P-III hemorrhagic metalloproteinases from Russell's viper venom: cloning, characterization, phylogenetic and functional site analyses

Two homologous P-III hemorrhagic metalloproteinases were purified from Russell's viper venoms from Myanmar and Kolkata (eastern India), and designated as daborhagin-M and daborhagin-K, respectively. They induced severe dermal hemorrhage in mice at a minimum hemorrhagic dose of 0.8-0.9 microg. Daborhagin-M specifically hydrolyzed an Aalpha-chain of fibrinogen, fibronectin, and type IV collagen in vitro. Analyses of its cleavage sites on insulin chain B and kinetic specificities toward oligopeptides suggested that daborhagin-M prefers hydrophobic residues at the P(1), P(1)', and P(2)' positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65kDa, which correlated with the high risk of systemic hemorrhagic symptoms elicited by Daboia envenoming in both regions. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 P-IIIs revealed the co-evolution of two major P-III classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.     

Venom phospholipases of Russell's vipers from Myanmar and eastern India--cloning, characterization and phylogeographic analysis

Venoms of Russell's vipers (genus Daboia) are known for their deadly coagulopathic and other effects. We herein studied various isoforms of venom phospholipases A(2) (PLAs) from two Daboia species at their geographic boundary. From Myanmar Daboia siamensis venom (designated as DsM), four PLAs (designated DsM-aI, aI', aII' and bI') were purified, and the cDNAs encoding two acidic (DsM-aI and aII) and two basic PLAs (DsM-bI and S1) were also cloned from its venom-glands. DsM-S1 is identical to the major venom PLA of southern India Daboia russelii, but the protein is absent from the venom. Additionally, four PLAs (designated DrK-aI, aII, bI and bII) were cloned from cDNA obtained from venom glands of a Kolkata D. russelii, and the PLAs were purified from the pooled venom (designated as DrK). The acidic DrK-aI is the most neurotoxic and lethal among these PLAs; DsM-aI which differs from DrK-aI by only the Phe2 substitution shows greatly reduced enzymatic activity and lethality. Both acidic PLAs do not form dimeric complex with basic PLAs in the same venoms. DsM-bI' is neurotoxic and lethal but its orthologous DrK-bI (97% identical to DsM-bI') is a much weaker toxin. Given the fact that most of the orthologous PLAs of DrK and DsM share 97-100% sequence identity, Daboia vipers of Myanmar and Kolkata must be closely related. Molecular phylogenetic analyses on 30 venom PLAs of Eurasian vipers' revealed co-evolution of five subtypes of venom PLAs in both Daboia and Vipera genera. Our results shed light on the intra- and inter-species variations and structure-function relationships of viperid venom PLAs.     

Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.     

Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1kDa), PLA(2) (13kDa), and trypsin inhibitor (6.5kDa) peaks. The complex showed an LD(50) of 5.06mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

Purification,characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis

One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77 nM determined. Cloning of BBPTI-1 including the open reading frame and 3′ untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3′ RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation.     

Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom.

A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. The N-terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short-chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.     

圆斑蝰蛇毒七个C-型凝集素样蛋白亚基的cDNA克隆与序列分析

按照Promega公司的mRNA提取试剂盒操作手册,从圆斑蝰蛇（Daboia russellii siamensis）的毒腺中提取mRNA;利用RT-PCR的方法进行体外扩增,获得C-型凝集素蛋白的基因,克隆到pMD18-T载体中。随机挑选14个阳性克隆进行核酸测序,获得7个编码不同蛇毒C-型凝集素样蛋白亚基的cDNA,分别命名为DRS-L1、DRS-L2、DRS-L3、DRS-L4、DRS-L5、DRS-L6和DRS-L7。由基因序列推导出的氨基酸序列表明,克隆到的7个蛇毒C-型凝集素样蛋白的亚基中均有糖识别结构域存在。BLAST分析显示,仅有DRS-L1的蛋白序列与目前已知的蛇毒C-型凝集素样蛋白的α亚基相似。序列同源性比较并结合半胱氨酸位点分析,推测DRS-L1和DRS—L2可能分别是圆斑蝰蛇毒X因子激活剂的轻链LC2和LC1。DRS-L3和DRS-L4可能是高分子量的蛇毒C-型凝集素样蛋白的β亚基,而DRS-L5和DRS-L6可能是低分子量的蛇毒C-型凝集素样蛋白的β亚基。DRS—L7可能是类似于血小板膜糖蛋白Ib结合蛋白的β亚基。     

Purification,Characterization, and Chemical Modification of Neurotoxic Peptide from Daboia russelii Snake Venom of India

Comprehensive knowledge of venom composition is very important for effective management of snake envenomation and antivenom preparation. Daboia russelii venom from the eastern region of India is the most neurotoxic among the four venom samples investigated. From the eastern D. russelii venom sample, neurotoxic peptide has been purified by combined method of ion exchange gel permeation chromatography and reversed phase high performance liquid chromatography. Molecular weight of Daboia neurotoxin III (DNTx‐III) found to be 6,849 Da (as measured on matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometer), and N‐terminal amino acid sequences is I K C F I T P D U T S Q A. Approximate LD₅₀ dosage was 0.24 mg/kg body weight. It produced concentration‐ and time‐dependent inhibition of indirectly stimulated twitches of Rana hexadactyla sciatic nerve gastrocnemius muscle preparations. Chemical modification of DNTx‐III tryptophan residue(s) reduced the twitch height inhibition property of toxin, signifying the importance of tryptophan residues for the neurotoxic function. This type of neurotoxic peptide is unique to east Indian regional D. russelii venom. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:295‐304, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21486     

