1

Revised M2:11282

Red cell ICAM-4 is a novel ligand for platelet activated αIIbβ3-integrin

Patricia Hermand#, Pierre Gane#, Martine Huet#, Vincent Jallu+, Cécile Kaplan+, HH

Sonneborn°, Jean-Pierre Cartron#§ and Pascal Bailly#

From #INSERM U76 and +Unité d’Immunologie plaquettaire, Institut National de la

Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France, °Biotest AG,

63276 Dreieich, Germany

*This investigation was supported in part by the Institut National de la Transfusion Sanguine (INTS),

the Institut National de la Santé et de la Recherche Médicale (INSERM. This article must therefore be

hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

§ correspondence : Dr Jean-Pierre Cartron

INSERM U76

Institut National de la Transfusion Sanguine

6 rue Alexandre Cabanel, 75015 Paris, France.

Phone : (33) 1.44.49.30.00

Fax : (33) 1.43.06.50.19

e-mail : cartron@idf.inserm.fr

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 10, 2002 as Manuscript M211282200 by guest on M

arch 22, 2018http://w

ww

.jbc.org/D

ownloaded from

2

Running title : ICAM4/αIIbβ3-integrin interaction, RBC, platelet GPIIb-IIIa, CHO

transfectants, hemostasis, thrombosis.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

ABSTRACT

ICAM-4 (LW blood group glycoprotein) is an erythroid specific membrane

component that belongs to the family of intercellular adhesion molecules and

interacts in vitro with different members of the integrin family, suggesting a potential

role in adhesion or cell interaction events, including hemostasis and thrombosis. To

evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized

RBCs, platelets and ICAM-Fc fusion proteins to a plastic surface and analyzed their

interaction in cell adhesion assays with RBCs and platelets from normal individuals

and patients, as well as with cell transfectants expressing the αIIbβ3 integrin. The

platelet fibrinogen receptor αIIbβ3 (platelet GPIIb-IIIa) in high affinity state following

GRGDSP peptide activation was identified for the first time as the receptor for RBC

ICAM-4. The specificity of the interaction was demonstrated by showing that (i)

activated platelets adhered less efficiently to immobilized ICAM-4-negative than to

ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the β3-chain alone and

for a complex-specific epitope of the αIIbβ3 integrin, and specific for ICAM-4 to a

lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb,

CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I

glanzmann’s thrombasthenia patients did not bind to coated ICAM-4. Further

support to RBC-platelet interaction was provided by showing that DTT-activated

αIIbβ3-CHO transfectants strongly adhere to coated ICAM-4-Fc protein but not to

ICAM-1-Fc, and was inhibitable by specific antibodies. Deletion of individual Ig-

domains of ICAM-4 and inhibition by synthetic peptides showed that the αIIbβ3

integrin binding site encompassed the first and second Ig-domains, and that the G65-

V74 sequence of domain D1 might play a role in this interaction. Although normal

RBCs are considered non adhesive during coagulation and are reported passively

entrapped in fibrin polymers, these studies identify ICAM-4 as the first RBC protein

ligand of platelets that may have relevant physiological significance.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

INTRODUCTION

The main physiological function of red blood cells (RBCs)1 which encapsulate

hemoglobin is to ensure the respiratory gases transport throughout of the human

body. However, the recent demonstration that mature RBCs express a growing

number of adhesion molecules, many of which exhibit blood group specificities (1-3),

reinforces the necessity to revisit the functional interaction of RBCs with leukocytes,

platelets and vascular endothelium in normal and pathological conditions.

It is interesting that many RBC adhesion molecules contain protein domains

characteristic of the immunoglobulin superfamily, suggesting some recognition

function. These molecules might participate to the normal RBC physiology, by

playing a role during erythropoiesis (differentation, maturation, enucleation, release),

self recognition mechanisms, red cell turnover and cell aging through cellular

interactions with counter receptors present on macrophages from bone marrow or

reticuloendothelial system in spleen and liver (1,4-9). Along this process, some

adhesion molecules are rapidly down-regulated and others are expressed at different

stages and remain on RBCs (10,11, and references herein). Finally, mature RBCs still

express adhesion molecules which are usually associated to leukocytes (CD44, CD47,

CD58) and others which have potential adhesion properties such as LW/ICAM-4

(CD242), Lu (CD239), Oka (CD147), CD99/Xg, JMH (CD108 ) and DO (1-3).

Nevertheless, normal RBCs do not adhere to circulating cells and vessel walls under

normal circumstances, suggesting that the RBC adhesion molecules are inaccessible

to their ligands. In contrast, the conversion of non adherent RBCs to adherent state

arise in several diseases. In such circumstances, adhesion molecules might be

involved in the pathophysiology of malaria (12,13), sickle cell disease (14-17) and

diabetes (18,19), mainly through an abnormal adhesion to the vascular endothelium

(1,20). Additionally, both phosphatidylserine exposure at the RBC surface and

adhesion molecules on these cells might also play a role in hemostasis and

thrombosis, for instance through interaction with cells expressing integrins, like

activated leukocytes, monocytes, platelets and endothelial cells (21,22). Interestingly

also, RBCs have the necessary signal transduction pathways to mediate these

functions (23).

Among RBC adhesion molecules, ICAM-4 (LW blood group glycoprotein, CD242)

emerges from the others by its structure similarities with ICAM-family and its

interaction characterized in vitro with different members of the β-integrin subfamilies

(αLβ2 (LFA-1), αMβ2 (Mac-1), α4β1 (VLA-4) (24-26), αV-integrins (αVβ1 and αVβ5) (27)).

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

These two families of proteins are well known to play crucial role in cell-cell

interactions and to be involved in large range of biological functions (28-31). For

instance, ICAM-4/integrin interaction might play a role during erythroid maturation

in bone-marrow or in the red cell turnover by spleen macrophages that express the

αdβ2 integrin (25,27,32). Additionally, ICAM-4 as well as the Lu blood group protein

might be involved in adhesion of sickle RBCs to TNF-α activated endothelial cells

(HUVEC) (7) and to laminin (33,34), respectively. It is suspected that abnormal

adhesion of sickle RBCs to endothelial cells and extracellular matrix proteins might

be responsible for the painful crisis of the disease that result from vaso-occlusive

episodes (35).

