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INTRODUCTION |
Hemostasis, prevention of blood loss from damaged blood vessels,
is dependent upon the activation of platelets by subendothelial collagens (types I and III) of the vessel wall. Platelet activation involves shape change, adhesion, aggregation, and secretion of granule
contents. These events lead to the formation of a clot at the site of
injury (1).
Platelets possess several receptors for collagen, recently reviewed
(2-4), including the integrin
2
1
(glycoproteins Ia-IIa), CD361 (glycoprotein IV), and
glycoprotein VI (GpVI). Platelet activation by collagen is a two-stage
process involving sites in collagen, which support platelet adhesion,
and others, which support both platelet adhesion and activation (5, 6).
At present,
2
1 is considered primarily an
adhesive co-receptor (7), whereas other collagen receptors, notably
glycoprotein VI, activate platelets (8). This study was designed to
clarify which collagen receptors transmit signals to the platelet
interior, information which is crucial for the development of
anti-platelet therapy based on collagen receptor antagonism (9).
Recent evidence suggests that CD36,
2
1,
and GpVI each contribute to both signaling and adhesion to collagen
(10). GpVI, recently cloned (11), acts with the Fc receptor
-chain
(12, 13) as a crucial signaling receptor complex, and platelets
deficient in GpVI fail to aggregate in response to collagen, although
the tyrosine kinase c-Src, but not
p72syk, is activated (14). The role of
2
1 in platelet signaling is unclear:
2
1-reactive snake venoms fuel the debate
on the integrin's role in platelet signaling (15, 16), and
overexpression of
2
1 has recently been
advanced as a risk factor in myocardial infarction and stroke (17,
18).
We have synthesized a collagen-related peptide (CRP) recognized by
GpVI, which shares both the triple-helical structure and activatory
characteristics of collagen (19). CRP comprises a repeating GPO motif,
a sequence representing about 10% of the primary structure of
collagen. CRP, when cross-linked (CRP-XL), is a potent platelet agonist
depending only upon GpVI for its activity. Several lines of evidence
show that CRP-XL is not recognized by
2
1
(19-24).
Recently, we have developed a triple-helical peptide containing the
sequence GFOGER, which is a high affinity binding motif for the
2 I domain (25). This peptide,
GPC-[GPP]5-GFOGER-[GPP]5-GPC, designated
GFOGER-GPP, supports platelet adhesion, as well as collagen, but does
not activate platelets in suspension even when cross-linked
(GFOGER-GPP-XL), nor does it stimulate obvious tyrosine phosphorylation
(23). Its binding to platelets is fully abrogated by the
2-specific monoclonal antibody 6F1. The sequence GFOGER has been co-crystallized with the
2 I domain,
demonstrating its binding to the metal ion-dependent
adhesion site (26).
CRP-XL and collagen elicit very similar signals from platelets,
activating protein kinase C (PKC) (27), mobilizing arachidonic acid
from platelet membranes (19), and Ca2+ from intracellular
stores (27); CRP-XL activates p38 mitogen-activated protein kinase (28)
and p72syk and leads to tyrosine
phosphorylation of many platelet proteins, including phospholipase
C
2 (29) and the Fc receptor
-chain (12). CRP-XL, like collagen,
activates platelet procoagulant expression (24). These studies
emphasize the importance of GpVI as a collagen receptor in platelets.
Platelet activation leads to the up-regulation of tyrosine kinases,
including FAK (30), p72syk (31), and members of
the c-Src family (32). FAK, a 125-kDa cytosolic non-receptor tyrosine
kinase, is associated with focal adhesion plaques of adherent cells
such as fibroblasts and platelets (33). FAK is of particular interest,
because it is considered a key intermediary of signaling through
integrins (34-36).
Phosphorylation of FAK occurs at five tyrosine residues and correlates
with an increase in FAK tyrosine kinase activity. Autophosphorylation of tyrosine 397 allows it to bind the c-Src family member Fyn (37, 38),
whereas phosphorylation of tyrosine 407 and the C-terminal tyrosine 861 may support the interaction of FAK with other signaling molecules (39).
Tyrosines 576 and 577 are phosphorylated by c-Src (40) and contribute
to the regulation of the catalytic activity of FAK. Another possible
regulatory mechanism for FAK is proteolytic cleavage by the
Ca2+-dependent protease calpain, which leads to
a reduction in its autophosphorylation (41).
Evidence from fibroblasts suggests that occupation of
1
integrins is a sufficient stimulus to activate FAK (35, 42, 43). Collagen binding to
2
1 in T cells
protects them from apoptosis in a FAK-dependent manner
(36). Adhesion of platelets to monomeric collagen occurs through
2
1. A causal relationship has been
proposed between
2
1 and FAK activation in
platelets adherent to monomeric collagens (44-46). This does not
exclude the operation of the two-step, two-site model (6), because
other (lower affinity) collagen receptors may come into play only after
platelet adhesion via
2
1. Secondary
binding of sequences within collagen, such as that of the GPO motif to
platelet GpVI, is increasingly viewed as an obligatory activatory event
(10, 22, 23).
