 |
INTRODUCTION |
During the formation of a platelet thrombus, the binding of
fibrinogen and von Willebrand factor to integrin
IIb
3 triggers outside-in signals. These
signals promote secretion of
- and dense granules, the secondary
wave of aggregation, filamentous actin formation, cytoskeletal
rearrangement, and the formation of platelet membrane vesicles with
procoagulant activities (1). Defects in outside-in signaling through
IIb
3 cause abnormal platelet-mediated
clot retraction and aggregation and excessive bleeding in the
hereditary disorder Glanzmann's thrombasthenia (2, 3) and in mice
engineered to express integrin
3 in which the
cytoplasmic tyrosines have been replaced with phenylalanine (4).
A number of molecules and pathways have been identified to be involved
in the earliest integrin-mediated outside-in signaling events (5).
Several downstream effects and mediators have also been identified,
including mitogen-activated protein kinases
(MAPKs) and myosin light chain kinases
(MLCKs). MAPKs are a family of serine/threonine kinases activated by
diverse extracellular stimuli like growth factors, cytokines, hormones,
and other stress factors (6). The MAPK cascade consists of a
three-kinase module; MAPK is the most distal, being activated by MEK,
which, in turn, is activated by a MEK kinase. Activation of MAPK
requires dual phosphorylation at threonine and tyrosine residue in a
Thr-X-Tyr motif. There are four known members of the MAPK
family: 1) extracellular signal-regulated kinase (p44ERK1
and p42ERK2), 2) c-Jun N-terminal kinase or
stress-activated protein kinase (p46JNK1 and
p55JNK2), 3) the p38 kinase, and 4) ERK5/big MAPK (BMK1).
ERK1 and ERK2 are involved in cell growth, proliferation, and adhesion,
whereas ERK5 is important for angiogenesis (6, 7). JNK and p38 have a
role in apoptosis (6). All the MAPK family members except BMK1 have
been identified in human platelets, but their function is
inadequately understood.
MLCK is a Ca2+/calmodulin-dependent enzyme that
phosphorylates Thr18 and Ser19 on the
regulatory light chain of myosin (8). MLCK contains multiple MAPK
consensus phosphorylation sites (PX(S/T)P) (9) and is
directly phosphorylated by ERK2 (10). Phosphorylation of myosin light
chains (MLCs) by MLCK is a critical regulatory step in myosin function
and regulates cell migration, cytoskeletal clustering of integrins, and
shape change and secretion in platelets (11).
Integrin
3 is polymorphic at residue 33 (Leu33 or Pro33; also known as PlA1
or PlA2, respectively), and the Pro33 form has
been associated with an enhanced adhesive phenotype in cell lines and
platelets (12, 13) and with acute coronary syndromes in some studies
(14). This polymorphism is not rare, and 25% of individuals of
Northern European descent express Pro33 isoforms on their
platelets. Compared with Leu33, Pro33 cells
exhibit enhanced integrin
IIb
3-mediated
outside-in signaling to focal adhesion kinase and
cytoskeletal-dependent cellular functions like fibrin clot
retraction and cell adhesion (12). This raises the possibility that
compared with Leu33 cells, Pro33 cells can
provide more efficient
IIb
3 outside-in
signaling. Evidence supporting a role for focal adhesion kinase and the
cytoskeleton in integrin-mediated MAPK activation (15, 16) led us to
explore whether the Leu-to-Pro substitution at amino acid 33 could
modulate
IIb
3 signaling through MAPK.
Using Chinese hamster ovary cells (CHO) and 293 cell lines
overexpressing equivalent levels of the two isoforms of
IIb
3 and human platelets, we found that
compared with the Leu33 isoform, the Pro33
variant of
3 induced greater outside-in activation of
ERK2 and/or MLCK. Inhibition of MLCK and ERK2 activation abolished the
increased adhesion to fibrinogen and clot retraction associated with
Pro33 cells, and MLCK inhibition abolished the increased
P-selectin secretion in Pro33 platelets.
 |
MATERIALS AND METHODS |
Reagents--
Human fibronectin was obtained from Invitrogen.
Human fibrinogen was from Enzyme Research Laboratories Inc. (South
Bend, IN). Cytochalasin, wortmannin, bovine serum albumin (BSA),
phosphatase inhibitor mixture, phorbol 12-myristate 13-acetate (PMA),
and sorbitol were from Sigma. Fluorescein isothiocyanate-labeled
anti-
IIb
3 (P2) and anti-P-selectin
antibodies were from Immunotech (Marseilles, France). Fluorescein
isothiocyanate-labeled anti-mouse antibody was from Pierce.
Anti-
v
3 antibody (LM609) was from
Chemicon International, Inc. (Temecula, CA). Antibodies specific for
the phosphorylated forms of ERK, JNK, and p38; anti-ERK1/2; and
inhibitors PD98059 (2'-amino-3'-methoxyflavone) and U0126 were obtained
from Promega (Madison, WI). Antibody specific for diphosphorylated MLC
was a generous gift from Dr. James Staddon (Eisai London Research, London). Antibodies to JNK and MLC were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-p38 antibody was obtained from Cell
Signaling Technology (Beverly, MA). Antibody LIBS6 was a gift from Dr.
Mark Ginsberg (Scripps Research Institute, San Diego, CA), and
antibodies 10E5 and c7E3 were gifts from Dr. Barry Coller (Rockefeller
University, New York).