The purpose of this report was to examine the potential role of ICAM-4 in RBC-

platelet interaction and to demonstrate that this protein interacts in vitro with the

high affinity state of activated platelet αIIbβ3-integrin.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

MATERIALS AND METHODS

Blood samples, reagents and antibodies

RBC from donors with common and rare phenotypes (Donull, Lunull of the Lu(a-b-)

type, LWnull, JMHnull) came from the frozen RBC collection of the Centre National de

Référence pour les Groupes Sanguins (Paris, France). Fresh blood samples from two

unrelated type-I glanzmann’s thrombasthenia patients were obtained after informed

consent. Apyrase, prostaglandin-E1 (PGE1), thrombin from human origin and anti-

glycophorin-A mAb (clone E4) and the peptide Arg-Gly-Glu (RGE) were purchased

from Sigma (St. Louis, MO). Peptides Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-

Asp (RGD) and the Fibrinogen Binding Inhibitor (FBI) peptide (residues 400-411 of

the fibrinogen γ chain) were from Bachem (Budendorf, Switzerland). Other peptides

Gly-Trp-Val-Ser-Tyr-Gln-Leu-Leu-Asp-Val (G-V, residues 65-74 of ICAM-4), Cys-

His-Ala-Arg-Leu-Asn-Leu-Asp-Gly-Leu-Val-Val-Arg (C-R, residues 180-192 of

ICAM-4) and corresponding random (rd) peptides used were synthesized and

purified by Neosystem (Strasbourg, France). Specific mAbs used in this study include

clones P2 and SZ22 recognizing the αIIb-chain (CD41) in the presence and the absence

of the β3-chain, respectively, clones SZ21 and SZ2 specific for the β3-chain (CD61) and

GpIb protein (CD42b), respectively, clone FA6.152 specific for CD36 and clone

AICD58 specific for CD58, which were purchased from Coulter/Immunotech

(Marseille, France). The mAb AP-2 specific for a complex-specific epitope of the αIIbβ3

integrin came from GTI (Brookfield, WI). PAC-1 and AK-4 mAbs specific for

activated αIIbβ 3 complex and P-selectin (CD62P), respectively, came from BD-

Pharmingen (SanDiego, CA). The mAb 3E12 to CD47 was from BioAtlantique

(Nantes, France). The murine mAb BS56 to ICAM-4/LWab was previously described

(36). ImmunoPure mouse IgG from Pierce (Rockford, IL, USA) was used as negative

control IgG. Chimeric ICAM-pIgI constructs derived from intact ICAM-4 (LWa allele)

carrying the two Ig-like domains D1 and D2 (residues 1-208), or deletion mutants D1-

ICAM-4 (residues 1-101) or D2-ICAM-4/ (residues 102-208) were used to produce

soluble Fc-fusion proteins as described (26). ICAM-1- and ICAM-2-pIgI constructs

(kindly provided by Dr. D. Simmons and E. Ferguson, Oxford, UK) were used to

produce ICAM-Fc soluble fusion proteins as above.

GRGDSP-activated platelets

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

Human platelets were obtained from fresh ACD-anticoagulated blood from

volunteers not taking any medication and were washed three times in modified

Tyrode’s albumin buffer (5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3,

5.5 mM glucose, 0.1% (w/v) bovine serum albumin (pH 6.5), 250 ng/ml PGE1, 25

mg/ml apyrase) by centrifugation at 1,200 x g for 10 min. Platelets were activated as

previously described (37,38). Briefly, 1x108 washed platelets resuspended in 0.1 ml

Tyrode’s-albumin buffer (pH 7.4) containing 2 mM CaCl2 and 1 mM MgCl2, were

incubated at 22°C for 5 min with 1 mM GRGDSP peptide. Then, an equal volume of

phosphate buffered saline (PBS, 10 mM phosphate buffer in 0.15 M NaCl, pH 7.2)

containing PGE1 (250 ng/ml), apyrase (25 mg/ml), 2 mM CaCl2, 1 mM MgCl2 and

1% (w/v) paraformaldehyde, was added and the mixture was incubated for 1 hour at

22°C . Then, 0.2 ml of 500 mM NH4Cl was added to stop the reaction in PBS. Fixed

activated platelets were washed several times to remove the activating peptide prior

to assays and resuspended in modified Tyrode’s buffer pH 7.4 containing divalent

cations. Fixed unactivated platelets used as control, were prepared by omitting

divalent cations and the activating peptide in the different buffers.

Platelet adhesion assays to immobilized RBCs

RBCs were immobilized on microtiter plates through binding to coated anti-

glycophorin A. Briefly, mAb E4 at 20 µg/ml (50 µl/well) in 25 mM Tris pH 8, 150 mM

NaCl, was adsorbed overnight at 4°C on flat-bottom 96-well microtiter plates (Nunc

A/S, Roskilde, Denmark). After two washes of wells with the same buffer, RBCs

(2.0x106/well in a final volume of 300 µl) resuspended in modified Tyrode’s buffer

pH 7.4 with or without cations (2 mM MgCl2 and 2 mM CaCl2) were added. After 1

hour incubation at 22°C, fixed GRGDSP-activated or unactivated platelets

(5.0x106/well in a final volume of 100 µl) in modified Tyrode’s buffer pH 7.4 with or

without divalent cations, respectively, were added to RBC coated wells. After 90 min

at 22°C , non adherent cells were removed by filling the wells with binding buffer,

and the microplates were put to float upside down in a PBS solution. Cells that

sticked to the plastic wells were recovered by vigorous shaking in 400 µl of PBS and

were counted by flow cytometric analysis using a FACSCalibur (Becton Dickinson,

San Diego, CA). Platelets and RBCs were distinguished by forward scatters and

platelet staining with the FITC-anti-human CD61 mAb (clone VI-PL2, BD

Biosciences).

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

RBCs adhesion to adherent platelets

Following isolation, unactivated platelets (1x107/well in a final volume of 100 µl)

resuspended in RPMI 1640, 10 mM Hepes containing PGE1 and apyrase were added

to wells to adhere overnight at 37°C. After washing, adherent platelets were

stimulated with thrombin (0.5 unit in 100 µl/well) diluted in Hank’s Balanced Salts

(HBSS) containing 2 mM CaCl2 during 20 min at room temperature. After another

washing, RBCs (3.3x106/well in a final volume of 300 µl) resuspended in HBSS with 2

mM CaCl2, 1 mM MgCl2, were added to each well. After 90 min at 22°C , non

adherent RBCs were removed by filling the wells with binding buffer, and the

microplates were put to float upside down in a PBS solution. Then RBCs numeration

was done using a Nikon Eclipse TE300 microscope (Nikon, Paris, France) (10 x

objective) coupled to a Biocom informatic system of images integration (Biocom, les

Ulis, France). For blocking experiments, RBCs and adherent platelets stimulated by

thrombin were pretreated with specific mAbs (2.5 µg/well) and ICAM-Fc protein (2.5

µg/well), respectively, for 30 min at 22°C.

