©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Substitutions of Proline 42 to Alanine and Methionine 46 to Asparagine around the RGD Domain of the Neurotoxin Dendroaspin Alter Its Preferential Antagonism to That Resembling the Disintegrin Elegantin (*)

(Received for publication, August 7, 1995; and in revised form, October 5, 1995)

Xinjie Lu (§) Salman Rahman (¶) Vijay V. Kakkar Kalwant S. Authi

From the Platelet Section, Thrombosis Research Institute, London SW3 6LR, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that the neurotoxin dendroaspin and the disintegrin kistrin, which show little overall sequence homology but similar residues around RGD (PRGDMP), preferentially inhibited platelet adhesion to fibrinogen. In contrast, the elegantin which has different amino acids around RGD (ARGDNP) preferentially inhibited platelet adhesion to fibronectin. To investigate further the role of amino acids around RGD in disintegrins, we have constructed the genes of a wild-type and of two mutant dendroaspins with substitutions around the RGD, namely [Asn]- and [Ala,Asn]dendroaspins. Proteins were expressed in Escherichia coli as glutathione S-transferase fusion recombinants and purified to homogeneity by affinity chromatography and reversed phase high performance liquid chromatography. Platelet aggregation studies revealed that wild-type dendroaspin showed an IC value similar to that of native dendroaspin, with [Ala,Asn]dendroaspin showing an IC value similar to that of elegantin. Interestingly, in platelet adhesion assays, the mutants showed a progressive shift in inhibitory preference, in particular, [Ala,Asn]dendroaspin showed nearly identical behavior as elegantin when fibronectin was the immobilized ligand (IC = 0.33 µM and 0.6 µM, respectively, compared with 20 µM for native dendroaspin). Native and recombinant wild-type dendroaspin bound to a single class of binding site exhibiting a K = 67 nM; [Asn]- and [Ala,Asn]dendroaspins, however, both produced biphasic isotherms with Kvalues = 87 nM and 361 nM for [Asn]dendroaspin and 33 nM and 371 nM for [Ala,Asn]dendroaspin, which are close to those of elegantin (K values = 18 nM and 179 nM). These studies prove that the amino acids flanking RGD provide an extended locus that regulate the affinity and selectivity of RGD protein dendroaspin.


INTRODUCTION

Integrins are a family of cell surface receptors that mediate adhesion of cells to each other or to extracellular matrix substrate (1, 2, 3, 4, 5) . They are composed of noncovalently associated alpha and beta transmembrane subunits selected from among 16 alpha and 8 beta subunits that heterodimerize to produce 20 receptors(6) . Among the integrins, the platelet membrane alphabeta(3) is the best characterized(3, 5) . Upon cell activation, the alphabeta(3) integrin binds several glycoproteins, predominantly through the Arg-Gly-Asp (RGD) tripeptide sequence (6, 7, 8) present in fibrinogen(9) , fibronectin(10) , von Willebrand factor(11) , vitronectin(12) , and thrombospondin(13) . The nature of the interactions between these glycoprotein ligands and their integrin receptors is known to be complex, and conformation changes occur in both the receptor (14) and the ligand(15) .

Recently, many proteins from a variety of snake venoms have been identified as potent inhibitors of platelet aggregation and integrin-dependent cell adhesion. The majority of these proteins which belong to the disintegrin family share a high level of sequence homology, are small (4-8 kDa), cysteine-rich, and contain the sequence RGD (16) or KGD(17) . In addition to the disintegrin family, a number of non-disintegrin RGD proteins of similar inhibitory potency, high degree of disulfide bonding, and small size have been isolated from both the venoms of the Elapidae family of snakes (18, 19) and leech homogenates(20) . All of these proteins are approximately 1000 times more potent inhibitors of the interactions of glycoprotein ligands with the integrin receptors than simple linear RGD peptides, a feature that is attributed to an optimally favorable conformation of the RGD motif held within the protein scaffold. The NMR structures of several inhibitors including kistrin(21, 22, 23) , flavoridin(24) , echistatin(25, 26, 27, 28) , albolabrin(29) , decorsin(30) , and dendroaspin (^1)(31, 32) have been reported, and the only common structural feature elucidated so far is the positioning of the RGD motif at the end of a solvent exposed loop, a characteristic of prime importance to their inhibitory action.

