From the Hematology-Oncology Division and the Department of
Medicine, the University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
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INTRODUCTION |
Exposure of a binding site for ligands such as fibrinogen and von
Willebrand factor on the platelet integrin
IIb
3 is a prerequisite for platelet aggregation (1). The process by which this binding site is
exposed has been termed "inside-out" signaling and is initiated
when agonist-generated intraplatelet signals induce a conformational
change in
IIb
3 by interacting with its cytoplasmic tails (2). To
study the process of
IIb
3 activation in vitro, we have
developed a model system in which wild-type and mutant
IIb
3
expressed in GM1500 cells, an Epstein-Barr virus-transformed human B
lymphocyte line, can be induced to interact with soluble and
immobilized fibrinogen by the phorbol ester phorbol 12-myristate 13-acetate (PMA)1 (3, 4).
Using this system, we observed that the cytoplasmic tail of
IIb is
not required for
IIb
3 function in lymphocytes, that the conserved
GFFKR motif in the
IIb tail is required for
IIb to interact with
3, and that signals interacting with the
3 cytoplasmic tail are
responsible for the ability of agonists to stimulate
IIb
3
function (4).
Phorbol esters such as PMA activate the conventional and novel isoforms
of protein kinase C (PKC) (5). While phorbol esters are a potent
stimulus for ligand binding to
IIb
3 in platelets (6), they bypass
the more proximal signaling events that are initiated when agonists
bind to their cognate platelet membrane receptors. Most of the known
platelet receptors for agonists are seven-transmembrane domain proteins
that are coupled to various G proteins (7). Stimulation of these
receptors on platelets is known to activate phospholipase C
,
generate diacylglycerol and inositol triphosphate, increase cytosolic
calcium, and activate several isoforms of PKC (7). We were interested
in determining whether stimulation of a G protein-coupled receptor on B
lymphocytes would also expose the ligand-binding domain of
IIb
3.
Honda et al. (8) have reported that stimulation of the human
N-formyl peptide chemoattractant receptor (fPR) in murine B
lymphocytes induces the
4
1-mediated adherence of these cells to
VCAM-1. The fPR is a seven-transmembrane domain protein coupled to the G protein G
i in leukocytes (9). Accordingly, we
coexpressed the human fPR and
IIb
3 in GM1500 cells and tested the
ability of the transfected cells to interact with immobilized and
soluble fibrinogen. We found that like PMA, the chemoattractant peptide formyl Met-Leu-Phe (fMLP), an fPR agonist, increased the avidity of
IIb
3 for immobilized fibrinogen and its affinity for soluble fibrinogen. Moreover, using a number of metabolic inhibitors, we
outlined an
IIb
3 activation pathway involving the G-protein Gi, PKC, and the actin cytoskeleton.
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EXPERIMENTAL PROCEDURES |
Reagents and Materials--
The anti-FLAG monoclonal antibody
(mAb) M1 and the fluorescent Ca2+ indicator fluo-3 AM were
obtained from IBI-Kodak and Molecular Probes, respectively. Immulon 2 flat bottom microtiter plates were purchased from Dynatech
Laboratories. PMA, fMLP, pertussis toxin (PTX), bovine serum albumin,
ionomycin, Arg-Gly-Asp-Ser (RGDS), and cytochalasin D were purchased
from Sigma. Human fibrinogen was obtained from Enzyme Research Labs.
Bisindolylmaleimide I (BIM I), bisindolylmaleimide V (BIM V), and
genistein were purchased from Calbiochem. Recombinant C3 exoenzyme was
purchased from Upstate Biotechnology. Lipofectin reagent and Opti-MEM
media were obtained from Life Technologies, Inc. G418 was purchased
from Mediatech. Piceatannol and hygromycin were obtained from
Boehringer Mannheim.
Coexpression of the Human fPR and
IIb
3 in Human B
Lymphocytes--
Because GM1500 cells express
3, introduction of a
cDNA for
IIb results in the expression of
IIb
3 on the cell
surface (4). Therefore, to coexpress the fPR and
IIb
3 in GM1500
cells, a cDNA for
IIb in the plasmid pREP4 containing a gene for
resistance to the antibiotic hygromycin (3) and a FLAG
octapeptide-tagged cDNA for the human fPR in the plasmid pRc/CMV
containing a gene for resistance to the antibiotic neomycin (a gift of
Drs. James J. Campbell and Eugene Butcher, Stanford University) (10)
were sequentially introduced into 7.5 × 106 cells by
electroporation (250 V and 960 microfarads). Stable co-transfectants
were selected by growth in RPMI media containing 20% fetal calf serum
and both G418 (750 µg/ml) and hygromycin (200 µg/ml). The
simultaneous presence of
IIb
3 and the fPR on the lymphocyte
surface was confirmed by flow cytometry after staining the cells with
either the
IIb
3-specific mAb A2A9 (11) or the anti-FLAG mAb M1,
followed by staining with fluorescein (FITC)-conjugated goat
anti-murine IgG. Flow cytometry was performed using a FACScan flow
cytometer (Becton-Dickinson) as described previously (12).
