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Red Cell ICAM-4 Is a Novel Ligand for Platelet-activated αIIbβ3 Integrin

2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês

10.1074/jbc.m211282200

1083-351X

Patricia Hermand, Pierre Gane, Martine Huet, Vincent Jallu, Cécile Kaplan, H.‐H. Sonneborn, Jean‐Pierre Cartron, Pascal Bailly,

Signaling Pathways in Disease

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitrowith 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 αIIbβ3 integrin. The platelet fibrinogen receptor αIIbβ3 (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 β3-chain alone and for a complex-specific epitope of the αIIbβ3integrin, 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 αIIbβ3-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 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 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. ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitrowith 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 αIIbβ3 integrin. The platelet fibrinogen receptor αIIbβ3 (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 β3-chain alone and for a complex-specific epitope of the αIIbβ3integrin, 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 αIIbβ3-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 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 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. The main physiological function of red blood cells (RBCs), 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, human umbilical vein endothelium cells; DTT, dithiothreitol; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline 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 (1Telen M.J. Semin. Hematol. 2000; 37: 130-142Crossref PubMed Scopus (77) Google Scholar, 2Spring F.A. Parsons S.F. Transfus. Med. Rev. 2000; 14: 351-363Crossref PubMed Scopus (18) Google Scholar, 3Cartron J.P. Colin Y. Transfus. Clin. Biol. 2001; 8: 163-199Crossref PubMed Scopus (74) Google Scholar), 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 (1Telen M.J. Semin. Hematol. 2000; 37: 130-142Crossref PubMed Scopus (77) Google Scholar, 4Verfaillie C. Hurley R. Bhatia R. McCarthy J.B. Crit. Rev. Oncol. Hematol. 1994; 16: 201-224Crossref PubMed Scopus (113) Google Scholar, 5Hanspal M. Curr. Opin. Hematol. 1997; 4: 142-147Crossref PubMed Scopus (40) Google Scholar, 6Bratosin D. Mazurier J. Tissier J.P. Estaquier J. Huart J.J. Ameisen J.C. Aminoff D. Montreuil J. Biochimie (Paris). 1998; 80: 173-195Crossref PubMed Scopus (303) Google Scholar, 7Parsons S.F. Spring F.A. Chasis J.A. Anstee D.J. Baillieres Best Pract. Res. Clin. Haematol. 1999; 12: 729-745Crossref PubMed Scopus (55) Google Scholar, 8Oldenborg P.A. Zheleznyak A. Fang Y.F. Lagenaur C.F. Gresham H.D. Lindberg F.P. Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1313) Google Scholar, 9Chan J.Y. Watt S.M. Br. J. Haematol. 2001; 112: 541-557Crossref PubMed Scopus (94) Google Scholar). Along this process, some adhesion molecules are rapidly down-regulated and others are expressed at different stages and remain on RBCs (Refs.10Bony V. Gane P. Bailly P. Cartron J.P. Br. J. Haematol. 1999; 107: 263-274Crossref PubMed Scopus (99) Google Scholar and 11Southcott M.J. Tanner M.J. Anstee D.J. Blood. 1999; 93: 4425-4435Crossref PubMed Google Scholar, 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 (1Telen M.J. Semin. Hematol. 2000; 37: 130-142Crossref PubMed Scopus (77) Google Scholar, 2Spring F.A. Parsons S.F. Transfus. Med. Rev. 2000; 14: 351-363Crossref PubMed Scopus (18) Google Scholar, 3Cartron J.P. Colin Y. Transfus. Clin. Biol. 2001; 8: 163-199Crossref PubMed Scopus (74) Google Scholar). 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 (12Ward T.M. Chitnis C.E. Miller L.H. Baillère's Clin. Infect. Dis. 1994; 1: 155-190Google Scholar, 13Oh S.S. Chishti A.H. Palek J. Liu S.C. Curr. Opin. Hematol. 1997; 4: 148-154Crossref PubMed Scopus (38) Google Scholar), sickle cell disease (14Wick T.M. Eckman J.R. Curr. Opin. 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Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar). 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 (21Marcus A.J. Safier L.B. FASEB J. 1993; 7: 516-522Crossref PubMed Scopus (243) Google Scholar, 22Andrews D.A. Low P.S. Curr. Opin. Hematol. 1999; 6: 76-82Crossref PubMed Scopus (319) Google Scholar). Interestingly also, RBCs have the necessary signal transduction pathways to mediate these functions (23Minetti G. Low P.S. Curr. Opin. Hematol. 1997; 4: 116-121Crossref PubMed Scopus (53) Google Scholar). 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 β integrin subfamilies (αLβ2(LFA-1), αMβ2 (Mac-1) (24Bailly P. Hermand P. Callebaut I. Sonneborn H.H. Khamlichi S. Mornon J.P. Cartron J.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5306-5310Crossref PubMed Scopus (94) Google Scholar, 25Bailly P. Tontti E. Hermand P. Cartron J.P. Gahmberg C.G. Eur. J. Immunol. 1995; 25: 3316-3320Crossref PubMed Scopus (109) Google Scholar, 26Hermand P. Huet M. Callebaut I. Gane P. Ihanus E. Gahmberg C.G. Cartron J.P. Bailly P. J. Biol. Chem. 2000; 275: 26002-26010Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), α4β1(VLA-4), αV integrins (αVβ1 and αVβ5); Ref. 27Spring F.A. Parsons S.F. Ortlepp S. Olsson M.L. Sessions R. Brady R.L. Anstee D.J. Blood. 2001; 98: 458-466Crossref PubMed Scopus (95) Google Scholar). 