**2. Snake venom prothrombin activators**

As mentioned above, the circulatory system is one of the main targets of snake venom toxins. These toxins affect heart function (e.g., cardiotoxins), vasculature and blood pressure (e.g., sarafotoxins, natriuretic peptides and hemorrhagic toxins), blood coagulation (e.g., procoagulant and anticoagulant proteins), platelet aggregation (e.g., agonists and antagonists) and fibrinolysis (e.g., direct and indirect fibrinolytic proteinases) (Braud et al. 2000; Chow and Kini 2001; Hutton and Warrell 1993; Kini 2004; Kini and Chow 2001; Kini and Evans 1990; Markland 1997; Markland 1998; Morita 2004). All procoagulant proteins are generally proteinases that promote blood coagulation by activating zymogens of specific plasma coagulation factors (Davie 2003). Prothrombin activators are a group of procoagulant proteins that specifically activate prothrombin to thrombin, which then induces blood coagulation through fibrin clot formation.

Several prothrombin activators have been identified and characterized from snake venoms (Gao et al. 2002; Hasson et al. 2003; Joseph and Kini 2001; Kornalik and Blomback 1975; Morita and Iwanaga 1978; Rosing and Tans 1991; Rosing and Tans 1992; Schieck et al. 1972; Silva et al. 2003; Speijer et al. 1986; St Pierre et al. 2005; Yamada and Morita 1997), and based on their properties (structure, cofactor requirements and end-products formed), these snake venom prothrombin activators have been classified into four groups (Kini et al. 2001) (Table 1). Group A and B prothrombin activators, such as ecarin from *Echis carinatus* venom (Kornalik and Blomback 1975; Morita and Iwanaga 1978; Nishida et al. 1995; Schieck et al. 1972) and multactivase from *E. multisquamatus* venom (Yamada and Morita 1997), are metalloproteinases which induce coagulation by converting prothrombin to meizothrombin. These proteins are structurally distinct from blood coagulation factors. Group C and D prothrombin activators, such as oscutarin from *Oxyranus scutellatus* venom (Owen and Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and Burnell 2005) and notecarin from *Notechis scutatus scutatus* venom (Tans et al. 1985), are serine proteinases that induce coagulation by converting prothrombin to thrombin. These proteins exhibit functional similarity to mammalian plasma coagulation factors. However, no detailed structural information of these proteins was available. To fill this void, we initiated structural studies of these prothrombin activators. We characterized one representative each of group C (pseutarin C) and group D (trocarin D) prothrombin activators. Our results show that snake venom prothrombin activators are structurally and functionally similar to mammalian plasma coagulation factors.


Table 1. Classification of snake venom prothrombin activators

## **2.1 Group D prothrombin activators**

258 Gene Duplication

As mentioned above, the circulatory system is one of the main targets of snake venom toxins. These toxins affect heart function (e.g., cardiotoxins), vasculature and blood pressure (e.g., sarafotoxins, natriuretic peptides and hemorrhagic toxins), blood coagulation (e.g., procoagulant and anticoagulant proteins), platelet aggregation (e.g., agonists and antagonists) and fibrinolysis (e.g., direct and indirect fibrinolytic proteinases) (Braud et al. 2000; Chow and Kini 2001; Hutton and Warrell 1993; Kini 2004; Kini and Chow 2001; Kini and Evans 1990; Markland 1997; Markland 1998; Morita 2004). All procoagulant proteins are generally proteinases that promote blood coagulation by activating zymogens of specific plasma coagulation factors (Davie 2003). Prothrombin activators are a group of procoagulant proteins that specifically activate prothrombin to thrombin, which then

Several prothrombin activators have been identified and characterized from snake venoms (Gao et al. 2002; Hasson et al. 2003; Joseph and Kini 2001; Kornalik and Blomback 1975; Morita and Iwanaga 1978; Rosing and Tans 1991; Rosing and Tans 1992; Schieck et al. 1972; Silva et al. 2003; Speijer et al. 1986; St Pierre et al. 2005; Yamada and Morita 1997), and based on their properties (structure, cofactor requirements and end-products formed), these snake venom prothrombin activators have been classified into four groups (Kini et al. 2001) (Table 1). Group A and B prothrombin activators, such as ecarin from *Echis carinatus* venom (Kornalik and Blomback 1975; Morita and Iwanaga 1978; Nishida et al. 1995; Schieck et al. 1972) and multactivase from *E. multisquamatus* venom (Yamada and Morita 1997), are metalloproteinases which induce coagulation by converting prothrombin to meizothrombin. These proteins are structurally distinct from blood coagulation factors. Group C and D prothrombin activators, such as oscutarin from *Oxyranus scutellatus* venom (Owen and Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and Burnell 2005) and notecarin from *Notechis scutatus scutatus* venom (Tans et al. 1985), are serine proteinases that induce coagulation by converting prothrombin to thrombin. These proteins exhibit functional similarity to mammalian plasma coagulation factors. However, no detailed structural information of these proteins was available. To fill this void, we initiated structural studies of these prothrombin activators. We characterized one representative each of group C (pseutarin C) and group D (trocarin D) prothrombin activators. Our results show that snake venom prothrombin activators are structurally and functionally similar to mammalian

**2. Snake venom prothrombin activators** 

induces blood coagulation through fibrin clot formation.

