Biochimie 107 (2014) 376e384

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Research paper

Isolation and characterization of four medium-size disintegrins from the venoms of Central American viperid snakes of the genera Atropoides, Bothrops, Cerrophidion and Crotalus Yamileth Angulo a, *, Adriana Castro a, Bruno Lomonte a, Alexandra Rucavado a,  María Gutie rrez a n Ferna ndez a, Juan J. Calvete b, Jose Julia a b

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos e, Costa Rica Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, Valencia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 July 2014 Accepted 12 October 2014 Available online 19 October 2014

Four disintegrins were isolated from the venoms of the Central American viperid snakes Atropoides mexicanus (atropoimin), Bothrops asper (bothrasperin), Cerrophidion sasai (sasaimin), and Crotalus simus (simusmin). Purifications were performed by reverse-phase HPLC. The four disintegrins have biochemical characteristics, i.e. molecular mass and location of Cys, which allow their classification within the group of medium-size disintegrins. All of them present the canonical RGD sequence, which determines their interaction with integrins in cell membranes. The disintegrins inhibited ADP and collagen-induced human platelet aggregation, with similar IC50s in the nM range. In addition, disintegrins inhibited the adhesion of an endothelial cell line and a melanoma cell line to the extracellular matrix proteins type I collagen, laminin, fibronectin, and vitronectin, albeit showing variable ability to exert this activity. This study expands the inventory of this family of viperid venom proteins, and reports, for the first time, disintegrins from the venoms of species of the genera Atropoides and Cerrophidion.  te  française de biochimie et biologie Mole culaire (SFBBM). All rights © 2014 Elsevier B.V. and Socie reserved.

Keywords: Disintegrins Snake venom Platelet aggregation Cell adhesion Integrins

1. Introduction Viperid snake venoms are complex mixtures containing many different proteins, both enzymatic and non-enzymatic [1]. Proteomic analysis of viperid venoms has revealed the presence of a limited number of protein families, with predominance of metalloproteinases (SVMPs), phospholipases A2 (PLA2s), and serine proteinases, in addition to other families such as L-amino acid oxidases, C-type lectin/lectin-like proteins, cysteine-rich secretory proteins (CRISPs), disintegrins, and vasoactive peptides, such as bradykinin-potentiating peptides, and other minor components [2]. SVMPs are key components of viperid snake venoms, and participate in the pathogenesis of envenomings owing to their ability to disrupt the microvasculature and induce hemorrhage [3]. SVMPs belong to the M12 family of reprolysins, and have been classified into three classes according to their domain structure: 1) P-I, 20e30 kDa enzymes containing the metalloproteinase domain * Corresponding author. Tel.: þ506 25117888; fax: þ506 22920485. E-mail address: [email protected] (Y. Angulo).

only; 2) P-II, 30e60 kDa proteins containing a disintegrin domain at the C-terminus; and 3) P-III, 60e100 kDa enzymes comprising an N-terminal metalloproteinase domain, followed by a disintegrinlike domain and a cysteine-rich domain [4]. Some P-III SVMPs present, in addition to the main chain, a smaller subunit constituted by a C-type lectin-like protein, linked to the main chain through disulfide bonds [4]. Various subclasses have been described in P-II and P-III SVMPs on the basis of post-translational cleavage processes and dimerization [4]. Many P-II SVMPs undergo a post-translational cleavage of the disintegrin domain from the main polypeptide chain, with the release of disintegrins [5,6]. Venom disintegrins have received a great deal of attention owing to their ability to bind to integrins in cell membranes, inducing a variety of effects which largely depend on the identity of the target integrin. In turn, integrins comprise a superfamily of receptors that play critical roles in the processes of cellecell adhesion and cell-matrix adhesion, platelet aggregation, inflammatory reactions, cell migration, angiogenesis, cell signaling, and others [7,8]. These proteins are heterodimers of transmembrane a and b subunits [9]. Various extracellular matrix and plasma proteins serve as ligands for integrins, such as fibronectin,

http://dx.doi.org/10.1016/j.biochi.2014.10.010  te  française de biochimie et biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2014 Elsevier B.V. and Socie

