Lipid Products of Phosphoinositide 3-Kinase and
Phosphatidylinositol 4',5'-Bisphosphate Are Both Required for
ADP-dependent Platelet Spreading*
Jean-Michel
Heraud
,
Claire
Racaud-Sultan
§,
Daisy
Gironcel
,
Corinne
Albigès-Rizo¶,
Thierry
Giacomini
,
Séverine
Roques
,
Véronique
Martel¶,
Monique
Breton-Douillon
,
Bertrand
Perret
, and
Hugues
Chap
From the
Institut Fédératif de Recherche
en Immunologie Cellulaire et Moléculaire, INSERM, Unité
326, Hôpital Purpan, F 31059 Toulouse Cedex, the
¶ Laboratoire d'Etudes de la Différenciation et de l
Adherenee Cellulaires/Unité Mixte de Recherches
CNRS-Université Joseph Fourier 5538, Institut Albert Bonniot,
Faculté de Médecine, F38706 La Tronche Cedex, and the
Ecole Nationale Supérieure de l'Aéronautique et de
l'Espace, 10 Avenue Edouard-Belin, 31055 Toulouse Cedex, France
 |
ABSTRACT |
We have shown previously that ADP released upon
platelet adhesion mediated by
IIb
3
integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D.,
Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer,
N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore,
in order to study a possible role of phosphoinositide 3-kinase in
platelet morphological changes following adhesion, human platelets were
pretreated with specific phosphoinositide 3-kinase inhibitors LY294002
and wortmannin. Under conditions where PtdIns-3,4-P2
synthesis was totally inhibited (25 µM LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix,
extended pseudopodia, but did not spread. Moreover, addition of ADP to
the medium did not reverse the inhibitory effects of phosphoinositide
3-kinase inhibitors on platelet spreading. Although synthetic
dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl
phosphatidylinositol 3',4',5'-trisphosphate restored only partially
platelet spreading, phosphatidylinositol 4',5'-bisphosphate
(PtdIns-4,5-P2) was able to trigger full spreading of
wortmannin-treated adherent platelets. Following 32P
labeling of intact platelets, the recovery of
[32P]PtdIns-4,5-P2 in anti-talin
immunoprecipitates from adherent platelets was found to be decreased
upon treatment by wortmannin. These results suggest that the lipid
products of phosphoinositide 3-kinase are required but not sufficient
for ADP-induced spreading of adherent platelets and that
PtdIns-4,5-P2 could be a downstream messenger of this
signaling pathway.
 |
INTRODUCTION |
Platelets play a key role in hemostasis by their capacities to
adhere and to aggregate in response to vascular injury. The most
abundant platelet integrin, the
IIb
3
complex, is largely responsible for platelet aggregation after binding
of soluble fibrinogen. Moreover,
IIb
3
integrin is required for a complete and irreversible platelet adhesion
to the subendothelial matrix (1). In this case, its preferential ligand
is the von Willebrand factor, but under certain conditions fibrinogen
or fibrin could also act as adhesion substrates. In its resting state,
IIb
3 integrin is able to recognize
immobilized fibrinogen. However, the interaction of soluble fibrinogen
with
IIb
3 complex requires a previous
conformational change of the integrin due to an inside-out signaling
pathway. When platelets adhere in vitro to a fibrinogen matrix, they undergo several irreversible morphological changes such as
rounding and spreading. These responses are sustained by a cytoskeletal
reorganization including extension of filopodia, lamellipodia, and
controlled orientation of stress fibers. It has been shown that a
concomitant granular secretion of ADP from adherent platelets was
necessary for spreading (2), and that it controlled specific signals,
i.e. p125FAK and PtdIns
3-kinase1 activations (2, 3).
Data from Haimovich et al. (4) show that tyrosine
phosphorylation of p125FAK tyrosine kinase seems to be
correlated with cell spreading upon platelet adhesion to a fibrinogen
matrix. On the other hand, PtdIns 3-kinase activity has been involved
in cytoskeletal rearrangements occurring during cell motility or
platelet aggregation (5-9). Moreover, studies in whole cells have
demonstrated an association of PtdIns 3-kinase with p125FAK
(10, 11) and the small G proteins Rac and Cdc42 (12), all of them being
involved in the regulation of cytoskeleton organization. Taking
advantage of specific PtdIns 3-kinase inhibitors, LY294002 and
wortmannin (8, 13, 14), we herein demonstrate that PtdIns 3-kinase is
involved in the ADP-signaling pathway that controls platelet spreading.
