Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin.

Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1ß1, α2ß1, α10ß1 and α11ß1. TC-I-15 is a small molecule inhibitor of α2ß1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1ß1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1ß1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1ß1 and α11ß1 as well as α2ß1. TC-I-15 was 100-fold more potent against α2ß1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.

Intravenous Vipera berus Venom-Specific Fab Fragments and Intramuscular Vipera ammodytes Venom-Specific F(ab')2 Fragments in Vipera ammodytes-Envenomed Patients.

Vipera ammodytes (V. ammodytes) is the most venomous European viper. The aim of this study was to compare the clinical efficacy and pharmacokinetic values of intravenous Vipera berus venom-specific (paraspecific) Fab fragments (ViperaTAb) and intramuscular V. ammodytes venom-specific F(ab')2 fragments (European viper venom antiserum, also called "Zagreb" antivenom) in V.ammodytes-envenomed patients. This was a prospective study of V.ammodytes-envenomed patients that were treated intravenously with ViperaTAb or intramuscularly with European viper venom antiserum that was feasible only due to the unique situation of an antivenom shortage. The highest venom concentration, survival, length of hospital stay and adverse reactions did not differ between the groups. Patients treated with intravenous Fab fragments were sicker, with significantly more rhabdomyolysis and neurotoxicity. The kinetics of Fab fragments after one or more intravenous applications matched better with the venom concentration in the early phase of envenomation compared to F(ab')2 fragments that were given intramuscularly only on admission. F(ab')2 fragments given intramuscularly had 25-fold longer apparent total body clearance and 14-fold longer elimination half-time compared to Fab fragments given intravenously (2 weeks vs. 24 h, respectively). In V.ammodytes-envenomed patients, the intramuscular use of specific F(ab')2 fragments resulted in a slow rise of antivenom serum concentration that demanded their early administration but without the need for additional doses for complete resolution of all clinical signs of envenomation. Intravenous use of paraspecific Fab fragments resulted in the immediate rise of antivenom serum concentration that enabled their use according to the clinical progress, but multiple doses might be needed for efficient therapy of thrombocytopenia due to venom recurrence, while the progression of rhabdomyolysis and neurotoxic effects of the venom could not be prevented.

Comparison of Preclinical Properties of Several Available Antivenoms in the Search for Effective Treatment of Vipera ammodytes and Vipera berus Envenoming.

Snakebites are a relatively rare medical emergency in Europe. In more than half of the annual cases caused by Vipera ammodytes, Vipera berus, and Vipera aspis, immunotherapy with animal-derived antivenom is indicated. Among eight products recently identified as available against European medically relevant species, only Zagreb antivenom, Viperfav, and ViperaTAb have been used almost exclusively for decades. Zagreb antivenom comprises V. ammodytes-specific F(ab')2 fragments. Viperfav is a polyspecific preparation based on F(ab')2 fragments against V. aspis, V. berus, and V. ammodytes venoms. ViperaTAb contains Fab fragments against the venom of V. berus. In 2014 the production of Zagreb antivenom was discontinued. Additionally, in the period of 2017 to 2018 a shortage of Viperfav occurred. Due to a lack of the product indicated for the treatment of V. ammodytes bites, other antivenoms were implemented into clinical practice without comparative assessment of their eligibility. The aim of our work was to identify a high-quality antivenom that might ensure the successful treatment of V. ammodytes and V. berus bites at the preclinical level. Differentiation between bites from these two species is difficult and unreliable in clinical practice, so the availability of a unique antivenom applicable in the treatment of envenoming caused by both species would be the most advantageous for Southeastern Europe. Zagreb antivenom, Viperfav, and ViperaTAb, as well as Viper venom antitoxin for V. berus envenoming and the in-development Inoserp Europe, which was designed to treat envenoming caused by all medically important European snakes, were comparatively tested for the first time. Emphasis was placed on their physicochemical properties, primarily purity and aggregate content, as well as their in vivo protective efficacies. As Zagreb antivenom is no longer available on the European market, Viperfav is the highest-quality product currently available and the only antivenom whose neutralisation potency against V. ammodytes and V. berus venoms was above regulatory requirements.

Moderate-to-severe Vipera berus envenoming requiring ViperaTAb antivenom therapy in the UK.

