Inhibition of human platelet aggregation by -amino acid oxidase purified from Naja naja kaouthia venom

-Amino acid oxidase (LAO) widely exists in snake venoms. Purification of LAO from the Naja naja kaouthia (monocellate cobra) venom has been reported (Tan and Swaminathan, 1992), but its structural characterization and physiological function remained to be determined. The function of snake venom LAOs in hemostasis, especially their effect on platelet aggregation, has been controversial. We determined the N-terminal amino acid sequence of the N. n. kaouthia LAO named K–LAO to be DDRRSPLEECFQQNDYEEFLEIAKNGLKKTxNPKHVXxV (38 residues). The protein data base search revealed that the enzyme had high similarities with other snake venom LAOs. Further, platelet aggregation studies revealed that K–LAO functionally did not induce platelet aggregation in a platelet-rich plasma system, but that it inhibited platelet aggregation induced by agonists such as ADP, collagen and ristocetin in a dose-dependent manner. K–LAO diminished platelet aggregation more intensely under low than high shear stress. This inhibitory activity of K–LAO on either ristocetin-induced or shear-induced platelet aggregation was quenched by addition of catalase. These results indicate that K–LAO functions as an inhibitor to platelet aggregation through the formation of hydrogen peroxide. The enzyme may contribute to the development of a severe hematological disorder due to cobra envenomation.     

L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification,characterization, platelet aggregation-inducing and antibacterial effects

An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography. The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits. The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme. The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs. Homology with elapid venom LAO is lower. TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium. The antibacterial effect associated with LAO activity was elminated with the addition of catalase. Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO. The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation. These results showed H2O2 formation is important for the biological effects of LAO.     

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.     

Development of a polymerase chain reaction to distinguish monocellate cobra (Naja khouthia) bites from other common Thai snake species, using both venom extracts and bite-site swabs.

A PCR technique was used in this study to identify and distinguish monocellate cobra snake bites using snake venoms and swab specimens from snake bite-sites in mice from bites by other common Thai snakes. The sequences of nucleotide primers were selected for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) since the sequences of monocellate cobra (Naja kaouthia) venom are still unknown. However, the 113-bp fragment of cDNA of the cobrotoxin-encoding gene was detected in the monocellate cobra venom using RT-PCR. This gene was not found in the venoms of Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), Daboia russelii siamensis (Siamese Russell's Viper, and Calloselasma rhodostoma (Malayan pit viper). Moreover, direct PCR could detect a 665-bp fragment of the cobrotoxin-encoding gene in the monocellate cobra venom but not the other snake venoms. Likewise, this gene was only observed in swab specimens from cobra snake bite-sites in mice. This is the first report demonstrating the ability of PCR to detect the cobrotoxin-encoding gene from snake venoms and swab specimens. Further studies are required for identification of this and other snakes from the bite-sites on human skin.     

Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom.

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.     

Humanized-Single Domain Antibodies (VH/V(H)H) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.     

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.     

Proteome and immunome of the venom of the Thai cobra, Naja kaouthia.

The proteome of the Thai cobra, Naja kaouthia, venom, revealed by two-dimensional liquid chromatography/tandem mass spectrometry, was found to consist of peptides which could be matched with 61 proteins in the database. These proteins were classified into 12 groups according to the differences in their biological activities: cardiotoxins, cobra venom factors, a cysteine-rich toxin, cytotoxins, kaouthiagin, mocarhagin, muscarinic toxin-like proteins, neurotoxins, an oxoglutarate dehydrogenase, phospholipases, serum albumin, and a weak toxin. Horse derived- anti-N. kaouthia venom hyperimmune serum currently used for the treatment of cobra ophitoxaemia reacted only to the cobra venom factors and phospholipases in the cobra holovenom by two-dimensional gel electrophoresis based-immunoblotting. The venom proteomic insight of this study should pave the way for preparing a therapeutic anti-venom of improved quality, i.e. also containing antibodies to the newly revealed toxic, but poorly immunogenic, minor venom components. It is expected that such a preparation should have a higher effectiveness than the currently used anti-venom in resuscitating cobra-bite victims.     

Development of reversed passive latex agglutination for detection of Thai cobra (Naja kaouthia) venom.

A simple, rapid, and sensitive diagnostic kit for detecting Thai cobra (Naja kaouthia) venom was developed using latex particles sensitized with venom specific immunoglobulin. The kit is capable of detecting 25-50 ng/ml of Thai cobra venom. The capability was not affected by human plasma. Specificity of the kit was proven using snake venoms from Vipera russelli, Calloselasma rhodostoma, Trimeresurus albolabris, Naja siamensis, Ophiophagus hannah, and Bungarus fasciatus. The diagnostic kit does not lose its capability under refrigeration for two months and by lyophilization.     

