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Article

Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms

by
Francisco C. P. Coimbra
1,
Elda E. Sanchez
2,
Bruno Lomonte
3,
José María Gutiérrez
3,
Juan J. Calvete
4 and
Bryan G. Fry
1,*
1
Adaptive Biotoxicology Lab, School of the Environment, University of Queensland, St Lucia, QLD 4072, Australia
2
National Natural Toxins Research Center, Department of Chemistry, Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA
3
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica
4
Laboratorio de Venómica Evolutiva y Traslacional, Instituto de Biomedicina de Valencia, CSIC, 46010 Valencia, Spain
*
Author to whom correspondence should be addressed.
Toxins 2024, 16(7), 291; https://doi.org/10.3390/toxins16070291
Submission received: 28 May 2024 / Revised: 23 June 2024 / Accepted: 25 June 2024 / Published: 26 June 2024
(This article belongs to the Special Issue Venom Components Acting on the Hemostatic System: Structural and Mechanistic Insights)
Browse Figures
Versions Notes

Abstract

:
This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.
Keywords:
Agkistrodon; viper; venom; evolution; coagulation
Key Contribution: Most reports on the venom effects of Agkistrodon species focus on A. contortrix contortrix, with the other subspecies and species comparably neglected. The genus Agkistrodon is shown to have dynamic variation in the anticoagulation effects of its venoms. The implications span evolution, clinical medicine, and biodiscovery.

Graphical Abstract

1. Introduction

The genus Agkistrodon within the Viperidae snake family evolved from Asian pit vipers that crossed the Bering Strait. These snakes are found in North America, having adapted to a wide range of habitats from wetlands to dry forests and ranging from the southeastern United States down to the northern province of Guanacaste in Costa Rica [1]. The Agkistrodon genus is divided into two clades; the copperhead clade, consisting of A. contortrix contortrix, A. contortrix mokasen, A. contortrix phaeogaster, A. laticinctus pictigaster, and A. laticinctus laticinctus, are specialists of wooded areas and rocky terrains, while the moccasin clade, consisting of A. bilineatus, A. conanti, A. howardgloydi, A. piscivorus leucostoma, A. piscivorus piscivorus, and A. taylori, are specialists to aquatic environments like swamps, streams, and marshes, with the exception of the southernmost species, A. howardgloydi, which has secondarily adapted to tropical dry forests [2]. Agkistrodon venoms have been shown to be dominant by snake venom metalloproteases (SVMPs), Group II phospholipase A2 (PLA2), and kallikrein-type serine proteases, with conserved proteolytic and phospholipase activities but differential haemorrhagic and myotoxic activities [3]. Agkistrodon envenomations may produce local and systemic effects; local symptoms include swelling, severe pain, bruising, blistering, and potentially significant tissue damage around the bite area, while the systemic signs and symptoms in severe cases include bleeding disorders, shock, or even organ failure, particularly if the venom components interfere with blood clotting or cardiovascular functions [4,5,6,7,8,9,10]. Most reports, however, are on A. c. contortrix, with few on other A. contortrix subspecies and scant reports for other species.
Snake venoms have long been studied for their complex biochemical properties and are primarily made up enzymatic and non-enzymatic proteins evolutionarily honed to alter any physiological pathway reachable by the bloodstream [11]. They represent an abundant source of bioactive molecules with a yet unrealised scope of potential medical applications in the leading areas of human death and disability, such as cardiovascular disease, diabetes, cancer, and chronic pain [12]. The coagulotoxic effects of snake venoms, while being critical for prey immobilisation and therefore predatory success and snakes’ survival, are of particular significant interest in medical research for potential therapeutic applications, especially in the treatment of conditions related to thrombosis and cardiovascular disease. A myriad of toxin types have been isolated and characterised from snake venoms that disrupt blood coagulation, with the venoms of viperid snakes being particularly rich in such toxins. The effects of anticoagulant toxins include the inhibition of the clotting enzymes thrombin, VIIa, Xa, IXa, XIa, and XIIa and the depletion of fibrinogen levels, leading to death from haemorrhagic shock or internal bleeding [13,14,15,16,17,18,19,20,21,22].
Platelet-mediated primary clotting is disrupted by the binding of venom toxins to a variety of platelet receptors or destruction of platelet cofactors such as the von Willebrand factor (vWF). In primary haemostasis, platelets are activated and alter their shape in a way that presents the fibrinogen binding receptor, thus facilitating platelet cross-linking and aggregation. The main families of toxins that interfere with platelet aggregation are 3FTxs (three-finger toxins) [23], L-amino acid oxidases [24], the lectin framework (including classical lectins and derived SNACLECS lectin-like proteins) [25], regions of the prepro region of the natriuretic peptide precursor [26], Group I PLA2 proteins [27], Group II PLA2 proteins [28], and the snake venom metalloproteinase (SVMP) disintegrin domain, which is released from P-II SVMPs by post-translational proteolytic cleavage and expressed as a monodomain in extremely derived forms [29].
The few direct inhibitors of the secondary clotting’s extrinsic and intrinsic pathways that have been characterised from snake venom include a dimeric 3FTx complex from Hemachatus haemachatus that inhibits FVIIa [30]; a monomeric 3FTx, also from H. haemachatus, that inhibits the extrinsic pathway activation of FX [31]; and a Kunitz peptide from Bungarus fasciatus that inhibits FXIa [32]. FIXa has been shown to be inhibited by lectin-like toxins from viperid snake venoms [25,33].
In contrast to the paucity of described inhibitors of the extrinsic or intrinsic pathways, a myriad of snake venom enzymes have been described that promote coagulopathic effects upon blood coagulation through deleterious actions upon the common pathway, which may contribute to the characteristic haemorrhagic syndrome in snakebite envenomings. The activation of Protein C by kallikrein-type serine proteases would inhibit FVa and FVIIIa upstream and consequently inhibit FX activation and prothrombinase formation indirectly downstream [34,35,36,37,38]. Direct inhibition of FXa has been shown for Group I PLA2 from Naja (cobra) venoms [27,39,40,41] in addition to lectin toxins from viperid snake venoms [25,33]. Lectin toxins that are inhibitors of prothrombin or thrombin (or both) have been isolated and characterised [25,42,43,44,45,46,47].
A major target for anticoagulant venoms acting upon the common pathway is fibrinogen depletion, either through destructive cleavage of fibrinogen, or cleavage in a peudo-procoagulant (aka: thrombin-like) manner such that the fibrin clots that are directly formed by the venom are weak and transient, thereby quickly breaking down [38,48]. The latter are pseudo-procoagulant in that the clots are transient and result in a net outcome of an anticoagulative state. We speculate this to be a consequence of a compromised capacity of the venom-cleaved fibrinogen chains to correctly polymerise into stable, interlinked fibrin meshes. These actions should not be confused with true procoagulant actions in which the cleavage of fibrinogen into fibrin retains its full polymerisation capabilities and functional potential due to the venom activating an upstream clotting enzyme, such as converting Factor VII into FVIIa, Factor X into FXa, or Factor II (prothrombin) into FIIa (thrombin) [16,21,48,49,50,51,52]. Both non-clotting and pseudo-procoagulant (transient clotting) cleavage of fibrinogen lead to a depletion of the levels of normal and intact fibrinogen available for clotting. In addition, the fibrinogen degradation products may also be bound by thrombin and thus also reduce the amount of thrombin available for clot formation [29,38]. Both outcomes would directly contribute to the haemorrhagic effects of these kinds of snake venoms.
Fibrinogenolysis in a non-clotting manner has been described for kallikrein-type serine proteases in addition to metalloproteases (SVMPs) in snake venoms [29,38]. Kallikrein-type enzymes may degrade both the Aα- and Bβ-chains or display selectivity for the Aα-chain or Bβ-chain [38]. Consistent with the single early origin of the Toxicofera venom system, the kallikrein-type serine proteases shared between the venoms of anguimorph lizards and those of snakes share this plesiotypic activity [38,53]. The plesiotypic (basal) state is the ability to degrade both chains, and the specialisation of one or the other chain is an apotypic (derived) activity. Degrading both the Aα- and Bβ-chains is also the plesiotypic state of P-III SVMP, but chain-specific versions have been described [29,54]. Thus, venom-induced fibrinogenolysis is a consequence of the action of pseudo-procoagulant enzymes, which form feeble fibrin clots that are readily degraded, or the action of enzymes that degrade fibrinogen and render it unclottable.
In addition to fibrinogenolysis, some snake venom enzymes have been shown to directly lyse fibrin clots [38,55]. Indirect clot lysis is also accomplished by kallikrein-type serine proteases activating plasminogen into plasmin [38,56,57,58,59]. Unlike plasminogen activation by bacteria [60,61,62,63], the snake venom activation of plasminogen occurs via proteolytic cleavage of the zymogen into the active enzymatic form. Other mechanisms of snake venoms for increasing the amount of available plasmin in order to destroy any clots formed in response to damage to the vascular bed are mediated by kallikrein-type serine proteases and metalloproteases that cause the release of the endogenous plasminogen activator from endothelial cells [29,38].
Despite its evolutionary novelty and medical importance, there is a notable deficiency in studies examining intra-species and inter-species variability in venom coagulotoxicity within the Agkistrodon genus [3], with questions remaining as to the contribution of such diversity to the variability in reported clinical coagulotoxic effects [4,64]. This incomplete analysis limits our understanding of the venom’s evolutionary history, potential clinical effects, and biomedical potential. Addressing these gaps through targeted research is critical not only for advancing our fundamental scientific knowledge but also for harnessing Agkistrodon venoms’ therapeutic utility.