BACKGROUND: Bites by the European adder (Vipera berus) in the UK are uncommon but potentially life threatening, and can be associated with marked limb swelling and disability. Following an interruption in Zagreb Imunoloski zavod antivenom supply around 2012, the UK changed its national choice of antivenom for Vipera berus to ViperaTAb, an ovine Fab monospecific antivenom. In the absence of randomised controlled trials, we established an audit to review its use in clinical practice. METHODS: A prospective audit of ViperaTAb use was conducted from March 2016 until November 2020 by the UK National Poison Information Service (NPIS). Users of the NPIS online toxicology database, TOXBASE, considering the use of antivenom for V. berus envenoming were invited to discuss the case with the on-call clinical toxicology consultant. Information was collected prospectively on indications, administration, adverse reactions and outcome of patients administered ViperaTAb antivenom. RESULTS: One hundred and seventy patients were administered ViperaTAb antivenom over five years. One hundred and thirty-two were adults and 38 children (median age and range: 38, 2-87 years). Bites occurred across the UK, but most commonly in coastal regions of Wales and of South-West and East England. Median time to presentation was 2.1 (IQR 1.5-4.0) h and to antivenom administration from presentation was 2.0 (IQR 0.9-3.6) h. A minority of patients presented to hospital more than 12 h after being bitten (n = 19, 11.2%) or received antivenom more than 12 h after presenting to hospital (n = 17, 10.0%). Features of systemic envenoming were present in 64/170 (37.6%) patients, including 23 (13.5%) with anaphylaxis and 26 (15.3%) with hypotension (nine with both). Clinician assessment considered the initial antivenom to have been effective in 122/169 (72.2%) patients. Repeated dosing was common, occurring in 55/169 (32.5%), predominantly due to persisting or worsening local effects (46/51, 90.2%). There were three cases of probable early adverse reaction. No deaths occurred during the study. Complications of envenoming were rare but included four patients that underwent surgery, three patients each with acute kidney injury, mild coagulopathy, or thrombocytopenia (one severe). The median duration of hospital stay was 43.7 (IQR 22.5-66.5) h, longer for children than adults (52.5 vs 41.3 h). CONCLUSION: ViperaTAb antivenom appears to be effective and safe and should be administered as soon as possible for patients meeting clinical criteria. Patients require close observation following antivenom to detect adverse reactions and progression or recurrence of envenoming. Close collaboration with expert NPIS consultant advice can help optimise antivenom timing, ensure repeated dosing is given appropriately, and avoid unnecessary surgical intervention. All hospitals, particularly those located in areas of relatively high incidence, should stock sufficient antivenom available at short notice, 24 h a day.

Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram).

Echis carinatus (EC) is known as saw-scaled viper and it is endemic to the Indian subcontinent. Envenoming by EC represents a major cause of snakebite mortality and morbidity in the Indian subcontinent. Zinc (Zn++) dependent snake venom metalloproteases (SVMPs) present in Echis carinatus venom (ECV) is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that ECV activates neutrophils and releases neutrophil extracellular traps (NETs) that blocks blood vessels leading to severe tissue necrosis. However, the direct involvement of SVMPs in the release of NETs is not clear. Here, we investigated the direct involvement of EC SVMPs in observed pathological symptoms in a preclinical setup using specific Zn++ metal chelator, Tetraethyl thiuram disulfide (TTD)/disulfiram. TTD potently antagonizes the activity of SVMPs-mediated ECM protein degradation in vitro and skin hemorrhage in mice. In addition, TTD protected mice from ECV-induced footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of peptidyl arginine deiminase (PAD) 4, citH3, MPO, and p-ERK. Further, we demonstrated that ECV-induced NETosis and tissue necrosis are mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV).

Utilising venom activity to infer dietary composition of the Kenyan horned viper (Bitis worthingtoni).