αIIbβ3-CHO transfectants and DTT activation

Chinese hamster ovary cell line (CHO) was grown in Iscove’s modified Dulbecco

medium with Glutamax-1 (Life Technologies, Inc.) supplemented with amphotericin-

B-penicillin-streptamycin and 10% fetal calf serum (FCS). CD41 (αIIb chain) and CD61

(β 3 chain) cDNAs subcloned into pcDNA3.1 vector (Invitrogen, Leek, The

Netherlands), kindly provided by Dr. P.J. Newman (Blood Center of Southeastern

Wisconsin, Milwaukee, WI), were cotransfected into CHO cells using the Lipofectin-

Reagent according to the manufacturer’s instructions (Life Technologies,

Gaithersburg, MD). Stable transformants resistant to G418 (0.6 mg/ml of geneticin)

were selected for CD41 and CD61 expression by immuno-magnetic separation using

mAb AP-2 and magnetic beads coated with anti-mouse IgG (Dynabeads-M-450,

DYNAL, Oslo, Norway). CD41 and CD61 expression of stable clones was analyzed

and quantified by flow cytometric analysis with Qifikit calibration beads, used

according to manufacturer’s instructions (Dako, Denmark). One clone with the

strongest expression of αIIbβ3-integrin was selected. For adhesion assays, αIIbβ3-CHO

transfectant and wild-type (parental) CHO cells were treated with or without 10 mM

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

DTT in RPMI 1640, 10 mM Hepes, at 22°C for 20 min to activate the αIIbβ3 complex

receptor (39).

Cell adhesion assays to immobilized proteins

Purified ICAM-Fc proteins diluted in 25 mM Tris pH 8.0, 150 mM NaCl, 2 mM MgCl2

and 2 mM CaCl2, were absorbed to flat-bottom 96-well microtiter plates overnight at

4°C, at 2.5-20 µg/ml (50 µl/well in triplicate). The wells were then blocked for 2 hrs at

22°C with 1% non-fat milk in the same buffer. For adhesion assays, either fixed

GRGDSP-activated or unactivated platelets (5x106/well in a final volume of 100 µl) in

modified Tyrode’s buffer pH 7.4 with or without divalent cations, respectively, wild-

type CHO cells, DTT-activated or unactivated αIIbβ3-CHO transfectants (1x105/well

in a final volume of 100 µl) resuspended in RPMI 1640, 10 mM Hepes containing 2

mM MgCl2 and 2 mM CaCl2, were added to the coated wells and incubated for 90

mins at 22°C. Non adherent cells were removed by washings before microscopic

observation and CHO cell numeration was done as indicated above. Platelets were

counted by flow cytometric analysis as above. For blocking experiments, the cells

were pretreated with specific peptides and their corresponding random counterpart

(125 µM final concentration) or with different mAbs (5 µg for 5x106 platelets or 1x105

CHO cells/100 µl) for 30 min at 22°C prior addition to protein-coated wells.

RESULTS

RBCs interact with activated platelets

To analyze molecular events occurring during RBC-platelet interaction, in vitro

cell adhesion assays were developed using RBCs from donors of common and rare

phenotypes immobilized to plastic surface via anti-GPA binding and platelets from

normal healthy donors, pretreated or not with the synthetic GRGDSP peptide in the

presence of inhibitors of platelet activation, thus resulting in specific αIIbβ3-integrin

activation and the acquisition of high affinity Fg-binding state without addition of a

cellular agonist (37). Accordingly, in addition to bind Fg, GRGDSP-treated platelets

reacted strongly with the mAb PAC-1, which binds to the activated αIIbβ3 complex

(40), but no reactivity with the mAb AK-4 (41), which binds to P-selectin normally

contained in intracellular α-granules (not shown). As shown in Fig. 1, GRGDSP-

activated platelets adhered more efficiently than unactivated platelets to immobilized

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

ICAM-4 positive RBCs from control donors. The 100% relative binding was

equivalent to 220 ± 100 GRGDSP-activated platelets adhered to 1.0 x 103 immobilized

RBCs. When unactivated platelets were used as control, a 69% reduced adhesion was

noted that corresponds to a mean background of 31±12%. As preliminary assays

showed that similar results were obtained with fresh and unfrozen RBCs (not

shown), the following studies were performed with unthawed RBCs since rare RBC

variants lacking different membrane proteins were available from our frozen

collection.

Activated platelets bind to coated RBCs lacking the blood group proteins Lu (CD239,

laminin receptor of 78-85 kDa), JMH (CD108, 80 kDa) and DO (ADP-

ribosyltransferase 4 of 47-67 kDa) but expressing normal levels of ICAM-4, as

efficiently as would normal ICAM-4 positive RBCs. Interestingly, when ICAM-4

negative (LWnull) RBCs lacking of the ICAM-4/LW glycoprotein (42 kDa) from three

unrelated donors were coated to plastic wells, a 40 % decrease binding of GRGDSP-

activated platelets was observed after deduction of the mean background

corresponding to the unactivated platelet adhesion to all types of RBCs (p<0.001 vs

unactivated platelets and p<0.05 vs controls).

To confirm that ICAM-4 plays a role in RBC-platelet interactions, RBC adhesion on

adherent platelets stimulated by thrombin, a more physiologically relevant platelet

activator than the RGDS peptide, was also analyzed although in this assay platelets

are more activated with α-granule release than GRGDSP-activated platelets (see

above). Although ICAM-4-positive RBCs did not bind to unstimulated adherent

platelets in the presence of PGE1 and apyrase (not shown), they bind strongly to

thrombin-stimulated platelets (Fig. 2). This binding was efficiently decrease to 50 ±

9% and 11 ± 1% by mAb BS56 and soluble ICAM-4-Fc protein, respectively, whereas

the mAb AICD58 reacting with the erythroid membrane CD58 protein and the

soluble ICAM-2-Fc protein had only a minor inhibitory effect (88 ± 4 and 85 ± 7%,

respectively). Similarly, mAbs anti-RhD (LOR-15C9), anti-Fy6 (BAM9917) and anti-

MER2 (1D12 or 2F7) directed against various RBC surface membrane proteins did not

exhibit any effect (not shown). Unfortunately, the non specific adherence of frozen

RBCs in this assay made impossible the comparative analysis between the ICAM-4

positive and negative RBCs. Altogether, these data suggests that ICAM-4 might take

a significant part (about 50%) in the adhesion of RBCs to activated platelets. As the

GRGDSP peptide is a trigger of a high affinity state of αIIbβ3-integrin which mediates

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

Fg binding and platelet aggregation (37), our data suggested that ICAM-4 might

interact with αIIbβ3-integrin but also with other adhesive molecules.

