Structure and function of snake venom toxins interacting with human von Willebrand factor

Hemostatic plug formation is a complex event mediated by platelets, subendothelial matrices and von Willebrand factor (VWF) at the vascular injury. Snake venom proteins have an excellent potency to regulate the interaction between VWF and platelet membrane receptors in vitro. Two protein families, C-type lectin-like proteins and Zn²⁺-metalloproteinases, have been found to affect platelet–VWF interaction. Botrocetin and bitiscetin from viper venom are disulfide-linked heterodimers with C-type lectin-like motif, and modulate VWF to elicit platelet glycoprotein Ib (GPIb)-binding activity via the A1 domain of VWF leading to the platelet agglutination. The crystal structures of botrocetin and bitiscetin together with complex from the VWF A1 domain indicate the following: (1) a central concave domain formed by two subunits of botrocetin or bitiscetin provides the binding site for VWF, (2) these modulators directly bind to the A1 domain of VWF in close proximity to the GPIb binding site, (3) both modulators induce no significant conformational change on the GPIb-binding site of the A1 domain but could provide a supplemental platform fitting for GPIb. These results suggest that the modulating mechanisms of these venoms are different from those performed by either antibiotic ristocetin in vitro or extremely high shear stress in vivo.

Other modulator toxins include kaouthiagin and jararhagin, chimeric proteins composed of metalloproteinase, disintegrin-like and Cys-rich domains. These toxins cleave VWF and reduce its platelet agglutinating or collagen-binding activity. Kaouthiagin from cobra venom specifically cleaves between Pro708 and Asp709 in the C-terminal VWF A1 domain resulting in the decrease of the multimer structure of VWF. Recently a plasma proteinase, which specifically cleaves VWF into a smaller multimer, has been elucidated to be a reprolysin-like metalloproteinase with thrombospondin motif family (ADAMTS). This endogenous metalloproteinase (ADAMTS-13) specifically cleaves between Tyr842 and Met843 in the A2 domain of VWF regulating its physiological hemostatic activity.

These VWF-binding snake venom proteins are suitable probes for basic research on platelet plug formation mediated by VWF, for subsidiary diagnostic use for von Willebrand disease or platelet disorder, and might be potently applicable to the regulation of VWF in thrombosis and hemostasis. Structural information of these venom proteins together with recombinant technology might strongly promote the construction of a new antihemostatic drug in the near future.     

Snake venom fibrin(ogen)olytic enzymes

Snake venoms contain a number of serine and metalloproteinases and included among these are the fibrin(ogen)olytic proteinases. Some years ago it was postulated that the fibrin(ogen)olytic enzymes may be clinically useful. Over the past 150 years a substantial body of literature has been generated on the identification and characterization of fibrin(ogen)olytic enzymes from a broad spectrum of snake species. In this review we describe the two different classes of fibrin(ogen)olytic enzymes isolated from snake venom and we summarize a number of studies aimed at characterizing the purified enzymes and/or their derivatives.

Two distinct classes of venom fibrin(ogen)olytic enzymes have been previously identified, the metalloproteinases and serine proteinases. These two classes of proteinases differ in their mechanism of action and they target different amino acid sequences in fibrin(ogen), but each perform the same role in nature. When a snake envenomates its prey it needs a mechanism to facilitate the spread of the toxic components throughout the circulation. Fibrin(ogen)olytic enzymes break down fibrin rich clots and help to prevent further clot formation by their action on fibrinogen. This characteristic feature has led to development of fibrin(ogen)olytic snake venom enzymes as potential clinical agents to treat occlusive thrombi. Fibrolase, a fibrinolytic metalloproteinase isolated from Agkistrodon contortrix contortrix venom and the serine β-fibrinogenolytic proteinase from Vipera lebetina have been chosen as representative enzymes from the two classes, and their biochemical and physiochemical properties will be described in detail.

Finally, the characterization and development of alfimeprase, a recombinant fibrinolytic enzyme derived from fibrolase, as a clinical agent is described citing the progression from the laboratory bench to its current status as having successfully completed Phase II clinical trials.     

Practical applications of snake venom toxins in haemostasis

Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac^®, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin^® from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve ‘therapeutic defibrination’. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders.     

