From 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
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ABSTRACT |
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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 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 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 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 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.
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
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
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
RBC-Platelet Interaction Is Mediated via ICAM-4--
To obtain
further evidence that ICAM-4 might interact with a high affinity state
of
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 RBC-Platelet Interaction Is Mediated via
ICAM-4/ Putative Domains on ICAM-4 That Interact with the
Further blocking experiments with synthetic peptides were performed.
Adhesion of activated-platelet was efficiently inhibited to 14 and 58%
by the FBI, Fg 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
Further analysis with platelets,
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
As LWnull RBCs still adhere to activated platelets and RBCs
adhere to adherent platelets stimulated by thrombin, which have release
their All these findings indicate that ICAM-4 is an unusual adhesion molecule
that has a broad ligand binding specificity, including at least some
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
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
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.
IIb
3 integrin. The platelet
fibrinogen receptor
IIb
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
3-chain alone and for a
complex-specific epitope of the
IIb
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
IIb
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
IIb
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
integrin subfamilies (
L
2 (LFA-1),
M
2 (Mac-1) (24-26),
4
1
(VLA-4),
V integrins (
V
1 and
V
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
d
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-
-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).
IIb
3 integrin.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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
IIb-chain (CD41) in the presence and the absence of the
3-chain, respectively, clones SZ21 and
SZ2 specific for the
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
IIb
3 integrin came from
GTI (Brookfield, WI). PAC-1 and AK-4 mAbs specific for activated
IIb
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.
IIb
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 (
IIb-chain) and CD61
(
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
IIb
3 integrin was
selected. For adhesion assays,
IIb
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
IIb
3
complex receptor (39).
IIb
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IIb
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
IIb
3 complex (40),
but no reactivity with the mAb AK-4 (41), which binds to P-selectin
normally contained in intracellular
-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 p < 0.05 or less versus control.
-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
IIb
3 integrin, which mediates Fg binding and platelet aggregation (37), our data
suggested that ICAM-4 might interact with
IIb
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.
IIb
3 integrin, type-I glanzmann's
thrombastenia platelets from two unrelated patients who both exhibit a
6-bp deletion in exon 7 of the
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
IIb
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), 3-chain (SZ21),
IIb-chain
(SZ22 and P2),
IIb
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.
IIb-chain in the presence of the
3-chain and the complex-specific epitope of the
IIb
3 integrin, respectively. SZ21 and
SZ22 mAbs that recognize the
3- and
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
IIb
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.
IIb
3 Integrin--
To
provide further evidence that ICAM-4 may interact with the
IIb
3 integrin, stable CHO transfectants
expressing recombinant human
IIb
3 were
generated and used in cell adhesion assays (Fig. 4). Several
IIb
3-CHO transfectants were obtained, and
one clone expressing a high level of
IIb
3
integrin (
IIb, 18,600 molecules/cell and
3, 67,000 molecules/cell, as estimated by flow
cytometric analysis with specific mAbs) was chosen for further studies.
The
IIb
3 integrin of these cells was
activated by DTT treatment and the adhesion of DTT-activated and
unactivated
IIb
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
IIb
3 integrin
complex (not shown). DTT-activated
IIb
3-CHO transfectants
dose-dependently bind to coated ICAM-4-Fc protein, whereas
untreated
IIb
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
IIb
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
IIb
3-CHO transfectants were
activated by the GRGDSP-peptide instead of DTT (not shown). The binding
of DTT-activated
IIb
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
IIb
3 integrin (AP2), but not with mAbs to
the
3-chain (SZ21) and
IIb-chain (SZ22
and P2) of the
IIb
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
IIb
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
IIb
3-CHO
transfectants to immobilized ICAM-4-Fc. A,
dose-dependent cell adhesion of DTT-activated
IIb
3-CHO transfectants (
) and of
control including parental CHO cells (DTT-treated or not) and
unactivated
IIb
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
IIb
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),
3-chain (SZ21),
IIb-chain (SZ22 and P2), and
IIb
3 complex (AP2). The results are
expressed as the relative percentage of activated
IIb
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.
IIb
3 Integrin--
As an attempt to
localize the
IIb
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
IIb
3-CHO transfectants
via the
IIb
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).
View larger version (26K):
[in a new window]
Fig. 5.
ICAM-4 domains interacting with platelet
IIb
3
integrin. Adhesion of DTT-activated
IIb
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
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.
-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/
IIb
3 integrin interaction
(Fig. 5B). When DTT-activated
IIb
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
IIb
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
IIb
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IIb
3 integrin (platelet fibrinogen
receptor GPIIb-IIIa) was identified as the receptor for RBC ICAM-4.
However, we found that the
IIb
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
IIb
3 integrin or to the
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
IIb
3 integrin (and
vitronectin receptor
v
3) did not bind to coated ICAM-4-Fc
protein, and (iv) DTT-activated
IIb
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
IIb
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
2
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.
IIb
3-CHO transfectants and ICAM-4 Fc
mutant proteins have shown that the two Ig-like domains of ICAM-4 are
required for
IIb
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
M
2 (Mac-1) integrin, whereas interaction
with the
L
2 (LFA-1) integrin requires
predominantly the first Ig domain D1 (26). The binding of adhesive
proteins to
IIb
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/
IIb
3 interaction. The platelet
IIb
3 integrin
also binds to the carboxyl-terminal end of the Fg
-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/
IIb
3 interaction. Moreover, the
peptide FBI also inhibited platelet binding to ICAM-4-Fc. These
findings suggest that the platelet
IIb
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/
IIb
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
IIb
3 integrin binding site on ICAM-4.
Therefore, binding inhibition by RGD and FBI peptides, which bind to
the
3 chain (GPIIIa) and
IIb chain
(GPIIb), respectively (47), suggest that ICAM-4 binds to the same or
overlapping site(s) on the
IIb
3 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,2 suggesting that this
polymorphism is neutral with regard to
ICAM-4/
IIb
3 integrin interaction.
IIb
3 integrin receptor with its RBC
ICAM-4 ligand is currently unknown.
-granule contents, it is assumed that other factors critical
for interaction may exist.
IIb
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
IIb
3 integrin on activated-platelet and
ICAM-1 and/or
v
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
4
1 (VLA-4), and to
v
1 and
v
5 integrins present on hematopoietic cells and might also account for the
binding of sickle RBCs to vascular endothelium (27).
1,
2,
3 (this report), and
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).
IIb
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.
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
IIb chain (CD41) of the
IIb
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
IIb-chain in the presence of the
3-chain, or by the monoclonal
antibody AP-2 specific for a complex-specific epitope of the
IIb
3 integrin, respectively. Monoclonal
antibodies SZ22 and SZ21 to the
IIb-chain and
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
integrins is calcium-dependent (26). Obviously, distinct
experimental conditions (platelet activation, flow conditions) and
monoclonal antibodies used may explain the reported differences.
![]() |
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-, tumor necrosis factor-
;
HUVEC, human umbilical vein endothelium cells;
DTT, dithiothreitol;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline.
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