RBC-platelet interaction is mediated via ICAM-4

To obtain further evidence that ICAM-4 might interact with a high affinity

state of α IIbβ3-integrin, type-I glanzmann's thrombastenia platelets from two

unrelated patients who both exhibit a 6 bp deletion in exon 7 of the β3 gene (42), were

used for cell adhesion assay to coated ICAM-4-Fc protein. Fig. 3A shows that

unactivated platelets from normal control donors did not bind to immobilized ICAM-

4-Fc, as expected from above data, whereas the same platelets activated by the

GRGDSP peptide bound readily to coated ICAM-4-Fc, but not to immobilized ICAM-

1. The 100% relative binding of GRGDSP-activated platelets to ICAM-4-Fc was

equivalent to 12.5±3.0% of the total added platelets. Conversely, platelets from the

thrombasthenic patients type 1 with a severe defect of αIIbβ3-integrin surface

expression, either unactivated (not shown) or GRGDSP-activated, failed to bind to

coated ICAM-4-Fc (Fig. 3A).

In order to determine the specificity of these interactions, the effect of different mAbs

and synthetic peptides on the platelet adhesion to immobilized ICAM-4-Fc protein

was investigated (Fig. 3B). Adhesion of activated platelets from normal control

donors was efficiently blocked (approximately, 70% and 60%, respectively) by P2 and

AP2 mAbs specific for the αIIb-chain in the presence of the β3-chain and the complex-

specific epitope of the αIIbβ3 integrin, respectively. SZ21 and SZ22 mAbs which

recognize the β3- and αIIb-chains alone, respectively, and the BS56 mAb specific for

ICAM-4, partially but significantly inhibited the interaction between ICAM-4 and

activated platelets, whereas the SZ2 mAb directed against the GPIb platelet

glycoprotein and the control mouse IgG had no significant effect (Fig. 3B). In

addition, mAbs FA6 and 3E12 directed against CD36 and CD47, respectively, did not

inhibit the platelet-ICAM-4 interaction. Blocking experiments by synthetic peptides

revealed that the RGD peptide that binds to αIIbβ3-integrin and inhibits Fg binding,

strongly reduced by 75 % the adhesion of activated platelets to ICAM-4, whereas the

RGE peptide had no effect.

RBC-platelet interaction is mediated via ICAM-4 /αIIbβ3 –integrin

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

To provide further evidence that ICAM-4 may interact with the αIIbβ3 integrin,

stable CHO transfectants expressing recombinant human αIIbβ3 were generated and

used in cell adhesion assays (Fig. 4). Several αIIbβ3-CHO transfectants were obtained

and one clone expressing a high level of αIIbβ3-integrin (αIIb, 18,600 molecules/cell

and β 3 , 67,000 molecules/cell, as estimated by by flow cytometric analysis with

specific mAbs) was chosen for further studies. The αIIbβ3 integrin of these cells was

activated by DTT treatment and the adhesion of DTT-activated and unactivated

αIIbβ3-CHO transfectants to immobilized ICAM-4-Fc was examined (Fig. 4). In a

preliminary experiment we found that these cells also reacted with PCA-1 Mab that

recognized the activated αIIbβ3 integrin complex (not shown). DTT-activated αIIbβ3-

CHO transfectants dose-dependently bind to coated ICAM-4-Fc protein, whereas

untreated αIIbβ3-CHO transfectants as well as parental CHO cells, either or not

treated with DTT, did not bind at all. About 32% of the total added DTT-activated

αIIbβ3-CHO transfectants adhered to coated ICAM-4-Fc, but there was no binding to

immobilized ICAM-1-Fc protein used as control (Fig. 4B). Identical results were

obtained when the αIIbβ3-CHO transfectants were activated by the GRGDSP-peptide

instead of DTT (not shown). The binding of DTT-activated αIIbβ3-CHO transfectants

to immobilized ICAM-4-Fc could be blocked by approximately 50 % by mAbs

specific for ICAM-4 (BS56) or for the complex-specific epitope of the αIIbβ3 integrin

(AP2), but not with mAbs to the β3-chain (SZ21) and αIIb-chain (SZ22 and P2) of the

αIIbβ3 integrin, as shown on Fig. 4B. The absence of inhibition noted with the mAb P2,

which efficiently blocked activated platelet adhesion (see Fig. 3) might result from

conformational changes and/or glycosylation variations of αIIbβ3 integrin in platelets

and the CHO-transfectants independently of the mode of integrin activation. As

expected, control mouse IgG had no effect.

Putative domains on ICAM-4 that interact with the αIIbβ3 integrin

As an attempt to localize the αIIbβ3 integrin binding site on ICAM-4, domain deletion

mutants lacking either extracellular Ig-like domain D1 or domain D2 were produced

and used in cell adhesion assays to chimeric Fc-proteins. Fig. 5A showed that the

binding of DTT-activated αIIbβ3-CHO transfectants via the αIIbβ3 integrin required the

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

presence of both domains D1 and D2, since a 50% decrease binding was observed in

the absence of either domain D1 or D2. Similar effects with less amplitude were

observed using GRGDSP-activated platelets (see Fig. 5B).

Further blocking experiments with synthetic peptides were performed. Adhesion of

activated-platelet was efficiently inhibited to 14% and 58% by the Fibrinogen Binding

Inhibitor (FBI, Fg γ chain residues 400-411) peptide and the ICAM-4 peptide G-V

(residues 65-74), respectively, two peptides exhibiting a QxxDV motif involved in the

fibrinogen/αIIbβ3-integrin interaction (Fig. 5B). When DTT-activated α IIbβ3-CHO

transfectants were used, a 78% decrease binding was observed in the presence of the

ICAM-4 derived peptide G-V whereas the peptide FBI failed to inhibit (Fig. 5A). The

lack of inhibition by the peptide FBI, might result from some changes of αIIbβ3

integrin when expressed in CHO-transfectants, as suspected for the reactivity of mAb

P2 (see above). Of not, neither the random peptides of FBI and G65-V74 nor a control

ICAM-4 peptide (C-R, residues 180-192) had any inhibitory effect. Altogether, these

results demonstrate that the αIIbβ3 binding site on ICAM-4 encompassed domains D1

and D2, and that it seems to reside at the tip of the E strand of domain D1 which is in

contact with the loop C’-E of domain D2.