**Type of proteinase** 

Metalloproteinase Meizothrombin

B Ca2+ Two subunits of

Serine proteinase Thrombin

Table 1. Classification of snake venom prothrombin activators

**Product** 

**formed Estimated Size**

~47 kDa

~25 and ~60 kDa

Two subunits of ~60 and ~220 kDa

~60 kDa FXa

**Similar to Plasma Coagulation Factors** 

None

"FXa-FVa" complex

**Examples** 

Ecarin

Carinactivase, multactivase

Pseutarin C oscutarin,

Trocarin D, hopsarin D, notanarin D

plasma coagulation factors.

Group **Cofactor Requirements**

A None

<sup>C</sup>Ca2+ *plus* phospholipids

> Ca2+ *plus* phospholipids *plus* factor Va

D

Group D prothrombin activators are found exclusively in the venom of Australian elapid snakes (Rosing and Tans 1991). Notecarin from *Notechis scutatus scutatus* venom was the first member of this group to be isolated and characterized (Tans et al. 1985). Since then, similar prothrombin activators have been characterized from several other snake venoms. They are glycoproteins with a molecular weight of ~50 kDa (Table 1). As a group of proteins, they share striking resemblances and requirements for optimal activity with activated mammalian plasma coagulation factor X (FXa) (Table 1) (Joseph et al. 1999; Marsh et al. 1997; Rao and Kini 2002; Stocker et al. 1994; Tans et al. 1985).

Venom of the Australian elapid *Tropidechis carinatus* (rough-scaled snake) was documented to have procoagulant properties 29 years ago (Chester and Crawford 1982). A prothrombin activator was isolated using gel filtration and benzamidine-based affinity chromatography and was partially characterized (Marsh et al. 1997). Our laboratory purified a prothrombin activator, trocarin D, from *T. carinatus* venom to homogeneity using a series of high performance liquid chromatography techniques including gel filtration, ion-exchange and reverse-phase chromatographies (Joseph et al. 1999). This purification procedure was refined to a single-step reverse-phase chromatographic method and was subsequently used for the purification of several other group D prothrombin activators such as notanarin D from *N. ater niger* venom, notecarin D from *N. scutatus* venom and hopsarin D from *Hoplocephalus stephensi* venom (Rao et al. 2003a). Our laboratory characterized trocarin D for its functional and structural properties in detail as a representative of group D prothrombin activators.

Functionally, trocarin D has properties which are similar to mammalian plasma coagulation FXa. They both promote blood coagulation by activating prothrombin to thrombin (Joseph et al. 1999). Trocarin D and FXa achieve activation of prothrombin by cleaving the same peptide bonds (Arg274-Thr275 and Arg323-Ile324). Both proteins have identical co-factor requirements of Ca2+ ions, phospholipids and activated factor V (FVa) for their optimal activities (Joseph et al. 1999). We determined the amino acid sequence of trocarin D and its precursor using Edman degradation (Joseph et al. 1999) and cDNA sequencing (Reza et al. 2005a), respectively. Trocarin D and mammalian FXa share significant sequence identity (~53-60%) and exhibit identical domain architecture (Joseph et al. 1999; Rao et al. 2003a) (Figure 1). Both proteins comprise two chains: a heavy chain, which has a serine proteinase with the characteristic catalytic triad (His42, Asp88 and Ser185), and a light chain, which has a Gla domain followed by two epidermal-growth factor-like domains (EGF-I and EGF-II). These two chains are held together by a single inter-chain disulfide bond (Joseph et al. 1999) (Figure 1). The differences between trocarin D and mammalian FXa reside in an insertion in the heavy chain, the size of the activation peptide and post-translational modifications. Firstly, there is a 12-residue insert in the heavy chain of trocarin D (Reza et al. 2005a). However, the functional importance of this insertion is not clear. Secondly, the activation peptide of trocarin D precursor is only 27 residues long (Reza et al. 2005a) compared to the activation peptides of mammalian FXs which ranges from 48 to 52 residues (Figure 1). Lastly, post-translational modifications show that trocarin D is glycosylated, but mammalian FXa is not. In addition, trocarin D also contains a *O*-linked carbohydrate at Ser52 of the light chain and a *N*-linked carbohydrate at Asn45 of the heavy chain (Joseph et al. 1999) (Figure 1). Interestingly, the *O*-linked carbohydrate moiety has a *N*-acetylglucosamine moiety, which is found commonly in nuclear and cytoplasmic proteins but rarely in secreted proteins (Hanover et al. 1987; Holt et al. 1987; Holt and Hart 1986; Snow et al. 1987). The

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 261

Pseutarin C was purified from *P. textilis* venom, and it activates prothrombin to thrombin. For its optimal activity, pseutarin C requires only Ca2+ ions and phospholipids (Rao and Kini 2002). These functional characteristics are similar to that of the mammalian "FXa-FVa" complex. As with other group C prothrombin activators, pseutarin C comprises two subunits of ~60 kDa and ~220 kDa (Rao and Kini 2002) (Table 1). The smaller subunit, with serine proteinase activity, was termed the pseutarin C catalytic subunit (PCCS) and the larger subunit, which has no enzymatic activity, was termed the pseutarin C nonenzymatic subunit (PCNS) (Rao and Kini 2002). A comparison of the protein quantities in the venom and plasma revealed that pseutarin C is expressed ~4,200 times higher in the venom than the amount of FV and FX in the plasma (Rao and Kini 2002). We purified pseutarin C and its subunits and characterized them both functionally and structurally as representatives of the