Cell Lines and Flow Cytometric Analysis--
Stable cell lines
overexpressing
IIb
3 were generated by
flow cytometric sorting using monoclonal antibody specific for
IIb
3 as previously described (12). These
included the "vector-only" control CHO cells (designated LK) and
CHO cells overexpressing the Leu33 and Pro33
isoforms of
IIb
3 (designated
Leu33 and Pro33, respectively). To address
concerns about clonal variation that may have occurred in the CHO cell
lines, a second set of cell lines was also generated in the human
embryonic kidney 293 cell line: line PC/Z, vector-only control; line
293Leu33, stably expressing the Leu33 isoform
of
IIb
3; and line 293Pro33,
stably expressing the Pro33 isoform of
IIb
3. Cell-surface expression of
IIb
3 on and 293 cells was analyzed by
flow cytometry using antibody P2, followed by fluorescein
isothiocyanate-labeled anti-mouse antibody (12). The mean channel
number corresponding to cell fluorescence intensity was used as a
measure of
IIb
3 surface expression.
Assessment of
IIb
3 expression levels was
performed within 24 h of each experiment to assure equivalent
expression between the Leu33 and Pro33 cell lines.
Adhesion to Immobilized Ligands and Cross-linking of
IIb
3 Receptors--
Cells were grown to
70-80% confluence and detached using 0.05% trypsin. After
neutralization, the cells were suspended in Tyrode's buffer (138 mM NaCl, 2.9 mM KCl, 12 mM
NaHCO3, 0.36 mM Na2HPO4, and 5.5 mM glucose, pH
7.4) containing 1.8 mM CaCl2 and 0.49 mM MgCl2 for adhesion studies with fibrinogen.
For studies with fibronectin, cells were suspended in Hanks' balanced
salt solution (136 mM NaCl, 5.3 mM KCl, 0.33 mM Na2HPO4, 0.44 mM
KH2PO4, and 5.5 mM glucose, pH 7.4)
with 1.8 mM CaCl2 and 0.49 mM
MgCl2. 24-well tissue culture plates were coated with 12.5 µg/ml fibrinogen, 12.5 µg/ml fibronectin, 10 µg/ml
anti-
IIb
3 antibody P2, 10 µg/ml anti-
v
3 antibody LM609, or 2.5 mg/ml
heat-treated BSA. 200 µl of 5 × 105 cells/ml
were added to each well and incubated for various time points (2.5, 5, and 10 min) at 37 °C in 5% CO2. In some experiments, cells were incubated for 30 min with 10 µM cytochalasin
D, 100 nM wortmannin, 20 µM PD98059, 10 µM U0216, or Me2SO (control) prior to the
adhesion experiments. The unbound cells were removed by washing, and
the cells bound to fibrinogen or fibronectin were lysed in ice-cold
lysis buffer (15 mM HEPES, pH 7.0, 145 mM NaCl, 0.1 mM MgCl2, 10 mM EGTA, 1%
Triton X-100, 2 mM Na3VO4, 250 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 15 µg/ml protease
inhibitors (chymostatin, antipain, pepstatin, and leupeptin), and a
phosphatase inhibitor mixture (Sigma)) as previously described (12).
The non-adherent cells from the BSA-coated wells were collected,
diluted 1:1 in Tyrode's buffer, and centrifuged at 100 × g for 5 min; and the pellet was solubilized in lysis buffer.
The lysates were incubated for 30 min on ice and clarified by
centrifugation at 3750 × g for 30 min, and the protein
concentration was determined using a Bio-Rad protein assay kit. Cells
incubated with 0.5 M sorbitol or 100 nm PMA served as a
positive control for MAPK activation. In some experiments, MAPK
activation was assessed after monoclonal antibody clustering of the
IIb
3 receptor. 5 × 105
cells suspended in 50 µl of Tyrode's buffer containing 1.8 mM CaCl2 and 0.49 mM
MgCl2, pH 7.4, were first incubated with 10 µg/ml
F(ab')2 fragment from antibody 10E5 or c7E3 for 30 min at 4 °C, followed by a 1:200 dilution of Fab-specific goat anti-mouse IgG for 20 min at 37 °C as described in other systems (17). The
cells were washed once with 200 µl of Tyrode's buffer and lysed.
Adhesion of Platelets to Immobilized Fibrinogen--
Blood was
obtained in acid/citrate/dextrose from normal, healthy, and fasting
donors of known PlA genotype. Washed platelets were
prepared as described (13), suspended in Tyrode's buffer, and allowed
to recover for 2 h at 37 °C. Fibrinogen (12.5 µg/ml) or
heat-denatured BSA was immobilized on six-well plates as described
above. 1 ml containing 1 × 108 platelets was added to
each well and incubated for 15 min at 37 °C in 5% CO2.
The fibrinogen-bound platelets and the non-adherent platelets from the
BSA-coated well were lysed in ice-cold lysis buffer, and the protein
content was determined.
MAPK and MLCK Activation--
MAPK activation was assessed by
immunoblotting using monoclonal antibodies specific for the active
(dual tyrosine- and threonine-phosphorylated) forms of activated
p44/42, p38, and JNK. MLC activation was determined by immunoblotting
using antibody specific for diphosphorylated (Thr18 and
Ser19) MLC. For these studies, 20-50 µg of protein
obtained from the lysates described above were separated by 7-10%
reducing SDS-PAGE; transferred to nitrocellulose membrane; blocked with
5% nonfat milk in Tris-buffered saline (20 mM Tris-HCl, pH
7.6, and 150 mM NaCl) containing 1% Tween 20 (TBS-T)
overnight at 4°C; and incubated with anti-phospho-ERK1/2
antibody (1:5000 dilution), anti-phospho-JNK antibody (1:5000
dilution), anti-phospho-p38 antibody (1:1000), or
anti-diphosphorylated MLC antibody (1:500) for 2.5 h at room
temperature. The blots were washed with TBS-T and incubated with
horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000)
for 1 h, and the immunoreactive bands were visualized using an ECL
system (Amersham Biosciences). To confirm equal loading of MAPK, the
membrane was stripped in buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol
for 30 min at 50°C and blocked with 5% nonfat milk. The
blots were reprobed with antibody to ERK1/2 (1:5000), JNK (1:3000), p38
(1:1000), or MLC (1:1000) as described above. The signals were
scanned using Photoshop Version 5.5 software, and densitometric
quantitation was performed using NIH Image software (Scion Image
beta Version 4.0.2, Scion Corp., Frederick, MD).
