Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

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

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

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG‐75, DEAE‐Sephadex A‐50, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). VLCII as a thrombin‐like enzyme was able to hydrolyze Nα‐CBZ L‐arginine‐p‐nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10‐phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration‐dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.     

Purification and characterization of a fibrinogenase from the venom of Western Diamondback rattlesnake (Crotalus atrox)

A fibrinogenolytic enzyme was isolated from the venom of Western Diamondback rattlesnake (Crotalus atrox) by a three-step procedure involving gel filtration and anion-exchange chromatography. The molecular weight was estimated as 22 900 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 4.65. The enzyme rapidly destroyed the ability of bovine fibrinogen to form a clot on incubation with thrombin. Incubation of fibrinogen with the fibrinogenolytic enzyme for 5 min resulted in the disappearance of the beta-chain of fibrinogen and the appearance of lower molecular weight fragments. Thus the enzyme can be classified as a beta-fibrinogenase. However, on prolonged incubation of the fibrinogen there was also a partial digestion of the alpha-chain. The fibrinogenase showed no activity towards fibrin or casein or arginine esters. The fibrinogenolytic activity was inhibited by phenylmethanesulphonyl fluoride (PMSF) but was unaffected by EDTA.     

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.     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).     

Purification and characterization of a coagulant enzyme, okinaxobin I, from the venom of Trimeresurus okinavensis (Himehabu snake) which releases fibrinopeptide B

A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.     

Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster)

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-α-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-α and B-β chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.     

Purification and properties of the thrombin-like enzyme from the venom of Lachesis muta muta

1. The coagulating enzyme of the Lachesis muta muta venom was purified to homogeneity by a combination of a gel filtration in Sephadex G-100 and affinity chromatography on agarose-agmatine resin. 2. Several forms of the enzyme were prepared by isoelectric focusing with pIs ranging from 3.1 to 5.0; the asialoenzyme focused as a narrow band at pH 8.7. SDS-PAGE analysis of the purified enzyme revealed a single broad band with apparent Mr of 41-47 kDa. 3. The enzyme cleaves only fibrinopeptide A from fibrinogen; it does not activate factor XIII and is devoid of kallikrein-like activity. 4. Kinetic properties of the enzyme were determined for representative synthetic chromogenic substrates and inhibitors.     

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.     

Kinin-releasing enzyme from the venom of Bitis arietans (puff adder)

A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu⁶-Cys⁷, His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵ bonds of the B chain of oxidized insulin and the Aα and Bβ chain of fibrinogen. Kinin-releasing and benzoyl-l-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.     

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.     

Purification, characterization and gene cloning of a novel phospholipase A2 from the venom of Agkistrodon blomhoffii ussurensis

A new phospholipase A2 with Gln at the site 49, abbreviated as Gln49-PLA2, has been purified from the venom of Agkistrodon blomhoffii ussurensis by using ion-exchange chromatography, gel filtration chromatography and reversed-phase HPLC, and behaves as a single-band on SDS-PAGE. Its molecular weight is 13881.85+/-0.33 Da given by mass spectrometry and pI is about 8.56 given by isoelectric focusing. Gln49-PLA2 does not show phospholipase A2 and hemorrhagic activity, whereas shows weak toxic and apparent anticoagulant activity. Based on the N-terminal sequencing and peptide mass fingerprint analysis, Gln49-PLA2 cDNA has been cloned by means of RT-PCR. Gln49-PLA2 consists of 122 amino acid residues and has the structural features of class II of snake venom phospholipase A2.     

Molecular properties of the Factor V-activating enzyme from Russell's viper venom.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom

Snake venoms of the Viperidae family contain a numberof proteins that cause hemostatic disturbances. Enveno-mation of this family is characterized by hemorrhage,edema, local tissue damage, myonecrosis, fibrinolytic andkinin releasing activities [1]. In southeastern Brazil, theviper Bothrops jararaca (Viperidae) is responsible for 90%of snakebite accidents [2]. The enzymes that have proteolytic, coagulate andhemorraghic activities can activate or interfere withthe process of coagulation, and…     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.     

Isolation of bothrasperin, a disintegrin with potent platelet aggregation inhibitory activity, from the venom of the snake Bothrops asper

The venom of Bothrops asper induces severe coagulation disturbances in accidentally envenomed humans. However, only few studies have been conducted to identify components that interact with the hemostatic system in this venom. In the present work, we fractionated B. asper venom in order to investigate the possible presence of inhibitors of platelet aggregation. Using a combination of gel filtration, anion-exchange chromatography, and reverse-phase high performance liquid chromatography, we isolated an acidic protein which shows a single chain composition, with a molecular mass of approximately 8 kDa, estimated by SDS-polyacrylamide gel electrophoresis. Its N-terminal sequence has high similarity to disintegrins isolated from different snake venoms, which are known to bind to cellular integrins such as the GPIIb/IIIa fibrinogen receptor on platelets. The purified protein exerted potent aggregation inhibitory activity on ADP-stimulated human platelets in vitro, with an estimated IC50 of 50 nM. This biological activity, together with the biochemical characteristics observed, demonstrate that the protein isolated from B. asper venom is a disintegrin, hereby named "bothrasperin". This is the first disintegrin isolated from Central American viperid snake species.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.