Báo cáo khoa học: Mapping the binding domains of the aIIb subunit A study performed on the activated form of the platelet integrin aIIbb3 pot

Tselepis1, Lambros Michalis2, Dimitrios Sideris2, Georgia Konidou3, Ketty Soteriadou3and Vassilios Tsikaris1 1 Department of Chemistry and2Medical School, University of Ioannina, Ioannin

Mapping the binding domains of the aIIb subunit

A study performed on the activated form of the platelet integrin aIIbb3

Nikolaos Biris1, Morfis Abatzis1, John V Mitsios1, Maria Sakarellos-Daitsiotis1, Constantinos Sakarellos1, Demokritos Tsoukatos1, Alexandros D Tselepis1, Lambros Michalis2, Dimitrios Sideris2,

Georgia Konidou3, Ketty Soteriadou3and Vassilios Tsikaris1

1

Department of Chemistry and2Medical School, University of Ioannina, Ioannina, Greece; and3Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece

aIIbb3, 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 aIIb

sub-unit is presented, using overlapping 20-peptides, in order to

identify the binding sites of aIIbpotentially involved in the

platelet-aggregation event.Regions aIIb313–332, aIIb265–

284 and aIIb 57–64 of aIIbb3 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 aIIbb3.Furthermore, aIIb313–332 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 aIIb 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: aIIb-binding domains; aIIb mapping; platelet-aggregation inhibitors; aIIbb3receptor; 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 aIIbb3is 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

aIIbb3, 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 aIIbb3, 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 aIIbb3that 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 aIIbb3, expression of recombinant truncated aIIbb3, 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)

[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 aIIbb3integrin.Therefore, in the context of this

study, the fine mapping of the fibrinogen-binding domains

on the aIIb subunit was accomplished and their potential

role in platelet aggregation was determined.More

speci-fically, a detailed mapping of the aIIbsubunit 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 aIIbb3-mediated outside-in signaling

Experimental procedures

Synthesis of peptides covering the extracellular region

of the aIIbsubunit

Eighty-two 20-peptides (overlapping by eight residues)

covering the extracellular region (1–992) of the aIIbsubunit

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) [aIIb

(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 aIIbpeptide 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 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 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,

H2O/0.1% trifluoroacetic acid; solvent B, CH3CN/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 aIIbsubunit The hydrophilicity profile of aIIb, 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· 108ÆmL)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 lM), was meas-ured in aliquots of 0.5 mL of PRP, in a platelet aggregometer (model 560; Chronolog, Corp.) at 37C, 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

FEBS 2003 aIIb-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/Pi (PBS),

pH 8.3–8.5, was incubated with 1 mgÆmL)1celite-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/Pi, at 4C, 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) at 280

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 C and

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· 108ÆmL)1to 4.5· 108ÆmL)1was diluted 10-fold with Walsh-albumin buffer [34].Diluted PRP was then mixed with FITC-Fg (500 nMfinal concentration), in the presence or absence of the peptides.Platelet activation was performed with 100 lMADP 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 actinonacti-vated 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 mMfinal 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 aIIb313–332 peptide to fibrinogen Binding of the aIIb 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.

poly(vinyl chloride) flat-bottomed microdilution plates

(150 ngÆmL)1) and incubated overnight at 4C.The plates

were then washed and incubated for a minimum of 1 h at

room temperature with NaCl/Picontaining 3% BSA.After

further washes, different concentrations of the aIIb313–332

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-(aIIb 313–332)] that was generated by immunizing

BALB/c mice with 1 mgÆmL)1of 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· 108ÆmL)1) were incubated with 0.025 lgÆmL)1of

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 lMfinal

concentration).Activa-tion was performed for 10 min at 37C.Platelets were then

diluted with NaCl/Pi(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 aIIb (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 aIIb, were inhibitors of platelet

aggregation induced by 5 lM ADP (inhibition achieved

by each of these five peptides was‡ 40%, whereas all the

others inhibited platelet aggregation by < 10%).The

identified inhibitory peptides, ETGGVFLCPWRAE GGQCPSL(G) (residues 49–68), GAVEILDSYYQRL HRLRAEQ(G) (residues 265–284), LHRLRAEQMASY FGHSVAVT(G) (residues 277–296), YMESRADRKLAE VGRVYLFL(G) (residues 313–332) and AVKSCV LPQTKTPVSCFNIQ(G) (residues 469–488), designated

aIIb49–68, aIIb256–284, aIIb277–296, aIIb313–332 and aIIb 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 PWRAEGGQ (residues 57–64), included in aIIb 49–68 and designated as aIIb 57–64, and the 21-peptide AVTDVNGDGRHDLLVGAPLYM (residues 294–314), designated as aIIb 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 aIIb 313–332 and aIIb 265–284 were the most potent inhibitors, because they exhibited the lowest IC50 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 IC50values

