ORIGINAL PAPER

Pascal Maurice Æ Ludovic Waeckel Æ Viviane Pires

Pascal Sonnet Æ Monique Lemesle Æ Brigitte Arbeille

Jany Vassy Æ Jacques Rochette Æ Chantal LegrandFrancoise Fauvel-Lafeve

The platelet receptor for type III collagen (TIIICBP) is presentin platelet membrane lipid microdomains (rafts)

Accepted: 8 September 2005 / Published online: 5 October 2005� Springer-Verlag 2005

Abstract Platelet interactions with collagen are orches-trated by the presence or the migration of plateletreceptor(s) for collagen into lipid rafts, which are spe-cialized lipid microdomains from the platelet plasmamembrane enriched in signalling proteins. Electronmicroscopy shows that in resting platelets, TIIICBP, areceptor specific for type III collagen, is present on theplatelet membrane and associated with the open cana-licular system, and redistributes to the platelet mem-brane upon platelet activation. After platelet lysis by 1%Triton X-100 and the separation of lipid rafts on a dis-continuous sucrose gradient, TIIICBP is recovered inlipid raft-containing fractions and Triton X-100 insolu-ble fractions enriched in cytoskeleton proteins. Plateletaggregation, induced by type III collagen, was inhibitedafter disruption of the lipid rafts by cholesterol deple-tion, whereas platelet adhesion under static conditionsdid not require lipid raft integrity. These results indicatethat TIIICBP, a platelet receptor involved in plateletinteraction with type III collagen, is localized withinplatelet lipid rafts where it could interact with otherplatelet receptors for collagen (GP VI and a2b1 integrin)for efficient platelet activation.

Keywords Platelets Æ Lipid rafts Æ Collagen Æ Plateletreceptors

Abbreviations BSA: Bovine serum albumin Æ FcR-cchain: c-Chain of the immunoglobulin Fc receptor ÆGEMs: Glycolipid-enriched membrane domains Æ GP:Glycoprotein Æ HEPES: N-(2-hydroxyethyl) piperazine-N¢-(2-ethanesulfonic acid) Æ HRP: Horse radishperoxidase Æ IgG: Immunoglobulin G Æ IgM: Immunoglobulin M Æ ITAM: Immunoreceptor tyrosineactivation motif Æ LAT: Linker for activation of Tcells Æ MbCD: Methyl-b-cyclodextrin Æ MES: 2-(N-morpholino)ethanesulfonic acid Æ PBS: Phosphatebuffered saline Æ SDS: Sodium dodecylsulfate Æ SDS/PAGE: Sodium dodecylsulfate/polyacrylamide gelelectrophoresis Æ TIIICBP: Type III collagen-bindingprotein Æ TBS: Tris-buffered saline Æ TRAP-6:Thrombin-related aggregating protein

Introduction

The platelet plasma membrane allows signal transduc-tion between extracellular stimuli and proteins at thecytoplasmic side of the plasma membrane. These eventsare essential for physiological platelet responses such asshape change, spreading, secretion, and aggregation.

Lipids and proteins in plasma membranes are un-equally distributed and form distinct microdomainscalled glycolipid-enriched membrane domains (GEMs),or rafts, identified in almost all cells and characterizedby their specific composition in lipids and proteins (Si-mons and Ikonen 1997). These microdomains are rich inglycosphingolipids, saturated phospholipids, and cho-lesterol (Brown and London 2000). In resting platelets,rafts are uniformly distributed over the plasma mem-brane and platelet stimulation by collagen or thrombinresults in their rapid clustering into larger domains(Kono et al. 2002; Bodin et al. 2003a).

Pascal Maurice and Ludovic Waeckel have contributed equally tothis work.