To ensure that the expressed fPR was functional, 5 × 107 cells were loaded with 5 µM fluo-3 AM at
room temperature for 30 min. The cells were then incubated with either
30 nM ionomycin or 100 nM fMLP for 30 s
and Ca2+ flux-induced fluorescence was measured with a
FACScan flow cytometer as described previously (8).
Measurement of
IIb
3 Function in Human B
Lymphocytes--
The ability of
IIb
3 expressed by lymphocytes to
interact with fibrinogen was tested by measuring agonist-stimulated
lymphocyte adherence to immobilized fibrinogen (3) and
agonist-stimulated binding of soluble FITC-fibrinogen using flow
cytometry as described previously (4).
To measure lymphocyte adherence to fibrinogen, the wells of microtiter
plates were coated with 10 µg/ml purified human fibrinogen in 50 mM NaHCO3 buffer, pH 8.0, containing 150 mM NaCl. Unoccupied protein-binding sites on the wells were
blocked with 5 mg/ml bovine serum albumin dissolved in the same buffer.
1.5 × 105 B lymphocytes, metabolically labeled
overnight with [35S]methionine, were suspended in 100 µl of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl2, 0.1%
glucose, and 1% bovine serum albumin, stimulated with either PMA or
fMLP, and added to the protein-coated wells. Following an incubation
for 30 min at 37 °C without agitation, the plates were vigorously washed four times with the suspension buffer and adherent cells were
dissolved using 2% SDS. The SDS solutions were counted for 35S in a liquid scintillation counter.
To measure the binding of soluble FITC-fibrinogen to agonist-stimulated
lymphocytes, purified human fibrinogen (13) was labeled with FITC using
a CalbiochemTM-FITC Labeling Kit as described by the manufacturer.
Fibrinogen labeled with FITC in this manner remained monomeric as
assessed by gel-filtration chromatography, supported platelet
aggregation as well as unlabeled fibrinogen, and was 95% clottable
with thrombin (13). 1.5 × 105 B lymphocytes were then
suspended in 100 µl of 10 mM sodium phosphate buffer, pH
7.4, containing 137 mM NaCl, 1 mM
CaCl2, and 1% bovine serum albumin (suspension buffer) and
incubated with 0.25 µM FITC-fibrinogen in the presence or
absence of PMA or fMLP for 30 min at room temperature. The cells were
washed once with suspension buffer and resuspended in a fixation
solution consisting of 10 mM sodium phosphate buffer, pH
7.4, containing 137 mM NaCl and 0.37% formalin. Following
a 10-min incubation on ice, the cells were again washed once with the
suspension buffer, and analyzed by flow cytometry as described previously (4).
Effect of Botulinum C3 Exoenzyme on
IIb
3 Function in GM1500
Cells--
Recombinant C3 exoenzyme from Clostridium
botulinum was introduced in GM1500 cells using Lipofectin Reagent.
1 × 107 GM1500 cells were suspended in 1 ml of
Opti-MEM media containing 40 µg of Lipofectin and 12 µg of C3
exoenzyme and incubated for 2 h at 37 °C. The cell suspension
was then divided into two aliquots. One aliquot was resuspended in
complete media for 1 h and the ability of these cells to adhere to
fibrinogen was tested as described above. ADP-ribosylation of the small
GTPase RhoA during the incubation was examined using the second aliquot
as described previously (14). Briefly, the cells were washed with
Opti-MEM, resuspended in 50 µl of 20 mM Tris-HCl buffer,
pH 7.5, containing 0.25 M sucrose, 5 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 2 mM benzamidine, and 0.5 mM
phenylmethylsulfonyl fluoride, and homogenized by sonication. Following
centrifugation of the homogenate at 1000 × g for 5 min, 15 µl of the supernatant were incubated with 50 ng of C3
exoenzyme and 10 µM [32P]NAD+
(Amersham) for 1 h at 30 °C in 100 mM Tris-HCl
buffer, pH 8.0, containing 20 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, and 5 mM MgCl2 in a total reaction volume of 100 µl. Sufficient SDS and dithiothreitol were added to make their final
concentrations 3% and 200 mM, respectively, and the
solution was heated at 100 °C for 3 min. Following 0.1% SDS-10%
polyacrylamide gel electrophoresis, ADP-ribosylated RhoA was visualized
by autofluorography.
