Albolabris Venom Research Articles

Diagnostic Tests for Hypofibrinogenemia Resulting from Green Pit Viper (Trimeresurus albolabris) Envenomation: A Simulated In Vitro Study.

The green pit viper (GPV) Trimeresurus albolabris is found in Southeast Asia. Its venom has a thrombin-like activity that can cause hypofibrinogenemia. Fibrinogen measurement is not always available. We aimed to establish a more available diagnostic tool indicating hypofibrinogenemia caused by GPV envenomation. This was an in vitro study, in which healthy subjects aged 20 to 45 y were enrolled. There were 2 experiments. In Experiment 1, blood samples from 1 subject had varying amounts of T albolabris venom added to determine its effect on the fibrinogen level (FL). In Experiment 2, 3 sets of blood samples were obtained from another 25 subjects. The 2 venom doses established in Experiment 1 were used on 2 sets of the samples to simulate severe (FL <1.0 g·L-1) and mild hypofibrinogenemia (FL 1.0-1.7 g·L-1). The third set of samples was venom-free. All samples were used for platelet counts, prothrombin time (PT)/international normalized ratio (INR)/activated partial thromboplastin time (aPTT), and 2 bedside clotting tests. Diagnostic parameters were calculated against the target FL of <1.0 g·L-1 and <1.7 g·L-1. Twenty-five subjects were enrolled in Experiment 2. On referencing normal cutoff values (platelet count >150,000 cells/mm3, venous clotting time <15 min, normal 20-min whole blood clotting time, INR <1.2, aPTT <30), we found abnormalities of 5, 0, 0, 3, and 22%, respectively. The highest correlation with hypofibrinogenemia was provided by PT/INR. For an FL of <1.0 g·L-1, PT and INR revealed the highest areas under the receiver operating characteristic curve, 0.76 (95% CI, 0.55-0.97) and 0.76 (95% CI, 0.57-0.97), respectively. The highest accuracy and the highest sensitivity were provided by PT/INR. PT/INR could be used as a diagnostic test for severe hypofibrinogenemia in GPV envenomation because of its high accuracy and area under the receiver operating characteristic curve.

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms.

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T. albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

Toxicity and cross-neutralization of snake venoms from two lesser-known arboreal pit vipers in Southeast Asia: Trimeresurus wiroti and Trimeresurus puniceus

The lesser-known Sundaic lance-headed pit vipers Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia) contribute to the disease burden of snakebite envenomation in Southeast Asia, but their venom toxicity and neutralization remain insufficiently investigated. This study demonstrated that both venoms were procoagulant (involving thrombin-like activity), hemorrhagic, and lethal to mice, with T. wiroti venom being more lethal (LD50 = 0.78 μg/g c.f. 1.21 μg/g). The hetero-specific antivenom from Thailand, Green Pit Viper Antivenom (GPVAV, raised against Trimeresurus albolabris) cross-reacted with T. wiroti and T. puniceus venoms with a higher efficacy of immunological binding activity for the latter. The antivenom was also effective in cross-neutralizing the procoagulant, hemorrhagic and lethal effects of the venoms. In lethality neutralization, GPVAV showed a potency of 0.79–1.05 mg venom per mL antivenom, corresponding to the complete neutralization of approximately 8–10 mg venom per unit vial of antivenom for T. wiroti and T. puniceus venoms. Taken together, it was inferred that T. wiroti, T. puniceus, and T. albolabris venoms share common toxin epitopes, thus enabling the cross-neutralization observed. These findings suggest that GPVAV may be potentially useful in the management of envenomation by T. wiroti and T. puniceus while awaiting clinical trial and validation.

Trimeresurus albolabris snakebite treatment implications arising from ontogenetic venom comparisons of anticoagulant function, and antivenom efficacy

Does the venom of Trimeresurus albolabris (white-lipped pit viper) differ between neonate and adults? This species is responsible for most snakebites within south and southeast Asia, yet it is unknown whether ontogenetic variation in venom composition occurs in this species, or how this might affect antivenom efficacy. Using a coagulation analyser robot, we examined the anticoagulant activity of T. albolabris venom from eight individuals across multiple age classes. We then compared the efficacy of Thai Red Cross Green Pit Viper Antivenom across these age classes. Venoms from all age classes were equally potent in their pseudo-procoagulant, fibrinogenolytic activity, in that fibrinogen was cleaved to form weak, unstable fibrin clots that rapidly broke down, thus resulting in a net anticoagulant state. Similarly, this coagulotoxic activity was well neutralised by antivenom across all venoms. Given that coagulotoxicity is the primary serious pathology in T. albolabris envenomations, we conclude that Thai Red Cross Green Tree Pit Viper Antivenom is a valid treatment for envenomations by this species, regardless of age or sex of the offending snake. These results are relevant for clinical treatment of envenomations by T. albolabris.

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom ‘Thai Red Cross Green Pit Viper antivenin’, varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy.

BackgroundSnakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens.MethodsIgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice.ResultsChickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg).ConclusionsIgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabris venom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Biological activities of Peristrophe bivalvis extracts: promising potential for anti-snake venoms against Naja kaouthia and Trimeresurus albolabris venoms

This study evaluates the in vitro anti-snake venom potential of Peristrophe bivalvis (PB) extracts against Naja kaouthia (NK) and Trimeresurus albolabris (TA) venoms, including inhibition of cytotoxic effects and enzymatic activities, and the binding-precipitation of extracts and venom proteins analysis. In addition, the antioxidant, cytotoxic and in vivo acute oral toxic activities of PB extracts are also reported. The in vitro cytotoxic and enzymatic analysis reveals that the ethanol extracts of stems and leaves of PB showed good anti-snake venom activity against NK and TA venoms. In addition, the antioxidant result indicated that only the ethanol extract of leaves exhibited weak DPPH radical-scavenging activity. The ethanol whole-plant extract of PB also showed no cytotoxicity against four cell lines. Moreover, the in vivo acute oral toxicity result of the ethanol whole-plant extract showed that all treated rats did not exhibit abnormal toxic signs or deaths.

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)4. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC50 of 0.25nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.

Recombinant Russell’s viper venom-factor X activator (RVV-X)-specific antibody: neutralization and crossreactivity with Cryptelytrops albolabris and Calloselasma rhodostoma venoms

Background: Russell’s viper venom-factor X activator (RVV-X) is a major procoagulant in Russell’s viper venom, and is composed of a heavy chain (RVV-XH) and two light chains (RVV-XL). It directly activates factor X in the final common coagulation pathway, which leads to rapid formation of blood clots. Objective: Produce rabbit anti-recombinant protein antibodies and identify their cross-reactivity with two viperine snake venoms. Methods: cDNA clones encoding RVV-XH and one of the light chains (RVV-XL; LC1) were recombinantly expressed in E. coli BL21 and used as antigens for rabbit immunization. The cross-reactivity of these anti-recombinant protein antibodies with two viperine snake venoms was determined using Western blot analysis. Results: rRVV-XH was more immunogenic than rRVV-XL. Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies bind specifically to RVV-X, but they do not neutralize purified RVV-X. In addition, rabbit anti-rRVV-XH IgG antibody also bind to an 18-kDa protein in C. rhodostoma venom, and many proteins in C. albolabris venom. Rabbit antirRVV- XL IgG antibody recognized protein bands of crude venoms of C. rhodostoma and C. albolabris at about 25-kDa and 23-kDa, respectively. Conclusion: Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies cross-reacted with molecules in other viperine venoms, which could have molecules with similar antigenic determinants. These antibodies could be useful to purify snake venom molecules by affinity chromatography as the first step in purification of factor X activator and other cross reacting molecules. Keywords: Anti-rRVV-XH IgG antibody, anti-rRVV-

Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper ( Trimeresurus albolabris) venom

Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an α fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure–function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.

Molecular cloning and sequence analysis of alboaggregin B

Green pit viper (Trimeresurus albolabris) venom contains a variety of C-type lectin-like proteins (CLPs) causing platelet aggregation and consumptive thrombocytopenia in biting victims. Alboaggregin B (AL-B), a heterodimeric glycoprotein (Gp) Ib-binding protein, was purified from the venom, but there is no reported cDNA sequence and the platelet agglutination mechanism is poorly understood. The full-length AL-B β clone was obtained from T. albolabris venom gland cDNA library. AL-B α was, later, derived using 3′-RACE based on the conserved sequence. In this study, purified AL-B dimer agglutinated human platelets with the EC50 of 180 nM and was completely inhibited by anti GpIb antibody. MALDI ToF mass spectroscopy found no glycosylation. The peptide mass fingerprints were matched with deduced amino acid sequences of cloned genes. AL-B α and β contained 156 and 146 amino acid, respectively, including 23-residue signal peptides. AL-B β showed the conserved hydrophilic patches, putative sites for GpIb binding. Furthermore, there was another conserved motif (SRTY) exclusively in platelet-agglutinating AL-B, TSV-GPIb-BP and Mamushigin. We propose that these three CLPs may function as bivalent adhesive proteins linking two GpIb molecules on adjacent platelets.

Molecular cloning of novel serine proteases and phospholipases A 2 from green pit viper ( Trimeresurus albolabris) venom gland cDNA library

Green pit viper ( Trimeresurus albolabris) is the most common venomous snake responsible for bites in Bangkok. It causes local edema and systemic hypofibrinogenemia resulted from the thrombin-like, as well as the fibrinolytic effects of the venom. However, the amino acid sequences of these venom proteins have never been reported. In this study, we have cloned five novel serine proteases from the Thai T. albolabris venom gland cDNA library. They were all closely homologous to the corresponding serine proteases from Chinese green viper ( Trimeresurus stejnegeri), suggesting the evolutionary proximity of the two species. In addition, their functional activities could be deduced. There were predicted to be two thrombin-like enzymes (GPV-TL1 and GPV-TL-2), two isoforms of a fibrinogenolytic enzyme (albofibrase) and a plasminogen activator (GPV-PA), suggesting that defibrination syndrome in patients is a combination of these enzymatic effects. By multiple sequence alignment, no conserved residue or motif responsible for distinct functions of snake venom serine proteases could be observed. Moreover, one Lys 49 and one Asn 49 phospholipase A 2 (PLA 2) genes were cloned. Lys 49 PLA 2 was predicted to devoid of catalytic activity, but showed a carboxy terminal cytotoxic region. No Asp 49 PLA 2 was found in 150 clones screened. This explains the marked limb edema but no hemolysis in patients. These novel serine proteases have potentials to be therapeutic anti-thrombotic and thrombolytic agents in the future.