Jararhagin and its multiple effects on hemostasis

Jararhagin is a 52 kDa hemorrhagic P-III metalloproteinase isolated from the venom of the medically important Brazilian pit-viper Bothrops jararaca. It is a member of the reprolysin family of zinc metalloproteinases containing a catalytic metalloproteinase domain followed by a disintegrin-like and a cysteine-rich domain. The impact of jararhagin on hemostasis has been extensively studied using in vitro and in vivo model systems as well as in clinical studies. Jararhagin-induced hemorrhage is the result of the degradation of sub-endothelial matrix proteins leading to the disruption of the blood vessel endothelium, with accompanying disturbances in platelet function. The versatility of jararhagin is further demonstrated by its direct action on von Willebrand factor, the degradation of fibrinogen, by its inhibition of platelet adhesion to collagen and by its inability to be affected by the plasma inhibitor ₂-macroglobulin. Collagen-induced platelet aggregation is inhibited by jararhagin though the binding of the molecule to the ₂ subunit I domain of the platelet surface ₂β₁ integrin (collagen receptor). Jararhagin also cleaves the β₁ subunit of the same integrin, inhibiting platelet interaction and ultimately causing impairment of signal transduction. The effect of jararhagin on cell systems other than platelets is evaluated; in fibroblasts, jararhagin functions as a collagen-mimetic substrate and, in endothelial cells, it causes apoptosis and indirectly inhibits cell proliferation by release of angiostatin-like compounds. Jararhagin induces a strong pro-inflammatory response characterized by intense leukocyte accumulation at the site of the injection. Although hemorrhage and edema are a response to the direct effect of jararhagin, jararhagin-induced inflammation and necrosis are dependent on macrophages and key pro-inflammatory cytokines or their receptors. Some data also indicate that the toxin possesses anti-tumorgenic properties. Methods for inhibiting jararhagin are reviewed; this encompasses the use of synthetic peptides to the isolation of naturally occurring mammalian peptides and the development of toxin-specific antibodies through DNA immunisation and monoclonal antibody technologies. The availability of jararhagin makes it an important tool for research into the mechanisms of action of similar toxins, for insights into cellular interactions and for clinical investigations into the treatment of envenomings from B. jararaca.     

Stejnihagin, a novel snake metalloproteinase from Trimeresurus stejnegeri venom, inhibited L-type Ca channels

Snake venom metalloproteinases (SVMPs) mainly distribute in Crotalid and Viperid snake venom and are classified into the Reprolysin subfamily of the M12 family of metalloproteinases. Previous function investigations have suggested that SVMPs are the key toxins involved in a variety of snake venom-induced pathogenesis including systemic injury, local damage, hemorrhage, edema, hypotension, hypovolemia, inflammation and necrosis. However, up to now, there is no report on ion channels blocking activity about SVMPs. Here, from Trimeresurus stejnegeri venom we purified a component Stejnihagin containing a mixture of Stejnihagin-A and -B, with 86% sequences identity, both as members of SVMPs. In the study, whole-cell patch clamp and vessel tension measurement were employed to identify the effect of Stejnihagin on L-type Ca²⁺ channels and vessel contraction. The results show that Stejnihagin inhibited L-type Ca²⁺channels in A7r5 cells with an IC₅₀ about 37 nM and simultaneously blocked 60 mM K⁺-induced vessel contraction. Besides, the inhibitory effect of Stejnihagin on L-type Ca²⁺ channels was also independent of the enzymatic activity. This finding offers new insight into the snake venom metalloproteinase functions and provides a novel pathogenesis of T. stejnegeri venom. Furthermore, it may also provide a clue to study the structure-function relationship of animal toxins and voltage-gated Ca²⁺ channel.     

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin–Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by -thrombin ( ₅₀=28 μg/ml). A single band of 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation ( ₅₀=0.19 μg/ml) compared to bothrojaracin ( ₅₀=0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Assessment of metalloproteinase inhibitors clodronate and doxycycline in the neutralization of hemorrhage and coagulopathy induced by Bothrops asper snake venom

Snake venom metalloproteinases (SVMPs) play a prominent role in the local and systemic manifestations of viperid snakebite envenomations. Thus, the possibility of using metalloproteinase inhibitors in the treatment of these envenomations is a promising therapeutic alternative. This study assessed the ability of two metalloproteinase inhibitors, the biphosphonate clodronate and the tetracycline doxycycline, to inhibit proteolytic, hemorrhagic, coagulant and defibrinogenating effects of Bothrops asper venom. Both compounds were able to inhibit these activities, at concentrations in the mM range, when incubated with venom prior to testing. However, when inhibition of hemorrhage was assessed in assays involving independent injection of venom and drug, inhibition was poor, even when these compounds were injected immediately after envenomation. These findings support the concept that the effectiveness of compounds, such as clodronate and doxycycline, whose inhibitory action on SVMPs is based on zinc chelation alone, is limited, and stress the view that more specific molecules are required for an effective inhibition of SVMPs in vivo.     