Recent studies have implied a role for the amino acids around the tripeptide RGD in regulating the ligand binding specificity shown by snake venom proteins. Scarborough et al.(33) examined a range of disintegrins and observed that those containing RGDW were very effective at inhibiting the interactions of fibrinogen to purified alphabeta(3) but not of vitronectin and fibronectin to purified alpha(v)beta(3) and alpha(5)beta(1), respectively, whereas the converse was true for disintegrins containing the sequence RGDNP. Other regions of amino acid sequence divergence may also be contributory(33) . We have reported that dendroaspin, a short chain neurotoxin analogue containing the RGD sequence, and the disintegrin kistrin, which show little overall sequence homology but have similar amino acids flanking the RGD sequence (PRGDMP), are both potent inhibitors of platelet adhesion to fibrinogen but poor antagonists of the binding of platelets to immobilized fibronectin(34) . In contrast, elegantin, which has 65% sequence homology to kistrin but markedly different amino acids around RGD (ARGDNP), preferentially inhibited platelet adhesion to fibronectin as opposed to fibrinogen and binds to an allosterically distinct site on alphabeta(3) complex. These studies suggested that the amino acids around the RGD determine the affinity and selectivity of these RGD proteins. In addition to RGD domains, a number of recent studies have suggested that amino acids at the carboxyl terminus of these proteins may affect their interactions with integrins. Deletion of the PRNP sequence from echistatin has been reported to reduce its ability to inhibit platelet aggregation, implying a reduction in the binding affinity(16) . Furthermore, the complete carboxyl-terminal peptide (PRNPHKGPAT) of echistatin not only competed with the binding of echistatin to the alphabeta(3) complex but also enhanced the binding of fibronectin and vitronectin to the purified alphabeta(3), indicating that this non-RGD component of the protein was able to alter the integrin affinity for glycoprotein ligands(35) . However, the mechanism by which amino acids at the carboxyl terminus of these proteins interact with their receptors and their binding characteristics are not yet understood.

In this study we examined the role of amino acids flanking the RGD sequence by expressing the neurotoxin variant dendroaspin in Escherichia coli and using site-directed mutagenesis. Dendroaspin, unlike echistatin, does not have any appreciable sequence at its carboxyl-terminal after Cys making it an excellent model to study the functional role of the nature of the amino acids flanking the sequence RGD. We show that recombinant dendroaspin has inhibitory properties identical with native dendroaspin indicating that the expressed protein has the correct folding and disulfide bonding and that substituting Met Asn (PRGDMP PRGDNP) or Met Asn and Pro Ala (PRGDMP ARGDNP) dramatically altered the preferential inhibitory properties and binding characteristics of the protein to the alphabeta(3) complex to that of the disintegrin elegantin containing the sequence ARGDNP. These studies prove that the amino acid flanking sequence RGD provide an extended locus that determines the preferential selectivity of dendroaspin.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, isopropyl-1-thio-beta-D-galactopyranoside, and DH5alpha competent cells were purchased from Life Technologies Ltd. (Paisley, UK) or Promega Ltd. (Southampton, UK). Vent (exo-) DNA polymerase was supplied by New England Biolabs Ltd. (Hitch, UK). Proteinase Factor Xa was purchased from Boehringer Mannheim (Sussex, UK). Human fibrinogen (grade L) was purchased from Kabi (Stockholm, Sweden). Human fibronectin was supplied by Bioproducts Laboratories (Herts, UK). Lyophilized snake venoms were obtained from either Latoxan (Rosans, France) or Sigma Ltd. (Dorset, UK). The monoclonal reagents PM6/248 and PM6/13, which have specificities for the native alphabeta(3) complex and beta(3) subunit, respectively, have been described previously(36) . Oligonucleotides were made either in King's College School of Medicine and Dentistry (London, UK) or in Cruachem Ltd. (Glasgow, UK) and further purified by denaturing polyacrylamide gel electrophoresis on a 15% acrylamide, 8 M urea gel. Deoxynucleotide triphosphates (dNTPs), dideoxynucleotide triphosphates (ddNTPs), and plasmid pGEX-3X, a vector that expresses a cloned gene as a fusion protein linked to glutathione S-transferase (GST), and glutathione-Sepharose CL-4B were purchased from Pharmacia Biotech Ltd. (Herts, UK). The ``Geneclean'' kit and plasmid maxi kit were purchased from Bio 101 (La Jolla, CA) and Qiagen Ltd. (Surrey, UK), respectively. The sequencing kit (Sequenase 2.0) was obtained from Cambridge Bioscience (Cambridge, UK). [S]dATPalphaS and I (15.3 mCi/µg iodine) were supplied by Dupont NEN (Herts, UK) and Amersham International Plc (Amersham, Bucks, UK), respectively.

