Red Cell ICAM-4 Is a Novel Ligand for Platelet-activated alpha IIbbeta 3 Integrin*

Patricia HermandDagger , Pierre GaneDagger , Martine HuetDagger , Vincent Jallu§, Cécile Kaplan§, H. H. Sonneborn, Jean-Pierre CartronDagger ||, and Pascal BaillyDagger

From Dagger  INSERM U76 and the § Unité d'Immunologie plaquettaire, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France and  Biotest AG, 63276 Dreieich, Germany

Received for publication, November 5, 2002, and in revised form, December 10, 2002

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 red blood cells (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 alpha IIbbeta 3 integrin. The platelet fibrinogen receptor alpha IIbbeta 3 (platelet GPIIb-IIIa) in a 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 beta 3-chain alone and for a complex-specific epitope of the alpha IIbbeta 3 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 dithiothreitol-activated alpha IIbbeta 3-Chinese hamster ovary 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 alpha IIbbeta 3 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 passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main physiological function of red blood cells (RBCs),1 which encapsulate hemoglobin, is to ensure the respiratory gases transport throughout 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 under 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 in the normal RBC physiology by playing a role during erythropoiesis (differentiation, 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 (Refs. 10 and 11, and references therein). Finally, mature RBCs still express adhesion molecules which are usually associated with leukocytes (CD44, CD47, CD58) and others that 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 arises 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 structural similarities to the ICAM family and its interaction characterized in vitro with different members of the beta  integrin subfamilies (alpha Lbeta 2 (LFA-1), alpha Mbeta 2 (Mac-1) (24-26), alpha 4beta 1 (VLA-4), alpha V integrins (alpha Vbeta 1 and alpha Vbeta 5); Ref. 27). These two families of proteins are well known to play crucial role in cell-cell interactions and to be involved in a 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 alpha dbeta 2 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-alpha -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 alpha IIbbeta 3 integrin.

    MATERIALS AND METHODS
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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. 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 gamma -chain; Fg) were from Bachem (Budendorf, Switzerland). Other peptides Gly-Trp-Val-Ser-Tyr-Gln-Leu-Leu-Asp-Val (Gly-Val, 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 alpha IIb-chain (CD41) in the presence and the absence of the beta 3-chain, respectively, clones SZ21 and SZ2 specific for the beta 3-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 alpha IIbbeta 3 integrin came from GTI (Brookfield, WI). PAC-1 and AK-4 mAbs specific for activated alpha IIbbeta 3 complex and P-selectin (CD62P), respectively, came from BD PharMingen (San Diego, 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 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-- 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 µg/ml apyrase) by centrifugation at 1,200 × g for 10 min. Platelets were activated as previously described (37, 38). Briefly, 1 × 108 washed platelets resuspended in 0.1 ml of 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 µg/ml), 2 mM CaCl2, 1 mM MgCl2, and 1% (w/v) paraformaldehyde, was added, and the mixture was incubated for 1 h 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 (2.0 × 106/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 h of incubation at 22 °C, fixed GRGDSP-activated or unactivated platelets (5.0 × 106/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 adhered to the plastic wells were recovered by vigorous shaking in 400 µl of PBS and were counted by flow cytometric analysis using a FACSCalibur. Platelets and RBCs were distinguished by forward scatters and platelet staining with the fluorescein isothiocyanate (FITC)-anti-human CD61 mAb (clone VI-PL2, BD Biosciences).

RBCs Adhesion to Adherent Platelets-- Following isolation, unactivated platelets (1 × 107/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 Hanks' Balanced Salts (HBSS) containing 2 mM CaCl2 for 20 min at room temperature. After another washing, RBCs (3.3 × 106/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 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.

