Mapping the binding domains of the a IIb subunit A study performed on the activated form of the platelet integrin a IIb b 3 Nikolaos Biris 1 , Morfis Abatzis 1 , John V. Mitsios 1 , Maria Sakarellos-Daitsiotis 1 , Constantinos Sakarellos 1 , Demokritos Tsoukatos 1 , Alexandros D. Tselepis 1 , Lambros Michalis 2 , Dimitrios Sideris 2 , Georgia Konidou 3 , Ketty Soteriadou 3 and Vassilios Tsikaris 1 1 Department of Chemistry and 2 Medical School, University of Ioannina, Ioannina, Greece; and 3 Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece a IIb b 3 , a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand- binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the a IIb sub- unit is presented, using overlapping 20-peptides, in order to identify the binding sites of a IIb potentially involved in the platelet-aggregation event. Regions a IIb 313–332, a IIb 265– 284 and a IIb 57–64 of a IIb b 3 were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this lig- and. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of a IIb b 3 . Furthermore, a IIb 313–332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and con- sequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encom- passed in a IIb 313–332, 265–284 and 57–64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets. Keywords: a IIb -binding domains; a IIb mapping; platelet- aggregation inhibitors; a IIb b 3 receptor; integrin inhibitors. The integrin family of adhesive protein receptors, composed of noncovalently associated a and b subunits, participates in a number of diverse functions ranging from embryogenesis to cellular aggregation, and differentiation to tumor cell growth and metastasis [1–5]. Integrin receptors consist of at least 20 members composed of different combinations of a and b subunits with distinct ligand-recognition specificity [6]. The integrin receptor a IIb b 3 is the most abundant glyco- protein on platelet plasma-membranes. This receptor binds to adhesive proteins, such as fibrinogen, von Willebrand factor, fibronectin, and vitronectin, via the recognition of short amino acid sequences, including the ubiquitous motif RGD, as well as the HHLGGAKQAGDV sequence of the fibrinogen c-chain [7,8]. Binding studies suggest that platelet activation (e.g. by ADP) induces conformational changes of a IIb b 3 , which result in higher affinity to fibrinogen, an event essential for platelet aggregation and thrombus formation [9,10]. mAbs recognizing specific epitopes on the extracellu- lar domains of both subunits are also able to induce/stabilize conformational changes of a IIb b 3 , which increase the affinity of the receptor for its ligands [11–13]. The discovery that the RGD sequence is present in a surprisingly large number of adhesive proteins, serving diverse functions, has led to extensive research in the development of small RGD-containing peptides as anti- thrombotic agents. Elucidation of the pharmacophoric nature of the Asp and Arg side-chains allowed new strategies, largely based on bioactive RGD conformations, to be developed for the rational design of peptide hybrids and nonpeptide mimetics as potential therapeutic drugs against platelet aggregation [14–19]. Recently, it has been proposed that binding of the RGD peptide leads to changes in a IIb b 3 that are associated with acquisition of high-affinity fibrinogen-binding function and subsequent platelet activation, despite the initial RGD- inhibitory effect [20]. Consequently, an alternative approach would be to inhibit RGD-mediated platelet activation by defining the ligand-binding sites on the receptor. Peptides modelled from these domains could be potent receptor competitors, thus bypassing the function of RGD and other ligand mimetic peptides as partial agonists. Ligand-binding sites in integrins have been investigated utilizing a combination of immunological, biochemical, and mutational approaches. For instance, proteolysis of a IIb b 3 , expression of recombinant truncated a IIb b 3 , or cross-linking studies suggest that ligand-recognition sites are present in the N-terminal portion of both subunits and support the concept that multiple ligand contact points are involved Correspondence to V. Tsikaris, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece. Fax: + 30 2651 098799, Tel.: + 30 2651 098383, E-mail: btsikari@cc.uoi.gr Abbreviations: FITC-Fg, FITC-labelled fibrinogen; PRP, platelet-rich plasma; SPPS, solid-phase peptide synthesis. (Received 29 May 2003, revised 15 July 2003, accepted 21 July 2003) Eur. J. Biochem. 