Evolutionary Adaptation of Factor V from the Venom of the Common Brown Snake into a Potent Procoagulant
Authors: Bos Mettine H.A. , Boltz Michael , Pierre Liam St. , de Jersey John , Masci Paul P , Lavin Martin L , Camire Rodney M. 
Published: April 2020

Abstract
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.