The platelet fibrinogen receptor, integrin
IIb
3, is required for FAK activation in
platelets stimulated with thrombin (46), although some stimuli, such as
cross-linking of Fc
RIIA, may activate FAK or other focal
adhesion-associated proteins without involving
IIb
3 (47, 48). FAK phosphorylation in
platelets has been dissociated from both
IIb
3 occupancy and focal adhesion
formation centering on
IIb
3 in the
absence of fibrinogen binding (49, 50).
We considered that FAK tyrosine phosphorylation in platelets might be
an early event after occupancy of
2
1 by
collagen fibers, and that we could determine the relative importance of
2
1 and GpVI in this process by comparing
the capacity of collagen, CRP-XL, and GFOGER-GPP-XL to elicit FAK
tyrosine phosphorylation. Synthetic triple-helical peptides have not
hitherto been examined in this context. We have applied these ligands
to human platelets, immunoprecipitated FAK with specific anti-FAK
antibodies, and determined the tyrosine phosphorylation state of the
enzyme as an index of its activity.
Furthermore, we have examined the role of intracellular
Ca2+ and PKC in platelet FAK activation using the
Ca2+ ionophore, ionomycin, to increase the internal
platelet Ca2+ concentration and the Ca2+
chelator, BAPTA, to buffer platelet cytosolic Ca2+; PKC was
either directly activated using the phorbol ester, TPA, or specifically
inhibited using Ro31-8220.
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EXPERIMENTAL PROCEDURES |
Materials--
Collagen, as native type I fibers isolated from
bovine tendon, was donated by Ethicon Inc., Somerville, NJ. CRP and
GFOGER-GPP (Gly-Pro-Cys-[Gly-Pro-Pro]5-Gly-Phe-Hyp-Gly-Glu-Arg-[Gly-Pro-Pro]5-Gly-Pro-Cys) were synthesized and cross-linked using 3-(2-pyridyldithio)propionic acid N-hydroxysuccinamide ester (P-3415, Sigma, UK) as
described previously (19, 25). Anti-phosphotyrosine (clone 4G10) was from Upstate Biotechnology Inc., Lake Placid, NY. Anti-FAK (catalog no.
F15020/L4) was from Affiniti Research Products, Nottingham, UK, and
C-20 (catalog no. sc-558) was from Santa Cruz Biotechnologies, Inc.,
Santa Cruz, CA. Anti-
2 mAbs, 6F1 and P1E6, were a kind gift from Dr B. S. Coller, School of Medicine, State University of
New York, Stony Brook, NY, and obtained from Calbiochem-Novabiochem (UK) Ltd., Nottingham, UK, respectively. The anti-
1 mAbs, 2A4 and
mAb13, were from Genosys Biotechnologies, Cambridge, UK, and from
Becton Dickinson, Oxford, UK, respectively. Horseradish
peroxidase-linked anti-mouse whole antibody from sheep (NA931), Rainbow
molecular weight markers (RPN756), [32P]orthophosphate,
and Hybond C nitrocellulose (RPN303C) were from Amersham Pharmacia
Biotech, UK. Thrombin (T-4265), apyrase (A-6535), aspirin (A-5376),
12-O-tetradecanoylphorbol-13-acetate (TPA, P-8139), bovine albumin fraction V (A-4503), Ponceau S (P-3504), leupeptin (L-2884), phenylmethylsulfonyl fluoride (P-7626), benzamidine (B-6506),
and luminol (A-4685) were all from Sigma, UK. Chemicals for
electrophoresis were Electran grade from BDH Laboratory Supplies, Poole, UK. 4-Iodophenol (I1, 020-1) was from Aldrich Chemical Co.,
Gillingham, UK. H2O2 (H/1800/07) was from
Fisons Scientific Equipment, Loughborough, UK, and RX medical x-ray
film was from Fuji Photo Film Co. Ltd., Japan. X-ray developer (LX24)
and fixer (FX40) were from Kodak Scientific, Cambridge, UK. BAPTA-AM
(1,2-[bis-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy-methyl ester; 196419), Pansorbin (507858), and
Ro31-8220 (557520) were from Calbiochem-Novabiochem, Nottingham, UK,
and Fura2-AM was from Molecular Probes, Eugene, OR. The fibrinogen receptor antagonist and RGD peptidomimetic, GR144053F, was a generous gift from GlaxoWellcome, Stevenage, UK. All other chemicals were of
standard reagent grade.