DISCUSSION

In this report in vitro cell adhesion assays have been developed to evaluate the

capacity of red cell ICAM-4 to interact with platelets and to identify the molecular

basis of the interaction. The αIIbβ3 integrin (platelet fibrinogen receptor GPIIb-IIIa)

was identified as the receptor for RBC ICAM-4. However, we found that the αIIbβ3

integrin had to be in its high affinity state to bind ICAM-4, as the interaction occurred

only after synthetic GRGDSP peptide activation, but not with untreated resting

platelets. This was based on the following evidence: (i) activated platelets adhered

less efficiently to immobilized ICAM-4-negative (LWnull) than to ICAM-4-positive

RBCs, (ii) monoclonal antibodies specific for a complex-specific epitope of the αIIbβ3

integrin or to the β3-chain alone and specific for ICAM-4 to a lesser extent, inhibited

platelet adhesion, (iii) activated platelets from two unrelated type-I glanzmann’s

thrombasthenia patients that are deficient for the αIIbβ3 integrin (and vitronectin

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

receptor αvβ3) did not bind to coated ICAM-4-Fc protein, and (iv) DTT-activated

αIIbβ3-CHO transfectants strongly adhere to coated ICAM-4-Fc protein but not to

coated ICAM-1-Fc, and this was inhibitable by specific antibodies. It should be

mentioned that αIIbβ3 integrin activation occurred in the absence of any signaling or

secretion (37,39) and that antibodies specific for GPIb, the von Willebrand receptor of

platelets, for CD36 (platelet GPIV) and CD47 (IAP, integrin-associated protein), two

multifunctional membrane proteins acting as thrombospondin receptors (43,44), did

not inhibit the platelet adhesion to immobilized ICAM4-Fc protein. As

thrombasthenic platelets that expressed normal levels of other platelet receptors like

the α2β1 integrin (collagen receptor), and the fibronectin and laminin receptors

(GPIa-IIa and GPIc’-IIa, respectively), did not adhere to ICAM4-Fc protein, it is

assumed that these proteins do not play a significant role in RBC-platelet interaction

under the experimental conditions used.

Further analysis with platelets, αIIbβ3–CHO transfectants and ICAM-4 Fc mutant

proteins have shown that the two Ig-like domains of ICAM-4 are required for αIIbβ3

integrin interaction, since domain deletion mutants lacking either the first (D1) or

second (D2) Ig-domain exhibited significant reduced binding (see Fig. 5A and B). A

similar effect has been observed when ICAM-4 mutant proteins interact with the

leukocyte αMβ2 (Mac-1) integrin, whereas interaction with the αLβ2 (LFA-1) integrin

requires predominantly the first Ig domain D1 (26). The binding of adhesive proteins

to αIIbβ3 integrin is predominantly mediated by the RGD peptide motif present on the

respective adhesive ligands (45), but this peptide which is absent from ICAM-4,

blocks the ICAM-4/αIIbβ3 interaction. The platelet αIIbβ3 integrin also binds to the

carboxy-terminal end of the Fg γ chain via a dodecapeptide sequence (peptide FBI,

residues 400-411) containing the motif QAGDV (46). Interestingly, ICAM-4 contains a

similar motif at position 70-74 (QLLDV) located in the first ICAM-4 Ig-like domain

(26) and the ICAM-4 peptide G-V (residues 65-74) including this motif is a potent

inhibitor of the ICAM-4/αIIbβ3 interaction. Moreover, the peptide FBI also inhibited

platelet binding to ICAM-4-Fc. These findings suggest that the platelet αIIbβ3 integrin

might interact with the G65-V74 sequence of ICAM-4 that includes a QxxDV motif

known to be involved in the fibrinogen/αIIbβ3-integrin interaction. The G65-V74

sequence motif which forms the tip of the E strand of domain D1 and is in contact

with the loop C’-E of domain D2 (26), most probably constitutes a major part of the

αIIbβ3 integrin binding site on ICAM-4 Therefore, binding inhibitions by RGD and FBI

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

peptides, which binds to the β3 chain (GPIIIa) and αIIb chain (GPIIb) respectively (47),

suggest that ICAM-4 binds to the same or overlapping site(s) on the αIIbβ3 complex. It

should be noticed that the G70R substitution responsible for the blood group LWa-

>LWb polymorphism (48) which corresponds to the first position of the QxxDV

motif, had no effect on RBC-platelet adhesion reported here (unpublished

observation), suggesting that this polymorphism is neutral with regards to ICAM-

4/αIIbβ3 integrin interaction.

Our studies therefore indicate that adhesion of normal RBCs to activated platelets

occur through a specific ligand/receptor interaction. Whether or not signaling events

across the platelet and/or RBC membranes are triggered by the interaction of the

αIIbβ3 integrin receptor with its RBC ICAM-4 ligand is currently unknown.

As LWnull RBCs still adhere to activated platelets and RBCs adhere to adherent

platelets stimulated by thrombin, which have release their α-granule contents, it is

assumed that other factors critical for interaction may exist. αIIbβ3 integrin activation

with either RGD-containing peptide or DTT induces Fg binding (37,39), suggesting

that indirect RBC-platelet contacts via this adhesive macro molecule might occur.

However, although ICAM-1 binds to Fg (49), interaction of the structurally related

ICAM-4 protein with Fg has not yet been documented. If such interaction exists, Fg

could form RBC-platelet and RBC-endothelium cross-bridges via RBC ICAM-4 and

αIIbβ3 integrin on activated-platelet and ICAM-1 and/or αvβ3-integrin present on the

stimulated vascular endothelium (50,51). Still other adhesion pathways mediating

cross-bridges of RBCs with platelets and/or endothelial cells with a variety of

adhesive proteins might also be operating, but this need further investigations.

Additionally, erythroid receptors for adhesive molecules, like the Lutheran (CD239,

laminin receptor) (33,34) and sulfated glycolipids (receptors of laminin, TSP, and

vWF) (52,53) are present on the RBCs and might also take part in the RBC-platelet

and endothelial cell interactions. Moreover, direct RBC-endothelial cell interaction

might also occur as ICAM-4 has been reported to bind to α4β1 (VLA-4), and to αvβ1-

and αvβ5-integrins present on hematopoietic cells and might also account for the

binding of sickle RBCs to vascular endothelium (27).

All these findings indicate that ICAM4 is an unusual adhesion molecule which has a

broad ligand binding specificity, including at least some β1, β2, β3 (this report), and β5

integrins, but the binding affinity for these ligands may vary widely. Another

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

example of receptor with a promiscuous specificity is the DARC protein (Duffy

Antigen receptor for Chemokines) which binds to CC and CXC families of

chemokines (54). Therefore, it is anticipated that ICAM-4 may have a potential role in

a number of physiological processes, including hemostasis and thrombosis (21,22).

Although further investigation of RBC interaction with blood cells and vascular cells

under various flow conditions should delineate more precisely the physiological

relevance of these interactions, RBC interaction with activated platelets, is supported

by several observations : (i) an active role in hemostasis and thrombosis (21,22) as

interaction between metabolically active RBCs and platelets is known to enhance

platelet reactivity (21), including the enhancement of αIIbβ3 integrin activation and P-

selectin expression (55), (ii) the presence of RBCs as well as of leukocytes in a

developing thrombus (56-58), in which platelets are activated and may interact with

RBCs, (iii) the presence of platelet-erythrocyte aggregates in patients with sickle cell

anemia (59,60) and end-stage renal disease (61). However, the molecular target(s)

responsible for RBCs-platelet interaction have not been characterized. Our results

provide the first direct characterization of a molecular interaction between normal

RBCs and platelets and together with the findings discussed above they strongly

suggest that RBCs may play an active role in hemostasis and thrombosis.