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RESULTS |
Introduction of a Functional fPR into GM1500
Cells--
Previously, we demonstrated that the phorbol ester PMA
induces the adherence of B lymphocytes expressing
IIb
3 to
immobilized fibrinogen (3). To determine whether adherence could also
be induced by stimulating a receptor on the lymphocyte surface, we introduced plasmids containing a cDNA for the human fPR tagged at
its 5' end with the FLAG epitope and a cDNA for
IIb into GM1500 B lymphocytes and selected for cells that stably expressed both proteins using the antibiotics G418 and hygromycin. As shown in Fig.
1A, the simultaneous presence
of
IIb
3 and the fPR on the lymphocyte surface was confirmed by
flow cytometry after staining the cells with the
IIb
3-specific
mAb A2A9 (11) and the anti-FLAG mAb M1. The cells also stained with a
FITC-labeled derivative of fMLP (data not shown).

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Fig. 1.
Co-expression of IIb 3 and the human fPR
in GM1500 cells. cDNAs encoding a FLAG epitope-tagged human
fPR and IIb were introduced sequentially into GM1500 cells by
electroporation. Stable co-transfectants were selected by growth in the
presence of both G418 and hygromycin as described under "Experimental
Procedures." The presence of the fPR and IIb 3 on the surface of
the selected cells was confirmed by flow cytometry after staining the
cells with the class-matched control antibody OKT3, the
IIb 3-specific mAb A2A9, and the anti-FLAG mAb M1, followed by
staining with FITC-conjugated goat anti-murine IgG (A).
Function of the expressed fPR was confirmed by loading the cells with 5 µM fluo-3 AM at room temperature for 30 min. The cells
were then incubated with either 30 nM ionomycin or 100 nM fMLP for 30 s and Ca2+ flux-induced
fluorescence was measured with a flow cytometer (B). The
effect of PTX on fMLP-stimulated Ca2+ flux was determined
by preincubating the cells with 100 ng/ml PTX for 2 h.
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Measurement of Ca2+ Flux in GM1500 Cells Expressing the
fPR--
fMLP induces a Ca2+ flux in murine B lymphocytes
expressing the human fPR (8). To verify that the fPR we expressed was
functional, transfected GM1500 cells were loaded with the fluorescent
Ca2+ indicator fluo-3 AM and exposed to either 30 nM ionomycin or 100 nM fMLP. The resulting
change in fluo-3 fluorescence was then measured by flow cytometry. As
shown in Fig. 1B, ionomycin and fMLP induced comparable
changes in fluo-3 fluorescence, confirming that fPR stimulation could
induce a flux of Ca2+ in the transfected cells (Fig.
1B). Moreover, the fMLP-stimulated Ca2+ flux was
inhibited by preincubating the cells for 2 h with 100 ng/ml PTX,
indicating that the fPR in GM1500 cells is coupled to a PTX-sensitive G
protein.
Comparison of PMA and fMLP-stimulated
IIb
3
Function--
Next, we compared the ability of fMLP and PMA to
stimulate the adherence of lymphocytes expressing
IIb
3 to
immobilized fibrinogen. As shown in Fig.
2A, there was a
concentration-dependent increase in lymphocyte adherence
following exposure of the cells to fMLP. In multiple experiments,
maximal adherence was observed at 300 nM fMLP, but was
never more than one-half to two-thirds that induced by 200 ng/ml PMA.
Moreover, no additive effect was seen when cells were stimulated
simultaneously with fMLP and PMA, suggesting that these agonists were
acting through the same signaling pathway.

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Fig. 2.
Comparison of PMA- and
fMLP-stimulated lymphocyte adherence to immobilized fibrinogen.
A, 1.5 × 105 B lymphocytes, metabolically
labeled with [35S]methionine and co-expressing IIb 3
and the fPR, were added to the wells of microtiter plates coated with
fibrinogen at 10 µg/ml and incubated for 30 min at 37 °C in the
presence or absence of 200 ng/ml PMA, the indicated concentrations of
fMLP, or both 200 ng/ml PMA and 1000 nM fMLP together.
Lymphocyte adherence to the immobilized fibrinogen was quantitated as
described under "Experimental Procedures." B, the effect
of soluble fibrinogen on lymphocyte adherence to immobilized fibrinogen
stimulated by 300 nM fMLP was measured by performing the
assay described in A in the presence of increasing
concentrations of soluble fibrinogen. The data in A and
B are expressed relative to the adherence of PMA-stimulated
cells in the absence of soluble fibrinogen; adherence of the
PMA-stimulated cells is designated 1.0. The data are also expressed as
the mean and S.E. of quadruplicate determinations.
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The adherence of PMA-stimulated lymphocytes to immobilized fibrinogen
is inhibited by soluble fibrinogen (4). As shown in Fig. 2B,
the adherence of fMLP-stimulated cells also decreased as the
concentration of soluble fibrinogen in the suspending buffer increased.
Moreover, like cells stimulated by PMA (4), inhibition was maximal at a
concentration of soluble fibrinogen of 40-50 µM.
