**3. Parallel prothrombin activator system in Australian elapid snakes**

As described above, groups C and D snake venom prothrombin activators are functional and structural homologues of mammalian blood coagulation factors. As snakes are vertebrates, their hemostatic system should contain plasma coagulation factors. Thus, Australian elapid snakes should possess parallel prothrombin activating systems: one in their venom, which is used as an offensive weapon to attack the hemostatic system of the prey, and the other in their plasma, which is used for their own hemostatic purpose. We examined the presence of plasma coagulation factors in the snake's hemostatic system and determined the relationship between the snake venom and plasma coagulation factors.

### **3.1 Trocarin D and FX from** *Tropidechis carinatus* **(TrFX)**

Since the liver mainly expresses plasma coagulation factors, the cDNA encoding *T. carinatus* FX (TrFX) was sequenced from liver tissue (Reza et al. 2005a). The deduced amino acid sequence of TrFX is similar to mammalian FX (~50%) and trocarin D (~80%) (Figure 5). Structurally, TrFX is similar to trocarin D. They both have conserved cysteine residues and identical domain architecture. However, there are some differences between TrFX and trocarin D. The activation peptide of TrFX is similar to the mammalian FXs and not to that of

Fig. 4. APC resistance assay (Rao et al. 2003b). Varying concentrations of APC were added either to pseutarin C (E; 8 nM) or bovine FXa-FVa (F; FXa 42 nM, FVa 2 nM) complex which was diluted in 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 5 mM CaCl2, and 0.5 mg/mL BSA. The reaction mixture was incubated for 30 minutes at room temperature. Prothrombin was added to a final concentration of 2.8 µM and thrombin formed was assayed using thrombin-specific chromogenic substrate S-2238. Each point represents an

0 100 200 300 400 500

BFXa-FVa Pseutarin C

**nM APC**

average of 2 independent experiments each carried out in triplicates.

0

20

40

60

**FVa Activity (%)**

80

100

120

**3.1 Trocarin D and FX from** *Tropidechis carinatus* **(TrFX)** 

**3. Parallel prothrombin activator system in Australian elapid snakes** 

As described above, groups C and D snake venom prothrombin activators are functional and structural homologues of mammalian blood coagulation factors. As snakes are vertebrates, their hemostatic system should contain plasma coagulation factors. Thus, Australian elapid snakes should possess parallel prothrombin activating systems: one in their venom, which is used as an offensive weapon to attack the hemostatic system of the prey, and the other in their plasma, which is used for their own hemostatic purpose. We examined the presence of plasma coagulation factors in the snake's hemostatic system and determined the relationship between the snake venom and plasma coagulation factors.

Since the liver mainly expresses plasma coagulation factors, the cDNA encoding *T. carinatus* FX (TrFX) was sequenced from liver tissue (Reza et al. 2005a). The deduced amino acid sequence of TrFX is similar to mammalian FX (~50%) and trocarin D (~80%) (Figure 5). Structurally, TrFX is similar to trocarin D. They both have conserved cysteine residues and identical domain architecture. However, there are some differences between TrFX and trocarin D. The activation peptide of TrFX is similar to the mammalian FXs and not to that of the venom prothrombin activators (Reza et al. 2005b). It is 57 residues long compared to 27 residues in trocarin D (Figure 5). In addition, there is no 12-residue insert in the heavy chain of TrFX as was observed to be present in the trocarin D precursor (Reza et al. 2005b). These differences in amino acid sequences, and the lengths of activation peptides and insertion in the heavy chain, suggest that TrFX and trocarin D are encoded by two independent genes. Hence, this confirms the presence of a parallel prothrombin activator system. TrFX is more similar to trocarin than to mammalian FX in terms of post-translational modifications (Reza et al. 2005a). TrFX and trocarin D both have *N*- and *O*-glycosylation modifications that are not found in mammalian FXs (as described previously).

Fig. 5. Alignment of deduced amino acid sequences of FX-like proteins from *T. carinatus* (TrFX and trocarin D) and *P. textilis* (PCCS, PFX1 and PFX2) snakes.

Trocarin D and TrFX differ in their physiological roles. Trocarin D plays an offensive role as a toxin in the venom that is used for killing prey. Upon envenomation, like other prothrombin activators (Masci et al. 1988; Rao et al. 2003a), it induces cyanation and death in experimental animals (Joseph et al. 1999) through disseminated intravascular coagulopathy. On the other hand, TrFX plays a crucial role in the coagulation cascade and prevents excessive blood loss by promoting blood coagulation when there is a vascular injury. Trocarin D is an active enzyme and is found in large quantities in the venom. In contrast, TrFX is found as a zymogen, which gets activated only when required and is found in much smaller concentrations in the plasma. Real-time polymerase chain reaction (RT-PCR) was used to determine the amount of expression of these two closely related proteins in the liver and venom gland. The results indicate that trocarin D is expressed in the venom gland but