alpha IIbbeta 3-CHO Transfectants and DTT Activation-- The Chinese hamster ovary cell line (CHO) was grown in Iscove's modified Dulbecco medium with Glutamax-1 (Invitrogen) supplemented with amphotericin-B-penicillin-streptamycin and 10% fetal calf serum. CD41 (alpha IIb-chain) and CD61 (beta 3-chain) cDNAs subcloned into pcDNA3.1 vector (Invitrogen), 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 (Invitrogen). 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 the manufacturer's instructions (Dako, Denmark). One clone with the strongest expression of alpha IIbbeta 3 integrin was selected. For adhesion assays, alpha IIbbeta 3-CHO transfectant and wild-type (parental) CHO cells were treated with or without 10 mM DTT in RPMI 1640, 10 mM Hepes, at 22 °C for 20 min to activate the alpha IIbbeta 3 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 h at 22 °C with 1% nonfat milk in the same buffer. For adhesion assays, either fixed GRGDSP-activated or unactivated platelets (5 × 106/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 alpha IIbbeta 3-CHO transfectants (1 × 105/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 min 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 5 × 106 platelets or 1 × 105 CHO cells/100 µl) for 30 min at 22 °C prior addition to protein-coated wells.

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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 alpha IIbbeta 3 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 alpha IIbbeta 3 complex (40), but no reactivity with the mAb AK-4 (41), which binds to P-selectin normally contained in intracellular alpha -granules (not shown). As shown in Fig. 1, GRGDSP-activated platelets adhered more efficiently than unactivated platelets to immobilized ICAM-4-positive RBCs from control donors. The 100% relative binding was equivalent to 220 ± 100 GRGDSP-activated platelets adhered to 1.0 × 103 immobilized RBCs. When unactivated platelets were used as control, a 69% reduced adhesion was noted that corresponded 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.


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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 represents 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 ± S.E. from at least six experiments is shown. By Student's t test analysis: ***, p < 0.001 versus unactivated platelet and Delta p < 0.05 or less versus control.

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 versus unactivated platelets and p < 0.05 versus 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 alpha -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 nonspecific 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 alpha IIbbeta 3 integrin, which mediates Fg binding and platelet aggregation (37), our data suggested that ICAM-4 might interact with alpha IIbbeta 3 integrin but also with other adhesive molecules.


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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. 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.

RBC-Platelet Interaction Is Mediated via ICAM-4-- To obtain further evidence that ICAM-4 might interact with a high affinity state of alpha IIbbeta 3 integrin, type-I glanzmann's thrombastenia platelets from two unrelated patients who both exhibit a 6-bp deletion in exon 7 of the beta 3 gene (42), were used for cell adhesion assays 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 alpha IIbbeta 3 integrin surface expression, either unactivated (not shown) or GRGDSP-activated, failed to bind to coated ICAM-4-Fc (Fig. 3A).


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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), beta 3-chain (SZ21), alpha IIb-chain (SZ22 and P2), alpha IIbbeta 3 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. 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 antibody (ctrl IgG), ICAM-1-Fc and wells without coated protein. The mean ± S.E. from three experiments is shown. By Student's t test analysis: **, p < 0.01, and *, p < 0.05.

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 alpha IIb-chain in the presence of the beta 3-chain and the complex-specific epitope of the alpha IIbbeta 3 integrin, respectively. SZ21 and SZ22 mAbs that recognize the beta 3- and alpha IIb-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 alpha IIbbeta 3 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/alpha IIbbeta 3 Integrin-- To provide further evidence that ICAM-4 may interact with the alpha IIbbeta 3 integrin, stable CHO transfectants expressing recombinant human alpha IIbbeta 3 were generated and used in cell adhesion assays (Fig. 4). Several alpha IIbbeta 3-CHO transfectants were obtained, and one clone expressing a high level of alpha IIbbeta 3 integrin (alpha IIb, 18,600 molecules/cell and beta 3, 67,000 molecules/cell, as estimated by flow cytometric analysis with specific mAbs) was chosen for further studies. The alpha IIbbeta 3 integrin of these cells was activated by DTT treatment and the adhesion of DTT-activated and unactivated alpha IIbbeta 3-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 alpha IIbbeta 3 integrin complex (not shown). DTT-activated alpha IIbbeta 3-CHO transfectants dose-dependently bind to coated ICAM-4-Fc protein, whereas untreated alpha IIbbeta 3-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 alpha IIbbeta 3-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 alpha IIbbeta 3-CHO transfectants were activated by the GRGDSP-peptide instead of DTT (not shown). The binding of DTT-activated alpha IIbbeta 3-CHO transfectants to immobilized ICAM-4-Fc could be blocked by ~50% by mAbs specific for ICAM-4 (BS56) or for the complex-specific epitope of the alpha IIbbeta 3 integrin (AP2), but not with mAbs to the beta 3-chain (SZ21) and alpha IIb-chain (SZ22 and P2) of the alpha IIbbeta 3 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 alpha IIbbeta 3 integrin in platelets and the CHO transfectants independently of the mode of integrin activation. As expected, control mouse IgG had no effect.


