e at SciVerse ScienceDirect

Toxicon 60 (2012) 512–519

Contents lists availabl

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

C-type lectin-like proteins from snake venoms

Franziska T. Arlinghaus, Johannes A. Eble*

Center for Molecular Medicine, Department of Vascular Matrix Biology, Excellence Cluster Cardio-Pulmonary System, Frankfurt University Hospital,Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

a r t i c l e i n f o

Article history:Received 31 January 2012Received in revised form 28 February 2012Accepted 1 March 2012Available online 10 March 2012

Keywords:C-type lectin-like proteinsCoagulation factorIntegrinRhodocetin

* Corresponding author. Tel.:þ4969630187652; faE-mail addresses: arlinghaus@med.uni-frankfurt

eble@med.uni-frankfurt.de (J.A. Eble).

0041-0101/$ – see front matter � 2012 Elsevier Ltddoi:10.1016/j.toxicon.2012.03.001

a b s t r a c t

C-type lectin-like proteins (CTLs) as found in snake venoms fulfill various physiologicalfunctions. They play a role in hemostasis and have helped elucidate mechanisms involvedin blood coagulation and platelet activation. Their basic structure consists of the subunitsa and b, which form heterodimers via a typical domain-swapping motif. These hetero-dimers can form oligomers such as the tetrameric flavocetin-A and convulxin, whicharrange into cyclic structures. Rhodocetin is a selective a2b1 integrin antagonist consistingof four distinct subunits forming a novel cruciform structure. Along with EMS16 and VP12,rhodocetin inhibits collagen-binding to the a2A-domain. These integrin-specific antago-nists are lead structures for the development of antimetastatic and antiangiogenic drugs.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Snake venoms contain a variety of proteins withdifferent biological activities (Morita, 2005a). They belongto different protein families such as phospholipases, serineproteinases, metalloproteinases, C-type lectins, and C-typelectin-like proteins (CTL). CTLs have opposite functions:Some induce platelet aggregation/agglutination, whileothers inhibit this effect (Ogawa et al., 2005).

C-type lectin-like proteins take their name from theirhigh sequence homology (15–40%) with the carbohydraterecognition domains of C-type lectins (Morita, 2005b).These are non-enzymatic, Ca2þ-dependent (“C”) proteinswhich bind sugar residues, as inferred by the term “lectin”(Clemetson et al., 2005). In contrast, most CTLs have losttheir carbohydrate-binding function along with their Ca2þ-binding function (Koh et al., 2006). Structurally, CTLs areheterodimers composed of homologous a and b subunitswith molecular weights of 14–15 and 13–14 kDa, respec-tively. Both covalent and non-covalent multimerization ofthese heterodimers are possible, giving yield to ab, (ab)2

x:þ4969630187656..de (F.T. Arlinghaus),

. All rights reserved.

and (ab)4 structures (Morita, 2005b). In contrast to CTLs,classic C-type lectins form exclusively homodimers linkedby an interchain disulfide bridge; these can also multi-merize to oligomers (Clemetson, 2010). CTL ab hetero-dimers are formed by domain-swapping, where a domainfrom one subunit replaces the corresponding domain of theother subunit. The result is a concave interface interactingwith the targets either electrostatically or by shape fitting(Morita, 2005b).

Classical C-type lectins are divided into sevensubgroups, according to their structural characteristics.They play a role in adhesion, endocytosis and pathogenneutralization (Ogawa et al., 2005). Some of the maintargets of CTLs are membrane receptors, coagulationfactors, and proteins essential to hemostasis. Adhesionreceptors of platelets, such as the von Willebrand factor(vWF)-binding GPIb-complex, the collagen-binding GPVIand integrin a2b1, and the fibrinogen receptor integrinaIIbb3, play important roles in platelet activation andaggregation. Accordingly, some CTLs act antithromboti-cally, others function as anticoagulants by inhibitingcoagulation factors, thereby eliciting hemorrhages, whileyet others activate coagulation factors without any neces-sity for protein cofactors (Lu et al., 2005) (Table 1). Due toan excessive consumption of coagulation factors, such

Table 1Function of selected C-type lectin-related proteins.

Ligand CLRP Structure Function

Factor IX IX-bp ab AnticoagulantIX/X-bp ab Anticoagulant

Factor X X-bp ab AnticoagulantIX/X-bp ab Anticoagulant

vWF Botrocetin ab AgonistBitiscetin ab Agonist

GPIb Alboaggregin-B ab AgonistEchicetin ab AgonistFlavocetin-A (ab)4 Antagonist

GPVI Convulxin (ab)4 Agonist

a2b1 EMS16 ab AntagonistRhodocetin abgd AntagonistVP12 ab Antagonist

CLEC-2 Aggretina ab Agonist

a Aggretin has also been shown to bind GPIb, GPVI, and a2b1.

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519 513

procoagulant CTLs lead to blood clotting, resulting in a lossof hemostasis and finally in hemorrhage.

2. CTLs as GPIb-mediated platelet activators

Snake venom proteins targeting the hemostasis systeminteract with one or more coagulation factors, causingcoagulation, or with the von Willebrand factor (vWF)(Clemetson et al., 2001). The vWF is a multimeric proteinwith four different types of modules: A, B, C, and D domain.Four A domains (A1–A4) play an important role in thefunction of vWF as they interact with the platelet receptorGPIb and with collagen. The vWF subunits oligomerize viadisulfide bridges in a head-to-head and tail-to-tail fashion.Usually, vWF adheres to collagen when the sub-endothelium is exposed at a site of injury. The collagen-binding site is located on the A1 and the A3 domain(Matsui and Hamako, 2005). vWF binding to exposedcollagen results in conformational changes, exposing thenormally inaccessible binding site for the platelet receptorglycoprotein (GP)-Ib on the A1 domain (Nuyttens et al.,2011). Only then does plasma vWF bind to platelets andlead to arterial thrombus formation.