Platelet stimulation induces a change in the affinity of
IIb
3 for
ligands and enables it to bind soluble fibrinogen (13). Previously, we
found that PMA stimulation enabled transfected lymphocytes expressing
IIb
3 to bind soluble fibrinogen (4). To determine if fPR
stimulation would do likewise, we stimulated lymphocytes coexpressing
IIb
3 and the fPR with either 200 ng/ml PMA or 300 nM
fMLP and used flow cytometry to compare the binding of FITC-fibrinogen.
As we had seen previously (4), the fluorescence histogram of
PMA-stimulated lymphocytes was shifted substantially to the right of
the histogram of unstimulated cells, indicating that fibrinogen was
bound to the stimulated cells (Fig.
3A). The fluorescence
histogram of fMLP-stimulated lymphocytes was also shifted to the right,
although again fMLP was a less potent agonist than PMA (Fig.
3B). Preincubating both sets of stimulated cells with the
mAb A2A9 prevented the shifts in fluorescence, confirming that the
FITC-fibrinogen was bound to
IIb
3. Moreover, like the binding of
fibrinogen to stimulated platelets (13, 15), fibrinogen binding to PMA
and fMLP-stimulated lymphocytes reached saturation at a fibrinogen
concentration of
200 µg/ml and was abolished by either a 15-fold
excess of unlabeled fibrinogen or 200 µM RGDS (data not
shown).

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Fig. 3.
FITC-fibrinogen binding to PMA- and
fMLP-stimulated lymphocytes. Purified human fibrinogen was labeled
with fluorescein isothiocyanate as described under "Experimental
Procedures." GM1500 cells expressing IIb 3 were suspended in a
10 mM sodium phosphate buffer, pH 7.4, containing 1 mM CaCl2 and incubated with 0.25 µM fluorescein-labeled fibrinogen in the absence or
presence of 200 ng/ml PMA (A) or 300 nM fMLP
(B) for 30 min at room temperature. The cells were then
washed with suspension buffer, fixed in buffer contain 0.37% formalin,
and examined by flow cytometry. The specificity of FITC-fibrinogen
binding was determined by performing the incubation in the presence of
the inhibitory mAb A2A9 at a concentration of 50 µg/ml.
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Effect of Signaling Pathway Inhibitors on
IIb
3 Function in
Lymphocytes--
Because PTX inhibits fMLP-stimulated Ca2+
flux in GM1500 cells expressing the fPR, we asked whether PTX would
inhibit fMLP-stimulated
IIb
3 function in these cells. As
expected, preincubating the cells with PTX had no effect on
PMA-stimulated adherence to fibrinogen (Fig.
4). However, PTX reduced fMLP-stimulated
adherence to nearly baseline levels. On the other hand, no inhibition
was observed when the cells were stimulated with both fMLP and PMA,
indicating that PMA was able to bypass the PTX effect. Identical
results were seen when FITC-fibrinogen binding, rather than cell
adherence, was used as the indicator of
IIb
3 function (data not
shown).

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Fig. 4.
Inhibition of PMA- and fMLP-stimulated
lymphocyte adherence to fibrinogen by PTX. To determine the effect
of PTX on IIb 3-mediated lymphocyte adherence to fibrinogen,
GM1500 cells coexpressing the fPR and IIb 3 were preincubated with
either buffer or 100 ng/ml PTX for 2 h. Lymphocyte adherence to
immobilized fibrinogen stimulated by 200 ng/ml PMA, 100 nM
fMLP, or both agonists together was measured as described in the legend
to Fig. 2 and under "Experimental Procedures." The data are
expressed as the mean and S.E. of quadruplicate determinations.
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The fMLP- and interleukin 8-stimulated adherence of murine B cells
mediated by
4
1 is unaffected by inhibiting PKC (16). To determine
the effect of PKC inhibitors on fMLP-stimulated
IIb
3 function in
human B cells, we incubated transfected GM1500 cells overnight with
nanomolar concentrations of either the high affinity PKC inhibitor BIM
I (17) or the low affinity inhibitor BIM V (18) and measured
agonist-stimulated lymphocyte adherence to fibrinogen. As expected, BIM
I reduced PMA-stimulated adherence to baseline levels, whereas the same
concentrations of BIM V had no effect (Fig.
5). However, to our surprise, BIM I, but
not BIM V, also reduced fMLP-stimulated adherence to baseline levels. Identical results were seen when FITC-fibrinogen binding was measured instead of adherence (data not shown). Thus, these experiments suggest
that fPR stimulation regulates
IIb
3 function in human B cells via
a signaling pathway that includes PKC.

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Fig. 5.