Nevertheless, our results suggest that PtdIns-4,5-P2, a
phospholipid tightly associated with actin-binding proteins in focal
contacts and a key regulator of actin polymerization (15), could be a
downstream messenger of this signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human fibrinogen, ADP, human thrombin, phorbol
12-myristate 13-acetate (PMA), apyrase, pyruvate kinase,
phosphoenolpyruvate, wortmannin, PtdIns-4-P, PtdIns-4,5-P2,
fatty acid-free bovine serum albumin, and phosphate-buffered saline
(PBS) were from Sigma. Lysophosphatidic acid (LPA), the
thrombospondin-1 cell binding domain peptide H-RFYVVMWK-OH, and the
TXA2 analog U46619 used were, respectively, from
Sigma, Bachem (Voisins-le Bretonneux, France), and Calbiochem
(Meudon, France). LY294002 and GF109203X were obtained, respectively,
from Biomol (Plymouth Meeting, PA) and Glaxo (Les Ulis, France).
Synthetic Di-C16-PtdIns-3,4-P2 (dipalmitoyl L-
-phosphatidyl-D-myo-inositol 3, 4-bisphosphate) and Di-C16-PtdIns-3,4,5-P3 were
purchased from Matreya (Pleasant Gap, PA).
Preparation of Platelets--
Human platelets were isolated from
fresh platelet concentrates (Centre Régional de Transfusion
Sanguine, Toulouse, France) by centrifugation as described previously
(16). All washing procedures were performed at 37 °C in the presence
of apyrase (1 unit/ml) as an ADP scavenger. In some experiments,
platelet-rich plasma was incubated with 100 µM aspirin
for 20 min to block cyclooxygenase activity. Platelets were labeled for
90 min with 0.4 mCi/ml [
-32P]phosphate (Amersham
Pharmacia Biotech, Bucks, United Kingdom), as described previously
(16). They were finally resuspended in modified Tyrode's buffer (pH
7.4) containing 2.5 mM CaCl2.
Cell Adhesion Assays and Lipid Extract Analysis--
Cell
culture flasks (75 cm2, Greiner Labortechnik, Poitiers,
France) were precoated or not (control) with 100 µg/ml of fibrinogen and were then blocked with fatty acid-free bovine serum albumin (3).
The cell adhesion assay was performed using 5 ml of human platelets
(3 × 107 platelets/ml) that were added for 60 min at
37 °C to the fibrinogen-coated flasks or to the control flasks. In
some experiments, the ADP scavenger pyruvate kinase plus
phosphoenolpyruvate (14.3 units/ml and 1 mM,
respectively; 10 min) or the protein kinase C (PKC) inhibitor GF109203X
(12 µM; 60 min) or the PtdIns 3-kinase inhibitors LY294002 (0-25 µM; 10 min) or wortmannin (0-100
nM; 15 min) were added to the platelet suspension before
adhesion. GF109203X, wortmannin, and LY294002 were dissolved in
Me2SO, which did not exceed 0.06% (v/v). Recovering of
adherent cells, evaluation of the extent of cell adhesion, and lipid
extract analysis by HPLC were performed as described previously
(3).
Spreading Restoration Assays--
After elimination of
unattached platelets and two washes with PBS, adherent
wortmannin-treated platelets were incubated with different agonists (20 µM ADP, 5 µM LPA, 50 µM
H-RFYVVMWK-OH, 5 µM U46619, 10 nM PMA, or 1 unit/ml thrombin) or phosphoinositides (PtdIns-4-P,
PtdIns-4,5-P2,
Di-C16-PtdIns-3,4-P2,
Di-C16-PtdIns-3,4,5-P3, or a mixture of these
lipids, 10-30 µM) in Tyrode's buffer for 30 min at
37 °C. Before use, phosphoinositides were dried, suspended in 10 mM Hepes (pH 7.0), and sonicated in the absence of carrier phospholipids.