Bitis are well known for being some of the most commonly encountered and medically important snake species in all of Africa. While the majority of species possess potently anticoagulant venom, only B. worthingtoni is known to possess procoagulant venom. Although known to be the basal species within the genus, B. worthingtoni is an almost completely unstudied species with even basic dietary information lacking. This study investigated various aspects of the unique procoagulant effects of B. worthingtoni venom. Coagulation assays determined the primary procoagulant effect to be driven by Factor X activating snake venom metalloprotease toxins. In addition to acting upon the mammalian blood clotting cascade, B. worthingtoni venom was also shown to clot amphibian plasma. As previous studies have shown differences in clotting factors between amphibian and mammalian plasmas, individual enzymes in snake venoms acting on plasma clotting factors can be taxon-selective. As venoms evolve under purifying selection pressures, this suggests that the procoagulant snake venom metalloprotease toxins present in B. worthingtoni have likely been retained from a recent common ancestor shared with the related amphibian-feeding Proatheris superciliaris, and that both amphibians and mammals represent a substantial proportion of B. worthingtoni current diet. Thus, taxon-specific actions of venoms may have utility in inferring dietary composition for rare or difficult to study species. An important caveat is that to validate this hypothesis field studies investigating the dietary ecology of B. worthingtoni must be conducted, as well as further investigations of its venom composition to reconstruct the molecular evolutionary history of the toxins present.

ET-B receptors involvement in peripheral opioid analgesia induced by light-emitting diode photobiomodulation in male and female mice.

Currently, photobiomodulation therapy (PBMT) is gaining space in the scientific and clinical environment. To help elucidate the importance of irradiance, this study evaluated the effect of two different PBMT irradiances (3.5 and 90 mW/cm2), given a fixed wavelength of 630 nm and a dose of 2 J/cm2, on mechanical hyperalgesia following Complete Freund's Adjuvant (CFA) intraplantar (i.pl.) injection in mice. Additionally, we investigated the role of peripheral opioid and endothelin-B receptors (ETB-R), as well as sex differences in treatment outcome. Different groups of male or female mice were evaluated 6 and 96 h after CFA. Mechanical hyperalgesia was evaluated 30 min after treatments. Naloxone or Bq-788 administration, fifteen minutes before PBMT or Sarafotoxin S6c, helped determine the involvement of peripheral opioid and ETB-Rs on PBMT. Lastly, ETB-Rs skin immunocontent in both sexes was quantified after PBMT consecutive daily treatments. PBMT at an irradiance of 90 mW/cm2, was more effective than 3.5 mW/cm2. Bq-788 and naloxone administration prevented the effects of PBMT and SRTX S6c; however, PBMT did not influence peripheral ETB-Rs immunocontent. The results suggest that irradiance influences PMBT effect; and that activation of ETB-R play a role in peripheral PBMT opioid induced analgesia. Lastly, PMBT effects do not appear to be sex-dependent.

Bitis arietans Snake Venom Induces an Inflammatory Response Which Is Partially Dependent on Lipid Mediators.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO-/-) or platelet-activating factor (PAF) receptor (PAFR-/- the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators' functions. However, 5-LO-/- mice did not show a reduction of inflammatory response induced by the venom, while PAFR-/- mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

Evolutionary Interpretations of Nicotinic Acetylcholine Receptor Targeting Venom Effects by a Clade of Asian Viperidae Snakes.

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

RGD-independent binding of Russell's Viper venom Kunitz-type protease inhibitors to platelet GPIIb/IIIa receptor.

This study elucidates the platelet-modulating properties of two snake venom Kunitz-type serine protease inhibitors, Rusvikunin and Rusvikunin-II, from Russell's Viper venom, their native and reconstituted complexes, and two synthetic custom peptides (developed from the platelet-binding region of Rusvikunin-II) against mammalian platelet-rich plasma (PRP) and washed platelets. The Rusvikunins and their complexes demonstrated concentration-dependent deaggregation and aggregation of washed platelets independent of von Willebrand factor and/or fibrinogen requirement. At lower concentrations they abolished collagen and ADP-induced platelet aggregation, but at higher concentrations, they progressively decreased the inhibition of ADP-induced aggregation and potentiated the effect of collagen on PRP. Rusvikunin complex/Rusvikunin-II bound to and induced RGD-independent aggregation of α-chymotrypsin-treated platelets. Molecular docking studies suggested interaction of Rusvikunin-II and custom peptides with platelet GPIIb/IIIa receptor, which was validated by spectrofluorometry analysis and ELISA. This study reports, for the first time, an RGD-independent binding of a snake venom component to the platelet GPIIb/IIIa receptor.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Characterization of a new member of kunitz-type protein family from the venom of Persian false-horned viper, Pseudocerastes persicus.