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, aIIb 294–

314, inhibited platelet aggregation, it was a less potent inhibitor under our experimental conditions than either aIIb 313–332 or aIIb265–284.Finally, our aggregation studies revealed that the eight-peptide aIIb57–64, that represents a fragment of the 20-peptide aIIb 49–68, retained the inhi-bitory potency of aIIb49–68 (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 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

FEBS 2003 aIIb-Binding domains (Eur J Biochem 270) 3763

fibrinogen binding to activated platelets; however, the

20-peptides aIIb 313–332 and aIIb 265–284 exhibited the

most potent inhibitory effect, as revealed by the lower IC50

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 aIIb 313–332 and aIIb 57–64 are illustrated in

Fig.3A,B Of importance is also the finding that the

observed inhibitory potency of the aIIb 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 aIIbb3.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

any concentration tested up to 4 0 mM (Table 1).By

contrast, the RGDS peptide almost completely inhibited

PAC-1 binding to activated platelets at a concentration of

1 mM(Table 1).Representative histograms illustrating the

effect of aIIb 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 aIIbb3.To address this, we performed solid-phase

binding assays on fibrinogen-coated plates.In these

experi-ments we used the 20-peptide aIIb313–332, 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-(aIIb313–332) mAb recognized the 20-peptide that had interacted with the coated fibrinogen, in a dose-dependent manner, suggesting that aIIb313–332 can bind to fibrinogen

Discussion

The aim of the present study was to map the fibrinogen-binding domains on the aIIbsubunit of the platelet aIIbb3 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) The effect of 500 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.

and testing the effect of 20-peptides on the activated form of

aIIbb3in situ, i.e.on intact platelets, was pursued.In total,

82 overlapping synthetic 20-peptides, derived from aIIb

(1–992), were tested.It was clearly shown that among them,

five 20-peptides (aIIb49–68, aIIb265–284, aIIb277–296, aIIb

313–332 and aIIb469–488) 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 surroundsuggest-ings

and thus could be available for ligand association.Among

the inhibitory 20-peptides, aIIb 313–332 and aIIb 265–284

were the most effective antagonists of platelet aggregation

To gain further insight into the fibrinogen-recognition sites

of aIIb, 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, aIIb313–332 and

aIIb265–284 were the most potent inhibitors

The finding that aIIb49–68 inhibited platelet aggregation

and fibrinogen binding, although to a lesser extent than aIIb

313–332 and aIIb265–284, 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 aIIbsubunit [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 aIIb49–68

The N-terminal region of the integrin a subunit is

composed of seven repeats (W1–W7), 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) aIIb57–64 corresponds to the loop connecting

strands 3 and 4 of W1, (b) aIIb265–284 comprises strands

3 and 4 of W4, including the loops (273–274) and (283–285)

and (c) aIIb313–332 incorporates strands 2 (313–318) and 3

(319–332) of W5, 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 (W2, W3,

W4and W5).The regions identified in our study (aIIb265–

284 and aIIb313–332) incorporate W4and W5.Interestingly,

in the same study it was shown, using loop swapping and

site-directed mutagenesis, that fibrinogen binding to

mutants of W5(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

W5-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 aIIbb3 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 aIIbwas

swapped with that of a5 (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 aIIb and induces aIIbb3conformational change and platelet aggrega-tion [50,51].Binding of this peptide to aIIb also induces platelet secretion and further activation [50,51] through an

aIIbb3-mediated outside-in signal transduction [52].Overall, the results of our study, in addition to the above observa-tions, suggest that the aIIb313–332 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 aIIbsubunit 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 aIIbb3-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 aIIb 49–68, aIIb 265–284, and aIIb313–332 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 aIIbsubunit, 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 aIIb

(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-aIIbb3 that contains the RYD sequence (an RGD mimic) in the CDR3 region of the heavy chain.PAC-1 binds to the activated form of aIIbb3 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 aIIbb3and affect its conformational state and function during ADP-induced platelet activation

It is also noteworthy that both aIIb313–332 and aIIb265–

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 (aIIb294–314) [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

FEBS 2003 aIIb-Binding domains (Eur J Biochem 270) 3765

aIIb, 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 aIIb296–306 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 aIIbdomains, owing to

their proximity to the presumptive fibrinogen- and

calcium-binding sites, play an important role in the ligand

inter-action with aIIbb3through 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 aIIbsubunit of aIIbb3and 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 aIIb313–332, 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.Ruoslahti, E.& Pierschbacker, M D.(1987) New perspectives in

cell adhesion: RGD integrins Science 238, 491–497.