Construction of Synthetic Dendroaspin and Mutant Dendroaspin Genes and the Expression Vectors

On the basis of the known amino acid sequence of dendroaspin(18, 19) , we designed a synthetic dendroaspin gene and mutant dendroaspin genes with high codon usage in E. coli(37) utilizing 5 oligonucleotides each for the coding and the complementary strands. The two strands of the dendroaspin gene were expanded by 7 codons at the 5` end to generate a BamHI restriction site and by 6 codons at the 3` to produce an EcoRI restriction site, thereby allowing directional cloning. In addition, two oligonucleotides were designed to contain the nucleotide sequence coding for the tripeptide RGD and flanking amino acid residues, allowing introduction of specific replacement codons for the production of mutant dendroaspin variants. Each purified oligonucleotide was phosphorylated at 37 °C for 60 min in the presence of 1 mM ATP and T4 polynucleotide kinase. Each pair of overlapping phosphorylated oligonucleotides were annealed separately on a Perkin Elmer thermal cycler. The following program was used: 95 °C, 5 min, 70 °C, 30 s, then slowly cooling to room temperature. Ligation was performed at 16 °C for 15 h in a total volume of 50 µl containing approximately 1 nM concentration of each annealed fragment, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl(2), 1 mM ATP, and 5% PEG 8000 and 5 units of T4 ligase. After ligation, the gene of dendroaspin (or mutant dendroaspin) was amplified by polymerase chain reaction, using 1 µl of ligation mixture as a template, 1 µl of two 5`-overhanging oligonucleotides as primers, and 2 units of vent polymerase. The following program was applied: one cycle of 3 min at 94 °C and 1 min at 72 °C, followed by 39 cycles of 30 s at 94 °C, and 2 min at 72 °C. The amplification product was checked and found to be of the expected size (216 bp) as ascertained on a 2% agarose gel and further purified on a 2% low-melting-point agarose gel. The gene of dendroaspin (or of mutant dendroaspins) was digested with EcoRI and BamHI and then cloned into the restricted vector pGEX-3X at the carboxyl terminus of the glutathione S-transferase (GST) gene. The Factor Xa cleavage sequence was positioned 5` to the gene coding for the recombinant proteins to produce recombinant plasmid pGEX-dendroaspin gene and pGEX-mutant-dendroaspin gene. The correct orientation and sequence of the genes of dendroaspin and mutant dendroaspins were confirmed by DNA sequencing using the method of Sanger et al.(38) .

Transformation and Protein Expression

The cloned vector was used to transform 50 µl of E. coli DH5alpha competent cells by standard methods(39) . Bacterial culture was carried out as follows; the culture was inoculated with an overnight seed culture (1%, v/v) and grown in LB/ampicillin medium (100 µg/ml) and shaken at 37 °C until it reached an A of 0.7, then isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.1 mM for induction. The cells were grown for an additional 4 h at 30 °C and harvested by centrifugation. In contrast to noninduced transformants, analysis of isopropyl-1-thio-beta-D-galactopyranoside-treated cell lysates by SDS-polyacrylamide gel electrophoresis showed an emergence of a 32-kDa protein corresponding to the GST-fusion protein.