*1. Pseutarin C catalytic subunit (PCCS) –* Functionally, PCCS is similar to mammalian FXa and group D prothrombin activators (Rao and Kini 2002). They have the same co-factor requirements, including Ca2+ ions, phospholipids and FVa, for their optimal activity and activate prothrombin by cleaving the same two peptide bonds (Arg274-Thr275 and Arg323- Ile324). The enzymatic activity (*Vmax*) of PCCS is enhanced by the presence of FVa (Rao and Kini 2002). The amino acid sequence of PCCS and its precursor was determined using both Edman degradation and cDNA sequencing (Rao et al. 2004; Rao and Kini 2002). Structurally, PCCS is also similar to mammalian FXa and group D prothrombin activators (Figure 1). Its sequence shows ~42% identity to mammalian FXa and 74-83% identity to group D prothrombin activators (Rao et al. 2004). Like mammalian FXa and group D prothrombin activators (Rao et al. 2004), the domain architecture of PCCS consists of a light and a heavy chain that are linked by a single disulfide bond (Rao et al. 2004). The light chain has a Gla domain followed by two EGF-like domains, and the heavy chain contains a serine proteinase domain (Figure 1). Despite such functional and structural similarities, the differences between PCCS and mammalian FXa reside in the size of the activation peptide and post-translational modifications (Rao et al. 2004). Similar to trocarin D precursor, the activation peptide of PCCS precursor is 27 residues long and is significantly shorter than those of mammalian FXs (Figure 1). Like trocarin D, it has an insertion in its heavy chain. Interestingly, the PCCS insert is 13 residues long and is distinctly different from the 12 residue insert in trocarin D (Rao et al. 2004; Reza et al. 2005b). This strongly indicates that the evolution of groups C and D prothrombin activators are independent. Like trocarin D, the Ser52 and Asn45 residues of the light and heavy chains of PCCS are *O*- and *N*glycosylated, respectively (Figure 1). However, as mentioned previously, these two residues have no post-translational modifications in mammalian FX/FXa. These functional and structural characteristics suggest that PCCS is a homologue of mammalian FXa and group D

*2. Pseutarin C nonenzymatic subunit (PCNS) –* Structurally, PCNS is similar to mammalian FV. The amino acid sequence of PCNS and its precursor was determined using Edman degradation and cDNA sequencing (Rao et al. 2003b; Rao and Kini 2002). PCNS shares ~50% identity with the mammalian FV and has identical domain architecture with mammalian FV (Rao et al. 2003b). Both PCNS and mammalian FV have six domains: A1, A2, B, A3, C1 and C2 (Figure 2). Domains A and C are functionally important, and these domains are highly conserved in PCNS and FVs of other species (Rao et al. 2003b). These structural similarities

suggest that PCNS is a homologue of mammalian FV (Rao et al. 2003b).

group C prothrombin activators.

prothrombin activators.

function of glycosylation is hypothesized to protect trocarin D from inactivation by proteolysis and confer it with thermal stability (Rao et al. 2003a; Wang et al. 1996). Although human FX contains two *O-*glycosylation and two *N-*glycosylation moieties (Inoue and Morita 1993), all the carbohydrate moieties are exclusively found on the activation peptide, which is removed when FX is activated. Therefore, activated mammalian FXa is not glycosylated. In contrast, the short activation peptide of trocarin D does not have any glycosylation sites. The last difference in post-translational modification is that the Asp63 of FX light chain EGF-I domain is *β*-hydroxylated (McMullen et al. 1983; Stenflo et al. 1987), but the corresponding residue in trocarin D is not (Joseph et al. 1999) (Figure 1).

Fig. 1. Domain architecture of mammalian FX, and precursors of trocarin D and PCCS. The Ys represent Gla residues; open triangles represent the *O-*linked carbohydrate at Ser52 on trocarin D and PCCS; solid triangles represent the *N-*linked carbohydrate at Asn45 on trocarin D and PCCS; the solid diamond represents *β-*hydroxylation at Asp62 on mammalian FXa.

## **2.2 Group C prothrombin activators**

The group C snake venom prothrombin activators are also found exclusively in the venoms of Australian elapid snakes (Rosing and Tans 1991). Oscutarin from *Oxyuranus scutellatus* venom was the first member of the group C prothrombin activators to be isolated and characterized (Owen and Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and Burnell 2005). This group of proteins is generally ~300 kDa in size and comprises two subunits (~60 and ~220 kDa) (Table 1). The smaller enzymatic subunit is a serine proteinase and has characteristics of FXa, whereas the nonenzymatic subunit resembles activated mammalian plasma coagulation factor V (FVa). Overall, group C prothrombin activators have striking resemblances to and similar co-factor requirements as the mammalian plasma coagulant "FXa-FVa" complex (Filippovich et al. 2005; Masci et al. 1988; Rao and Kini 2002; Speijer et al. 1986; Walker et al. 1980) (Table 1).