One C-type lectin-like protein that has been discoveredto bind to the vWF domain A1 is botrocetin from the SouthAmerican snake Bothrops jararaca. It is a heterodimericprotein with sequence similarity to Ca2þ-dependent C-typelectins whose subunits a and b have molecular masses of14 kDa and 14.5 kDa, respectively (Andrews et al., 1989).The subunits are connected via a single disulfide bond,while each subunit contains three internal disulfide bonds(Usami et al., 1993). Initially, botrocetin was thought toinduce platelet agglutination by triggering a conforma-tional change within the vWFA1 domain to an active formbinding to the platelet receptor GPIb; antibodies againstbotrocetin, vWF, and GPIb individually preventedbotrocetin-induced agglutination (Read et al., 1989). Later,a mutant of the vWF A1 domain with increased affinity for

the GPIb receptor was shown to convert back into a low-affinity conformation upon botrocetin-binding, implyingthat botrocetin preferentially binds to a less active vWF andenlarges the GPIb binding surface on the A1 domain(Fukuda et al., 2002).

Another vWF-binding CTL was purified from Africanpuff adder venom (Bitis arietans). The a and b subunits ofthis disulfide-linked heterodimeric CTL measure 16 kDaand 13 kDa, respectively (Hamako et al., 1996). Like botro-cetin, bitiscetin binds both the vWF and the GPIb receptoron platelets. Several studies with different antibodies haveshown that bitiscetin binds to the a4 and a5 helices of thevWF A1 domain (Matsui et al., 2002) (Fig. 1). Crystalstructure analysis has revealed that the a5 loop of the A1domain does in fact lie within the concave surface ofbitiscetin, resulting from domain-swapping (Hirotsu et al.,2001). The vWF A1 domain is equipped with additionalbinding sites at each end of the concave surface (Maitaet al., 2003). Furthermore, while a specific antibodyagainst the bitiscetin a subunit prevents platelet aggluti-nation, it does not prevent binding to the vWF. Theseresults suggest that the a subunit is situated closer to theGPIb-binding site (Matsui et al., 2002).

3. CTLs as anti-coagulants

Many snake venom C-type lectin-like proteins targetingcoagulation factors have been described, such as factor IX-and factor X-binding proteins. These are Ca2þ-dependentCTLs, although their Ca2þ binding sites differ from those oftraditional C-type lectins (Suzuki et al., 2005). The Ca2þ

ions enable binding to the gamma-carboxyglutamic acid(Gla) domain found in the coagulation factor. Usually, theGla domain is required for Ca2þ-dependent coagulationfactor binding to phospholipid membranes (Mizuno et al.,2001). The first snake venom protein discovered to bindto a coagulation factor and described as a CTL was the IX/X-binding protein, isolated from the venom of the Japanesehabu snake Trimeresurus flavoviridis (Atoda et al., 1991). Thesubunits a and b of this heterodimer are characterized bya 47% sequence identity. At the same time, they arehomologous to the vWF-binding protein botrocetin (Sekiyaet al., 1993). Binding to both coagulation factor IX andcoagulation factor X, this CTL is designated as IX/X-bindingprotein (IX/X-BP). Recently, habu IX/X-BP has been shownto block the interaction between activated factor X and itscofactor in the prothrombinase complex (Ishikawa et al.,2009). To date, IX/X-BPs have also been purified fromBothrops jararaca, Agkistrodon halys Pallas, and Echis car-inatus leucogaster (Chen and Tsai, 1996; Ishikawa et al.,2009; Sekiya et al., 1993).

Another example of a coagulation factor-binding CTLthat is independent of any cofactor is the factor IX-bindingprotein (IX-BP), also purified from Trimeresurus flavoviridis.It inhibits activated factor IX-induced blood coagulation at0.4 nM (Atoda et al., 1995). As with all CTLs, IX-BP ishomologous to the carbohydrate recognition domain (CRD)of classical C-type lectins. The a (16.8 kDa) and b (15.7 kDa)subunits of this heterodimer are linked by a disulfide bridge(Mizuno et al., 1999). The loop-swapping of central portionsof the subunits resembles the loop-exchange of monomeric

Fig. 1. Binding of bitiscetin and botrocetin to vWF A1-domain and GPIba. The bitiscetin/botrocetin a and b subunits are shown in blue and purple, respectively;vWF A1-domain (red) is displayed from the same angle in both A and B, with the a-helices 5 and 6 facing the front of the globule-shaped A-domain; GPIba isshown in green. Note that bitiscetin (A) and botrocetin (B) bind in a different orientation to the vWF A1-domain despite their highly homologous structure. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519514

C-type lectins. It has been suggested that this exchangeleads to the loss of the original Ca2þ-binding site and toa disruption in the carbohydrate-binding site. Mizuno et al.(1999), describes the loop-swapping in detail. Of the 129amino acid residues constituting the a subunit, only 19differ from those in the IX/X-binding protein (Mizuno et al.,1999); the b subunits of these coagulation factor-bindingproteins are identical. It appears that the different aminoacid residues bring about the functional differences ofthese two proteins (Atoda et al., 1995).

In 1998, Atoda et al. purified another anticoagulantsnake venom CTL from the Southeast Asian pit viperDeinagkistrodon acutus which showed factor X-bindingactivity (Atoda et al., 1998). The 16 kDa a subunit has 68%sequence identity with the IX/X-binding protein; the15 kDa b subunit has 87% sequence identity with the IX/X-BP. Most of the differing amino acid residues are situated onthe surface of the X-binding protein, especially at theconcave interface, the binding site of the protein formed bythe loop-swapping of the subunits (Mizuno et al., 2001).