Inhibition of PMA- and fMLP-stimulated
lymphocyte adherence to fibrinogen by the PKC inhibitors BIM I and BIM
V. The effect of BIM I ( ) and BIM V ( ) on
IIb 3-mediated lymphocyte adherence to fibrinogen was determined
by incubating GM1500 cells coexpressing the fPR and IIb 3
overnight with the indicated concentrations of the inhibitors.
Lymphocyte adherence to immobilized fibrinogen was stimulated by either
200 ng/ml PMA or 100 nM fMLP and measured as described in
the legend to Fig. 2 and under "Experimental Procedures." The data
are expressed as the mean and S.E. of quadruplicate
determinations.
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Stimulation of integrin function is associated with the activation of a
number of protein tyrosine kinases (19). To determine whether tyrosine
phosphorylation regulates
IIb
3 function in lymphocytes, we
incubated transfected GM1500 cells overnight with micromolar
concentrations of the tyrosine kinase inhibitor genistein (20) and
measured agonist-stimulated lymphocyte adherence to fibrinogen. As
shown in Fig. 6, genistein concentrations
as high as 150 µM had no effect on either PMA- or
fMLP-stimulated lymphocyte adherence. A small degree of inhibition (29 and 16%, respectively) was observed at a genistein concentration of
300 µM, a concentration at which genistein also affects
the activity of serine/threonine kinases (20). Activation of the
non-receptor tyrosine kinase Syk is an early event after platelet
stimulation by agonists such as collagen and thrombin (21, 22),
although in Epstein-Barr virus-transformed lymphocytes, Syk activity is
constitutively inhibited by the Epstein-Barr virus-encoded protein LMP2
(23). Nevertheless, to determine whether residual Syk activity in
GM1500 cells could regulate
IIb
3 function, we incubated
transfected cells with the Syk inhibitor piceatannol (24) and measured
both PMA and fMLP-stimulated lymphocyte adherence to fibrinogen. As shown in Fig. 6, 30 µg/ml piceatannol, a concentration that
completely inhibited collagen-induced platelet aggregation, had no
effect on
IIb
3 function in cells stimulated by either PMA or
fMLP.

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Fig. 6.
Effect of protein tyrosine kinase inhibitors
on PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen.
GM1500 cells coexpressing the fPR and IIb 3 were incubated
overnight with either the protein tyrosine kinase inhibitor genistein
at 150 µM or the Syk inhibitor piceatannol at 30 µg/ml.
Lymphocyte adherence to immobilized fibrinogen was stimulated by either
200 ng/ml PMA ( ) or 100 nM fMLP ( ) and measured as
described in the legend to Fig. 2 and under "Experimental
Procedures." The data are expressed as the mean and S.E. of
quadruplicate determinations. , no agonist.
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Role of the Actin Cytoskeleton in Regulating
IIb
3 Function in
Lymphocytes--
Inhibitors of actin polymerization impair
1 and
2 integrin function in leukocytes, suggesting that the actin
cytoskeleton regulates integrin function in these cells (14, 16, 25). Whether the actin cytoskeleton regulates
IIb
3 function in
platelets is less certain, although it was recently reported that the
actin polymerization inhibitor cytochalasin E inhibits
thrombin-stimulated
IIb
3 function (26). To determine if the actin
cytoskeleton regulates
IIb
3 function in GM1500 cells, we measured
the effect of cytochalasin D on unstimulated and PMA-stimulated
lymphocyte adherence to fibrinogen. We found that increasing
concentrations of cytochalasin D inhibited PMA-stimulated adherence,
with few adherent cells remaining at a cytochalasin D concentration of 10 µM (Fig. 7). By
contrast, submicromolar concentrations unexpectedly, but consistently,
increased unstimulated adherence, such that adherence in the presence
0.01 µM cytochalasin D was 2-3-fold greater than in its
absence. We also examined the effect of cytochalasin D on
FITC-fibrinogen binding to both PMA-stimulated and unstimulated GM1500
cells. As shown in Fig. 8A,
0.1 µM cytochalasin D, but not 0.01 µM
cytochalasin D, completely inhibited FITC-fibrinogen binding to
PMA-stimulated cells. Conversely, whereas cytochalasin D concentrations
of 0.1 µM or greater did not influence the interaction of
FITC-fibrinogen with unstimulated lymphocytes, 0.01 µM
cytochalasin D consistently induced FITC-fibrinogen binding to these
cells, albeit to a limited degree (Fig. 8B).

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Fig. 7.
Effect of cytochalasin D on PMA-stimulated
and unstimulated lymphocyte adherence to fibrinogen. The adherence
assay described in the legend to Fig. 2 and under "Experimental
Procedures" was performed in the presence of the indicated
concentration of cytochalasin D using either unstimulated GM1500 cells
expressing IIb 3 or the same cells stimulated with 200 ng/ml PMA.
The data shown are the mean and S.E. of three separate experiments.