function of glycosylation is hypothesized to protect trocarin D from inactivation by proteolysis and confer it with thermal stability (Rao et al. 2003a; Wang et al. 1996). Although human FX contains two *O-*glycosylation and two *N-*glycosylation moieties (Inoue and Morita 1993), all the carbohydrate moieties are exclusively found on the activation peptide, which is removed when FX is activated. Therefore, activated mammalian FXa is not glycosylated. In contrast, the short activation peptide of trocarin D does not have any glycosylation sites. The last difference in post-translational modification is that the Asp63 of FX light chain EGF-I domain is *β*-hydroxylated (McMullen et al. 1983; Stenflo et al. 1987),

Fig. 1. Domain architecture of mammalian FX, and precursors of trocarin D and PCCS. The Ys represent Gla residues; open triangles represent the *O-*linked carbohydrate at Ser52 on trocarin D and PCCS; solid triangles represent the *N-*linked carbohydrate at Asn45 on trocarin D and PCCS; the solid diamond represents *β-*hydroxylation at Asp62 on mammalian

The group C snake venom prothrombin activators are also found exclusively in the venoms of Australian elapid snakes (Rosing and Tans 1991). Oscutarin from *Oxyuranus scutellatus* venom was the first member of the group C prothrombin activators to be isolated and characterized (Owen and Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and Burnell 2005). This group of proteins is generally ~300 kDa in size and comprises two subunits (~60 and ~220 kDa) (Table 1). The smaller enzymatic subunit is a serine proteinase and has characteristics of FXa, whereas the nonenzymatic subunit resembles activated mammalian plasma coagulation factor V (FVa). Overall, group C prothrombin activators have striking resemblances to and similar co-factor requirements as the mammalian plasma coagulant "FXa-FVa" complex (Filippovich et al. 2005; Masci et al. 1988; Rao and Kini 2002;

FXa.

**2.2 Group C prothrombin activators** 

Speijer et al. 1986; Walker et al. 1980) (Table 1).

but the corresponding residue in trocarin D is not (Joseph et al. 1999) (Figure 1).

Pseutarin C was purified from *P. textilis* venom, and it activates prothrombin to thrombin. For its optimal activity, pseutarin C requires only Ca2+ ions and phospholipids (Rao and Kini 2002). These functional characteristics are similar to that of the mammalian "FXa-FVa" complex. As with other group C prothrombin activators, pseutarin C comprises two subunits of ~60 kDa and ~220 kDa (Rao and Kini 2002) (Table 1). The smaller subunit, with serine proteinase activity, was termed the pseutarin C catalytic subunit (PCCS) and the larger subunit, which has no enzymatic activity, was termed the pseutarin C nonenzymatic subunit (PCNS) (Rao and Kini 2002). A comparison of the protein quantities in the venom and plasma revealed that pseutarin C is expressed ~4,200 times higher in the venom than the amount of FV and FX in the plasma (Rao and Kini 2002). We purified pseutarin C and its subunits and characterized them both functionally and structurally as representatives of the group C prothrombin activators.

*1. Pseutarin C catalytic subunit (PCCS) –* Functionally, PCCS is similar to mammalian FXa and group D prothrombin activators (Rao and Kini 2002). They have the same co-factor requirements, including Ca2+ ions, phospholipids and FVa, for their optimal activity and activate prothrombin by cleaving the same two peptide bonds (Arg274-Thr275 and Arg323- Ile324). The enzymatic activity (*Vmax*) of PCCS is enhanced by the presence of FVa (Rao and Kini 2002). The amino acid sequence of PCCS and its precursor was determined using both Edman degradation and cDNA sequencing (Rao et al. 2004; Rao and Kini 2002). Structurally, PCCS is also similar to mammalian FXa and group D prothrombin activators (Figure 1). Its sequence shows ~42% identity to mammalian FXa and 74-83% identity to group D prothrombin activators (Rao et al. 2004). Like mammalian FXa and group D prothrombin activators (Rao et al. 2004), the domain architecture of PCCS consists of a light and a heavy chain that are linked by a single disulfide bond (Rao et al. 2004). The light chain has a Gla domain followed by two EGF-like domains, and the heavy chain contains a serine proteinase domain (Figure 1). Despite such functional and structural similarities, the differences between PCCS and mammalian FXa reside in the size of the activation peptide and post-translational modifications (Rao et al. 2004). Similar to trocarin D precursor, the activation peptide of PCCS precursor is 27 residues long and is significantly shorter than those of mammalian FXs (Figure 1). Like trocarin D, it has an insertion in its heavy chain. Interestingly, the PCCS insert is 13 residues long and is distinctly different from the 12 residue insert in trocarin D (Rao et al. 2004; Reza et al. 2005b). This strongly indicates that the evolution of groups C and D prothrombin activators are independent. Like trocarin D, the Ser52 and Asn45 residues of the light and heavy chains of PCCS are *O*- and *N*glycosylated, respectively (Figure 1). However, as mentioned previously, these two residues have no post-translational modifications in mammalian FX/FXa. These functional and structural characteristics suggest that PCCS is a homologue of mammalian FXa and group D prothrombin activators.