Y. Angulo et al. / Biochimie 107 (2014) 376e384

vitronectin, fibrinogen, laminin, some types of collagen, and von Willebrand factor. Several of these molecules interact with integrins through an internal sequence, which often comprises the tripeptide RGD [7] or the triple-helical GFOGER sequence in the major collagens [9]. In the case of endothelial cells, integrins are essential for their adhesion to the basement membrane, thus contributing to the integrity and stability of blood vessels [8]. Integrin a5b1 functions as a fibronectin receptor, whereas integrin a3b1 binds to laminin, collagen and fibronectin; integrin a2b1 is a collagen and laminin receptor, and integrin avb3 is a vitronectin receptor [10,11]. Integrin aIIbb3 occurs in the plasma membrane of platelets, where it contributes to platelet aggregation after the binding to fibrinogen or von Willebrand factor [12,13]. Snake venom disintegrins have been classified into four different groups according to their polypeptide length and the number of disulfide bonds [14,6]. The first group comprises the long disintegrins with 84 amino acids and 7 disulfide bonds [15]. The second group is formed by medium-size disintegrins containing about 70 amino acids and 6 disulfide bonds; the majority of disintegrins characterized belong to this group. The third group is composed of homodimeric and heterodimeric molecules. Dimeric disintegrins contain subunits of about 67 residues with 10 cysteines involved in the formation of 4 intra-chain disulfide bonds and 2 interchain cystine linkages [16e18]. The fourth group is constituted by short disintegrins composed of 41e51 residues and 4 disulfide bonds [19,20]. The binding of disintegrins to integrins is primarily mediated by a sequence of three amino acids located in a loop near the C-terminus [21e23]; such interaction precludes the binding of integrins to their physiological ligands in the extracellular matrix. The majority of disintegrins contain the sequence RGD which represents the ancestral integrin-recognition motif [6,22,24,25]. The disulfide bonds around the RGD sequence determine the formation of a loop [20,26e29]. Therefore both the amino acids adjacent to the RGD tripeptide and the location of disulfide bonds determine the conformation and, consequently, the affinity of the disintegrin for receptors [15,25,30,31]. RGD disintegrins bind to the aIIbb3 integrin on the platelet membrane, thus inhibiting platelet aggregation [14]; moreover, RGD blocks integrins a8b1, a5b1,avb1 and avb3. Some disintegrins contain variations in the RGD sequence, generated by at least three mutations, such as KGD, that inhibits aIIbb3 integrin, MGD and VGD, which bind to integrin a5b1, and WGD, that binds to a5b1 and avb3 integrins. In addition, MLD targets a4b1, a4b7, a3b1, a6b1, a7b1 and a9b1 integrins and is characteristic of heterodimeric disintegrins [23,31e35]. The conserved aspartate residue of these tripeptides might be responsible for the binding to the b subunit of integrins, whereas the other two residues determine the binding to the a subunit [36]. Another group of disintegrins, characterized by the sequences KTS and RTS, is comprised by short, monomeric molecules containing about 40 amino acids in its polypeptide chain; they bind a1b1 integrins [35,37e39]. The threonine present in the tripeptide KTS of the disintegrin obtustatin is key for the binding to a1b1 integrin [40]. It has been suggested that the RTS/KTS short disintegrins may have been recruited into the venom gland of Eurasian vipers independently of the canonical neofunctionalization pathway characteristic of the RGD disintegrins [41]. In addition to the well-characterized effect of inhibition of platelet aggregation, through the binding to aIIbb3 integrin, other activities of potential pharmacological applications have been described for RGD disintegrins. For example, interaction with integrin avb3 affects cell migration, with impact in angiogenesis, tumor metastasis, and atherosclerosis [42,43]. Various disintegrins have been shown to reduce experimental metastasis in melanomas [44,45], and others inhibit endothelial cell adhesion to the