Optical Microscopy--
At the end of the adhesion step or the
spreading restoration assay, unattached platelets were removed by
washing with PBS and the buffer was replaced by 1% glutaraldehyde in
0.1 M Na2HPO4. Fixation was
continued at room temperature for 15 min. After washing, adherent
platelets were examined by interference light microscopy with a
Reichert EMF4 microscope. Micrographs were taken at original magnification ×1250.
Immunoprecipitation of Talin--
Adherent platelets (4.5 × 108 platelets) were scraped off at 4 °C in a lysis
buffer containing 20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 mM PMSF, 10 µM pepstatin, 10 µg/ml
leupeptin, and 1% (v/v) Triton X-100. Resting platelets in suspension
(4.5 × 108 platelets) were centrifuged and
resuspended in 600 µl of the lysis buffer. After sonication (20 kHz
for 2 × 10 s) and centrifugation (12,000 × g for 10 min at 4 °C), the soluble fraction was collected and subsequently precleared for 30 min at 4 °C with protein
G-Sepharose 4B fast flow (Sigma). Precleared suspensions were then
incubated overnight at 4 °C with the polyclonal anti-talin antibody
prepared as described previously at a 1:50 dilution (17). Capture of immune complex was performed by adding 50 µl of protein G-Sepharose 4B fast flow. The immunoprecipitates were then washed once with PBS
without calcium and magnesium, supplemented with anti-proteases as
described above and 0.1% (v/v) Triton X-100, and twice with the same
buffer without Triton.
Lipid Extraction and Western Blotting on Talin
Immunoprecipitates--
For lipid analysis, immunoprecipitation of
talin was performed after plating of 32P-labeled platelets
as described above. Lipids were extracted as described previously (3)
and separated by TLC following the procedure established by Pignataro
and Ascoli (18). Briefly, lipid extracts were applied on
oxalate-EDTA-impregnated silica gel plates, which were developed twice
for 120 min with CHCl3, CH3OH, 9.15 M NH4OH (40:40:15). Individual lanes containing
commercial standards PtdIns-4-P or PtdIns-4,5-P2 were
stained with iodine vapors. After exposition of the plates for 3-7
days, the radioactive spots were visualized and quantitated by a
PhosphorImager 445 SI (Molecular Dynamics, Inc). Quantification was
also performed after scraping the appropriate areas of the plate and
counting in a liquid scintillation counter.
For protein analysis, anti-talin immunoprecipitates were solubilized,
separated on 7.5% SDS-polyacrylamide gels, and blotted onto
nitrocellulose as described previously (11). Immunodetection of talin
was performed with the mouse monoclonal anti-talin antibody 8d4 from
Sigma. Antibody reaction was visualized using the ECL chemiluminescence
system (Amersham Pharmacia Biotech). Quantification of the different
bands was performed by a densitometric analysis, which determines the
pixel volume in each area (Gel Doc 1000, Bio-Rad).
 |
RESULTS |
Wortmannin and LY294002 Inhibit PtdIns-3,4-P2 Synthesis
Triggered upon Platelet Adhesion--
Wortmannin and LY294002 have
been largely used in platelets as specific inhibitors of PtdIns
3-kinase, at nanomolar (10-100 nM) and micromolar (25 µM) concentrations, respectively (8, 9, 19, 20). Since we
have shown previously that platelet adhesion triggers accumulation of
[32P]PtdIns-3,4-P2 (3), we first assessed the
inhibitory effects of wortmannin and LY294002 on the production of
[32P]PtdIns-3,4-P2 as a reflection of PtdIns
3-kinase activation. P-Labeled platelets were
preincubated with increasing doses of wortmannin or LY294002, and cells
were then plated for 60 min on the fibrinogen matrix before lipid
extraction and analysis by HPLC. As shown in Fig.
1, LY294002 and wortmannin inhibited [32P]PtdIns-3,4-P2 synthesis in a
dose-dependent manner with 80% inhibition achieved at 12 µM and 50 nM, respectively. The production of
[32P]PtdIns-3,4-P2 was totally abrogated at
25 µM LY294002 and 100 nM wortmannin, at
which concentrations platelet adhesion was not significantly affected
(Table I). At these concentrations, among other phosphoinositides (PtdIns, PtdOH, PtdIns-4-P, and
PtdIns-4,5-P2), only the PtdIns-4-P level was found to be
somewhat decreased, but not significantly in comparison with control
Me2SO-treated platelets (Fig.
2).

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Fig. 1.