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Fig. 4.   Adhesion of alpha IIbbeta 3-CHO transfectants to immobilized ICAM-4-Fc. A, dose-dependent cell adhesion of DTT-activated alpha IIbbeta 3-CHO transfectants () and of control including parental CHO cells (DTT-treated or not) and unactivated alpha IIbbeta 3-CHO transfectants () to ICAM-4-Fc protein coated to plastic wells at varying concentrations. The results are expressed as mean percentage of bound cells ± S.E. of three experiments. B, effect of different mAbs on the adhesion of DTT-activated alpha IIbbeta 3 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), beta 3-chain (SZ21), alpha IIb-chain (SZ22 and P2), and alpha IIbbeta 3 complex (AP2). The results are expressed as the relative percentage of activated alpha IIbbeta 3-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 ± S.E. from three experiments is shown. By Student's t test analysis: ***, p < 0.001.

Putative Domains on ICAM-4 That Interact with the alpha IIbbeta 3 Integrin-- As an attempt to localize the alpha IIbbeta 3 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 alpha IIbbeta 3-CHO transfectants via the alpha IIbbeta 3 integrin required the 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).


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Fig. 5.   ICAM-4 domains interacting with platelet alpha IIbbeta 3 integrin. Adhesion of DTT-activated alpha IIbbeta 3-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 gamma  chain and ICAM-4 peptides on cells binding. Cells were pretreated with FBI (residues 400-411) and peptides Gly-Val (residues 65-74) and Cys-Arg (residues 180-192) derived from ICAM-4 at the final concentration of 125 µM. After 30 min of 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. The Cys-Arg 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 ± S.E. from three experiments is shown. By Student's t test analysis: ***, p < 0.001, and **, p < 0.01.

Further blocking experiments with synthetic peptides were performed. Adhesion of activated-platelet was efficiently inhibited to 14 and 58% by the FBI, Fg gamma -chain residues 400-411) peptide and the ICAM-4 peptide Gly-Val (residues 65-74), respectively, two peptides exhibiting a QXXDV motif involved in the fibrinogen/alpha IIbbeta 3 integrin interaction (Fig. 5B). When DTT-activated alpha IIbbeta 3-CHO transfectants were used, a 78% decrease in binding was observed in the presence of the ICAM-4-derived peptide Gly-Val whereas the peptide FBI failed to inhibit (Fig. 5A). The lack of inhibition by the peptide FBI, might result from some changes of alpha IIbbeta 3 integrin when expressed in CHO transfectants, as suspected for the reactivity of mAb P2 (see above). Neither the random peptides of FBI and Gly-65---Val-74 nor a control ICAM-4 peptide (Cys-Arg, residues 180-192) had any inhibitory effect. Altogether, these results demonstrate that the alpha IIbbeta 3 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.