270, 3760–3767 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03762.x [5,21–24]. Electron microscopy and biophysical analysis have also been applied to identify the ligand-binding sites of integrins [25,26]. Integrins are conformationally labile, and easily subjected to proteolysis and disulfide bond rearrange- ment during purification and storage [24]. This limitation has often led to inconsistent results in studies of ligand- binding sites between different research groups. In this study, we aimed to develop compounds that bound to fibrinogen at sites that were recognized by the activated a IIb b 3 integrin. Therefore, in the context of this study, the fine mapping of the fibrinogen-binding domains on the a IIb subunit was accomplished and their potential role in platelet aggregation was determined. More speci- fically, a detailed mapping of the a IIb subunit was performed using synthetic 20-peptides, which overlapped by eight residues and covered the extracellular region of the subunit. Subsequently, the inhibitory effect of all peptides was deter- mined on ADP-induced platelet activation. These peptides are expected to inhibit fibrinogen binding to the receptor, thus blocking platelet aggregation and further activation through a IIb b 3 -mediated outside-in signaling. Experimental procedures Synthesis of peptides covering the extracellular region of the a IIb subunit Eighty-two 20-peptides (overlapping by eight residues) covering the extracellular region (1–992) of the a IIb subunit were synthesized according to the Multiblock method [27]. Syntheses were performed on Wang resin (p-alkoxybenzyl alcohol resin) [28] and the protocols were based on the principles of the solid-phase peptide synthesis (SPPS) [29– 31]. A spare glycine was incorporated as the C-terminal residue (shown in parenthesis in the peptide sequences below) to simplify and reduce the cost of the syntheses. Peptides were obtained by treatment of the resin for 3.0 h with a mixture of trifluoroacetic acid/triisopropylsilane/ water (95 : 2.5 : 2.5; v/v/v). Cleavage of cysteine-containing peptides was performed by treatment with a mixture of trifluoroacetic acid/triisopropylsilane/water/dimethylsulfide (94 : 2.5 : 1 : 2.5; v/v/v/v). After removal of the resin, the filtrate was evaporated and the peptides precipitated by cold ether. Yields ranged from 15 to 30 mg. The Kaiser test was applied in each step of the coupling/deprotection, mainly in peptide sequences predicted as difficult according to the peptide companion software of Multiblock, as, for example, the 20-peptide ERAIPIWWVLVGVLGGLLLL(G) [a IIb (961–980)]. The purity of the crude peptides, in statistical samples, tested by ESI-MS, ranged from 60 to 80% (Fig. 1A). The crude peptides were used in a first screening, aiming to investigate their inhibitory effect on ADP-induced platelet aggregation. Synthesis of the a IIb peptide analogues that exhibit the best inhibitory effect towards platelet aggregation The peptides, identified through the screening process to exhibit the greatest inhibitory effects on platelet aggregation, were synthesized on Fmoc-Gly-Wang resin (0.8 mmolÆg )1 of resin) following SPPS [29–31]. Aspartic acid and glutamic acid were introduced as Fmoc-Asp-(t-Butoxy)-OH and Fmoc-Glu-(t-Butoxy)-OH, respectively; asparagine and glutamine as Fmoc-Asn-(trityl group)-OH and Fmoc- Gln-(trityl group)-OH, respectively; arginine as Fmoc-Arg- (2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-OH; serine and threonine