The coagulation factors targeted by these CTLs areencountered half-way through the platelet activationcascade. They are responsible for amplifying the coagula-tion cascade and ensuing hemostatic plug formation afterinjury. Anticoagulant snake venom proteins are exploitedto shed light on these hemostatic processes, both inresearch and diagnostics.

4. Antithrombotic CTLs

Platelets are one main target of antithrombotic drugsbeing developed. The chief receptors considered are thecollagen receptors GPVI and a2b1 integrin as well as thevWF receptor GPIb (Clemetson and Clemetson, 2008). GPIbis an important platelet receptor, as it has key functions inboth the hemostatic and thrombotic pathways. One of thefirst CTLs discovered to bind directly to GPIb wasalboaggregin-B (Peng et al., 1991) from the Southeast AsianTrimeresurus albolabris. Its a and b subunits are constitutedof 133 and 123 amino acid residues, respectively; thesehave 41.1% identity and are homologous to botrocetin andthe IX/X-BP (Usami et al., 1996). Alboaggregin-B inducesplatelet agglutination, which can be blocked by

a monoclonal antibody against the 45 kDa-N-terminaldomain of the GPIb receptor (Peng et al., 1991; Yoshidaet al., 1993). A tetrameric variant of this protein,alboaggregin-A, was discovered by Kowalska et al., whichwas able to induce platelet aggregation via GPIb (Kowalskaet al., 1998). Alboaggregin-A-induced aggregation resultedin phospholipase-C activity in platelets as well as in tyro-sine kinase activity (Andrews et al., 1996).

Shortly after the discovery of alboaggregin-B, a GPIb-binding CTL was identified in the Asian viper Echis car-inatus. In contrast to the platelet-agglutinating CTLalboaggregin-B, echicetin inhibits platelet agglutination(Peng et al., 1993). Later, crosslinked as well as clusteredechicetin were shown to induce aggregation, similar toalboaggregin-A (Navdaev et al., 2001). Seven cysteinylresidues stabilize the a and b subunits, which havemolecular weights of 16 kDa and 14 kDa, respectively (Penget al., 1994). Binding to platelets was measured with a Kd of30 nM; this binding was inhibited by a monoclonal anti-body against the 45 kDa-N-terminal domain of the GPIbreceptor (Peng et al., 1993). Furthermore, thrombin wasshown to be a competitor for the echicetin binding site onGPIb (Peng et al., 1995). Later, echicetinwas used to identifypurpureotin as another GPIb-binding CTL. This heterodimerobtained from Trimeresurus purpureomaculatus has 16 kDaa and 14.5 kDa b subunits which are non-covalently joinedby hydrophobic and electrostatic interactions (Li et al.,2004).

Another antithrombotic CTL which targets the plateletreceptor GPIb and induces small platelet aggregates isflavocetin-A from Trimeresurus flavoviridis (Taniuchi et al.,1995). Both subunits of this protein contain an additionalcysteine residue as compared with other CTLs (Shin et al.,2000). These residues form interchain disulfide bonds ina head-to-tail fashion, resulting in the first known tetra-meric structure of a CTL (Fukuda et al., 1999). It has beenproposed that this tetrameric structure crosslinks platelets,thereby initiating a platelet response (Taniuchi et al., 2000),which is possibly subject to cooperative binding activity(Fukuda et al., 2000).

Convulxin, obtained from the South American pit viperCrotalus durissus terrificus, is also a tetrameric protein,although initially described as a trimer (Batuwangala et al.,

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519 515

2004; Francischetti et al., 1997; Leduc and Bon, 1998). Itinduces platelet aggregation which can be inhibited byanti-a2b1 (6F1) and anti-GPVI antibodies (Jandrot-Perruset al., 1997). The tetrameric structure permits clusteringof the GPVI receptor on platelets, thus activating these andleading to an increase in intracellular Ca2þ levels(Batuwangala et al., 2004).

One CTLwhich has causedmuch controversy is aggretin,also known as rhodocytin, isolated from the venom of theMalayan pit viper (Calloselasma rhodostoma). It is a hetero-dimer with the domain-swapping typical for snake C-typelectins which leads to a loss of the carbohydrate-bindingsite (Huang et al., 1995). The two subunits show highidentity with other CTLs and contain seven conservedcysteine residues; subunits are connected by a singledisulfide bond, while each has three intercatenary disulfidebonds (Chung et al., 1999). Aggretin induces plateletaggregation, which early studies surmised to be due to itsbinding to the collagen receptor a2b1, since aggregationwas inhibited by the monoclonal antibody 6F1 (Huanget al., 1995). In another study, GPIb was proposed to benecessary to block aggregation, as the a2 antibody A2IIE10was able to prevent aggregation solely in combinationwiththe GPIb antagonist agkistin. When employed separately,these two antibodies only partially inhibited or delayed theaggregation response (Chung et al., 2001). Althoughaggretin seems to provoke cell and platelet responses, it didnot bind to a2b1 integrin in a cell-free protein interactionassay (Eble et al., 2001). Nonetheless, using humanumbilical vein endothelial cells (HUVEC), Chung et al.(2004) could show aggretin to induce proliferation andmigration of cells, which can be inhibited by an a2b1antibody (Chung et al., 2004). Binding of aggretin to a2b1also induces proliferation of vascular smooth muscle cells(VSMC) (Chung et al., 2009).