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DISCUSSION
REFERENCES

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 alpha IIbbeta 3 integrin (platelet fibrinogen receptor GPIIb-IIIa) was identified as the receptor for RBC ICAM-4. However, we found that the alpha IIbbeta 3 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 alpha IIbbeta 3 integrin or to the beta 3-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 alpha IIbbeta 3 integrin (and vitronectin receptor alpha vbeta 3) did not bind to coated ICAM-4-Fc protein, and (iv) DTT-activated alpha IIbbeta 3-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 alpha IIbbeta 3 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 ICAM-4-Fc protein. As thrombasthenic platelets that expressed normal levels of other platelet receptors like the alpha 2beta 1 integrin (collagen receptor), and the fibronectin and laminin receptors (GPIa-IIa and GPIc'-IIa, respectively), did not adhere to ICAM-4-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, alpha IIbbeta 3-CHO transfectants and ICAM-4 Fc mutant proteins have shown that the two Ig-like domains of ICAM-4 are required for alpha IIbbeta 3 integrin interaction, since domain deletion mutants lacking either the first (D1) or second (D2) Ig-domain exhibited significant reduced binding (see Fig. 5, A and B). A similar effect has been observed when ICAM-4 mutant proteins interact with the leukocyte alpha Mbeta 2 (Mac-1) integrin, whereas interaction with the alpha Lbeta 2 (LFA-1) integrin requires predominantly the first Ig domain D1 (26). The binding of adhesive proteins to alpha IIbbeta 3 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/alpha IIbbeta 3 interaction. The platelet alpha IIbbeta 3 integrin also binds to the carboxyl-terminal end of the Fg gamma -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 Gly-Val (residues 65-74), including this motif, is a potent inhibitor of the ICAM-4/alpha IIbbeta 3 interaction. Moreover, the peptide FBI also inhibited platelet binding to ICAM-4-Fc. These findings suggest that the platelet alpha IIbbeta 3 integrin might interact with the Gly-65---Val-74 sequence of ICAM-4 that includes a QXXDV motif known to be involved in the fibrinogen/alpha IIbbeta 3 integrin interaction. The Gly-65---Val-74 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 alpha IIbbeta 3 integrin binding site on ICAM-4. Therefore, binding inhibition by RGD and FBI peptides, which bind to the beta 3 chain (GPIIIa) and alpha IIb chain (GPIIb), respectively (47), suggest that ICAM-4 binds to the same or overlapping site(s) on the alpha IIbbeta 3 complex. It should be noticed that the G70R substitution responsible for the blood group LWaright-arrowLWb polymorphism (48), which corresponds to the first position of the QXXDV motif, had no effect on RBC platelet adhesion reported here,2 suggesting that this polymorphism is neutral with regard to ICAM-4/alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha -granule contents, it is assumed that other factors critical for interaction may exist. alpha IIbbeta 3 integrin activation with either RGD-containing peptide or DTT induces Fg binding (37, 39), suggesting that indirect RBC-platelet contacts via this adhesive macromolecule 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 alpha IIbbeta 3 integrin on activated-platelet and ICAM-1 and/or alpha vbeta 3 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 needs further investigation. 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 alpha 4beta 1 (VLA-4), and to alpha vbeta 1 and alpha vbeta 5 integrins present on hematopoietic cells and might also account for the binding of sickle RBCs to vascular endothelium (27).

All these findings indicate that ICAM-4 is an unusual adhesion molecule that has a broad ligand binding specificity, including at least some beta 1, beta 2, beta 3 (this report), and beta 5 integrins, but the binding affinity for these ligands may vary widely. Another 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 alpha IIbbeta 3 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 article 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 rate 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 beta 2 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 alpha IIb chain (CD41) of the alpha IIbbeta 3 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 alpha IIb-chain in the presence of the beta 3-chain, or by the monoclonal antibody AP-2 specific for a complex-specific epitope of the alpha IIbbeta 3 integrin, respectively. Monoclonal antibodies SZ22 and SZ21 to the alpha IIb-chain and beta 3-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 beta  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 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.

    FOOTNOTES

* This work was supported in part by the Institut National de la Transfusion Sanguine (INTS) and INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: INSERM U76, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France. Tel.: 33-1-44-49-30-00; Fax: 33-1-43-06-50-19; E-mail: cartron@idf.inserm.fr.

Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M211282200

2 J. P. Cartron, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: RBCs, red blood cells; FBI, fibrinogen binding inhibitor; TSP, thrombospondin; vWF, von Willebrand Factor; mAb, monoclonal antibody; PGE1, prostaglandin-E1; TNF-alpha , tumor necrosis factor-alpha ; HUVEC, human umbilical vein endothelium cells; DTT, dithiothreitol; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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