Snake Venom Proteins Research Articles

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 Zn 2+-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 probes of platelet adhesion receptors and their ligands

Snake venom proteins that modulate platelet adhesive interactions are chiefly from either of two main structural families: the C-type lectin-like family, or the metalloproteinase-disintegrins. Snake venom probes from both families selectively target platelet adhesion receptors, including glycoprotein (GP) Ib-IX-V, GP VI, α2β1 and αIIbβ3 (GP IIb-IIIa). These receptors act together to mediate platelet adhesion, activation and aggregation (thrombus formation) under hydrodynamic shear stress in flowing blood. The receptors are members of the leucine-rich repeat family (GP Ib-IX-V), the immunoglobulin superfamily (GP VI), or integrins (α2β1, αIIbβ3). In addition, adhesive glycoproteins in matrix and/or plasma such as von Willebrand factor (that binds GP Ibα and αIIbβ3), collagen (that binds GP V, GP VI and α2β1), or fibrinogen (that binds αIIbβ3), are also targeted by C-type lectin family or metalloproteinase-disintegrin snake venom proteins. Emerging structural and functional evidence is beginning to explain how interactions between the conserved structural module-domains that make up these mammalian and snake proteins are regulated. Whether homologous adhesion/counter-receptors on platelets and other vascular cells are also potential snake venom targets is as yet largely unexplored.

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.

Ohanin, a Novel Protein from King Cobra Venom, Induces Hypolocomotion and Hyperalgesia in Mice

We have identified, purified, and determined the complete amino acid sequence of a novel protein, ohanin from Ophiophagus hannah (king cobra) venom. It is a small protein containing 107 amino acid residues with a molecular mass of 11951.47 +/- 0.67 Da as assessed by electrospray ionization-mass spectrometry. It does not show similarity to any known families of snake venom proteins and hence is the first member of a new family of snake venom proteins. It shows similarity to PRY and SPRY domain proteins. It is nontoxic up to 10 mg/kg when injected intraperitoneally in mice. Ohanin produced statistically significant and dose-dependent hypolocomotion in mice. In a pain threshold assay, it showed dose-dependent hyperalgesic effect. The ability of the protein to elicit a response at greatly reduced doses when injected intracerebroventricularly as compared with intraperitoneal administration in both the locomotion and hot plate experiments strongly suggests that ohanin acts on the central nervous system. Since the natural abundance of the protein in the venom is low (approximately 1 mg/g), a synthetic gene was constructed and expressed. The recombinant protein, which was obtained in the insoluble fraction in Escherichia coli, was purified under denaturing condition and was refolded. Recombinant ohanin is structurally and functionally similar to native protein as determined by circular dichroism and hot plate assay, suggesting that it will be useful in future structure-function relationship studies.

Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A 2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms

The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A 2 (PLA 2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

The snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation

Botrocetin is a snake venom protein that enhances the affinity of the A1 domain of plasma von Willebrand factor (vWF) for the platelet receptor glycoprotein Ibalpha (GPIbalpha), an event that contributes to bleeding and host death. Here we describe a kinetic and crystallographic analysis of this interaction that reveals a novel mechanism of affinity enhancement. Using high-temporal-resolution microscopy, we show that botrocetin decreases the GPIbalpha off-rate two-fold in both human and mouse complexes without affecting the on-rate. The key to this behavior is that, upon binding of GPIbalpha to vWF-A1, botrocetin prebound to vWF-A1 makes no contacts initially with GPIbalpha, but subsequently slides around the A1 surface to form a new interface. This two-step mechanism and flexible coupling may prevent adverse alterations in on-rate of GPIbalpha for vWF-A1, and permit adaptation to structural differences in GPIbalpha and vWF in several prey species.

