From Venom to Vein: Factor VII Activation as a Major Pathophysiological Target for Procoagulant Australian Elapid Snake Venoms

Abstract

Australian elapid snake venoms are uniquely procoagulant, utilizing blood clotting enzyme Factor Xa (FXa) as a toxin, which evolved as a basal trait in this clade. The subsequent recruitment of Factor Va (FVa) as a toxin occurred in the last common ancestor of taipans (Oxyuranus species) and brown snakes (Pseudonaja species). Factor II (prothrombin) activation has been stated as the primary mechanism for the lethal coagulopathy, but this hypothesis has never been tested. The additional activation of Factor VII (FVII) by Oxyuranus/Pseudonaja venoms has historically been considered as a minor, unimportant novelty. This study aimed to investigate the significance of toxic FVII activation relative to prothrombin activation by testing a wide taxonomical range of Australian elapid species with procoagulant venoms. The activation of FVII or prothrombin, with and without the Factor Va as a cofactor, was assessed, along with the structural changes involved in these processes. All procoagulant species could activate FVII, establishing this as a basal trait. In contrast, only some lineages could activate prothrombin, indicating that this is a derived trait. For species able to activate both zymogens, Factor VII was consistently more strongly activated than prothrombin. FVa was revealed as an essential cofactor for FVII activation, a mechanism previously undocumented. Species lacking FVa in their venom utilized endogenous plasma FVa to exert this activity. The ability of the human FXa:FVa complex to activate FVII was also revealed as a new feedback loop in the endogenous clotting cascade. Toxin sequence analyses identified structural changes essential for the derived trait of prothrombin activation. This study presents a paradigm shift in understanding how elapid venoms activate coagulation factors, highlighting the critical role of FVII activation in the pathophysiological effects upon the coagulation cascade produced by Australian elapid snake venoms. It also documented the novel use of Factor Va as a cofactor for FVII activation for both venom and endogenous forms of FXa. These findings are crucial for developing better antivenoms and treatments for snakebite victims and have broader implications for drug design and the treatment of coagulation disorders. The research also advances the evolutionary biology knowledge of snake venoms.

Keywords: Factor VII; Factor Va; adaptation; coagulation; evolutionary biology; molecular evolution; prothrombin; venom; zymogen.

Figure 5

Ancestral reconstruction of prothrombin and Factor VII zymogen activation. Organismal phylogeny based on Lee [63]. Toxin recruitment phylogenetic positions are shown. Values (% relative to control enzyme) are area under the curve, N = 4, calculated as mean and standard deviation. Branch color scheme has a colder color for weaker activity and a warmer color for stronger activity. Non-procoagulant lineages within the Australian snake radiation are shown with gray branches in order to place convergent amplification of the procoagulant trait into the full evolutionary context. N.D. = not detectable.

Figure 1

The clotting cascade showing the extrinsic and intrinsic pathways, which both ultimately trigger the formation of fibrin clots (shown in red). The two zymogens examined in this study (FVII and prothrombin) are shown in blue.

Figure 2

Plasma clotting effects of venoms containing only weaponized FXa (blue) and those containing both weaponized FXa and weaponized FVa (red). Lower values indicate faster clotting and thus greater potency. Values are N = 4 calculated as mean and standard deviation. Spontaneous clotting control time was 431.5 ± 7.7 s. Graphed values are in Supplementary File S1.

Figure 3

Zymogen activation effects of venoms containing only weaponized FXa (blue) and those containing both weaponized FXa and weaponized FVa (red). Higher values indicate greater potency. Note: for allow a comparison across venoms, both graphs are scaled relative to the point of the greatest impact across the graphs. Values are area under the curve, N = 4, calculated as mean and standard deviation. Y-axis values are % relative to the positive control (FVIIa or thrombin), which are set to 100%. Graphed values are in Supplementary File S1.

Figure 4

Relative ability to activate the prothrombin (brown) and Factor VII (green) zymogen activation effects of venoms containing only weaponized FXa (blue lines above taxa names) and those containing both weaponized FXa and weaponized FVa (red lines above taxa names). Higher values indicate greater potency. Relative differences between pairs for each species were statistically significant (p < 0.001). Y-axis values are Figure 3 values normalized relative to the most potent venom (Pseudonaja nuchalis). Graphed values are in Supplementary File S1.

Figure 6

Sequence alignment of FXa enzymes. Gray is a liver produced Factor X used in circulating plasma. Brown are the venom forms: unable to activate prothrombin in brown with Demansia vestigiata acting as a proxy for the D. papuensis and D. psammophis venoms examined in this study as sequences are currently not available for these species or only weakly active (Pseudechis porphyriacus). Green are the venom forms able to strongly activate prothrombin (Cryptophis, Hoplocephalus, Notechis, Tropidechis, Pseudonaja, and Oxyuranus). Cysteines are highlighted in black. The NX (S or T) glycosylation tri-amino acid motif shown in blue highlight (where X = any amino acid except for cysteine or proline). Note: signal peptide sequences removed to save space.