Platelet Preparation--
Platelet concentrates, less than 24 h-old, pooled from four donors, were obtained from the National Blood
Service, Long Road, Cambridge, UK, centrifuged at 250 × g for 15 min to remove red blood cells, leaving
platelet-rich plasma, from which the platelets were centrifuged at
700 × g for 15 min. The platelet pellet was resuspended in loading buffer (LB; 145 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM
MgSO4, 0.5 mM EGTA, 10 mM HEPES, pH
7.36). The platelets were pelleted at 700 × g for 10 min and resuspended in LB at 109/ml for immunoprecipitation
and at 5 × 108/ml for other work. Aspirin (100 µM) and apyrase (0.25 units/ml) were used where indicated.
Immunoprecipitation--
Platelet-agonist suspensions (500 µl)
were mixed with an equal volume of 2 × radioimmune precipitation
buffer (2% Triton X-100, 2% sodium deoxycholate, 0.2% SDS (each
w/v), 316 mM NaCl, 2 mM EGTA, 20 mM
Tris/HCl, pH 7.2, with 10 mg/ml leupeptin, 10 mM
benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4), and incubated on ice
for 30 min before centrifugation (13,000 × g) for 5 min at 4 °C. Pansorbin (60 µl/ml lysate) was added to each sample tube and rotated at 4 °C for 60 min. Before use, stock Pansorbin was
centrifuged (13,000 × g) for 1 min at 4 °C,
resuspended in the original volume of 1 × radioimmune
precipitation buffer, and allowed to stand at room temperature for 15 min. It was then centrifuged again and resuspended in 1× radioimmune
precipitation buffer containing bovine serum albumin (1% w/v). Samples
were centrifuged (13,000 × g) for 1 min at 4 °C,
the supernatant was removed, and to it anti-FAK polyclonal antibody
(Santa Cruz) was added at 1 µg per ml of platelet lysate, and the
sample was rotated for 20 h at 4 °C. Pansorbin (60 µl) was
then added to each sample, and the samples were mixed and rotated at
4 °C for 60 min before being centrifuged (13,000 × g) for 1 min at 4 °C. Pellets were washed three times
with 800 µl of ice-cold 1× radioimmune precipitation buffer before
being resuspended in 80 µl of 1× SDS sample buffer (10% glycerol,
0.002% bromphenol blue, 2% SDS, each w/v, 70 mM Tris/HCl,
pH 7.2, with 1% 2-mercaptoethanol, v/v) and boiled for 5 min. Samples
were divided in 2× 40 µl, and proteins were separated by 8%
SDS-polyacrylamide gel electrophoresis then blotted to nitrocellulose (2 h at 1 mA/cm2, Hoefer TE77 semi-dry blotter). Uniform
protein transfer was verified by Ponceau S staining. One blot was
incubated with 4G10 (1:2500) and washed with TBST (20 mM
Tris/HCl, 136 mM NaCl, 0.1% (w/v) Tween 20, pH 7.6), and
anti-phosphotyrosine was detected using horseradish peroxidase-linked
anti-mouse antibody (1:10,000) and enhanced chemiluminescence (1.24 mM luminol, 1.63 mM 4-iodophenol, 2.71 mM H2O2). Phosphorylation was
quantitated densitometrically using a Leica Q500 image analyzer (51)
and is expressed as a percentage change relative to control values. The
other blot was probed with monoclonal anti-FAK (Affiniti, 1:1000), to
verify uniform recovery of FAK. Each experiment was performed using a different platelet preparation on three separate occasions.
Platelet Aggregation--
Platelets were prepared as for
immunoprecipitation and resuspended to 109/ml in LB. 150 µl of suspension was stirred (1100 rpm) in an aggregometer at
30 °C as described (19), and inhibitors or solvent were added and
followed 5 min later by ligand in a volume of 3 µl as indicated.
To verify the inhibitory properties of the anti-
1
antibody, 2A4, washed platelets were prepared from whole blood (19), and preincubated in the aggregometer as above for 1 min with 2A4 (20 µg/ml), before addition of just sufficient collagen fibers to cause
maximal aggregation.
Protein Kinase C Activity--
Platelets were prepared as for
immunoprecipitation and resuspended to 109/ml in LB. They
were labeled with 32Pi at 100 µCi/ml for
1 h at 30 °C, centrifuged to remove excess radiolabel, and
resuspended to 109/ml. Samples (20 µl) were treated with
ligand (5 µl), and the reaction was stopped after 2 min using 25 µl
of Laemmli buffer (51). Proteins were separated on a 10%
polyacrylamide gel, and the phosphorylation of a 47-kDa protein band
(p47, presumed to be pleckstrin, the major protein kinase C substrate
in platelets (52)) was detected by autoradiography.
Intracellular Ca2+ Measurement--
Platelet
concentrates were centrifuged to remove red cells as above and loaded
with 2 µM Fura2-AM at room temperature for 45 min.