PMA-stimulated adherence in the absence of cytochalasin D was
designated 100% adherence. , no PMA; , PMA.
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Fig. 8.
Effect of cytochalasin D on FITC-fibrinogen
binding to PMA-stimulated and unstimulated lymphocytes. The effect
of the indicated concentrations of cytochalasin D (CD) on
the binding of FITC-fibrinogen to either PMA-stimulated (A)
or unstimulated (B) GM1500 cells expressing IIb 3 was
determined using flow cytometry as described in the legend to Fig. 3
and under "Experimental Procedures." The dashed line in
B corresponds to the peak fluorescence intensity of
unstimulated cells incubated with 10 µM cytochalasin D. The data shown are representative of three separate experiments.
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The Rho family of small GTPases regulates a number of cellular
functions such as shape, motility, and adhesion by reorganizing the
actin cytoskeleton (27). In lymphocytes, inhibiting RhoA with C3
exoenzyme from C. botulinum impairs
agonist-stimulated cell adhesion mediated by
L
2 (14) and
4
1
(16). C3 exoenzyme has also been reported to inhibit thrombin-induced
platelet aggregation (28). To determine whether RhoA plays a role in
the regulation of
IIb
3 function in GM1500 cells, we introduced C3
exoenzyme into the cells using Lipofectin and measured its effect on
PMA and fMLP-stimulated adherence to fibrinogen. To verify that C3 exoenzyme had ADP-ribosylated RhoA in the Lipofectin-treated cells, an
aliquot of these cells was homogenized and re-exposed to the enzyme in
the presence of [32P]NAD+. As shown in Fig.
9A, there was a substantial
reduction in the incorporation of 32P into RhoA from cells
that had been treated with Lipofectin in the presence of C3 exoenzyme
compared with cells that had been treated with Lipofectin in its
absence. This indicates that RhoA in the former cells had been
ADP-ribosylated during the first incubation with C3 exoenzyme,
rendering it resistant to ADP-ribosylation during the second.
Nevertheless, as shown in Fig. 9B, inhibiting RhoA with C3
exoenzyme had essentially no effect on the ability of the cells to
adherence to fibrinogen following stimulation with either low or high
concentrations of PMA and fMLP.

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Fig. 9.
Effect of C3 exoenzyme on PMA- and
fMLP-stimulated lymphocyte adherence to fibrinogen. 12 µg of C3
exoenzyme was introduced into 1 × 107 GM1500 cells
coexpressing the fPR and IIb 3 using Lipofectin as described under
"Experimental Procedures." A, inhibition of RhoA was
determined by incubating homogenates of cells incubated with Lipofectin
in the absence (lane 1) or presence of C3 exoenzyme
(lane 2) with 50 ng of C3 exoenzyme and 10 µM
[32P]NAD+ (Amersham) for 1 h at
30 °C. 32P-Labeled RhoA was then visualized by 0.1%
SDS-10% polyacrylamide gel electrophoresis and autofluorography.
B, the effect of RhoA inhibition on PMA- and fMLP-stimulated
lymphocyte adherence to fibrinogen was measured using the adherence
assay described in the legend to Fig. 2 and under "Experimental
Procedures." Control cells were incubated with Lipofectin in the
absence of C3 exoenzyme and C3 cells were incubated with Lipofectin in
the presence of 12 µg of C3 exoenzyme. The data are expressed as the
mean and S.E. of quadruplicate determinations.
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DISCUSSION |
To delineate signaling pathways that can convert
IIb
3 from
an inactive to a ligand binding conformation, we have expressed recombinant
IIb
3 in human B lymphocytes. Initially, we found that
the phorbol ester PMA not only induced the adherence of these cells to
fibrinogen, but enabled
IIb
3 to bind soluble fibrinogen (4). In
the current work, we asked whether an extracellular agonist acting
through its cognate membrane receptor could induce
IIb
3 function
in these cells. Stimulation of G protein-coupled receptors in platelets
results in
IIb
3 activation (7). Similarly, stimulation of these
receptors leads to integrin activation in lymphocytes and other cells
of the hematopoietic lineage (9). We found that by coexpressing
IIb
3 with the human formyl peptide receptor, a
seven-transmembrane domain G protein-coupled receptor (9), we could
induce B cell adherence to immobilized fibrinogen and soluble
fibrinogen binding to
IIb
3 using the chemoattractant peptide
fMLP. Moreover, we found that the signaling pathway initiated by fPR
stimulation involved PKC and similar to signaling initiated by PMA,
resulted in a change in the actin cytoskeleton.