Adhesion Using a Parallel Plate Flow Chamber--
Glass
coverslips were coated with 200 µl of 12.5 µg/ml fibrinogen and
incubated for 3 h in a humidified chamber. Before using each
coverslip, excess fibrinogen was rinsed with Tyrode's buffer, and then
the parallel plate flow chamber was assembled with the coverslip
forming the base of the chamber. Measurements of cell adhesion under
flow conditions were as we have previously described (18). During the
experiments, the parallel plate flow chamber was mounted on an
inverted-stage microscope (Eclipse TE300, Nikon Instruments, Melville,
NY) equipped with a ×20 phase objective and a high-speed camera
(Quantix Photometrics, Photometrics Ltd., Tucson, AZ) connected to a
computer terminal. In most experiments, 5 × 105 cells
were perfused through the chamber for 5 min at a constant flow rate of
25 s
1, which produces a constant wall shear stress. In
some cases, cells were pretreated for 30 min with 20 µM
MAPK inhibitor PD98059 or 10 µg/ml ML-7 and control Me2SO
before perfusion. Four fixed fields of observation were identified, and
the number of cells adhering to these regions in each experiment was
counted using the Metamorph imaging system.
Clot Retraction Assay--
Clot retraction was measured as we
have previously described (12). Briefly, CHO cells (4 × 106) were pretreated with either Me2SO or 20 µM PD98059 for 1 h. Cells were washed once with
Tyrode's buffer; resuspended in 300 µl of
-minimal
essential medium containing 28 mM CaCl2 and 25 mM HEPES, pH 7.4; and mixed with 200 µl of
fibronectin-depleted plasma, 250 µg of fibrinogen, and 5 µg of
aprotinin. Clot formation was initiated by the addition of 2.5 units of
thrombin. After incubation at 37 °C for varying time periods, the
volume of liquid not incorporated into the clot was measured.
Platelet Secretion Studies--
Secretion of
-granules was
studied by assaying for P-selectin expression using a modification of
our previously described whole blood flow cytometric assay (13).
Briefly, 10 µl of washed platelets (2 × 106) of
known PlA genotype were incubated with 10 µl of 0.2 mg/ml
fluorescein isothiocyanate-labeled anti-P-selectin antibody in a total
of 40 µl of Tyrode's buffer containing 10 mM HEPES and
activated using varying concentrations of thrombin for 2 min. Samples
were fixed with 0.5% paraformaldehyde and analyzed for P-selectin
expression by flow cytometry. In some experiments, platelets were
incubated with Me2SO or 10 µg/ml ML-7 before thrombin stimulation.
 |
RESULTS |
Immobilized Fibrinogen Induces Outside-in Signaling to ERK2, but
Not to JNK and p38--
To determine whether integrin
IIb
3 can induce outside-in signaling to
MAPK, CHO cells overexpressing
IIb
3 were
allowed to adhere to immobilized fibrinogen or maintained in suspension over a BSA substrate. Substantially greater levels of phosphorylated ERK2 were detected in adherent Pro33 cells compared with
Leu33 cells at 2.5, 5, and 10 min (Fig.
1A, lane 3 versus lane 2). Compared with Leu33 cells,
Pro33 cells exhibited an ~10-fold increase in
phosphorylated ERK2 at 2.5 min and an ~5-fold increase at 5 and 10 min (Fig. 1B). The levels of total ERK in Leu33
and Pro33 cells were equivalent and could not account for
the signaling differences (Fig. 1A). ERK2 signaling was
dependent upon
IIb
3 and fibrinogen
because 1) no phosphorylated ERK2 was detected in the vector control LK
cells not expressing
IIb
3 (Fig.
1A, lane 1); 2) cells maintained in suspension
over the BSA substrate did not trigger activation of ERK2 (lanes
4-6); and 3) adhesion to fibrinogen under these conditions is
completely inhibited with integrelin and
IIb
3-specific function-blocking antibody
10E5 (12). A 2.5-fold increase in ERK2 phosphorylation was observed in
Pro33 cells compared with Leu33 cells in
response to soluble fibrinogen binding (data not shown).

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 1.
Activation of ERK2 in CHO and 293 cells
adhering to immobilized fibrinogen. A, LK (vector-only
parental), Leu33 (designated A1), and
Pro33 (designated A2) CHO cells were allowed to
adhere to fibrinogen (FGN; lanes 1-3) or
maintained in suspension over a BSA matrix (lanes 4-6) for
2.5, 5, and 10 min, after which the cells were solubilized, and 20 µg
of protein were separated by 10% SDS-PAGE and blotted with
anti-phospho-ERK antibody (pERK2 panels). ERK2 migrated at
the expected molecular mass of 42 kDa as determined with molecular mass
markers. The same blot was stripped and reprobed with anti-ERK antibody
(ERK1/2 panels) to assess equivalency of total ERK1/2. This
blot is representative of four different experiments. B,
shown are the results of densitometric quantification of ERK2
activation (ratio of phosphorylated ERK2 to total ERK2 in arbitrary
units) in cells that adhered to fibrinogen at 2.5, 5, and 10 min. The
enhanced ERK2 activation in Pro33 over Leu33
cells was significant (p = 0.01) as determined by
repeated measure analysis of variance. C, shown is the
activation of ERK2 in 293 cells adhering to immobilized
fibrinogen. Vector-only 293 (lanes 1 and 4),
293Leu33 (lanes 2 and 5), and
293Pro33 (lanes 3 and 6) cells
were studied as described for A. The 2.5-min time point is
shown. This blot is representative of three different experiments.
