Detection of Venom after Antivenom Is Not Associated with Persistent Coagulopathy in a Prospective Cohort of Russell's Viper (Daboia russelii) Envenomings

Background

Venom recurrence or persistence in the circulation after antivenom treatment has been documented many times in viper envenoming. However, it has not been associated with clinical recurrence for many snakes, including Russell''s viper (Daboia spp.). We compare the recovery of coagulopathy to the recurrence or persistence of venom in patients with Russell''s viper envenoming.

Methodology/Principal Findings

The study included patients with Russell''s viper (D. russelii) envenoming presenting over a 30 month period who had Russell''s viper venom detected by enzyme immunoassay. Demographics, information on the snake bite, and clinical effects were collected for all patients. All patients had serum collected for venom specific enzyme immunoassay and citrate plasma to measure fibrinogen levels and prothrombin time (international normalised ratio; INR). Patients with venom recurrence/persistence were compared to those with no detectable recurrence of venom. There were 55 patients with confirmed Russell''s viper envenoming and coagulopathy with low fibrinogen concentrations: 31 with venom recurrence/persistence, and 24 with no venom detected post-antivenom. Fibrinogen concentrations increased and INR decreased after antivenom in both the recurrence and non-recurrence patients. Clinical features, laboratory parameters, antivenom dose and length of hospital were similar for both groups. Pre-antivenom venom concentrations were higher in patients with venom recurrence/persistence with a median venom concentration of 385 ng/mL (16–1521 ng/mL) compared to 128 ng/mL (14–1492 ng/mL; p = 0.008).

Conclusion

Recurrence of Russell''s viper venom was not associated with a recurrence of coagulopathy and length of hospital stay. Further work is required to determine if the detection of venom recurrence is due to the venom specific enzyme immunoassay detecting both venom-antivenom complexes as well as free venom.     

SjAPI,the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Background

Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.

Principal Findings

Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.

Conclusions/Significance

To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.     

SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom

Background

Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized.

Principal Findings

A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (K_i = 1.6×10⁻⁷ M) and thermostability.

Conclusions

The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides.     

Allopurinol reduces the lethality associated with acute renal failure induced by Crotalus durissus terrificus snake venom: comparison with probenecid

Background

Acute renal failure is one of the most serious complications of envenoming resulting from Crotalus durissus terrificus bites. This study evaluated the relevance of hyperuricemia and oxidative stress and the effects of allopurinol and probenecid in renal dysfunction caused by direct nephrotoxicity of C. d. terrificus venom.

Methodology/Principal Findings

Hematocrit, protein, renal function and redox status were assessed in mice. High ratio of oxidized/reduced glutathione and hyperuricemia induced by C. d. terrificus venom were ameliorated by both, allopurinol or probenecid, but only allopurinol significantly reduced the lethality caused by C. d. terrificus venom. The effectiveness of probenecid is compromised probably because it promoted hypercreatinemia and hypocreatinuria and worsed the urinary hypo-osmolality in envenomed mice. In turn, the highest effectiveness of allopurinol might be due to its ability to diminish the intracellular formation of uric acid.

Conclusions/Significance

Data provide consistent evidences linking uric acid with the acute renal failure induced by C. d. terrificus venom, as well as that this envenoming in mice constitutes an attractive animal model suitable for studying the hyperuricemia and that the allopurinol deserves to be clinically evaluated as an approach complementary to anti-snake venom serotherapy.     