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 (28Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5860) Google Scholar, 29Luscinskas F.W. Lawler J. FASEB J. 1994; 8: 929-938Crossref PubMed Scopus (265) Google Scholar, 30Gahmberg C.G. Tolvanen M. Kotovuori P. Eur. J. Biochem. 1997; 245: 215-232Crossref PubMed Scopus (192) Google Scholar, 31Hayflick J.S. Kilgannon P. Gallatin W.M. Immunol. Res. 1998; 17: 313-327Crossref PubMed Scopus (124) Google Scholar). 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 αdβ2integrin (25Bailly P. Tontti E. Hermand P. Cartron J.P. Gahmberg C.G. Eur. J. Immunol. 1995; 25: 3316-3320Crossref PubMed Scopus (109) Google Scholar, 27Spring F.A. Parsons S.F. Ortlepp S. Olsson M.L. Sessions R. Brady R.L. Anstee D.J. Blood. 2001; 98: 458-466Crossref PubMed Scopus (95) Google Scholar, 32Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (232) Google Scholar). 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) (7Parsons S.F. Spring F.A. Chasis J.A. Anstee D.J. Baillieres Best Pract. Res. Clin. Haematol. 1999; 12: 729-745Crossref PubMed Scopus (55) Google Scholar) and to laminin (33Udani M. Zen Q. Cottman M. Leonard N. Jefferson S. Daymont C. Truskey G. Telen M.J. J. Clin. Invest. 1998; 101: 2550-2558Crossref PubMed Scopus (171) Google Scholar,34El Nemer W. Gane P. Colin Y. Bony V. Rahuel C. Galacteros F. Cartron J.P. Le Van Kim C. J. Biol. Chem. 1998; 273: 16686-16693Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), 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 (35Hebbel R.P. Mohandas N. Embury S.H. Hebbel R.P. Mohandas N. Steinberg M.H. Sickle Cell Disease: Basic Principles and Clinical Practice. Raven Press, New York1994: 217-230Google Scholar). 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. RBC from donors with common and rare phenotypes (Donull, Lunullof 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 γ-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 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 αIIbβ3 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 (36Sonneborn H.H. Ernst M. Voak D. Vox Sanguinis. 1994; 67: 114Google Scholar). 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 (26Hermand P. Huet M. Callebaut I. Gane P. Ihanus E. Gahmberg C.G. Cartron J.P. Bailly P. J. Biol. Chem. 2000; 275: 26002-26010Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 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. 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 (37Du X.P. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 38Xia Z. Frojmovic M.M. Biophys. J. 1994; 66: 2190-2201Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Briefly, 1 × 108washed platelets resuspended in 0.1 ml of Tyrode's-albumin buffer (pH 7.4) containing 2 mm CaCl2 and 1 mmMgCl2, 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. 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 mmCaCl2) 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). 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 mmMgCl2, 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. 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 (αIIb-chain) and CD61 (β3-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 αIIbβ3 integrin was selected. For adhesion assays, αIIbβ3-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 αIIbβ3complex receptor (39Zucker M.B. Masiello N.C. Thromb. Haemost. 1984; 51: 119-124Crossref PubMed Scopus (75) Google Scholar). 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 αIIbβ3-CHO transfectants (1 × 105/well in a final volume of 100 μl) resuspended in RPMI 1640, 10 mm Hepes containing 2 mmMgCl2 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. To analyze molecular events occurring during RBC-platelet interaction, in vitrocell 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 (37Du X.P. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (419) Google Scholar). Accordingly, in addition to bind Fg, GRGDSP-treated platelets reacted strongly with the mAb PAC-1, which binds to the activated αIIbβ3 complex (40Abrams C.S. Ellison N. Budzynski A.Z. Shattil S.J. Blood. 1990; 75: 128-138Crossref PubMed Google Scholar), but no reactivity with the mAb AK-4 (41Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar), 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 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. 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 versuscontrols). 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 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 αIIbβ3 integrin, which mediates Fg binding and platelet aggregation (37Du X.P. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (419) Google Scholar), our data suggested that ICAM-4 might interact with αIIbβ3 integrin but also with other adhesive molecules. To obtain further evidence that ICAM-4 might interact with a high affinity state of αIIbβ3 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 (42Morel-Kopp M.C. Kaplan C. Proulle V. Jallu V. Melchior C. Peyruchaud O. Aurousseau M.H. Kieffer N. Blood. 1997; 90: 669-677Crossref PubMed Google Scholar), were used for cell adhesion assays to coated ICAM-4-Fc protein. Fig. 3 A 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. 3 A). 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. 3 B). 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 that 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. 3 B). 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. 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 αIIbβ3integrin (αIIb, 18,600 molecules/cell and β3, 67,000 molecules/cell, as estimated 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 trans