D, shown is the mean fluorescence intensity of antibody P2
( IIb 3-specific) binding to the three CHO
cell lines and the three 293 cell lines used in these experiments
performed within 24 h of the adhesion experiments.
|
|
Although CHO cell lines were generated by cell sorting and should not
have been subject to clonal variation, we generated a second set of 293 cell lines to confirm the effects of the substitution of Leu with Pro
at amino acid 33. Essentially the same results were obtained with the
293 cells as with the CHO cells (Fig. 1, C and
D). Densitometry revealed that compared with
293Leu33 cells, 293Pro33 cells demonstrated
7-fold greater phosphorylation of ERK2 at 2.5 min (data not shown).
Relative to CHO cells, in 293 cells, the
Pro33-dependent difference in ERK2 activation
appeared to lessen over time (data not shown), perhaps reflecting
cell-type specificities. Nevertheless, at early time points in both
cell lines, the
3 Pro33 substitution
exhibited greater activation of ERK2. For the data shown in the rest of
this report, we show studies with CHO cells, although similar results
were obtained in 293 cells.
Under similar conditions, adhesion of Leu33 and
Pro33 cells to fibronectin did not cause activation of ERK2
(Fig. 2A), indicating that the
differential signaling due to the Pro33 polymorphism of
integrin
3 is ligand-specific. The cells used in these
experiments were fully capable of activating ERK2 because PMA and
sorbitol caused robust activation in all three cell lines (Fig.
2B, lanes 1-6). The absence of ERK signaling on
fibronectin might reflect trans-dominant inhibition of
integrin signaling, perhaps due to the cross-talk between the
overexpressed
IIb
3 and the endogenous
5
1 integrins.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 2.
Activation of ERK2 in CHO cells adhering to
immobilized fibronectin. A, immunoblot of phospho-ERK2
and total ERK1/2. CHO cells were allowed to adhere to fibronectin
(FN; lanes 2-4) or maintained in suspension over
a BSA substrate (lanes 5-7) for 2.5, 5, and 10 min and
processed as described in the legend to Fig. 1. Lane 1 contains lysates of LK cells treated with 100 nM PMA as a
positive control for the anti-phospho-ERK antibody blotting
(pERK2 panels). The same blot was stripped and reprobed with
anti-ERK antibody (ERK1/2 panels). B, immunoblot
showing ERK1/2 activation in all three cell lines treated with 100 nM PMA or 0.5 M sorbitol for 10 min. The blot
was probed with anti-phospho-ERK antibody (upper panel) or
anti-ERK antibody (lower panel). This blot is representative
of two different experiments. Surface expression of
IIb 3 was not detectably different between
the Leu33 (A1) and Pro33
(A2) cell lines (not shown).
|
|
To determine whether the enhanced phosphorylation of ERK2 was mediated
primarily through
IIb
3, ERK2 activation
was examined in cells adhering to wells coated with
anti-
IIb
3 antibody P2, anti-
v
3 antibody LM609, or BSA. There was
an ~10-fold greater phosphorylation of ERK2 in Pro33
cells compared with Leu33 cells that adhered to antibody P2
(Fig. 3, A and B).
No ERK2 activation was detected in LK cells (Fig. 3A,
lane 4) and in cells maintained in suspension over BSA
substrate (lanes 7-9). Adhesion to LM609-coated wells did
not trigger activation of ERK2 in either Leu33 or
Pro33 cells (Fig. 3A), indicating that low
levels of chimeric hamster-human
v
3
expressed in these cell lines did not contribute to ERK2 signaling. We
next examined whether other related MAPK members are activated by the
integrin
IIb
3-fibrinogen interaction. In contrast to ERK2 activation, no phosphorylated JNK or phosphorylated p38 was observed in response to cell adhesion to fibrinogen at 2.5, 5, and 10 min (Fig. 4, A and
B). However, JNK and p38 in these cells were fully activated
in response to 0.5 M sorbitol treatment (Fig. 4,
A and B, lanes 7-9). These studies
indicate that 1) integrin
IIb
3 is capable
of inducing outside-in signaling to ERK2, but not to JNK or p38; and 2)
compared with the Leu33 isoform, the Pro33
variant of
IIb
3 confers early and
efficient ERK2 signaling.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
Activation of ERK2 in CHO cells adhering to
immobilized antibodies. A, immunoblot showing
phosphorylated ERK2 from the three CHO cell lines that adhered to
antibody LM609 (lanes 1-3) or antibody P2 (lanes
4-6) or that were maintained in suspension over a BSA matrix
(lanes 7-9) for 5 min. The blot was probed with
anti-phospho-ERK antibody (pERK2; upper panel) or
anti-ERK antibody (lower panel). B, densitometric
quantification of ERK2 activation in cells that adhered to antibody P2
at 5 min in three different experiments. The increased ERK2 activation
in Pro33 (A2) over Leu33
(A1) cells was significant (p = 0.03) as
determined by repeated measure analysis of variance. C, mean
fluorescence intensity of antibody P2 binding to the three CHO cell
lines as determined by flow cytometry. These signaling differences were
not due to either differences in total ERK levels (A,
lower panel) or differences in the surface expression of
IIb 3 (C).