*2. Pseutarin C nonenzymatic subunit (PCNS) –* Structurally, PCNS is similar to mammalian FV. The amino acid sequence of PCNS and its precursor was determined using Edman degradation and cDNA sequencing (Rao et al. 2003b; Rao and Kini 2002). PCNS shares ~50% identity with the mammalian FV and has identical domain architecture with mammalian FV (Rao et al. 2003b). Both PCNS and mammalian FV have six domains: A1, A2, B, A3, C1 and C2 (Figure 2). Domains A and C are functionally important, and these domains are highly conserved in PCNS and FVs of other species (Rao et al. 2003b). These structural similarities suggest that PCNS is a homologue of mammalian FV (Rao et al. 2003b).

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 263

is not conserved in PCNS (Rao et al. 2003b). Secondly, FVa is inactivated by phosphorylation at Ser692 (Kalafatis et al. 1994). This phosphorylation site is not present in PCNS (Rao et al. 2003b). Lastly, PCNS is shielded from APC inactivation (Nesheim et al. 1982; Rao et al. 2003b) and is kept activated through its constant and stable association with FXa-like PCCS (Rao et al. 2003a; Rao et al. 2004; Thorelli et al. 1998) in the venom. We have shown experimentally that pseutarin C is unaffected by APC, while bovine FXa-FVa complex is completely inactivated (Bos et al. 2009; Rao et al. 2003b) (Figure 4). Overall, PCNS is a good example of how a toxin gene is duplicated from an ancestral gene and undergoes modifications to gain unique characteristics that allow it to function efficiently as a toxin.

Fig. 3. Functionally important proteolytic sites in PCNS and FV. (A) Activation sites.

in purple arrow. Inactivation sites are missing in PCNS.

Thrombin and FXa cleavage sites of PCNS, PFV and bovine FV are shown in blue, green and red, respectively. Critical activation sites are conserved in PCNS and PFV. (B) Inactivation sites. Activated protein C (APC) cleavage sites of PFV and bovine FV are shown in green and red, respectively. In addition to Arg505, PFV has a primitive inactive site at Arg316 shown

Fig. 2. Domain architecture of bovine FV, PCNS precursor and PFV. *N-*glycosylation sites are shown as colored knobs. Black knobs are conserved, while red knobs are found only in PCNS and PFV, and blue knobs are found only in PFV.

Despite being a FV homologue, PCNS shows several differences with FV from other species. Firstly, the domain B size of PCNS is significantly smaller (127 residues) than that of fishes (fugu: 530 residues and zebrafish: 756 residues) and mammals (murine: 843 residues, bovine: 869 residues, and human: 882 residues) (Rao et al. 2003b) (Figure 2). During FV activation, domain B is removed by thrombin or FXa by cleavage at three activation sites: Arg709, Arg1018 and Arg1545 (bovine FV numbering) (Foster et al. 1983; Nesheim et al. 1979; Suzuki et al. 1982) (Figure 3B). Although only two of these sites (Arg709 and Arg1545) are conserved in PCNS (Figure 3B), the complete domain B can still be cleaved off during the activation of PCNS (Rao et al. 2003b). Thus, the difference in domain B size should not have any effect on the function of PCNS. Secondly, PCNS and FV of other species have different post-translation modifications. While bovine FV has 29 glycosylation sites, PCNS has only 11 potential N-glycosylation sites (Rao et al. 2003b) (Figure 2). Mammalian FV is phosphorylated at the Ser692 residue, but PCNS is not (Rao et al. 2003b). In addition, there are six sulfation sites in human FV that are absent in PCNS (Rao et al. 2003b). This difference in post-translation modifications is interesting, as sulfation and phosphorylation are important for regulating the activation of human FV by thrombin (Kalafatis et al. 1994; Pittman et al. 1994).

Aside from these differences, it is noted that PCNS possesses certain modifications and associations that allow it to function efficiently as a toxin (Bos et al. 2009). Firstly, PCNS has evolved a way to evade inactivation by protein C. In the human coagulation system, protein C is activated by thrombin in the presence of thrombomodulin, and activated protein C (APC) subsequently inactivates FVa in a negative feedback loop (Esmon 2001). This inactivation occurs by proteolytic cleavage at three sites on the FVa heavy chain: Arg306, Arg506 and Arg662 (Kalafatis et al. 1994; Mann et al. 1997) in bovine FVa (Figure 3B). PCNS is able to evade APC inactivation, as it does not have any of the three APC cleavage sites (Rao et al. 2003b). Even an alternate less efficient cleavage site at Arg316 (van der Neut et al. 2004b)

Fig. 2. Domain architecture of bovine FV, PCNS precursor and PFV. *N-*glycosylation sites are shown as colored knobs. Black knobs are conserved, while red knobs are found only in