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extracellular matrix [25]. Thus, the broad pharmacological scope of disintegrins underscores the need for a continuous exploration in search of novel disintegrins and the identification of their targets and pharmacological activities. The proteomes of venoms from Central American viperid species have been studied in the last years [46e53]. This intensive characterization has highlighted the presence of disintegrins in the venoms of several of these species, thus raising the possibility of isolating and characterizing novel members of this family of proteins from these venoms. In the present work we describe the isolation and structural and functional characterization of three novel disintegrins from the venoms of the Mesoamerican species Atropoides mexicanus, Cerrophidion sasai and Crotalus simus. In addition, the characterization of a disintegrin previously isolated from the venom of Bothrops asper [54] is now described. 2. Materials and methods 2.1. Venoms The venoms of A. mexicanus, B. asper, C. simus and C. sasai were obtained and pooled from adult specimens collected at various locations in Costa Rica and kept at the serpentarium of Instituto Clodomiro Picado, Universidad de Costa Rica. Immediately after extraction, the venoms were centrifuged to remove insoluble debris, lyophilized and stored at 20  C. 2.2. Purification of disintegrins Batches of 20 mg of freeze-dried venoms from A. mexicanus, B. asper, C. simus and C. sasai were dissolved in 250 mL of solution A (5% acetonitrile, 94.9% water and 0.1% trifluoroacetic acid). Insoluble material was discarded after centrifugation at 3000  g for 3 min, and the clear supernatants were fractionated by reversephase HPLC on a Vydac C-18 (250  10 mm; 5 mm particle size) using an Agilent Model 1200 chromatograph, eluting at a flow rate of 2 mL/min with a gradient that utilized 0.1% TFA and 5% acetonitrile in water (solution A) to 0.1% TFA in acetonitrile (solution B), as follows: 0e15% B over 30 min, 15e20% B to 42 min, 20e80 % B to 43 min, maintained at 80% B to 59 min, and 0% B to 60 min. Separations were monitored at 215 nm, and fractions were collected manually and dried using a Speed-Vac system (Thermo Scientific, Minneapolis, USA). HPLC-separated fractions were dissolved in water and their final protein concentration was determined with the “DC Protein Assay” (BioRad, California, USA) using bovine serum albumin as standard. 2.3. Homogeneity and molecular mass determination Homogeneity of the final preparations was evaluated by electrophoresis on a discontinuous triphasic polyacrylamide gel system [55], in the presence of sodium dodecylsulphate and tricine (SDSPAGE), with a 4% spacer gel, a 10% first separating gel, and a 16.5% second separating gel. A mixture of low molecular weight markers was included. Separations were performed with a constant voltage of 120 V for 1e2 h, and the proteins were visualized by staining with Coomassie Blue R-250. The molecular mass of each isolated disintegrin was determined by MALDI-TOF on a model 4800-Plus Proteomics Analyzer (Applied Biosystems). The proteins were mixed with an equal volume of a saturated solution of a-cyanohydroxycinnamic acid in 50% acetonitrile and applied (1 mL) onto an OptiTof-384 plate (ABSciex). After drying, the samples were analyzed in linear positive mode using 500 shots/spectrum and laser intensity of 4200, in the m/z range 2000 to 18,000. Calibration