Purification of Elegantin, Dendroaspin, Recombinant Wild-type Dendroaspin, and Mutant Dendroaspins

Elegantin and dendroaspin were purified using reverse-phase HPLC as described previously(40) . Recombinant dendroaspins were purified as follows: the cell pellets were suspended in PBS buffer (pH 7.4) containing 1% Triton X-100 and the protease inhibitors phenylmethylsulfonyl fluoride (1 µM), pepstatin (5 µg/ml), aprotinin (5 µg/ml), trypsin inhibitor (1 µg/ml), 1 mM EDTA, and sonicated on ice. The sonicated mixture was centrifuged at 7,800 times g at 4 °C for 10 min to pellet the cell debris and insoluble material. Recombinant GST-dendroaspin and GST-mutant-dendroaspins from supernatants were purified by affinity chromatography on glutathione-Sepharose CL-4B columns by adsorption in PBS containing 150 mM NaCl and elution with 50 mM Tris-HCl containing 10 mM reduced glutathione (pH 8.0). Elution of the absorbed material with glutathione resulted in the appearance of a major band migrating at 32 kDa (GST-dendroaspin fusion protein) in 12.5% polyacrylamide gels. The appropriate fractions comprising the 32-kDa fusion proteins were then digested in the presence of 150 mM NaCl, 1 mM CaCl(2), and Factor Xa (1:100, w/w, Factor Xa:fusion protein) at 4 °C for 24 h. Treatment of the purified GST-proteins with Factor Xa released recombinant proteins migrating as 7-kDa bands, approximating the size of dendroaspin, and free GST appearing as an intensification of a 28-kDa band on SDS-polyacrylamide gel electrophoresis. The digested mixture was loaded onto a Vydac C(18) reverse-phase HPLC analytical column (TP104) and eluted with a linear gradient of 0-26% acetonitrile (1.78% per min) containing 0.1% trifluoroacetic acid, followed by 26-36% acetonitrile in 0.1% trifluoroacetic acid (0.25% per min). When necessary, further analytical columns were run under the same conditions. The fractions from HPLC were freeze-dried, dissolved in water, and assayed for inhibition of ADP-induced platelet aggregation.

Electrospray Ionization Mass Spectrometry of the Dendroaspin Proteins

Electrospray ionization mass spectroscopy was used to determine the molecular sizes of native and mutant dendroaspins. Samples (approximately 50 pmol) were lyophilized and dissolved in 20 µl of acetonitrile/water (1:1). Ion-mass spectral analysis was performed with a SSQ 710 mass analyzer (Finnigan Mat, UK) using an injection rate of 5 µl/min. There were observed molecular masses of 6746.0 ± 2 for native dendroaspin, 6746.6 ± 2 for recombinant wild-type dendroaspin, 6728.2 ± 2 for [Asn]dendroaspin and 6704.2 for [Ala,Asn]dendroaspin. These are in good agreement with the molecular masses of 6745.66, 6745.66, 6728.5, and 6702.4 (respectively) calculated from their complete amino acid sequences on the basis that all cysteinyl residues participate in intrachain disulfides.

Measurement of Platelet Aggregation

Platelet aggregation was measured by the increase in light transmission as described previously(34, 40) . Briefly, platelet-rich plasma was prepared from citrated human blood obtained from healthy individuals by centrifugation at 200 times g for 15 min. Washed platelets were prepared from platelet-rich plasma and resuspended in adhesion/aggregation buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl(2), 2 mM CaCl(2), 10 mM glucose, 3.5 mg/ml bovine serum albumin, and 10 mM HEPES, pH 7.35) and adjusted to a count of 3 times 10^8/ml. Platelet aggregation (320-µl incubations) was induced with 10 µM ADP in the presence of 1.67 mg/ml fibrinogen and measured using a Payton dual-channel aggregometer linked to a chart recorder.

Measurement of Platelet Adhesion

Platelet adhesion was measured as described previously(34) . Briefly, 96-well plates were coated overnight at 4 °C with either human fibrinogen or fibronectin reconstituted in PBS (pH 7.4) at appropriate concentrations (2-10 µg/ml, 100 µl). Platelets were treated with antagonists at appropriate concentrations for 3 min before the addition (90 µl) to the microtiter plates which were preloaded with 10 µl of 500 µM ADP. After 60 min, the number of adherent platelets was determined by measurement of platelet acid phosphatase activity using 130 µl of the developing buffer (sodium acetate, pH 5.5, 10 mMp-nitrophenyl phosphate, 0.1% Triton X-100)/well, and the absorbance values were read at 410/630 nm on an automated plate reader(34) .

