Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

INTRODUCTION: Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. MATERIALS AND METHODS: Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. RESULTS: Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig. 1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig. 1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig. 2A), N. kaouthia = 0.38 mg/kg (Fig. 2B), and D. russelii = 7.92 mg/kg (Fig. 2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig. 3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig. 4). CONCLUSION: Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Snake venom cysteine-rich secretory protein from Mojave rattlesnake venom (Css-CRiSP) induces acute inflammatory responses on different experimental models.

Snake venoms contain various molecules known for activating innate immunity and causing local effects associated with increased vascular permeability, such as vascular leakage and edema, common symptoms seen in snakebite envenomings. We have demonstrated that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability. This study aimed to explore the functional role of CRiSP isolated from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom (Css-CRiSP) on the activation of inflammatory responses in different models. We measured the release of inflammatory mediators in cultured human dermal blood endothelial cells (HDBEC), lymphatic endothelial cells (HDLEC) and monocyte-derived macrophages (MDM) at 0.5, 1, 3, 6, and 24 h after treatment with Css-CRiSP (1 µM). We also determined the acute inflammatory response in BALB/c mice 30 min after intraperitoneal injection of the toxin (2 µg/mouse). Css-CRiSP induced the production of IL-8 and IL-6, but not TNF-α, in HDBEC and HDLEC in a time-dependent manner. In addition, Css-CRiSP significantly enhanced the production of IL-6, TNF-α, IL-8, and IL-1ß in MDM. Moreover, it caused a remarkable increase of chemotactic mediators in the exudates of experimental mice. Our results reveal that Css-CRiSPs can promote a sustained release of inflammatory mediators on cell lines and an acute activation of innate immunity in a murine model. These findings contribute to the growing body of evidence supporting the involvement of svCRiSPs in the augmentation of envenomation effects, specifically, the role of svCRiSPs in inducing vascular dysfunction, initiating early inflammatory responses, and facilitating the activation of leukocytes and releasing mediators. These findings will lead to a better understanding of the pathophysiology of envenoming by Mojave rattlesnakes, allowing the development of more efficient therapeutic strategies.

Proteomic Profiling of Extracellular Vesicles Isolated from Plasma and Peritoneal Exudate in Mice Induced by Crotalus scutulatus scutulatus Crude Venom and Its Purified Cysteine-Rich Secretory Protein (Css-CRiSP).

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

Evaluation of Signaling Pathways Profiling in Human Dermal Endothelial Cells Treated by Snake Venom Cysteine-Rich Secretory Proteins (svCRiSPs) from North American Snakes Using Reverse Phase Protein Array (RPPA).

Cysteine-Rich Secretory Proteins (CRiSPs) are typically found in many snake venoms; however, the role that these toxins play in the pathophysiology of snakebites is still unclear. Herein, we compared the effects of snake venom CRiSPs (svCRiSPs) from the most medically important species of North American snakes on endothelial cell permeability and vascular permeability. We used reverse phase protein array (RPPA) to identify key signaling molecules on human dermal lymphatic (HDLECs) and blood (HDBECs) endothelial cells treated with svCRiSPs. The results showed that Css-CRiSP isolated from Crotalus scutulatus scutulatus and App-CRiSP from Agkistrodon piscivorus piscivorus are the most potent causes of increase vascular and endothelial permeability in comparison with other svCRiSPs used in this study. We examined the protein expression levels and their activated phosphorylation states in HDLECs and HDBECs induced by App-CRiSP and Css-CRiSP using RPPA. Interestingly, both App-CRiSP and Css-CRiSP induced caveolin-1 expression in HDBECs. We also found that stimulating HDBECs with Css-CRiSP and App-CRiSP significantly induced the phosphorylation of mTOR and Src, respectively. In HDLECs, Css-CRiSP significantly downregulated the expression of N-Cadherin and phospholipase C-gamma, while App-CRiSP significantly enhanced Akt and JNK phosphorylation. These results suggest that the increased endothelial permeability in HDLECs and HDBECs by Css-CRiSP and App-CRiSP may occur through different pathways.

Proteomic Identification and Quantification of Snake Venom Biomarkers in Venom and Plasma Extracellular Vesicles.

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV's from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri, and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5' nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism.

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Contribution of endothelial cell and macrophage activation in the alterations induced by the venom of Micrurus tener tener in C57BL/6 mice.

An acute inflammatory response, cellular infiltrates, anemia, hemorrhage and endogenous fibrinolysis activation were previously described in C57BL/6 mice injected with M. tener tener venom (Mtt). As the endothelium and innate immunity may participate in these disturbances and due to our poor understanding of the alterations produced by these venoms when the neurotoxic component is not predominant, we evaluated the effects in an in vitro model. At 24 h, the release of pro-inflammatory mediators was detected in peritoneal macrophages. At different times, the release of pro-inflammatory (TNF-α, IL-6, NO and E-Selectin), pro-coagulant (vWF and TF) and pro-fibrinolytic (uPA) mediators were seen in liver sinusoidal endothelial cells (LSECs). These results suggest that Mtt venom activates macrophages and endothelium, thus inducing the release of mediators, such as TNF-α, that orchestrate the acute inflammatory response and the later infiltration of mononuclear cells into liver in C57BL/6 mice. In addition, endothelium activation promotes TF expression, which may in turn modulate the inflammatory and hemostatic response. These findings suggest crosstalk between inflammation and hemostasis in the alterations observed in Micrurus envenomation, where the neurotoxic manifestations do not predominate.