Dose-dependent inhibition of
adhesion-induced [32P]PtdIns-3,4-P2
synthesis by wortmannin and LY294002. Washed platelets were
pretreated with wortmannin (15 min) or LY294002 (10 min) at different
concentrations and then were plated on the fibrinogen matrix for
60 min. After washing, adherent cells were scraped off and their lipid
extract was analyzed by HPLC after deacylation.
[32P]PtdIns-3,4-P2 was quantified as
described under "Experimental Procedures."
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Table I
Effects of wortmannin and LY294002 on platelet adhesion
Platelets were incubated in absence (A) or presence of Me2SO
(0.03%; 15 min), wortmannin (100 nM; 15 min) or LY294002
(25 µM; 10 min) and plated on the fibrinogen matrix for
60 min. Calculation of adhesion percentage was as described under
"Experimental Procedures." Data are from five independent
experiments.
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Fig. 2.
Effects of wortmannin and LY294002 on
32P labeling of various phospholipids from resting and
adherent platelets. Washed platelets were pretreated in absence
(A) or presence of wortmannin (100 nM; 15 min),
LY294002 (25 µM; 10 min), or Me2SO (vehicle;
0.03%; 15 min) and were plated on the fibrinogen matrix for 60 min.
Control resting platelets (C) were added to a flask without
fibrinogen. Control and adherent platelets were then recovered, and
their lipid extracts were analyzed by HPLC after deacylation as
described under "Experimental Procedures." 32P
radioactivity incorporated into various phosphoinositides is expressed
in counts/min from 3 × 108 platelets, and data are
means ± S.E. from three independent experiments. Radioactivity of
[32P]PtdIns-3,4-P2 was undetectable in
samples of non-adherent platelets or platelets treated with PtdIns
3-kinase inhibitors and is not represented.
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PtdIns 3-Kinase Activation Is Necessary for
ADP-dependent Platelet Spreading--
As described
previously (21), upon adhesion, adherent platelets undergo the
following steps of morphological changes: disk to sphere shape change,
extension of pseudopodia, and a much slower process, cell spreading
(Fig. 3, A and B).
Pretreatment of platelets with wortmannin or LY294002 inhibited
platelet spreading on fibrinogen (Fig. 3, C and
D). However, it should be noted that, although pretreated
platelets did not fully spread, they still extended pseudopodia. The
inhibitory effect of LY294002 and wortmannin on cell spreading was
already detectable at 6 µM and 25 nM,
respectively. Concentrations of 25 µM LY294002 and 100 nM wortmannin completely prevented platelet spreading.
After removing wortmannin and LY294002 from the adhesion medium by two
washes, we observed that only the inhibitory effect of LY294002 was
reversible after 30 min (data not shown). Indeed, LY294002 is a
competitive inhibitor at the ATP-binding site of PtdIns 3-kinase (13),
whereas wortmannin induces a covalent modification of the catalytic
site of the enzyme (14). In agreement with Haimovich et al.
(2, 4), pretreatment of platelets with the ADP scavenger pyruvate
kinase plus phosphoenolpyruvate just before the adhesion assay induced
the same effects as treatment with the PtdIns 3-kinase inhibitors,
i.e. absence of spreading but persistence of pseudopodal
extension (Fig. 3E). After washing, ADP (20 µM) was added to adherent platelets to overcome the ADP scavenging system. Addition of ADP restored full spreading of all
adherent platelets as shown in Fig. 3F. On the other hand, addition of 20 µM ADP to adherent platelets pretreated
with wortmannin did not reverse inhibition of platelet spreading (Fig.
3G). These data demonstrate that PtdIns 3-kinase signaling
pathway is required for ADP-induced spreading of adherent
platelets.

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Fig. 3.
Effects of wortmannin, LY294002, and ADP on
platelet spreading. Platelets were incubated in absence
(A) or presence of Me2SO (0.03%; B),
wortmannin (100 nM; C and G),
LY294002 (25 µM; D) as described previously in
the legend to Fig. 2. In some experiments, the ADP scavenger pyruvate
kinase plus phosphoenolpyruvate was added to the platelet suspension
before adhesion (E and F) as described under
"Experimental Procedures." Adhesion was performed for 60 min, and
after two washes, ADP (20 µM) was added to the adherent
platelets for 30 min (F and G). After fixation,
adherent cells were observed by interference light microscopy.