After this paper was submitted, adhesion of normal RBCs to fMLP (formyl-Met-Leu-

Phe peptide)-activated neutrophils and collagen-activated platelets, as well as to

fibrin, was shown under low shear rates conditions (62). Interestingly, the data

suggested that adhesion of RBCs to neutrophils might be mediated through Mac-1

(CD11b/CD18) and ICAM-4, supporting recent findings indicating that β2 integrins

and ICAM-4 interact with each other (26). Additionally, RBC-platelet interaction was

strongly reduced by soluble fibrinogen and EDTA and was partially inhibited by

antibodies to CD36 and GPIb, but no inhibition was noted with a single antibody

against the α IIb chain (CD41) of the α IIbβ3 integrin (62). Although no effect of

monoclonal antibodies to GPIb and CD36 was found in static conditions of assay, our

results indicate that the interaction of thrombin-activated platelets with intact RBCs

(Fig. 2) and of GRGDSP-activated platelets with immobilized ICAM-4 (Fig. 3B) could

be inhibited by soluble ICAM-4 (by 89%) and to at least 50% by the monoclonal

antibody P2 recognizing the αIIb-chain in the presence of the β3-chain, or by the

monoclonal antibody AP-2 specific for a complex-specific epitope of the αIIbβ3

integrin, respectively. Monoclonal antibodies SZ22 and SZ21 to the αIIb-chain and β3-

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

chain alone, respectively, were weak inhibitors in the latter condition (Fig. 3B).

Consistent with the above results, our studies have shown that ICAM-4 interaction

with β -integrins is calcium-dependent (26). Obviously, distinct experimental

conditions (platelet activation, flow conditions) and monoclonal antibodies used may

explain the reported differences.

In conclusion, although passive entrapment of RBCs during coagulation or

thrombosis is commonly accepted, these data provide independent evidence

indicating that a physiological interaction between RBCs and activated platelets (and

neutrophils) mediated by specific receptor/ligand interactions can occur in a variety

of biological process, notably during normal hemostatic conditions (clot formation),

pathological occlusion conditions (deep vein thrombosis, sickle cell disease) and

possibly inflammation, particularly under low blood flow conditions, close to static,

which may facilitate RBC adhesion events. Although ICAM-4 may play a significant

role, clearly other receptor/ligand interactions are likely to occur which deserves

further analysis.

Acknowledgements.

We would like to thank Dr David Simmons and Dr Elaine Ferguson for the supply of

the ICAM-pIgI constructs, and Dr P.J Newman for the CD41 and CD61 cDNAs in

pcDNA3.1 vector.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

FOOTNOTES

1the abbreviations used are : RBCs, red blood cells; FBI, Fibrinogen Binding Inhibitor; TSP, thrombospondin ; vWF, von Willebrand Factor ; mAb, monoclonal antibody ;PGE1, prostaglandin-E1 ; TNF-α, Tumor Necrosis Factor-α ; HUVEC, HumanUmbilical Vein Endothelium Cells.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

REFERENCES

1. Telen, M. J. (2000) Semin Hematol 37, 130-1422. Spring, F. A., and Parsons, S. F. (2000) Transfus Med Rev 14, 351-3633. Cartron, J. P., and Colin, Y. (2001) Transfus Clin Biol 8, 163-1994. Verfaillie, C., Hurley, R., Bhatia, R., and McCarthy, J. B. (1994) Crit Rev Oncol

Hematol 16, 201-2245. Hanspal, M. (1997) Curr Opin Hematol 4, 142-1476. Bratosin, D., Mazurier, J., Tissier, J. P., Estaquier, J., Huart, J. J., Ameisen, J. C.,

Aminoff, D., and Montreuil, J. (1998) Biochimie 80, 173-1957. Parsons, S. F., Spring, F. A., Chasis, J. A., and Anstee, D. J. (1999) Baillieres Best

Pract Res Clin Haematol 12, 729-7458. Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., and

Lindberg, F. P. (2000) Science 288, 2051-20549. Chan, J. Y., and Watt, S. M. (2001) Br J Haematol 112, 541-55710. Bony, V., Gane, P., Bailly, P., and Cartron, J. P. (1999) Br J Haematol 107, 263-27411. Southcott, M. J., Tanner, M. J., and Anstee, D. J. (1999) Blood 93, 4425-443512. Ward, T. M., Chitnis, C. E., and Miller, L. H. (1994) Baillère’s Clinical Infectious

Diseases 1, 155-19013. Oh, S. S., Chishti, A. H., Palek, J., and Liu, S. C. (1997) Curr Opin Hematol 4, 148-

15414. Wick, T. M., and Eckman, J. R. (1996) Curr Opin Hematol 3, 118-12415. Hebbel, R. P. (1997) J Clin Invest 99, 2561-256416. Serjeant, G. R. (1997) Lancet 350, 725-73017. Bunn, H. F. (1997) N Engl J Med 337, 762-76918. Wautier, J. L., Wautier, M. P., Schmidt, A. M., Anderson, G. M., Hori, O.,

Zoukourian, C., Capron, L., Chappey, O., Yan, S. D., Brett, J., and et al. (1994) ProcNatl Acad Sci U S A 91, 7742-7746

19. Schmidt, A. M., Hofmann, M., Taguchi, A., Yan, S. D., and Stern, D. M. (2000)Semin Thromb Hemost 26, 485-493

20. Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A., McEver, R.P., Pober, J. S., Wick, T. M., Konkle, B. A., Schwartz, B. S., Barnathan, E. S.,McCrae, K. R., Hug, B. A., Schmidt, A. M., and Stern, D. M. (1998) Blood 91, 3527-3561

21. Marcus, A. J., and Safier, L. B. (1993) Faseb J 7, 516-52222. Andrews, D. A., and Low, P. S. (1999) Curr Opin Hematol 6, 76-8223. Minetti, G., and Low, P. S. (1997) Curr Opin Hematol 4, 116-12124. Bailly, P., Hermand, P., Callebaut, I., Sonneborn, H. H., Khamlichi, S., Mornon, J. P.,

and Cartron, J. P. (1994) Proc Natl Acad Sci U S A 91, 5306-531025. Bailly, P., Tontti, E., Hermand, P., Cartron, J. P., and Gahmberg, C. G. (1995) Eur J

Immunol 25, 3316-332026. Hermand, P., Huet, M., Callebaut, I., Gane, P., Ihanus, E., Gahmberg, C. G., Cartron,