A new member of kunitz-type protein family, PPTI (PseudocerastesPersicusTrypsin Inhibitor), was isolated from the venom of Persian false horned viper Pseudocerastes persicus and characterized. Mass spectrometry and amino acid sequencing revealed that PPTI is a 68 amino acid protein with molecular weight of about 7.6 kDa. The first amino acid residue of PPTI is N-terminally blocked via a post translational modification to pyroglutamyl. Sequence comparison against UniProtKB shows a high sequence similarity of PPTI with kunitz-type proteins, especially serine protease inhibitors and dendrotoxins (DTXs). The number of cysteines and disulfide bonding pattern of PPTI are the same as kunitz-type proteins. Based on sequence derive information, anti-protease activity of PPTI against trypsin was experimentally examined. The constructed homology models of PPTI confirmed the ability of PPTI to fold similarly to kunitz domain. The presence of characteristic basic-hydrophobic functional dyad of DTXs in PPTI supports its inhibitory potential against potassium channels. In summary, this study hypothesized the dual functionality of PPTI according to its inhibitory effect on trypsin and its potential ability in blocking potassium channel.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Comparative Profiling of Three Atheris Snake Venoms: A. squamigera, A. nitschei and A. chlorechis.

A proteomic and transcriptomic comparative analysis of the venoms of three Atheris species (A. squamigera, A. nitschei and A. chlorechis) was carried out by size exclusion liquid chromatography, gel electrophoresis, mass spectrometry, and mRNA sequencing. The improved proteomic profiling utilised in this work was combined with transcript studies, advancing our insights into venom composition, protein distribution and inter-species variation among the three bush vipers. Crude venoms of all three samples contained at least 10-20 protein components, ranging in size from ≤ 3 to > 98 kDa. Both approaches yielded converging overall information, pointing to phospholipases, disintegrins, serine proteases and metalloproteases as the major toxin classes, which are likely to explain the local and systemic symptoms observed in envenomation by Atheris genus. Being considered as the main factors involved in the distinct venom-induced pathologies, these identified snake venom proteins are of particular interest in terms of understanding their physiological and biological function as well as for their contribution in potential medical treatments.

Vasomotor dysfunction in human subcutaneous arteries exposed ex vivo to food-grade titanium dioxide.

Animal studies have shown that titanium dioxide (TiO2) exposure affects arterial vasomotor function, whereas little is known about the effects in arteries from humans. This study investigated vasomotor responses after direct exposure of human subcutaneous arteries to food-grade TiO2 (E171) (14 or 140 µg/ml) for 30 min and 18 h. Vasomotor responses to bradykinin, 5-hydroxytryptamine (5-HT), sarafotoxin 6c (S6c) and nitroglycerin were recorded in wire-myographs. Vasoconstrictor responses to 5-HT were increased in arteries exposed to E171 for 18 h (P < 0.05). Furthermore, an increase in S6c responses was seen in low concentration E171 exposed arteries (30 min exposure; P < 0.05). The vasorelaxation response to nitroglycerin was increased in low concentration E171 exposed arteries (30 min exposure; P < 0.05). Vasorelaxation responses to bradykinin were unaffected after treatment with E171. There was no difference in gene expression levels of intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, 5-hydroxytryptamine receptor 1B, 5-hydroxytryptamine receptor 2A, endothelin receptor A and endothelin receptor B in E171 exposed arteries after exposure to TiO2 for 30 min or 18 h. In conclusion, this study shows that the same type of vasomotor dysfunction is found in artery segments of rats and humans following ex vivo exposure to E171.

Targeting α1 inserted domain (I) of α1ß1 integrin by Lebetin 2 from M. lebetina transmediterranea venom decreased tumorigenesis and angiogenesis.