as Fmoc-Ser-(t-butyl group)-OH and Fmoc-Thr-(t-butyl group)-OH, respectively; lysine as Fmoc-Lys-(t-butoxycarbonyl group)-OH; tyrosine as Fmoc-Tyr-(t-butoxycarbonyl group)-OH; cysteine as Fmoc-Cys-(trityl group)-OH; and histidine as Fmoc- His-(trityl group)-OH. Fmoc groups were removed using 20% piperidine in dimethylformamide. Couplings were performed by using an amino acid/2-(1H-benzotriazole-1- yl)1,1,3,3 tetramethyluronium tetrafluoroborate/N-hydro- xybenzotriazole/N-ethyldiisopropylamine/resin molar ratio of 3 : 2.9 : 3 : 3 : 1. Dimethylformamide, used for cou- plings, was previously distilled to remove traces of amines. Deprotection and coupling reactions were monitored by using the Kaiser test. The crude peptides were obtained by treatment of the peptidyl resin for 3 h with a mixture of trifluoroacetic acid/triisopropylsilane/water (95 : 2.5 : 25; v/v/v) or trifluoroacetic acid/triisopropylsilane/water/ dimethylsulfide (94 : 2.5 : 1 : 2.5; v/v/v/v) in the case of cysteine-containing peptides. The resin was eliminated by filtration, the filtrate was evaporated under reduced pres- sure, and the product precipitated by cold ethyl ether (yields ranged from 75 to 90%). Peptides were purified by preparative reverse-HPLC on a C18 column (solvent A, H 2 O/0.1% trifluoroacetic acid; solvent B, CH 3 CN/0.1% trifluoroacetic acid) programmed gradients. Yields ranged from 35 to 45%. The purity of the peptides and their molecular masses were assessed by analytical HPLC and ESI-MS, respectively (Fig. 1B). Hydrophilicity profile of the a IIb subunit The hydrophilicity profile of a IIb , based on its primary structure, was analysed according to the method of Hopp & Woods [32]. Platelet-aggregation studies Platelet-aggregation studies were performed in platelet-rich plasma (PRP) prepared from peripheral venous blood of apparently healthy normolipidemic volunteers, as previ- ously described [33]. The platelet count of PRP was adjusted to a final platelet concentration of 2.5 · 10 8 ÆmL )1 with homologous platelet-poor plasma. The PRP was then preincubated with each of the synthetic 20-peptides or with the RGDS peptide (used as a positive control) for 1 min before the initiation of aggregation. Platelet aggre- gation, in the presence of ADP (1.0–5.0 l M ), was meas- ured in aliquots of 0.5 mL of PRP, in a platelet aggregometer (model 560; Chronolog, Corp.) at 37 °C, with continuous stirring at 1200 r.p.m. The maximal aggregation, achieved within 3 min after addition of the agonist, was determined and expressed as a percentage of 100% light transmission calibrated for each specimen (maximal percentage of aggregation). All aggregation assays were conducted within 3 h after venepuncture. All peptides were dissolved in normal saline or in 5% (v/v) dimethylsulfoxide/normal saline. Peptides that were insol- uble in the above solutes were excluded from the study. Ó FEBS 2003 a IIb -Binding domains (Eur. J. Biochem. 270) 3761 For peptides containing Cys residues, 1,4-dithiothreitol was used to avoid oxidation. Fluorescein labelling of fibrinogen Fluorescein labelling of fibrinogen was perfomed as previ- ously described [34]. In brief, freshly thawed fibrinogen (20 mgÆmL )1 ), diluted to 2 mgÆmL )1 in NaCl/P i (PBS), pH 8.3–8.5, was incubated with 1 mgÆmL )1 celite-FITC for 60 min at room temperature in the dark with intermittent vortexing. The celite-FITC was separated from the conju- gated fibrinogen by centrifugation in a microfuge (10 000 g) for 5 min. The FITC-labelled fibrinogen (FITC-Fg) in the supernatant was normally separated from unreacted free FITC by exhaustive dialysis in NaCl/P i ,at4°C, and any remaining celite-FITC was removed by subsequent centri- fugation at 10 000 g for 5 min. The concentration of FITC- Fg was determined by measuring the absorbance (A)at280 and 495 nm. The molar ratio of fluorescein to protein in our preparations, calculated as previously described [34], was 4.7 ± 0.5. Aliquots of FITC-Fg were stored at )80 °Cand freshly thawed at room temperature