Characterization of the anticoagulants from Taiwan cobra (Naja naja atra) snake venom

Taiwan cobra (Naja naja atra) snake venom was separated into 19 fractions by means of CM-Sephadex C-50 column chromatography. Anticoagulant Fractions V-VII were refractionated by gel filtration on Sephadex G-50 and the purified component possessed phospholipase A2 activity and an inhibitory effect on collagen-induced platelet aggregation. The anticoagulant action could be antagonized by phospholipid or platelet factor 3. Anticoagulant Fraction XVII was also further refractionated by gel filtration on Sephadex G-50 and the purified component was shown to be cardiotoxin. It was a weak anticoagulant, caused direct hemolysis and potentiated collagen-induced platelet aggregation. Thromboelastographic studies showed that the anticoagulant action of cobra venom is due to the synergistic effects of phospholipase A2 and cardiotoxin.     

中国眼镜蛇毒蛋白酶的分离、纯化及其对血小板聚集的影响

目的：从中国眼镜蛇毒中分高纯化出可水解纤维蛋白原的蛇毒蛋白酶,并观察其体内给药对血小板聚集的影响。方法：使用超滤的方法以及肝素亲和层析柱HeparinSepharoseCL－6B和凝胶层析柱SephadexG150分高纯化中国眼镜蛇毒蛋白酶。SDS-聚丙烯酰胺凝胶电泳（SDS－PAGE）检测其纯度和分子量。比浊法测定血小板聚集率。结果：从中国眼镜蛇毒中纯化出具有水解纤维蛋白原活性的蛋白酶,经SDS－PAGE电泳测定为一条带,分子量为47.1kD。该蛋白酶低剂量（0．025mg／kg）、中剂量（0．05mg／kg）以及高剂量（0．1mg／kg）体内给药后,剂量依赖性地抑制ADP（10μmol／L）、胶原（100μg／ml）诱导的兔血小板聚集。结论：具有水解纤维蛋白原活性的中国眼镜蛇毒蛋白酶在体内表现出抑制血小板聚集的作用。     

Crystal structures of an acidic phospholipase A(2) from the venom of Naja Kaouthia.

A phospholipase A(2) purified from the venom of Naja kaouthia (Guangxi cobra) exhibits anticoagulant activities. The structures of two crystal forms were determined by X-ray crystallography at 2.8A resolution with the Naja naja (India cobra) PLA(2) as an initial model. The enzyme exhibits a trimer structure, which is similar to that of India cobra PLA(2). This reinforces the physiological relevance of the oligomer. The trimer has a wide cavity, which allows the substrate to enter and interact with the catalytic site. The formation of the trimer may serve as a storage method to improve the solubility at high concentration in the venom gland. The Ca2+ binding loop in the absence of the cation can exist in different conformations depending on its surroundings.     

蛇毒蛋白与血小板血栓形成

多种蛇毒蛋白具有与血小板表面整合素、膜糖蛋白Ⅰb（GPⅠb）或血管性血友病因子（VWF）相互作用而影响血栓形成的功能。影响血小板血栓形成的蛇毒蛋白目前分为3类：去整合素、VWF调节蛇毒蛋白及GPⅠb结合蛋白。其中,去整合素具有高效抑制血小板聚集的功能;VWF调节蛇毒蛋白具有体外介导VWF依赖型血小板聚集的功能;而GPⅠb结合蛇毒蛋白又分为2组：GPⅠb激动剂和GPⅠb拮抗剂,分别起诱导血小板聚集与抑制VWF介导的血小板聚集的功能。本文将对上述影响血小板血栓形成的蛇毒蛋白的结构、功能及其在临床上的研究与应用进行综述。     

Polyclonal antibodies against native weak toxin Naja kaouthia discriminate native weak toxins and some other three-fingered toxins against their denaturated forms.