2. Results and Discussion

Plasma clotting time was significantly delayed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus, with no clotting within a machine maximum time of 999 seconds for all four A. conanti replicates and all but one replicate for A. contortrix mokasen and A. howardgloydi (Figure 1A), with significant shifts relative to the control (Figure 1B). As the three most potent venoms (A. conanti, A. contortrix mokasen, and A. howardgloydi) do not represent a monophyletic clade (Figure 1C), this suggests that independent amplification of net anticoagulation potency on clotting time has occurred on at least three separate occasions within the genus (Figure 1). This represents convergent evolution, suggesting selection forces geared towards the same functional outcome. Subsequent tests to ascertain sites of action included incubation with activated enzymes or incubation with plasma followed by the addition of a specific enzyme to determine if downstream zymogens or cofactors were affected.
The incubation tests of the activated enzymes Factors XIa, IXa, Xa, and IIa (also known as thrombin) revealed that FXIa was not significantly inhibited by any of the venoms; FIXa was significantly inhibited (p < 0.05) by A. bilineatus, A. contortrix contortrix, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, and A. laticinctus laticinctus, with A. howardgloydi dramatically the most potent; FXa was significantly inhibited (p < 0.05) for all venoms, with A. conanti, A. contortrix contortrix, A. conanti, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorous the most potent; and FIIa (thrombin) was not significantly affected by any of the venoms (Figure 2 and Figure 3). As the taxa that most potently inhibited FXa do not form a monophyletic clade (Figure 4), this suggests that FXa inhibition is a basal trait within this genus that has been amplified on three occasions and therefore an indication of convergence in adaptation. This is consistent with FXa inhibition having been documented in other pit vipers from the Americas, including the Bothriechis, Bothrops, Cerrophidion, Crotalus, and Ophryacus species [21,22,65,66]; Asian pit vipers, including the Deinagkistrodon and Gloydius species [65,67,68]; true vipers, including the Cerastes, Daboia, Pseudocerastes, and Vipera species [65,69,70,71,72,73]; and elapid snakes including the Naja and Pseudechis species [14,41,74,75]. The toxin types responsible are diverse, including Kunitz peptides, lectin-framework toxins, and phospholipases A2.
To investigate the depletion of the phospholipid cofactor or zymogen degradation, plasma incubation was followed by the addition of an activated enzyme (Figure 2 and Figure 3). This revealed that the clotting cascade steps between that of FXIa and FIXa were affected by A. bilineatus, A. conanti, A. contortrix contortrix, A. contortrix mokasen, A. contortrix phaeogaster, and A. howardgloydi; that those of FIXa and FXa were affected by A. bilineatus, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, and A. piscivorus leucostoma; and those of FXa and FIIa were affected by A. contortrix mokasen, A. contortrix phaeogaster, and A. howardgloydi (Figure 2 and Figure 3). The depletion of phospholipid levels by PLA2 toxins has been shown to inhibit the activity of FXa due to this enzyme’s requirement of phospholipids as binding cofactors [76]. Therefore, the lessened activity of FXIa and FIXa when added to plasma preincubated with venom may also be due to lowered phospholipid levels. In this case, there would be an equal distribution of effect for the FXIa, FXIa, and FXa additions after preincubation, a pattern seen for A. bilineatus, A. contortrix mokasen, A. contortrix phaeogaster, and A. howardgloydi. The most potent effects were noted for A. contortrix mokasen and A. howardgloydi, indicative of the convergent amplification of this trait. Therefore, for these species, the effects upon the activity of FXIa, FIXa, and FXa when added to the plasma preincubated with venom appear to be due to phospholipid degradation. However, for A. conanti, only the addition of FXIa was affected, not the downstream FIXa or FXa pathways. This therefore suggests that A. conanti degrades the FIX zymogen. Zymogen destruction has been previously described for pit vipers from the Americas, such as the destruction of the FX zymogen by Crotalus mictlantecuhtli mictlantecuhtli and Crotalus mictlantecuhtli oaxacus [51]. Future work, however, would be required to confirm the hypothesis that A. conanti venom destroys the FIX zymogen.
Thromboelastography was utilised to examine the direct effects upon fibrinogen (Figure 5). None of the venoms clotted fibrinogen in the pseudo-procoagulant manner seen in diverse pit vipers such as the Crotalus, Gloydius, and Protobothrops species [17,18,19,77] and true vipers such as the Bitis species [78]. Subsequently, we ascertained the effects upon fibrin clot formation and strength by incubating fibrinogen with the venoms and subsequently adding thrombin to trigger clot formation. A. piscivorus leucostoma was the only venom to delay clot formation (Figure 5A) relative to the control (Figure 5B). This is consistent with depletion of fibrinogen levels due to destructive cleavage. This is in contrast to the delay of the onset of clot formation and the diminishing of the speed of clot growth when whole plasma is used [79], which is due to contributory effects observed upon upstream clotting factors (Figure 2 and Figure 3). The direct degradation of fibrinogen is a trait that has been documented in the venoms of other pit vipers, such as the Crotalus, Gloydius, and Protobothrops species [17,19,20], and true vipers, such as the Bitis and Causus species [15,78]. However, clot strength was affected by all species (Figure 5C) relative to the control (Figure 5D). This is consistent with cleaving fibrinogen in a non-clotting manner but one that produced damaged fibrinogen still able to be cleaved by thrombin and form into a weak, unstable fibrin clot. Therefore, species that did not impede coagulation can still produce pathophysiological outcomes by damaging fibrinogen such that any clots formed (by prey or a human bite victim) would be weak and short-lived, resulting in an anticoagulative state of reduced or corrupted clotting potential, thereby promoting or facilitating pathologies such as internal bleeding and haemorrhagic shock.
Subsequent assays were undertaken to ascertain the relative conversion of the Protein C zymogen into an activated form (Figure 6), as this is of interest not only in the context of venomous snake predatory ecology but also due to such venom enzymes having been of significant drug design and development interest, exemplified by their use in the development of the drug Protac® isolated from A. contortrix contortrix [80]. Protein C, once activated to Activated Protein C (APC), inactivates clotting factors Va and VIIIa. This inactivation prevents the overproduction of thrombin and thus controls the formation of blood clots. Protein C also influences fibrinolysis (the process of breaking down clots) by modulating the activity of plasminogen activator inhibitor-1 (PAI-1), thereby promoting the breakdown of fibrin clots.