before use. Fibrinogen binding The effect of 20-peptides on FITC-Fg binding to platelets was studied by flow cytometry, using a FACsCaliber flow cytometer (Becton-Dickinson, San Jose, CA, USA), as previously described [35,36]. PRP with platelet number ranging from 2.5 · 10 8 ÆmL )1 to 4.5 · 10 8 ÆmL )1 was diluted 10-fold with Walsh-albumin buffer [34]. Diluted PRP was then mixed with FITC-Fg (500 n M final concentration), in the presence or absence of the peptides. Platelet activation was performed with 100 l M ADP at room temperature for 60 min in the dark. Then platelets were immediately analysed by flow cytometry, using 10 000 cell events. The mean fluorescence intensity values for both the nonacti- vated and activated platelets, in the presence or absence of the 20-peptide, were calculated. The mean fluorescence intensity values of nonactivated platelets, in the presence or absence of the 20-peptide (nonspecific binding), were subtracted from those obtained after platelet activation (total binding), respectively, thus obtaining the specific binding of FITC-Fg [37]. The effect of an RGDS peptide (1 m M final concentration) on FITC-Fg binding to activa- ted platelets was also studied using the same procedure. Numeric data were processed using CELLQUEST software (Becton-Dickinson). Binding of the a IIb 313–332 peptide to fibrinogen Binding of the a IIb 313–332 20-peptide to fibrinogen was assessed by a solid-phase immunoassay. Briefly, fibrinogen diluted in bicarbonate buffer (pH 9.6) was plated in Fig. 1. ESI-MS of the crude (A) and purified (B) a IIb 313–332. Calculated M r , 2473.90; found M r , 2474.49. 3762 N. Biris et al. (Eur. J. Biochem. 270) Ó FEBS 2003 poly(vinyl chloride) flat-bottomed microdilution plates (150 ngÆmL )1 ) and incubated overnight at 4 °C. The plates were then washed and incubated for a minimum of 1 h at room temperature with NaCl/P i containing 3% BSA. After further washes, different concentrations of the a IIb 313–332 peptide were added to the coated wells and the plates were incubated for 2 h at room temperature. Plates were then washed and incubated overnight with an IgM mouse mAb [anti-(a IIb 313–332)] that was generated by immunizing BALB/c mice with 1 mgÆmL )1 of the 20-peptide conjugated to mouse serum albumin by means of 0.1% glutaraldehyde. Fusion was carried out by the direct cloning method [38]. Binding of the mAb to the 20-peptide was assessed using horseradish peroxidase-conjugated anti-mouse immuno- globulins, as previously described [39]. PAC-1 binding Platelets, in PRP, were labeled with FITC/PAC-1 (Becton- Dickinson) using a modification of the technique previ- ously described by Golden et al. [40]. Briefly, platelets (2.5–4.5 · 10 8 ÆmL )1 ) were incubated with 0.025 lgÆmL )1 of FITC/PAC-1 in the presence or absence of the peptides, or the RGDS peptide (used as a positive control), prior to activation with ADP (100 l M final concentration). Activa- tion was performed for 10 min at 37 °C. Platelets were then diluted with NaCl/P i (1 : 5; v/v) and immediately analyzed by flow cytometry. Results Eighty-two 20-peptides, overlapping by eight residues, covering the entire extracellular sequence of a IIb (1–992), were synthesized as described above [27]. The purity of these crude peptides, as estimated by ESI-MS, ranged from 60 to 80% (Fig. 1A). The synthetic peptides were subsequently screened as possible inhibitors of platelet aggregation induced by ADP. All peptides were used at a final concentration of 1 mgÆmL )1 . Through this screening pro- cedure, it was found that five peptides spanning sequences within the 1–488 region of a IIb , were inhibitors of platelet aggregation induced by 5 l M ADP (inhibition achieved by each of these five peptides was ‡ 40%, whereas all the others inhibited platelet aggregation by < 10%). The identified inhibitory peptides, ETGGVFLCPW RAE GGQCPSL(G) (residues 49–68), GAVEILDSYYQRL HRL RAEQ(G) (residues 265–284), LHRLRAEQMASY FGHSVAVT(G) (residues 277–296), YMESRADRKLAE VGRVYLFL(G) (residues 