Polyclonal antibodies obtained by immunization of rabbits with native form of weak toxin (WTX) from cobra Naja kaouthia venom efficiently interacted with WTX and a weak toxin from Naja oxiana venom, but not so with their denaturated forms. These antibodies could also bind with lower affinity other groups of three-fingered toxins: long-chain alpha-neurotoxins, muscarinic toxins and cytotoxins, but practically did not bind short-chain alpha-neurotoxins. The efficiency of toxin-antibody interaction depends on the group (weak toxins, long or short alpha-neurotoxins, cytotoxins etc.) to which the toxin belongs, but not on species of snake from which the toxin originates. There is a correlation between the results obtained and phylogenetic analysis of the three-fingered toxins which revealed that WTX is very close to other weak toxins, relatively close to long alpha-neurotoxins, cytotoxins and muscarinic toxins, but is distant from the short alpha-neurotoxins.     

Purification, characterization and potent lung lesion activity of an l-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom

L-amino acid oxidases (LAOs) are one of the major components of snake venoms, which possess numerous biological functions. However, little is known of the influence of LAOs on organ lesions. In the present study, a unique LAO from Agkistrodon blomhoffii ussurensis snake venom named ABU-LAO was purified by Heparin-Sepharose FF chromatography followed by an ion-exchange chromatography procedure. The purified ABU-LAO appears a dimer with a molecular mass of approximately 108.8kDa. Kinetics studies showed that ABU-LAO is very active towards its substrates L-Asn, L-Phe, L-Tyr, L-Leu, L-Ile and L-Trp. The most striking observation in the present study is that ABU-LAO causes severe pneumorrhagia, pulmonary interstitial edema, fusion of pulmonary alveoli, cardiac interstitial edema and bleeding when being intravenously injected into BALB/c mice. ABU-LAO also induces liver cell necrosis and release of cytokines including IL-6, IL-12 and IL-2 from highly purified human peripheral blood monocytes and T cells, respectively. In conclusion, ABU-LAO potently induces lesions in lungs and livers. The ability of ABU-LAO will contribute to the understanding of the pathogenesis of snakebite wound.     

尖吻蝮蛇毒小分子多肽的分离及抗血小板聚集作用

目的从尖吻蝮蛇毒中分离纯化一种抗血小板聚集小分子多肽,研究其理化性质以及对ADP、胶原、凝血酶诱导的血小板聚集反应的影响。方法经SephadexG-75凝胶过滤,超滤,DEAE-SepharoseCL-6B离子交换层析法分离纯化蛇毒组分,采用高效液相鉴定纯度,用SDS-凝胶电泳(SDS-PAGE)测定其分子量,用比浊法测定其抗血小板聚集活性。结果从尖吻蝮蛇毒中分离相对分子质量约为7862u等电点为4.29的组分,该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性。结论此法成功地从尖吻蝮蛇毒中纯化出抗血小板聚集组分。该组分与去整合素比较相似,可能属于去整合素家族。     

A novel natriuretic peptide from the cobra venom

Natriuretic peptides (NPs) play crucial roles in human physiology and pathophysiology through natriuresis, dieresis and vasorelaxation. NPs are also one of the important components of snake venoms. However, the low abundance in snake venom hampered the investigation. Here, a novel natriuretic peptide named Na-NP was purified from the cobra Naja atra venom. Na-NP consists of 45 amino acid residues and its molecular weight is 4618.5 Da. A full-length cDNA encoding Na-NP was obtained from the cDNA library constructed from the venom gland. The open reading frame of cloned Na-NP was composed of 498 bp and coded for a 165-amino acid residue protein precursor. The nucleotide and deduced protein sequences of Na-NP were remarkably conserved with other elapid NPs while significant different from the viperid NPs. Na-NP showed weak activity to relax the aortic rings precontracted with phenylephrine. Meanwhile, Na-NP showed cGMP-promotion activity against primary cultured rabbit endocardial endothelial cells, but had no effect on human platelet aggregation. In conclusion, this is the first report of a natriuretic peptide from the cobra N. atra venom. Na-NP might be served as a useful tool for the study of human NPs and the development of novel therapeutic drugs.     

Structure and function of snake venom proteins affecting platelet plug formation

Many snake venom proteins have been isolated that affect platelet plug formation by interacting either with platelet integrins, membrane glycoprotein Ib (GPIb), or plasma von Willebrand factor (VWF). Among them, disintegrins purified from various snake venoms are strong inhibitors of platelet aggregation. Botrocetin and bitiscetin derived from Bothrops jararaca and Bitis arietans venom, respectively, induce VWF-dependent platelet agglutination in vitro. Several GPIb-binding proteins have also been isolated from snake venoms. In this review, we focus on the structure and function of those snake venom proteins that influence platelet plug formation. These proteins are potentially useful as reagents for the sub-diagnosis of platelet disorder or von Willebrand disease, as well as for clinical and basic research of thrombosis and hemostasis.     

Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.