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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 500-507
Platelet/Endothelial Cell Adhesion Molecule-1 Serves as a
Costimulatory Agonist Receptor That Modulates Integrin-Dependent
Adhesion and Aggregation of Human Platelets
By
David Varon,
Denise E. Jackson,
Boris Shenkman,
Rima Dardik,
Ilya Tamarin,
Naphtali Savion, and
Peter J. Newman
From The National Hemophilia Center and the Institute of Thrombosis
and Hemostasis, Tel-Hashomer, Israel; the Blood Research Institute, The
Blood Center of Southeastern Wisconsin, Milwaukee, WI; The Goldschleger
Eye Research Institute, Tel-Aviv University, Tel-Aviv, Israel; and the
Departments of Cellular Biology and Pharmacology, Medical College of
Wisconsin, Milwaukee, WI.
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ABSTRACT |
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD
member of the Ig gene superfamily that is expressed on the surface of
circulating platelets, monocytes, neutrophils, and selective T-cell
subsets. It is also a major component of the endothelial cell
intercellular junction. Previous studies have shown that cross-linking
PECAM-1 on the surface of leukocytes results in the activation of
adhesion molecules of both the  1 and 2
integrin family. In addition, the process of leukocyte transendothelial
migration appears to be mediated, at least in part, by homophilic
adhesive interactions that take place between leukocyte and endothelial
cell junctional PECAM-1 molecules. However, little is known about the
functional role of this membrane glycoprotein in human platelets. In
the present study, we examined the effects of PECAM-1 engagement on
integrin-mediated platelet-extracellular matrix or platelet-platelet
interactions. Bivalent, but not monovalent, anti-PECAM-1 monoclonal
antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface
coverage) and aggregation (increased average size) onto extracellular
matrix, under both oscillatory or defined low shear flow conditions
(200 s 1) in a modified cone and plate viscometer.
Moreover, bivalent anti-domain 6 MoAbs were capable of serving as
costimulatory agonists to markedly enhance both adenosine diphosphate
(ADP)- and platelet activating factor (PAF)-induced platelet
aggregation responses. These antibodies appeared to act via outside-in
signal transduction through PECAM-1, as evidenced by the fact that
their binding (1) led to conformational changes in the
 IIb3 integrin complex, (2) induced
surface expression of P-selectin, and (3) resulted in the tyrosine
phosphorylation of PECAM-1. Together, these data support a role for
PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a
costimulatory agonist receptor capable of modulating integrin function
in human platelets during adhesion and aggregation.
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INTRODUCTION |
PLATELET/ENDOTHELIAL cell adhesion
molecule-1 (PECAM-1) is a 130-kD transmembrane glycoprotein
that is expressed on the surface of circulating platelets, monocytes,
neutrophils, and selected T-cell subsets. It is also a major
constituent of the endothelial cell intercellular junction, where up to
106 PECAM-1 molecules concentrate after the formation of
cell-cell contact (for a review, see Newman1). The 574 amino acid extracellular domain of PECAM-1 is organized into six
Ig-like homology units,2 followed by a single-pass
transmembrane domain, and a 118 amino acid cytoplasmic tail that
contains specific sites for palmitoylation, phosphorylation, and
assembly of cytosolic signaling molecules.3-6 Approximately
30% of the molecular mass of PECAM-1 is composed of carbohydrate
residues whose influence on the adhesive properties of PECAM-1 is as
yet unknown.
A great deal has been learned in the past several few years about the
participation of PECAM-1 in the process of leukocyte transendothelial
migration. The expression and/or distribution of PECAM-1 on the
cell surface has been shown to be modulated in transmigrating
leukocytes7-9 and on endothelial cells exposed to
inflammatory cytokines.9-13 Moreover, Zocchi et
al14 have shown that PECAM-1- but not ICAM-1-transfected
fibroblasts support chemokine-independent transmigration of activated T
lymphocytes.14 Finally, anti-PECAM-1 antibodies have been
shown to inhibit leukocyte migration across an endothelial cell barrier
both in vitro15 and in vivo,16-18 and the
potential clinical relevance of interfering with PECAM-1
function has recently been shown in two different animal models of
cardiac ischemia/reperfusion injury.19,20
The precise mechanism by which PECAM-1 mediates cell-cell interactions
is not clear. Several studies have shown that PECAM-1 is capable of
interacting with other PECAM-1 molecules expressed on the cell
surface21-23; a homophilic process that is mediated by
N-terminal Ig-homology domains 1 and 2.22,23 The integrin
v324,25 and a yet to be
characterized 120-kD glycoprotein found on activated T
lymphocytes26 have also been implicated as heterophilic
counter receptors for PECAM-1. However, in addition to its intrinsic
adhesive properties, a number of investigators have shown that
engagement of PECAM-1 can upregulate the function of adhesion receptors
other than PECAM-1, most notably members of the integrin family. Tanaka et al27 provided the first experimental evidence that
PECAM-1 might be involved in transducing signals to other adhesion
receptors. They showed that cross-linking of PECAM-1 on the surface of
selected T-lymphocyte subsets resulted in the upregulation of
1 integrin function. T cells treated with certain
bivalent anti-PECAM-1 monoclonal antibodies (MoAbs) exhibited
increased adherence to plastic wells coated with fibronectin (via
51) or vascular cell adhesion molecule-1 (VCAM-1; via 41). Additional
cross-linking of the anti-PECAM-1 MoAb using goat antimouse IgG
further augmented adhesion, but monovalent Fab fragments were
ineffective, suggesting that dimerization of PECAM-1 on the cell
surface might be required for integrin affinity modulation. Similarly,
Leavesley et al28 reported that the binding of a bivalent
anti-PECAM-1 MoAb to CD34+ hematopoietic progenitor cells
enhanced their adhesion to VCAM-1-transfected CHO cells,28
a process presumably mediated by 41.