The human fPR is normally expressed in phagocytic cells where it
transduces signals by activating the PTX-sensitive G proteins G
i2 or G
i3 (9). We found that PTX
inhibited the fMLP-stimulated interaction of co-transfected GM1500
cells with both immobilized and soluble fibrinogen, implying that
G
i activation can regulate
IIb
3 function in these
cells. Whether G
i can also regulate
IIb
3 function
in platelets is uncertain. However, PTX inhibits thrombin-stimulated
phosphoinositide hydrolysis in saponin-permeabilized human platelets,
suggesting that at least the thrombin receptor in human platelets can
couple to G
i (29). On the other hand, platelets from
G
q-deficient mice fail to aggregate in response to
thrombin, ADP, collagen, arachidonic acid, and U46619, despite normal
levels of G
i, indicating that PTX-insensitive G protein G
q couples agonist receptors to
IIb
3 in murine
platelets (30). Nonetheless, it is possible that the difference between
human lymphocytes and murine platelets may simply reflect a difference in the types of signaling pathways that are present in human and murine
cells, similar to a difference in the types of thrombin receptors
expressed by human and murine platelets (31, 32).
The ability of inhibitors of PKC (33) to inhibit platelet aggregation
and/or ligand binding to
IIb
3 implies that protein phosphorylation by PKC regulates
IIb
3 activity. Moreover, the PKC
activator PMA is a potent stimulus for ligand binding to
IIb
3 on
both human platelets (6) and G
q-deficient murine
platelets (30). The identity of the proteins phosphorylated by PKC in platelets to regulate
IIb
3 function is unknown. The
3
cytoplasmic tail has been found to contain phosphorylated threonine
residues after platelet stimulation by thrombin, PMA, or the
thromboxane analogue U46619 (34). However, the fraction of
3
containing phosphorylated threonine in both resting and stimulated
platelets was low and unlikely to affect the function of more than a
few
IIb
3 heterodimers. We found that the fMLP-stimulated
interaction of
IIb
3 with either immobilized or soluble fibrinogen
in B cells was prevented by the specific PKC inhibitor BIM I. Thus,
activation of PKC, either directly with PMA or via fPR activation of
G
i, is sufficient to induce
IIb
3 function in
lymphocytes. On the other hand, Laudanna et al. (16)
observed that inhibiting PKC had no effect on fMLP-stimulated
4
1
function in murine B cells (16). Again, it is possible that this
difference simply reflects differences between human and murine cells.
It is also possible that at least two signaling pathways can be
initiated by fPR stimulation and that these pathways can differentiate
between
1 and
3 integrins. In support of this possibility, Weber
et al. (35) found that signaling pathways arising from the
receptors for the chemoattractants RANTES, MCP-3, and C5a in
eosinophils can differentially regulate the function of
4
1 and
L
2. Whether PKC-independent pathways also exist in B cells that
can regulate
IIb
3 function remains to be determined.
Phosphorylation of tyrosines 747 and 759 in the
3 cytoplasmic tail
has also been detected after thrombin-stimulated platelet aggregation
(36). However, this phosphorylation was not observed after thrombin
stimulation in the absence of aggregation, suggesting that it is a
consequence of ligand binding to
IIb
3 ("outside-in" signaling), rather than being part of the process of
IIb
3
activation. Blystone et al. (37) also detected
phosphorylation of
3 Tyr-747 after monocytes were exposed to
Mn2+ or platelets were treated with either Mn2+
or thrombin. When they expressed
v
3 heterologously in K562 cells,
they found Tyr-747 phosphorylation necessary, but not sufficient, to
support either PMA and thrombin-stimulated cell adhesion, and like
Tyr-747 phosphorylation in platelets (36), required ligand binding to
v
3. We found that the protein tyrosine kinase inhibitor genistein
had no effect on the ability of
IIb
3 in B lymphocytes to interact
with fibrinogen. Similarly, we found that piceatannol, an inhibitor
reportedly specific for the tyrosine kinase Syk found in lymphocytes
and platelets (38), had no effect on either PMA- or fMLP-stimulated
IIb
3 function in lymphocytes. Thus, our data, combined with the
inability to detect significant amounts of phosphorylated serine or
threonine on the
3 of stimulated platelets (34), suggest that
phosphorylation of
IIb
3 is not required to regulate its
interaction with ligands.
The biochemical events that follow PKC activation in lymphocytes and
platelets are uncertain. However, one consequence of PKC-mediated
signaling is regulation of membrane-cytoskeletal interactions (39). For
example, Kucik and co-workers (40) found that exposing human B
lymphocytes to PMA increased the diffusion of
L
2 in the plane of
the lymphocyte membrane and augmented lymphocyte adherence to ICAM-1.