P. Maurice Æ L. Waeckel Æ J. Vassy Æ C. LegrandF. Fauvel-Lafeve (&)INSERM, U 553, IFR 105, Institut d’Hematologie, UniversiteParis VII Denis Diderot, 75475 Paris, FranceE-mail: f.lafeve@chu-stlouis.frTel.: +33-1-53724026Fax: +33-1-53724027

V. Pires Æ P. Sonnet Æ J. RochetteUPRES EA 3901 Unite INERIS, Universite de Picardie JulesVernes, Pole Sante, 80037 Amiens, France

M. Lemesle Æ B. ArbeilleLaboratoire de Microscopie Electronique, Faculte de Medecine,Hopital Bretonneau, Universite Francois Rabelais, 37032 Tours,France

Histochem Cell Biol (2006) 125: 407–417DOI 10.1007/s00418-005-0076-y

The presence of a variety of membrane protein recep-tors within platelet lipid rafts such asCD36,GPVI,GPIb-IX-V, and FccRIIa (Dorahy et al. 1996; Locke et al. 2002;Shrimpton et al. 2002; Wonerow et al. 2002; Bodin et al.2003b), as well as proteins involved in cell signalling suchas the transmembrane adaptor LAT, tyrosine kinases ofthe Src family (Fyn and Lyn) and phosphatidylinositolphosphates (Bodin et al. 2003a), has led to the consensusthat these lipid domains play an important role in theprocess of signal transduction. Indeed, platelet stimula-tion by thrombin or collagen is largely dependent on theintegrity of rafts (Kono et al. 2002; Bodin et al. 2003a, b),as their disruption by methyl-b-cyclodextrin (MbCD)results in the inhibition of platelet aggregation (Ezumiet al. 2002; Grgurevich et al. 2003).

Type I and type III collagens are predominant vas-cular thrombogenic molecules that are located withinthe subendothelium. They are rapidly exposed to plate-lets after a vascular lesion and participate in early hae-mostasis phases. Several platelet receptors for collagenhave been described in the literature, the most physio-logically important being GPIaIIa (a2b1 integrin) andGPVI (Nieswandt and Watson 2003).

We have described a new receptor for type III collagen,named TIIICBP for Type III Collagen Binding Protein(MM 68–72 kDa) (Monnet and Fauvel-Lafeve 2000),that specifically recognizes an octapeptide sequence,KOGEOGPK (O for hydroxyproline), within the a(1)III-CB4 fragment (residues 655–662) of human type III col-lagen. This octapeptide represents a minimal bindingsequence from type III collagen able to interact withplatelets (Fauvel et al. 1979) that prevents platelet contactand spreading on type III collagen and subendothelium,under both static and flow conditions but does not affectplatelet interactions with type I collagen (Monnet andFauvel-Lafeve 2000). Moreover, the binding of the octa-peptide to the platelets induces the phosphorylation ofTIIICBP and other signalling proteins involved in plateletactivation by collagen, such as the c-chain of the immu-noglobulin Fc receptor and LAT (Maurice et al. 2004).

In this study, we have investigated the localization ofTIIICBP in platelets and demonstrated that TIIICBP isassociated with the plasma membrane and the opencanalicular system in resting platelets, and redistributesto the outside plasma membrane upon platelet activa-tion by collagen or TRAP-6. After separation of thelipid-enriched microdomains, TIIICBP appears associ-ated with lipid rafts and the cytoskeleton, in both restingand activated platelets, thus strengthening a role forTIIICBP in platelet activation and signal transductioninduced by type III collagen.

Materials and methods

Reagents and antibodies

Calf skin type III collagen was purchased from Chem-icon International (Temecula, CA). The KOGEOGPK

octapeptide was synthesized on an Applied BiosystemsModel 433A peptide synthesizer, using standard auto-mated continuous-flow solid-phase peptide synthesismethods. Synthesis was initiated with commerciallyavailable Fmoc (9-fluorophenylmethoxy)-Lys-Sasrin(Bachem, Germany). Coupling reactions were mediatedby 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate and diisopropylethylamine in N-methyl-2-pyrrolidone solvent, using a standard Fast-Moc protocol. The terminal Fmoc group of the growingpeptide chain was removed using 20% piperidine in N-methyl-2-pyrrolidone. The peptide was then cleavedfrom the resin, using a 90% trifluoro acetic solution indichloromethane, and purified by reversed-phase highperformance liquid chromatography. Electrospray massspectrometric sequence analysis was used to check theexpected sequence (KOGEOGPK). The biotinylatedoctapeptide was prepared as previously described(Monnet and Fauvel-Lafeve 2000). The protease inhib-itor cocktail Complete was from Roche (Basel, Swit-zerland). The anti-TIIICBP monoclonal antibody 1G2was obtained as previously described (Monnet et al.2001). The anti-a2 integrin chain monoclonal antibody6F1 was provided by Dr. B. Coller (Mount Sinai Hos-pital, New York, NY). The anti-CD36 monoclonalantibody FA6, the anti-vinculin, and anti-FcR c-chainpolyclonal antibodies were purchased, respectively, fromBeckman (Midland, ON), Sigma (St. Louis, MO), andUpstate Biotechnologies (Lake Placid, NY).