|
|

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of JNK and p38 in CHO cells
adhering to immobilized fibrinogen. CHO cells were studied, and
lysates were processed as described in the legend to Fig. 1 and
immunoblotted for active and total JNK (A) and p38
(B). A, immunoblot probed with anti-phospho-JNK
antibody (pJNK2/pJNK1) recognizing activated JNK1 (p46) and
JNK2 (p54). The same blot was stripped and reprobed with anti-JNK1
antibody. Cells treated with 0.5 M sorbitol (lanes
7-9) was included in every experiment to confirm the ability to
detect phospho-JNK and to demonstrate that JNK can be activated in
these cells. Data shown are representative of three experiments.
B, immunoblot probed with anti-phospho-p38 (pP38)
and anti-p38 antibodies. Lanes 7-9 show cells treated with
0.5 M sorbitol. FGN, fibrinogen; A1,
Leu33 cells; A2, Pro33 cells.
|
|
IIb
3 Cross-linking Activates
ERK2--
Adhesive processes often involve multivalent receptor-ligand
interaction and the clustering of integrins. We used antibody-mediated cross-linking to further assess the role of this mechanism in the
Pro33 signaling effect. Antibody cross-linking of
IIb
3 resulted in greater activation of
ERK2 in Pro33 cells compared with Leu33 cells
(Fig. 5A, lane 3 versus lane 2), whereas incubation with either
antibody 10E5 or the secondary antibody alone did not cause activation
in either cell line (lanes 4-9). Similar results were obtained in both CHO cells (Fig. 5B, lane 3 versus lane 2) and 293 cells (data not shown) using
anti-
3 antibody c7E3. Cross-linking with antibody c7E3
showed a small basal level of ERK2 phosphorylation even in vector-only
(LK) cells (Fig. 5B), perhaps due to the ability of the Fab
fragment from antibody c7E3 to bind other endogenous integrins on CHO
cells. In these cross-linking studies, densitometry showed
1.5-3.1-fold greater ERK2 phosphorylation in Pro33 cells
compared with Leu33 cells (data not shown).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 5.
Activation of ERK2 in CHO cells using
antibody-mediated cross-linking of integrin
3. Integrin
IIb 3 in CHO cells was cross-linked using
either antibody 10E5 (A) or c7E3 (7E3;
B), followed by goat anti-mouse antibody (GAM).
Total ERK levels were not different in each lane (A,
lower panel). This blot is representative of three different
experiments. Shown in C is the mean fluorescence intensity
of antibody P2 binding to the three CHO cell lines as determined by
flow cytometry. Surface expression of
IIb 3 was not detectably different between
the Leu33 (A1) and Pro33
(A2) cells and could not account for the signaling
difference. pERK2, phospho-ERK2.
|
|
ERK2 Activation Is Mediated by MEK and Requires Post-ligand Binding
Events--
Sequential activation of the Ras GTPase and the kinases
Raf and MEK is the best characterized pathway for activation of the ERKs (15). To determine whether activation of ERK2 in CHO cells adherent to fibrinogen was mediated through MEK, we preincubated cells
in the presence and absence of the MEK inhibitors PD98059 and U0216 and
studied cell interactions with fibrinogen. Adhesion of
Me2SO-treated (control) cells to immobilized fibrinogen
caused greater activation of ERK2 in Pro33 cells than in
Leu33 cells (Fig. 6,
A, lane 3 versus lane 2; and
B, lane 5 versus lane 3).
In contrast, treatment with PD98059 (Fig. 6A) or U0216 (Fig.
6B) completely prevented the induction of ERK2 activity in
both Leu33 and Pro33 cells. Because PD98059
binds to inactive MEK and prevents Raf from phosphorylating MEK, these
data demonstrate that activation of ERK in Leu33 and
Pro33 cells is mediated through upstream MEK/Raf.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
Activation of ERK2 in CHO cells is mediated
through upstream MEK and requires post-ligand binding events.
Cells were incubated with Me2SO (DMSO) or
inhibitors, allowed to adhere to fibrinogen for 5 min, and
immunoblotted as described in the legend to Fig. 1. The MEK
inhibitors used were PD98059 (20 µg/ml; A) and U0126 (10 µg/ml; B). In C, cells were treated with 10 µg/ml cytochalasin D (CYTO-D) or 100 nM
wortmannin (WORTMAN.) prior to adhesion to immobilized
fibrinogen. The data are representative of two different experiments.
ERK2 was not phosphorylated in any experiment in which cells were
maintained in suspension over a BSA matrix (not shown). Surface
expression of IIb 3 was not detectably
different between the Leu33 (A1) and
Pro33 (A2) cell lines in these experiments (not
shown). pERK2, phospho-ERK2.
|
|
To examine whether cytoskeleton assembly has a role in ERK2 activation,
cells were preincubated with cytochalasin D. Cytochalasin D completely
prevented the induction of ERK2 activity in both Leu33 and
Pro33 cells (Fig. 6C, lanes 2 and
3 versus lanes 5 and 6). Cytoskeletal reorganization in response to integrin activation is activated by lipid
kinases like phosphoinositide 3-kinase (19), and we used the
phosphoinositide 3-kinase inhibitor wortmannin to examine the possible
effect of phosphoinositide 3-kinase on the
IIb
3-mediated activation of ERK2.
