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Snake venoms are excellent sources of pharmacologically active proteins and peptides, and hence are potential sources of leads for drug developments. It has been previously established that krait (Bungarus genus) venoms contain mainly neurotoxins. A screening for anticoagulants showed that Bungarus fasciatus venom exhibits potent anticoagulant effect in standard clotting assays. Through sequential fractionation of the venom by size exclusion and high performance liquid chromatographies, coupled with functional screening for anticoagulant activities, we have isolated and purified two anticoagulant proteins, termed BF-AC1 (Bungarus fasciatusanticoagulant 1) and BF-AC2. They have potent inhibitory activities (IC50 of 10 nM) on the extrinsic tenase complex. Structurally, these proteins each has two subunits covalently held together by disulfide bond(s). The N-terminal sequences of the individual subunits of BF-AC1 and BF-AC2 showed that the larger subunit is homologous to phospholipase A2, while the smaller subunit is homologous to Kunitz type serine proteinase inhibitor. Functionally, in addition to their anticoagulant activity, these proteins showed presynaptic neurotoxic effects in both in vivo and ex vivo experiments. Thus, BF-AC1 and BF-AC2 are structurally and functionally similar to β-bungarotoxins, a class of neurotoxins. The enzymatic activity of phospholipase A2 subunit plays a significant role in the anticoagulant activities. This is the first report on the anticoagulant activity ofβ-bungarotoxins and these results expand on the existing catalogue of haemostatically active snake venom proteins.

It is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows approximately 80% identity with trocarin D but approximately 50% identity with the mammalian FX. Our present study confirms the presence of two separate genes--one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.

Pseutarin C, a group C prothrombin activator from Pseudonaja textilis venom, is a large protein complex consisting of catalytic and nonenzymatic subunits, which are functionally similar to the mammalian FXa-FVa complex. Here, we present the complete cDNA sequence of the catalytic subunit of pseutarin C. The cDNA of the catalytic subunit encodes a protein of 449 amino acids, which includes a 22-residue signal peptide, 18-residue propeptide and a mature protein of 409 amino acids. The deduced amino acid sequence shows 74-83% identity to group D prothrombin activators from snake venom and approximately 42% identity to mammalian FX and has identical domain structure. The precursor of the catalytic subunit of pseutarin C has several unique features. The activation peptide of the catalytic subunit of pseutarin C is significantly smaller (27 as compared to 52 residues in mammalian FX) and does not contain any glycosylation sites. Unlike coagulation FXa, Ser52 and Asn45 of the light and heavy chains are O- and N-glycosylated in pseutarin C catalytic sub-unit. There is a 12-residue insertion in pseutarin C catalytic sub-unit close to the region that is implicated in binding to FVa. This is the first sequence of the catalytic subunit of a group C prothrombin activator.

The three phospholipase A2 isoenzymes from Naja nigricollis crawshawii snake venom inhibit both blood coagulation and platelet aggregation. To investigate the correlation between phospholipid splitting ability of these enzymes and their inhibitory activities, the effects of various preincubation times and the inclusion of EDTA were examined. Preincubation of plasma and thromboplastin with the phospholipase isoenzymes resulted in an increase in Ca2+-initiated clotting time with time of preincubation. Incubation of the isoenzymes with EDTA before their addition to the plasma-thromboplastin mixture reduced the anticoagulant effect. These results indicate that the catalytic activity contributes at least partially to the anticoagulant effect. However, inhibition of platelet aggregation appears to be independent of enzymatic activity since there is no increase in inhibition with time of preincubation of platelets and phospholipases, and inclusion of EDTA has no significant effect on inhibition of aggregation. All three enzymes appear to belong to class B of the platelet affector PLA2 enzymes as determined by platelet effects, since they do not initiate platelet aggregation.