313–332) and AVKSCV LPQTKTPVSCFNIQ(G) (residues 469–488), designated a IIb 49–68, a IIb 256–284, a IIb 277–296, a IIb 313–332 and a IIb 469–488, respectively, were selected for further study. To achieve this they were synthesized, in relatively larger quantities, purified and characterized by ESI-MS (Fig. 1B). The inhibitory effect of different concentrations of these peptides on platelet aggregation induced by ADP was further evaluated. In addition, the eight-peptide PW RAEGGQ (residues 57–64), included in a IIb 49–68 and designated as a IIb 57–64, and the 21-peptide AVTDVNGDGRHDLLVGAPLYM (residues 294–314), designated as a IIb 294–314, which has been proposed by D’Souza et al. to comprise the binding site for the 12-peptide of the fibrinogen c-chain [41], were also synthes- ized, purified and tested for their inhibitory effects on platelet aggregation. All purified peptides inhibited platelet aggregation in a dose-dependent manner. However, as shown in Table 1, the 20-peptides a IIb 313–332 and a IIb 265–284 were the most potent inhibitors, because they exhibited the lowest IC 50 values (the concentration that induces 50% inhibition of platelet aggregation). Typical aggregation curves illustrating the inhibitory effect of these peptides on ADP-induced platelet aggregation, as well as typical sigmoidal curves for the estimation of the IC 50 values of these peptides, are presented in Fig. 2. It is important to note that the inhibitory effect of these 20-peptides, described above, towards platelet aggregation, was comparable to that exhibited by the RGDS peptide (Table 1). Our results also demonstrated that although the 21-peptide, a IIb 294– 314, inhibited platelet aggregation, it was a less potent inhibitor under our experimental conditions than either a IIb 313–332 or a IIb 265–284. Finally, our aggregation studies revealed that the eight-peptide a IIb 57–64, that represents a fragment of the 20-peptide a IIb 49–68, retained the inhi- bitory potency of a IIb 49–68 (Table 1). The above results prompted us to further investigate the inhibitory activity of our synthetic peptides on fibrinogen binding to ADP-activated platelets by FACS analysis using FITC-Fg. As shown in Table 1, all peptides inhibited Table 1. Inhibitory features of the purified peptide analogues derived from a IIb amino acid sequence on ADP-induced platelet activation. Selection of the peptides listed was based on the results obtained from the initial screening of the crude peptides. Peptide analogue of a IIb Inhibition of platelet aggregation (IC 50 values, l M ) Inhibition of fibrinogen binding (IC 50 values, l M ) Inhibition of PAC-1 binding (%) a IIb 49–68 5623 3910 0 a IIb 57–64 3451 1122 0 a IIb 265–284 800 530 0 a IIb 277–296 2844 2116 0 a IIb 294–314 a 2510 1762 0 a IIb 313–332 300 130 0 a IIb 469–488 7490 4288 0 RGDS 210 113 78.0 ± 6.0 b a For details, see the Results. b Values represent the mean ± SD from four different platelet preparations and show the inhibitory effect of RGDS at a final concentration of 1 m M . Ó FEBS 2003 a IIb -Binding domains (Eur. J. Biochem. 270) 3763 fibrinogen binding to activated platelets; however, the 20-peptides a IIb 313–332 and a IIb 265–284 exhibited the most potent inhibitory effect, as revealed by the lower IC 50 values of FITC-Fg binding to activated platelets. This finding is in accordance with our aggregation experiments. Representative histograms of the inhibition of FITC-Fg binding by a IIb 313–332 and a IIb 57–64 are illustrated in Fig. 3A,B. Of importance is also the finding that the observed inhibitory potency of the a IIb 313–332 was comparable with that of the RGDS, used as a positive control (Table 1). We next investigated whether the above inhibitory effects of our peptides are a result of their interaction with the activated form of a IIb b 3 . To address this question we studied, by FACS analysis, the effect of these peptides on PAC-1 binding to platelets activated with ADP. This analysis revealed that binding of PAC-1 to stimulated