Platelet isolation

Blood from consenting healthy human donors was an-ticoagulated with acid-citrate dextrose. The plateletswere washed as previously described (Fauvel-Lafeveet al. 1993), and resuspended into a pH 7.5 Tyrode’sbuffer (137 mM NaCl, 2.7 mM KCl, 1.2 mM NaHCO3,0.36 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 5 mMHEPES, 5.5 mM glucose) to the required final concen-tration.

Electron microscopy

The washed platelets, either unstimulated or stimulatedwith 10 lM TRAP-6 (Calbiochem, San Diego, CA), for5 min at 37�C, were fixed by the addition of 2% para-formaldehyde and 0.1% glutaraldehyde in 100 mM so-dium phosphate buffer pH 7.4, for 10 min at 20�C. Thefixed platelets were washed twice with PBS–0.35% BSAby centrifugation for 5 min at 1,300 g, dehydrated ingraded ethanol, and embedded in LR White mediumresin (Taab Lab Equipment, Kent, UK). Polymerizationwas performed for 48 h at �20�C. Ultrathin sections(70 nm) were cut with a Reichert OM-U3 ultramicro-tome (Reichert Scientific Instruments, Oxford, UK) andmounted on collodion-coated 200- or 300-mesh, thin-bargold grids (Biocell Research Lab, Copenhagen, OE).Labelling was carried out by an indirect immunogold

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procedure essentially as previously described (Arbeilleet al. 1991). The platelet sections were incubated with theanti-TIIICBP monoclonal antibody 1G2 (Monnet et al.2001) diluted to 100 lg/ml in PBS–1% BSA, for 60 minat 20�C. The grids were washed several times with PBS–0.1% BSA, then incubated with a goat anti-mouse IgMconjugated to 20 nm gold particles (Biocell ResearchLab) (1:30) for 40 min at 20�C, and washed extensivelywith PBS–0.1% BSA and distilled water.

Confocal microscopy

The washed platelets were deposited either on polyly-sine- or octapeptide-coated glass slides, for 30 min atroom temperature. After washes in PBS, the slides werefixed with 4% paraformaldehyde, washed again in PBSand incubated first with 3% BSA for 1 h, then with10 lg/ml FA6 or 50 lg/ml 1G2 antibodies or both, forone night at 4�C. The slides were washed in PBS andincubated with the Alexa fluor 488-anti-mouse IgG(Molecular Probes, San Francisco, CA) (1:50) and Texasred-anti-mouse IgM antibodies (Calbiochem) (1:100),for 30 min at room temperature. After washes in PBS,the slides were mounted in Moviol and observed with aconfocal microscope as previously described (Vassyet al. 2001).

Cholesterol depletion and repletion

Cholesterol depletion was performed by incubatingplatelets (109/ml) in pH 6.5 buffer A (140 mM NaCl,5 mM KCl, 5 mM KH2PO4, 1 mM MgSO4, 10 mMHEPES, 5 mM glucose, 0.2% BSA), containing 5 mMMbCD (Sigma), for 15 min at 37�C. For cholesterolrepletion, the cholesterol-depleted platelets were centri-fuged and resuspended in buffer A, without BSA, con-taining 2 mM water-soluble cholesterol (Sigma), for30 min at 37�C. The platelets were washed and used foraggregation experiments.

Platelets adhesion and aggregation

Washed platelet adhesion to the immobilized octapep-tide or type III collagen was performed in coated wellsas previously described (Maurice et al. 2004). Kineticstudies were carried out by incubating control or cho-lesterol-depleted platelets on immobilized type III col-lagen or octapeptide for 0–20 min at room temperature.The number of adherent platelets was determinedaccording to Bellavite et al. (1994). The results are themean of three assays run in triplicate ± SEM.

Aggregation was monitored by the turbidimetricmethod, using a dual-channel Beckman Chrono-Logaggregometer (Chrono-Log, Claremont, CA). The wa-shed platelets (3·108 ml) were activated by type IIIcollagen (20 lg/ml) at 37�C and the change in lighttransmission was continuously recorded.

