The most striking difference between the sequences of the Australian elapid venom FXa-like proteins and blood coagulation FX is the length of the activation peptide. Even though the length of the activation peptide varies throughout evolution from 43 residues in the rat to 52 in human and 65 in zebrafish, the activation peptide of snake venom FX is considerably shorter, being 27 residues [15,17,18]. This is also considerably shorter than the FX expressed in the liver of P. textilis, which has an activation peptide of 56 residues (Figure 1) [24]. It is unclear if the FXa-like venom component comprises an intact activation peptide, as it was not detected upon N-terminal sequencing [4]. This would suggest that the activation peptide is either removed upon intracellular proteolysis of FX, or following its secretion into the venom. There is some evidence that glycosylation of the human FX activation peptide may contribute to substrate recognition by the intrinsic or extrinsic FX activating complex [25,26]. Although the activation peptide comprises several putative N-glycosylation sites (Figure 1), whether or not it plays a role in the proteolytic activation of venom FX similar to mammalian FX remains to be determined.

The light chain of the venom FXa-like component is very similar to that of blood coagulation FX. The Gla domain of venom FXa comprises eleven conserved Glu residues, which have been shown by chemical Gla analysis to be fully γ-carboxylated in P. textilis FXa (Figure 1) [27]. Furthermore, the structural elements that mediate anionic phospholipid binding are also present in the FXa-like subunit [21], which implies that, similar to mammalian FXa, venom FXa is capable of membrane binding. The structure of the EGF domains seems to be maintained as well, given that the six cysteines that form disulfide bridges characterizing the individual EGF domains are conserved. In addition, the cysteines that connect the heavy and light chains are also present in venom FXa (Figure 1).

Other important regions of FXa that are well conserved in the snake venom FXa-like subunit include the N-terminal heavy chain sequence Ile¹⁶–Val¹⁷, which is essential to protease maturation [23,28], and the catalytic triad His⁵⁷, Asp¹⁰², and Ser¹⁹⁵ (Figure 1). Some of the regions that undergo a conformational change upon activation are highly conserved in venom FXa, such as active site loop Ala¹⁸³–Asp¹⁹⁴, which includes Asp¹⁹⁴ that forms a salt bridge with Ile¹⁶, and the Na⁺ binding site Glu²¹⁶–Glu²²⁶. In contrast, most of the residues in the catalytic domain important for Ca²⁺ coordination are lost. More intriguingly, the Ca²⁺-interactive region is followed by a 13 residue insert, Pro²⁵¹–Leu²⁶³ (regular numbering of venom FX), that has been found in the venom FXa of all three elapids and, to some extent, in the group D prothrombin activators [17,24]. However, this sequence is not present in liver-expressed P. textilis FX, nor in other serine proteases involved in blood coagulation, such as FVIIa, FIXa, or activated protein C (APC). The functional role of this insertion is unclear.

A remarkable feature of the FV homologs expressed in the elapid venom is that their B domain is extraordinarily short: 46 versus ~600–800 residues in mammals (the FV B domain is defined as the region removed following thrombin cleavage to generate the heavy and light chains; Figure 2) [30]. Our laboratory has previously shown that the B domain plays an important function by interfering with functional binding interactions of FV through imposing steric and/or conformational constraints, thereby preventing constitutive cofactor function [31,32]. In addition, we have identified a discrete region of the B domain that plays a critical role in stabilizing the procofactor state [32]. Part of this region (residues 963–1008 of human FV) is unusually basic with 18 of 46 residues being Arg or Lys, and is well conserved across the vertebrate lineage [30]. Given that venom FV lacks the majority of the B domain sequence, it is not surprising that the so called basic region is absent as well. This intriguing observation prompted us to assess the cofactor capacity of purified recombinant venom-derived P. textilis FV (pt-FV) [27]. Consistent with our previous observations, we were able to show that the absence of the basic region correlates with the expression of procoagulant FV activity, indicating that venom FV is expressed as a constitutionally active FV variant [27]. As such, this is the first FV species observed thus far that exists as a constitutively active cofactor. Interestingly, liver-expressed P. textilis FV has a similar short B domain that lacks the basic region [19], which would suggest that the FV circulating in the elapid’s plasma is constitutionally active as well; however, biochemical studies are needed to confirm this possibility.