Fig. 2. The cruciform structure of rhodocetin shown from different angles. The subunstructure with marked disulfide bonds as well as schematically. The ab and gd heteinto a cruciform structure. (For interpretation of the references to color in this figu

In contrast, a study was published in 2001 in whichaggretin was shown to induce platelet aggregation whenneither the a2b1 integrin, nor GPIb, nor GPVI were presenton platelets (Bergmeier et al., 2001). Subsequently, a studywas published showing that aggretin binds to the C-typelectin receptor CLEC-2, activating a novel signaling pathwayvia the single tyrosine phosphorylation site of its cytoslic tailand inducing platelet aggregation (Suzuki-Inoue et al.,2006). In macrophages, aggretin-binding to CLEC-2 leads tothe release of TNF-a and IL-6 through activation of theMAPKand NF-kB signaling pathways (Chang et al., 2010). Crystalstructure analysis of aggretin has revealed a tetramericstructure with two heterodimers in antiparallel alignment.Modeling suggested that aggretin interacts either with twoCLEC-2monomersorwithoneor twoCLEC-2dimers (Hooleyet al., 2008; Watson and O’Callaghan, 2011).

The collagen-binding integrin a2b1 is yet anotherreceptor that leads to platelet activation. Reduced expres-sion of a2b1 results in dysfunction of platelet responses(Nuyttens et al., 2011). It is the only integrin inhibited by C-type lectin-like proteins; other integrins require dis-integrins, a functionally and structurally distinct class ofintegrin inhibitors (Calvete et al., 2005). To date, there areonly three known CTLs which are antagonists of a2b1integrin. These have been shown to bind to the a2A-domain, which is homologous to the vWF A-domain andcontains the collagen-binding site. Upon collagen binding,the integrin a2A-domain undergoes a substantial changefrom an inactive/closed to an active/open conformation(Emsley et al., 2000). This conformational change isconveyed throughout the integrin, eventually leading tointegrin signaling (Arnaout et al., 2005).

The first selective a2b1 antagonist discovered wasEMS16, a heterodimeric protein obtained from the Asianviper Echis multisquamatus (Marcinkiewicz et al., 2000). It

its a (blue), b (purple), g (red), and d (yellow) are depicted in their secondaryrodimers are formed via loop-swapping motifs and associate non-covalentlyre legend, the reader is referred to the web version of this article.)

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519516

inhibits collagen-induced platelet aggregation as well ascell migration (Okuda et al., 2003). Its crystal structure,resolved at 1.9 Å, reveals the typical domain-swapping CTLinterface. The concave surface formed by this loopexchange is characterized by a distinctive positivelycharged electrostatic potential patch which may serve asthe a2A-domain binding site (Horii et al., 2003). A secondcrystal structure of EMS16, complexed with the a2A-domain, shows that EMS16 spatially blocks the collagen-binding site independently from divalent cations. It hasalso been suggested that EMS16 stabilizes the closedconformation of the a2A-domain (Horii et al., 2004).

Rhodocetin is a platelet aggregation inhibitor purifiedfrom the snake venom of the Malayan pit viper Callos-elasma rhodostoma. It was initially believed to be a hetero-dimer with non-covalently bound subunits (Eble et al.,2001; Wang et al., 1999). The cysteinyl residues whichusually form an intersubunit disulfide bridge areexchanged by Ser79 and Arg75 in the a and b subunits,respectively (Wang et al., 1999). A crystal structure resolvedat 1.9 Å revealed an additional b sheet in each rhodocetinsubunit; the backbones of the two b sheets form hydrogenbonds with each other, thus stabilizing the dimeric struc-ture (Paaventhan et al., 2005). The a (15.9 kDa) andb subunits (15.1 kDa) dose-dependently inhibited plateletaggregation induced by collagen only when paired; plateletaggregation was not prevented when the subunits wereapplied individually (Wang et al., 1999). Furthermore,rhodocetinwas found to be an antagonist of collagen type I-dependent cellular responses, such as cell adhesion andmigration (Eble et al., 2001). It was also able to impedeextravasation of tumor cells into the liver stroma, a cause ofmicrometastases (Rosenow et al., 2008). Moreover, it wasdiscovered that rhodocetin is unable to cluster the integrin

Fig. 3. Alignment of the four rhodocetin subunits. Within the hetero-tetramer the binding interface of the ab- and gd-heterodimer pairs is formedby special peptide turns in the a and g subunits. The backbones of therhodocetin subunits and the sequences of the peptide turns are color-coded.(For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

a2b1, and therefore unable to generate focal adhesions anda firm attachment of cells to this CTL (Eble et al., 2002). Inthe presence of Ca2þ ions, rhodocetin binds more readily toa2b1 than does collagen, suggesting it prefers a less activeconformation of the integrin. Additionally, the rhodocetinbinding site on the integrin was located on the a3 and a4loops and adjacent helices, which form part of the collagen-binding site on the a2A-domain (Eble and Tuckwell, 2003).More recently, rhodocetin has been shown to be a hetero-tetramer composed of four homologous subunits a, b, g,and d (Fig. 2). Heterodimers are formed via a domain-swapping motif, with the dimers ab and gd associatingwith one another and arranging into a cruciform molecule,

Fig. 4. Interaction of rhodocetin with the collagen-binding integrin a2b1.The rhodocetin tetramer binds to the inactive conformation of integrin a2b1via the gd heterodimer. gd associates tightly with the a2A-domain whichsubsequently undergoes a slight conformational change. This in turn leads todissociation of the rhodocetin tetramer: while rhodocetin gd stays firmly atthe inactive a2A-domain, rhodocetin ab is released.