3 Boucaut, J.C., Darribere, T., Boulekbache, H & Thiery, J.P.

(1984) Prevention of gastrulation but not neurulation by

anti-bodies to fibronectin in amphibian embryos Nature 307, 364–367.

4 Humphries, M.J., Yamada, K.M & Olden, K (1988)

Investiga-tion of the biological effects of anti-cell adhesive synthetic peptides

that inhibit experimental metastasis of B16–F10 murine

melan-oma cells J Clin Invest 81, 782–790.

5 Smith, J.W & Cheresh, D.A (1990) Integrin (alpha v beta 3)–

ligand interaction.Identification of a heterodimeric RGD binding

site on the vitronectin receptor J Biol Chem 265, 2168–2172.

6 Loftus, J.C., Halloran, C.E., Ginsberg, M.H., Feigen, L.P.,

Zablocki, J.A & Smith, J.W (1996) The amino-terminal one-third

of alpha IIb defines the ligand recognition specificity of integrin

alpha IIb beta 3 J Biol Chem 271, 2033–2039.

7 D’Souza, S.E., Ginsberg, M.H & Plow, E.F (1991)

Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif Trends Biochem.

Sci 16, 246–250.

8 Kloczewiak, M , Timmons, S , Lukas, T J & Hawiger, J (1984)

Platelet receptor recognition site on human fibrinogen.Synthesis

and structure–function relationship of peptides corresponding to

the carboxy-terminal segment of the gamma chain Biochemistry

23, 1767–1774.

9 Sims, P.J., Ginsberg, M.H., Plow, E.F & Shattil, S.J (1991)

Effect of platelet activation on the conformation of the plasma

membrane glycoprotein IIb–IIIa complex J Biol Chem 266,

7345–7352.

10 Savage, B., Marzec, U.M., Chao, B.H., Harker, L.A.,

Maraganore, J.M & Ruggeri, Z.M (1990) Binding of the snake

venom-derived proteins applaggin and echistatin to the arginine–

glycine–aspartic acid recognition site(s) on platelet glycoprotein

IIb–IIIa complex inhibits receptor function J Biol Chem 265, 11766–11772.

11 Gulino, D., Ryckewaert, J.-J., Andrieux, A., Rabiet, M.-J & Marguerie, G.(1990) Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation J Biol Chem 265, 9575–9581.

12 Kouns, W C , Wall, C D , White, M M , Fox, C F & Jennings, L.K (1990) A conformation-dependent epitope of human platelet glycoprotein IIIa J Biol Chem 265, 20594–20601.

13 Kouns, W.C & Jennings, L.K (1991) Activation-independent exposure of the GPIIb-IIIa fibrinogen receptor Thromb Res 63, 343–354.

14 Pierschbacher, M.D.& Ruoslahti, E.(1984) Variants of the cell recognition site of fibronectin that retain attachment-promoting activity Proc Natl Acad Sci USA 81, 5985–5988.

15 Shebuski, R.J., Berry, D.E., Bennett, D.B., Romoff, T., Storer, B.L., Ali, F & Samanen, J (1989) Demonstration

of Ac–Arg–Gly–Asp–Ser–NH2 as an antiaggregatory agent in the dog by intracoronary administration Thromb Haemost 61, 183–188.

16 Samanen, J., Ali, F., Romoff, T., Calvo, R., Sorenson, E., Vasko,

J , Storer, B , Berry, D , Bennett, D , Strohsacker, M , Power, D , Stadel, J.& Nichols, A.(1991) Development of a small RGD peptide fibrinogen receptor antagonist with potent antiaggre-gatory activity in vitro J Med Chem 34, 3114–3125.

17 Haubner, R , Gratias, R , Diefenback, B , Goodman, S L , Jonczyk, A.& Kessler, H.(1996) Structural and functional aspects

of RGD-containing cyclic pentapeptides as highly potent and selective integrin a v b 3 antagonists J Am Chem Soc 118, 7461–7472.

18 Locardi, E., Mullen, D.G., Mattern, R.-H & Goodman, M (1999) Conformations and pharmacophores of cyclic RGD con-taining peptides which selectively bind integrin alpha (v) beta3.

J Pept Sci 5, 491–506.

19 Ojima, I , Chakravarty, S.& Dong, Q.(1995) Antithrombotic agents: from RGD to peptide mimetics Bioorg Med Chem 3, 337–360.