Modulation of integrin affinity does not appear to be limited to
1 integrins, because several groups have reported that
ligation of PECAM-1 also leads to upregulation of 2
integrin function in lymphokine-activated killer cells,29
monocytes and neutrophils,30 and natural killer cells.31 Together, these data suggest a mechanism by which
dimerization or oligomerization of PECAM-1 on the cell
surface results in the generation of specific signals that are capable
of modulating integrin affinity.
Human platelets offer an attractive model system to further examine the
relationship between PECAM-1 engagement and cellular activation.
Platelets normally exist in a resting, nonadhesive state, but can be
stimulated by multiple agonists, under well-controlled conditions, to
undergo a series of easily measurable cell biologic and biochemical
changes, including protein-tyrosine phosphorylation, -granule
release, cell-extracellular matrix interactions, and cell-cell
interactions (platelet aggregation). Moreover, platelets coexpress
approximately 10,000 copies of PECAM-132,33 and 80,000 copies of the well-characterized integrin,
IIb3.34 Finally, there are
specific MoAbs that are capable of detecting subtle conformational
changes in this integrin that report its conformational (affinity)
state, as well as MoAbs that specifically and sensitively measured the
exposure of the -granule-specific membrane protein, P-selectin, on
the platelet surface. The purpose of the present investigation,
therefore, was to test the hypothesis that PECAM-1 could
serve as a costimulatory agonist receptor whose engagement modulates
downstream cellular responses, including platelet adhesion, platelet
aggregation, and integrin affinity.
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MATERIALS AND METHODS |
MoAbs.
Well-characterized35,36 domain-specific MoAbs used in this
study included PECAM-1.3 (directed against Ig-homology domain 1),
PECAM-1.1 (domain 5), PECAM-1.2 (domain 6), and 4G6 (domain 6; kindly
provided by Dr Steven Albelda, University of Pennsylvania School of
Medicine, Philadelphia, PA). IV.3, a blocking MoAb
specific for the human Fc IIa receptor,37 was kindly
provided by Dr Clark Anderson (Ohio State University, Columbus, OH).
The ligand-induced binding site (LIBS) antibody, D3, which specifically
recognizes the active conformation of
IIb3,38 was kindly provided
by Dr Lisa Jennings (University of Tennessee, Memphis,
TN). The anti-P-selectin MoAb, S12,39 was
kindly provided by Dr Rodger McEver (University of Oklahoma, Oklahoma
City, OK). F(ab )2 and Fab fragments
were generated using immobilized pepsin or papain, respectively,
according to the manufacturer's (Pierce, Rockford, IL) instructions.
After overnight dialysis in phosphate-buffered saline (PBS), pH 7.4, all fragments were carefully analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both
reducing and nonreducing conditions to ensure that no intact IgG
remained. Before their use, the reactivity of all PECAM-1-specific
antibodies was confirmed by enzyme-linked immunosorbent assay against
an immobilized recombinant protein containing the complete
extracellular domain of human PECAM-1. The functional integrity of IV.3
Fab fragments was determined by measuring their ability to block
FcIIa receptor-mediated platelet activation induced by human
heparin-induced thrombocytopenia (HITP) antibodies.
Platelet deposition on extracellular matrix (ECM).
ECM-coated tissue culture wells were prepared according to the method
of Gospodarowicz et al.40 Briefly, bovine corneal endothelial cells were grown to confluence in 16-mm diameter tissue culture plates (Nunc, Roskilde, Denmark), washed with PBS, pH 7.4, and
dissolved by exposure to 0.5% Triton X-100 and 0.1 mol/L NH4OH, followed by extensive washing with distilled water.
Two hundred fifty microliters of citrated whole blood was preincubated with the indicated antibodies for 15 minutes at 37°C, and the mixture was added to the ECM-coated plates, which were then inserted into a modified rotating Teflon cone and plate viscometer described in
detail by Varon et al.41 The platelets were then subjected to either low (200 s1) or high (1,300 s1) shear rates for 2 minutes, thoroughly washed
with PBS, fixed, and stained by May-Grünwald stain. Platelet
interactions with extracellular matrix and with each other were
analyzed using an inverted Olympus light microscope (Olympus Corp, Lake
Success, NY). The image was captured by a video camera,
digitized, and quantitated using a computer-assisted image analysis
system.41 Data are expressed as the percentage of surface
coverage, the average size of the aggregate, and the total object
number. Statistical analysis of the data (mean ± SD) was performed
using a paired Student's t-test for single measurements or
repeated measures of variance for a series of measurements. P
values less than .05 were considered significant.