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 267

The cDNA sequence of *P. textilis* FV (PFV) was determined from its liver (Minh et al. 2005). The deduced amino acid sequence of PFV shows similarities to other mammalian and nonmammalian FVs (~50%) and PCNS (~96%) and shares identical domain architecture (Minh et al. 2005). Like the FVs of other species, PFV and PCNS comprise A1, A2, B, A3, C1 and C2 domains (Figure 3A). Functionally important domains A and C are highly conserved in both PFV and PCNS, whereas domain B is the most variable (Minh et al. 2005). The domain B (126 residues) of PFV is one residue shorter than that of PCNS (127 residues), and is much shorter than that of mammalian and non-mammalian FVs. A more detailed comparison shows that all the FXa and thrombin proteolytic cleavage sites (which are important for activation of these nonenzymatic proteins) are conserved in PFV and PCNS (Figure 3A). However, PFV has an additional FXa proteolytic cleavage site at Arg1765 (Minh et al. 2005; Rao et al. 2003b). This cleavage site also exists in mammalian FV but not in FVs of teleosts (Minh et al. 2005). This is evolutionarily interesting as this additional cleavage site may be a characteristic found only in tetrapod FVs. However, the functional implication of this

As mentioned previously, PCNS has evolved to be resistant to inactivation by activated protein C (APC), which is crucial to its function as a toxin. On the other hand, PFV is similar to other FV, as it can still be inactivated by APC. PFV can be inactivated by APC by cleavage at Arg316, a primitive inactive site (van der Neut et al. 2004a), and at Arg506 (Minh et al. 2005) (Figure 3B). The expression profiles of PFV and PCNS in the liver and the venom gland were determined using RT-PCR. As with other venom prothrombin activator genes, PCNS is expressed only in the venom gland, while PFV is expressed only in the liver. It was found that PCNS is expressed ~280 times higher in the venom gland than is PFV in the liver (Minh

et al. 2005). Thus, PCNS and PFV are have differential expressions (Minh et al. 2005).

Based on sequence comparisons, we confirmed the presence of parallel prothrombin activator systems in Australian elapid snakes and showed for the first time that groups C and D prothrombin activators in snake venom and their plasma coagulation factor counterparts are closely related. We also proposed that these venom prothrombin activators evolved from their plasma coagulation factor counterparts by gene duplication and were

**4. Phylogenetic relationship between snake venom and plasma prothrombin** 

A phylogenetic tree of the snake venom and plasma prothrombin activators with other known FX sequences was constructed to understand their evolutionary relationships using zebrafish FX as the out group (Reza et al. 2006; St Pierre et al. 2005). All the reptilian sequences form a monophyletic group (Reza et al. 2006) (Figure 7). Within the reptilian clade, group C and D prothrombin activators appear as two separate clades on the tree. This indicates that, despite their similarities, group C and D prothrombin activators have originated independently. Interestingly, the PFX2 sequence is found nested within the group C prothrombin activators. This supports the hypothesis that PFX2 is an evolutionary intermediate of PCCS from PFX1. Based on the topology of the phylogenetic tree, it is suggested that these snake venom prothrombin activators have been "recruited" through

**3.3 PCNS and FV from** *Pseudonaja textilis* **(PFV)** 

cleavage with regards to procoagulant activity of FV is not yet known.

subsequently modified to function efficiently as toxins.

independent evolutionary events (Reza et al. 2006) (Figure 7).

**activators** 

not in the liver, while TrFX is expressed in the liver but not in the venom gland. Further, the expression of trocarin D is ~30 times higher in the venom gland than TrFX in the liver (Reza et al. 2007). Such differential expression patterns of trocarin D and TrFX strongly support the distinct physiological roles of these two proteins.

## **3.2 PCCS and FX from** *Pseudonaja textilis* **(PFX)**

To understand the evolution of group C prothrombin activators, we also determined the cDNA sequence of the *P. textilis* FX (PFX) from the liver. Interestingly, two PFX isoforms (PFX1 and PFX2) were detected in the liver, and their cDNA sequences are ~85% similar (Reza et al. 2006). The domain architecture and cysteine residues of these two isoforms are also conserved compared to group D prothrombin activators. Amino acid sequence comparison shows that PFX1 is more similar to TrFX (~94%), while PFX2 is more similar PCCS and trocarin D (~90%) (Figure 5). Further, PFX1 has a longer activation peptide, similar to plasma FXs, whereas PFX2 has a shorter activation peptide, similar to PCCS and trocarin D. Also, PFX2 has a 9-residue insert, which is not present in PFX1. These structural differences suggest that PFX1, PFX2 and PCCS are encoded by three independent genes and that PFX2 is an evolutionary intermediate between PFX1 and PCCS (Reza et al. 2006) (Figure 5). This similarly confirms the presence of a parallel prothrombin activator system. The expression profiles of PFX1, PFX2 and PCCS were determined in liver and venom gland tissues by RT-PCR (Reza et al. 2006). The results show that PFX1 and PFX2 are expressed only in the liver, while PCCS is expressed only in the venom gland. PFX1 is also found to be expressed ~55,000 times higher than PFX2 in the liver, and PCCS is expressed ~80 times higher in the venom gland than is PFX1 in the liver (Reza et al. 2006). In summary, the sequence comparisons and expression profiles indicate that PCCS has evolved from PFX1 by gene duplication and PFX2 is an intermediary product of this "recruitment" process (Figure 6).

Fig. 6. Schematic diagram showing the probable evolutionary path in the recruitment of FX protein as toxin in the venom (Reza et al. 2006).