Induction of ERK2 activity in both Leu33 and
Pro33 cells was ablated by wortmannin (Fig. 6C,
lanes 2 and 3 versus lanes 8 and
9). These results indicate that activation of ERK2 is
dependent on post-ligand binding events such as actin
polymerization and phosphoinositide 3-kinase signaling.
Outside-in Signaling in CHO Cells and Platelets to
MLC--
Compared with Leu33-expressing CHO cells,
Pro33 cells show a dramatically increased reorganization of
the actin cytoskeleton when bound to immobilized fibrinogen (12).
Because myosin and MLCK are critical for reorganization of the actin
cytoskeleton and MLCK is a cytoplasmic substrate of ERK2 (10), we
examined outside-in signaling in CHO cells and human platelets to MLCK.
MLCK phosphorylates Thr18 and Ser19 on MLC, and
diphosphorylated MLC can be detected by a specific antibody (20).
Adhesion to immobilized fibrinogen caused greater phosphorylation of
MLC in Pro33 cells than in Leu33 cells (Fig.
7A, lane 3 versus lane 2). Compared with Leu33 cells,
Pro33 cells exhibited an ~1.8-fold increase in the levels
of diphosphorylated MLC. Because MLCK activation is required for
MLC phosphorylation at Thr18 and Ser19, we
interpret the data in Fig. 7 to mean that MLCK has been differentially activated in the Pro33 and Leu33 cell lines.
The difference in MLCK activation between Leu33- and
Pro33-expressing CHO cells was largely transient and
appeared to lessen at later time points (data not shown).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
Activation of MLCK in CHO cells and human
platelets adhering to immobilized fibrinogen. A, LK,
Leu33 (A1), and Pro33
(A2) CHO cells were allowed to adhere to fibrinogen
(FGN; lanes 1-3) or maintained in suspension
over a BSA matrix (lanes 4-6) for 2.5 min, after which the
cells were solubilized, and 50 µg of protein were separated by 7%
SDS-PAGE and blotted with anti-diphosphorylated MLC antibody
(ppMLC; upper panel) or anti-MLC antibody
(lower panel). Similar results were obtained in two other
experiments. The levels of total MLC in Leu33 and
Pro33 cells were equivalent (lower panel). Cells
maintained over the BSA substrate showed little-to-no diphosphorylation
of MLC. B, shown is an immunoblot of diphosphorylated MLC in
washed human platelets that were allowed to adhere to fibrinogen for 15 min or maintained in suspension over a BSA matrix. The blot was probed
with anti-diphosphorylated MLC antibody (upper panel) or
anti-MLC antibody (lower panel). C, shown are the
results from densitometric quantification of MLCK activation in four
PlA1,A1 (Pro33-negative (neg))
subjects and five PlA1,A2 (Pro33-positive
(pos)) subjects. Compared with PlA1,A1
platelets, PlA1,A2 platelets demonstrated 3.5-fold greater
MLCK activation upon binding to fibrinogen.
|
|
The above studies were conducted exclusively in
IIb
3-expressing CHO or 293 cells. We next
assessed MLCK activity in human platelets expressing the
Pro33 isoform of
3. Compared with platelets
lacking the Pro33 form, adhesion of
Pro33-positive platelets to fibrinogen resulted in an
enhanced diphosphorylation of MLC (Fig. 7B, lane
1 versus lane 3). No phosphorylation of MLC was
detected in platelets maintained in suspension over BSA substrate.
Compared with Pro33-negative platelets, adhesion of
Pro33-positive platelets to fibrinogen revealed a 3.5-fold
increase in the levels of diphosphorylated MLC (Fig.
7C).
Inhibition of ERK2 and/or MLC Activation Abolishes
Enhanced Functional Effects in Pro33-expressing Platelets
and Cells--
Fibrin clot retraction is a classic
IIb
3-mediated outside-in signaling
function. Compared with Leu33 cells, the Pro33
variant exhibited a small but significant increase (p = 0.02) in fibrin clot retraction at varying time points, and this
increase was abolished by the MEK inhibitor PD98059 (p = 0.63) (Fig. 8, A and
B). PD98059 was also able to abolish the greater adhesion of
Pro33 cells to fibrinogen under shear stress in a parallel
plate flow chamber (data not shown).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of inhibiting ERK2 activation on
the 3 Pro33-mediated
increase in clot retraction. CHO cells were treated with
Me2SO or the MEK inhibitor PD98059 (20 µg/ml) for 30 min
and washed, and thrombin-induced clot retraction was performed at the
time points indicated. Shown is the clot retraction of LK ( ),
Leu33 ( ), and Pro33 ( ) cells in the
presence of Me2SO (A) or 20 µM
PD98059 (B). Pro33 cells showed significantly
greater fibrin clot retraction compared with Leu33 cells
(p = 0.02), and this difference was abolished by the
MEK inhibitor PD98059 (p = 0.633; repeated measures
analysis of variance). The results are expressed as S.E. of three
independent experiments. Surface expression of
IIb 3 was not detectably different between
the Leu33 and Pro33 cells (not shown).
|
|
Because MLCK signaling modulates platelet secretion (11), we examined
the functional consequence of enhanced MLCK signaling in
Pro33 platelets. Compared with Leu33 platelets,
Pro33-positive platelets exhibited an ~3-fold increase in
-granule secretion (as reflected in P-selectin expression) in
response to 0.5 units of thrombin (p = 0.04), and this
increase was abolished by the MLCK inhibitor ML-7 (p = 0.35) (Fig. 9, A and
B). We also tested the effect of MLCK inhibition on cell
adhesion under fluid shear stress. Compared with Leu33
cells, significantly more Pro33 cells adhered to fibrinogen
under shear stress (p = 0.005) (Fig. 9C),
and MLCK inhibition abolished the difference in adhesion between
Leu33 and Pro33 cells (p = 0.792 in the presence of ML-7). The enhanced adhesion of
293Pro33 cells to fibrinogen could also be abolished by
treatment with ML-7 (Fig. 9D). These data indicate that
enhanced ERK2 and/or MLC signaling through the Pro33
isoform of integrin
3 regulates cellular functions of
IIb
3.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of inhibiting MLCK activation on
the 3 Pro33-mediated
increased secretion in platelets and increased adhesion in cells.