20 Du, X , Plow, E F , Frelinger, A L , O’Toole, T E , Loftus, J C & Ginsberg, M.H (1991) Ligands activate integrin IIb (platelet GpIIb–IIIa) Cell 65, 409–416.

21 Lam, S.C.-T (1992) Isolation and characterization of a chymo-tryptic fragment of platelet glycoprotein IIb–IIIa retaining Arg– Gly–Asp binding activity J Biol Chem 267, 5649–5655.

22 Wippler, J , Kouns, W C , Schlaeger, E J , Kuhn, H , Hadvary, P.

& Steiner, B.(1994) The integrin alpha IIb–beta 3, platelet gly-coprotein IIb–IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.

J Biol Chem 269, 8754–8761.

23 D’Souza, S.E., Ginsberg, M.H., Burke, T.A., Lam, S.C.-T & Plow, E.F (1988) Localization of an Arg–Gly–Asp recognition site within an integrin adhesion receptor Science 242, 91–93.

24 Loftus, J.C., Smith, J.W & Ginsberg, M.H (1994) Integrin-mediated cell adhesion: the extracellular face J Biol Chem 269, 25235–25238.

25 Nermut, M.V., Green, N.M., Eason, P., Yamada, S.S & Yamada, K.M (1988) Electron microscopy and structural model of human fibronectin receptor EMBO J 7, 4093–4099.

26 Hantgan, R.R., Braaten, J.V & Rocco, M (1993) Dynamic light scattering studies of alpha IIb beta 3 solution conformation Biochemistry 32, 3935–3941.

27.Krchnak, V.& Vagner, J.(1990) Color-monitored solid-phase multiple peptide synthesis under low-pressure continuous-flow conditions Pept Res 3, 182–193.

28.Wang, S S.(1973) p-Alkoxybenzyl alcohol resin and p-alkoxy-benzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments J Am Chem Soc 95, 1328–1333.

29 Stewart, J.M & Young, J.D (1984) Principles of Peptide Synthesis,

2nd edn.Pierce Chemical Co., Rockford, IL.

30.Atherton, E.& Sheppard, R C.(1989) Solid Phase Peptide

Synthesis: A Practical Approach.IRL Press, Oxford, England.

31.Bodansky, M & Bodansky, A.(1994) The Practice of Peptide

Synthesis, 2nd edn.Springer, Berlin.

32 Hopp, T.P & Woods, K.R (1981) Prediction of protein antigenic

determinants from amino acid sequences Proc Natl Acad Sci.

USA 78, 3824–3828.

33 Goudevenos, J., Tselepis, A.D., Tsoulatos, D., Grekas, G.,

Kritikakos, J.& Sideris, D.(1995) Platelet aggregability to platelet

activating factor at rest and after exercise in patients with coronary

artery disease Eur Heart J 16, 1036–1043.

34 Xia, Z , Wong, T , Liu, Q , Kasirer-Friede, A , Brown, E &

Frojmovic, M.M (1996) Optimally functional fluorescein

iso-thiocyanate-labelled fibrinogen for quantitative studies of binding

to activated platelets and platelet aggregation Br J Haematol 93,

204–214.

35 Frojmovic, M.M., Wong, T & Van de Ven, T (1991)

Dynamic measurements of the platelet membrane glycoprotein

IIb–IIIa receptor for fibrinogen by flow

cytometer.I.Methodo-logy, theory and results for two distinct activators Biophys J 59,

815–827.

36.Xia, Z.& Frojmovic, M M.(1994) Aggregation efficiency of

activated normal or fixed platelets in a simple shear fluid: effect of

shear and fibrinogen occupancy Biophys J 66, 2190–2201.

37 Frojmovic, M.M., Mooney, R.M & Wong, T (1994) Dynamics

of platelet glycoprotein IIb–IIIa receptor expression and

fibrino-gen binding.I.Quantal activation of platelet subpopulations

varies with adenosine diphosphate concentration Biophys J 67,

2060–2068.

38 Soteriadou, K.P., Tzinia, A.K., Hadziantoniou, M.G & Tzartos,

S.J (1988) Identification of monomeric and oligomeric forms of a

major Leishmania infantum antigen by using monoclonal

anti-bodies Infect Immun 56, 1180–1186.

39 Tsikaris, V., Sakarellos, C., Sakarellos-Daitsiotis, M., Cung, M.T.,

Marraud, M., Konidou, G., Tzinia, A & Soteriadou, K.P (1996)

Use of sequential oligopeptide carriers (SOCn) in the design of

potent Leishmania gp63 immunogenic peptides Pept Res 9,

240–247.

