Snake venom prothrombin activators can be classified in to four groups based on their structure, cofactor requirements and the products formed. Groups A and B are metalloproteases, whereas groups C and D are serine proteases. The latter two groups have been found only in Australian elapids. We showed that group D prothrombin activators are structural and functional homologs of blood coagulation factor Xa. Subsequently, we also showed that group C prothrombin activators are similar to mammalian factor Xa-factor Va complex. These prothrombin activators induce microclots leading to disseminated intravascular coagulopathy, cyanosis and death. Thus they are used as toxins. As snakes also have a blood coagulation system, they possess factor X and factor V which would play critical role in their hemostatic mechanism in their plasma. Our studies have linked origin and evolution of these two parallel prothrombin activator systems which play two distinct physiological roles in Australian elapids.

• We have shown that the two parallel prothrombin activator systems are structurally similar with subtle differences. For example, the activation peptide of the venom protein is much shorter than that of the liver factor X. Further, there is an insertion of 11-residue segment in the heavy chain. The implications of these changes are unclear. Interestingly, sites for proteolytic inactivation by activated protein C (APC) are modified in the venom factor V but not in plasma factor V protein making the venom prothrombin activator resistant to inactivation by APC. This provides a distinct advantage for their use as toxins. Thus these venom prothrombin activators (group C and D) have evolved by gene duplication of coagulation factors and recruited to be expressed in the venom glands.

• During our sequencing studies of factor X gene from Pseudonaja textilis liver, we found two sequences, PFX1 and PFX2. PFX2 has several characteristics similar to venom gene while PFX1 is similar to the factor X gene of other snake factor X. The activation peptide segment in PFX2 is shorter in size, similar to venom protein. There is a smaller insert in the heavy chain. Thus PFX2 appears as an intermediate in the evolution of the factor Xa-like enzymatic subunit of prothrombin activators.  

• Using real-time PCR we showed that the above proteins are expressed with high tissue-specificity in the liver and venom glands. To understand their expression and regulation, we completed the gene sequence of trocarin D, catalytic subunit of pseutarin C and factor Xs from Tropidechis carinatus and Pseudonaja textilis snakes. The venom genes have a insert containing three TATA-like boxes, a GATA box and Y-box in the 5’-upstream region. We identified this segment as VERSE (Venom Recruitment/Switch Element).In addition, the venom genes have three insertions and two deletions in their first introns compared to liver factor X gene. We are currently attempting understand the role of these differences in tissue-specific expression and regulation.

 

 

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