Similar effects were observed following exposure of the lymphocytes to
low concentrations of cytochalasin D. These data suggest that
cytoskeletal constraints, released by either PKC activation or
cytochalasin D, maintain
L
2 in a low avidity state. Lub et
al. (41) extended these observations by showing that maximum
L
2-mediated lymphocyte adherence required both
L
2
clustering and an increase in its affinity for ICAM-1. We found that
micromolar concentrations of cytochalasin D inhibited the
PMA-stimulated interaction of lymphocytes expressing
IIb
3 with
either immobilized or soluble fibrinogen. Conversely, we found that
nanomolar concentrations of cytochalasin D actually induced fibrinogen
binding to
IIb
3 on unstimulated cells. Thus, our observations
suggest that both PKC and the actin cytoskeleton play a role in
regulating both the avidity and affinity of
IIb
3 for ligands. How
this might occur is uncertain. Platelet stimulation results in a
conformational change in
IIb
3 that increases its affinity for
ligands (42, 43). It is also associated with the disassembly of
polymerized actin, followed by actin reassembly and a change in
platelet morphology (44). It is conceivable that low concentrations of
cytochalasin D can initiate actin disassembly, resulting in increases
in both integrin mobility and affinity. Fox et al. (44)
found that a variable amount of the
IIb
3 in detergent lysates of
unstimulated platelets was recovered with fragments of the membrane
skeleton and was redistributed to a detergent-insoluble fraction
containing a network of cytoplasmic actin filaments after ligand
binding. They also observed that high concentrations of cytochalasin E
inhibited the binding of the activation-dependent mAb PAC1
to
IIb
3 on ADP and thrombin-stimulated platelets (26). Thus, it
is possible that the membrane skeleton in platelets, or in our case in
lymphocytes, interacts with the cytoplasmic tails of
IIb
3 to
constrain the integrin in a low affinity configuration. Relief of this
constraint by agonists (or cytochalasins) could then be responsible for
an augmented interaction of
IIb
3 with immobilized fibrinogen and
for its ability to bind soluble ligands.
Platelet stimulation is also associated with the formation of clusters
of ligand-occupied
IIb
3 on the platelet surface (26). However,
ligand valency does not appear to be a factor in the ability of
IIb
3 to recognize soluble ligands (45). Moreover,
IIb
3 is a
univalent receptor (11) and electron microscopy of fibrinogen bound to
IIb
3 suggests that a fibrinogen molecule can only bind to one
IIb
3 heterodimer on the surface of a given platelet (46). Hence,
it is unlikely that clustering of
IIb
3 alone can account for its
ability to bind soluble fibrinogen. Nevertheless, it is possible, and
even likely, that agonist-induced clustering of
IIb
3 contributes
to the augmented adherence of stimulated lymphocytes and platelets to
immobilized fibrinogen.
Activity of members of the Ras family of small GTPases has major
effects on cytoskeletal organization. For example, microinjection of
activated forms of the small GTPases cdc42, Rac, and Rho into Swiss 3T3
fibroblasts results in the formation of filopodia, lamellopodia, and
stress fibers, respectively (27). Similar effects have been reported in
macrophages (47). Moreover, the small GTPase RhoA appears to play an
important role in regulating integrin function in leukocytes, and
perhaps in platelets as well. For example, inhibiting RhoA with C3
exoenzyme prevented PMA-stimulated
L
2-mediated homotypic
lymphocyte aggregation (14) and the fMLP-stimulated
4
1-mediated
adherence of murine lymphocytes to VCAM-1 (16). Morii and co-workers
(28) have reported that C3 exoenzyme completely inhibits thrombin and
PMA-stimulated human platelet aggregation, although they found little
correlation between the extent of RhoA inhibition by the C3 exoenzyme
and the extent to which platelet function was impaired. On the other
hand, in preliminary studies, Leng et al. (48) did not
observe the reported effect of C3 exoenzyme on platelet aggregation. We
found that C3 exoenzyme had no effect on either PMA and fMLP-stimulated
lymphocyte adherence to fibrinogen, despite substantial
ADP-ribosylation of RhoA. Thus, RhoA is unlikely to transduce the
signals that regulate
IIb
3 function, at least in our system. A
more likely candidate is Rac1. Microinjection of activated Rac1 into
Swiss 3T3 cells induces the submembranous accumulation of actin and the
formation of integrin-containing focal complexes at the cell margin
(49). Furthermore, activation of Rac1 by the exchange factor Tiam1 in
T-lymphoma cells induces the formation of submembranous actin
filaments, membrane ruffling, and an invasive phenotype (50). Thus, it
is conceivable that a Rac-mediated reorganization of the membrane
skeleton in lymphocytes, and by extrapolation in platelets, could be an
intermediary step in the regulation of
IIb
3 function by
agonists.
In summary, we have shown that the ability of integrin
IIb
3,
expressed in B lymphocytes, to interact with immobilized and soluble
fibrinogen can be stimulated by the G protein-coupled formyl peptide
receptor. Moreover, our studies begin to define a signaling pathway
that includes a PTX-inhibitable G-protein, PKC, and the actin
cytoskeleton.