J. P., and Bailly, P. (2000) J Biol Chem 275, 26002-2601027. Spring, F. A., Parsons, S. F., Ortlepp, S., Olsson, M. L., Sessions, R., Brady, R. L.,

and Anstee, D. J. (2001) Blood 98, 458-46628. Springer, T. A. (1990) Nature 346, 425-43429. Luscinskas, F. W., and Lawler, J. (1994) Faseb J 8, 929-93830. Gahmberg, C. G., Tolvanen, M., and Kotovuori, P. (1997) Eur J Biochem 245, 215-

23231. Hayflick, J. S., Kilgannon, P., and Gallatin, W. M. (1998) Immunol Res 17, 313-327

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

32. Van der Vieren, M., Le Trong, H., Wood, C. L., Moore, P. F., St John, T., Staunton,D. E., and Gallatin, W. M. (1995) Immunity 3, 683-690

33. Udani, M., Zen, Q., Cottman, M., Leonard, N., Jefferson, S., Daymont, C., Truskey,G., and Telen, M. J. (1998) J Clin Invest 101, 2550-2558

34. El Nemer, W., Gane, P., Colin, Y., Bony, V., Rahuel, C., Galacteros, F., Cartron, J. P.,and Le Van Kim, C. (1998) J Biol Chem 273, 16686-16693

35. Hebbel, R. P., and Mohandas, N. (1994) Sickle Cell Diease : Basic Principles andClinical Pracrice, 217-230

36. Sonneborn, H. H., Ernst, M., and Voak, D. (1994) Vox Sanguinis 67, 11437. Du, X. P., Plow, E. F., Frelinger, A. L., 3rd, O'Toole, T. E., Loftus, J. C., and

Ginsberg, M. H. (1991) Cell 65, 409-41638. Xia, Z., and Frojmovic, M. M. (1994) Biophys J 66, 2190-220139. Zucker, M. B., and Masiello, N. C. (1984) Thromb Haemost 51, 119-12440. Abrams, C. S., Ellison, N., Budzynski, A. Z., and Shattil, S. J. (1990) Blood 75, 128-

13841. Lasky, L. A. (1992) Science 258, 964-96942. Morel-Kopp, M. C., Kaplan, C., Proulle, V., Jallu, V., Melchior, C., Peyruchaud, O.,

Aurousseau, M. H., and Kieffer, N. (1997) Blood 90, 669-67743. Greenwalt, D. E., Lipsky, R. H., Ockenhouse, C. F., Ikeda, H., Tandon, N. N., and

Jamieson, G. A. (1992) Blood 80, 1105-111544. Brown, E. J., and Frazier, W. A. (2001) Trends Cell Biol 11, 130-13545. Pytela, R., Pierschbacher, M. D., Ginsberg, M. H., Plow, E. F., and Ruoslahti, E.

(1986) Science 231, 1559-156246. Kloczewiak, M., Timmons, S., Lukas, T. J., and Hawiger, J. (1984) Biochemistry 23,

1767-177447. Plow, E. F., D'Souza, S. E., and Ginsberg, M. H. (1992) Semin Thromb Hemost 18,

324-33248. Hermand, P., Gane, P., Mattei, M. G., Sistonen, P., Cartron, J. P., and Bailly, P. (1995)

Blood 86, 1590-159449. Tsakadze, N. L., Zhao, Z., and D'Souza, S. E. (2002) Trends Cardiovasc Med 12, 101-

10850. Altieri, D. C. (1999) Thromb Haemost 82, 781-78651. Byzova, T. V., Rabbani, R., D'Souza, S. E., and Plow, E. F. (1998) Thromb Haemost

80, 726-73452. Hillery, C. A., Du, M. C., Montgomery, R. R., and Scott, J. P. (1996) Blood 87, 4879-

488653. Roberts, D. D., Williams, S. B., Gralnick, H. R., and Ginsburg, V. (1986) J Biol Chem

261, 3306-330954. Neote, K., Darbonne, W., Ogez, J., Horuk, R., and Schall, T. J. (1993) J Biol Chem

268, 12247-1224955. Valles, J., Santos, M. T., Aznar, J., Martinez, M., Moscardo, A., Pinon, M.,

Broekman, M. J., and Marcus, A. J. (2002) Blood 99, 3978-398456. Saniabadi, A. R., Lowe, G. D., Barbenel, J. C., and Forbes, C. D. (1985) Thromb Res

38, 225-23257. Joseph, R., Welch, K. M., D'Andrea, G., and Riddle, J. M. (1989) Thromb Res 53,

485-49158. Bernstein, E., and Kairinen, E. (1971) Science 173, 76559. Wun, T., Paglieroni, T., Tablin, F., Welborn, J., Nelson, K., and Cheung, A. (1997) J

Lab Clin Med 129, 507-516

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

60. Wun, T., Paglieroni, T., Field, C. L., Welborn, J., Cheung, A., Walker, N. J., andTablin, F. (1999) J Investig Med 47, 121-127

61. Sirolli, V., Strizzi, L., Di Stante, S., Robuffo, I., Procopio, A., and Bonomini, M.(2001) Thromb Haemost 86, 834-839

62. Goel, M. S., and Diamond, S. L. (2002) Blood 100, 3797-3803

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

FIGURE LEGENDS

Fig. 1. Adhesion of platelets to immobilized RBCs.

Adhesion of GRGDSP-activated platelets (gray bars) from normal donors to ICAM-4

positive (ctrl, Lunull, JMHnull, Donull) and ICAM-4 negative (LWnull) frozen RBCs

immobilized onto plastic wells. Unactivated platelets (hatched bars) were used as

controls and the horizontal line visualized the mean background (31±12% )

corresponding to the unactivated platelet adhesion to all types of RBCs. The results

are expressed as the relative percentage of bound platelets, where 100% is calculated

from the total number of normal GRGDSP-activated platelets bound to immobilized

RBCs. The mean ± SEM from at least six experiments is shown. By Student's t test

analysis, ***p<0.001 versus unactivated platelet and ∆p<0.05 or less versus control.

Fig. 2. Adhesion of RBCs to adherent platelets.

Adhesion of ICAM-4 positive RBCs to adherent platelets stimulated by thrombin (0.5

unit/well). RBCs and stimulated adherent platelets were pretreated or not with

saturating concentration of mAbs to ICAM-4 (BS56) and ICAM-4-Fc protein,

respectively. The percentage of bound RBCs is indicated on the top right of each field

of view. The 100% corresponds to the total number of RBCs bound to adherent

platelets. Controls include mAb anti-CD58 (AICD58) which binds to RBCs, unrelated

mouse IgG antibody (ctrl IgG) and ICAM-2-Fc protein.

Fig. 3. Adhesion of platelets to immobilized ICAM-4-Fc protein.