Iodination of Ligands and Ligand Binding Studies

Iodination of all proteins used in this study was performed using Enzymobead Radioiodination Reagent (Bio-Rad Laboratories, Ltd., Hertfordshire, UK) according to the manufacturer's specifications. The binding of I-labeled disintegrins, dendroaspin, and mutant dendroaspins to washed platelets was performed under equilibrium conditions essentially as described previously(34) . Briefly, the incubation mixture was composed of 300 µl of washed platelets (3 times 10^8/ml), 10 µl of agonist (1.75 mM ADP giving a final concentration of 50 µM), 10 µl of I-labeled protein samples, 5-20 µl of resuspension buffer and made to a final volume of 350 µl. In antibody inhibition studies, platelet suspensions were treated with antibody for 30 min prior to exposure to ADP and then added to I-protein samples, and the mixture was incubated at room temperature for an additional 60 min. Incubations were terminated by loading onto a 25% (w/v) sucrose, 1% bovine serum albumin cushion and centrifugation at 12,000 times g for 10 min. Both platelet pellets and supernatants were counted to determine the levels of bound and free ligand. Background binding levels were determined in the presence of a 50-fold excess of unlabeled disintegrin or 10 mM EDTA.


RESULTS AND DISCUSSION

Design and Functional Characterization of Recombinant Wild-type and Mutant Dendroaspins

We had previously noted that dendroaspin and kistrin share similar amino acid residues at positions flanking the tripeptide RGD and speculated that this sequence similarity underpinned the similar functional and binding characteristics of these two structurally unrelated snake venom inhibitors (Table 1, (34) ). To test this hypothesis, in the present study, we generated dendroaspin variants with specific substitution of the residues at positions flanking RGD to those residues present in elegantin (Table 1), a well-defined disintegrin with functional and binding characteristics to the alphabeta(3) complex distinct from those of kistrin and dendroaspin.



The functional characterization of the recombinant wild-type and of the mutant dendroaspins purified from cell lysates of E. coli was determined by platelet aggregation and adhesion assays. In order to verify that the expression system generated was satisfactory, we first compared the functional properties of recombinant dendroaspin with that of native dendroaspin purified from snake venom. As shown in Table 2, recombinant dendroaspin showed platelet aggregation inhibition as potent as native dendroaspin and displayed similar inhibitory activity toward ADP-induced platelet aggregation both in platelet-rich plasma and washed platelets. This indicated that the protein folded correctly and formed the correct disulfide bondings. The mutant [Asn]dendroaspin showed an IC value similar to that of recombinant dendroaspin, while the mutant with two substitutions, [Ala,Asn]dendroaspin, showed an IC value similar to that observed with elegantin (Table 2).



We previously observed that measurement of ADP-activated platelet adhesion to immobilized glycoproteins highlights selective inhibitory preferences for RGD snake venom proteins that are less easily discernible using the platelet aggregation assay(34, 39) . In such experiments, we have shown that dendroaspin and kistrin are potent inhibitors of platelet adhesion to fibrinogen, whereas elegantin preferentially inhibits platelet adhesion to fibronectin(34) . Fig. 1and Table 3(showing IC values) illustrate the results obtained with the wild-type recombinant and mutant dendroaspins in comparison with the inhibitory properties of native dendroaspin and elegantin. Wild-type dendroaspin showed an identical inhibitory profile toward the inhibition of platelet adhesion to fibrinogen compared to native dendroaspin. Interestingly, the mutants [Asn]dendroaspin and [Ala,Asn]dendroaspin exhibited a progressive decrease in their ability to inhibit platelet adhesion to fibrinogen in both maximal inhibitory levels (using a maximum of 5 µM protein) and IC values. Indeed, appropriate substitutions at both flanking positions, i.e. [Ala,Asn]dendroaspin, showed an 8-fold lower IC that approached the value obtained with elegantin. Studies using fibronectin as the immobilized ligand showed an even more striking change with respect to antagonistic preference. Both recombinant and native dendroaspins were relatively poor inhibitors of activated platelet adhesion to fibronectin displaying only 40% inhibition at 15 µM. However, the singly substituted Met Asn of dendroaspin has a similar IC for platelet adhesion to both fibrinogen and fibronectin, whereas the doubly substituted (Pro Ala and Met Asn) mutant shows an approximately 4-fold preference for inhibition of binding to fibronectin. In particular, the latter showed a maximal extent of inhibition and IC values that were markedly similar with those displayed by elegantin. Thus, substituting Pro Ala and Met Asn in the residues immediately flanking the RGD in dendroaspin altered the inhibitory preferences of dendroaspin to that of elegantin. The presence of asparagine adjacent to aspartic acid would be particularly important in inhibiting the interactions of fibronectin with its receptor. This study strongly supports our previous studies(34) .