Placed into a phylogenetic context (Figure 7), both the ability to directly damage fibrinogen, such that thrombin forms weaker clots, and the ability to activate the endogenous enzyme Protein C (which inactivates clotting factors Va and VIIIa and promotes fibrinolysis) are traits basal to the genus, but both traits have each been independently amplified on several occasions. The damaging action upon fibrinogen is at the most potentiated in the last common ancestor of the A. laticinctus taxa within the copperhead clade and the last common ancestor of the A. piscivorus taxa within the moccasin clade (Figure 7). For Protein C activation, this has been potentiated in the last common ancestor of the A. contortrix taxa while being amplified on two separate occasions within the moccasin clade, once in A. bilineatus and again in A. conanti (Figure 7). Conspicuously, the two actions appear to be negatively correlated but not entirely mutually exclusive, with the venoms amongst the most potent for one action being amongst the least potent for the other (Figure 5, Figure 6 and Figure 7).
The intraspecific and interspecific variability in Agkistrodon observed in this study regarding increases in clotting time, decreases in fibrinogen levels, and decreases in fibrin clot strength and prior proteomics data [3] are consistent with clinical reports of intraspecific and interspecific variability in increased PT and aPTT times, hypofibrinogenemia, and bleeding complications [4,64]. From a clinical perspective, these variations may reflect the differential ability to cause haemostatic disorders in the envenomed patient, which may impact diagnosis and management plans, including relative antivenom efficacy. The clinical implications of these findings are significant for understanding snakebite envenomation and its treatment, especially when considering the various nuanced angles of attack that can result in the same or similar coagulopathies. Clinical reports have described alterations in clotting parameters in a proportion of cases inflicted by Agkistrodon species [8,81]. Variability in venom effects can lead to different clinical presentations, such as increased prothrombin time (PT), activated partial thromboplastin time (aPTT), hypofibrinogenemia, and bleeding complications. Clinicians need to be aware of the potential variability in envenomation effects to accurately diagnose and manage snakebite cases. This may involve tailored therapeutic approaches and supportive care based on the specific venom profile of the snake involved. However, the variability in venom composition also underscores the need for broad-spectrum antivenoms that can neutralise a range of venom effects by reinforcing current viperid antivenoms with relevant Agkistrodon species so as to widen their neutralising scope.
From an evolutionary perspective, these variations may reflect adaptive significance, natural selection, and genetic drift and gene flow. The observed variability in the venom impacts upon clotting time, fibrinogen levels, and fibrin clot strength may reflect evolutionary adaptations to different ecological niches or prey types. Future work should investigate effects on prey plasma, as this has been shown to be an area of dynamic variation [52,82,83,84,85]. The differences in venom effects may be the result of natural selection, where certain venom profiles confer a survival advantage in specific environments. Variability in venom properties might also arise from genetic drift or gene flow between populations, contributing to the diversity observed in clotting time and fibrinogen levels.
From a biodiscovery perspective, these variations reflect the potential to yield novel therapeutics or biotechnological applications such as diagnostic uses. The unique properties of snake venom proteins and enzymes, such as those affecting clotting time and fibrinogen levels, can inspire the development of new drugs for coagulation disorders, cancer, and other diseases. Studying the variability in venom effects can provide insights into the biochemical pathways involved in haemostasis and how they can be modulated. This can lead to the discovery of new therapeutic targets. Venom components can also be harnessed for biotechnological applications, such as developing diagnostic tools or novel biomaterials. Understanding the diversity in venom effects can expand the potential applications of these bioactive molecules.
In conclusion, the observed variability in venom effects across different species and populations of Agkistrodon highlights the intricate interplay between evolutionary adaptation, clinical implications, and biodiscovery potential. Evolutionarily, these variations underscore the adaptive significance of venom composition, driven by natural selection and genetic dynamics such as drift and gene flow. Clinically, this variability necessitates a nuanced approach to diagnosing and managing snakebite envenomation, emphasising the importance of the development of broader-spectrum antivenoms. From a biodiscovery perspective, the unique biochemical properties of snake venoms hold immense promise for the development of novel therapeutics and biotechnological applications. By harnessing the diversity in venom effects, we can unlock new pathways for medical and scientific advancements, ultimately benefitting a wide range of fields from haemostasis to cancer treatment and beyond. As such, the coagulotoxic properties of snake venoms represent a fascinating area of research that bridges evolutionary biology, pharmacology, and medicine.

3. Materials and Methods

3.1. Venoms

A. c. contortrix venom was donated to author J.C. by César Olmos (Cullera, Valencia, Spain).
A. c. conanti (Liberty County, FL, USA), A. c. mokasen (Powell County, KY, USA), A. laticinctus pictagaster (Pecos County, TX, USA), A. laticinctus laticinctus (Tarrant County, TX, USA), and A. p. leucostoma (Harris County, TX, USA) venoms were collected from specimens maintained in the serpentarium of the National Natural Toxins Research centre at Texas A&M University—Kingsville, USA and collected by author E.S.
A. c. phaeogaster and A. taylori venoms were collected by author E.S. from specimens caught in Cole County, Missouri (USA), and Sota La Marina, Tamaulipas (Mexico), respectively.
A. howardgloydi venom was collected by authors B.L. and J.C. from specimens collected in the Costa Rican Province of Guanacaste and maintained in captivity in the serpentarium of the Instituto Clodomiro Picado (University of Costa Rica).
A. bilineatus (Mexico) and A. p. piscivorus (USA) venoms were purchased from Latoxan (Valence, France).