Platelet aggregation.
Blood from healthy, nonaspirinated volunteer donors was collected into
plastic tubes containing 3.8% (wt/vol) trisodium citrate (9:1) or acid
citrate dextrose, pH 4.6 (9:1) and mixed gently by inversion.
Platelet-rich plasma (PRP) was prepared by centrifugation of the blood
at 140g for 15 minutes and collecting the upper layer. Platelet-poor plasma (PPP) was prepared by centrifugation of the remaining lower layer at 2,000g for 10 minutes. The
concentration of platelets in PRP was adjusted with PPP to 2 × 108/mL. Aggregation studies were performed using a four
channel platelet aggregometer (PAP-4 Bio-Data, Horsham,
PA). Preliminary studies showed that the anti-PECAM-1
MoAbs, by themselves, failed to induce platelet aggregation (not
shown), similar to previous reports of the functional effects of other
anti-PECAM-1 MoAbs.32,33,42 All aggregation studies were
performed in duplicate on at least three separate occasions.
Flow cytometry.
Platelets were washed twice in Ringer's citrate dextrose (RCD; 108 mmol/L NaCl, 38 mmol/L KCl, 1.7 mmol/L NaHCO3, 21.2 mmol/L Na citrate, 27.8 mmol/L glucose, and 1.1 mmol/L MgCl2, pH
6.5) containing 50 ng/mL prostaglandin E1
(PGE1), diluted to a final concentration of 5 × 106/mL, and then incubated with various agonists in a
microtiter well for 1 hour at room temperature in the presence of 2 mmol/L CaCl2. Control agonists included buffer alone,
normal mouse IgG1 (NM IgG), 5 mmol/L RGEW peptide, or 5 mmol/L RGDW peptide. Antibody agonists were used at 10 µg/mL final
concentration and included PECAM-1.3, PECAM-1.1, PECAM-1.2, and 4G6.
All antibodies were used as either intact IgGs,
F(ab)2 fragments, or monovalent Fab fragments.
Platelets were washed, incubated with fluorescein isothiocyanate (FITC)-conjugated D3 or S12 for 30 minutes at room temperature, washed
once more, transferred to 450 µL of RCD, pH 7.4, and analyzed on a
FACScan (Becton Dickinson, San Jose, CA). At least 5,000 platelets per
sample were examined. Fluorescence data were displayed as logarithmic
contour plots, dot plots, or logarithmic histograms using LYSYS II
software (Becton Dickinson). Mean fluorescence intensity of S12 or D3
binding (minus background fluorescence observed in the presence of
buffer alone) from at least three separate experiments (±SD) was
then plotted versus the agonist used.
Immunoprecipitation and antiphosphotyrosine immunoblots.
Washed human platelets (1 × 109/mL) were incubated
under stirring conditions with buffer, 7 µmol/L thrombin
receptor-activating peptide (TRAP; having the amino acid sequence
SFLLRN), NM IgG1 F(ab)2, or PECAM-1.2
F(ab)2 and then lysed for 1 hour at 4°C in an
equal volume of 2% Triton X-100, 10 mmol/L EGTA, 15 mmol/L HEPES, 145 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 20 µg/mL
leupeptin, and 2 mmol/L sodium orthovanadate, pH 7.4. After
centrifugation at 14,000 rpm for 5 minutes at 4°C, clarified supernatants were subjected to immunoprecipitation using 10 µg of NM
IgG1 or PECAM-1.3. Immune complexes were collected on
protein G Sepharose beads (Pharmacia Biotech AB, Uppsala, Sweden),
resolved on a 12.5% SDS-polyacrylamide gel, and transferred
electrophoretically to Immobilon polyvinylidene fluoride membrane
(Millipore, Bedford, MA). Detection of tyrosine-phosphorylated PECAM-1
using the horseradish peroxidase (HRP)-conjugated antiphosphotyrosine
MoAb, PY-20, was performed as previously described.43
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RESULTS |
Engagement of PECAM-1 Ig-domain 6 promotes platelet-extracellular
matrix and platelet-platelet interactions.