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PtdIns-4,5-P2 but Not Lipid Products of PtdIns 3-Kinase
Is Sufficient for a Full Platelet Spreading--
Our previous
measurements of PtdIns 3-kinase activation in adherent platelets (3)
have shown that, although the PtdIns-3,4,5-P3 level was not
significantly modified between 5 and 30 min of adhesion, PtdIns-3,4-P2 accumulated as a function of the adhesion
time. Time course of PtdIns-3,4-P2 production closely
paralleled platelet spreading upon adhesion (data not shown). In order
to determine whether products of PtdIns 3-kinase could be involved in
platelet spreading, we used
Di-C16-PtdIns-3,4-P2 and
Di-C16-PtdIns-3,4,5-P3, which were reported to
trigger biologic responses when added to whole cells (7, 22). As shown
in Fig. 4A, addition of
Di-C16-PtdIns-3,4-P2 (20 µM) on
adherent platelets pretreated with wortmannin only partially restored
platelet spreading. After 30 min of incubation with
Di-C16-PtdIns-3,4-P2, some adherent platelets
have lost their round shape and have undergone pseudopodal and
hyalomere extension. Nevertheless, these modifications concerned only a
small proportion of adherent platelets (5%), as compared with 35% of
spread platelets obtained with non-pretreated control cells (Fig.
4B). Using amounts of
Di-C16-PtdIns-3,4-P2 between 10 and 30 µM, we obtained a dose-dependent increase in
the response rate (detected as early as 15 min of adhesion) and in the
proportion of responsive cells (Fig. 4B). In no case did we
observe a full spreading of platelets, even after 60 min of incubation.
Addition of Di-C16-PtdIns-3,4,5-P3 or
PtdIns-4-P or both (data not shown) together with
Di-C16-PtdIns-3,4-P2 was not more efficient in
restoring full spreading (Fig. 4B). Surprisingly, addition
of PtdIns-4,5-P2 to adherent platelets pretreated with
wortmannin triggered full spreading (Fig. 4, A and
B). Moreover, the number of fully spread platelets was
increased by addition of both
Di-C16-PtdIns-3,4-P2 and
PtdIns-4,5-P2 (Fig. 4B). Nevertheless, under
these conditions, the amount of fully spread platelets was far below
that observed in the control situation.

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Fig. 4.
Effects of different phosphoinositides on
spreading of platelets pretreated with wortmannin. A,
platelets were incubated in presence of 0.03% Me2SO
(A) or 100 nM wortmannin (W,
PtdIns-3,4-P2, PtdIns-4,5-P2) as described
previously in the legend to Fig. 2. Adhesion was performed for 60 min
and after two washes, Di-C16-PtdIns-3,4-P2 or
PtdIns-4,5-P2 (20 µM) was added to the
adherent platelets for 30 min as described under "Experimental
Procedures." After fixation, adherent cells were observed by
interference light microscopy. Platelets in a fully spread stage are
indicated by large arrowheads, whereas those in a partial
spread stage are indicated by thin arrowheads. B,
different lipids were prepared and added to adherent platelets
pretreated with wortmannin (W) as described under
"Experimental Procedures." Adherent platelets pretreated with
0.03% Me2SO (A) were taken as a control. The
histogram was used to express the percentages of platelets in a partial
spread stage (black box) or in a full spread stage
(hatched box). One hundred randomly chosen platelets were
counted from each experiment (n = 2-6), and each
experiment has a paired control.
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PtdIns-4,5-P2 effects on cell spreading are unlikely to be
dependent on induction of platelet release reaction, as ADP (20 µM), LPA (5 µM), the thrombospondin-1 cell
binding domain peptide (H-RFYVVMWK-OH; 50 µM), could not
restore spreading of platelets treated with 100 nM
wortmannin (Table II). By contrast, the
TXA2 analog U46619 (5 µM) triggered full
spreading of adherent platelets pretreated with 100 nM
wortmannin. However, addition of PtdIns-4,5-P2 (20 µM) to adherent platelets pretreated with aspirin (100 µM) and wortmannin (100 nM) was still able to
restore full platelet spreading (Table II).