40 Golden, A., Brugge, J.S & Shattil, S.J (1990) Role of platelet

membrane glycoprotein IIb–IIIa in an agonist-induced tyrosine

phosphorylation of platelet proteins J Cell Biol 111, 3117–3127.

41 D’Souza, S.E., Ginsberg, M.H., Burke, T.A & Plow, E.F (1990)

The ligand binding site of the platelet integrin receptor GPIIb-IIIa

is proximal to the second calcium-binding domain of its alpha

subunit J Biol Chem 265, 3440–3446.

42 Calvete, J.J., Schafer, W., Mann, K., Henschen, A &

Gonzalez-Rodriguez, J.(1992) Localization of the cross-linking sites of

RGD and KQAGDV peptides to the isolated fibrinogen receptor,

the human platelet integrin glycoprotein IIb/IIIa.Influence of

peptide length Eur J Biochem 206, 759–765.

43 Tanaka, S., Hayashi, T., Hori, Y., Terada, C., Sup Han, K., Seop

Ahn, H., Bourre, F & Tani, Y (2002) A Leu55to Pro substitution

in the integrin a IIb is responsible for a case of Glanzmann’s

thrombasthenia Br J Haematol 118, 833–835.

44.Springer, T.(1994) Folding of the N-terminal, ligand-binding region of integrin a-subunits into ab-propeller domain Proc Natl Acad Sci USA 94, 65–72.

45 Kamata, T., Tieu, K.K., Irie, A., Springer, T.A & Takada, Y (2001) Amino acid residues in the alpha IIb subunit that are cri-tical for ligand binding to integrin alpha IIbbeta3 are clustered in the beta-propeller model J Biol Chem 276, 44275–44283.

46 Ambo, H., Kamata, T., Handa, M., Kawai, Y., Oda, A., Murata, M., Takada, Y & Ikeda, Y (1998) Novel point mutations in the

a IIb subunit (Phe289fiSer, Glu324fiLys and Gln747fiPro) causing thrombasthenic phenotypes in four Japanese patients.

Br J Haematol 102, 829–840.

47 Ferrer, M , Fernandez-Pinel, M , Gonzalez-Manchon, C , Gonzalez, J , Ayuso, M S.& Parrilla, R.(1996) A mutant (Arg327fiHis) GPIIb associated to thrombasthenia exerts a dominant negative effect in stably transfected CHO cells Thromb Haemost 76, 292–301.

48 Ruan, J., Peyruchaud, O., Alberio, L., Valles, G., Clemetson, K., Bourre, F.& Nurden, A.T.(1998) Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann’s thrombasthenia.

Br J Haematol 102, 918–925.

49 Tao, J , Arias-Sagado, E G , Gonzalez-Manchon, C , Iruin, G , Butta, N., Ayuso, M.S & Parrilla, R (2000) A 1063G fi A mutation in exon 12 of glycoprotein (GP) IIb associated with a thrombasthenic phenotype: mutation analysis of [324E] GPIIb.

Br J Haematol 111, 965–973.

50 Derrick, J.M., Taylor, D.B., Loudon, R.G & Gartner, T.K (1997) The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin a IIb b 3 Biochem J.

325, 309–313.

51 Derrick, J.M., Loudon, R.G & Gartner, T.K (1998) Peptide LSARLAF activates a IIb b 3 on resting platelets and causes resting platelet aggregate formation without platelet shape change Thromb Res 89, 31–40.

52 Derrick, J.M., Shattil, S.J., Poncz, M., Gruppo, R.A & Gartner, T.K (2001) Distinct domains of a IIb b 3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an a IIb b 3 interacting peptide Thromb Haemost 86, 894–901.

53 Soteriadou, K.P., Remoundos, M.S., Katsikas, M.C., Tzinia, A.K., Tsikaris, V., Sakarellos, C & Tzartos, S.J (1992) The Ser–Arg–Tyr–Asp region of the major surface glycoprotein of Leishmania mimics the Arg–Gly–Asp–Ser cell attachment region

of fibronectin J Biol Chem 267, 13980–13985.

54 D’Souza, S.E., Ginsberg, M.H., Matsueda, G.R & Plow, E.F (1991) A discrete sequence in a platelet integrin is involved in ligand recognition Nature 350, 66–68.

55 Taub, R , Gould, J , Garsky, V M , Ciccarone, T M , Hoxie, J , Friedman, P.A & Shattil, S.J (1989) A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen J Biol Chem.

264, 259–265.

56 Akiyama, S.K & Yamada, K.M (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.

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