Platelets were pelleted by centrifugation as above and resuspended to
108/ml in LB. The platelet suspensions were transferred to
a Spex Fluoromax DM3000CM fluorimeter, and fluorescence excited at 340 and 380 nm was used to calculate the intracellular calcium
concentration as described previously (53). Where indicated, BAPTA-AM
(20 µM) was preincubated after Fura2 loading for 20 min.
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RESULTS |
Immunoprecipitation of FAK--
FAK, immunoprecipitated from
platelets activated by collagen at 25 µg/ml, showed a
time-dependent increase in tyrosine phosphorylation (Fig.
1a). The increase in tyrosine
phosphorylation at 60 s was detectable but small; therefore, 5-min
incubation, causing a substantial increase in FAK tyrosine
phosphorylation, was chosen for subsequent assays. Fig. 1b
shows equal recovery of FAK in each sample.

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Fig. 1.
Collagen fibers and CRP-XL activate FAK in a
time-dependent fashion. a and b,
platelets treated with collagen (25 µg/ml) for the indicated times
were lysed, and FAK was precipitated and immunodetected using 4G10
anti-phosphotyrosine (a) or anti-FAK (b) as
described. c and d, platelets were treated with
CRP-XL (5 µg/ml) and handled otherwise as for a and
b.
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Immunoprecipitation of FAK from platelets activated by CRP-XL at 5 µg/ml showed a time-dependent increase in tyrosine
phosphorylation of FAK (Fig. 1c). Fig. 1d shows
immunoprecipitated FAK from CRP-XL-activated platelets, using the
Affiniti monoclonal anti-FAK for immunodetection. Again, equal amounts
of FAK were demonstrated in each sample.
Effect of Ligand Concentration--
Fig.
2, a and d, shows a
concentration-dependent increase in the tyrosine
phosphorylation of FAK immunoprecipitated from platelets activated by
different concentrations of collagen and CRP-XL. Equal recovery of FAK
was demonstrated in all cases (data not shown). Collagen fibers at 25 µg/ml and CRP-XL at 5 µg/ml caused near-maximal increases in FAK
tyrosine phosphorylation. Some experiments (data not shown) were
performed in the presence of apyrase, which scavenges ADP secreted by
activated platelets, and aspirin, which blocks the conversion of
arachidonate to thromboxane A2. The inhibitors had no
marked effect, indicating that tyrosine phosphorylation of FAK does not
depend upon these processes in platelets stimulated by collagen or
CRP-XL.

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Fig. 2.
Activation of FAK by collagen fibers and
CRP-XL is dose-dependent and independent of
2 1
occupancy. Platelets were treated with ligand, as indicated, for 5 min, then FAK tyrosine phosphorylation was determined in
immunoprecipitates as described in the legend to Fig. 1. Platelets were
treated in parallel experiments with collagen, up to 100 µg/ml, in
the absence (a) or presence (c) of 50 µM Ca2+ in excess of the 500 µM
EGTA in the buffer. In b, platelets were preincubated with
the anti- 2, P1E6 (2 µg/ml), or anti- 1,
2A4 (20 µg/ml) as indicated, for 5 min, then treated with collagen
fibers (25 µg/ml) for 5 min. In d, platelets were treated
with the indicated levels of CRP-XL and were otherwise handled exactly
as in a; in e platelets were treated with CRP-XL
(5 µg/ml) after preincubation with P1E6 or 2A4 as for
b.
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In some experiments the basal level of FAK tyrosine phosphorylation was
detectable, whereas others (e.g. Fig. 2e) showed
negligible FAK tyrosine phosphorylation. This may reflect variation
between donors, or in the activation state of resting platelets between experiments, as well as in the immunodetection procedure. Conclusions throughout this study are therefore based on comparisons made within an
experiment, and where possible, within immunoblots rather than between blots.
Role of
2
1 in FAK Tyrosine
Phosphorylation by Collagen and CRP-XL--
When FAK was
immunoprecipitated from platelets preincubated with
anti-
2 P1E6 or anti-
1 2A4 for 5 min
before activation with collagen for 5 min, there was no diminution,
confirmed by densitometry, in the level of tyrosine phosphorylation of
FAK induced by either ligand (Fig. 2b). Similar data were
obtained using the anti-
1 mAb13 (data not shown) or the
anti-
2, 6F1 (Fig. 5b).
CRP-XL induces platelet activation without involvement of
2
1 and caused substantial tyrosine
phosphorylation of FAK. This shows that ligation of GpVI, the receptor
for CRP-XL, induces phosphorylation of FAK. As anticipated, the
anti-
2 and anti-
1 antibodies had no
effect on the tyrosine phosphorylation of FAK by CRP-XL.