Washed platelets were incubated with varying thrombin concentrations
for 2 min in the presence of Me2SO (DMSO;
A) or the MLCK inhibitor ML-7 (10 µg/ml; B) and
evaluated for P-selectin expression. Results are expressed as S.E. from
seven PlA1,A1 (Pro33-negative (neg))
subjects and seven PlA1,A2 (Pro33-positive
(pos)) subjects. p = 0.04 ( ),
p = 0.03 (*), and p = 0.1 (#) for
Pro33-negative versus Pro33-positive
subjects in the presence of Me2SO.
p = 0.06 ( ), p = 0.35 (**), and
p = 0.1 (#) for Pro33-negative
versus Pro33-positive subjects in the presence
of ML-7. CHO (C) and 293 (D) cells were treated
with Me2SO or ML-7 (10 µg/ml) for 30 min, washed, and
perfused over immobilized fibrinogen in a parallel flow chamber at a
flow rate of 25 s 1. The number of cells was scored using
a camera linked to the Metamorph imaging system. In C,
compared with Leu33 cells, Pro33 cells
demonstrated 2-fold greater adhesion to fibrinogen (p = 0.005), and ML-7 abolished this difference in adhesion
(p = 0.792). The results are expressed as S.E. of three
independent experiments. In D, compared with
293Leu33 cells, 293Pro33 cells
demonstrated 3-fold greater adhesion to fibrinogen (p = 0.001), and ML-7 abolished this difference in adhesion
(p = 0.695). The results are expressed as S.E. of two
independent experiments. Surface expression of
IIb 3 was not detectably different between
the Leu33 and Pro33 receptors in the CHO and
293 cell lines (not shown).
|
|
 |
DISCUSSION |
Outside-in signaling is crucial for linking integrin ligation with
numerous cellular processes, including adhesion, spreading, migration,
and clot retraction. In this study, we used CHO and 293 cells and human
platelets to evaluate the impact of the
Leu33/Pro33 polymorphism on
IIb
3 outside-in signaling. The major
findings of this study demonstrate that the Pro33 form of
IIb
3 can induce enhanced outside-in
signaling via MAPK and MLCK and that these signaling pathways are
indispensable for the hyperfunctional responses shown by
Pro33 platelets and cells. This study identifies a novel
means for regulating integrin function through post-receptor occupancy, i.e. through ERK2 and MLCK signaling, and provides further
insights regarding the molecular mechanisms responsible for the
prothrombotic phenotype of a common inherited variation in integrin
3.
Integrin-mediated ERK2 Activation--
Several different
approaches and two different sets of
IIb
3-expressing cell lines were used to
address the impact of the Leu-to-Pro substitution at position 33 of
integrin
3 on post-receptor occupancy signaling.
Compared with Leu33 cells, Pro33 cells
demonstrated substantially greater activation of ERK2 when cells bound
to immobilized fibrinogen (Figs. 1 and 6). This enhanced ERK2
phosphorylation was substrate-specific (Fig. 2) and dependent upon
IIb
3 (Fig. 3) and an intact actin
cytoskeleton and signaling through MAPK kinase and phosphoinositide
3-kinase (Fig. 6). What are the consequences of this enhanced ERK2
phosphorylation? Integrin-mediated ERK2 activation has been most
intensively studied as a regulator of gene expression and cell
proliferation, but this pathway also regulates haptotactic and
chemotactic cell migration (21). Cell adhesion and spreading are
inhibited by dominant-negative ERK (22) and promoted by ERK activation
(23). Inhibition of ERK activation blocks the formation of peripheral
actin microspikes (24), and active ERK is targeted to newly forming
focal adhesions after integrin engagement (25). We observed the
Pro33 isoform to enhance actin polymerization, spreading,
adhesion, and clot retraction upon integrin engagement (12) and have
observed a Pro33-mediated increase in haptotactic migration
(26). MAPK kinase inhibition had little effect on Leu33
cell adhesion to immobilized fibrinogen (data not shown) or clot retraction (Fig. 8), but MAPK kinase inhibition abolished the Pro33 enhancement of these cellular functions. Thus, the
enhanced ERK2 activation seen in cells expressing the Pro33
variant of integrin
IIb
3 would be
predicted to have greater in vivo adhesive and migratory properties.
Integrin-mediated MLCK Activation--
Human platelets and CHO
cells expressing the Pro33 variant exhibited enhanced
activation of MLCK compared with Leu33-expressing cells
upon adhering to immobilized fibrinogen (Fig. 7). Phosphorylation of
MLCK is a critical regulatory step in myosin function, promoting myosin
ATPase activity and increasing an actinomyosin contractile
response that is involved in platelet shape change and secretion,
regulation of cell migration, and polymerization of actin cables (10).
