The Cytotoxic Mode of Action of the Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei (Fig.16.1) is rich in components which belong to different functional groups. Besides low molecular mass compounds, the venom contains several disulphide-rich peptides, also called mini-proteins, which act as neurotoxins on ion channels or as enhancers of neurotoxins. Likewise, a variety of small cytolytic peptides, which destroy membranes very efficiently, and enzymes are present in the venom. Neurotoxins with cytolytic activity, cytolytic a-helical small cationic peptides and enzymes most probably attacking connective tissue and phospholipid membranes cause the overall cytotoxic effect of this venom. Synergistic and enhancing interactions between components enable the spider to achieve a maximum of toxicity with a minimum of venom quantity.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Screening for lupus anticoagulant: improving the performance of the lupus-sensitive PTT-LA

Introduction: The aim of the present work was to verify whether calculating a ratio between clotting times obtained with the sensitive PTT-LA and a less sensitive activated partial thromboplastin time (aPTT)-reagent may represent a valuable aPTT-based screening strategy for lupus anticoagulants (LA). Methods: For the pilot study, plasma samples from normal subjects (n = 15) and from patients with LA (n = 10), therapeutic anticoagulation with vitamin K-antagonists (VKA) (n = 15) or unfractionated heparin (n = 15), coagulation factors deficiency (n = 16), and inhibitory antibodies against factor VIII or IX (n = 11) were studied. For the evaluation study, 1553 consecutive plasma samples from nonanticoagulated patients investigated for LA between January 2005 and December 2007 at our institution were studied. Following screening strategies were employed: Pathromtin-SL (aPTT-SL), PTT-LA (aPTT-LA), ratio aPTT-LA/aPTT-SL (aPTT-ratio), and Russell's viper venom (RVV) based LA-Check. LA positive samples were identified by mixing studies and diluted RVV confirmation test (LA-Check/LA-Sure). Results: Pilot study: All screening strategies had a 100% sensitivity, and the aPTT-ratio reached the highest specificity (82%; 95%CI: 74-90%). Within the evaluation study, following sensitivities for LA screening were observed: aPTT-SL 59.0% (95%CI: 57-61%), aPTT-LA 82.1% (95%CI: 80-84%), aPTT-ratio 92.3% (95%CI: 91-94), and LA-Check 83.3% (95%CI: 82-85%). Conclusion: Calculating a ratio between the LA-sensitive PTT-LA and the less sensitive Pathromtin-SL improves the performance of the PTT-LA itself and represents a simple and sensitive aPTT-based integrated strategy for LA screening.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Unpredicted adverse reaction to omalizumab

Despite promising reports of the use of omalizumab as add-on therapy in patients with systemic mastocytosis and recurrent anaphylaxis during specific venom immunotherapy (VIT), unpredicted adverse effects may lead to therapy failure. We present the case of a patient with systemic mastocytosis and Hymenoptera venom allergy who was administered omalizumab as add-on therapy to improve VIT tolerability after repeated severe adverse reactions despite H1/H2-antihistamine prophylaxis. We describe an unexpected discontinuation of omalizumab following successful initiation of VIT in a patient with systemic mastocytosis, with subsequent lack of tolerability of VIT. An interesting aspect of this case is the correlation of basophil activation test results with both clinical tolerability and VIT intolerance.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Increased level of antibodies cross-reacting with Ves v 5 and CRISP-2 in MAR-positive patients

Anti-sperm antibodies (ASA) have been described to be involved in immunological infertility. A possible antigen for ASA is the human cysteine-rich secretory protein 2 (CRISP-2), a sperm surface protein important in sperm-oocyte interaction. Furthermore, anti-CRISP-2 antibodies were shown to decrease fertility rates in vitro. Recently, we have reported cross-reacting antibodies recognizing CRISP-2 and antigen 5 from yellow jacket venom (Ves v 5) in human serum.

Snake C-type lectin-like proteins and platelet receptors

Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect haemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Snake venom components are classified into various families, such as serine proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases. Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-like proteins have now been characterized, as heterodimeric structures with alpha and beta subunits that often form large molecules by multimerization. They activate platelets by binding to VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to GPVI while another series affects platelet function via integrin alpha2beta1. Some act by inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their ligands, have been determined. Structure-activity studies show that these proteins are quite complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with more than one platelet receptor and have complex mechanisms of action.

Translocation of GPIb and Fc receptor gamma-chain to cytoskeleton in mucetin-activated platelets

Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

Snake venoms and hemostasis

Snake venoms are complex mixtures of biologically active proteins and peptides. Many of them affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Based on sequence, these snake venom components have been classified into various families, such as serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. For almost every factor involved in coagulation or fibrinolysis there is a venom protein that can activate or inactivate it. Venom proteins affect platelet function by binding or degrading vWF or platelet receptors, activating protease-activated receptors or modulating ADP release and thromboxane A2 formation. Some venom enzymes cleave key basement membrane components and directly affect capillary blood vessels to cause hemorrhaging. L-Amino acid oxidases activate platelets via H2O2 production.

Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

Platelet GPIb complex as a target for anti-thrombotic drug development

Specific inhibition of platelet function is a major target of anti-thrombotic drug research. Platelet receptors are both accessible and specific but have multiple functions often linked to a wide range of ligands. GPIb complex is best known as a major platelet receptor for von Willebrand factor essential for platelet adhesion under high shear conditions found in arteries and in thrombosis. Recent animal studies have supported inhibition of GPIb as a good candidate for anti-thrombotic drug development with several classes of proteins showing important specific effects and the required discrimination between roles in haemostasis and thrombosis is important to protect against bleeding complications. These include antibodies, several classes of snake venom proteins, mutant thrombin molecules and peptides affecting subunit interactions. However, due to the nature of its receptor-ligand interactions involving large protein-protein interfaces, the possibility of developing classic pharmaceutical inhibitors for long term (and perhaps oral) treatment is still unclear, and additional information about structural interactions and signalling mechanisms is essential.