To investigate the effects of PECAM-1 engagement on the process of
platelet adhesion and spreading, citrated whole blood was exposed to
extracellular matrix in a modified cone and plate viscometer at a shear
rate of 200 s1, as previously
described.41 Under these conditions, platelets adhere to
the matrix but do not form platelet aggregates. Application of higher
shear rate of 1,300 s1 or the addition of other
agonists is accompanied by more extensive adhesion as well as the
formation of platelet aggregates. As shown in
Fig 1, preincubation of platelets with the
anti-PECAM-1 Ig-domain 6-specific antibody, PECAM-1.2, before their
exposure to extracellular matrix coated surfaces increased by nearly
50% both surface coverage (from 14.47 ± 3.84 to 22.50 ± 5.66, P < .017) and the average size of the platelet aggregates
(from 26.10 ± 2.05 to 38.42 ± 7.12, P < .002). The
MoAb 4G6, which also maps to PECAM-1 Ig-domain 6,35 showed
similar effects (not shown). NM IgG1 or the anti-PECAM-1 Ig-domain 1-specific MoAb, PECAM-1.3, failed to augment platelet deposition on extracellular matrix, suggesting that this stimulatory effect was transmitted into the cell in a PECAM-1 domain-specific manner. In addition, PECAM-1.2 significantly enhanced both adenosine diphosphate (ADP)- and platelet activating factor
(PAF)-induced platelet-platelet interactions, as
measured by platelet aggregometry using platelet-rich plasma
(Fig 2A). This effect was not due to activation of the platelet FcIIa receptor, because preincubation of
platelets with saturating amounts of a blocking antibody to FcRII
(IV.3) did not abolish the costimulatory effect of PECAM-1.2 (Fig 2B).
The effect of PECAM-1.2 on both ADP- (2 µmol/L) and PAF- (40 nmol/L)
induced activation of platelets was dose-dependent at PECAM-1.2
concentrations ranging from 0.2 to 5 µg/mL (Fig 2C and D). Together,
these data suggest that engagement of PECAM-1 through Ig domain 6 lowers the threshold for platelet stimulation by a variety of agonists,
including ADP, PAF, and ECM under conditions of shear.
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| Fig 1.
Anti-PECAM-1 antibodies promote platelet deposition on
extracellular matrix. Two hundred fifty microliters of citrated whole blood was preincubated for 15 minutes at 37°C with either buffer alone or 5 µg/mL of normal mouse IgG1, PECAM-1.3, or
PECAM-1.2. After 8 minutes of exposure to ECM-covered plates under
conditions of low shear (200 s1), samples were washed
and stained and adherent platelets and aggregates were evaluated by
image analysis as described in the Materials and Methods. Data are
expressed as the percentage of ECM coverage (A) as well as average size
(in square micrometers) of the aggregates formed (B). Note that both
parameters were significantly increased (P = .017 and
P = .02, respectively) in the presence of PECAM-1.2, but not
PECAM-1.3.
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| Fig 2.
Anti-PECAM-1 MoAbs act as costimulatory agonists in ADP-
and PAF-induced platelet aggregation. PRP was preincubated with (1) buffer, (2) normal mouse IgG1, (3) PECAM-1.3, or (4)
PECAM-1.2. All antibodies were used at a final concentration of 5 µg/mL. Note that PECAM-1.2 augmented low-dose (1.25 µmol/L)
ADP-induced platelet aggregation, both in the absence (A) and presence
(B) of the MoAb IV.3 (a blocking MoAb specific for FcRIIa). The
degree of potentiation of aggregation induced by PECAM-1.2 varied
somewhat from experiment to experiment (compare [A] with [B]), and
this variation was not attributable to FcRIIa blockade. Incubation of PRP with increasing concentrations of PECAM-1.2 (shown in micrograms per milliliter), followed by the addition of either 2 µmol/L ADP (C)
or 40 nmol/L PAF (D), showed that the effects of antibody-mediated dimerization of PECAM-1 are dose-dependent.
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Dimerization of PECAM-1 on the platelet surface leads to
conformational changes in
IIb3 and
exposure of P-selectin on the platelet surface.
To investigate the molecular mechanism by which PECAM-1 Ig-domain 6 engagement promotes increased platelet adhesion (Fig 1) and aggregation
(Fig 2), we examined the ability of PECAM-1 antibodies, in either
monovalent or bivalent form, to induce (1) conformational changes in
the platelet integrin IIb3 and (2)
-granule secretion. As shown in Fig 3,
incubation of platelets with PECAM-1.2 (directed against Ig-domain 6),
but not PECAM-1.1 (Ig-domain 5) or PECAM-1.3 (Ig-domain 1), led to
activation of the IIb3 complex, as
reported by the binding of the conformationally sensitive LIBS
antibody, D3. This effect was dependent on PECAM-1 dimerization,
because PECAM-1.2 F(ab)2 fragments induced D3
binding, whereas monovalent Fab fragments did not
(Fig 4A). A second Ig-domain 6-specific MoAb, 4G6, had similar effects. In addition, incubation of resting platelets with either of these two MoAbs, either as intact IgGs or
F(ab)2 fragments, led to exposure of the -granule
membrane-specific protein, P-selectin, on the platelet surface (Fig
4B), indicating that PECAM-1 cross-linking also results in some degree
of cellular activation. These events do not appear to be due to ADP
release or thromboxane A2 generation, because, as shown in
Table 1, the addition of PGE1,
apyrase, indomethicin, or SQ 29.5 (a thromboxane receptor antagonist)
before stimulation with PECAM-1.2 had little to no effect on D3
exposure.
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| Fig 3.
