A Clot Twist: Extreme Variation in Coagulotoxicity Mechanisms in Mexican Neotropical Rattlesnake Venoms

Abstract

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus, and C. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors

“…The comparative testing of the enzyme inhibitors prinomastat and DMPS revealed highly contrasting differences in their specific abilities to neutralize the P-IIId SVMP responsible for FX activation by the venoms in this study. This is congruent with recent data published for FX activation of neonate Crotalus culminatus venom, which prinomastat neutralised but DMPS at the same concentration did not ( 59 ) and another report of DMPS requiring long incubation times to exert a discernable effect ( 60 ).…”

“…Such variation in relationship to altitude and prey type and prey escape potential has been noted for the rattlesnake species Crotalus helleri ( 79 ). In addition, as all venom samples used in this study from adult specimens, future work should examine ontogenetic shifts to ascertain if juvenile snakes have differentially procoagulant venoms, which has been noted for other species ( 59 , 78 , 80 , 81 ).…”

In vitro and in vivo preclinical venom inhibition assays identify metalloproteinase inhibiting drugs as potential future treatments for snakebite envenoming by Dispholidus typus

“…This was unexpected and hints at different levels of drug exposure and metabolism in this single dose intravenous-delivered model, as both matrix metalloproteinase inhibiting drugs showed potent inhibitory activity in our in vitro assays and have been shown in other studies to neutralise the in vivo lethal effects of E. ocellatus venom in murine models ( Arias et al, 2017 ; Albulescu et al, 2020a ). Studies by other groups have similarly shown the impressive in vitro inhibitory activities of marimastat and prinomastat against SVMP activity in rattlesnakes and a wide range of palearctic vipers ( Chowdhury et al, 2021a ; Seneci et al, 2021 ) and strong inhibitory effects of prinomastat against the anticoagulant activity of spitting cobra venoms ( Chowdhury et al, 2021b ).…”

Dynamic genetic differentiation drives the widespread structural and functional convergent evolution of snake venom proteinaceous toxins

“…12 ). Clotting factor activation has also been documented in the additional crotaline genera Calloselasma (Factor X) and Crotalus (Factor X by neonate Crotalus culminatus ) [ 116 , 117 ], but the toxins responsible have not been sequenced. Consequently, their phylogenetic affinity to the Bothrops -type procoagulant P-III SVMP is unknown, and it cannot be determined whether procoagulant SVMPs have evolved once or several times in the pit vipers.…”