The proposed investigation focuses on the mechanism by which the strongly anticoagulant phospholipases from cobra venoms interfere with hemostasis. A hypothesis that strong anticoagulant phospholipases found in cobra venoms have a non-catalytic mechanism will be tested by incubation of the strongest anticoagulant phospholipase with tissue factor deprived of lipid and reconstituted with non-cleavable phospholipid. The basic anticoagulant phospholipase from Naja nigricollis venom will be chemically and proteolytically modified in an attempt to eliminate catalytic activity while retaining the anticoagulant effect. The enzyme will also be cleavedto obtain peptides which will be tested for anticoagulation. All three phospholipase isoenzymes from N. nigricollis will be sequenced to tru to identify cationic and external hydrophobic domains which can contribute to the anticoagulant effect. Reversibility of the anitcoagulant effect with antibodies specific to the anitcoagulant phospholipase will be examined. The classes of phospholipids cleaved and the kinetics of their cleavage from tissue factor and platelets by the three phopholipase isoenzymes will be examined. The lipid analyses will be extended further by the identificaion of fatty acids released during enzyme treatments. All lipid analyses will be correlated with functional studies of coagulation and/or platelet aggregation. These studies should prove insights into the mechanism of action of the strong anticoagulant phospholipases. They should also give new information about structural requirements of lipids critical to the coagulation process and platelet aggregation. Such anticoagulant enzymes might ultimately have clinical applications and should prove to be valuable research tools for future studies on hemostasis, thrombosis and their regulation.