Hypotensive Agents from Snake Venoms

Many snake venoms contain toxins which produce profound cardiovascular effects. The site of action of these toxins includes cardiac muscle, vascular smooth muscle and the capillary vascular bed. Some snake venoms, for example, contain peptides that inhibit angiotensin converting enzyme and potentiate the biological actions of bradykinin. Other snake venoms contain structural and functional equivalents of mammalian natriuretic peptides. Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp. which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins which block L-type Ca(2+) currents (eg. calciseptine, FS2 toxins, C(10)S(2)C(2) and S(4)C(8)). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity. Some of these proteins and peptides have proven to be highly selective tools in the study of physiological processes. Others have been used as probes of potential therapeutic targets or as lead compounds in the development of therapeutic agents. Therefore these and other related snake venom proteins hold great promise in the future understanding and treatment of cardiovascular diseases.

C-type Lectin-related Proteins from Snake Venoms

C-type lectin-like proteins (CLPs) of snake venom have a variety of biological properties, acting for example as anticoagulants, procoagulants, and agonists/antagonists of platelet activation. The structural and functional studies of the first identified CLP, factor IX/factor X-binding protein, have led to an understanding how new functionally heterodimeric CLPs from monomeric C-type lectin related proteins may have evolved by 3D domain swapping, and have contributed to our understanding of the significance of magnesium ions in the blood coagulation cascade reaction. Two metallo-proteases, carinactivase and RVV-X (factor X activator of a snake venom), with C-type lectin-like domains, were isolated, characterized, and found to be useful in the study of the properties of prothrombin and coagulation factor X. There are also several unique CLPs belong to agonists and antagonists of platelet receptors, platelet glycoprotein Ib and glycoproteins Ia/IIa and VI, collagen receptors and the following CLPs that modulate platelet function. These CLPs may provide the new insights into platelet function: alboaggregin-B, echicetin, botrocetin, bitiscetin, flavocetin-A, aggretin/rhodocytin, convulxin, and agkistin.

Regulation of Syk Function by Ubiquitination in Human Platelets.

Platelets have an integral function in maintaining hemostasis. Signaling pathways are strictly regulated to ensure the cessation of bleeding without precipitating a thrombotic episode. An important initial physiological step in this process is the interaction of platelets with freshly exposed subendothelial collagen after vascular injury. A major platelet receptor involved is the GPVI/FcRγ-chain complex. Upon activation of this receptor, the immunoreceptor tyrosine-based activation motif (ITAM) present in the FcRγ-chain is phosphorylated by Src family kinases. This causes the tyrosine kinase Syk to bind to the ITAM where it is autophosphorylated, initializing a signaling cascade that activates a number of proteins including PLCγ2, PI-3 kinase and small G proteins. Syk appears to play an early pivotal role in GPVI/FcRγ-chain signal transduction and its regulation is crucial. Recent studies have shown that platelet aggregation, in response to collagen-related peptide (CRP), is potentiated in c-Cbl knockout mice. c-Cbl, a protein containing an E3 ligase responsible for substrate recognition for ubiquitin, appears to regulate numerous cytoplasmic kinases in other cell systems. This protein modification has emerged as one of the most common regulatory processes in all eukaryotes, second only to possibly phosphorylation. It appears to target proteins for proteosomal degradation although recently other mechanisms have been described, ranging from protein kinase activation to translation control. In the work presented here, we demonstrate that Syk associates with c-Cbl after GPVI/FcRγ-chain activation and therefore explored the possibility that Syk is ubiquitinated during GPVI/FcRγ-chain stimulation in human platelets. We have found that Syk is rapidly ubiquitinated upon activation by collagen, CRP and the snake venom protein convulxin, but not thrombin. PP2 and SU6656, two Src kinase inhibitors, prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases and probably requires Syk phosphorylation. The ubiquitination of Syk did not cause any apparent degradation of the protein as evidenced by the lack of effect of proteosomal and lysosomal inhibitors. We have been able to separate ubiquitinated Syk from its non-ubiquitinated counterpart. We have used an in vitro kinase assay to compare the activity of ubiquitinated Syk to non-ubiquitinated Syk. Surprisingly, when we compared the specific activity of the two Syk fractions, we found that the ubiquitinated Syk appeared to be about five-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that the majority (75%) of the active Syk is in the ubiquitinated fraction. In addition we found that Syk is not ubiquitinated in c-Cbl deficient mice. We therefore propose that c-Cbl plays a regulatory role in GPVI/FcRγ-chain stimulation through ubiquitination of Syk. The fact that 75% of active Syk is ubiquitinated suggests that ubiquitination plays an essential role in the regulation of its function. A possible function of ubiquitination may be targeting Syk to appropriate signaling molecules.

