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

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera, and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes (Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.

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

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.

Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine–binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Exosites expedite blood coagulation

A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a “dock and lock.” This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.

Exosite binding drives substrate affinity for the activation of coagulation factor X by the intrinsic Xase complex

Factor X activation by the intrinsic Xase complex, composed of factor IXa bound to factor VIIIa on membranes, is essential for the amplified blood coagulation response. The biological significance of this step is evident from bleeding arising from deficiencies in factors VIIIa or IXa in hemophilia. Here, we assess the mechanism(s) that enforce the distinctive specificity of intrinsic Xase for its biological substrate. Active-site function of IXa was assessed with a tripeptidyl substrate (PF-3688). The reversible S1 site binder, 4-aminobenzamidine (pAB), acted as a classical competitive inhibitor of PF-3688 cleavage by Xase. In contrast, pAB acted as a noncompetitive inhibitor of factor X activation. This disconnect between peptidyl substrate and protein substrate cleavage indicates a major role for interactions between factor X and extended sites on Xase in determining substrate affinity. Accordingly, an uncleavable factor X variant, not predicted to engage the active site of IXa within Xase, acted as a classical competitive inhibitor of factor X activation. Fluorescence studies confirmed the binding of factor X to Xase assembled with IXa with a covalently blocked active site. Our findings suggest that the recognition of factor X by the intrinsic Xase complex occurs through a multistep “dock-and-lock” pathway in which the initial interaction between factor X and intrinsic Xase occurs at exosites distant from the active site, followed by active-site docking and bond cleavage.

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre− mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Biochemical Characteristics of Emicizumab Activity with Factors IXa, X, and VIIa

Introduction. Emicizumab (Hemlibra) is a bivalent antibody that binds to factor IXa and factor X; this binding can promote factor IXa activation of factor X. In patients on prophylaxis with emicizumab, breakthrough bleeding has been treated successfully with factor VIIa (eptacog alfa (activated) (NovoSeven)). Objective. Our goal was to study the biochemistry of the interaction of emicizumab with factor IXa and factor X. We further wanted to study how binding of emicizumab to factor X would impact factor X activation by factor VIIa. Background. Data from surface plasmon resonance binding studies has shown that solution phase interactions between emicizumab and factors IX, IXa, X, and Xa are in the micromolar range (supplement to Kitazawa et al Nature Med 2012; 18: 1570-1574). That same publication showed that a lipid surface is required for activity. Other studies have shown that emicizumab binds to EGF1 of factor IX and EGF2 of factor X (Kitazawa et al Thromb Haemost 2017; 117: 1348-1357). Methods. Lipids were large unilamellar vesicles with 14% phosphatidylserine. Factors IX and X were purified from plasma. The basic design of an assay is to vary one element while holding other elements constant. To determine the apparent Km for factor X, factor X was varied with constant factor IXa (0.1 nM), lipid (100 µM), and emicizumab (400 nM). To determine the apparent Kd for factor IXa, factor IXa was varied with constant factor X (135 nM), lipid (100 µM), and emicizumab (3, 10, or 30 nM). The binding of factor IXa was determined in the presence and the absence of plasma concentration (80 nM) factor IX. Factor VIIa/tissue factor activation of factor X was measured with 0.25 nM VIIa/TF complex with factor X (135 nM) and either no emicizumab, 400 nM, or 50 µM. Factor VIIa activation of factor X in the absence of tissue factor was measured with varied factor VIIa (25-100 nM), fixed factor X (135 nM) and lipid (100 µM) and either no emicizumab, 400 nM, or 50 µM. Results. In the presence of lipid, the apparent binding constants for formation of the IXa-emicizumab-X complex were tighter than the solution phase reactions. Under the conditions studied, the Km,app for factor X was about 25 nM. The Kd,app for factor IXa was about 5 nM. Surprisingly, when lipid and factor X were present, factor IX did not compete with factor IXa for activation of factor X. Factor VIIa/tissue factor activation of factor X was slowed considerably by emicizumab. By contrast, factor VIIa activation of factor X in the absence of tissue factor was not slowed. Conclusions. The binding of factor X to the factor VIIa/tissue factor complex involves multiple domains in factor X. Since emicizumab reduced factor X activation by the factor VIIa/tissue factor complex, it appears that binding of emicizumab to the second EGF domain of factor X interfered with formation of the activating complex. By contrast, activation of factor X by factor VIIa alone was not reduced by emicizumab suggesting that interactions between factor VIIa and the second EGF domain of factor X are not essential for formation of that lipid bound complex. We would predict from the solution phase binding constants that high concentrations of emicizumab would be required to form the complex with factors IXa and X. This is in contrast to the observation that relatively low concentrations of emicizumab give significant shortening of an aPTT assay. The tight binding constants in the presence of lipid may explain the results seen in clotting assays. Further, this significant effect of emicizumab in clotting assays is consistent with the surprising observation that factor IX does not compete with factor IXa in formation of the lipid bound complex of IXa-emicizumab-X. So even a small amount of factor IXa can form functional complexes that activate factor X. Disclosures Monroe: Novo Nordisk A/S: Honoraria, Research Funding. Hoffman:Novo Nordisk A/S: Consultancy, Honoraria, Research Funding.

Coagulotoxicity of Bothrops (Lancehead Pit-Vipers) Venoms from Brazil: Differential Biochemistry and Antivenom Efficacy Resulting from Prey-Driven Venom Variation

Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter– and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.

Orphan Three-Finger Toxins Bind at Tissue Factor–Factor VIIa Interface to Inhibit Factor X Activation: Identification of Functional Site by Docking

Three-finger toxins (3FTxs) contribute to toxicity of venomous snakes belonging to the family Elapidae. Currently, functions of a considerable proportion of 3FTxs are still unknown. Here, we describe the function of orphan group I 3FTxs consisting of four members. We also identified a new member of this group by sequencing a transcript isolated from Naja naja venom. This transcript, named najalexin, is identical to that previously described 3FTx from Naja atra venom gland, and shared high sequence identity with ringhalexin from Hemachatus haemachatus and a hypothetical protein from Ophiophagus hannah (here named as ophiolexin). The three-dimensional structure, as predicted by molecular modeling, showed that najalexin and ophiolexin share the same conserved structural organization as ringhalexin and other 3FTxs. Since ringhalexin inhibits the activation of factor X by the tissue factor–factor VIIa complex (TF-FVIIa), we evaluated the interaction of this group of 3FTxs with all components using in silico protein–protein docking studies. The binding of orphan group I 3FTxs to TF-FVIIa complex appears to be driven by their interaction with TF. They bind to fibronectin domain closer to the 170-loop of the FVIIa heavy chain to inhibit factor X activation. The docking studies reveal that functional site residues Tyr7, Lys9, Glu12, Lys26, Arg34, Leu35, Arg40, Val55, Asp56, Cys57, Cys58, and Arg65 on these 3FTxs are crucial for interaction. In silico replacement of these residues by Ala resulted in significant effects in the binding energies. Furthermore, these functional residues are not found in other groups of 3FTxs, which exhibit distinct pharmacological properties.