This is consistent with our previous studies in which we identified 1)
greater actin polymerization, adhesion, and migration of
Pro33 cells on fibrinogen compared with Leu33
cells (12, 26) and 2) a lower threshold for platelet activation,
-granule release, and fibrinogen binding in Pro33
homozygous platelets compared with Leu33-expressing
platelets (13). Because MLCK is a substrate for ERK2, the results with
PD98059 and ML-7 (Figs. 8 and 9) strongly suggest that outside-in
signaling via ERK and MLCK controls the enhanced functions of
adhesion, clot retraction, and secretion in Pro33
cells and platelets. It is therefore conceivable that the enhanced ERK2/MLCK signaling in Pro33 cells following the ligation
of
IIb
3 leads to a greater cytoskeletal change and favors stronger and sustained adhesion compared with Leu33 cells. Our findings indicate that platelet physiology
and signaling will be altered between the Leu33 and
Pro33 forms of
IIb
3 and that
the potential prothrombotic consequences apply to a large number of individuals.
Integrin Cross-linking Enhances ERK Activation in Pro33
Cells--
Our studies show that cross-linking integrin
IIb
3 enhanced activation of ERK2 in
Pro33 cells (Fig. 5) in a manner similar to cell adhesion
to immobilized fibrinogen (Fig. 1). This suggests that clustering of
integrin
IIb
3 following adhesion to
fibrinogen may underlie the enhanced activation of ERK2 in
Pro33 cells. This idea is supported by the rapid remodeling
of cytoskeletal machinery in Pro33 cells (12) and
observations that identify the cytoskeletal apparatus as a key
component in the process of integrin clustering (27). The requirement
of intact cytoskeletal structures for ERK2 activation (Fig.
6C) is also consistent with earlier observations that
cytochalasin D blocks integrin-mediated MAPK and Raf activation (16).
It is likely that disruption of actin structure precludes the
formation of a highly ordered cytoskeletal system that is essential for
the recruitment of signaling molecules vital for activation of ERK2.
Indeed, phosphoinositide 3-kinase may be one such intermediate
signaling molecule because wortmannin completely abolished ERK2
activation (Fig. 6C). These results suggest that the
enhanced signaling to ERK2 in Pro33 cells is dependent on
post-fibrinogen binding events, involving clustering of
IIb
3 with subsequent actin rearrangement
and signaling through phosphoinositide 3-kinase.
Extracellular Structure and Intracellular Signaling--
Integrin
cytoplasmic domains have been shown to play an important role in
integrin signaling (28), and this study illustrates that residues in
the extracellular region of integrin
3 can also contribute to signal transduction. Similar regulation of intracellular signals by the extracellular domain of integrin
1 has
been reported (29). How could a conformational change in the
extracellular domain (30) of
3 alter intracellular
signaling? We are currently pursuing two major possibilities wherein
the altered extracellular conformation in the Pro33 isoform
might physically 1) alter the cytoplasmic domain for a more efficient
juxtaposition of
3 tails with proximal signaling molecules like Src and Syk and/or 2) induce or inhibit associations with transmembrane signaling molecules (e.g. PECAM
(platelet endothelial cell
adhesion molecule), Fc
receptor IIA, JAM1
(junctional adhesion molecule-1), etc.) that either enhance or
repress, respectively, intracellular signaling. In either case, ERK2
activation could be modulated through focal adhesion
kinase-dependent or -independent pathways.
ERK and MLCK Signaling in Platelets--
ERK signaling is
important for megakaryocyte differentiation and proplatelet formation
(31, 32), for the glycoprotein Ib-IX-dependent activation
of platelet integrin
IIb
3 (33), and for
regulating the release of stored Ca2+ (34). The effect of
ERK signaling on platelet aggregation and secretion appears to depend
on the concentration of agonists: inhibition of ERK activation blocks
aggregation to low doses of collagen, arachidonic acid, U46619,
and thrombin (33, 35), but high concentrations of agonists can induce
aggregation despite ERK inhibition (36, 37). Signaling through ERK also
regulates cell spreading (31, 38, 39). Perhaps the major effect of ERK
signaling in platelets is to regulate this post-receptor occupancy process that may be distinct from agonist-induced inside-out signaling. This would be quite consistent with previous data showing no difference in fibrinogen binding to the Pro33 and Leu33
isoforms of
3 (13, 40), but consistent differences in
outside-in processes such as bleeding times, spreading, actin
reorganization, and clot retraction (12, 41). Moreover, signaling
through MLCK is required for platelet aggregation and secretion in
response to ADP (42, 43). Pro33-expressing platelets showed
greater activation of MLCK (Fig. 7), and this correlates with the
increased aggregation and greater secretion of P-selectin in
Pro33-positive platelets in response to ADP (13, 44).
Importantly, in this study, we have demonstrated that the enhanced MLCK
signaling in Pro33 platelets is required for its increased
platelet reactivity, as determined by
-granule secretion (Fig. 9,
A and B). Finally, ERK and p38 phosphorylate and
activate cytoplasmic phospholipase A2 in platelets (45-47),
which would release more arachidonic acid in Pro33-positive
individuals during platelet aggregation. This hypothesis is supported
by data from our laboratory (13, 48, 49) and others (41) showing that
Pro33-expressing platelets and cell lines exhibit a greater
dependence on cyclooxygenase than do cells expressing only
Leu33.
In conclusion, we have shown that integrin
IIb
3 engagement with immobilized
fibrinogen induces outside-in signaling to ERK2 and MLCK and that the
Leu33/Pro33 polymorphism regulates the extent
of this signaling. Taken in the context of known functions of ERK and
MLCK, our findings support a mechanism whereby the Pro33
variant has little effect on direct agonist-induced integrin activation, but rather enhances signaling and cell adhesive functions after
IIb
3 has been engaged or
cross-linked by fibrinogen. This prothrombotic phenotype may partially
explain the reported arterial thrombotic risk for
Pro33-positive individuals in some clinical epidemiology studies.