Recent work in this laboratory has shown that the affinity of platelet
2
1 is dependent upon the presence of
micromolar Ca2+ in the suspending medium (54). For this
reason, the experiments shown above for collagen were repeated in the
presence of a small excess of Ca2+ over EGTA in the buffer,
conditions that support
2
1-dependent platelet
adhesion to immobilized collagens. FAK tyrosine phosphorylation was not
enhanced by the presence of Ca2+ compared with the parallel
incubation in the absence of Ca2+ (Fig. 2c).
We have recently shown the peptide sequence GFOGER to be a recognition
motif in type I collagen for the
2
1 I
domain (25). Application of the cross-linked triple-helical peptide,
GFOGER-GPP-XL, to platelets at up to 50 µg/ml caused no discernible
increase in FAK tyrosine phosphorylation (Fig.
3a). In contrast, in this experiment as in Fig. 2a, collagen fibers caused substantial
FAK tyrosine phosphorylation. The addition of micromolar
Ca2+ to the medium did not support FAK phosphorylation
stimulated by even high levels of the peptide (200 µg/ml).

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Fig. 3.
GFOGER-GPP-XL does not elicit FAK
phosphorylation in the presence or absence of Ca2+.
Platelets were treated with the indicated levels of the
2 1-specific peptide, GFOGER-GPP-XL, for 5 min in the absence (a) or presence (b) as
indicated, of an excess of Ca2+ as in Fig. 2c.
FAK phosphorylation was determined as for Fig. 1. The lane
marked col represents a control using collagen fibers at 25 µg/ml.
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Functional Verification of the
Anti-
2
1 Antibodies--
6F1 as used in
the present study completely blocked platelet adhesion to monomeric
collagen (54, 55). Similar experiments showed both P1E6 and mAb13 to be
effective inhibitors of platelet adhesion to monomeric collagen. The
anti-
2, P1E6, blocked the capacity of reconstituted type
I collagen fibers to induce platelet aggregation (7). We verified here
that both P1E6 and the anti-
1, 2A4, could attenuate the
platelet aggregation stimulated by threshold concentrations of native
collagen fibers (data not shown). Together, these data confirm the
functional activity of the antibodies used here.
Functional Verification of the
IIb
3
Antagonist GR144053F--
Fig. 4 shows
that CRP-XL (5 µg/ml) or thrombin (1 unit/ml), levels of agonist
consistent with the rest of the study, aggregated platelets suspended
in medium containing 0.5 mM EGTA, but that collagen fibers
(25 µg/ml) caused minimal platelet aggregation. Preincubation with
the fibrinogen receptor antagonist GR144053F (1 µM)
reduced the extent of aggregation to <15% of control values in
platelets stimulated by CRP-XL or thrombin.

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Fig. 4.
Platelet aggregation, which occurs in the
presence of EGTA with CRP-XL and thrombin, is sensitive to
GR144053F. Platelets were prepared as for immunoprecipitation
studies and suspended in LB at 1 × 109 per ml. They
were preincubated as indicated with GR144053F (GR; 1 µM) for 5 min, stirring at 1100 rpm in the aggregometer,
then ligands were added to elicit aggregation. a, thrombin
(Thr) 1 units/ml was added; b, CRP-XL
(CRP) was added at 5 µg/ml; c, collagen fibers
(Col) were added at 25 µg/ml, at times indicated by
arrows.
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Effect of GR144053F on FAK Tyrosine Phosphorylation--
Fig.
5a shows that preincubation of
platelets with 1 µM GR144053F, a level which causes
complete blockade of
IIb
3 (54), caused a
substantial reduction in FAK tyrosine phosphorylation in platelets
subsequently stimulated by CRP-XL (77% reduction over four trials) or
thrombin (63% over two trials). This effect was of similar order to
the inhibition (~85%) of aggregation by GR144053F for CRP-XL or
thrombin. In contrast, there was little observable inhibition (15%;
five trials) of the action of collagen by GR144053F, even when used in
conjunction with
2-blockade by 6F1 (Fig. 5b).
Basal phosphorylation of FAK was also inhibited to some extent (30%;
four trials), perhaps indicating a degree of activation of platelets
under resting conditions, consistent with the suggestion, above, that
the basal platelet preparations might to some extent be activated. This
effect of GR144053F was minor compared with the marked inhibition of
the action of CRP-XL or thrombin.

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Fig. 5.
FAK phosphorylation, stimulated by CRP-XL and
thrombin, but not collagen, is sensitive to GR144053F. Platelets
were preincubated with GR144053F (1 µM) for 5 min.
Ligands were then added for a further 5 min, and FAK was
immunoprecipitated as described. a, platelets were
stimulated with CRP-XL (5 µg/ml) or thrombin (1 unit/ml) as indicated
(bas represents basal controls). b, platelets
were stimulated with collagen fibers (25 µg/ml) as indicated. The
presence of GR144053F or of the anti- 2 monoclonal
antibody, 6F1 at 2 µg/ml in the preincubation is denoted by + beneath
the relevant lanes.
