Venomous snakes produce an array of toxic compounds, including procoagulants to defend themselves and incapacitate prey. The Australian snake Pseudonaja textilis has a venom-derived prothrombin activator homologous to coagulation factors V (FV) and Xa (FXa). Here we show that the FV component (pt-FV) has unique biologic properties that subvert the normal regulatory restraints intended to restrict an unregulated procoagulant response. Unlike human FV, recombinant pt-FV is constitutively active and does not require proteolytic processing to function. Sequence comparisons show that it has shed a large portion of the central B-domain, including residues that stabilize the inactive procofactor state. Remarkably, pt-FV functions in the absence of anionic membranes as it binds snake-FXa with high affinity in solution. Furthermore, despite cleavage in the heavy chain, pt-FV is functionally resistant to activated protein C, an anticoagulant. We speculate this stability is the result of noncovalent interactions and/or a unique disulfide bond in pt-FV linking the heavy and light chains. Taken together, these findings provide a biochemical rationale for the strong procoagulant nature of venom prothrombinase. Furthermore, they illustrate how regulatory mechanisms designed to limit the hemostatic response can be uncoupled to provide a sustained, disseminated procoagulant stimulus for use as a biologic toxin. IntroductionThe circulation of blood coagulation factors as precursors and their localization to the site of vascular injury after activation are important factors that maintain normal hemostasis and restrict indiscriminate clotting. 1 Bypassing these regulatory paradigms can be life-threatening, yet organisms have evolved strategies that exploit these systems for a selective advantage. For example, the venom of numerous snake species comprises a diverse array of proteases that activate and overwhelm the host hemostatic system in an uncontrolled fashion. 2 Some of the most potent venoms come from the Australian elapid family, including Pseudonaja textilis, Oxyuranus microlepidotus, and Oxyuranus scutellatus. 3 Their venom is considered the most toxic in the world and is unusual as it contains 2 coagulation proteins: factor V (FV) and a factor Xa (FXa)-like enzyme. [4][5][6][7][8][9][10] These proteins make up approximately 20% to 40% of the total venom and form a powerful procoagulant complex. 5,7 This complex converts prothrombin to thrombin, and its activity is enhanced, to various degrees, by calcium and phospholipids, but not by the cofactor FVa. [4][5][6][7]11 Venom-derived FV from these snakes share approximately 44% homology with mammalian FV and have a similar domain structure (A1-A2-B-A3-C1-C2). 8,10,12 However, we were surprised to learn that their B-domains are remarkably short, approximately 46 versus approximately 800 residues in mammals. (Amino acid numbering for pt-FV is as follows: the mature sequence starts at ϩ1 and the remaining sequence is numbered consecutively to 1430. The pt-FV B-domain is defined ...

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Some of the most toxic snakes in the world are those from the Australian Elapid family, including the three most venomous land snakes Inland Taipan, Coastal Taipan, and Common Brown snake. Their venom is strongly procoagulant and they are the only species known to have acquired a powerful prothrombin activator in their venom, which consists of a factor Xa (FXa)-like and factor V (FV)-like component. Venom-derived FV (pt-FV) from the Common Brown snake P. textilis shares 44% sequence homology with mammalian FV and has a similar domain organization. Remarkably, the B domain length of pt-FV is dramatically shortened compared to human FV (46 vs. 836 aa). This adaptation provides a unique opportunity to gain new insight into the function of the B domain and to examine the mechanistic basis for the strong procoagulant nature of the venom-derived prothrombinase complex. Pt-FV was expressed in BHK cells, purified, and characterized in functional assays employing FXa purified from P. textilis venom (pt-FXa). SDS-PAGE analysis revealed that pt-FV migrated as a single chain protein (~180 kDa). Thrombin completely processed pt-FV to pt-FVa, yielding the characteristic heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are connected by a unique disulfide bond. Functional analysis of prothrombin and prethrombin-1 conversion using a purified component assay in the presence of pt-FXa and negatively charged phospholipids revealed that pt-FV exhibits kinetic parameters comparable to human prothrombinase. Proteolytic processing of single chain pt-FV to the heterodimer did not significantly increase cofactor activity, indicating that pt-FV is expressed as a constitutively active cofactor that has bypassed the normal requirement for proteolytic activation. These results were confirmed using an uncleavable variant, pt-FV-QQ. We speculate that the mechanistic basis for this constitutive cofactor activity is related to the absence of a key cluster of conserved B domain residues, which we have recently shown to play an important role in maintaining FV as an inactive procofactor (JBC2007;282:15033). Additional experiments revealed that the pt-FV–pt-FXa complex does not require a membrane surface to optimally function, as the kinetics of prethrombin-1 activation were equivalent in the presence or absence of membranes. Binding measurements indicated that this was due to the high affinity interaction (Kd ~8 nM) of pt-FV with pt-FXa in solution. Interestingly, human FVa did not bind soluble pt-FXa with high affinity, suggesting that pt-FXa binding involves unique molecular features on pt-FV. Additional studies revealed that pt-FV does not lose activity following incubation with high concentrations of activated protein C (APC), even though the pt-FV heavy chain was fully proteolyzed. Collectively, our findings provide new insights into FV structure/function as well as a biochemical rationale for the powerful procoagulant nature of the prothrombinase complex from P. textilis venom. Remarkably, pt-FV has acquired at least three gain of function elements: first, it is constitutively active and as such the first example of a naturally occurring active FV variant. Second, pt-FV has a unique conformation as it bypasses the normal requirement for a membrane surface to achieve high affinity FXa binding. Finally, pt-FV is functionally resistant to APC which could be due to its unique disulfide bond. Taken together, venom-derived P. textilis FV represents an exceptional example of a protein that has adapted into a potent biological weapon for host defense and to incapacitate prey. Uncovering the mechanistic details of these gain of function elements will provide a new level of understanding of FV/FVa function.