Engagement of PECAM-1 Ig-domain 6 leads to conformational
changes in the integrin IIb3. Washed
human platelets were preincubated with the FcRIIa-specific antibody,
IV.3 (to prevent possible Fc-receptor activation), before the addition
of 10 µg/mL of normal mouse IgG (NM IgG), PECAM-1.1, PECAM-1.2, or
PECAM-1.3. Buffer or RGDW peptide was used as negative and positive
controls, respectively. After the addition of FITC-conjugated D3 for 30 minutes at room temperature, platelets were washed and transferred to
450 µL of RCD buffer, pH 7.4, and analyzed by flow cytometry. Note
that PECAM-1.2 binding resulted in the exposure of the D3 epitope to almost the same extent as that induced by RGD peptide, a known modulator of integrin conformational change.62 The ability
of PECAM-1.2 to induce conformational changes in
IIb3 varied somewhat from experiment to
experiment; sometimes PECAM-1.2 was more effective than RGDW in
inducing D3 binding (see Fig 4).
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| Fig 4.
Cross-linking PECAM-1 induces conformational changes in
IIb3 and exposure of P-selectin on the
platelet surface. Washed platelets (5 × 106/mL) were
incubated with the indicated agonists as described in the Materials and
Methods. All antibodies were used at a final concentration of 10 µg/mL and were used in the form of intact IgG (not shown),
F(ab)2 fragments, or as monovalent Fab fragments. After the addition of FITC-conjugated D3 (A) or FITC-conjugated S12 (B)
for 30 minutes at room temperature, platelets were washed and
transferred to 450 µL of RCD, pH 7.4, and analyzed by flow cytometry.
Results are expressed as the mean ± SD of fluorescence intensity for
three independent experiments. The observation that bivalent PECAM-1.2
or 4G6 F(ab)2 fragments, but not their monovalent Fab counterparts, induced conformational changes in the
IIb3 integrin complex and exposure of
P-selectin on the platelet surface suggests that receptor dimerization
is required for outside/in signal transduction mediated by PECAM-1.
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Engagement of PECAM-1 induces tyrosine phosphorylation of its
cytoplasmic domain.
Recent studies have shown that PECAM-1 becomes tyrosine phosphorylated
during platelet aggregation in an integrin-dependent process that
results in the association of the SH2 domain-containing protein-tyrosine phosphatase, SHP-2, with tyrosine residues 663 and 686 of the PECAM-1 cytoplasmic domain.43 To further examine the
mechanism by which PECAM-1.2 is able to act as a costimulatory agonist
to modulate integrin-mediated cellular adhesion and aggregation responses (Figs 1-3), we incubated resting platelets with
PECAM-1.2 F(ab)2 fragments, solubilized the cells,
immunocaptured PECAM-1, and probed Western blots of the resulting
immunoprecipitated proteins with the phosphotyrosine-specific MoAb,
PY-20. As shown in Fig 5, dimerization of
PECAM-1 induced by PECAM-1.2 F(ab)2 fragments initiated outside/in signal transduction, resulting in tyrosine phosphorylation of the PECAM-1 cytoplasmic domain to an extent similar
to that induced by the strong platelet agonist, TRAP. These data
suggest that dimerization or oligomerization of PECAM-1 on the platelet
surface promotes downstream cellular events, including increased
adhesion and aggregation, via the creation of specific docking sites
for SH2-containing signaling molecules such as SHP-2.
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| Fig 5.
PECAM-1 dimerization results in its tyrosine
phosphorylation. Washed platelets (1 × 109/mL) were
incubated at 37°C with the following agonists in the presence of 2 mmol/L CaCl2, 1 mmol/L MgCl2, and 100 µg/mL
fibrinogen: (1) buffer (in the presence of stirring), (2) 7 µmol/L
TRAP for 5 minutes under stirring conditions (fully aggregated), (3) 10 µg/mL normal mouse IgG1 F(ab)2
fragments for 30 minutes (stirred), and (4) 10 µg/mL PECAM-1.2
F(ab)2 for 30 minutes (stirred). After detergent
lysis, immunoprecipitations (IP) were performed using either normal
mouse IgG1 (NM, left panel) or PECAM-1.3 (right panel).
Bound proteins were resolved by 12.5% SDS-PAGE and analyzed by
immunoblotting using an HRP-conjugated antiphosphotyrosine antibody
(PY-20). Arrows indicate the positions of tyrosine phosphorylated PECAM-1 and the heavy chain of IgG.
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| |
DISCUSSION |
In addition to serving as a homophilic cell-cell adhesion
molecule,22 recent studies have shown that
antibody-mediated engagement or dimerization of PECAM-1 on the cell
surface can result in a number of downstream cellular events, including
synthesis and release of hydrogen peroxide44 and
proinflammatory cytokines45 by human monocytes, cell
spreading and cytoskeletal rearrangement of natural killer
cells,46 tyrosine phosphorylation of multiple cytoplasmic
proteins in human T lymphocytes,47 and differentiation of
cord blood progenitor cells.48 In addition, cross-linking PECAM-1 on the surface of leukocytes has been shown to result in the
activation of adhesion molecules of both the 1 and
2 integrin family.27-31 The purpose of the
present investigation was to examine whether PECAM-1-mediated
activation of integrins could be extended to the 3
integrin subfamily and to begin to understand the mechanism by which
PECAM-1 engagement leads to augmentation of integrin function.