Platelet raft isolation

Resting or activated platelets (2.109/0.5 ml) were lysedby adding an equal volume of ice-cold 50 mM MES-buffered saline pH 6.5 (50 mM MES, 150 mM NaCl,2 mM orthovanadate, 2 mM sodium fluorure, completeprotease inhibitors) containing 2% Triton X-100. Sub-sequent procedures were performed at 4�C. The lysatewas adjusted to 1.37 M sucrose (40%) by the addition ofan equal volume of 2.74 M sucrose prepared in 25 mMMES-buffered saline. A discontinuous sucrose gradient[1 M (30%), 0.8 M (25%), 0.6 M (20%), 0.5 M (15%),0.3 M (10%), 0.15 M (5%), 1.33 ml each] was layeredon the top of the 1.37 M (40%) homogenate. The raft-enriched fractions were obtained by flotation after ul-tracentrifugation (200,000 g for 16 h at 4�C) in a Beck-man SW41 rotor. Eight fractions of 1.37 ml wereharvested with a pipette from the top of the tube andthen diluted (9·) in 25 mM MES-buffered saline. Theproteins were separated from the sucrose solution byultracentrifugation (245,000 g for 45 min at 4�C) andrecovered in 100 ll of 10 mM Tris/HCl buffer pH 6.5containing 2% SDS. Proteins in the fractions werequantified in a BCA assay (Pierce, Rockford, IL).

Dot and western blots

Raft detection in gradient fractions was performed bydot blot. Ten micro litres from each fraction were ad-sorbed on 0.2 lm nitrocellulose membrane. The mem-branes were then saturated with 5% non-fat milk inTBS-buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl)containing 0.05% Tween 20.

Raft-containing fractions were revealed after incu-bation of the membrane, with HRP-conjugated choleratoxin B subunit (Calbiochem) that binds to the plateletraft GM1 ganglioside (Harder et al. 1998), for 1 h atroom temperature. The membrane was washed fivetimes with TBS and the reaction was detected with ECL(Amersham, Bucks, UK).

The presence of TIIICBP in raft fractions was de-tected by a ligand blot, using the biotinylated octapep-tide followed by HRP–streptavidin (Sigma) aspreviously described (Monnet and Fauvel-Lafeve 2000).For other proteins, the membranes were incubated, for1 h at room temperature, with anti-CD36 (2 lg/ml),anti-a2 integrin chain (2 lg/ml), anti-FcR c-chain(1:5,000), or anti-vinculin (1:400) antibodies, washedfour times in TBS, incubated with a 1:5,000 dilution ofHRP-conjugated anti-mouse or anti-rabbit IgG anti-bodies (Dako, Carpinteria, CA), for 1 h at room tem-perature and revealed as above. The dot blots wereanalysed by densitometry using the Image J program(http://rsb.info.nih.gov/ij/).

The presence of TIIICBP in raft-containing fractionswas verified after separation by SDS/PAGE (Laemmli1970) of proteins present in the gradient fractions fol-lowed by the ligand blot as above.

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Results

Electron microscopy

Figure 1 shows a labelling by the monoclonal antibody1G2, directed against TIIICBP of the platelet membraneand open canalicular system, in resting platelets (Fig. 1a).After platelet activation induced by TRAP-6, 1G2 deco-rated the platelet membrane more intensely (Fig. 1b).

Isolation of platelet membrane rafts

Resting or type III collagen-activated platelets werelysed by 1% Triton X-100 and fractionated through asucrose step gradient. After ultracentrifugation, anopaque band could clearly be seen at a density between10 and 18% sucrose (Fig. 2a). The sucrose gradient wasfractionated into eight fractions of 1.25 ml from the topof the gradient, and the opaque band was generallyrecovered in fractions 2–4. The position of lipid raftswithin the sucrose gradient (fractions 2–4) was deter-mined by a dot blot, using the cholera toxin B subunitthat specifically recognizes the raft marker gangliosideGM1 (Harder 1998) (Fig. 2b).