Despite being a FV homologue, PCNS shows several differences with FV from other species. Firstly, the domain B size of PCNS is significantly smaller (127 residues) than that of fishes (fugu: 530 residues and zebrafish: 756 residues) and mammals (murine: 843 residues, bovine: 869 residues, and human: 882 residues) (Rao et al. 2003b) (Figure 2). During FV activation, domain B is removed by thrombin or FXa by cleavage at three activation sites: Arg709, Arg1018 and Arg1545 (bovine FV numbering) (Foster et al. 1983; Nesheim et al. 1979; Suzuki et al. 1982) (Figure 3B). Although only two of these sites (Arg709 and Arg1545) are conserved in PCNS (Figure 3B), the complete domain B can still be cleaved off during the activation of PCNS (Rao et al. 2003b). Thus, the difference in domain B size should not have any effect on the function of PCNS. Secondly, PCNS and FV of other species have different post-translation modifications. While bovine FV has 29 glycosylation sites, PCNS has only 11 potential N-glycosylation sites (Rao et al. 2003b) (Figure 2). Mammalian FV is phosphorylated at the Ser692 residue, but PCNS is not (Rao et al. 2003b). In addition, there are six sulfation sites in human FV that are absent in PCNS (Rao et al. 2003b). This difference in post-translation modifications is interesting, as sulfation and phosphorylation are important for regulating the activation of human FV by thrombin (Kalafatis et al. 1994;

Aside from these differences, it is noted that PCNS possesses certain modifications and associations that allow it to function efficiently as a toxin (Bos et al. 2009). Firstly, PCNS has evolved a way to evade inactivation by protein C. In the human coagulation system, protein C is activated by thrombin in the presence of thrombomodulin, and activated protein C (APC) subsequently inactivates FVa in a negative feedback loop (Esmon 2001). This inactivation occurs by proteolytic cleavage at three sites on the FVa heavy chain: Arg306, Arg506 and Arg662 (Kalafatis et al. 1994; Mann et al. 1997) in bovine FVa (Figure 3B). PCNS is able to evade APC inactivation, as it does not have any of the three APC cleavage sites (Rao et al. 2003b). Even an alternate less efficient cleavage site at Arg316 (van der Neut et al. 2004b)

PCNS and PFV, and blue knobs are found only in PFV.

Pittman et al. 1994).

is not conserved in PCNS (Rao et al. 2003b). Secondly, FVa is inactivated by phosphorylation at Ser692 (Kalafatis et al. 1994). This phosphorylation site is not present in PCNS (Rao et al. 2003b). Lastly, PCNS is shielded from APC inactivation (Nesheim et al. 1982; Rao et al. 2003b) and is kept activated through its constant and stable association with FXa-like PCCS (Rao et al. 2003a; Rao et al. 2004; Thorelli et al. 1998) in the venom. We have shown experimentally that pseutarin C is unaffected by APC, while bovine FXa-FVa complex is completely inactivated (Bos et al. 2009; Rao et al. 2003b) (Figure 4). Overall, PCNS is a good example of how a toxin gene is duplicated from an ancestral gene and undergoes modifications to gain unique characteristics that allow it to function efficiently as a toxin.

Fig. 3. Functionally important proteolytic sites in PCNS and FV. (A) Activation sites. Thrombin and FXa cleavage sites of PCNS, PFV and bovine FV are shown in blue, green and red, respectively. Critical activation sites are conserved in PCNS and PFV. (B) Inactivation sites. Activated protein C (APC) cleavage sites of PFV and bovine FV are shown in green and red, respectively. In addition to Arg505, PFV has a primitive inactive site at Arg316 shown in purple arrow. Inactivation sites are missing in PCNS.

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 265

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

iver\_type\_1 90 iver\_type\_2 90 Cat 90

iver\_type\_1 180 iver\_type\_2 180 Cat 180

iver\_type\_1 270 iver\_type\_2 241 Cat 241

iver\_type\_1 347 iver\_type\_2 327 Cat 331

iver\_type\_1 437 iver\_type\_2 417 Cat 421

iver\_type\_1 483 iver\_type\_2 463 Cat 449

D D D D D

R R R R R

H H H H H

Y Y Y Y Y

V V V V V

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Q Q Q Q Q

N N N N A A A A A

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N N N N N

G G G G G

S S S S S

L L L L L

MAPQLLLCLILTFLWSLSEAESNVFLKSKVANRFLQRTKRANSLFEEFKAGNIERECIEERCSKEEAREAFEDNEKTETFWNVYVDGDQC MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLFEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLVEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLVEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRSNSLFEEIRPGNIERECIEEKCSKEEAREVFEDNEKTETFWNVYVDGDQC

SSNPCHYGGTCKDGIGSYTCTCLAGYEGKNCQYVLYQSCRVDNGNCWHFCKPVQNEIQCSCAESYLLGDDGYSCVAGGDFSCGRNIKARN SSNPCHYGGTCKDGIGSYTCTCLSGYEGKNCEYVLYKSCRVDNGDCWHFCKPVQNGIQCSCAESYLLGEDGHSCVAGGDFSCGRNIKTRN SSNPCHYRGICKDGIGSYTCTCLSGYEGKNCERVLYKSCRVDNGNCWHFCKHVQNDIQCSCAEGYLLGEDGHSCVAGGNFSCGRNIKTRN SSNPCHYRGICKDGIGSYTCTCWSGYEGKNCERVLYKSCRVDNGNCWHFCKSVQNDIQCSCAEGYLLGEDGHSCVAGGNFSCGRNIKTRN SSNPCHYRGTCKDGIGSYTCTCLPNYEGKNCEKVLYQSCRVDNGNCWHFCKRVQSETQCSCAESYRLGVDGHSCVAEGDFSCGRNIKARN