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Role of Protein Kinase C in FAK Tyrosine Phosphorylation--
To
investigate signaling pathways required for FAK tyrosine
phosphorylation, we examined the role of protein kinase C. FAK was
immunoprecipitated from platelets stimulated for 5 min with collagen
(25 µg/ml), CRP-XL (5 µg/ml), or TPA (400 nM). As
before, marked tyrosine phosphorylation of FAK was induced by collagen and CRP-XL, whereas control levels were undetectable (Fig.
6a). TPA caused a very minor
increase in FAK tyrosine phosphorylation: densitometry showed that
CRP-XL and collagen were each about 20 times more effective than
TPA.

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Fig. 6.
A role for PKC activity and
[Ca2+]i in the tyrosine phosphorylation of
FAK. Tyrosine phosphorylation was determined in immunoprecipitates
as described, from (a) platelets stimulated with collagen
(25 µg/ml), CRP-XL (5 µg/ml), or TPA (400 nM) for 5 min, as indicated. For b, platelets were preincubated for 10 min with Ro31-8220 (5 µM), denoted by +, then treated
with collagen (25 µg/ml) or CRP-XL (5 µg/ml) for 5 min. For
c FAK tyrosine phosphorylation was determined in platelets
treated with 1 µM ionomycin (Io) with or
without Ro31-8220 preincubation as above. For d, platelets
were preincubated for 20 min with BAPTA-AM (20 µM), then
stimulated with collagen or CRP-XL as indicated. e, a
Western blot of whole platelet lysates, treated with CRP or collagen
after preincubation with BAPTA as indicated, then probed for
phosphotyrosine. The position where FAK is expected to run in this blot
is indicated.
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Effect of Ro31-8220 or BAPTA on Tyrosine Phosphorylation of FAK
Stimulated by Collagen, CRP-XL, or Ionomycin--
Fig. 6b
shows complete inhibition of tyrosine phosphorylation of FAK
immunoprecipitated from platelets after pretreatment with the PKC
inhibitor Ro31-8220 (5 µM) prior to activation by collagen (25 µg/ml) or CRP-XL (5 µg/ml) for 5 min. We have shown 5 µM Ro31-8220 to cause complete inhibition of PKC,
measured as p47 phosphorylation (56). The calcium ionophore, ionomycin, also caused substantial tyrosine phosphorylation of FAK (Fig. 6c), suggesting a role for calcium signaling in FAK
activation, and again, as for collagen and CRP-XL, this action was
substantially attenuated by Ro31-8220.
Preincubation of platelets with the Ca2+-chelating agent,
BAPTA-AM, to buffer rises in intracellular Ca2+, markedly
attenuated the ability of both CRP-XL and collagen fibers to stimulate
tyrosine phosphorylation of FAK (Fig. 6d). For comparison,
in Western blots prepared from whole platelet lysates there was an
inhibition of overall tyrosine phosphorylation stimulated by collagen,
CRP-XL and in the control samples of 12, 21, and 7%, respectively
(Fig. 6e), when platelets were preincubated with BAPTA-AM.
This inhibition indicated that the effects on FAK are highly specific.
In contrast, one band of about 38 kDa increased in intensity
significantly after BAPTA pretreatment in both collagen- and
CRP-stimulated platelets. Note that the effects of BAPTA on the 120-kDa
region are minor for collagen, although much more apparent for CRP,
suggesting that other bands insensitive to BAPTA comigrate with
FAK.
Effect of Ionomycin on PKC Activity--
Fig.
7a shows that ionomycin, from
500 to 2000 nM, was an effective activator of PKC,
determined from the phosphorylation of p47. Higher ionomycin levels
caused no further increase in phosphorylation of p47 (data not
shown).

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Fig. 7.
Ionomycin, collagen, and CRP-XL stimulate PKC
activity. a, platelets were labeled with
32Pi as described then treated with increasing
levels of ionomycin, as indicated, and an autoradiograph was prepared
as under "Experimental Procedures." The position of p47 is
indicated on the left. For b and c,
platelets were prepared as above, then preincubated with 20 µM BAPTA-AM for 20 min, as indicated, before stimulating
with collagen (25 µg/ml), CRP-XL (5 µg/ml), ionomycin
(Io; 1 µM), or TPA (200 nM) as
shown. Autoradiographs were prepared as above.
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Effect of BAPTA-AM on PKC Activity--
Fig. 7b shows
the effect of BAPTA-AM (20 µM) on platelets activated by
collagen (25 µg/ml) or CRP-XL (5 µg/ml). With or without BAPTA
loading, both effectors caused marked activation of PKC, indicated by
p47 phosphorylation. The action of TPA (not shown) or CRP-XL, was
largely insensitive to the presence of BAPTA; only the action of
collagen was noticeably attenuated, but PKC activity persisted at
greater than 50% despite BAPTA loading. This suggests the presence of
Ca2+-sensitive and -insensitive PKC isoforms activated by
collagen receptors in human platelets.