3.2. Coagulation Methods

Human plasma work was performed under University of Queensland Biosafety Approval #IBC134BSBS2015 and Human Ethics Approval #2016000256. The Australian Red Cross (44 Musk Street, Kelvin Grove, QLD 4059, Australia) supplied human platelet—poor plasma (3.2% citrated) under research approval #16-04QLD-10.
The anticoagulant methods were as follows:
  • (Clotting enzyme + venom) + plasma
    ○
    Undertaken to ascertain sites of actions upon particular enzymes.
    ○
    Step 1: 50 µL of venom + 50 µL of 0.025 M calcium + 25 µL of OK buffer + 50 µL of PPL + 75 µL of human plasma.
    ○
    Step 2: 120 s incubation at 37 °C.
    ○
    Step 3: Addition of 25 µL of enzyme.
  • (Venom + plasma) + clotting factor
    ○
    Undertaken to ascertain sites of action upon zymogens downstream from a particular enzyme.
    ○
    Step 1: 50 µL of venom + 50 µL of 0.025 M calcium + 25 µL of OK buffer + 50 µL of PPL + 75 µL of human plasma.
    ○
    Step 2: 120 s incubation at 37 °C.
    ○
    Step 3: Addition of 25 µL of enzyme.
  • Enzymes used:
    ○
    Factor IIa (thrombin), Stago cat. #00611.
    ○
    Factor VIIa, 1.5 µg/mL, Haemonetics cat. #HCVIIA-0031.
    ○
    Factor IXa, 15 μg/mL, Haemonetics cat. #HCXIA-0160.
    ○
    Factor Xa, Stago kit; cat. # 00311.
    ○
    Factor Xia, 15 µg/mL, Haemonetics cat. #HCXIA-0160.
    ○
    Factor XII (activated with kaolin), 15 µg/mL, Haemonetics cat. #HCXII-0155.
Thromboelastography was undertaken using the TEG5000 platform to determine the venom effects upon the strength and elasticity of the fibrin clots. The methods were as follows:
  • Seven µL of venom was added to 72 µL of CaCl2, 72 µL of phospholipid, and 20 µL of Owren–Keller buffer, followed by 189 µL of fibrinogen and then run immediately for 30 min measurement of clot formation.
  • As the venoms did not directly clot the fibrinogen, assays were completed with an additional step by adding 7 µL of thrombin followed by a further 30 min to determine if the fibrinogen had been destroyed by the venom to prevent clotting.
Protein C zymogen activation was undertaken using a Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) as follows:
  • In total, 10 μL of phospholipid, 10 μL of venom (containing 1 μg of venom), and 10 μL of Protein C zymogen (containing 10 μg total of zymogen) were manually pipetted, followed by the automated pipetting of 70 μL of a mixture of buffer (5 mM CaCl2, 150 mM NaCl, and 50 mM Tris-HCl at pH 7.3) and fluorogenic peptide substrate (ES011 substrate Boc-Val-Pro-Arg-AMC; Boc: t-Butyloxycarbonyl; 7-Amino-4- methyl coumarin) into each experimental well of a 384-well plate.
  • Activated factors were used in place of zymogens as positive controls.
  • The zymogens were also replaced in the venom control wells by 10 μL of Fluoroskan buffer to determine the activity of the venom directly upon the substrate.
  • Fluorescence generated by the cleaving of the substrate was automatically recorded by the machine. The results were obtained by subtracting blank values from reactions, followed by the subtraction of the venom without zymogen from the venom with zymogen.

3.3. Phytools Methods

Ancestral state reconstruction was undertaken in R-studio (Version 2024.04.2+764) using the following script:
  • library(ape)
  • library(maps)
  • library(phytools)
  • data<-read.csv(file.choose())
  • dat<-data
  • mapvar<-dat$var
  • names(mapvar)<-dat$species
  • tree<-read.tree(file.choose())
  • asr<-contMap(tree,mapvar,plot=F)
  • plot(setMap(asr,col=c(1,4,5,3,7,2,6)),lwd=10)

3.4. Statistical Analyses

Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons were undertaken in Graphpad Prism 8.1.1 (GraphPad Prism Inc., La Jolla, CA, USA) to ascertain the statistical significance of the venom effects relative to the control values.

Author Contributions

Conceptualisation, methodology, investigation, writing—original draft preparation: F.C.P.C. and B.G.F.; resources, writing—review and editing: E.E.S., B.L., J.M.G. and J.J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Australian Research Council Discovery Project DP190100304. The National Institutes of Health fund the NNTRC out of the Office of Research Infrastructure Programs.