Figure 1: Inhibition of platelet adhesion to immobilized glycoproteins by RGD-containing proteins. Washed platelet suspensions were incubated with various concentrations of native dendroaspin (), recombinant wild-type dendroaspin (up triangle), [Asn]dendroaspin (), [Ala,Asn]dendroaspin (bullet), or elegantin (box) for 3 min prior to application to microtiter wells coated with either 10 µg/ml fibrinogen (A) or fibronectin (B). The number of adherent platelets were determined by measurement of endogenous acid phosphatase as described previously(34) . Results are expressed as percent inhibition relative to the number of adherent platelets observed in the absence of inhibitors. All points were performed in quadruplicate, and the mean ± S.E. are shown in Table 3(n = 2-4).





Binding of I-labeled recombinant and mutated dendroaspins and of I-elegantin to activated platelets was studied to determine whether the alterations in functional properties of the mutated dendroaspins were reflected in their binding characteristics. All four I-labeled proteins bound to ADP-activated platelets in a saturable and cation-dependent manner (Fig. 2, insets). Scatchard analysis of the data using the Kinetic, EBDA, Ligand, and Lowry version 4 software programs (BIOSOFT, Cambridge, UK) indicated that recombinant dendroaspin bound to a single class of binding site exhibiting a K(d) = 67 nM (Fig. 2A and Table 3) with a B(max) equal to approximately 29,100 sites per platelet. The [Asn]- and [Ala,Asn]dendroaspin, however, both produced biphasic isotherms again using the Kinetic, EBDA, Ligand, and Lowry version 4 software with K(d) values = 87 nM and 361 nM for [Asn]dendroaspin (Fig. 2B and Table 3) and 33 nM and 371 nM for [Ala,Asn]dendroaspin (Fig. 2C and Table 3). In agreement with the results obtained with the adhesion experiments, the mutant dendroaspins showed a progressive shift in their binding characteristics toward those of elegantin (K(d) values = 18 nM and 179 nM) as shown in Fig. 2D and Table 3. Considering that [Ala,Asn]dendroaspin and elegantin share little sequence homology ( Table 1and Table 2) and have structures unrelated, except for the (A)RGD(N) domain, the close similarity of the dissociation constants is striking.


Figure 2: Scatchard analysis of binding of I-labeled-proteins to ADP treated platelets. Scatchard analysis was performed with the RADLIG (radioligand) software version 4 of Kinetic, EBDA, Ligand, and Lowry (BIOSOFT, Cambridge, UK). Varying concentrations of I-labeled recombinant wild-type dendroaspin (A; one-site fit, R^2 = 0.990), I-labeled [Asn]dendroaspin (B; two-site fit, R^2 = 0.999 and 0.999, respectively), I-labeled [Ala,Asn]dendroaspin (C; two-site fit, R^2 = 0.996 and 0.983, respectively), and I-labeled elegantin (D; two-site fit, R^2 = 0.990 and 0.997, respectively) were incubated with ADP-treated, washed platelets (3 times 10^8/ml) for 30 min at room temperature in a volume of 350 µl. Bound and free levels of I-labeled RGD-containing proteins were determined by loading 300 µl of the platelet suspension onto a cushion of 25% (w/v) sucrose, 1% bovine serum albumin and centrifuged for 10 min at 12000 times g. Both the radioactivity in the platelet pellets and supernatants were determined. Insets, saturation isotherms of I-labeled RGD-containing proteins binding to ADP-treated washed platelets. The curves show nonspecific (up triangle), specific (box), and total (bound + free) binding (down triangle). The values are representive of three similar experiments with all points performed in duplicate. (S.E. were less than 10%.)