platelets was not affected by any of the purified peptides at anyconcentrationtestedupto4.0m M (Table 1). By contrast, the RGDS peptide almost completely inhibited PAC-1 binding to activated platelets at a concentration of 1m M (Table 1). Representative histograms illustrating the effect of a IIb 313–332 and RGDS on PAC-1 binding are presented in Fig. 3C. The above results suggest that our synthetic peptides do not interact with the activated receptor, although they significantly inhibit the binding of fibrinogen to the activated platelets as well as inhibiting platelet aggregation. We further investigated whether the inhibitory effect of our peptides could be a result of their interaction with fibrinogen at sites that are critical for the binding of this ligand to the activated a IIb b 3 . To address this, we performed solid-phase binding assays on fibrinogen-coated plates. In these experi- ments we used the 20-peptide a IIb 313–332, which was the most potent inhibitory peptide, as well as a mAb raised against this 20-peptide, as described above in the Experi- mental procedures. Results presented in Fig. 4 indicate that the anti-(a IIb 313–332) mAb recognized the 20-peptide that had interacted with the coated fibrinogen, in a dose- dependent manner, suggesting that a IIb 313–332 can bind to fibrinogen. Discussion The aim of the present study was to map the fibrinogen- binding domains on the a IIb subunit of the platelet a IIb b 3 receptor, in its activated form. To achieve this, a high- throughput screening approach, consisting of synthesizing Fig. 2. Aggregation curves. Representative aggregation curves illus- trating the inhibitory effect of different concentrations of a IIb 313–332 (A) and a IIb 265–284 (B) on platelet aggregation, and dose-dependent curves for both peptides demonstrating the inhibition of platelet aggregation (C). Fig. 3. Representative histograms, obtained by FACS analysis. The effect of 500 l M a IIb 313–332 (A) and 500 l M a IIb 57–64 (B) on FITC- fibrinogen (FITC-Fg) binding to platelets activated with 100 l M ADP. (C)Theeffectof500 l M a IIb 313–332 or 1 m M RGDS on FITC/PAC-1 binding to platelets activated with 100 l M ADP. Fig. 4. Binding of the anti-(a IIb 313–332) monoclonal antibody to fibrinogen-coated plates in the absence (dark bars) or presence (open bars) of different concentrations of the a IIb 313–332 peptide. Numbers below the bars represent the concentration (lgÆmL )1 ) of the 20-pep- tide. Data shown are representative of three independent experiments carried out in triplicate. 3764 N. Biris et al. (Eur. J. Biochem. 270) Ó FEBS 2003 and testing the effect of 20-peptides on the activated form of a IIb b 3 in situ, i.e. on intact platelets, was pursued. In total, 82 overlapping synthetic 20-peptides, derived from a IIb (1–992), were tested. It was clearly shown that among them, five 20-peptides (a IIb 49–68, a IIb 265–284, a IIb 277–296, a IIb 313–332 and a IIb 469–488) are capable of inhibiting platelet aggregation, although to different extents. Importantly, all these sequences are highly hydrophilic (3.5 score), suggest- ing that they are exposed to the extracellular surroundings and thus could be available for ligand association. Among the inhibitory 20-peptides, a IIb 313–332 and a IIb 265–284 were the most effective antagonists of platelet aggregation. To gain further insight into the fibrinogen-recognition sites of a IIb , we evaluated the inhibitory effect of the above peptides on fibrinogen binding to activated platelets. It was shown that all peptides inhibit fibrinogen binding; however, in accordance with the aggregation studies, a IIb 313–332 and a IIb 265–284 were the most potent inhibitors. The finding that a IIb 49–68 inhibited platelet aggregation and fibrinogen binding, although to a lesser extent than a IIb 313–332 and a IIb 265–284, is in agreement with previously published results, as a longer sequence (42–73, which includes 57–64) has been proposed as a ligand-binding site of the a IIb subunit [42]. In