A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sepharyl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release action in rabbit platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable molonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E₁ completely blocked both aggregoserpentine-induced aggregation and release reaction. Furthermore, platelet aggregoserpentine lowered basal and prostaglandin E₁-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A₂, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Purpureotin: a novel di-dimeric C-type lectin-like protein from Trimeresurus purpureomaculatus venom is stabilized by noncovalent interactions

Purpureotin, a novel di-dimeric C-type lectin-like protein (CLP) from Trimeresurus purpureomaculatus, was purified and sequenced. While its native molecular mass was determined to be 63kDa, purpureotin showed a single band of 30kDa on nonreducing SDS-PAGE and two polypeptide chains (16.0 and 14.5kDa) under reducing condition. These results were subsequently confirmed by mass spectrometric analyses. Based on these results, we postulate that purpureotin is a dimer of the alpha,beta-heterodimer which is held together by noncovalent interactions. Molecular modeling studies indicate that a dimer of alpha,beta-heterodimers can be formed where the alpha chains are held together by electrostatic charges and beta chains via hydrophobic interactions. Functionally, purpureotin induced platelet aggregation without any cofactor in a dose-dependent manner. However, the platelet aggregation effect was blocked by echicetin. Therefore, purpureotin is assumed to be a GPIb-binding protein which binds to the same or a closely related GPIb site on platelets as echicetin.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.     

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca²⁺ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.     

Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation

Halysase, a hemorrhagic metalloprotease, has an apparent molecular weight of 66kDa and belongs to the class P-III snake venom metalloprotease. Class P-III snake venom metalloproteases have multifunctional domains including a protease domain and a disintegrin-like domain. Halysase was able to preferentially hydrolyze the alpha-chain of fibrinogen. Proteolytic activity of the enzyme was completely inhibited by metal chelating agents but not by other typical protease inhibitors. The enzyme principally cleaves X-Leu, X-Tyr, X-Phe, and X-Ala peptide bonds of the oxidized insulin B-chain. Halysase strongly suppresses collagen-induced human platelet aggregation in a dose-dependent manner. Apohalysase that is devoid of its metalloprotease activity was also able to inhibit the platelet aggregation to a certain extent. Experimental evidence clearly indicates that each of the two distinct domains of halysase, the metalloprotease and the disintegrin-like domains, plays its characteristic role to inhibit human platelet aggregation.     

Characterization of ''basparin A,'' a prothrombin-activating metalloproteinase,from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis

A prothrombin activator, named 'basparin A,' was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors alpha(2) macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming.     

Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of -mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 59-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5–9.5 and the optimum temperature was 60°C, with activity being rapidly lost within 1 min at 70°C. The K_m of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and -mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.     

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20 kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Purification and properties of a glycerophosphocholine phosphodiesterase from bovine brain myelin

An enzyme releasing phosphocholine from glycerophosphocholine was purified to apparent homogeneity based upon SDS-PAGE. The enzyme was liberated from lyophilized bovine myelin by differential detergent extraction and final purification was accomplished with Q-Sepharose Fast Flow chromatography yielding an apparently homogenous protein. The molecular mass based upon PAGE was approximately 14 kDa. The enzyme was also capable of releasing p-nitrophenol from p-nitrophenyl-phosphocholine. Maximal activity was obtained with 0.2 mM ZnCl₂ or 1 mM CoCl₂. p-Nitrophenylphosphocholine and phosphocholine were competitive inhibitors of glycerophosphocholine hydrolysis with Ki's of 0.028 mM and 0.03 mM respectively. Glycerophosphocholine and phosphocholine were competitive inhibitors of p-nitrophenylphosphocholine hydrolysis with Ki's of 0.5 mM and 1.75 mM respectively.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - GPC glycerophosphocholine - pNPPC p-nitrophenylphosphocholine - OG octyl--glucoside - PMSF phenylmethylsulfonylfluoride - CNPase 23-cyclic nucleotide 3-phosphodiesterase     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil

Trapidil (N,N-diethyl-5-methyl[l,2,4]triazolo[l,5-α]pyrimidine-7-amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K_i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β-PGI_i). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55 kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation (“Met-turn”), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala₁₄-Leu₁₅ peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aα-chain of fibrinogen and the α-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca²⁺ and Mg²⁺ but inhibited by Zn²⁺ and Cu²⁺. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD = 30 ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.     

Geographic variations,cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis

Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A(2) (PLA(2)s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA(2)s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA(2)s induced edema, but had no anti-platelet activity. There was also a common basic PLA(2) myotoxin in all the samples. We have cloned five acidic PLA(2)s and several hybrid-like nonexpressing PLA(2)s. Molecular masses and N-terminal sequences of the purified PLA(2)s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA(2)s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA(2)s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA(2)s in pit viper venom.     

Crystal structure of the platelet activator convulxin, a disulfide-linked alpha4beta4 cyclic tetramer from the venom of Crotalus durissus terrificus

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.