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519 517

where gd is stabilized by a disulfide bridge between Cg81and Cd74 (Eble et al., 2009). The interface of this novelcruciform structure is chiefly made up by the additionalpeptide turns N74KGQR in the a and K77EQQC in the g chain(Fig. 3). The glycine76 residue of the a subunit fits neatlyinto a groove formed by the peptide turn of the g subunit(Eble et al., 2009). Using different monoclonal antibodies,rhodocetin was shown to dissociate into its two hetero-dimers after binding to the a2A-domain (Fig. 4); the gdheterodimer interacts with the a2A-domain, whilst the abheterodimer is released (Bracht et al., 2011). Recently, theab heterodimer was shown to bind the platelet receptorGPIb and inhibit platelet aggregation induced by vWF plusristocetin. Crosslinking of ab with avidin induced theformation of small platelet agglutinates and thereby thephosphorylation of the kinase p72SYK in platelets(Navdaev et al., 2011).

Not long ago, a third a2A-domain-binding CTL waspurified from Vipera palaestinae snake venom. VP12 isa heterodimer with molecular sizes of 15.9 for the a and15.8 kDa for the b subunit chain. This proteinwas shown toprevent cell migration of the melanoma cell line MV3(Staniszewska et al., 2009); it was also found to inhibitbinding of the a2b1-transfected cell line K562 to collagentype I with an IC50 of 0.5 nM (Momic et al., 2011).

5. Conclusion

Despite their similar structure, CTLs show an amazingversatility of functions as they target different platelet andcellular receptors as well as coagulation factors. Thegrowing number of isolated snake venom CTLs, revealinga continuously increasing variety of biological activities,suggests a huge biomedical potential. They have alreadybeen shown to constitute useful tools for studying hemo-stasis and thrombosis, but they may also serve as potentiallead structures for the development of synthetic antico-agulant and antithrombotic compounds. However, theirvariability makes it difficult to determine the plateletreceptor or coagulation factor which is the most effectivetarget. So far, CTLs have aided in elucidating some mecha-nisms in the coagulation cascade and in tracking signalingpathways. Here, the a2b1 integrin is of especially greatinterest, although its role in collagen-induced plateletactivation remains unclear. However, the distribution ofa2b1 in endothelial and smoothmuscle cells suggests otherapplications for integrin-targeting CTLs. EMS16, rhodoce-tin, and VP12 will most likely not be the last CTLs to bediscovered which inhibit collagen-dependent cell adhesionand migration. They will likely play a role in the treatmentof tumor metastasis and angiogenesis.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

References

Andrews, R.K., Booth, W.J., Gorman, J.J., Castaldi, P.A., Berndt, M.C., 1989.Purification of botrocetin from Bothrops jararaca venom. Analysis ofthe botrocetin-mediated interaction between von Willebrand factor

and the human platelet membrane glycoprotein Ib-IX complex.Biochemistry 28, 8317–8326.

Andrews, R.K., Kroll, M.H., Ward, C.M., Rose, J.W., Scarborough, R.M.,Smith, A.I., Lopez, J.A., Berndt, M.C., 1996. Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and relatedviper venom proteins to the platelet membrane glycoprotein Ib-IX-Vcomplex. Effect on platelet aggregation and glycoprotein Ib-mediatedplatelet activation. Biochemistry 35, 12629–12639.

Arnaout, M.A., Mahalingam, B., Xiong, J.P., 2005. Integrin structure, allo-stery, and bidirectional signaling. Annu. Rev. Cell Dev. Biol. 21, 381–410.

Atoda, H., Hyuga, M., Morita, T., 1991. The primary structure of coagula-tion factor IX/factor X-binding protein isolated from the venom ofTrimeresurus flavoviridis. Homology with asialoglycoprotein recep-tors, proteoglycan core protein, tetranectin, and lymphocyte Fcepsilon receptor for immunoglobulin E. J. Biol. Chem. 266, 14903–14911.

Atoda, H., Ishikawa, M., Mizuno, H., Morita, T., 1998. Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein. Biochemistry 37, 17361–17370.

Atoda, H., Ishikawa, M., Yoshihara, E., Sekiya, F., Morita, T., 1995. Bloodcoagulation factor IX-binding protein from the venom of Trimer-esurus flavoviridis: purification and characterization. J. Biochem. 118,965–973.

Batuwangala, T., Leduc, M., Gibbins, J.M., Bon, C., Jones, E.Y., 2004.Structure of the snake-venom toxin convulxin. Acta Crystallogr. DBiol. Crystallogr. 60, 46–53.

Bergmeier, W., Bouvard, D., Eble, J.A., Mokhtari-Nejad, R., Schulte, V.,Zirngibl, H., Brakebusch, C., Fassler, R., Nieswandt, B., 2001. Rhodo-cytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin,glycoprotein VI, and the ligand-binding domain of glycoproteinIbalpha. J. Biol. Chem. 276, 25121–25126.

Bracht, T., Figueiredo de Rezende, F., Stetefeld, J., Sorokin, L.M., Eble, J.A.,2011. Monoclonal antibodies reveal the alteration of the rhodocetinstructure upon alpha2beta1 integrin binding. Biochem. J. 440, 1–11.

Calvete, J.J., Marcinkiewicz, C., Monleon, D., Esteve, V., Celda, B., Juarez, P.,Sanz, L., 2005. Snake venom disintegrins: evolution of structure andfunction. Toxicon 45, 1063–1074.

Chang, C.H., Chung, C.H., Hsu, C.C., Huang, T.Y., Huang, T.F., 2010. A novelmechanism of cytokine release in phagocytes induced by aggretin,a snake venom C-type lectin protein, through CLEC-2 ligation. J.Thromb. Haemost. 8, 2563–2570.

Chen, Y.L., Tsai, I.H., 1996. Functional and sequence characterization ofcoagulation factor IX/factor X-binding protein from the venom ofEchis carinatus leucogaster. Biochemistry 35, 5264–5271.

