin with berus inhibitor and with other venom phospholipase A2- The inhibitor would complex phospholipid at its protein binding site, impairing the normal arrangement of coagulation protein factors and consequently their activation. The positive charges of the and factor could play an important part in the formation of the complex. The anticoagulant effect was reversible with addition of specific antibodies.