addition, a naturally occurring mutation (L55P) within this region has been reported in patients with Glanzmann thrombasthenia, suggesting that this region is important for platelet aggregation [43]. The present study further illustrates the importance of the eight- peptide sequence 57–64 in maintaining the inhibitory effect of the original 20-peptide a IIb 49–68. The N-terminal region of the integrin a subunit is composed of seven repeats (W 1 –W 7 ), which have been predicted to fold into a b-propeller domain. Strands 1, 2, 3 and 4 are connected by successive hairpin turns, and strand 4 of one sheet is connected to strand 1 of the next [44,45]. In this regard (a) a IIb 57–64 corresponds to the loop connecting strands 3 and 4 of W 1 ,(b)a IIb 265–284 comprises strands 3and4ofW 4 , including the loops (273–274) and (283–285) and (c) a IIb 313–332 incorporates strands 2 (313–318) and 3 (319–332) of W 5 , enclosing the loop (313–323). Kamata et al. [45] showed that mutations which disrupt fibrinogen binding are clustered to one side of the b-propeller (W 2 ,W 3 , W 4 and W 5 ). The regions identified in our study (a IIb 265– 284 and a IIb 313–332) incorporate W 4 and W 5 . Interestingly, in the same study it was shown, using loop swapping and site-directed mutagenesis, that fibrinogen binding to mutants of W 5 (residues 283–285) was completely abolished. This finding is consistent with our results as the reported mutations are within the region 265–284. In addition, in the same study it was shown that binding of fibrinogen to W 5 -swapping mutants (residues 313–323) was partially inhibited, suggesting that these residues play a moderate role in fibrinogen binding. However, in this study the activation of a IIb b 3 was performed using a mAb (mAb PT25-2) that recognizes residues 335–338 located in the close vicinity of the 313–323 domain. Thus, it is possible that the binding of this antibody to residues 335–338 could influence the interaction between 313–323 and fibrinogen. Further- more, the region YMESRADRKLA (313–323) of a IIb was swapped with that of a 5 (LMDRTPDGRPQ), which contains an DGR motif. This motif could contribute to ligand binding by its charged side-chains (discussed in the text below), thus diminishing the expected decrease in the binding of fibrinogen to this region. In support of our findings concerning the importance of region 313–332 in fibrinogen finding, two naturally occurring mutations (E324K and R327H) have been repor- ted in patients with Glanzmann thrombasthenia [46–49]. Moreover, it has been shown that the peptide LSARLAF [50] binds to complementary region 315–321 of a IIb and induces a IIb b 3 conformational change and platelet aggrega- tion [50,51]. Binding of this peptide to a IIb also induces platelet secretion and further activation [50,51] through an a IIb b 3 -mediated outside-in signal transduction [52]. Overall, the results of our study, in addition to the above observa- tions, suggest that the a IIb 313–332 region is important, not only for fibrinogen binding but also for platelet activation. The rationale of this study was based on the assumption that peptide fragments derived from the a IIb subunit could act as inhibitors of platelet aggregation through their direct interaction with fibrinogen. The development of such ligand-binding antagonists may be advantageous against the RGD-like antagonists [20] because they could inhibit platelet aggregation without inducing a IIb b 3 -mediated out- side-in signaling. The latter has been proposed to occur for the RGD-like antagonists [20], which bind to the receptor. However, as previously mentioned, the a IIb 49–68, a IIb 265–284, and a IIb 313–332 comprise the RAD and RAE sequences that mimic the RGD sequence. We and others have demonstrated that such substitutions do not significantly affect the adhesive properties of RGD [53]. Therefore, one could assume that the identified peptide- antagonists, although fragments of the a IIb subunit, could function via their RGD-like pattern by