Chung, C.H., Au, L.C., Huang, T.F., 1999. Molecular cloning and sequenceanalysis of aggretin, a collagen-like platelet aggregation inducer.Biochem. Biophys. Res. Commun. 263, 723–727.

Chung, C.H., Lin, K.T., Chang, C.H., Peng, H.C., Huang, T.F., 2009. Theintegrin alpha2beta1 agonist, aggretin, promotes proliferation andmigration of VSMC through NF-kB translocation and PDGF produc-tion. Br. J. Pharmacol. 156, 846–856.

Chung, C.H., Peng, H.C., Huang, T.F., 2001. Aggretin, a C-type lectin protein,induces platelet aggregation via integrin alpha(2)beta(1) and GPIb ina phosphatidylinositol 3-kinase independent pathway. Biochem.Biophys. Res. Commun. 285, 689–695.

Chung, C.H., Wu, W.B., Huang, T.F., 2004. Aggretin, a snake venom-derivedendothelial integrin alpha 2 beta 1 agonist, induces angiogenesis viaexpression of vascular endothelial growth factor. Blood 103, 2105–2113.

Clemetson, K.J., 2010. Snaclecs (snake C-type lectins) that inhibit oractivate platelets by binding to receptors. Toxicon 56, 1236–1246.

Clemetson, K.J., Clemetson, J.M., 2008. Platelet GPIb complex as a targetfor anti-thrombotic drug development. Thromb. Haemost. 99, 473–479.

Clemetson, K.J., Lu, Q., Clemetson, J.M., 2005. Snake C-type lectin-likeproteins and platelet receptors. Pathophysiol. Haemost. Thromb. 34,150–155.

Clemetson, K.J., Navdaev, A., Dormann, D., Du, X.Y., Clemetson, J.M., 2001.Multifunctional snake C-type lectins affecting platelets. Haemostasis31, 148–154.

Eble, J.A., Beermann, B., Hinz, H.J., Schmidt-Hederich, A., 2001. alpha 2beta1 integrin is not recognized by rhodocytin but is the specific, highaffinity target of rhodocetin, an RGD-independent disintegrin andpotent inhibitor of cell adhesion to collagen. J. Biol. Chem. 276, 12274–12284.

Eble, J.A., Niland, S., Bracht, T., Mormann, M., Peter-Katalinic, J.,Pohlentz, G., Stetefeld, J., 2009. The alpha2beta1 integrin-specificantagonist rhodocetin is a cruciform, heterotetrameric molecule.FASEB J. 23, 2917–2927.

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519518

Eble, J.A., Niland, S., Dennes, A., Schmidt-Hederich, A., Bruckner, P.,Brunner, G., 2002. Rhodocetin antagonizes stromal tumor invasionin vitro and other alpha2beta1 integrin-mediated cell functions.Matrix Biol. 21, 547–558.

Eble, J.A., Tuckwell, D.S., 2003. The alpha2beta1 integrin inhibitor rho-docetin binds to the A-domain of the integrin alpha2 subunit prox-imal to the collagen-binding site. Biochem. J. 376, 77–85.

Emsley, J., Knight, C.G., Farndale, R.W., Barnes, M.J., Liddington, R.C., 2000.Structural basis of collagen recognition by integrin alpha2beta1. Cell101, 47–56.

Francischetti, I.M., Saliou, B., Leduc, M., Carlini, C.R., Hatmi, M., Randon, J.,Faili, A., Bon, C., 1997. Convulxin, a potent platelet-aggregating proteinfrom Crotalus durissus terrificus venom, specifically binds to plate-lets. Toxicon 35, 1217–1228.

Fukuda, K., Doggett, T.A., Bankston, L.A., Cruz, M.A., Diacovo, T.G.,Liddington, R.C., 2002. Structural basis of von Willebrand factoractivation by the snake toxin botrocetin. Structure 10, 943–950.

Fukuda, K., Mizuno, H., Atoda, H., Morita, T., 1999. Crystallization andpreliminary x-ray studies of flavocetin-A, a platelet glycoprotein Ib-binding protein from the habu snake venom. Acta Crystallogr. DBiol. Crystallogr. 55, 1911–1913.

Fukuda, K., Mizuno, H., Atoda, H., Morita, T., 2000. Crystal structure offlavocetin-A, a platelet glycoprotein Ib-binding protein, revealsa novel cyclic tetramer of C-type lectin-like heterodimers. Biochem-istry 39, 1915–1923.

Hamako, J., Matsui, T., Suzuki, M., Ito, M., Makita, K., Fujimura, Y., Ozeki, Y.,Titani, K., 1996. Purification and characterization of bitiscetin, a novelvon Willebrand factor modulator protein from Bitis arietans snakevenom. Biochem. Biophys. Res. Commun. 226, 273–279.

Hirotsu, S., Mizuno, H., Fukuda, K., Qi, M.C., Matsui, T., Hamako, J.,Morita, T., Titani, K., 2001. Crystal structure of bitiscetin, a von Wil-lebrand factor-dependent platelet aggregation inducer. Biochemistry40, 13592–13597.

Hooley, E., Papagrigoriou, E., Navdaev, A., Pandey, A.V., Clemetson, J.M.,Clemetson, K.J., Emsley, J., 2008. The crystal structure of the plateletactivator aggretin reveals a novel (alphabeta)2 dimeric structure.Biochemistry 47, 7831–7837.

Horii, K., Okuda, D., Morita, T., Mizuno, H., 2003. Structural character-ization of EMS16, an antagonist of collagen receptor (GPIa/IIa) fromthe venom of Echis multisquamatus. Biochemistry 42, 12497–12502.