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Table II
Spreading restoration of wortmannin-treated platelets by different
agonists
Platelets were incubated with wortmannin (100 nM; 15 min)
and/or GF 109203X (12 µM; 60 min) and then plated on the
fibrinogen matrix for 60 min. In some experiments, platelet-rich plasma
was incubated with 100 µM aspirin. Spreading restoration
assays were performed by addition of 20 µM ADP, 5 µM LPA, 50 µM H-RFYVVMWK-OH, 1 unit/ml
thrombin (THR), 5 µM U46619, 10 nM PMA, or 20 µM PtdIns-4,5-P2 as described under
"Experimental Procedures." After 30 min, efficiency in full
spreading restoration was observed by microscopy and was expressed as
follows: percentage of adherent platelets in a full spread state
(100%) (++++), 50% (++), 10% (+), or 0% ( ).
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The fact that strong platelet agonists, such as thrombin (1 unit/ml),
the TXA2 analog U46619, and the PKC activator PMA (10 nM), were able to restore spreading of platelets pretreated
with 100 nM wortmannin (Table II) might suggest a PtdIns
3-kinase-independent pathway of platelet spreading. In order to
determine a possible activation of PKC isoforms by high concentrations
of PtdIns-4,5-P2 (23), we used the specific PKC inhibitor
GF109203X (24). Preincubation of platelets with GF109203X (12 µM) induced inhibition of platelet spreading as described
previously by Haimovich and co-workers (4). When platelets had been
pretreated by both GF109203X (12 µM) and wortmannin (100 nM) together, exogenous PtdIns-4,5-P2 was still
able to restore full spreading of adherent platelets with a similar
efficiency as in the case of a pretreatment with wortmannin alone
(Table II). These experiments argue in favor of a specific
PtdIns-4,5-P2 effect on the restoration of cell spreading
that is independent on PKC activation.
The partial rescue of platelet spreading with lipids suggests that
there may be additional components to the PtdIns
3-kinase-dependent pathway. We addressed the possibility
that the myosin light chain kinase (MLCK) could be involved in platelet
spreading. Indeed, inhibitors of MLCK have been shown to disassemble
focal adhesions and to reduce their phosphotyrosine staining (25), and
wortmannin is a MLCK inhibitor, but at micromolar concentrations (26). Preincubation of platelets with 10 µM ML-7 (specific
inhibitor of MLCK from Biomol; Ref. 27) during the 30 min before
adhesion did not modify platelet spreading (data not shown). We
concluded that MLCK does not seem to be involved in platelet
spreading.
In Vivo Association of PtdIns-4,5-P2 with Talin Is
Decreased upon Wortmannin Treatment--
Since the level of
[32P]PtdIns-4,5-P2 was not significantly
modified in vivo upon treatment of platelets with
wortmannin, we looked for possible changes of the lipid content of
focal adhesion sites. Talin is a prominent actin-binding protein that
links integrins and the cytoskeleton and has been shown to be required
for cell spreading (28, 29). Although some variations could be observed in the amount of talin immunoprecipitated from platelets (Fig. 5A), mean values obtained from
two experiments were essentially the same (Fig. 5B). Under
these conditions, only traces of PtdIns-4-P were recovered in
anti-talin immunoprecipitates from resting platelets, whereas, after
adhesion, the amount of PtdIns-4-P increased, PtdIns-4,5-P2 and traces of PtdIns-3,4-P2 becoming detectable (Fig.
5C). For instance, the amount of talin isolated from
activated platelets was 1.4- and 0.9-fold (two experiments) the amount
obtained from control platelets, whereas associated phospholipids were
increased by 7.5- and 11-fold, respectively. This indicated that
adhesion promoted specific association of phosphoinositides with talin. Moreover, by comparison with the relative amounts of
polyphosphoinositides found in total platelets, there was an enrichment
in PtdIns-4-P and PtdIns-3,4-P2 in the anti-talin
immunoprecipitates (compare Figs. 2 and 5D). Treatment of
platelets with wortmannin induced a strong decrease (60-100%) of
these three lipids associated with talin (Fig. 5, C and
D). Thus, we conclude that PtdIns 3-kinase inhibitors may
modify the association of PtdIns-4,5-P2 with talin and
therefore influence its possible function in platelet spreading.

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Fig. 5.