Unlike that which has been shown in other cell types, numerous
PECAM-1-specific antibodies have been examined for their effects on
platelet function over the past 10 years, and not one has been found,
by itself, to induce or inhibit in vitro platelet aggregation, adhesion, or granule secretion. In the present report, we found that
certain anti-PECAM-1 MoAbs were able to potentiate the ability of
human platelets to adhere both to ECM proteins (Fig 1) and to each
other (Fig 2). The findings suggest that, at least in human platelets,
PECAM-1 engagement is by itself insufficient to induce a measurable
cellular response and requires costimulation by either shear (Fig 1) or
another soluble agonist (Fig 2). We have extended these observations by
further showing that the mechanism by which this occurs appears to be
related to the generation of intracellular signals that follow
dimerization or oligomerization of PECAM-1 on the cell surface. Thus,
intact anti-PECAM-1 IgG as well as F(ab)2
fragments, but not monovalent Fab fragments, were shown to be capable
of inducing conformational changes in the platelet integrin
IIb3 and exposing P-selectin on the
platelet surface (Figs 3 and 4). Although we do not yet
fully understand the signal transduction pathway that leads from
PECAM-1 engagement to integrin activation, Levine et al47
have recently shown that anti-PECAM-1 MoAbs induce tyrosine
phosphorylation of multiple unidentified cellular
substrates.47 Our finding that PECAM-1 itself becomes
tyrosine phosphorylated after PECAM-1 antibody binding (Fig 5), coupled
with the recent demonstration by Jackson et al43 that
tyrosine phosphorylation of the PECAM-1 cytoplasmic domain creates
docking sites for cytosolic SH2-containing signaling molecules, offers
hints as to the molecular mechanism of PECAM-1/integrin cross-talk and
may help explain the link between PECAM-1 dimerization and
1 and 2 integrin activation seen
previously by other investigators.
Stockinger et al44,45 have clearly shown that, in some
instances, cellular activation induced by anti-PECAM-1 MoAbs can be
entirely explained by the secondary engagement of IgG Fc receptors. In
fact, the contribution of Fc receptor activation to signal transduction
events attributed to PECAM-1 engagement cannot be ruled out in several
other investigations in which intact anti-PECAM-1 IgG and/or
secondary antibodies were used to evoke a PECAM-1-specific cellular
response.28,29,49 The Fc region of IgG of many animal species, when in the correct conformation, can serve as a potent stimulatory agonist, as exemplified by the often life-threatening clinical syndrome, heparin-induced thrombocytopenia, in which human
antibodies specific for heparin/platelet factor 4 complexes bind, via
the Fc region of the resulting immune complex, to the platelet Fc
receptor (FcRIIa), resulting in both platelet activation (thrombosis) and clearance (thrombocytopenia).50,51 The
often-employed use of nonactivating, isotype-matched MoAbs alone is an
insufficient control for specificity of Fc receptor activation, because
unbound antibodies do not have nearly as high an affinity for the Fc
receptor as do bound antibodies,37 and many MoAbs, even
when bound via their Fab regions to their target antigens, are
spatially oriented in such a manner that precludes Fc receptor
activation (for a more detailed treatment of the critical role of
antigen topography in the interaction of antibodies with Fc receptors,
see Kumpel et al,52 Horsewood et al,53 and
Tomiyama et al54). In the present investigation, we
attempted to carefully exclude Fc receptor-mediated cellular activation
by using F(ab)2 antibody fragments and/or by
preblocking platelet FcRIIa (the only Fc receptor for IgG present in
platelets55) with saturating levels of the blocking FcRIIa-specific MoAb, IV.3. The fact that augmentation of platelet aggregation (Fig 2), LIBS and P-selectin exposure (Figs 3 and 4 and
Table 1), and PECAM-1 tyrosine phosphorylation (Fig 5) all occurred
even after these precautions had been taken strongly support the notion
that PECAM-1 receptor dimerization, and not secondary Fc receptor
engagement, is capable of leading directly to cellular activation.
It is not clear why some anti-PECAM-1 MoAbs are able to augment
integrin function and cellular activation, whereas others are not, or
why, in the present study, both potentiating anti-PECAM-1 antibodies,
PECAM-1.2 and 4G6, map to Ig-domain 6, the Ig-homology domain closest
to the membrane. Several previous observations may be relevant. First,
Ig-domain 6 contains two divalent cation binding sites,56
although the structural and functional consequences of cation occupancy
are not yet known. Second, Sun et al22 recently showed that
the homophilic adhesive properties of PECAM-1-containing proteoliposomes could be selectively increased by Fab fragments of the
same two antibodies, PECAM-1.2 and 4G6 (anti-PECAM-1 Fabs specific for
other Ig homology domains did not augment adhesion), and proposed that
engagement of domain 6 might induce LIBS-like long-range conformational
changes in the PECAM-1 molecule. Finally, another domain 6-specific
antibody, LYP21, as well as an Ig-domain 6 peptide corresponding to
amino acid residues 551-574 have been shown to inhibit T-cell
responses57 and delay the onset of graft-versus-host disease.58 Together with the data presented here on the
effect of these antibodies on integrin activation and P-selectin
exposure, it is tempting to speculate that Ig-domain 6 may play a
regulatory role in PECAM-1 function, mediating both outside-out as well
as outside-in signal transduction.