A, Microphotographs showing the comparative adhesion of GRGDSP-activated

platelets from normal or type-I glanzmann’s thrombasthenia (GT type-I) patients to

ICAM-Fc proteins coated to flat-bottom 96-well microtiter plates (1 µg/well). ICAM-

1-Fc protein and unactivated normal platelets were used as negative controls. B,

Adhesion of normal GRGDSP-activated platelets to coated ICAM-Fc proteins (1

µg/well) and pretreated or not with saturating concentrations of mAbs specific for

ICAM-4 (BS56), β3-chain (SZ21), αIIb-chain (SZ22 and P2), αIIbβ3 complex (AP2), CD47

(3E12) and CD36 (FA6.152), or pretreated with 125 µM (final concentration) of RGE

and RGD peptides. The results are expressed as the relative percentage of activated

platelets bound to coated ICAM-Fc proteins. The 100% value is calculated from the

total number of activated platelets bound in the absence of peptides or mAbs.

Negative controls include mAb SZ2 specific for platelet gpIb, unrelated mouse IgG

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

antibody (ctrl IgG), ICAM-1-Fc and wells without coated protein. The mean ± SEM

from three experiments is shown. By Student’s t test analysis, **p<0.01, and *p<0.05.

Fig. 4. Adhesion of αIIbβ3-CHO transfectants to immobilized ICAM-4-Fc.

A, Dose-dependent cell adhesion of DTT-activated αIIbβ3-CHO transfectants (�) and

of control including parental CHO cells (DTT-treated or not) and unactivated αIIbβ3-

CHO transfectants (����) to ICAM-4-Fc protein coated to plastic wells at varying

concentration. The results are expressed as mean percentage of bound cells ± SEM of

three experiments. B, Effect of different mAbs on the adhesion of DTT-activated

αIIbβ3 CHO-transfectant to ICAM-4-Fc coated (500 ng/well). Cells were pretreated or

not with saturating concentrations of indicated mAbs specific for ICAM-4 (BS56), β3-

chain (SZ21), αIIb-chain (SZ22 and P2) and αIIbβ3 complex (AP2). The results are

expressed as the relative percentage of activated αIIbβ3-CHO transfectants bound to

coated ICAM-Fc proteins as in Fig. 3. Controls included unrelated mouse IgG

antibody (ctrl IgG) and wells coated with either ICAM-1-Fc (500 ng/well) or no

protein at all. The mean ± SEM from three experiments is shown. By Student's t test

analysis, ***p<0.001.

Fig. 5. ICAM-4 domains interacting with platelet aIIbb3 integrin .

Adhesion of DTT-activated αIIbβ3-CHO transfectants (A) and GRGDSP-activated

platelets (B) to intact ICAM-4-Fc (+) and to ICAM-4-Fc domain deletion mutants (D1

or D2) coated to plastic surface (500 ng/well) and effects of Fg γ chain and ICAM-4

peptides on cells binding. Cells were pretreated with the Fibrinogen Binding

Inhibitor peptide (FBI, residues 400-411) and peptides G-V (residues 65-74) and C-R

(residues 180-192) derived from ICAM-4 at the final concentration of 125 µM. After 30

mins incubation, the cells were tested for binding to coated ICAM-4-Fc (+). For each

peptide tested, the corresponding random (rd) peptide was used as control. C-R

peptide was used as a control ICAM-4 sequence. The results are expressed as

indicated in Fig. 4. Negative adhesion controls include wells without coated protein.

The mean ± SEM from three experiments is shown. By Student’s t test analysis,

***p<0.001, and **p<0.01.

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Fig. 1

0

20

40

60

80

100

120

Rel

ativ

e b

ind

ing

of

pla

tele

ts (

%)

ICAM-4 negative RBCs

ctrl

.

Donull

Lunull

JMHnull

***

*********

***����

***����

***����

LWnull 1

LWnull 2

LWnull 3

ICAM-4 positive RBCs

GRGDSP-actived

normal platelets

unactivated

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

+ BS56

+ Ctrl. IgG

+ ICAM-4-Fc

+ ICAM-2-Fc

50±9 % 11±1 %

97±4 %

100±4 %

88±4 % 85±7 %

+ AICD58

ICAM-4 positive RBCs on thrombin-stimulated platelets

Fig. 2

by guest on March 22, 2018http://www.jbc.org/Downloaded from

Fig. 3

ICAM-1-Fc ICAM-4-Fc

Normal platelets GT (type-I) plateletsA

GRGDSP-activated

ICAM-4-Fc

unactivated

ICAM-4

- -

B

-

20

40

60

80

100

120

0

R

elat

ive

bin

din

g o

fG

RG

DS

P-a

ctiv

ated

pla

tele

ts (

%)

ICAM-1

Antibody or peptide

-

- - -

Fc-protein

20

40

60

80

100

120

0

Patien

t 1

Patien

t 2

RGE RGD

**

SZ21 SZ22 P2 AP2BS56 3E12ctrl.IgGSZ2FA6

**

*

****

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Fig. 4

- BS56 AP2SZ21 SZ22 ctrl.IgGP2

+ + + + + + +--

0

20

40

60

80

100

120

Rel

ativ

e b

ind

ing

of

DT

T-a

ctiv

ated

αααα

IIbββββ 3

CH

O-t

ran

sfec

tan

t (%

)

Antibody

Fc-protein

B

-+

ICAM-1 ICAM-4

***

***

DTT-activatedααααIIbββββ3 CHO-transfectant

Bo

un

d c

ells

(%

)

ICAM-4-Fc protein (ng)

10

15

20

25

30

35

40

0 200 400 600 800 1000

5

0

A

- unactivated CHO-cell- unactivated ααααIIbββββ3 CHO-transfectant

- DTT-activated CHO-cell

control

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Fig. 5

Rel

ativ

e b

ind

ing

of

DT

T-a

ctiv

ated

αααα

IIbββββ

3 C

HO

-tra

nsf

ecta

nt

(%)

-D1 D2 + +

--

peptide

Fc-protein

-

ICAM-4

0

20

40

60

80

100

120

+FBI rdFBI G-V rdG-V C-R-

+ + +

A

*** ***

***

Rel

ativ

e b

ind

ing

of

GR

GD

SP

-act

ivat

ed p

late

let

(%)

-D1 D2 + +

--

peptide

Fc-protein

-

ICAM-4

0

20

40

60

80

100

120

+FBI rdFBI G-V rdG-V C-R-

+ + +

B

***

***

** **

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Sonneborn, Jean-Pierre Cartron and Pascal BaillyPatricia Hermand, Pierre Gane, Martine Huet, Vincent Jallu, Cécile Kaplan, Hans H

(sub3}-integrinβIIbαRed cell ICAM-4 is a novel ligand for platelet activated

published online December 10, 2002J. Biol. Chem. 

  10.1074/jbc.M211282200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on March 22, 2018

http://ww

w.jbc.org/

Dow

nloaded from