Sequence alignment reveals that in comparison to human FV, venom FV has lost six cysteine residues: three in the A2 domain (Cys⁵⁷⁵–Cys⁶⁵⁶, Cys⁵⁸⁵; human residue numbering), one in the B domain (Cys¹⁰⁸⁵), and two free cysteines in the C1-C2 domains, of which Cys¹⁹⁶⁰ is nonconserved throughout vertebrate evolution, whereas Cys²¹¹³ is only present in mammals. Venom FV has two newly introduced cysteines that are unique to Australian elapids: Cys⁶⁴² in A2 and Cys¹⁰⁰² in A3 (snake residue numbering; Figure 2). Recent data from our laboratory show that either Cys⁵⁴⁰ or Cys⁶⁴² in the A2 domain is involved in a disulfide bond that connects the heavy and light chains of pt-FV [27]. This is consistent with previous observations made by Speijer et al., who obtained evidence suggesting the existence of such a linker between the heavy and light chains of O. scutellatus FV [2]. This disulfide bond is a unique feature of snake venom FV, since it has not been observed in any other FV species to date. Based on homology with the cysteines in human FV, Cys¹⁰⁰² could be the only free cysteine in the venom FV light chain; therefore, this residue may be part of this unique disulfide bond linking the heavy and light chains. This assumption would also imply that plasma P. textilis FV cannot form a similar disulfide bond, as it lacks Cys¹⁰⁰² [19].

We have recently shown that P. textilis venom-derived FV is functionally resistant to inactivation by APC, as no loss of cofactor activity was observed upon incubation with very high concentrations of APC [27]; a similar observation was made using the venom FV-FXa complex, pseutarin C [14]. However, while the FVa functional activity was unaffected following APC treatment, we did observe proteolytic degradation of pt-FV, indicative of cleavage by APC [27]. N-terminal sequencing of the degradation products indicated that human APC cleaves pt-FV at Lys⁵⁰⁷ and Arg⁷⁴². These sites are homologous to the APC cleavage site Arg⁵⁰⁶ in human FV and to Arg⁷⁰⁹, the thrombin cleavage site. Furthermore, our data suggested that APC cleaves pt-FV at some additional sites in the A2 region somewhere within Glu⁵⁰⁸–Arg⁷⁴²; however, we were unable to make the exact determination. Both the venom FV from O. scutellatus and O. microlepidotus as well as the FV expressed in the liver of P. textilis have similar residues at these sites, suggesting that they would be cleaved by APC in a comparable manner.

The mammalian FVa structure is stabilized by several interdomain contacts linking the C1-C2, A3-C1, and A3-C2 domains [33]. Metal ion binding further contributes to these interdomain interactions by providing stability to the local structure [33]. Analysis of the A3-C1-C2 residues involved in direct interdomain contacts indicates that all of the residues connecting the C1-C2 and A3-C2 domains are conserved in venom FV, while the hydrophobic interactions linking the A3-C1 domains are at least partially preserved. Of the residues implied in Cu²⁺ binding, the majority are found in venom FV, as well as several residues involved in essential interactions between A1 and A3. Furthermore, the high affinity Ca²⁺ binding site is completely conserved. This implies that heavy and light chains of venom FV are linked in a manner similar to mammalian FV.

Factor V undergoes multiple posttranslational modifications, including N-glycosylation, phosphorylation, and sulfation at multiple sites [29], which are not fully conserved in venom FV. Mammalian FV is heavily N-glycosylated, and 25 out of the 37 putative N-glycosylation sites are located within the B domain (Figure 1). Venom FV, on the other hand, has 16 potential N-glycosylation sites of which one is in the B domain (Figure 1) [34]. Some of these N-glysocylation sites are similar to human FV, whereas others are unique to snake FV. Interestingly, liver-expressed P. textilis FV has an additional putative N-glycosylation site in the B domain at Asn⁵⁷³ [19], which is not preserved in any of the venom FV species. Phosphorylation of the human FVa heavy chain at Ser⁶⁹² has been implied to enhance the rate of APC-dependent inactivation [35]. Although this phosphorylation site is absent in the venom FVa-like subunit, venom-derived P. textilis FV could be proteolyzed by human APC as discussed above [27]. This suggests that phosphorylation of the FVa heavy chain may not be critical to inactivation by APC per se. Sulfation of FV has been speculated to be of importance for the recognition of FV by thrombin and for full FVa cofactor activity [36]. However, out of the six sulfation sites present in human FV, the one homologous to human Tyr¹⁵⁹³ is conserved in snake venom FV. Furthermore, our data on the thrombin-mediated pt-FV activation indicate a minor role for sulfation in the recognition of venom FV by thrombin [27].