Effects of wortmannin treatment on
association of polyphosphoinositides with talin in
vivo. Platelets were labeled with
[32P]orthophosphate (C and D) or
not (A and B) and plated on the fibrinogen matrix
(A, W) or kept in suspension (C)
for 60 min as described in the experimental procedure. In some cases,
platelets were pretreated with 100 nM wortmannin before
plating (W). There was no detectable difference in the
amount of lipid-incorporated 32P between the adherent and
suspended samples. Immunoprecipitation of talin from 4.5 × 108 platelets was performed with a polyclonal anti-talin
antibody followed by Western blotting (A), and lipids were
extracted and separated by TLC (C) as described under "Experimental
Procedures." No lipid was precipitated by protein G-Sepharose alone.
Quantification of the amounts of talin immunoprecipitated was performed
by a densitometric analysis (B), and the lipid content
(D) is expressed taking the amount of PtdIns-4-P in assay
A as 100% (74 cpm). B and D are means
from two independent experiments.
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 |
DISCUSSION |
We have shown previously that synthesis of
PtdIns-3,4-P2 in adherent platelets is under the control of
the released ADP, since addition of ADP reversed the inhibitory effects
of an ADP scavenger on PtdIns-3,4-P2 synthesis (3). Our
present results, in agreement with those of Haimovich et al.
(2, 4), demonstrate that ADP release occurring upon platelet adhesion
is required for full platelet spreading. Thus, ADP released by adherent
platelets controls both PtdIns-3,4-P2 synthesis and cell
spreading. In conditions where PtdIns-3,4-P2 synthesis was
totally abolished by inhibitors of PtdIns 3-kinase, we observed an
inhibition of platelet spreading while pseudopodal extension was
maintained. We demonstrate here that the PtdIns 3-kinase signaling
pathway is required for ADP-induced spreading of adherent
platelets.
In order to determine which lipid is responsible for platelet
spreading, we have added synthetic PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 to adherent wortmannin-treated platelets.
PtdIns-3,4-P2 appears to be the most efficient in restoring
partial spreading, i.e. pseudopodal and hyalomere extension.
However, among all phosphoinositides tested, PtdIns-4,5-P2
alone was able to trigger the full spreading of wortmannin-treated
platelets. Although all phospholipid solutions were prepared under
similar conditions, their packing into micelles or vesicules was not
characterized. Moreover, differences in the acyl chains of
PtdIns-3,4-P2 (palmitate) and PtdIns-4,5-P2
(stearate and arachidonate) may have influenced their activity.
Nevertheless, our results suggest a role for both
PtdIns-4,5-P2 and lipid products of PtdIns 3-kinase in the
signal transduction pathway leading to platelet spreading.
We have observed a trend toward a decrease of
[32P]PtdIns-4-P in adherent platelets pretreated with
PtdIns 3-kinase inhibitors. This result suggests that a
wortmannin-sensitive PtdIns 4-kinase might exist in platelets, as has
been shown in other models (30, 31). Nevertheless, in a cell system,
wortmannin was reported to inhibit PtdIns 4-kinase at µM
concentration, and LY294002 has not been described as an inhibitor of
known PtdIns 4-kinases (13). Thus, PtdIns 3-kinase activity could be
upstream of a PtdIns 4-kinase and/or a PtdIns-4-P phosphatase. Finally,
even though we have not measured a significant decrease of
[32P]PtdIns-4,5-P2 in whole platelets
pretreated with PtdIns 3-kinase inhibitors, a decrease of PtdIns-4-P
level could impair the synthesis of a particular pool of
PtdIns-4,5-P2 required for platelet spreading.
Recent studies from the Schlessinger and Rhee laboratories (32, 33)
demonstrate that
isoforms of phospholipase C (PLC) could be
activated by PtdIns-3,4,5-P3, either by targeting to cell
membrane through their PH domain or by direct activation through their
SH2 domain. One could thus expect an increase of PtdIns-4,5-P2 level when adherent platelets have been
pretreated with PtdIns 3-kinase inhibitors. Nevertheless, at least two
reasons could explain why in our experiments this variation is not
observed. First, in our previous paper (3), we have shown that upon
platelet adhesion on a fibrinogen matrix a PLC active on
PtdIns-4,5-P2 was rapidly and transiently stimulated.