Through the recent development of knowledge in biotechnology and bioinformatics, snake venoms are widely used to develop new drugs to treat diseases such as hypertension and cancer. We have previously reported that Lebetin 2 isolated from Macrovipera lebetina transmediterranea venom displays a potent anti-platelet activity and exerts a cardioprotective effect in ischemia-reperfusion (IR) injury model. Here, we report that Lebetin 2 possess an anti-tumor effect by targeting the integrin receptor function. It was thus able to inhibit both adhesion and migration of pheochromocytoma cells (PC12) and α1ß1 integrin-expressing CHO cells (CHO-α1) to type I and IV collagens. Moreover, this peptide affects proliferation of PC12 cells by modulating AKT phosphorylation. Furthermore, Lebetin 2 exhibits a potent anti-angiogenic effect as assessed in vitro and ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Interestingly, the interaction mode of Lebetin 2 with the integrin α1ß1, assessed in silico, showed that the peptide represents a steric obstruction preventing the collagen from enforcing the interactions with the integrin.

Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) with short and long venom glands.

Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and inhibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evolution knowledge but also highlight the limited clinical management outcomes for Causus envenomations.

Functional characterization of recombinant snake venom rhodocytin: rhodocytin mutant blocks CLEC-2/podoplanin-dependent platelet aggregation and lung metastasis.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and ß-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. SUMMARY: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and ß-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTßWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTßWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTßK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

Snake venom VEGF Vammin induces a highly efficient angiogenic response in skeletal muscle via VEGFR-2/NRP specific signaling.

Vascular Endothelial Growth Factors (VEGFs) are promising molecules for the treatment of ischemic diseases by pro-angiogenic therapy. Snake venom VEGFs are a novel subgroup with unique receptor binding profiles and as such are potential new therapeutic agents. We determined the ligand-receptor interactions, gene regulation and angiogenic properties of Vipera ammodytes venom VEGF, Vammin, and compared it to the canonical angiogenic factor VEGF-A to evaluate the use of Vammin for therapeutic angiogenesis. Vammin efficiently induced VEGFR-2 mediated proliferation and expression of genes associated with proliferation, migration and angiogenesis. VEGF-A165 and especially VEGF-A109 induced less pronounced effects. Vammin regulates a number of signaling pathways by inducing the expression of NR4A family nuclear receptors and regulators of calcium signaling and MAP kinase pathways. Interestingly, MARC1, which encodes an enzyme discovered to catalyze reduction of nitrate to NO, was identified as a novel VEGFR-2 regulated gene. In rabbit skeletal muscle adenoviral delivery of Vammin induced prominent angiogenic responses. Both the vector dose and the co-receptor binding of the ligand were critical parameters controlling the type of angiogenic response from sprouting angiogenesis to vessel enlargement. Vammin induced VEGFR-2/NRP-1 mediated signaling more effectively than VEGF-A, consequently it is a promising candidate for development of pro-angiogenic therapies.

Understanding Russell's viper venom factor V activator's substrate specificity by surface plasmon resonance and in-silico studies.

Blood coagulation factor V (FV) is activated either by Factor X or thrombin, cleaving at three different sites viz., Site I (Arg709-Ser710), site II (Arg1018-Thr1019), and site III (Arg1545-Ser1546). Russell's viper venom factor V activator (RVV-V) is a thrombin-like serine proteinase that activates FV with selective, single cleavage at site III. A long lasting effort is being pending in understanding the 'selective' binding specificity of the RVV-V towards site III. Here, we present the binding kinetic study of RVV-V with two designed peptides corresponding to the regions from site I (Gln699-Asn713) and site II (1008Lys-Pro1022), respectively, that include 15 amino acids. Our investigation for justifying the binding efficacy and kinetics of peptides includes SPR method, protein-peptide docking, molecular dynamics simulation, and principal component analysis (PCA). Surprisingly, the SPR experiment disclosed that the Peptide II showed a lower binding affinity with KD of 2.775 mM while the Peptide I showed none. Docking and simulation of both the peptides with RVV-V engaged either rooted or shallow binding for Peptide II and Peptide I respectively. The peptide binding resulted in global conformational changes in the native fold of RVV-V, whereas the similar studies for thrombin failed to make major changes in the native fold. In support, the PCA analysis for RVV-V showed the dislocation of catalytic triad upon binding both the peptides. Hence, RVV-V, a serine protease, is incompetent in cleaving these two sites. This study suggests a transition in RVV-V from the native rigid to the distorted flexible structure and paves a way to design a new peptide substrate/inhibitor.