However, other explanations for the seemingly selective action of
domain 6 antibodies on cell function are possible. It could be that
MoAbs specific for Ig-domain 6 happen to bind with a topographical orientation that preferentially favors PECAM-1/PECAM-1 interactions on
the cell surface. Receptor dimerization, in turn, may be all that is
required to induce conformational changes in the molecule, leading to
its activation and initiating intracellular tyrosine phosphorylation,
augmenting integrin function, or increasing the homophilic binding
properties of PECAM-1 proteoliposomes. It is notable that augmentation
of 1 and 2 integrin function in
leukocytes does not appear to be limited to Ig-domain 6-specific
MoAbs27,35; perhaps the relative receptor density or its
lateral mobility within the plane of the membrane is sufficiently
different from that in platelets to permit PECAM-1 dimerization by a
less-selective set of anti-PECAM-1 MoAbs. Further studies using fixed
monomeric and dimeric membrane-bound forms of PECAM-1 are
planned to distinguish between these two models of PECAM-1-mediated
cellular activation.
Independent of the mechanism by which PECAM-1 MoAbs are exerting their
effects, it is becoming clear that PECAM-1 can both initiate, as well
as respond to, changes in cellular function. Osawa et al59
have recently shown that PECAM-1 becomes tyrosine phosphorylated in
endothelial cells subjected to mechanical shear stress and suggested
that PECAM-1 may be one of the junctional receptors responsible for
sensing and then communicating changes in fluid flow. Whether
mechanical shear contributes to the tyrosine phosphorylation of
platelet PECAM-1 that occurs in aggregating platelets43 is
not known. In addition, Sagawa et al60 showed that
aggregation of the high-affinity IgE receptor in rat basophilic leukemia cells also results in the tyrosine phosphorylation of PECAM-1.
Finally, the recent studies of Lu et al61 suggest that the
phosphorylation state of PECAM-1 may also be sensitive to cell-cell and
cell matrix interactions, because integrin engagement and/or
cell migration of cultured endothelial cells were shown to result in
tyrosine dephosphorylation of PECAM-1. Taken together, these data
implicate PECAM-1 as one of a growing number of cell surface receptors
that are able to mediate bidirectional signal transduction, serving
both as an agonist receptor as well as an adhesion molecule in blood
and vascular cells. Studies defining the cytosolic signaling molecules
that link these two interrelated cellular functions of PECAM-1
represent important areas of future investigation.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted September 8, 1997.
D.V. and D.E.J. contributed equally to this work.
Supported by Grants No. HL-44612 and HL-40926 (to P.J.N.) from the
National Institutes of Health and a postdoctoral fellowship award to
D.E.J. (#F96F-Post-34) from the Wisconsin Affiliate of the American
Heart Association. D.V. and N.S. were supported by grants from the
National Council for Research and Development, Israel and Deutsche
Forschungsanstalt, Fuer Luft und Raumfahrt. P.J.N. is an Established
Investigator of the American Heart Association.
Presented in abstract form at the 1996 European Granulocyte and
Platelet Meeting in Helsinki, Finland.
Address reprint requests to Peter J. Newman, PhD, Blood Research
Institute, The Blood Center of Southeastern Wisconsin, 638 N 18th St,
Milwaukee, WI 53233-2121.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Kevin Kupcho for technical support in performing flow
cytometric measurements, Dr Gian Visentin for his assistance in HITP
antibody-induced activation of platelets, and Dr William Campbell for
providing indomethicin and SQ 29.5.
| |
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R. D. Thompson, K. E. Noble, K. Y. Larbi, A. Dewar, G. S. Duncan, T. W. Mak, and S. Nourshargh
Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane
Blood,
March 15, 2001;
97(6):
1854 - 1860.
[Abstract]
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T. Zhao and P. J. Newman
Integrin Activation by Regulated Dimerization and Oligomerization of Platelet Endothelial Cell Adhesion Molecule (PECAM)-1 from Within the Cell
J. Cell Biol.,
January 8, 2001;
152(1):
65 - 74.
[Abstract]
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U. Naik, M. Naik, K Eckfeld, P Martin-DeLeon, and J Spychala
Characterization and chromosomal localization of JAM-1, a platelet receptor for a stimulatory monoclonal antibody
J. Cell Sci.,
January 2, 2001;
114(3):
539 - 547.
[Abstract]
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J. Werr, E. E. Eriksson, P. Hedqvist, and L. Lindbom
Engagement of {beta}2 integrins induces surface expression of {beta}1 integrin receptors in human neutrophils
J. Leukoc. Biol.,
October 1, 2000;
68(4):
553 - 560.
[Abstract]
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G. Montrucchio, G. Alloatti, and G. Camussi
Role of Platelet-Activating Factor in Cardiovascular Pathophysiology
Physiol Rev,
October 1, 2000;
80(4):
1669 - 1699.