In agreement with previous studies (Dorahy et al.1996), the low-density raft-containing fractions (frac-tions 2–4) contained less than 5% of total platelet pro-teins (Fig. 2c) and the majority of the proteins wererecovered into higher-density fractions (7–8) that cor-responded to the cytoskeleton Triton X-100 insolublefractions (see below).

Platelet protein distribution in sucrose gradient fractions

By a dot blot analysis, the greatest part of CD36, amajor raft protein (Dorahy et al. 1996), was recovered in

fractions 2–4 (Fig. 3a) in resting platelets and its local-ization was not modified after platelet stimulation bytype III collagen. In the resting platelets, vinculin, acytoskeleton protein, was principally recovered intoTriton X-100 insoluble fractions 7 and 8. A part ofvinculin moved to the raft-containing fractions afterplatelet stimulation (Fig. 3b), suggesting a close inter-action between the rafts and cytoskeleton proteins dur-ing platelet activation.

The presence of a platelet collagen receptor (integrina2-chain) and a signalling protein (FcR c-chain) in raftfractions was also revealed using specific antibodies. Theintegrin a2-chain was gathered into raft fractions andfraction 8, after platelet activation by type III collagen(Fig. 3c). In resting platelets, the FcR c-chain was moreor less present in all gradient fractions and relocalizedinto raft fractions after type III collagen-induced plateletactivation (Fig. 3d).

TIIICBP association with membrane rafts

Figure 4 shows that TIIICBP is present in raft fractions(fractions 2–4) and the Triton X-100 insoluble fraction(fraction 8) in resting platelets, using either dot blot orligand blot experiments, after the electrophoretic sepa-ration of proteins from the different sucrose gradientfractions (Fig. 4a, b). The stimulation of platelets bytype III collagen caused a moderate shift in the distri-bution of TIIICBP from the cytoskeleton to raft-con-taining fractions (Fig. 4c).

Raft disruption by cholesterol depletion

Membrane cholesterol depletion is now an establishedmethod for disrupting lipid rafts and raft-associatedfunctions. In this study, we selectively depleted plateletmembrane cholesterol using the cholesterol-complexing

Fig. 1 Electron microscopic localization of TIIICBP in platelets. Resting (a) or TRAP-6 activated platelets (b) were incubated with the1G2 monoclonal anti-TIIICBP antibody and the fixed antibody was revealed by a 12 nm gold-conjugated anti-mouse IgM antibody

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agent MbCD. The treatment of platelets with MbCDinduced a complete destruction of the rafts, verified bythe delocalization of the GM1 ganglioside (not shown),and led to a shift of TIIICBP into Triton X-100insoluble fractions (Fig. 4d).

Confocal microscopy

The resting platelets were labelled with the FA6 anti-CD36 (green fluorescence) and 1G2 anti-TIIICBP (redfluorescence) monoclonal antibodies (Fig. 5a, b). As

Fig. 2 Isolation of platelet lipid rafts. Platelets lysed by 1% TritonX-100 were deposited on a (0.15–1 M) discontinuous sucrosegradient. After ultracentrifugation (200,000 g for 16 h), the lipidraft-enriched fraction was observed as an opalescent ring in theupper part of the gradient (a). Height equal fractions were

harvested from the top of the tube and the presence of lipid raftsin the fractions was assessed by dot blot using the HRP-conjugatedcholera toxin B subunit. Lipid rafts were identified into fractions 2–4 (b). Platelet protein concentration in each gradient fraction wasmeasured using a BCA assay (c)

Fig. 3 Platelet protein distribution into sucrose gradient fractions.The distribution of CD 36 (a), vinculin (b), integrin alpha 2-chain(c), and FcR c-chain (d) into the sucrose gradient fractions fromresting (filled triangle) and collagen-activated platelets (filledsquare) was determined by densitometry analysis of dot blots

performed with each specific antibody. As control, the densito-metric profile of the dot blot staining with the HRP–cholera toxinB subunit is shown on the CD 36 profile (filled circle). Results arerepresentative of two independent experiments

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shown in the merge picture (Fig. 5c), the same plateletstructures were labelled with both antibodies, with asustained labelling of the plasma membrane in somehighly fluorescent patches. In cholesterol-depletedplatelets, the labellings were diffuse (Fig. 5d–f).