A protease in the venom of king cobra (Ophiophagus hannah): purification, characterization and substrate specificity on oxidized insulin B-chain

and . A protease in the venom of king cobra (Ophiophagus hannah): purification, characterization and substrate specificity on oxidized insulin B-chain. Toxicon 26, 1145–1155, 1988.—A protease in the venom of Ophiophagus hannah (king cobra) has been purified to a homogeneous state by successive chromatographies on Sephadex G-100 superfine, DEAE-cellulose, hydroxyapatite and CM-polyvinylalcohol copolymer columns. The mol.wt as determined by SDS-PAGE and gel filtration was approximately 70,000. The purified enzyme possessed a specific activity approximately 1/25 that of crystalline trypsin, whereas it had no hemorrhagic activity. The substrate specificity was determined using oxidized insulin B-chain as a substrate; the enzyme cleaved the Asn₃-Gln₄, Gln₄-His₅, His₁₀-Leu₁₁, Ala₁₄-Leu₁₅ and Tyr₁₆-Leu₁₇ positions. The sites cleaved by the protease were compared to proteases from other snake venoms.     

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis

cDNAs encoding 4 short chain -neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to -neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, ¹⁰E¹¹T and ¹⁰Q¹¹A, which could be further subdivided into ¹⁰E¹¹T²⁸S; ¹⁰E¹¹T²⁸G and ¹⁰Q¹¹A²⁸S; ¹⁰Q¹¹A²⁸G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.     

Differential effects of Co(2+) and Ni(2+) on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta

Growth, morphological changes, amino acid composition, total soluble protein, and protein electrophoretic pattern were monitored for Scenedesmus obliquus and Nitzschia perminuta grown in the presence of different concentrations of Co²⁺ and Ni²⁺. Lower concentrations of cobalt stimulated the dry mass production and total soluble protein content of the two algae, whereas higher concentrations were inhibitory. Generally, N. perminuta showed more tolerance to the phytotoxicity of the two metals than S. obliquus and more tolerance to nickel than cobalt. However, S. obliquus seems to be more tolerant to cobalt than nickel. Cobalt and nickel have induced an increase in cell volume, change and disorder in cell shape. The increase in cell volume was much observed in Ni²⁺ treated cells. At the same time, the two metals did not induce any distinct morphological abnormalities in N. perminuta. Co²⁺ has stimulated the biosynthesis of all free amino acids in S. obliquus, except aspartic acid and phenylalanine, whereas Ni²⁺ caused 22% inhibition in the content of total free amino acids, except cystine and arginine. On the other hand, Co²⁺ has reduced the content of free amino acids in N. perminuta, except cystine, methionine, valine, and lysine. On the other hand, Ni²⁺ stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. High cobalt concentration (4 ppm) resulted in the disappearance of 28.7 kDa protein, 3.5 ppm Ni²⁺ stimulated the appearance of 18 and 20 kDa proteins in S. Obliquus, while 37 kDa proteins disappeared from N. perminuta treated with high doses of Co²⁺ and Ni²⁺.     

Effects of snake venom proteins on blood platelets

R. M. and H. J. . Review article—Effects of snake venom proteins on blood platelets. Toxicon 28, 1387–1422, 1990.—Snake venoms are complex mixtures which contain pharmacologically active polypeptides and proteins. Several snake venom constituents interfere in platelet aggregation, an important cellular process in thrombosis and hemostasis. These components range in size from small molecular weight polypeptides to high molecular weight proteins. Some of the proteins are enzymes, such as phospholipase A₂, proteinases, nucleotidases, or -amino acid oxidase, while others do not exhibit enzymatic activity. These components may initiate and/or inhibit platelet aggregation. Some venom factors induce platelet agglutination. This review deals with the physical characteristics of these venom factors, the mechanisms of their platelet effects, structure-function relationships, and their physiological significance.     

A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: Isolation and characterization

Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K_m and V_max values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 μg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 °C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe²⁺, Mn²⁺, Cu²⁺ and heparin.     