Horii, K., Okuda, D., Morita, T., Mizuno, H., 2004. Crystal structure ofEMS16 in complex with the integrin alpha2-I domain. J. Mol. Biol. 341,519–527.

Huang, T.F., Liu, C.Z., Yang, S.H., 1995. Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom,activates phospholipase C by acting as a glycoprotein Ia/IIa agonist.Biochem. J. 309 (Pt 3), 1021–1027.

Ishikawa, M., Kumashiro, M., Yamazaki, Y., Atoda, H., Morita, T., 2009.Anticoagulant mechanism of factor IX/factor X-binding protein iso-lated from the venom of Trimeresurus flavoviridis. J. Biochem. 145,123–128.

Jandrot-Perrus, M., Lagrue, A.H., Okuma, M., Bon, C., 1997. Adhesion andactivation of human platelets induced by convulxin involve glyco-protein VI and integrin alpha2beta1. J. Biol. Chem. 272, 27035–27041.

Koh, D.C., Armugam,A., Jeyaseelan, K., 2006. Snake venomcomponents andtheir applications in biomedicine. Cell Mol. Life Sci. 63, 3030–3041.

Kowalska, M.A., Tan, L., Holt, J.C., Peng, M., Karczewski, J., Calvete, J.J.,Niewiarowski, S., 1998. Alboaggregins A and B. Structure and inter-action with human platelets. Thromb. Haemost. 79, 609–613.

Leduc, M., Bon, C., 1998. Cloning of subunits of convulxin, a collagen-likeplatelet-aggregating protein from Crotalus durissus terrificus venom.Biochem. J. 333 (Pt 2), 389–393.

Li, X., Zheng, L., Kong, C., Kolatkar, P.R., Chung, M.C., 2004. Purpureotin:a novel di-dimeric C-type lectin-like protein from Trimeresuruspurpureomaculatus venom is stabilized by noncovalent interactions.Arch. Biochem. Biophys. 424, 53–62.

Lu, Q., Navdaev, A., Clemetson, J.M., Clemetson, K.J., 2005. Snake venom C-type lectins interacting with platelet receptors. Structure-functionrelationships and effects on haemostasis. Toxicon 45, 1089–1098.

Maita, N., Nishio, K., Nishimoto, E., Matsui, T., Shikamoto, Y., Morita, T.,Sadler, J.E., Mizuno, H., 2003. Crystal structure of von Willebrandfactor A1 domain complexed with snake venom, bitiscetin: insightinto glycoprotein Ibalpha binding mechanism induced by snakevenom proteins. J. Biol. Chem. 278, 37777–37781.

Marcinkiewicz, C., Lobb, R.R., Marcinkiewicz, M.M., Daniel, J.L., Smith, J.B.,Dangelmaier, C., Weinreb, P.H., Beacham, D.A., Niewiarowski, S., 2000.Isolation and characterization of EMS16, a C-lectin type protein fromEchis multisquamatus venom, a potent and selective inhibitor of thealpha2beta1 integrin. Biochemistry 39, 9859–9867.

Matsui, T., Hamako, J., 2005. Structure and function of snake venom toxinsinteracting with human von Willebrand factor. Toxicon 45, 1075–1087.

Matsui, T., Hamako, J., Matsushita, T., Nakayama, T., Fujimura, Y., Titani, K.,2002. Binding site on human von Willebrand factor of bitiscetin,a snake venom-derived platelet aggregation inducer. Biochemistry 41,7939–7946.

Mizuno, H., Fujimoto, Z., Atoda, H., Morita, T., 2001. Crystal structure of ananticoagulant protein in complex with the Gla domain of factor X.Proc. Natl. Acad. Sci. U.S.A. 98, 7230–7234.

Mizuno, H., Fujimoto, Z., Koizumi, M., Kano, H., Atoda, H., Morita, T., 1999.Crystal structure of coagulation factor IX-binding protein from habusnake venom at 2.6 A: implication of central loop swapping based ondeletion in the linker region. J. Mol. Biol. 289, 103–112.

Momic, T., Arlinghaus, F.T., Arien-Zakay, H., Katzhendler, J., Eble, J.A.,Marcinkiewicz, C., Lazarovici, P., 2011. Pharmacological aspects ofviperaxantinapalestinae venom. Toxins (Basel) 3, 1420–1432.

Morita, T., 2005a. Structure–function relationships of C-type lectin-related proteins. Pathophysiol. Haemost. Thromb. 34, 156–159.

Morita, T., 2005b. Structures and functions of snake venom CLPs (C-typelectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities. Toxicon 45, 1099–1114.

Navdaev, A., Dormann, D., Clemetson, J.M., Clemetson, K.J., 2001. Echice-tin, a GPIb-binding snake C-type lectin from Echis carinatus, alsocontains a binding site for IgMkappa responsible for platelet agglu-tination in plasma and inducing signal transduction. Blood 97, 2333–2341.

Navdaev, A., Lochnit, G., Eble, J.A., 2011. The rhodocetin alphabeta subunittargets GPIb and inhibits von Willebrand factor induced plateletactivation. Toxicon 57, 1041–1048.

Nuyttens, B.P., Thijs, T., Deckmyn, H., Broos, K., 2011. Platelet adhesion tocollagen. Thromb. Res. 127 (Suppl. 2), S26–S29.

Ogawa, T., Chijiwa, T., Oda-Ueda, N., Ohno, M., 2005. Molecular diversityand accelerated evolution of C-type lectin-like proteins from snakevenom. Toxicon 45, 1–14.

Okuda, D., Horii, K., Mizuno, H., Morita, T., 2003. Characterization andpreliminary crystallographic studies of EMS16, an antagonist ofcollagen receptor (GPIa/IIa) from the venom of Echis multisquamatus.J. Biochem. 134, 19–23.