Platelet aggregation and adhesion

In washed platelets, type III collagen (20 lg/ml) in-duced 70% platelet aggregation after 5 min of stirringin the aggregometer (Fig. 6a). This aggregation wasdecreased to 40% after raft destruction by MbCD(Fig. 6b) and could be totally recovered after choles-terol repletion, with a slight enhancement of theplatelet reactivity (80% aggregation) and a shorteningof the lag phase (Fig. 6c).

In contrast to platelet aggregation, platelet adhesionto immobilized type III collagen or octapeptide was notinhibited by treatment with MbCD, as well the totalnumber of adherent platelet as the kinetics of adhesion(Fig. 7a, b). By confocal microscopy, control andcholesterol-depleted platelets adhered similarly to theoctapeptide. They were spread and did not present,anymore, the typical raft staining. The labellings werediffuse for both anti-TIIICBP and CD36 antibodies(Fig. 8).

Discussion

Lipid rafts are specific areas within plasma membranesthat result from the lateral organization of sphingo-lipid and cholesterol into moving platforms ontowhich specific proteins attach (Simons and Ikonen1997). Proteins such as GPI-anchored proteins (Brownand London 2000), transmembrane proteins (Skibbenset al. 1989; Sargiacomo et al. 1993; Danielsen and vanDeurs 1995) like LAT (Simons and Toomre 2000) anddoubly acylated tyrosine kinases of the Src familyare linked to these lipid rafts (Dorahy and Burns1998).

During platelet activation induced by collagen,GPVI, which plays an essential role in collagen-inducedsignal transduction, migrates into rafts and non-cova-lently binds to the FcR c-chain (Locke et al. 2002) thatcontains an immunoreceptor tyrosine activation motif(ITAM). ITAM is phosphorylated by the Src familytyrosine kinases, Fyn and Lyn, constitutively associatedwith the cytoplasmic domain of GPVI (Suzuki-Inoueet al. 2002). The association between GP VI and the FcRc-chain occurs in lipid rafts. Several studies have shownthat the absence of GPVI (Nieswandt et al. 2001; Katoet al. 2003; Kuijpers et al. 2003), its blockage by specificantibodies (Lecut et al. 2003, 2004; Siljander et al. 2004)

Fig. 4 Localization of TIIICBP into sucrose gradient fractions. aThe position of TIIICBP into the sucrose fraction was revealed bydot blot using the biotinylated octapeptide and HRP–streptavidin.As control, the raft-containing fractions were labelled by HRP–cholera toxin B subunit. b Platelet proteins in the sucrose gradientfractions were separated by SDS-PAGE and the presence ofTIIICBP was revealed by ligand blot as above. c Densitometricanalysis of dot blots showing TIIICBP position into sucrosegradient fractions from resting (filled triangle) and type III

collagen-activated platelets (filled square). Results are representa-tive of three independent experiments. d Platelets depleted incholesterol by incubation for 15 min at 37�C with 5 mM methyl-b-cyclodextrin (see ‘Materials and methods’) were analysed forTIIICBP position in the sucrose gradient fractions as above.MbCD-treated platelets (filled circle) and control platelets (filledtriangle). Results are representative of two independent experi-ments

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or the competition with a soluble form of GPVI(Massberg et al. 2004; Gruner et al. 2005) inhibit theformation of surface platelet aggregates under flowconditions. However, the blockage of GPVI does not

seem to affect the platelet primary adhesion to collagen(Lecut et al. 2003, 2004; Siljander et al. 2004), suggestingthat other receptors for collagen could be involved in theadhesion process.

Fig. 5 Localization of TIIICBP by confocal microscopy. Plateletswere deposited on polylysine coated glass slides and incubated withthe 1G2 anti-TIIICBP and FA6 anti-CD 36 antibodies followed byincubation with an anti-mouse IgM antibody coupled to Texas redand anti-mouse IgG antibody coupled to Alexa fluor 488. a Platelet

labelling with the anti-TIIICBP antibody. b Platelet labelling withthe anti-CD 36 antibody. c Merge picture. d Cholesterol-depletedplatelets labelled with the anti-TIIICBP antibody. e Cholesterol-depleted platelets labelled with the anti-CD 36 antibody. f Mergepicture

Fig. 6 Effect of cholesterol depletion on platelet aggregation induced by 20 lg/ml type III collagen. a Control platelets. b Plateletsdepleted in cholesterol by incubation for 15 min at 37�C with 5 mM methyl-b-cyclodextrin. c Cholesterol-depleted platelets incubated for30 min at 37�C with 2 mM water-soluble cholesterol

