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.     

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA₂, C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA₂. The cross-reactivity with anti-PLA₂ revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA₂. These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.     

Purification, characterization and biological activities of the l-amino acid oxidase from Bungarus fasciatus snake venom

l-Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55 kDa in SDS-PAGE and an apparent molecular weight of 70 kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against l-Tyr, l-Asp, l-Phe, l-Glu, l-Trp, l-His, l-Gln, l-Ile, l-Met, l-Leu and moderately active against l-Lys, l-Arg, l-Ala and l-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12 h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3 h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.     

Insights of local tissue damage and regeneration induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom

Envenomations caused by Bothrops snake venoms are characterized by prominent local tissue damage due to myonecrosis, hemorrhage, edema and acute muscle damage which is widely correlated with phospholipases A₂ (PLA₂). In the present study, the progression of local tissue damage and inflammation induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom, was evaluated. Local tissue damages characterized by edema, necrosis and inflammation were evaluated until 24 h after inoculation of BnSP-7. The regeneration of myofibers, analyzed by light microscopy, was observed from 72 h to 2 weeks post-inoculation of toxin. MMP-2 was expressed in gastrocnemius muscle at all time points tested, while the expression of MMP-9 increased expressively at the same time interval of regenerating muscle, suggesting the involvement of MMP-9 in the regeneration process. The production of pro-inflammatory cytokines was also increased, whereas IL-1β showed the highest level. Modification of BnSP-7 with BPB decreased the release of IL-8, IL-6 and IL-1β when compared to native BnSP-7. These data suggest that BnSP-7 acts as pro-inflammatory incentives (mediators), inducing MMP and cytokine production from the inflammatory and satellite cells, and thus it may play an important role in inflammatory process and, consequently, in the evolution of local tissue damage and regeneration.     

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M_r = 34,000 under reducing conditions and pI  6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (−70 to 37 °C), pH values (3–9) or in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺ and Mn²⁺). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.     

Isolation and functional characterization of a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA(2), a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA(2) is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA(2) with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA(2) induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA(2)s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Local inflammatory response induced by scorpionfish Scorpaena plumieri venom in mice

The Scorpaena plumieri fish venom induces a severe pain and edema, observed both clinically and experimentally. In order to understand more about the envenomation syndrome, the present study characterized experimentally the local acute inflammatory response induced by S. plumieri venom (SpV) in a mouse model of tissue injury. Our results demonstrated that the local inflammatory response provoked after 2 h of SpV injection in footpad of mice is characterized by release of pivotal pro-inflammatory mediators (TNF, IL-6 and MCP-1). These mediators could be associated with histopathological changes observed into paw tissue, characterized by cellular infiltration, mainly neutrophils. Additionally, an investigation of edema formation pathways involved in inflammatory response was performed. SpV-induced edema was reduced significantly by previous administration of aprotinin or icatibant (HOE-140). However, the pre-treatment with diclofenac sodium and promethazine had less effect on this response. These results demonstrate that the kallikrein-kinin system (KKS) plays a major role in the edema formation. Despite the whole venom hydrolyzed the kallikrein synthetic substrate S-2302 (Pro-Phe-Arg-pNA), its main pro-inflammatory fraction was devoid of kininogenase activity. Our results demonstrate that SpV evokes a complex inflammatory reaction stimulating a secretion of TNF, IL-6, MCP-1 and leukocytes recruitment at the site of venom injection. In addition provide clear evidence of the involvement of the KKS in inflammatory response induced by S. plumieri venom.     

Histamine improves survival and protects against interleukin-2-induced pulmonary vascular leak syndrome in mice

The therapeutic efficacy in the treatment of metastatic cancer with high doses of interleukin-2 (IL-2) has been limited by the onset of vascular leak syndrome (VLS) and related toxicities. VLS is characterized by an increase in vascular permeability and severe hypotension resulting in interstitial edema and organ failure. This study explores the protective effects of histamine dihydrochloride (HDC) against IL-2-induced toxicities in mice. Treatment with HDC administered before or after IL-2 (1.25 x 10(6) IU, BID) was shown to protect mice from VLS-related toxicities and mortality in a dose-dependent manner. Survival rates when HDC was added were 56, 75 and 81% at doses of 0.47, 4.7 and 47.0 mg/kg, respectively, compared to 42% survival with IL-2 alone. HDC protected against IL-2-induced macroscopic pulmonary lesions, reduced edema (up to 62% reduction in lung wet/dry weight ratio) and reduced capillary leakage into the lungs as measured by a reduction in Evans Blue dye content. In addition, the systemic effect on serum cytokine levels showed that HDC only moderately lowered IL-2 induced IFN-gamma, IL-6, IL-10, IL-18 and TNF-alpha. Serum levels of IL-1beta, IL-4 and IL-12 were not measurably induced by IL-2 treatment. HDC modulates many cellular functions including regulating cytokines and blocking immune-suppression caused by reactive oxygen species (ROS) generated by the NADPH oxidase. However, the protective effect of HDC on alleviating IL-2-induced pulmonary edema was not related to ROS inhibition. Our data indicate that HDC treatment improves survival and protects against IL-2 induced VLS independent of ROS regulation in mice.     

Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom

A phospholipase A₂ inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A₂ activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A₂ (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of crotoxin.In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA₂ activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA₂ active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA₂, but extended to the three acidic forms as well.     

大叶藤黄中三个新酮类成分

为了研究大叶藤黄树皮中酮类成分，运用正相和反相硅胶柱色谱法对大叶藤黄树皮乙酸乙酯萃取物进行分离纯化，并用波谱技术鉴定化合物结构。共分离得到3个新酮类化合物，其结构分别鉴定为1,2,5-三羟基-6-甲氧基酮(1)，1,4,6-三羟基-5-甲氧基酮(2)，1,2,7-三羟基-4-(1,1-二甲基烯丙基)酮(3)。     

Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions

In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.     

彭泽贝母中二萜类成分

为了研究彭泽贝母的化学成分。利用多种柱色谱方法进行分离纯化，根据光谱数据和理化性质进行结构鉴定。共分离并鉴定了6个对映-贝壳杉类二萜化合物： ent-kauran-15-en-17-ol (I)， ent--kauran-15-en-3α,17-diol (II)， 鄂贝新醇(III)， ent--kauran-16α,17-diol (IV)， ent--kauran-3α，16α,17-triol (V)， ent--16,17-epoxy-kauran-3α-ol (VI)。6个化合物均为首次从彭泽贝母中分得，其中ent--16,17-epoxy-kauran-3α-ol (VI)为新化合物。     

Bitis arietans Snake Venom and Kn-Ba,a Snake Venom Serine Protease,Induce the Production of Inflammatory Mediators in THP-1 Macrophages

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB₄, LTC₄, TXB₂, and PGE₂, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE₂ was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.     

Biochemical and biological properties of phospholipases A(2) from Bothrops atrox snake venom

Phospholipases A(2) (PLA(2)s), of molecular mass 13-15kDa, are commonly isolated from snake venom. Two myotoxins with PLA(2) activity, BaPLA(2)I and BaPLA(2)III, with estimated molecular masses of 15kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA(2)I was basic, with a pI of 9.1, while BaPLA(2)III was neutral with a pI of 6.9. On a molecular basis, BaPLA(2)III exhibited higher catalytic activity on synthetic substrates than BaPLA(2)I. Comparison of the N-terminal residues of BaPLA(2)I with other PLA(2) proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA(2) Lys(49) from B. atrox (89%). In contrast, BaPLA(2)III demonstrated 75, 72, and 71% homology with PLA(2) from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA(2)I and BaPLA(2)III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA(2)s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA(2)I- and BaPLA(2)III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA(2) edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA(2) enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA(2) enzyme.     

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.     

Oral exposure to culture material extract containing fumonisins predisposes swine to the development of pneumonitis caused by Pasteurellamultocida

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides and F. proliferatum that commonly occurs in maize. In swine, consumption of contaminated feed induces liver damage and pulmonary edema. Pasteurella multocida is a secondary pathogen, which can generate a respiratory disorder in predisposed pigs. In this study, we examined the effect of oral exposure to fumonisin-containing culture material on lung inflammation caused by P. multocida. Piglets received by gavage a crude extract of fumonisin, 0.5mg FB(1)/kg body weight/day, for 7 days. One day later, the animals were instilled intratracheally with a non toxin producing type A strain of P. multocida and followed up for 13 additional days. Pig weight and cough frequency were measured throughout the experiment. Lung lesions, bronchoalveolar lavage fluid (BALF) cell composition and the expression of inflammatory cytokines were evaluated at the autopsy. Ingestion of fumonisin culture material or infection with P. multocida did not affect weight gain, induced no clinical sign or lung lesion, and only had minimal effect on BALF cell composition. Ingestion of mycotoxin extract increased the expression of IL-8, IL-18 and IFN-gamma mRNA compared with P. multocida infection that increased the expression of TNF-alpha. The combined treatment with fumonisin culture material and P. multocida delayed growth, induced cough, and increased BALF total cells, macrophages and lymphocytes. Lung lesions were significantly enhanced in these animals and consisted of subacute interstitial pneumonia. TNF-alpha, IFN-gamma and IL-18 mRNA expression was also increased. Taken together, our data showed that fumonisin culture material is a predisposing factor to lung inflammation. These results may have implications for humans and animals consuming FB(1) contaminated food or feed.     

A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom

Phospholipase A₂ (PLA₂, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A₂ from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA₂s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA₂s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.     

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.

Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.