interacting with the receptor, as is probably the case for the DGR sequence of the reported putative fibrinogen-binding site of a IIb (296–306) [54], located at the proximity of 313–332. To test this hypothesis, inhibition experiments were performed in the presence of PAC-1, a ligand-mimetic anti-a IIb b 3 that contains the RYD sequence (an RGD mimic) in the CDR3 region of the heavy chain. PAC-1 binds to the activated form of a IIb b 3 and is inhibited by RGD peptides [55]. We thus demonstrated that PAC-1 binding to the receptor was not affected by any peptide tested, in contrast to RGDS, which, as expected, signifi- cantly inhibited PAC-1 binding. Consequently, the identified peptides do not influence the binding of RGD- containing ligands, thus suggesting that the inhibition of fibrinogen binding to the activated receptor, as well as platelet aggregation, could be caused by their interaction with fibrinogen. The latter is further supported by the results of the solid-phase binding experiments. The identified peptides appear to be potent competitors of the receptor for fibrinogen and hence are not expected to interact with a IIb b 3 and affect its conformational state and function during ADP-induced platelet activation. It is also noteworthy that both a IIb 313–332 and a IIb 265– 284 sequences are adjacent to the region that has been proposed to comprise the binding site for the 12-peptide of the fibrinogen c-chain (a IIb 294–314) [41]. This site, identi- fied using a chemical cross-linking approach, is proximal to the second calcium-binding domain [41]. The same authors subsequently demonstrated that the 12-peptide TDVNGDGRHDL, corresponding to residues 296–306 of Ó FEBS 2003 a IIb -Binding domains (Eur. J. Biochem. 270) 3765 a IIb , inhibited ADP-induced aggregation of washed platelets in Tyrode’s buffer supplemented with divalent ions [54]. In the same study, it was shown that the a IIb 296–306 peptide binds directly to fibrinogen, an interaction that depends on divalent ions and can be inhibited by RGD-containing peptides [54]. It was also suggested that its inhibitory potency could be related to the presence of the DGR motif (the invert of RGD), as peptides with this motif act as inhibitors to RGD-containing ligands to certain integrins [56]. It is probable that these two a IIb domains, owing to their proximity to the presumptive fibrinogen- and calcium- binding sites, play an important role in the ligand inter- action with a IIb b 3 through its c-chain 12-peptide. In conclusion, our findings indicate that sequences 313– 332, 265–284 and 57–64 are potential fibrinogen-binding domains on the a IIb subunit of a IIb b 3 and the corresponding peptides inhibit platelet aggregation and antagonize fibrino- gen association, possibly by interacting with this ligand. We hypothesize that RAD and RAE adhesive motifs, encom- passed in a IIb 313–332, 265–284 and 57–64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets. Acknowledgements This work was supported by the Greek General Secretariat for Research and Technology. References 1. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell 48, 549–554. 2. 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(1985) Synthetic peptides com- petitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin. J. Biol. Chem. 260, 10402–10405. Ó FEBS 2003 a IIb -Binding domains (Eur. J. Biochem. 270) 3767 . Mapping the binding domains of the a IIb subunit A study performed on the activated form of the platelet integrin a IIb b 3 Nikolaos Biris 1 , Morfis Abatzis 1 , John V. Mitsios 1 , Maria Sakarellos-Daitsiotis 1 ,. fibrinogen -binding domains on the a IIb subunit was accomplished and their potential role in platelet aggregation was determined. More speci- fically, a detailed mapping of the a IIb subunit was performed using. platelet activation. The rationale of this study was based on the assumption that peptide fragments derived from the a IIb subunit could act as inhibitors of platelet aggregation through their direct interaction