Venom proteomic analysis of medically important Nigerian viper Echis ocellatus and Bitis arietans snake species

Snakebite envenoming remains a neglected tropical disease which poses severe health hazard, especially for the rural inhabitants in Africa. In Nigeria, vipers are responsible for the highest number of deaths. Hydrophilic interaction liquid chromatography coupled with LC-MS/MS was used to analyze the crude venoms of Echis ocellatus (Carpet viper) and Bitis arietans (Puff adder) in order to understand their venom proteomic identities. Results obtained revealed that gel-free proteomic analysis of the crude venoms led to the identification of 85 and 79 proteins, respectively. Seventy-eight (78) proteins were common between the two snake species with a 91.8% similarity score. The identified proteins belong to 18 protein families in E. ocellatus and 14 protein families in B. arietans. Serine proteases (22.31%) and metalloproteinases (21.06%) were the dominant proteins in the venom of B. arietans; while metalloproteinases (34.84%), phospholipase A₂s (21.19%) and serine proteases (15.50%) represent the major toxins in the E. ocellatus venom. Other protein families such as three-finger toxins and cysteine-rich venom proteins were detected in low proportions. This study provides an insight into the venom proteomic analysis of the two Nigerian viper species, which could be useful in identifying the toxin families to be neutralized in case of envenomation.     

Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction

Background

Proteases have been shown to degrade airway mucin proteins and to damage the epithelium impairing mucociliary clearance. There are increased proteases in the COPD airway but changes in protease-antiprotease balance and mucin degradation have not been investigated during the course of a COPD exacerbation. We hypothesized that increased protease levels would lead to mucin degradation in acute COPD exacerbations.

Methods

We measured neutrophil elastase (NE) and alpha 1 protease inhibitor (A1-PI) levels using immunoblotting, and conducted protease inhibitor studies, zymograms, elastin substrate assays and cigarette smoke condensate experiments to evaluate the stability of the gel-forming mucins, MUC5AC and MUC5B, before and 5–6 weeks after an acute pulmonary exacerbation of COPD (n = 9 subjects).

Results

Unexpectedly, mucin concentration and mucin stability were highest at the start of the exacerbation and restored to baseline after 6 weeks. Consistent with these data, immunoblots and zymograms confirmed decreased NE concentration and activity and increased A1-PI at the start of the exacerbation. After recovery there was an increase in NE activity and a decrease in A1-PI levels. In vitro, protease inhibitor studies demonstrated that serine proteases played a key role in mucin degradation. Mucin stability was further enhanced upon treating with cigarette smoke condensate (CSC).

Conclusion

There appears to be rapid consumption of secreted proteases due to an increase in antiproteases, at the start of a COPD exacerbation. This leads to increased mucin gel stability which may be important in trapping and clearing infectious and inflammatory mediators, but this may also contribute acutely to mucus retention.     

Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network

Background

Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity.

Methodology/Principal Findings

In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn₁₄) which displayed both a 125-fold increased capacity to inhibit KLK4 (K _i = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn₁₄ was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion.

Conclusion/Significance

These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.     

A genomic survey of proteases in Aspergilli

Background

Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far.

Results

We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli.

Conclusions

Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-523) contains supplementary material, which is available to authorized users.     

Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell’s Viper Bites with Coagulopathy

Background

Russell’s viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell’s viper envenoming.

Methodology/Principal Findings

In a prospective cohort of 146 patients with Russell’s viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39y (16–82y) and 111 were male. The median peak INR was 6.8 (interquartile range[IQR]:3.7 to >13), associated with low fibrinogen [median,<0.01g/L;IQR:<0.01–0.9g/L), low factor V levels [median,<5%;IQR:<5–4%], low factor VIII levels [median,40%;IQR:12–79%] and low factor X levels [median,48%;IQR:29–67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48h post-antivenom. The median INR remained >3 at 6h post-antivenom but had reduced to <2, by 24h. The aPTT had also returned to close to normal (<50sec) at 24h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis).

Conclusions

Russell’s viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48h. Severity of clotting abnormalities was associated with venom concentrations.     

Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.     

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr

Background

The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician''s toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and Principal Findings

As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions

This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.     

Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation,Cytokine Production and Mast Cell Degranulation

Background

Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell''s viper) and antivenom treatment.

Methodology/Principal Findings

Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor α (TNFα), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine.

Conclusions/Significance

We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation. Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations, as well as a priming effect from the immune response to the venom itself.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.