Institutional Review Board Statement

All work was undertaken under animal ethics approval, University of Queensland, Animal Ethics Approval 15 March 2021/AE000075. Frozen human platelet-poor plasma (3.2% citrated) was supplied by the Australian Red Cross (44 Musk Street, Kelvin Grove, QLD 4059, Australia) under research approval #16-04QLD-10.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are presented in the figures.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Campbell, J.A.; Lamar, W.W. The Venomous Reptiles of the Western Hemisphere; Cornell University Press: Ithaca, NY, USA, 2004; Volume 2. [Google Scholar]
  2. Burbrink, F.T.; Guiher, T.J. Considering gene flow when using coalescent methods to delimit lineages of North American pitvipers of the genus Agkistrodon. Zool. J. Linn. Soc. 2015, 173, 505–526. [Google Scholar] [CrossRef]
  3. Lomonte, B.; Tsai, W.C.; Urena-Diaz, J.M.; Sanz, L.; Mora-Obando, D.; Sanchez, E.E.; Fry, B.G.; Gutierrez, J.M.; Gibbs, H.L.; Sovic, M.G.; et al. Venomics of New World pit vipers: Genus-wide comparisons of venom proteomes across Agkistrodon. J. Proteom. 2014, 96, 103–116. [Google Scholar] [CrossRef]
  4. Wills, B.K.; Billet, M.; Rose, S.R.; Cumpston, K.L.; Counselman, F.; Shaw, K.J.; Cresswell, K.G.; Charlton, N. Prevalence of hematologic toxicity from copperhead envenomation: An observational study. Clin. Toxicol. 2020, 58, 262–265. [Google Scholar] [CrossRef]
  5. Domanski, K.; Kleinschmidt, K.C.; Greene, S.; Ruha, A.M.; Bebarta, V.S.; Onisko, N.; Campleman, S.; Brent, J.; Wax, P.; Tox, I.C.N.A.S.R.G. Cottonmouth snake bites reported to the ToxIC North American snakebite registry 2013–2017. Clin. Toxicol. 2020, 58, 178–182. [Google Scholar] [CrossRef]
  6. Gerardo, C.J.; Vissoci, J.R.; Brown, M.W.; Bush, S.P. Coagulation parameters in copperhead compared to other Crotalinae envenomation: Secondary analysis of the F(ab’)(2) versus Fab antivenom trial. Clin. Toxicol. 2017, 55, 109–114. [Google Scholar] [CrossRef] [PubMed]
  7. Thorson, A.; Lavonas, E.J.; Rouse, A.M.; Kerns, W.P., 2nd. Copperhead envenomations in the Carolinas. J. Toxicol. Clin. Toxicol. 2003, 41, 29–35. [Google Scholar] [CrossRef] [PubMed]
  8. Kopec, K.T.; Yen, M.; Bitner, M.; Evans, C.S.; Gerardo, C.J. Marked hypofibrinogenemia and gastrointestinal bleeding after copperhead (Agkistrodon contortrix) envenomation. Wilderness Environ. Med. 2015, 26, 488–490. [Google Scholar] [CrossRef]
  9. Lawrence, W.T.; Giannopoulos, A.; Hansen, A. Pit viper bites: Rational management in locales in which copperheads and cottonmouths predominate. Ann. Plast. Surg. 1996, 36, 276–285. [Google Scholar] [CrossRef]
  10. Lonati, D.; Butera, R.; Cima, M.; Cozzio, S.; Locatelli, C.; Manzo, L. Exotic snakes in Europe. A case of Mexican Moccasin (Agkistrodon bilineatus) snakebite. Presse Med. 2004, 33, 1582–1584. [Google Scholar] [CrossRef]
  11. Casewell, N.R.; Wuster, W.; Vonk, F.J.; Harrison, R.A.; Fry, B.G. Complex cocktails: The evolutionary novelty of venoms. Trends Ecol. Evol. 2013, 28, 219–229. [Google Scholar] [CrossRef]
  12. Bedraoui, A.; Suntravat, M.; Mejjad, S.E.; Enezari, S.; Oukkache, N.; Sanchez, E.E.; Galan, J.A.; Fatimy, R.E.; Daouda, T. Therapeutic potential of snake venom: Toxin distribution and opportunities in deep learning for novel drug discovery. Med. Drug Discov. 2024, 21, 100175. [Google Scholar] [CrossRef]
  13. Bittenbinder, M.A.; Dobson, J.S.; Zdenek, C.N.; Op den Brouw, B.; Naude, A.; Vonk, F.J.; Fry, B.G. Differential destructive (non-clotting) fibrinogenolytic activity in Afro-Asian elapid snake venoms and the links to defensive hooding behavior. Toxicol. Vitr. 2019, 60, 330–335. [Google Scholar] [CrossRef] [PubMed]
  14. Bittenbinder, M.A.; Zdenek, C.N.; Op den Brouw, B.; Youngman, N.J.; Dobson, J.S.; Naude, A.; Vonk, F.J.; Fry, B.G. Coagulotoxic cobras: Clinical implications of strong anticoagulant actions of african spitting Naja venoms that are not neutralised by antivenom but are by LY315920 (Varespladib). Toxins 2018, 10, 516. [Google Scholar] [CrossRef] [PubMed]
  15. Coimbra, F.C.P.; Dobson, J.; Zdenek, C.N.; Brouw, O.d.B.; Hamilton, B.; Debono, J.; Masci, P.; Frank, N.; Ge, L.; Kwok, H.F.; et al. Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) with short and long venom glands. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2018, 211, 7–14. [Google Scholar] [CrossRef] [PubMed]
  16. Debono, J.; Bos, M.H.A.; Coimbra, F.; Ge, L.; Frank, N.; Kwok, H.F.; Fry, B.G. Basal but divergent: Clinical implications of differential coagulotoxicity in a clade of Asian vipers. Toxicol. Vitr. 2019, 58, 195–206. [Google Scholar] [CrossRef] [PubMed]
  17. Debono, J.; Bos, M.H.A.; Do, M.S.; Fry, B.G. Clinical implications of coagulotoxic variations in Mamushi (Viperidae: Gloydius) snake venoms. Comp. Biochem. Physiol. Toxicol. Pharmacol. CBP 2019, 225, 108567. [Google Scholar] [CrossRef] [PubMed]
  18. Debono, J.; Bos, M.H.A.; Frank, N.; Fry, B. Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus. Toxicol. Lett. 2019, 316, 35–48. [Google Scholar] [CrossRef] [PubMed]
  19. Debono, J.; Bos, M.H.A.; Nouwens, A.; Ge, L.; Frank, N.; Kwok, H.F.; Fry, B.G. Habu coagulotoxicity: Clinical implications of the functional diversification of Protobothrops snake venoms upon blood clotting factors. Toxicol. Vitr. 2018, 55, 62–74. [Google Scholar] [CrossRef] [PubMed]
  20. Dobson, J.; Yang, D.C.; Op den Brouw, B.; Cochran, C.; Huynh, T.; Kurrupu, S.; Sanchez, E.E.; Massey, D.J.; Baumann, K.; Jackson, T.N.W.; et al. Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus. Comp. Biochem. Physiol. Toxicol. Pharmacol. CBP 2017, 205, 62–69. [Google Scholar] [CrossRef]
  21. Jones, L.; Youngman, N.J.; Neri-Castro, E.; Guadarrama-Martinez, A.; Lewin, M.R.; Carter, R.; Frank, N.; Fry, B.G. Differential antivenom and small-molecule inhibition of novel coagulotoxic variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American viperid snake venoms. Toxins 2022, 14, 511. [Google Scholar] [CrossRef]
  22. Youngman, N.J.; Carlsson, D.J.; Jones, L.; Neri-Castro, E.; Alagon, A.; Fry, B.G. Cloud serpent coagulotoxicity: The biochemical mechanisms underpinning the anticoagulant actions of Mixcoatlus and Ophryacus venoms. Toxicon 2022, 211, 44–49. [Google Scholar] [CrossRef] [PubMed]
  23. Utkin, Y.; Sunagar, K.; Jackson, T.N.W.; Reeks, T.; Fry, B.G. Three-Finger Toxins (3FTxs). In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 215–227. [Google Scholar]
  24. Tan, N.H.; Fry, B.G.; Sunagar, K.; Jackson, T.N.W.; Reeks, T.; Fung, S.Y. L-amino acid oxidase enzymes. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 215–227. [Google Scholar]
  25. Arlinghaus, F.T.; Fry, B.G.; Sunagar, K.K.; Jackson, T.N.W.; Eble, J.A.; Reeks, T.; Clemetson, K.J. Lectin proteins. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 299–311. [Google Scholar]
  26. Fry, B.G.; Jackson, T.N.W.; Takacs, Z.; Reeks, T.; Sunagar, K. C-type natriuretic peptides. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 318–326. [Google Scholar]