Maximal increase of PtdOH production and PtdIns-4,5-P2
decrease was observed as early as 5 min of adhesion. Thereafter, these
two metabolites returned gradually to their basal level, and that
corroborates the absence of PtdOH and PtdIns-4,5-P2
variations after 60 min of adhesion, as shown in Fig. 2 of our present
article. We thus believe that in the late steps of platelet adhesion
PLC activity is not involved. However, it should be of importance to
check PLC activity during the early steps of adhesion of platelets
treated with PtdIns 3-kinase inhibitors. Second, since our present data
show a decrease of PtdIns-4-P level upon platelet treatment with PtdIns
3-kinase inhibitors, an eventual increase of the
PtdIns-4,5-P2 level might be impaired.
PtdIns-4,5-P2 regulates several actin-binding proteins as
profilin, gelsolin,
-actinin, and vinculin (34). One of the major proteins of focal adhesions, talin, has been shown to be involved in
cell spreading (28, 29). Its interaction with lipids has been
documented in vitro and could be of importance for talin nucleated actin polymerization (34). Here, we show that
PtdIns-4,5-P2 as well as PtdIns-4-P and
PtdIns-3,4-P2 become associated with talin upon platelet
adhesion. Moreover, treatment of platelets by wortmannin strongly
reduces the amounts of polyphosphoinositides recovered in the
anti-talin immunoprecipitate. Even though it remains to be determined
whether this association is direct or not, our results support the
notion of a possible regulation by PtdIns 3-kinase of a pool of
PtdIns-4,5-P2 potentially involved in cell spreading.
Hartwig et al. (35) have reported that D3 and D4
polyphosphoinositides uncap F-actin in resting permeabilized platelets. At low concentrations (10 µM), PtdIns-4,5-P2
and PtdIns-3,4-P2 are more effective than
PtdIns-3,4,5-P3. Synthesis of PtdIns-4-P and
PtdIns-4,5-P2, which are correlated with the exposure of
barbed filament ends, seem to be under the control of the small G
protein Rac (35). This small G protein has been shown to regulate
extension of peripheral lamellipodia (36), to associate in
vivo with both PtdIns 3-kinase and PtdIns-4-P 5-kinase (12), and
it was suggested that PtdIns 3-kinase functions upstream of Rac (37,
38). Moreover, PtdIns-4,5-P2 and PtdIns-3,4-P2
both regulate, in vitro, the severing and capping of the
protein gelsolin (9), whose genetic defect is responsible for the
absence of lamellae although the filopod formation is maintained, upon
platelet activation (39). Thus, in our model, PtdIns 3-kinase could
regulate actin remodeling directly through PtdIns-3,4-P2
synthesis and/or indirectly through PtdIns-4,5-P2
synthesis.
Recent results from King et al. (40), showing that spreading
of COS 7 cells attached to fibronectin is delayed after treatment by
wortmannin and LY294002, support the view that the PtdIns 3-kinase signaling pathway is required for cell spreading, as controlled by
integrins and/or by tyrosine kinase receptors (41). It has been
suggested that ADP released from adherent platelets supports some
specific signals such as Vav phosphorylation via an indirect mechanism
involving activation of
IIb
3 (42).
Furthermore, an integrin-associated protein agonist peptide triggers
activation of
IIb
3 integrin resulting in
platelet spreading on immobilized fibrinogen (43). Thus, upon platelet
adhesion to immobilized fibrinogen, it remains to be clarified whether
platelet spreading is secondary to the
IIb
3 integrin engagement.
 |
ACKNOWLEDGEMENTS |
We thank Dr. B. Payrastre and D. Bacqueville
for helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by the Association pour la
Recherche sur le Cancer, Paris, and the Conseil Régional
Midi-Pyrénées, Toulouse, France.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Fax: 33-5-61-77-94-01;
E-mail: racaud{at}purpan.inserm.fr.
1
The abbreviations used are: PtdIns 3-kinase,
phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PtdIns-4-P,
phosphatidylinositol 4'-phosphate; PtdIns-3,4-P2,
phosphatidylinositol 3',4'-bisphosphate; PtdIns-4,5-P2,
phosphatidylinositol 4',5'-bisphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3',4',5'-trisphosphate;PtdOH, phosphatidic acid;
LPA, lysophosphatidic acid; PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; MLCK, myosin light chain kinase; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; TXA2,
thromboxane A2; Di-C16, dipalmitoyl.
 |
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