Effect of Ro31-8220 on [Ca2+]i--
Fig.
8 shows time courses for the rise in
[Ca2+]i evoked by collagen (a) or ionomycin (b),
with and without pre-incubation of platelets with the PKC inhibitor,
Ro31-8220. Inhibition of PKC caused a marked increase in both the peak
amplitude and duration of Ca2+ signals observed under these
conditions. Parallel measurement of [Ca2+]i using
Fura2 showed that calcium signaling was abolished by BAPTA loading
(data not shown).

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Fig. 8.
Ro31-8220 enhances Ca2+ signals
elicited by collagen and ionomycin. Platelets were loaded with
Fura2-AM, as described, then stirred in the cuvette of a fluorimeter,
after preincubation with Ro31-8220 for 10 min, where indicated.
Collagen (a, 90 µg/ml) or ionomycin (b, 1 µM) were added as indicated by the arrows.
Intracellular calcium levels were calculated as described under
"Experimental Procedures."
|
|
 |
DISCUSSION |
Our aim in this study was to explore the capacity of the platelet
integrin
2
1 to activate FAK, comparing
the efficacy of the synthetic analogue of collagen, CRP-XL, with that
of native type I collagen fibers and with the
2
1-specific peptide, GFOGER-GPP-XL. Thus,
we intended to determine whether
2
1 acts
as a signaling receptor for collagen in platelets, working from the
premise that FAK phosphorylation is an event likely to be
integrin-dependent in platelets as well as in other cells.
The first part of the present work addresses the role of the collagen
receptor
2
1 in the regulation of FAK.
Both collagen and CRP-XL activate FAK, as indicated by its tyrosine
phosphorylation state, in a concentration- and
time-dependent manner. The failure of antibodies against
the
2 and
1 integrin subunits, which
prevent adhesion to collagen (validated as described under
"Results") to block FAK activation demonstrates that
2
1 occupancy by collagen fibers does not
regulate FAK. This result contrasts with the proposed general role of
1 integrins in FAK activation (43). These experiments were performed in the presence of micromolar Ca2+,
conditions where the integrin is known to be competent to bind collagen
(54). The potency of CRP-XL, which does not bind
2
1, in stimulating tyrosine
phosphorylation of FAK suggests that another collagen receptor, GpVI,
initiates FAK activation in platelets.
Recently, we have identified the sequence GFOGER within collagen type
I, which binds to the I domain of the integrin
2 subunit (25). This peptide sequence, in triple-helical conformation, binds
platelets in
2
1-dependent
manner and supports purified
2
1 binding.
By co-crystallization with the recombinant
2 I domain,
we have shown that the E residue of the peptide coordinates the
divalent cation in the metal ion-dependent adhesion site of the integrin
2 subunit (26). This indicates that the
peptide properly replicates the
2
1-binding properties of collagen.
However, even at levels up to 200 µg/ml with or without micromolar
Ca2+ (Fig. 3b), GFOGER-GPP-XL caused no
discernible increase in tyrosine phosphorylation of platelet FAK. This
shows that neither
2
1 occupancy nor
clustering by the cross-linked peptide is sufficient to activate FAK in
platelets in suspension.
The fibrinogen receptor
IIb
3 has
attracted most attention as a means of regulating FAK activity in
platelets. Collagen and CRP-XL were added to unstirred suspensions of
platelets in the presence of EGTA, conditions where
IIb
3 is not competent and aggregation is
not anticipated (57); therefore, we did not expect
IIb
3 to regulate FAK in these
experiments. To verify this, we added collagen fibers to platelets
stirred in an aggregometer, causing, as anticipated, no significant
aggregation. However, despite the presence of EGTA, both CRP-XL and
thrombin under similar conditions caused some aggregation, which was
highly sensitive to the
IIb
3 antagonist,
GR144053F (Fig. 4). This indicates that GR144053F as used here is a
good antagonist of
IIb
3 occupancy.
Partial inhibition of FAK phosphorylation by GR144053F in platelets
treated with either CRP-XL or thrombin showed that
IIb
3 activation is important in the
regulation of FAK, as has been shown previously for thrombin (44). But
GR144053F had little effect on the activation of FAK by collagen,
consistent with collagen's failure to cause much aggregation under
these conditions. (It should be noted that aggregation is more likely
to occur during stirring in the aggregometer than in all other
components of the study, where platelets were not stirred for more than
a second after the addition of ligand.) The inclusion of both GR144053F and 6F1 (Fig. 5b), to provide simultaneous blockade of
2
1 and
IIb
3, had little effect on FAK tyrosine
phosphorylation stimulated by collagen fibers, which is therefore shown
to proceed in platelets without the involvement of either integrin
under these conditions. Recently, the use of mutant
IIb
3 showed that FAK activation could be
dissociated from
IIb
3 occupancy (49), as
we propose here for the regulation of FAK by collagen fibers.