[Abstract]
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S. Mahooti, D. Graesser, S. Patil, P. Newman, G. Duncan, T. Mak, and J. A. Madri
PECAM-1 (CD31) Expression Modulates Bleeding Time in Vivo
Am. J. Pathol.,
July 1, 2000;
157(1):
75 - 81.
[Abstract]
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R. D. Thompson, M. W. Wakelin, K. Y. Larbi, A. Dewar, G. Asimakopoulos, M. A. Horton, M. T. Nakada, and S. Nourshargh
Divergent Effects of Platelet-Endothelial Cell Adhesion Molecule-1 and {beta}3 Integrin Blockade on Leukocyte Transmigration In Vivo
J. Immunol.,
July 1, 2000;
165(1):
426 - 434.
[Abstract]
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C Johnson-Leger, M Aurrand-Lions, and B. Imhof
The parting of the endothelium: miracle, or simply a junctional affair?
J. Cell Sci.,
January 3, 2000;
113(6):
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[Abstract]
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S. Yang, J. Graham, J. W. Kahn, E. A. Schwartz, and M. E. Gerritsen
Functional Roles for PECAM-1 (CD31) and VE-Cadherin (CD144) in Tube Assembly and Lumen Formation in Three-Dimensional Collagen Gels
Am. J. Pathol.,
September 1, 1999;
155(3):
887 - 895.
[Abstract]
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D. K. Newton-Nash and P. J. Newman
A New Role for Platelet-Endothelial Cell Adhesion Molecule-1 (CD31): Inhibition of TCR-Mediated Signal Transduction
J. Immunol.,
July 15, 1999;
163(2):
682 - 688.
[Abstract]
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G. S. Duncan, D. P. Andrew, H. Takimoto, S. A. Kaufman, H. Yoshida, J. Spellberg, J. Luis de la Pompa, A. Elia, A. Wakeham, B. Karan-Tamir, et al.
Genetic Evidence for Functional Redundancy of Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1): CD31-Deficient Mice Reveal PECAM-1-Dependent and PECAM-1-Independent Functions
J. Immunol.,
March 1, 1999;
162(5):
3022 - 3030.
[Abstract]
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I. Bird, V Taylor, J. Newton, J. Spragg, D. Simmons, M Salmon, and C. Buckley
Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration
J. Cell Sci.,
January 6, 1999;
112(12):
1989 - 1997.
[Abstract]
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F. Pellegatta, S. L. Chierchia, and M. R. Zocchi
Functional Association of Platelet Endothelial Cell Adhesion Molecule-1 and Phosphoinositide 3-Kinase in Human Neutrophils
J. Biol. Chem.,
October 23, 1998;
273(43):
27768 - 27771.
[Abstract]
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C. T. Hua, J. R. Gamble, M. A. Vadas, and D. E. Jackson
Recruitment and Activation of SHP-1 Protein-tyrosine Phosphatase by Human Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1). IDENTIFICATION OF IMMUNORECEPTOR TYROSINE-BASED INHIBITORY MOTIF-LIKE BINDING MOTIFS AND SUBSTRATES
J. Biol. Chem.,
October 23, 1998;
273(43):
28332 - 28340.
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Q.-H. Sun, C. Paddock, G. P. Visentin, M. M. Zukowski, W. A. Muller, and P. J. Newman
Cell Surface Glycosaminoglycans Do Not Serve as Ligands for PECAM-1. PECAM-1 IS NOT A HEPARIN-BINDING PROTEIN
J. Biol. Chem.,
May 8, 1998;
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M. Cicmil, J. M. Thomas, T. Sage, F. A. Barry, M. Leduc, C. Bon, and J. M. Gibbins
Collagen, Convulxin, and Thrombin Stimulate Aggregation-independent Tyrosine Phosphorylation of CD31 in Platelets. EVIDENCE FOR THE INVOLVEMENT OF Src FAMILY KINASES
J. Biol. Chem.,
August 25, 2000;
275(35):
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M.-H. Ruchaud-Sparagano, T. R. Walker, A. G. Rossi, C. Haslett, and I. Dransfield
Soluble E-selectin Acts in Synergy with Platelet-activating Factor to Activate Neutrophil beta 2-Integrins. ROLE OF TYROSINE KINASES AND Ca2+ MOBILIZATION
J. Biol. Chem.,
May 19, 2000;
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G. Ji, C. D. O'Brien, M. Feldman, Y. Manevich, P. Lim, J. Sun, S. M. Albelda, and M. I. Kotlikoff
PECAM-1 (CD31) regulates a hydrogen peroxide-activated nonselective cation channel in endothelial cells
J. Cell Biol.,
April 1, 2002;
157(1):
173 - 184.
[Abstract]
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G. Cao, C. D. O'Brien, Z. Zhou, S. M. Sanders, J. N. Greenbaum, A. Makrigiannakis, and H. M. DeLisser
Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration
Am J Physiol Cell Physiol,
May 1, 2002;
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Abstract
Introduction
Abstract