  27. Sunagar, K.; Jackson, T.N.W.; Reeks, T.; Fry, B.G. Group I Phospholipase A2 Enzymes. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 327–334. [Google Scholar]
  28. Sunagar, K.; Tsai, I.H.; Lomonte, B.; Jackson, T.N.W.; Fry, B.G. Group II Phospholipase A2 Enzymes. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 335–340. [Google Scholar]
  29. Casewell, N.R.; Sunagar, K.; Takacs, Z.; Calvete, J.J.; Jackson, T.N.W.; Fry, B.G. Snake venom metalloprotease enzymes. In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 347–363. [Google Scholar]
  30. Banerjee, Y.; Mizuguchi, J.; Iwanaga, S.; Kini, R.M. Hemextin AB complex, a unique anticoagulant protein complex from Hemachatus haemachatus (African Ringhals cobra) venom that inhibits clot initiation and factor VIIa activity. J. Biol. Chem. 2005, 280, 42601–42611. [Google Scholar] [CrossRef] [PubMed]
  31. Girish, V.M.; Kini, R.M. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus. Sci. Rep. 2016, 6, 32036. [Google Scholar] [CrossRef] [PubMed]
  32. Chen, W.; Carvalho, L.P.; Chan, M.Y.; Kini, R.M.; Kang, T.S. Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity. J. Thromb. Haemost. 2015, 13, 248–261. [Google Scholar] [CrossRef] [PubMed]
  33. Lu, Q.; Clemetson, J.M.; Clemetson, K.J. Snake venoms and hemostasis. J. Thromb. Haemost. 2005, 3, 1791–1799. [Google Scholar] [CrossRef] [PubMed]
  34. Bakker, H.M.; Tans, G.; Yukelson, L.Y.; Janssen-Claessen, T.W.; Bertina, R.M.; Hemker, H.C.; Rosing, J. Protein C activation by an activator purified from the venom of Agkistrodon halys halys. Blood Coagul. Fibrinolysis 1993, 4, 605–614. [Google Scholar] [CrossRef]
  35. Kisiel, W.; Kondo, S.; Smith, K.J.; McMullen, B.A.; Smith, L.F. Characterization of a protein C activator from Agkistrodon contortrix contortrix venom. J. Biol. Chem. 1987, 262, 12607–12613. [Google Scholar] [CrossRef]
  36. Kogan, A.E.; Bashkov, G.V.; Bobruskin, I.D.; Romanova, E.P.; Makarov, V.A.; Strukova, S.M. Protein C activator from the venom of Agkistrodon blomhoffi ussuriensis retards thrombus formation in the arterio-venous shunt in rats. Thromb. Res. 1993, 70, 385–393. [Google Scholar] [CrossRef] [PubMed]
  37. Nakagaki, T.; Kazim, A.L.; Kisiel, W. Isolation and characterization of a protein C activator from tropical moccasin venom. Thromb. Res. 1990, 58, 593–602. [Google Scholar] [CrossRef]
  38. Vaiyapuri, S.; Sunagar, K.; Gibbins, J.M.; Jackson, T.N.W.; Reeks, T.; Fry, B.G. Kallikrein Enzymes In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery; Fry, B.G., Ed.; Oxford University Press: New York, NY, USA, 2015; pp. 267–280. [Google Scholar]
  39. Kerns, R.T.; Kini, R.M.; Stefansson, S.; Evans, H.J. Targeting of venom phospholipases: The strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex. Arch. Biochem. Biophys. 1999, 369, 107–113. [Google Scholar] [CrossRef]
  40. Kini, R.M. Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms. Toxicon 2005, 45, 1147–1161. [Google Scholar] [CrossRef] [PubMed]
  41. Mukherjee, A.K.; Kalita, B.; Thakur, R. Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism. PLoS ONE 2014, 9, e101334. [Google Scholar] [CrossRef] [PubMed]
  42. Monteiro, R.Q. Targeting exosites on blood coagulation proteases. Acad. Bras. Cienc. 2005, 77, 275–280. [Google Scholar] [CrossRef] [PubMed]
  43. Monteiro, R.Q.; Zingali, R.B. Inhibition of prothrombin activation by bothrojaracin, a C-type lectin from Bothrops jararaca venom. Arch. Biochem. Biophys. 2000, 382, 123–128. [Google Scholar] [CrossRef] [PubMed]
  44. Oliveira-Carvalho, A.L.; Guimaraes, P.R.; Abreu, P.A.; Dutra, D.L.; Junqueira-de-Azevedo, I.L.; Rodrigues, C.R.; Ho, P.L.; Castro, H.C.; Zingali, R.B. Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach. Toxicon 2008, 51, 659–671. [Google Scholar] [CrossRef] [PubMed]
  45. Zingali, R.B.; Bianconi, M.L.; Monteiro, R.Q. Interaction of bothrojaracin with prothrombin. Haemostasis 2001, 31, 273–278. [Google Scholar] [CrossRef] [PubMed]
  46. Zingali, R.B.; Ferreira, M.S.; Assafim, M.; Frattani, F.S.; Monteiro, R.Q. Bothrojaracin, a Bothrops jararaca snake venom-derived (pro)thrombin inhibitor, as an anti-thrombotic molecule. Pathophysiol. Haemost. Thromb. 2005, 34, 160–163. [Google Scholar] [CrossRef]
  47. Zingali, R.B.; Jandrot-Perrus, M.; Guillin, M.C.; Bon, C. Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: Characterization and mechanism of thrombin inhibition. Biochemistry 1993, 32, 10794–10802. [Google Scholar] [CrossRef] [PubMed]
  48. Oulion, B.; Dobson, J.S.; Zdenek, C.N.; Arbuckle, K.; Lister, C.; Coimbra, F.C.P.; Op den Brouw, B.; Debono, J.; Rogalski, A.; Violette, A.; et al. Factor X activating Atractaspis snake venoms and the relative coagulotoxicity neutralising efficacy of African antivenoms. Toxicol. Lett. 2018, 288, 119–128. [Google Scholar] [CrossRef]
  49. Chowdhury, A.; Lewin, M.R.; Carter, R.W.; Casewell, N.R.; Fry, B.G. Keel venom: Rhabdophis subminiatus (Red-Necked Keelback) venom pathophysiologically affects diverse blood clotting pathways. Toxicon 2022, 218, 19–24. [Google Scholar] [CrossRef]
  50. Chowdhury, A.; Zdenek, C.N.; Dobson, J.S.; Bourke, L.A.; Soria, R.; Fry, B.G. Clinical implications of differential procoagulant toxicity of the palearctic viperid genus Macrovipera, and the relative neutralization efficacy of antivenoms and enzyme inhibitors. Toxicol. Lett. 2021, 340, 77–88. [Google Scholar] [CrossRef] [PubMed]
  51. Seneci, L.; Zdenek, C.N.; Chowdhury, A.; Rodrigues, C.F.B.; Neri-Castro, E.; Benard-Valle, M.; Alagon, A.; Fry, B.G. A clot twist: Extreme variation in coagulotoxicity mechanisms in Mexican neotropical rattlesnake venoms. Front. Immunol. 2021, 12, 612846. [Google Scholar] [CrossRef] [PubMed]
  52. Sousa, L.F.; Zdenek, C.N.; Dobson, J.S.; Op den Brouw, B.; Coimbra, F.; Gillett, A.; Del-Rei, T.H.M.; Chalkidis, H.M.; Sant’Anna, S.; Teixeira-da-Rocha, M.M.; et al. Coagulotoxicity of Bothrops (Lancehead Pit-Vipers) Venoms from Brazil: Differential biochemistry and antivenom efficacy resulting from prey-driven venom variation. Toxins 2018, 10, 411. [Google Scholar] [CrossRef] [PubMed]
  53. Koludarov, I.; Jackson, T.N.; Brouw, B.O.D.; Dobson, J.; Dashevsky, D.; Arbuckle, K.; Clemente, C.J.; Stockdale, E.J.; Cochran, C.; Debono, J.; et al. Enter the dragon: The dynamic and multifunctional evolution of Anguimorpha lizard venoms. Toxins 2017, 9, 242. [Google Scholar] [CrossRef] [PubMed]
  54. Sanchez, E.F.; Magalhes, A.; Mandelbaum, F.R.; Diniz, C.R. Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta). Biochim. Biophys. Acta 1991, 1074, 347–356. [Google Scholar] [CrossRef] [PubMed]
  55. Vaiyapuri, S.; Harrison, R.A.; Bicknell, A.B.; Gibbins, J.M.; Hutchinson, G. Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros. PLoS ONE 2010, 5, e9687. [Google Scholar] [CrossRef] [PubMed]
  56. Muanpasitporn, C.; Rojnuckarin, P. Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom. Toxicon 2007, 49, 1083–1089. [Google Scholar] [CrossRef] [PubMed]
  57. Park, D.; Kim, H.; Chung, K.; Kim, D.S.; Yun, Y. Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom. Toxicon 1998, 36, 1807–1819. [Google Scholar] [CrossRef] [PubMed]