5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.     

Molecular cloning and sequence analysis of cDNA encoding flavoridin, a disintegrin from the venom of Trimeresurus flavoviridis.

We isolated a cDNA of 2001bp encoding the full-length precursor of flavoridin, which is one of the four disintegrins in the venom of Trimeresurus flavoviridis, and analyzed the cDNA nucleotide sequence. The deduced amino acid sequence of the open reading frame consisted of a pro-domain (190 residues), a metalloproteinase domain (205 residues), a spacer domain (18 residues) and a disintegrin (flavoridin) domain (70 residues), thus indicating that the flavoridin precursor belongs to the P-II class of snake venom metalloproteinases. The unknown metalloproteinase domain shared strong sequence similarity with HR2a (71.2% identity) and H(2)-proteinase (74.1% identity), a low molecular mass hemorrhagic metalloproteinase and a non-hemorrhagic metalloproteinase in the same snake venom, respectively.     

Purification and biological effects of C-type lectin isolated from Bothrops insularis venom

Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10 μg/mL increased perfusion pressure (PP; control₆₀=108.27±4.9; BiLec₆₀=112.9±5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control₆₀=5.38±0.51; BiLec₆₀=6.01±0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control₁₂₀=0.160±0.020; BiLec₁₂₀=0.082±0.008 mL g⁻¹ min⁻¹; *p<0.05). Glomerular filtration rate (GFR; control₁₂₀=0.697±0.084; BiLec₁₂₀=0.394±0.063 mL g⁻¹ min⁻¹; *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa⁺), potassium (TK⁺) and chloride tubular transport (TCl⁻). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.     

A snake venom phospholipase A2 with high affinity for muscarinic acetylcholine receptors acts on guinea pig ileum

A group of small proteins, designated as muscarinic toxins (MTs), have been isolated from the venom of African green mamba (Dendroaspis angusticeps) and documented to bind selectively to individual muscarinic acetylcholine receptor (mAChR) subtypes. These components have less been reported to be isolated from other snake venoms. In this study, we have isolated a snake factor with high affinity for mAChR from the venom of Naja atra (Chinese cobra) by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow and Poros^®CM 4.6/100 Perfusion Chromatography Column. The final preparation was homogeneous as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and HPLC. The isolated active component, which was designated muscarinic protein (MP), was found to displace [³H]quinuclidinyl benzilate binding to rat cortex synaptosomes in a dose-dependent manner, and the K_i value estimated was 10.1 nM. The isolated MP was determined to have a molecular weight of 13.3 kDa and an N-terminal amino acid sequence of NLYQFKNMIQCTVPSR, which is highly homologous with phospholipase A₂ from the venoms of genus Naja. The N. atra MP could hydrolyze phosphatidylcholine in a dose-dependent manner. In guinea-pig ileum, MP produced an onset and dose-dependent contraction, which could be reversed by atropine indicating the involvement of mAChR. The EC₅₀ value of MP for guinea-pig ileum contraction was estimated as 30 nM, and the maximum contraction caused by MP was approximately 43% of that obtained from carbachol. These results seem to suggest that the snake venom phospholipase A₂ may not only have high affinity for mAChRs but also have the ability to activate mAChRs. However, it is possible that the toxin caused the contraction in the guinea-pig ileum by inducing acetylcholine release via another mechanism.     

Purification, cloning and characterization of a metalloproteinase from Naja atra venom

The complement system is a very important part of the immune system. Many snake venoms possess activities that influence the complement. A new metalloproteinase (termed atrase B) with anticomplementary activity was purified from Naja atra venom. Atrase B is a single chain glycoprotein with a molecular mass of 49.4 kDa and an isoelectric point of 9.7. Its N-terminal sequence shows high homology to those of metalloproteinases from cobra venoms. The cDNA sequence reveals that atrase B is a PIII class metalloproteinase. Atrase B slowly cleaves the Aα chain of fibrinogen. It also exhibits edema-inducing activity, but has no hemorrhagic activity and proteolytic activity against fibrin, azocasein, and N-benzoyl-l-arginine ethyl ester. Interestingly, atrase B inhibits activation of the complement classical and alternative pathways in a dose- and time-dependent manner. Complement components factor B and C6 are major targets for atrase B to cleave. Atrase B is the first identified SVMP that cleaves complement components factor B, C6, C7, and C8.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

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.