The neutralization efficacy of expired polyvalent antivenoms: An alternative option.

The expense of production and distribution of snakebite antivenom, as well as its relatively infrequent use, has caused antivenom to be increasingly difficult to obtain and ultimately producing an alarming global shortage. Unused, expired antivenom may represent a significant, untapped resource to ameliorate this crisis. This study examines the efficacy of expired antivenom over time using in vitro, whole blood clotting, and platelet function statistics. Representatives from three years for four different global brands of polyvalent antivenom were chosen and tested against their corresponding venoms as well as other venoms that could display cross-reactivity. These antivenoms include Wyeth Polyvalent (U.S.; exp. 1997, 2001, 2003), Antivipmyn® (Mexico; exp. 2005, 2013, 2017), Biotecfars Polyvalent (Venezuela; exp. 2010, 2014, 2016), and SAIMR (South Africa; exp. 1997, 2005, 2017). Venoms of species tested were Crotalus atrox against Wyeth; C. atrox and Crotalus vegrandis against Antivipmyn®; C. atrox, C. vegrandis and Bothrops colombiensis against Biotecfar; and Bitis gabonica and Echis carinatus against South African Institute for Medical Research (SAIMR). Parameters recorded were activated clotting time (ACT), clotting rate (CR), and platelet function (PF). Preliminary results are encouraging as the antivenoms maintained significant efficacy even 20 y after their expiration date. We anticipate these results will motivate further studies and provide hope in the cases of snakebite emergencies when preferable treatments are unavailable.

The isolation and characterization of a new snake venom cysteine-rich secretory protein (svCRiSP) from the venom of the Southern Pacific rattlesnake and its effect on vascular permeability.

A novel snake venom cysteine-rich secretory protein (svCRiSP), Hellerin, was purified from C. o. helleri venom using sequential reverse phase and cation-exchange chromatography. Gel electrophoresis, N-terminal sequencing, and LC-MS/MS sequencing identified a single protein with a molecular mass of approximately 24.8 kDa and confirmed its identity as a svCRiSP. Hellerin had cytotoxic effects on human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but not in human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs). Hellerin produced a dramatic increase in both blood vascular permeability in vivo, and in the trans-epithelial permeability of cultured HDLEC and HDBEC cells. This is the first study that describes the effect of a svCRiSP on vascular, blood and lymphatic permeability.

Pro-inflammatory response and hemostatic disorder induced by venom of the coral snake Micrurus tener tener IN C57BL/6 mice.

Micrurus venoms are known to induce mainly neurotoxicity in victims. However, other manifestations, including hemorrhage, edema, myotoxicity, complement activation, and hemostatic activity have been reported. In order to develop a more complete pharmacological profile of these venoms, inflammatory responses and hemostasis were evaluated in C57BL/6 mice treated with a sub-lethal dose of M. t. tener (Mtt) venom (8 µg/mouse), inoculated intraperitoneally. The venom induced moderate bleeding into the abdominal cavity and lungs, as well as infiltration of leukocytes into the liver. After 30 min, the release of pro-inflammatory mediators (TNF-α, IL-6, and NO) were observed, being most evident at 4 h. There was a decrease in hemoglobin and hematocrit levels at 72 h, a prolongation in coagulation times (PT and aPTT), a decrease in the fibrinogen concentration and an increase in fibrinolytic activity. In this animal model, it was proposed that Mtt venom induces inflammation with the release of mediators such as TNF-α, in response to the toxins. These mediators may activate hemostatic mechanisms, producing systemic fibrinolysis and hemorrhage. These findings suggest alternative treatments in Micrurus envenomations in which neurotoxic manifestations do not predominate.

Role of the inflammatory response in the hemorrhagic syndrome induced by the hemolymph of the caterpillar Lonomia achelous.

INTRODUCTION: Contact with the caterpillar of Lonomia achelous causes a hemorrhagic syndrome in humans prompted by two processes, an initial mild DIC that is later masked by overwhelming fibrinolytic activity. Although the venom affects both the hemostatic and inflammatory systems separately, it is not clear whether the hematological and hemostatic disturbances may in part be due to an indirect effect via inflammatory mediators. Here we report results on the crosstalk between these systems, particularly the effect of the pro-inflammatory cytokine TNF-α on hemostatic parameters. MATERIALS AND METHODS: the nitric oxide and TNF-α responses, as well as activation of the coagulation and fibrinolytic systems, were measured in macrophages and endothelial cells treated with Lonomia achelous hemolymph (LAH). The same responses were then determined, in a mouse model of LAH envenomation, after treatment with an anti-TNF-α antibody. RESULTS: Both macrophages and endothelial cells responded strongly to LAH in terms of pro-inflammatory mediator release and fibrinolytic activities as well as pro-coagulant activity (TF activity) in endothelial cells. Treatment with antibody against TNF-α decreased both TNF-α and NO3-/NO2- serum levels in the mice, after LAH injection. Blocking TNF-α also modified significantly the serum levels of plasminogen, fibrinogen and FXIII in mice, as well as decreased TF activity in endothelial cells. CONCLUSIONS: LAH may induce a hemostatic effect through endothelial and macrophage activation. These activated cell release hemostatic enzymes as well as pro-inflammatory mediators, principally TNF-α, that potentiate this release in an autocrine fashion, amplifying the fibrinolytic effect, which may in turn exacerbate the hemorrhagic manifestations. As far as we are aware, this is the first report of the relationship between the hemostatic system and the inflammatory responses in a hemorrhagic syndrome induce by animal secretions.