  58. Sanchez, E.F.; Santos, C.I.; Magalhaes, A.; Diniz, C.R.; Figueiredo, S.; Gilroy, J.; Richardson, M. Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom. Arch. Biochem. Biophys. 2000, 378, 131–141. [Google Scholar] [CrossRef]
  59. Zhang, Y.; Wisner, A.; Xiong, Y.; Bon, C. A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J. Biol. Chem. 1995, 270, 10246–10255. [Google Scholar] [CrossRef]
  60. Collen, D. Staphylokinase: A potent, uniquely fibrin-selective thrombolytic agent. Nat. Med. 1998, 4, 279–284. [Google Scholar] [CrossRef] [PubMed]
  61. Haiko, J.; Laakkonen, L.; Juuti, K.; Kalkkinen, N.; Korhonen, T.K. The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1. J. Bacteriol. 2010, 192, 4553–4561. [Google Scholar] [CrossRef] [PubMed]
  62. Korhonen, T.K.; Haiko, J.; Laakkonen, L.; Jarvinen, H.M.; Westerlund-Wikstrom, B. Fibrinolytic and coagulative activities of Yersinia pestis. Front. Cell Infect. Microbiol. 2013, 3, 35. [Google Scholar] [CrossRef] [PubMed]
  63. Peetermans, M.; Vanassche, T.; Liesenborghs, L.; Lijnen, R.H.; Verhamme, P. Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity. Crit. Rev. Microbiol. 2016, 42, 866–882. [Google Scholar] [CrossRef] [PubMed]
  64. Keyler, D.E.; Vandevoort, J.T. Copperhead envenomations: Clinical profiles of three different subspecies. Vet. Hum. Toxicol. 1999, 41, 149–152. [Google Scholar] [PubMed]
  65. Faure, G.; Gowda, V.T.; Maroun, R.C. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics. BMC Struct. Biol. 2007, 7, 82. [Google Scholar] [CrossRef]
  66. Youngman, N.J.; Peng, Y.H.; Harris, R.J.; Jones, L.; Llinas, J.; Haworth, M.; Gillett, A.; Fry, B.G. Differential coagulotoxic and neurotoxic venom activity from species of the arboreal viperid snake genus Bothriechis (palm-pitvipers). Comp. Biochem. Physiol. Toxicol. Pharmacol. CBP 2022, 256, 109326. [Google Scholar] [CrossRef]
  67. Chen, M.; Ye, X.; Ming, X.; Chen, Y.; Wang, Y.; Su, X.; Su, W.; Kong, Y. A Novel Direct Factor Xa inhibitory peptide with anti-platelet aggregation activity from Agkistrodon acutus venom hydrolysates. Sci. Rep. 2015, 5, 10846. [Google Scholar] [CrossRef]
  68. Zhang, Y.; Xu, X.; Shen, D.; Song, J.; Guo, M.; Yan, X. Anticoagulation factor I, a snaclec (snake C-type lectin) from Agkistrodon acutus venom binds to FIX as well as FX: Ca2+ induced binding data. Toxicon 2012, 59, 718–723. [Google Scholar] [CrossRef]
  69. Fatah, C.; Samah, S.; Fatima, L.D. Antiplatelet and anticoagulant activities of two phospholipase A2s purified from Cerastes cerastes venom: Structure-function relationship. J. Biochem. Mol. Toxicol. 2018, 32, e22219. [Google Scholar] [CrossRef]
  70. Mukherjee, A.K.; Dutta, S.; Mackessy, S.P. A new C-type lectin (RVsnaclec) purified from venom of Daboia russelii russelii shows anticoagulant activity via inhibition of FXa and concentration-dependent differential response to platelets in a Ca2+-independent manner. Thromb. Res. 2014, 134, 1150–1156. [Google Scholar] [CrossRef] [PubMed]
  71. Samah, S.; Fatah, C.; Jean-Marc, B.; Safia, K.T.; Fatima, L.D. Purification and characterization of Cc-Lec, C-type lactose-binding lectin: A platelet aggregation and blood-clotting inhibitor from Cerastes cerastes venom. Int. J. Biol. Macromol. 2017, 102, 336–350. [Google Scholar] [CrossRef] [PubMed]
  72. Thakur, R.; Kumar, A.; Bose, B.; Panda, D.; Saikia, D.; Chattopadhyay, P.; Mukherjee, A.K. A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa. Biochimie 2014, 105, 149–158. [Google Scholar] [CrossRef] [PubMed]
  73. Yasmin, R.; Chanchal, S.; Ashraf, M.Z.; Doley, R. Daboxin P, a phospholipase A(2) of Indian Daboia russelii venom, modulates thrombin-mediated platelet aggregation. J. Biochem. Mol. Toxicol. 2023, 37, e23476. [Google Scholar] [CrossRef] [PubMed]
  74. Stefansson, S.; Kini, R.M.; Evans, H.J. The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymatic mechanism. Biochemistry 1990, 29, 7742–7746. [Google Scholar] [CrossRef] [PubMed]
  75. Zdenek, C.N.; Youngman, N.J.; Hay, C.; Dobson, J.; Dunstan, N.; Allen, L.; Milanovic, L.; Fry, B.G. Anticoagulant activity of black snake (Elapidae: Pseudechis) venoms: Potency, mechanisms, and antivenom efficacy. Comp. Biochem. Physiol. Part. C Toxicol. Pharmacol. 2020, 330, 176–184. [Google Scholar] [CrossRef] [PubMed]
  76. Dashevsky, D.; Benard-Valle, M.; Neri-Castro, E.; Youngman, N.J.; Zdenek, C.N.; Alagon, A.; Portes-Junior, J.A.; Frank, N.; Fry, B.G. Anticoagulant Micrurus venoms: Targets and neutralization. Toxicol. Lett. 2021, 337, 91–97. [Google Scholar] [CrossRef] [PubMed]
  77. Seneci, L.; Zdenek, C.N.; Bourke, L.A.; Cochran, C.; Sanchez, E.E.; Neri-Castro, E.; Benard-Valle, M.; Alagon, A.; Frank, N.; Fry, B.G. A symphony of destruction: Dynamic differential fibrinogenolytic toxicity by rattlesnake (Crotalus and Sistrurus) venoms. Comp. Biochem. Physiol. Toxicol. Pharmacol. CBP 2021, 245, 109034. [Google Scholar] [CrossRef] [PubMed]
  78. Youngman, N.J.; Debono, J.; Dobson, J.S.; Zdenek, C.N.; Harris, R.J.; Op den Brouw, B.; Coimbra, F.C.P.; Naude, A.; Coster, K.; Sundman, E.; et al. Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis. Toxins 2019, 11, 422. [Google Scholar] [CrossRef]
  79. Nielsen, V.G.; Redford, D.T.; Boyle, P.K. Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species. Basic. Clin. Pharmacol. Toxicol. 2016, 118, 390–395. [Google Scholar] [CrossRef]
  80. Stocker, K.; Fischer, H.; Meier, J.; Brogli, M.; Svendsen, L. Characterization of the protein C activator Protac from the venom of the southern copperhead (Agkistrodon contortrix) snake. Toxicon 1987, 25, 239–252. [Google Scholar] [CrossRef]
  81. Maharaj, S.; Seegobin, K.; Chang, S. Coagulopathy following Crotalipiae snakebites in northeast Florida. Blood Coagul. Fibrinolysis 2022, 33, 220–223. [Google Scholar] [CrossRef]
  82. Youngman, N.J.; Llinas, J.; Fry, B.G. Evidence for resistance to coagulotoxic effects of Australian elapid snake venoms by sympatric prey (blue tongue skinks) but not by predators (monitor lizards). Toxins 2021, 13, 590. [Google Scholar] [CrossRef] [PubMed]
  83. Youngman, N.J.; Llinas, J.; Haworth, M.; Gillett, A.; Jones, L.; Walker, A.A.; Fry, B.G. Untangling interactions between Bitis vipers and their prey using coagulotoxicity against diverse vertebrate plasmas. Toxicon 2022, 216, 37–44. [Google Scholar] [CrossRef] [PubMed]
  84. Youngman, N.J.; Zdenek, C.N.; Dobson, J.S.; Bittenbinder, M.A.; Gillett, A.; Hamilton, B.; Dunstan, N.; Allen, L.; Veary, A.; Veary, E.; et al. Mud in the blood: Novel potent anticoagulant coagulotoxicity in the venoms of the Australian elapid snake genus Denisonia (mud adders) and relative antivenom efficacy. Toxicol. Lett. 2019, 302, 1–6. [Google Scholar] [CrossRef]
  85. Zdenek, C.N.; Chowdhury, A.; Haw, G.Y.H.; Violette, A.; Fourmy, R.; Christ, T.; Vonk, F.J.; Fry, B.G. Taxon-selective venom variation in adult and neonate Daboia russelii (Russell’s Viper), and antivenom efficacy. Toxicon 2022, 205, 11–19. [Google Scholar] [CrossRef]