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was performed using a mixture of standards (CalMix ABSciex) placed on the same plate. 2.4. Amino acid sequence The N-terminal sequences of the disintegrins were obtained by direct Edman degradation of the reduced and alkylated proteins in a Shimadzu sequencer PPSQ-33A. The position of the cysteines was confirmed by identification of carboxymethylated residues. Completion of the amino acid sequences was obtained by tandem mass spectrometry of peptides obtained by hydrolysis of the reduced and alkylated proteins after digestion with endopeptidases (trypsin, chymotrypsin). De novo sequencing of peptides was performed by MALDI-TOF-TOF in positive reflector mode, using acyanohydroxycinnamic acid as matrix, or by electrospray ionization MS/MS on a QTrap 3200 instrument (Applied Biosystems), under previously described conditions [56]. Amino acid sequences were deduced by manual interpretation of collision-induced dissociation spectra or with the aid of ProteinPilot v.4.0.8 (ABSciex). Amino acid sequence alignments with known disintegrins were performed using the BLASTp tool (http://blast.ncbi.nlm.nih.gov). 2.5. Inhibition of platelet aggregating activity To evaluate inhibition of platelet aggregation by disintegrins, platelet-rich plasma (PRP) was prepared by centrifugation, at 50  g for 15 min, of citrated blood from human healthy volunteers. Platelet counts were performed using a Sysmex XE automated equipment, and PRP was diluted with platelet poor plasma (PPP), prepared by centrifuging blood at 850  g for 30 min, to obtain a concentration of 3  108 platelets/mL. Aliquots of 225 mL of PRP were incubated at 37  C with various concentrations of each disintegrin (37, 75, 150 and 300 nM) for 5 min with stirring at 600 rpm. Then, platelet aggregation was initiated by the addition of 25 mL of the agonists (to achieve final concentrations of either 20 mM ADP or 10 mg/mL collagen), and monitored by the increase in light transmittance signal using a model AggRAM aggregometer (Helena Laboratories, Texas, USA) interfaced to a chart recorder during 5 min. PPP (225 mL) alone was utilized as a blank, whereas PRP (225 mL) incubated only with ADP or collagen, was utilized as positive control for aggregation. 2.6. Cell adhesion inhibition assays In order to assess the percentage of inhibition of cell adhesion by the disintegrins, a B16 melanoma (ATCC- CRL-6322) and a capillary endothelial cell line of murine origin (tEnd) [57] were used. The following adhesion proteins were immobilized on 96-well microtiter plates (Falcon) in phosphate buffered saline (PBS; 0.12 M NaCl, 0.04 M sodium phosphate buffer, pH 7.2) overnight at 4  C: 0.5 mg/ well fibronectin (MP Biomedicals, Ohio, USA), 1 mg laminin (Roche, Mannheim, Germany), 0.05 mg vitronectin (SigmaeAldrich), and 0.5 mg collagen I (MP Biomedicals). Wells were blocked with 5% bovine serum albumin (BSA) for one hour at 37  C. In parallel, the two cells lines were grown in 25 cm2 bottles using Dulbecco's Modified Eagle's Medium (DMEM, SigmaeAldrich), supplemented with 10% fetal calf serum (FCS) (SigmaeAldrich), 2 mM glutamine, 1 mM pyruvic acid, penicillin (100 U/mL), streptomycin (0.1 mg/mL), and amphotericin B (0.25 mg/mL), in a humidified atmosphere with 7% CO2 at 37  C. Subconfluent monolayers were treated for 5 min at 37  C with trypsin (1500 U/mL), containing 5.3 mM EDTA, and resuspended in DMEM with 1% FCS and 5% BSA. Then the cell density was quantified in a Neubauer chamber and adjusted to 5.0  105 cells/mL. Disintegrins, at concentrations of 1.25, 2.5, and 5 nM, were added to 200 mL of cell suspensions, and incubated for 1 h at 37  C.

Then, cells were added to the adhesion proteins-coated plates, and incubated for 2 h at 37  C. The wells were washed with a sterile solution of 1% BSA in PBS. Then, 25 mL/well of a fresh solution of 5 mg/ mL MTT in basic medium were added and incubated for 2 h at 37  C. Dimethylsulfoxide (SigmaeAldrich) was added to each well (100 mL), and absorbances were recorded in a dual wavelength microplate reader (Thermo Scientific) at 560 nm with reference at 690 nm filter. Reference control for 100% cell adhesion consisted of cells incubated without disintegrins. All analyses were performed in triplicate. 2.7. Statistical analyses The significance of the differences between mean values of experimental groups in the cell adhesion inhibitory assays was determined by ANOVA, followed by Tukey's test. Values of p < 0.05 were considered significant. 3. Results 3.1. Isolation and molecular masses of novel disintegrins Fig. 1 shows the RP-HPLC chromatograms obtained for the purification of disintegrins from the four venoms studied. In the case of B. asper (Fig. 1A), the peak corresponding to the disintegrin eluted at 41.0 min. Disintegrins in the venoms of C. simus and A. mexicanus eluted at 39.1 and 39.3 min, respectively (Fig. 1B and C). In the case of the venom of C. sasai, two peaks containing disintegrins eluted at 42.0 and 42.7 min, corresponding to isoforms of a disintegrin (Fig. 1D). Homogeneity of purified disintegrins was assessed by SDS-PAGE using a triphasic gel system. As estimated by this technique, all disintegrins had a molecular mass of ~8 kDa. The spectra obtained by MALDI-TOF evidenced the presence of isoforms with slightly different masses (Fig. 2), probably related to variations in the in the length of the final sequences resulting from the proteolytic processing of their precursors. The main masses of the disintegrins corresponded to 7473 (B. asper), 7106 (C. simus), 7450 (C. sasai), and 7342 (A. mexicanus). 3.2. Amino acid sequence of disintegrins The reconstruction of the amino acid sequences of the isolated disintegrins is shown in Fig. 3. The sequences of B. asper and C. sasai