Complement Inactivation by Recombinant Human C3 Derivatives

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.

Immobilized betulinic acid column and its interactions with phospholipase A2 and snake venom proteins

Betulinic acid (BA) is a plant-derived pentacyclic triterpenoid. Although BA has been found to have diverse pharmacological effects, including anti-tumor and anti-inflammatory actions and potential as inhibitor of phospholipase A2 (PLA2), its cellular targets remain unclear. In this study, BA was immobilized onto an acrylamide matrix. The immobilized-BA column could retain the purified PLA2 of bovine pancreas or the PLA2 of snake venom from Naja nigricollis. The bound PLA2 were not eluted by high salt concentrations but were eluted by either acid or calcium free buffer. Besides the PLA2, a group of basic proteins of snake venom with molecular weights of about 7 kDa were also strongly bound by immobilized BA. One of these proteins was identified as gamma-cardiotoxin. The usefulness of immobilized BA for exploring the cellular targets of BA is discussed.

Identification and cloning of snake venom vascular endothelial growth factor (svVEGF) from Bothrops erythromelas pitviper

Vascular endothelial growth factors (VEGFs) are among the most important angiogenic proteins found on vertebrates. In the last years, some reports of the occurrence of such proteins in snake venoms are rising the importance of this family of proteins as toxins, since they appear to be involved in many features of Viperidae envenoming, such as hypotension and venom spread through increase in vascular permeability. Here we describe the occurrence of snake venom VEGF in Bothrops erythromelas, a clinical important snake from Northeast of Brazil, through immunodetection and cloning of its cDNA and briefly provide an overview comparison of all recent described svVEGF sequences.

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma.

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3.

Rhodostomin, a Disintegrin, Inhibits Adhesion of Neutrophils to Fibrinogen and Attenuates Superoxide Production

Disintegrins are a group of Arg (or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of α_IIbβ₃ integrin. Here, we studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils could reach saturation in a dose-dependent manner, and its binding was increased in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) and N-formyl-Met-Leu-Phe. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, α_Mβ₂), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against β₃ integrin, or mAbs raised against α_M and β₂ integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. Taken together, our results indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory properties.

Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.

Rhodostomin, a disintegrin, inhibits adhesion of neutrophils to fibrinogen and attenuates superoxide production.

Disintegrins are a group of Arg (or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of alpha(IIb)beta(3) integrin. Here, we studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils could reach saturation in a dose-dependent manner, and its binding was increased in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) and N-formyl-Met-Leu-Phe. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, alpha(M)beta(2)), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against beta(3) integrin, or mAbs raised against alpha(M) and beta(2) integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. Taken together, our results indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory properties.

The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom.

There are different glycosylated proteins in snake venoms, but no glycosylated representatives of a large family of three-fingered toxins have previously been detected. A new glycoprotein was isolated from the venom of the Thai cobra Naja kaouthia. MALDI MS of the glycoprotein contained an array of peaks in the range from approximately 8900 to approximately 9400 Da indicating its microheterogeneity. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine, fucose and neuraminic acid. The N-terminal sequence of the glycoprotein was identical to that of cytotoxin 3 (CX3) from N. kaouthia, and CD spectra of the glycoprotein and CX3 were almost the same. Cleavage of a glycan moiety by N-glycosidase F gave a protein of molecular mass practically coinciding with that of CX3. MALDI MS of the tryptic digest of reduced glycoprotein S-pyridylethylated at cysteine residues, contained peaks corresponding to all tryptic fragments of CX3, with the exception of fragment 24-30. The peak corresponding to this peptide appeared in the mass-spectrum of similarly treated deglycosylated glycoprotein. These data show that the potential N-glycosylation site at Asn29 in CX3 is utilized for glycan attachment and that the glycoprotein is glycosylated CX3. In vivo toxicity of the glycoprotein to the cricket Gryllus assimilis was twofold lower than that of CX3. The cytotoxic activity of the glycoprotein towards HL60 cells was about two orders of magnitude lower than that of CX3, but could be made equal to the CX3 cytotoxicity by deglycosylation. Thus for the first time we have isolated a glycosylated three-fingered snake venom toxin wherein glycosylation appears to modulate its biological activity.

Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom.

A snake-venom protein named AHP-LAAO has been purified from Agkistrodon halys pallas venom using four-stage chromatography. AHP-LAAO is a novel member of the snake-venom L-amino-acid oxidase family. Its amino-acid sequence shows high homology to other members of this family. For L-leucine, the values of k(cat) and K(M) are 31.1 s(-1) and 0.25 mM, respectively. The molecular weight of AHP-LAAO is about 60.7 kDa as determined by MALDI-TOF mass spectrometry. AHP-LAAO can also induce apoptosis of cultured Hela cells. Two sets of diffraction data with similar resolution limits (about 2.5 A) were collected independently at MacCHESS (Cornell High Energy Synchrotron Source, USA) and IHEP (Institute of High Energy Physics, Beijing, China). The crystals belong to space group I2(1)3, with unit-cell parameter a = 169.31 A, corresponding to one molecule in the asymmetric unit and a volume-to-weight ratio of 3.33 A(3) Da(-1). The final structural model is similar to that of L-amino-acid oxidase from Calloselasma rhodostoma venom.

Use of snake venom inhibitors in studies of the function and tertiary structure of coagulation factors.

C-type lectin-like proteins (CLPs) of snake venom have a variety of biological properties, acting for example as anticoagulants, procoagulants, and agonists/antagonists of platelet activation. The structural and functional studies of the first identified venom CLP, factors IX/X-binding protein (IX/X-bp), have contributed to our understanding of the roles of magnesium ions in the blood coagulation cascade reaction. The crystal structures of gamma-carboxyglutamic acid (Gla) domains of cpagulation factors X and IX have recently been clarified in structural studies of complexes between the Gla domain of factor X and X-bp (a venom CLP) and between the Gla domain of factor IX and IX-bp (a venom CLP).

Snake venom toxins affecting platelet function.

Botrocetin from the South American pit viper Bothrops jararaca was described as an activator of von Willebrand factor-dependent platelet aggregation by Read, Shermer, and Brinkhous in 1978 (1). Subsequently, botrocetin has been widely used as an important in vitro modulator in the analysis of von Willebrand factor and platelet aggregation. Botrocetin has since been identified as a heterodimer of the C-type lectin family of snake venom proteins (~25 kDa nonreduced, ~14 kDa reduced), the primary sequence and crystal structure have been determined, and specific binding sites within the A1 domain of von Willebrand factor have been identified (2–8). Interestingly, members of the metalloproteinase-disintegrin family of snake venom proteins—jararhagin, jaracetin, and one-chain botrocetin—are functionally related to two-chain botrocetin, and also interact with the von Willebrand factor A1 domain (4,9,10). Jaracetin and one-chain botrocetin are variably processed forms of jararhagin and are found in the same viper species as the C-type lectin family, two-chain botrocetin. In this regard, recent evidence suggests that C-type lectin proteins and metalloproteinase-disintegrins may be derived from a common gene encoding a much larger precursor protein (11). An increasing number of C-type lectin proteins and metalloproteinases from cobra or viper venoms have been reported that selectively target either von Willebrand factor or its platelet receptor, glycoprotein (GP) Ibα of the GPIb-IX-V complex (some examples are shown in Table 1). These include the cobra venom metalloproteinase-disintegrin, mocarhagin from the Mozambiquan spitting cobra Naja mocambique mocambique (Naja mossambica mossambica) that cleaves GP Ibα within an anionic sequence containing three sulfated tyrosines (19,24,25). Mocarhagin also cleaves the neutrophil receptor PSGL-1 (P-selectin glycoprotein ligand-1) within an analogous sequence, and therefore has antiinflammatory activity in addition to being antithrombotic (18). In contrast, kaouthiagin from Naja kaouthia cleaves von Willebrand factor (21). A variety of C-type lectin proteins including alboaggregin-B (Table 1) bind to GPIbα and inhibit von