Integrin-independent activation of FAK has also been reported in
platelets activated using immobilized human IgG (48), an event that
depends instead on Fc
RIIA.
The identity of the collagen receptors responsible for FAK activation
remains to be resolved. Our experiments demonstrate that GpVI occupancy
alone, resulting from treating platelets with CRP-XL, is not sufficient
to elicit full tyrosine phosphorylation of FAK that is independent of
IIb
3. In this respect, CRP-XL shows some
similarity to thrombin. Using specific antibodies to cross-link CD36, a
candidate receptor along with GpVI, others have discounted CD36 as a
regulator of FAK (58), although this technique provides clustering only
of CD36 populations rather than of CD36 with other receptors, as we
expect will occur with the native collagen fibers used here.
Investigation of whether GpVI acts as a co-receptor in regulating FAK
in platelets stimulated with collagen fibers, and the possible role of
CD36 in these events, must await the development of receptor-specific antagonists.
We sought to identify intracellular signaling events that are involved
in the regulation of FAK activity during platelet activation. PKC has
been implicated in FAK activation in both platelets (48, 59) and other
cells (60, 61) that were adherent to non-collagenous substrates. We
found that TPA stimulated only a slight increase in tyrosine
phosphorylation of FAK in platelet suspensions. However, pretreatment
of platelets with the PKC inhibitor, Ro31-8220, virtually abolished
tyrosine phosphorylation of FAK caused by collagen or CRP-XL. This
suggests that, although direct stimulation of PKC itself is
insufficient to cause major stimulation of FAK, PKC is an important
mediator of the tyrosine phosphorylation of FAK stimulated by either
collagen or CRP-XL.
Next, we showed that [Ca2+]i is also important in
the control of FAK tyrosine phosphorylation, by using ionomycin to elicit Ca2+ mobilization, and BAPTA-AM loading to buffer
[Ca2+]i. Ionomycin stimulated tyrosine
phosphorylation of FAK, whereas BAPTA-AM completely abolished tyrosine
phosphorylation of FAK in platelets stimulated with collagen or CRP-XL,
confirming a role for Ca2+. These results contrast with the
work of Haimovich et al. (48) who showed that, in
IgG-adherent platelets, exposure to BAPTA-AM caused, if anything,
increased FAK phosphorylation. The same group reported no effect of
BAPTA-AM on FAK phosphorylation in platelets adherent to fibrinogen
(59), but in the same paper, they show that BAPTA-AM abolishes the
action of thrombin in stimulating FAK in fibrinogen-adherent platelets.
A requirement for increased [Ca2+]i in the
regulation of FAK was similarly proposed for epinephrine-stimulated
platelet suspensions (59). Possibly, the role of Ca2+ in
regulating FAK activity may be ligand-specific.
Ionomycin treatment also activated PKC. To resolve the roles of
[Ca2+]i and PKC, platelets were first
preincubated with Ro31-8220 to inactivate PKC and then treated with
ionomycin. FAK tyrosine phosphorylation was virtually abolished, as in
platelets stimulated with collagen or CRP-XL after PKC blockade. It is
important to note that Ro31-8220 enhanced the increase in
[Ca2+]i stimulated by either collagen or
ionomycin, very likely as a consequence of inhibiting
PKC-dependent Ca2+ ATPases, which export
Ca2+ from the cytosol. Reciprocal experiments showed that,
although BAPTA blocks FAK phosphorylation, it had little effect on PKC activity. Hence, neither elevated [Ca2+]i or
increased PKC activity is sufficient to support FAK phosphorylation,
but each is necessary for FAK activation by collagen, CRP-XL, or
ionomycin. Such a role for Ca2+ has been proposed for
endothelial cell FAK activation consequent to spreading on type IV
collagen (62).
In conclusion, our data suggest that the regulation of platelet FAK by
native collagen fibers is independent of integrins, occurring despite
blockade of
2
1 or
IIb
3 or both. The activation of
phospholipase C
2 via GpVI (29, 63) causes Ca2+ and PKC
signals essential for the regulation of FAK. Yet these signals
together, activated by either CRP-XL or thrombin, are not sufficient to
elicit full FAK phosphorylation without
IIb
3 occupancy. Coordination of signals
from
IIb
3 and other receptors have been
proposed to regulate FAK (34). Our data suggest that collagen, perhaps
because it is recognized by different platelet receptor populations in
addition to GpVI and
2
1, is able to bypass the requirement for integrins. The role and identity of these
co-receptors for collagen remain to be elucidated.