Fig. 1. Purification of dimeric disintegrins. Reverse-phase HPLC separation of proteins from venoms of (A) Bothrops asper, (B) Crotalus simus, (C) Atropoides mexicanus and (D) Cerrophidion sasai. The peaks corresponding to disintegrins are labeled with asterisks.

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Fig. 2. Determination of the molecular mass of the disintegrins by MALDI-TOF mass spectrometry analysis in positive linear mode. Disintegrins from (A) Bothrops asper, (B) Crotalus simus, (C) Cerrophidion sasai (D) Atropoides mexicanus.

disintegrins were established using automated Edman degradation of the reduced and carboxymethylated proteins, and of peptides obtained after hydrolysis of the proteins with trypsin and chymotrypsin. In the case of C. simus disintegrin, the sequence was determined entirely by analyzing six peptides by mass spectrometry. Finally, the sequence of the disintegrin of A. mexicanus was partially determined through the analysis of tryptic fragments by mass spectrometry, leaving a probable 12 amino acid segment undetermined. The observation that disintegrins from B. asper, C. simus and C. sasai comprise 73, 71, and 72 residues, respectively, and that the partial sequence of A. mexicanus likely comprises 72 amino acids, indicates that the four disintegrins isolated belong to the medium-size disintegrin family. In agreement, the position of the twelve cysteine residues is also characteristic of this group of disintegrins [19,58]. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession numbers C0HJM3 (bothrasperin in B. asper), C0HJM4 (simusmin in C. simus), C0HJM5 (sasaimin in C. sasai). Fig. 4 shows the alignment of amino acid sequences of disintegrins purified from the venoms of a variety of viperid snake species; the Cys residues as well as the canonical RGD sequence are highlighted.

used as agonist, IC50s ranged from 56 to 66 nM for the four disintegrins (Table 1). 3.4. Inhibition of cell adhesion in vitro Fig. 5 shows the inhibition of endothelial cells (tEnd) adhesion to vitronectin, laminin, collagen I, and fibronectin, by different concentrations of the disintegrins. Although the binding to all substrates was inhibited by the disintegrins, highest inhibition was observed in the binding to vitronectin, as in all cases an inhibition of 70e80% was achieved at the concentrations used. In addition, inhibition of tEnd cells binding to collagen type I was observed for all disintegrins with percentages higher than 50% (Fig. 5). A lower extent of inhibition of the binding of tEnd cells to fibronectin and laminin was observed for the disintegrins, when compared to vitronectin and type I collagen. On the other hand, the inhibition by disintegrins of the adhesion of B16 melanoma cell line to vitronectin, laminin, collagen I and fibronectin is shown in Fig. 6. The four disintegrins had an inhibitory activity on the binding of melanoma cells to different proteins. Highest inhibition was observed in the cases of binding to vitronectin and laminin (Fig. 6). 4. Discussion

3.3. Inhibition of human platelet aggregation The four isolated disintegrins are potent inhibitors of ADP- and collagen-induced human platelet aggregation. Inhibitory concentrations 50% (IC50) for each disintegrin are shown in Table 1. IC50s of disintegrins from B. asper, C. simus and A. mexicanus are ~50 nM, when using collagen as agonist, while in the case of C. sasai disintegrin the IC50 was 100 nM. On the other hand, when ADP was