KREASLPDFQTDFSDDYDAIDENNFVETPTNFSGLVPTVQSQNATLLKKSDNPSPDIRVVNGTDCKLGECPWQALLINDQGDGFCGGTIL KREANLPDFQTDFSDDYDEIDENNFVETPTNFSGLVLTVQSQNATLLKKSDNPSPDIRVVNGTDCKLGECPWQALLLNDEGDGFCGGTIL KREANLPDF.............................VQSQNATLLKKSDNPSPDIRIVNGMDCKLGECPWQAALVDEKEGVFCGGTIL KREASLPDF.............................VQSQNAPLLKISDNPSPDIRIVNGMDCKLGECPWQAALVDDKKGVFCGGTIL KREASLPDF.............................VQSQKATLLKKSDNPSPDIRIVNGMDCKLGECPWQAVLINEKGEVFCGGTIL

SPIYVLTAAHCINQTKYIRVVVGEIDISRKKTGRLLSVDKIYVHQKFVP.............STYDYDIALIQMKTPIQFSENVVPACLP SPIYVLTAAHCINQTKYITVVVGEIDISSKKTGRLHSVDKIYVHQKFVP.............ATYDYDIAIIQLKTPIQFSENVVPACLP SPIYVLTAAHCINETETISVVVGEIDKSRIETGPLLSVDKIYVHKKFVPPQKAY....KFDLAAYDYDIAIIQMKTPIQFSENVVPACLP SPIYVLTAAHCINETETISVVVGEIDRSRAETGPLLSVDKVYVHKKFVPPKKSQEFYEKFDLVSYDYDIAIIQMKTPIQFSENVVPACLP SPIHVLTAAHCINQTKSVSVIVGEIDISRKETRRLLSVDKIYVHTKFVPPN.YYYVHQNFDRVAYDYDIAIIRMKTPIQFSENVVPACLP

TADFANQVLMKQDFGIVSGFGRTRERGQTSNTLKVVTLPYVDRHTCMLSSNFPITQNMFCAGYNTLPQDACQGDSGGPHITAYRDTHFIT TADFANQVLMKQNFGIVSGFGRTRERGKTSNTLKVVTLPYVDRHTCMLSSNFPITQNMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT TADFANQVLMKQDFGIVSGFGRIFEKGPKSKTLKVLKVPYVDRHTCMVSSETPITPNMFCAGYDTLPRDACQGDSGGPHTTVYRDTHFIT TADFANQVLMKQDFGIVSGFGGIFGRGPNSKTLKVLKVPYVDRHTCMLSSNFPITPTMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT TADFANEVLMKQDSGIVSGFGRIQFKQPTSNTLKVITVPYVDRHTCMLSSDFRITQNMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT

C C C C C

M M M M M

L L V L L

S S S S S

S S S S S

N N E N D

F F F F

P P P P I I I I I

T T T T T

Q Q Q

N N N N

M M M M M

F F F F F

C C C C C

A A A A A

G G G G G

Y Y Y Y Y

N D D D D

T T T T T

L L L L L

P P P P P

Q Q Q Q

D D D D D

A A A A A

C C C C C

Q Q Q Q Q

G G G G G

D D D D D

S S S S S

G G G G G

G G G G G

P P P P P

H H H H H

I I I I

T T T T T

A A A A

Y Y Y Y Y

R R R R R

D D D D D

T T T T T

H H H H H

F F F F F

I I I I I TTTTT 483

T T T T T

K K K K K

F F F F F

V V V V V

P P P P P P P P

L L L L L

L L L L L

K K K K K

K K K K

S S S S S

D D D D D

N N N N N

P P P P P

S S S S S

P P P P P

D D D D D

I I I I I

R R R R R

V V I I I

V V V V V

F F F D D D

N N N N N

G G G G G M M M

D D D D D

C C C C C

Y Y Y Y Y

D D D D D

Y Y Y Y Y

D D D D D

I I I I I

A A A A A

L I I I I

I I I I I

Q Q Q Q M L M M M

K K K K K

T T T T T

P P P P P

I I I I I

Q Q Q Q Q

F F F F F

S S S S S

E E E E E

N N N N N

V V V V V

V V V V V

P P P P P

A A A A A

C C C C C

L L L L L PPPPP 437

K K K K K

L L L L L

G G G G G

E E E E E

C C C C C

P P P P P

W W W W W

Q Q Q Q Q

A A A A A L L L L L N N D D N

D D E D E K K K

G G E G V V V

T T T T

C C C C C

W W W W W

H H H H H

F F F F F

C C C C C

K K K K K V V V V V

Q Q Q Q Q

N N N N I I I I Q Q Q Q Q

C C C C C

S S S S S

C C C C C

A A A A A

E E E E E

S S G G S

Y Y Y Y Y

L L L L L L L L L

G G G G G

D E E E D D D D D

G G G G G H H H H

S S S S S

C C C C C

V V V V V

A A A A A

G G G G E

G G G G G

D D N N D

F F F F F

S S S S S

C C C C C

G G G G G

R R R R R

F F F F F

C C C C C

G G G G G

G G G G G

T T T T T

I I I I I LLLLL 347

N N N N N

I I I I I

K K K K K T T T R R R R R NNNNN 270

N D N N N

E E E E E

E E E E E

F F F F I

K K K K R S S S G G G G G

N N N N N

I I I I I

E E E E E

R R R R R

E E E E E

C C C C C

I I I I I

E E E E E

E E E E E

R R R R K

C C C C C

S S S S S

K K K K K

E E E E E

E E E E E

A A A A A

R R R R R

E E E E E

A A A A F F F F F

E E E E E

D D D D D

N D D D N

E E E E E

K K K K K

T T T T T

E E E E E

T T T T T

F F F F F

W W W W W

N N N N N

V V V V V

Y Y Y Y Y

V V V V V

D D D D D

G G G G G

D D D D D

Q Q Q Q Q CCCCC 180

F F F 90

90

180

241

330

420

455

Fig. 5. Alignment of deduced amino acid sequences of FX-like proteins from *T. carinatus*

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

(TrFX and trocarin D) and *P. textilis* (PCCS, PFX1 and PFX2) snakes.

not found in mammalian FXs (as described previously).

M M M M M

**TrFX PFX1 PFX2 PCCS Trocarin D**

**TrFX PFX1 PFX2 PCCS Trocarin D**

**TrFX PFX1 PFX2 PCCS Trocarin D**

**TrFX PFX1 PFX2 PCCS Trocarin D**

**TrFX PFX1 PFX2 PCCS Trocarin D**

**TrFX PFX1 PFX2 PCCS Trocarin D** S S S S S

K K K K K

S S S S S

T T T T T

G G G G G

I I I I I

I V V V I

S S S S S

W W W W

G G G G G

E E E E E

G G G G G

C C C C C

A A A A A R R R

A A A A A

D D D D D

F F F F F

A A A A A

N N N N N

Q Q Q Q E

V V V V V

L L L L L

M M M M M

K K K K K

Q Q Q Q Q

D N D D D

G G G G G

K K K R K

Y Y Y Y Y

G G G G G Y Y Y Y Y

T T T T T

K K K K K

V V L L V

S S S S S

R K K K K

F F F F F

F F F F G G G G G

I I I I I

V V V V V

S S S S S

G G G G G

F F F F F

G G G G G

R R R R I I I

P P P P P

I I I I I

Y Y Y Y V V V V V

L L L L L

T T T T T

A A A A A

A A A A A

H H H H H

C C C C C

I I I I I

N N N N N

Q Q E E Q

T T T T T

K K K I I I I V S S S

V V V V V

V V V V I

V V V V V

G G G G G

E E E E E

I I I I I

E E E G R R K R K

GIISWGEGCAQTGKYGAYTKVSRFILWIKRIMRLKLPSTESSTGRL... GIVSWGEGCAQTGKYGVYTKVSKFILWIKRIIRQKQPSTESSTGRL... GIVSSGEGCARNGKYGIYTKLSKFIPWIKRIMRQKLPSTESSTGRL... GIVSWGEGCARKGRYGIYTKLSKFIPWI..................... GIISWGEGCARKGKYGVYTKVSKFIPWIKKIMSLK..............

I I I I I P P P

W W W W W

I I I I I

K K K K

R R R K

I I I I

M I M M

R R R K K K K

G G G G P P P

T T T

S S S S S

N N N

T T T T T

L L L L L

K K K K K

V V V V V T T T

P P P S S S T T T E E E S S S S S S T T T G G G R R R L L L

L L V V V

P P P P P

Y Y Y Y Y

V V V V V

D D D D D

I I I

S S S S S

R R R R

K K K E E E

T T T T T

G G G G R R R

L L L L L

L L L L

S S S S S

V V V V V

D D D D D

K K K K K

I I I V I

R R R R R

E E E E E

A A A A A

S S S

L L L L L

P P P P P

D D D D D

F F F F F

S S S S S

N N N N N

P P P P P

C C C C C

H H H H H

Y Y Y Y Y R R R

G G G G G

T T T

C C C C C

K K K K K

D D D D D

G G G G G

I I I I I

G G G G G

S S S S S

Y Y Y Y Y

T T T T T

C C C C C

T T T T T

C C C C C

L L L W L S S S G G G G Y Y Y Y Y

A A A A A

P P P P P

Q Q Q Q Q

L L L L L

L L L L L

L L L L L

C C C C C

L L L L L

I I I I I

L L L L L

**Signal peptide**

T T T T T

F F F F F

L L L L L

W W W W W

S S S S S

L L L L L P P P P

E E E E E

A A A A A

E E E E E

S S S S S

N N N N N

V V V V V

F F F F F

L L L L L

K K K K K

E E E E E

G G G G G

K K K K K

**Activation peptide**

N N N N N

C C C C C

Q E E E E V V V V V

L L L L L

Y Y Y Y Y K K K S S S S S

S S S S S

K K K K K

V V V V V

**Propeptide**

A A A A A

N N N N N

R R R R R

F F F F F

L L L L L

Q Q Q Q Q

R R R R R

T T T T T

K K K K K

C C C C C

V V V V V

Q Q Q Q Q

S S S S S

R R R R R

V V V V V

R R R R R

A A A A N N N N N

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 average of 2 independent experiments each carried out in triplicates.