Paaventhan, P., Kong, C., Joseph, J.S., Chung, M.C., Kolatkar, P.R., 2005.Structure of rhodocetin reveals noncovalently bound heterodimerinterface. Protein Sci. 14, 169–175.

Peng, M., Emig, F.A., Mao, A., Lu, W., Kirby, E.P., Niewiarowski, S.,Kowalska, M.A., 1995. Interaction of echicetin with a high affinitythrombin binding site on platelet glycoprotein GPIb. Thromb. Hae-most. 74, 954–957.

Peng, M., Holt, J.C., Niewiarowski, S., 1994. Isolation, characterization andamino acid sequence of echicetin beta subunit, a specific inhibitor ofvon Willebrand factor and thrombin interaction with glycoprotein Ib.Biochem. Biophys. Res. Commun. 205, 68–72.

Peng, M., Lu, W., Beviglia, L., Niewiarowski, S., Kirby, E.P., 1993. Echicetin:a snake venom protein that inhibits binding of von Willebrand factorand alboaggregins to platelet glycoprotein Ib. Blood 81, 2321–2328.

Peng, M., Lu, W., Kirby, E.P., 1991. Alboaggregin-B: a new platelet agonistthat binds to platelet membrane glycoprotein Ib. Biochemistry 30,11529–11536.

Read, M.S., Smith, S.V., Lamb, M.A., Brinkhous, K.M., 1989. Role of botro-cetin in platelet agglutination: formation of an activated complex ofbotrocetin and von Willebrand factor. Blood 74, 1031–1035.

Rosenow, F., Ossig, R., Thormeyer, D., Gasmann, P., Schluter, K., Brunner, G.,Haier, J., Eble, J.A., 2008. Integrins as antimetastatic targets of RGD-independent snake venom components in liver metastasis [cor-rected]. Neoplasia 10, 168–176.

Sekiya, F., Atoda, H., Morita, T., 1993. Isolation and characterization of ananticoagulant protein homologous to botrocetin from the venom ofBothrops jararaca. Biochemistry 32, 6892–6897.

Shin, Y., Okuyama, I., Hasegawa, J., Morita, T., 2000. Molecular cloning ofglycoprotein Ib-binding protein, flavocetin-A, which inhibits plateletaggregation. Thromb. Res. 99, 239–247.

Staniszewska, I., Walsh, E.M., Rothman, V.L., Gaathon, A., Tuszynski, G.P.,Calvete, J.J., Lazarovici, P., Marcinkiewicz, C., 2009. Effect of VP12 andviperistatin on inhibition of collagen-receptor-dependent melanomametastasis. Cancer Biol. Ther. 8, 1507–1516.

Suzuki-Inoue, K., Fuller, G.L., Garcia, A., Eble, J.A., Pohlmann, S.,Inoue, O., Gartner, T.K., Hughan, S.C., Pearce, A.C., Laing, G.D.,Theakston, R.D., Schweighoffer, E., Zitzmann, N., Morita, T.,Tybulewicz, V.L., Ozaki, Y., Watson, S.P., 2006. A novel Syk-dependent mechanism of platelet activation by the C-type lectinreceptor CLEC-2. Blood 107, 542–549.

F.T. Arlinghaus, J.A. Eble / Toxicon 60 (2012) 512–519 519

Suzuki, N., Shikamoto, Y., Fujimoto, Z., Morita, T., Mizuno, H., 2005.Crystallization and preliminary X-ray analysis of coagulation factorIX-binding protein from habu snake venom at pH 6.5 and 4.6. ActaCrystallogr. Sect. F Struct. Biol. Cryst. Commun. 61, 147–149.

Taniuchi, Y., Kawasaki, T., Fujimura, Y., 2000. The high molecular mass,glycoprotein Ib-binding protein flavocetin-A induces only smallplatelet aggregates in vitro. Thromb. Res. 97, 69–75.

Taniuchi, Y., Kawasaki, T., Fujimura, Y., Suzuki, M., Titani, K., Sakai, Y.,Kaku, S., Hisamichi, N., Satoh, N., Takenaka, T., et al., 1995. Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteinswith high affinity purified from Trimeresurus flavoviridis venom,inhibit platelet aggregation at high shear stress. Biochim. Biophys.Acta 1244, 331–338.

Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., Titani, K.,1993. Primary structure of two-chain botrocetin, a von Willebrandfactor modulator purified from the venom of Bothrops jararaca. Proc.Natl. Acad. Sci. U.S.A. 90, 928–932.

Usami, Y., Suzuki, M., Yoshida, E., Sakurai, Y., Hirano, K., Kawasaki, T.,Fujimura, Y., Titani, K., 1996. Primary structure of alboaggregin-Bpurified from the venom of Trimeresurus albolabris. Biochem. Bio-phys. Res. Commun. 219, 727–733.

Wang, R., Kini, R.M., Chung, M.C., 1999. Rhodocetin, a novel plateletaggregation inhibitor from the venom of Calloselasma rhodostoma(Malayan pit viper): synergistic and noncovalent interaction betweenits subunits. Biochemistry 38, 7584–7593.

Watson, A.A., O’Callaghan, C.A., 2011. Molecular analysis of the interactionof the snake venom rhodocytin with the platelet receptor CLEC-2.Toxins (Basel) 3, 991–1003.

Yoshida, E., Fujimura, Y., Miura, S., Sugimoto, M., Fukui, H., Narita, N.,Usami, Y., Suzuki, M., Titani, K., 1993. Alboaggregin-B and botro-cetin, two snake venom proteins with highly homologous aminoacid sequences but totally distinct functions on von Willebrandfactor binding to platelets. Biochem. Biophys. Res. Commun. 191,1386–1392.