Many disintegrins have been isolated and characterized from snake venoms of species of the family Viperidae [6,14]. These studies have highlighted a complex pattern of structural and functional diversity which, in turn, has paved the way for the exploration of disintegrins as potentially useful lead compounds for the blockade of the binding of endogenous ligands to integrins. Despite the plethora of disintegrins described, the characterization

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Fig. 3. Amino acid sequences of disintegrins isolated from the venoms of (A) Bothrops asper, (B) Crotalus simus, (C) Cerrophidion sasai, and (D) Atropoides mexicanus. The sequences were established by automated Edman degradation of the protein and of overlapping peptides isolated by reverse-phase HPLC after digestion of the proteins with trypsin or chymotrypsin endopeptidases.

of disintegrins present in viperid taxa typical of Central America has received little attention, although their presence in many viperid venoms from this region has been demonstrated in proteomic studies [46,48,49,51,52]. In this work, four disintegrins have been isolated and characterized from the venoms of species classified in the genera Bothrops, Crotalus, Cerrophidion and Atropoides. The disintegrin from B. asper venom corresponds to bothrasperin, previously isolated by Pinto et al. [54]. Disintegrins are divided into five different groups according to the length of their polypeptide chain and the number of disulfide bonds [14,59]. The four disintegrins isolated in this study have molecular masses of about 8 kDa and comprise single polypeptide chains of about 70 amino acid residues. This observation, together with the pattern of Cys, indicates that these proteins belong to the medium-size group of disintegrins [14]. Other disintegrins belonging to this group are trigramin, albolabrin, elegantin, flavouridin, barbourin and rhodostomin [15]. Many disintegrins are characterized for having the canonical sequence RGD at the tip of a loop [14,30,60]. In addition, the specificity and affinity of disintegrins for various integrins is influenced by the residues located in the vicinity of this tripeptide, as well as by the characteristics of the C-terminal region, which is located nearby the loop [14]. The disintegrin from

B. asper venom presents Pro and Asn flanking the RGD sequence. This arrangement characterizes disintegrins showing high selectivity for avb3 and a5b1 present in many cell types [61]. These integrins have been associated with angiogenesis and are involved in vascular diseases [62]. In the case of C. sasai disintegrin, an Asp is present flanking the RGD sequence. This has been associated with selectivity to avb3 integrin [36]. On the other hand, Trp, present in this region in the disintegrins of C. simus and A. mexicanus venoms, is known to contribute to selectivity to the integrin aIIbb3 [61]. Thus, differences observed in the residues that flank the canonical RGD tripeptide in the disintegrin loop are likely to contribute to variable selectivity of these four disintegrins to different integrins. In agreement with the presence of RGD, the four disintegrins hereby described induced a potent inhibition of ADP- and collageninduced platelet aggregation, with IC50s in the nM range. The binding of these agonists to their receptors, i.e. integrin a2b1 and glycoprotein (GP) VI in the case of collagen, and ADP receptor in the case of ADP, induces a series of cellular changes that result in conformational modifications and exposure of the integrin aIIbb3, which binds to fibrinogen and fibronectin, hence inducing aggregation [63]. Thus, both agonists enhance the expression of aIIbb3 integrin, albeit through different receptors and pathways [64,65].

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Fig. 4. Amino acid sequence comparison of disintegrins. The one-letter code for amino acid nomenclature is used. Cysteine residues are shadowed in pale gray. RGD and non-RGD binding tripeptide motifs are underlined and shown in bold.

This action, characteristic of many disintegrins, might play a role in platelet hypoaggregation in vivo, with the possible potentiation of SVMP-induced hemorrhagic activity. Likewise, such inhibitory activity might find applications in the search for novel antithrombotic agents, as in the case of the disintegrin tablysin-15 [66]. The ability of disintegrins to bind several types of integrins has prompted the analysis of their inhibition of adhesion of various cell types to substrates composed by different extracellular matrix proteins. In this study we have explored the inhibition of the adhesion of an endothelial cell line and a melanoma cell line to type I collagen, laminin, fibronectin and vitronectin. Regarding endothelial cells, highest inhibition of adhesion to substrates by the four disintegrins was observed with vitronectin and collagen, although an effect on the binding to laminin and fibronectin was also observed. Noteworthy, B. asper disintegrin exerted a higher inhibition of binding to fibronectin, when compared to the other disintegrins. This might be related to a higher affinity of this disintegrin to integrins a5b1 and aVb3, since these are fibronectin receptors [20]. On the other hand, the sequence RGDNP, present in B. asper disintegrin, has been associated with inhibition of binding to endothelial cell integrin [61]. Table 1 Inhibitory concentrations 50% (IC50 nM) of disintegrins on human platelet aggregation. Agonist