To confirm that both binding sites occupied by the two dendroaspin mutants on ADP-treated platelets were present on the alphabeta(3) integrin complex, the effects of two inhibitory antibodies on radioligand binding were monitored. PM6/248, a monoclonal antibody with specificity for the native alphabeta(3) complex(36) , effectively inhibited in a dose-related manner the binding of all these I-labeled recombinant dendroaspins by 80-100% (Fig. 3). In contrast, an anti-alpha(5)beta(1) antibody was comparatively ineffective, confirming that the binding parameters observed were specifically associated with the alphabeta(3) complex.


Figure 3: Inhibition of I-labeled proteins to ADP-treated platelets by antibodies. Platelet suspensions were incubated with various concentrations of the alphabeta(3) specific monoclonal antibody PM6/248 (filled symbols) or a polyclonal antibody raised against the integrin alpha(5)beta(1) (open symbols) for 30 min before addition of I-labeled recombinant dendroaspin (, down triangle), I-labeled [Asn]dendroaspin (, ), I-labeled [Ala,Asn]dendroaspin (, up triangle), and I-labeled elegantin (, box) at 30 nM for monoclonal antibody PM6/248 and 170 nM for the polyclonal antibody raised against the integrin alpha(5)beta(1) and ADP. Platelet suspensions were then incubated at room temperature for an additional 60 min. Levels of bound I-labeled proteins were determined as described in the legend to Fig. 2. The results are expressed as percent inhibition.



Further evidence for the close similarity in the binding of [Ala,Asn]- and [Asn]dendroaspin and of elegantin to the alphabeta(3) complex was obtained by examining the association kinetics of three ligands (Fig. 4). Native and recombinant dendroaspin show simple and rapid binding, reaching equilibrium by 5 min. However, elegantin, [Asn]- and [Ala,Asn]dendroaspin showed complex association kinetics with approximately 3- to 4-fold higher binding before equilibrium than at equilibrium. The reasons for this complex association pattern are not known at present, but are not due to internalization of the ligand as the binding was fully reversible (data not shown). That [Asn]- and [Ala,Asn]dendroaspin, but not native dendroaspin, behaved in this manner points to this property being solely due to the presence of the ARGDN sequence, and whether other ARGDN-containing disintegrins, e.g. viridin, jararacin, cotiarin (Table 1), behave in a similar manner remains to be examined.


Figure 4: Association kinetics of I-labeled proteins to ADP-treated platelets. I-labeled [Asn]dendroaspin (up triangle), I-labeled [Ala,Asn]dendroaspin (box), I-labeled dendroaspin (), I-labeled recombinant dendroaspin (), and I-labeled elegantin () (all at 40 nM) were incubated with ADP-treated platelets for various lengths of time before determination of the bound and free ligand concentrations as described previously. Results are expressed as percent ligand bound relative to bound levels at equilibrium. All data shown are either a representive experiment or a compilation of two independent determinations with points performed in duplicate.



These studies report that the amino acids around the RGD motif regulate the affinity and selectivity of the RGD protein dendroaspin and support our earlier studies (34) and those of Scarborough et al.(33) . Further details of the mechanisms of integrin/ligand interactions will benefit greatly from the analysis of both wild-type and mutant dendroaspins by x-ray crystallography or NMR spectroscopy. Until the receptor-ligand complexes are available for such structural studies, the further structure/function evaluation of snake venom adhesive ligands may allow us to engineer potent antagonists that show not only ligand specificity but also receptor specificity.


FOOTNOTES

*
This work was supported by the Thrombosis Research Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Platelet Section, Thrombosis Research Institute, Manresa Rd., London SW3 6LR, United Kingdom.

Present address: Coagulation Research Laboratory, Haemophilia Centre, St. Thomas' Hospital, Lambeth Palace Rd., London SE1 7EH, U.K.

(^1)
The abbreviations used are: dendroaspin, Dendroaspis jamesonii kaimose platelet aggregation inhibitor; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; GST, glutathione S-transferase; ATPalphaS, adenosine 5`-O-(1-thiotriphosphate).


ACKNOWLEDGEMENTS

We are grateful to Drs. E. I. Hyde, F. Lupu, and V. Ellis for valuable discussion, Drs. David S. Millar and Deborah A. White for technical assistance, and Dr. John D. Deadman and Emmanuel Skordlakes for mass spectrometry.


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