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Figure 1. Venom impacts upon clotting of human plasma. (A) Raw time numbers (machine maximum: 999 s). p-values for each venom relative to control are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. (B) Proportional increases in clotting time relative to the control (no venom effect = 0%). (C) Ancestral reconstruction of the relative effects upon clotting time; phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
Figure 1. Venom impacts upon clotting of human plasma. (A) Raw time numbers (machine maximum: 999 s). p-values for each venom relative to control are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. (B) Proportional increases in clotting time relative to the control (no venom effect = 0%). (C) Ancestral reconstruction of the relative effects upon clotting time; phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
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Figure 2. Copperhead clade proportional increase in clotting time relative to the control for each test (no venom effect = 0%). Note: to allow for comparison across venoms, all graphs are scaled relative to the point of greatest impact, which is (plasma + venom) + FIXa for A. conanti in Figure 3. Data are n = 4 mean ± standard deviation.
Figure 2. Copperhead clade proportional increase in clotting time relative to the control for each test (no venom effect = 0%). Note: to allow for comparison across venoms, all graphs are scaled relative to the point of greatest impact, which is (plasma + venom) + FIXa for A. conanti in Figure 3. Data are n = 4 mean ± standard deviation.
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Figure 3. Moccasin clade proportional increase in clotting time relative to the control for each test (no venom effect = 0%). Note: to allow for comparison across venoms, all graphs are scaled relative to the point of greatest impact, which is (plasma + venom) + FIXa for A. conanti in this figure. Data are n = 4 mean ± standard deviation.
Figure 3. Moccasin clade proportional increase in clotting time relative to the control for each test (no venom effect = 0%). Note: to allow for comparison across venoms, all graphs are scaled relative to the point of greatest impact, which is (plasma + venom) + FIXa for A. conanti in this figure. Data are n = 4 mean ± standard deviation.
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Figure 4. Ancestral reconstruction of elative inhibition of Factor Xa, showing the proportional increase in clotting time relative to the control (no venom effect = 0%). Phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
Figure 4. Ancestral reconstruction of elative inhibition of Factor Xa, showing the proportional increase in clotting time relative to the control (no venom effect = 0%). Phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
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Figure 5. Thromboelastography tests where venom was incubated with fibrinogen without forming a clot, with thrombin subsequently added to form. (A) Thromboelastography R values (reaction time) representing the time taken from the start of the test until initial fibrin formation began. This measurement reflects the speed at which clotting starts. As the fibrinogen test was run under Claussian conditions, in which an excess of thrombin was added, any delay in R is reflective of depletion of fibrinogen levels. (B) R values as proportional increases in clotting time relative to the control (no venom effect = 0%). (C) Thromboelastography G values, which each represent the shear elastic modulus strength of a clot, which quantifies the clot’s firmness. The G value is expressed in dynes per square centimetre (d/sc) and provides a direct measure of the strength and stability of the blood clot formed during the test. Lower values indicate weaker clots. (D) G values as proportional increases in clotting time relative to the control (no venom effect = 0%; negative values indicate decreases in clot strength). p-values in (A,C) for each venom relative to control are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. Data are n = 4 mean ± standard deviation.
Figure 5. Thromboelastography tests where venom was incubated with fibrinogen without forming a clot, with thrombin subsequently added to form. (A) Thromboelastography R values (reaction time) representing the time taken from the start of the test until initial fibrin formation began. This measurement reflects the speed at which clotting starts. As the fibrinogen test was run under Claussian conditions, in which an excess of thrombin was added, any delay in R is reflective of depletion of fibrinogen levels. (B) R values as proportional increases in clotting time relative to the control (no venom effect = 0%). (C) Thromboelastography G values, which each represent the shear elastic modulus strength of a clot, which quantifies the clot’s firmness. The G value is expressed in dynes per square centimetre (d/sc) and provides a direct measure of the strength and stability of the blood clot formed during the test. Lower values indicate weaker clots. (D) G values as proportional increases in clotting time relative to the control (no venom effect = 0%; negative values indicate decreases in clot strength). p-values in (A,C) for each venom relative to control are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. Data are n = 4 mean ± standard deviation.
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Figure 6. Relative activations of Protein C. p-values are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. Data are n = 4 mean ± standard deviation.
Figure 6. Relative activations of Protein C. p-values are from Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett T3 multiple comparisons. Data are n = 4 mean ± standard deviation.
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Figure 7. Ancestral reconstruction of the inverse relationships between the relative thromboelastographic determination of effects upon clot strength and the relative ability to activate Protein C. Phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
Figure 7. Ancestral reconstruction of the inverse relationships between the relative thromboelastographic determination of effects upon clot strength and the relative ability to activate Protein C. Phylogeny based upon Burbrink [2]. Data are n = 4 mean ± standard deviation.
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Coimbra, F.C.P.; Sanchez, E.E.; Lomonte, B.; Gutiérrez, J.M.; Calvete, J.J.; Fry, B.G. Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms. Toxins 2024, 16, 291. https://doi.org/10.3390/toxins16070291

AMA Style

Coimbra FCP, Sanchez EE, Lomonte B, Gutiérrez JM, Calvete JJ, Fry BG. Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms. Toxins. 2024; 16(7):291. https://doi.org/10.3390/toxins16070291

Chicago/Turabian Style

Coimbra, Francisco C. P., Elda E. Sanchez, Bruno Lomonte, José María Gutiérrez, Juan J. Calvete, and Bryan G. Fry. 2024. "Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms" Toxins 16, no. 7: 291. https://doi.org/10.3390/toxins16070291

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