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We have previously described a platelet receptorspecific for type III collagen (TIIICBP) that recognizesthe KOGEOGPK octapeptide sequence within type III

collagen (Monnet and Fauvel-Lafeve 2000). Theblockage of this receptor by the octapeptide or specificmonoclonal antibodies inhibits platelet contact, adhe-

Fig. 7 Effect of cholesterol depletion on platelet adhesion inducedby type III collagen or the KOGEOGPK peptide. a Adhesionnumber: control platelets (white bars), platelets depleted incholesterol (grey bars) or platelets repleted in cholesterol (hatchedbars) were deposited onto type III collagen- or octapeptide-coatedwells for 1 h at room temperature. After washings, adherent

platelets were measured by colorimetry. b Adhesion kinetics:control platelets (white bars) or cholesterol-depleted platelets (greybars) were incubated on type III collagen- or octapeptide-coatedwells for time varying from 0 to 20 min. Adherent platelet numberwas determined as above. Results are the mean of threeindependent assays run in triplicate ± SEM

Fig. 8 Confocal microscopy of adherent platelets. Control orcholesterol-depleted platelets were deposited on octapeptide-coatedglass slides and incubated with the anti-TIIICBP and the anti-

CD36 antibodies followed by secondary antibodies as in Fig. 5. aPlatelet labelling with the anti-TIIICBP antibody. b Plateletlabelling with the anti-CD 36 antibody. c Merge picture

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sion, and aggregation induced by type III collagen orsubendothelial matrix under static or flow conditions(Monnet and Fauvel-Lafeve 2000; Monnet et al. 2001).Using flow cytometry, we have observed that TIIICBPis expressed at the cell surface in resting platelets andits expression is increased after platelet activation,suggesting the existence of an intra-platelet pool ofTIIICBP (Monnet et al. 2001). In this study, electronmicroscopy revealed the presence of TIIICBP in theplasma membrane’s outer surface and also in theplatelet cytosol, as TIIICBP appeared linked to theopen canalicular system formed by invaginations of theplatelet membrane. The open canalicular system un-folds after platelet activation, which allows the redis-tribution of the internal pool of TIIICBP to the plateletsurface.

We previously reported (Maurice et al. 2004) that the68 kDa protein of TIIICBP was tyrosine phosphory-lated during platelet adhesion to the immobilized KO-GEOGPK peptide, together with signalling proteinssuch as the FcR c-chain, LAT, Syk, and PLCc2.Moreover, the FcR c-chain and LAT co-immunopre-cipitated with TIIICBP in non-activated platelets, indi-cating a physical link between these three proteins in theplatelet membrane (Maurice et al. 2004). Since the FcRc-chain and LAT are localized in platelet lipid rafts(Bodin et al. 2003b) we have searched for the presence ofTIIICBP in platelet membrane microdomains that weisolated after lysis of platelets with Triton X-100. Pre-vious works have shown that detergent insoluble lipidrafts are truly the unique product of the method bywhich they have been prepared (Pike 2003). Using sev-eral detergents (Triton X-100, Triton X-114, Brij35,Brij58, Brij98, or NP 40) at concentrations between 0.1and 1%, TIIICBP, as revealed by dot blot with thebiotinylated octapeptide, was always found in the su-crose gradient floating fractions (not shown). However,in our hands, 1% Triton X-100 appeared to be the bestchoice allowing an optimal separation of rafts, as at-tested by the position of the GM1 ganglioside intofractions 2–4 (Fig. 2). According to Baglia et al. (2003),the position of the GM1 ganglioside is not affected bythe detergent concentration or by platelet activation.Our raft separation procedure was also validated by thepresence of CD 36, another protein known to be locatedin membrane microdomains, in the same three fractions.In platelets, anti-CD 36 antibodies are considered asgood markers for confocal microscopy (Shrimpton et al.2004). In our experiments, we observed a colocalizationof CD 36 and TIIICBP, forming patches in unstimulatedplatelet plasma membrane. These patches disappearedafter disruption of the lipid rafts by MbCD. Thesepatches were not visible in platelets adherent to collagenor octapeptide, indicating a disruption of lipid raftsduring platelet adhesion and spreading.