B. asper

C. sasai

C. simus

A. mexicanus

ADP (20 mM) Collagen (10 mg/mL)

59 49

66 100

56 49

63 53

IC50 value is the disintegrin concentration (nM) that induces 50% inhibition of ADPor collagen-induced platelet aggregation, as compared to controls in which agonists were added to platelets in the absence of disintegrins (see Materials and methods for details).

In the case of B16 melanoma cell line, disintegrins inhibited adhesion to the four extracellular matrix proteins studied, showing highest inhibition when using laminin and vitronectin as substrates. B16 melanoma cells express multiple receptors to extracellular matrix proteins, such as fibronectin, laminin and collagen. In agreement, the integrin a6b1, which is largely responsible for binding to laminin, is highly expressed in metastatic B16 cells [67]. The strong inhibition of adhesion of the two cell lines to vitronectin might be associated with the ability of disintegrins to block aVb3 integrin [20]. Thus, subtle differences in the affinity of these disintegrins to the various integrin molecules in the plasma membrane of endothelial cells or melanoma cells are likely to determine the variable ability of these venom proteins to inhibit adhesion to various extracellular matrix molecules. This may have implications in terms of affecting cellular functions which depend on the interaction of integrins to these matrix molecules, a hypothesis that needs to be tested by analyzing inhibition of cellular movement in matrices. The fact that these four RGD disintegrins inhibit the adhesion of both cell lines to collagen and laminin is noteworthy, since these extracellular matrix proteins do not have exposed RGD sequences. This finding is likely to be due to the fact that preparations of laminin and collagen are partially denatured during isolation. In the case of collagen, it has been described that the denaturation of the triple-helix can lead to the exposure of cryptic RGD motif, thus explaining the binding of this protein to a5b1 and av-integrins [68]. In conclusion, four novel medium-size disintegrins harboring the canonical RGD sequence have been isolated from the venoms of the Central American viperids A. mexicanus, B. asper, C. simus and C. sasai. The proteins inhibit human platelet aggregation in nanomolar range concentrations and are also able to inhibit, at varying degrees, the adhesion of endothelial and melanoma cells to type I collagen, laminin, fibronectin and vitronectin. These disintegrins should be

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Fig. 5. Inhibition of endothelial cell adhesion (t-End) to fibronectin, laminin, vitronectin and type I collagen. Cells were treated with various concentrations of disintegrins of A. mexicanus (A), B. asper (B), C. simus (C) and C. sasai (D) prior to seeding and were allowed to adhere for 2 h at 37  C. Reference control for 100% cell adhesion consisted of cells incubated without disintegrins. Results are presented as mean ± SD (n ¼ 3). Values within each disintegrin concentration having different superscripts are significantly (p < 0.05) different between them.

Fig. 6. Inhibition of melanoma cell adhesion (t-End) to fibronectin, laminin, vitronectin and type I collagen. Cells were treated with various concentration of disintegrins of A. mexicanus (A), B. asper (B), C. simus (C) and C. sasai (D) prior to seeding, and were allowed to adhere for 2 h at 37  C. Reference control for 100% cell adhesion consisted of cells incubated without disintegrins. Results are presented as mean ± SD (n ¼ 3). Values within each disintegrin concentration having different superscripts are significantly (p < 0.05) different between them.

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Isolation and characterization of four medium-size disintegrins from the venoms of Central American viperid snakes of the genera Atropoides, Bothrops, Cerrophidion and Crotalus.

Four disintegrins were isolated from the venoms of the Central American viperid snakes Atropoides mexicanus (atropoimin), Bothrops asper (bothrasperin...
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