ossil remains from ~100 million years ago (Ma) show that snakes were widely distributed across the world by the late Cretaceous period 1. During the course of their evolution, snakes lost their limbs, acquiring a serpentine body 2. Some also evolved or co-opted venom systems to help subdue, capture and digest their prey 2,3. The Colubroides clade of advanced snakes encompasses >3,000 extant species including >600 venomous species 4. The most venomous snakes include the true vipers and pit vipers, both members of the Viperidae family, and cobras, kraits, mambas and sea snakes from the Elapidae family 5. Although humans are not an intended target, accidental contact with venomous snakes can be deadly. Snakebite envenoming is a serious neglected tropical disease that affects ~5 million people worldwide annually, leading to ~400,000 amputations and >100,000 deaths 6. In India alone, the high rural population density combined with the presence of the 'big four' deadly snakes, namely the Indian cobra (Naja naja), Russell's viper (Daboia russelli), sawscaled viper (Echis carinatus) and common krait (Bungarus caeruleus), results in >46,000 snakebite-related deaths annually 7. Snake venom is a potent lethal cocktail rich in proteins and peptides, secreted by specialized venom gland cells. Venom components can be broadly classified as neurotoxic, cytotoxic, cardiotoxic or hemotoxic, and the composition can vary both between and within species 8-11. Currently, snake antivenom is the only treatment effective in the prevention or reversal of the effects of envenomation. Since 1896, antivenom has been developed by immunization of large mammals, such as the horse, with snake venom to generate a cocktail of antibodies that are used for therapy 12. Given the heterologous nature of these antibodies, they often elicit adverse immunological responses when treating snakebite victims 13. Moreover, the antivenom composition is not well defined and its ability to neutralize the venom

SummarySeveral snake venoms contain procoagulant proteins that can activate prothrombin. We have purified pseutarin C, a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilis). It converts prothrombin to thrombin by cleaving both the peptide bonds Arg274 – Thr275 and Arg323 – Ile324, similar to mammalian factor Xa. It is a protein complex (∼250 Kd) consisting of an enzymatic and a nonenzymatic subunit. These subunits were separated by reverse phase HPLC and their interactions with bovine factor Xa and factor Va were studied. The enzymatic subunit of pseutarin C has a ∼13 fold higher affinity for bovine factor Va (K d of 11.4 nM for pseutarin C enzymatic subunit – bovine factor Va interaction as compared to a K d of 147.4 nM for the bovine factor Xa-Va interaction). The non-enzymatic component, however, was unable to activate bovine factor Xa. N-terminal sequence analysis of the catalytic subunit of pseutarin C showed ∼ 60% homology to mammalian factor Xa and ∼78% homology to trocarin, a group D prothrombin activator from Tropidechis carinatus venom. Structural information for the non-enzymatic subunit of pseutarin C was obtained by amino terminal sequencing of several internal peptides. The sequence data obtained indicates that the non-enzymatic subunit of pseutarin C has similar domain architecture like the mammalian factor Va and the overall homology is ∼55%. Thus pseutarin C is the first venom procoagulant protein that is structurally and functionally similar to mammalian factor Xa-Va complex.

Among snake venom procoagulant proteins, group II prothrombin activators are functionally similar to blood coagulation factor Xa. We have purified and partially characterized the enzymatic properties of trocarin, the group II prothrombin activator from the venom of the Australian elapid, Tropidechis carinatus (rough-scaled snake). Prothrombin activation by trocarin is enhanced by Ca2+, phospholipids, and factor Va, similar to that by factor Xa. However, its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The light chain possesses an N-terminal Gla domain containing 11 γ-carboxyglutamic acid residues, followed by two epidermal growth factor (EGF)-like domains; the heavy chain is a serine proteinase. Both chains are likely glycosylated: the light chain at Ser 52 and the heavy chain at Asn 45. Unlike other types of venom procoagulants, trocarin is the first true structural homologue of a coagulation factor. It clots snake plasma and thus may be similar, if not identical, to snake blood coagulation factor Xa. Unlike blood factor Xa, it is expressed in high quantities and in a nonhepatic tissue, making snake venom the richest source of factor Xa-like proteins. It induces cyanosis and death in mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.