Molecular cloning, expression and purification of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma

A cDNA encoding LAAO from the Malayan pit viper (Calloselasma rhodostoma) was cloned into an expression vector of the methylotropic yeast Pichia pastoris. The LAAO open reading frame was inserted after the alpha-MF-signal sequence. Upon induction soluble and active LAAO is produced and exported into the culture supernatant at a concentration of up to 0.4 mg/L. Recombinant LAAO was purified from this by ion exchange and molecular sieve chromatography to yield apparently homogeneous protein in quantities of approximately 0.25 mg/L growth medium. Expressed LAAO exhibits the same electrophoretic mobility as native LAAO (62 kDa) and exhibits approximately the same extent of glycosylation as authentic LAAO from snake venom. Catalytic properties and substrate specificity of recombinant LAAO are similar to those of native enzyme.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Purification,Partial Characterizations,and N‐Terminal Amino Acid Sequence of a Procoagulant Protein FV‐2 from Daboia Russelli Siamensis (Myanmar) Venom

In this study, we purified and characterized the procoagulant protein FV‐2 from Daboia russelli siamensis (Myanmar) venom using ion‐exchange chromatography on CM‐Sephadex C‐50 and gel filtration on Superdex^TM G‐75 column. The activation of factor X and prothrombin was determined, respectively, by specific chromogenic substrates. The fibrinogen‐clotting activity, thermal stability, and pH stability were also determined. The N‐treminal sequence was determined by the National Center of Biomedical Analysis of China. In the end, FV‐2 was achieved with a molecular weight of 13,608.0 Da. It could activate factor X, but did not affect prothrombin or fibrinogen. The suitable pH was 6.5–7.5, and the suitable temperature ranged from 25 to 60°C. The N‐terminal sequence was Asn‐Phe‐Phe‐Gln‐Phe‐Ala‐Glu‐Met‐Ile‐Val‐Lys‐Met‐Thr‐Gly‐Lys. Taken together, our studies suggest that FV‐2 is a factor X–activating enzyme, which can activate factor X to factor Xa, but it has no effect on prothrombin and fibrinogen.     

A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms

Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.     

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators

The present study describes the purification and partial characterization of a basic anticoagulant PLA₂ enzyme named as Rv(i) PLA₂ from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA₂ family. The peptide sequence showed similarity to uncharacterized basic PLA₂ enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA₂ exhibited strong phospholipase A₂ and anticoagulant activity. It also induced expression of COX‐2 and TNF‐α mRNA in a dose‐dependent manner in phorbol 12‐myristate 13‐acetate differentiated THP‐1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA₂ with p‐bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA₂ might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom

An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.     

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.     

Inhibition of Naja naja venom hyaluronidase: role in the management of poisonous bite

Hyaluronidase is present virtually in all snake venoms and has been known as a "spreading factor." The enzyme damages the extracellular matrix at the site of the bite, leading to severe morbidity. In this study, the benefits of inhibiting the hyaluronidase activity of Indian cobra (Naja naja) venom have been investigated. Anti-NNH1 and aristolochic acid both inhibited the in vitro activity of the purified hyaluronidase, (NNH1) and the hyaluronidase activity of whole venom in a dose-dependent manner. Both anti-NNH1 and aristolochic acid abolished the degradation of hyaluronan in human skin tissue sections by NNH1 and by whole venom. Aristolochic acid quenched the fluorescent emission of NNH1. A non-competitive mechanism of NNH1 inhibition was observed with aristolochic acid. NNH1 potentiates the toxicity of Daboia russellii VRV-PL-VIII myotoxin and hemorrhagic complex-I. However, the potentiation of toxicity was inhibited dose-dependently by anti-NNH1 and aristolochic acid. Further, mice injected with whole venom which had been preincubated with anti-NNH1/aristolochic acid, showed more than a two-fold increase in survival time, compared to mice injected with venom alone. A more moderate increase in survival time was observed when mice were injected with anti-NNH1/aristolochic acid 10 min after whole venom injection. This study illustrates the significance of venom hyaluronidase in the pathophysiology of snake venom poisoning and the therapeutic value of its inhibition.     

Transcobalamin II expression is regulated by transcription factor(s) binding to a hexameric sequence (TGGTCC) in the promoter region of the gene

An isoenzyme of glutathione S-transferase (adGST) was purified from liver intestine of the seashell (Asaphis dichotoma) by GST-Sepharose 4B affinity chromatography followed by reverse-phase HPLC. The enzyme has a pI value of 4.6 and is composed of two subunits each with a molecular weight of 23kDa. It exhibits different catalytic activities toward the substrates 1-chloro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, ethacrynic acid, and p-nitrophenyl acetate and, fascinatingly, shows high activity toward CDNB. The amino acid composition of adGST was determined and found to be very similar to the Sloane squid GSTs. N-terminal analysis of the first 15 residues of adGST revealed that it has 73% sequence identity with the pig roundworm GSTs. The adGST shows characteristics similar to those of class sigma GSTs, as was indicated by its substrate specificity, N-terminal amino acid sequence, and amino acid composition.     

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential

Background

Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates.

Methods

A 51.3 kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice.

Results

Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3 kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α₂-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α₂MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity.

Conclusions

Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy.

General significance

Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.