In agreement with a previous work (Locke et al.2002), we found that the FcR c-chain was present in allsucrose gradient fractions and redistributed to rafts afterplatelet activation by type III collagen. Locke et al.

(2002) have shown that only the phosphorylated form ofthe FcR c-chain was localized in rafts, independently ofthe FcR c-chain binding to GP VI. In resting platelets,we also identified the integrin a2-chain in all sucrosegradient fractions and observed its relocalization intorafts upon platelet activation by type III collagen. Suchredistribution of this integrin chain has been observed inJurkat cells adherent to type IV collagen (Holleran et al.2003).

The cytoskeleton protein vinculin was recovered intofractions 7–8 containing Triton X-100 insoluble proteins.After platelet activation by type III collagen, a part ofvinculin redistributed from fractions 7–8 to raft-con-taining fractions. A similar shift of actin, anothercytoskeleton protein was observed after Fc�RI receptorcross-linking inmast cells (Holowka et al. 2000) andmorerecently in human platelets activated by TRAP-6 (Bodinet al. 2005). Thus, in activated platelets, raft-associatedactin and vinculin could be highly linked to raft receptorproteins and remain associated after Triton lysis.

Together, these results demonstrated that our raftseparation procedure was in agreement with data of theliterature and could be used to localize TIIICBP. Thisreceptor was found in raft-containing fractions, asshown by its co-localization with the GM1 ganglioside,and also in the cytoskeleton-containing fraction 8. Thissuggests that TIIICBP would be present into rafts,highly associated with cytoskeleton proteins and thusunable to float in the gradient. This hypothesis is sup-ported by the partial re-localization of TIIICBP intoraft-containing fractions that followed vinculin shifting,after platelet activation by type III collagen.

Platelet membrane integrity is important for plateletaggregation induced by collagen. In agreement with re-sults of the literature obtained with type I collagen(Ezumi et al. 2002; Grgurevich et al. 2003), we demon-strate here that platelet membrane depletion in choles-terol results in 30% inhibition of platelet aggregationinduced by 20 lg/ml type III collagen. On the otherhand, repletion in cholesterol enhanced platelet aggre-gation induced by type III collagen, probably byincreasing platelet membrane fluidity.

In contrast to aggregation, platelet adhesion to typeIII collagen or to the octapeptide was not modified bycholesterol depletion, as shown by the number (Fig. 7)and the morphology of the adherent platelets (Fig. 8).We have previously reported that TIIICBP is involved inearly platelet contact with type III collagen, a stage thatdoes not require platelet activation (Monnet et al. 2000).In this study, we observed that platelet adhesion tocollagen or octapeptide is independent of lipid rafts.This could be explained by an artefact inherent to theadhesion assay. In this assay, collagen reactive- oroctapeptide-sequences are highly concentrated on asmall area, which may increase the number of interactivereceptors and favour their clustering, subsequent plateletactivation, and spreading.

In conclusion, the presence of TIIICBP in plateletlipid rafts, in association with the FcR c-chain, suggests

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a potential cooperation between TIIICBP and GPVI.TIIICBP could associate with GPVI that moves to raftsduring platelet aggregation induced by type III collagen,and contribute to full activation of PLCc2, calciummobilization, and activation of GPIIbIIIa. The presenceof TIIICBP in lipid rafts could also explain the inhibi-tory effect of anti-TIIICBP antibodies or the octapeptideon platelet aggregation induced by CRP or convulxin,two specific agonists for GPVI (Maurice et al. 2004).The binding of TIIICBP antagonists could impair GPVItransfer to rafts and its association with the FcR c-chain.

Finally, the localization of TIIICBP in rafts and itsassociationwith the cytoskeleton give some indications onthe physical nature of this receptor,whichwould contain ahighly hydrophobic transmembrane domain and a cyto-plasmic tail able to bind to cytoskeleton proteins.

Acknowledgements The authors are grateful to Mrs J. Treton andl’Etablissement Francais du Sang from Hopital Saint-Louis, Paris,for blood collection. This work was financed by funding from theINSERM, Universite Paris-7, and the Conseil Regional D’Ile-de-France (inter-regional program ‘‘Regulation de la matrice extra-cellulaire pathologique’’).

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