A Selective Role for Phosphatidylinositol 3,4,5-Trisphosphate in
the Gi-dependent Activation of Platelet
Rap1B*
Paolo
Lova
§,
Simona
Paganini
,
Emilio
Hirsch¶,
Laura
Barberis¶,
Matthias
Wymann
,
Fabiola
Sinigaglia§,
Cesare
Balduini
, and
Mauro
Torti
**
From the
Department of Biochemistry, University of
Pavia, via Bassi 21, 27100 Pavia, Italy, the § Department
of Medical Sciences, University "A. Avogadro," via Solaroli 17, 28100 Novara, Italy, the ¶ Department of Genetics, Biology,
and Biochemistry, University of Turin, via Santena 5bis, 10126 Turin,
Italy, and the
Institute of Biochemistry, Department of
Medicine, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland
Received for publication, May 16, 2002, and in revised form, October 17, 2002
 |
ABSTRACT |
The small GTP-binding protein Rap1B is activated
in human platelets upon stimulation of a
Gi-dependent signaling pathway. In this
work, we found that inhibition of platelet adenylyl cyclase by
dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did
not restore Rap1B activation in platelets stimulated by cross-linking
of Fc
receptor IIA (Fc
RIIA) in the presence of ADP scavengers.
Moreover, elevation of the intracellular cAMP concentration did not
impair the Gi-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of Fc
RIIA, by stimulation of
the P2Y12 receptor for ADP, or by epinephrine. However, in platelets from PI3K
-deficient mice, both ADP and epinephrine were
still able to normally stimulate Rap1B activation through a
PI3K-dependent mechanism, suggesting the involvement of a
different isoform of the enzyme. Moreover, the lack of PI3K
did not
prevent the ability of epinephrine to potentiate platelet aggregation through a Gi-dependent pathway. The inhibitory
effect of wortmannin on Rap1B activation was overcome by addition of
phosphatidylinositol 3,4,5-trisphosphate
(PtdIns(3,4,5)P3), but not PtdIns(3,4)P2, although both lipids were found to support phosphorylation of Akt.
Moreover, PtdIns(3,4,5)P3 was able to relieve the
inhibitory effect of apyrase on Fc
RIIA-mediated platelet
aggregation. We conclude that stimulation of a
Gi-dependent signaling pathway causes
activation of the small GTPase Rap1B through the action of the PI3K
product PtdIns(3,4,5)P3, but not PtdIns(3,4)P2,
and that this process may contribute to potentiation of platelet aggregation.
 |
INTRODUCTION |
Rap1B is a small GTP-binding protein highly expressed in human
platelets (1). In resting cells, it is mainly located at the membrane,
but it translocates to the cytosol upon phosphorylation by protein
kinase A (2). In activated platelets, Rap1B rapidly interacts with the
reorganized actin-based cytoskeleton (3). As other GTPases, Rap1B is
activated by binding of GTP. Platelet stimulation by different
agonists, such as thrombin, collagen, and ADP, induces the rapid
binding of GTP to Rap1B (4, 5). An increase in the intracellular
Ca2+ concentration in stimulated platelets has been shown
to be sufficient to promote Rap1B activation, and specific
Ca2+/calmodulin-sensitive guanine nucleotide exchange
factors for Rap1B have been identified (5, 6). We (7) and others (8)
have recently described a new pathway for Rap1B activation that is
initiated by stimulation of membrane Gi-coupled receptors and that is independent of intracellular Ca2+ increases. In
fact, the sole binding of ADP to the P2Y12 receptor, as
well as the interaction of epinephrine with the
2A-adrenergic receptor, is sufficient to trigger Rap1B
activation. Moreover, we have found that agonists that activate
platelets through stimulation of Gq-coupled receptors, such
as the thromboxane A2 analog U46619, or through stimulation
of a tyrosine kinase-based pathway, such as in the case of
cross-linking of Fc
RIIA,1
totally rely on binding of secreted ADP to the Gi-coupled
P2Y12 receptor to activate Rap1B (7). Finally, activation
of Rap1B induced by ADP or epinephrine is prevented in
G
i2- and G
z-deficient mice, respectively
(8).
During the last few years, activation of a
Gi-dependent signaling pathway has been
recognized to represent a crucial event absolutely required to elicit
full platelet activation. For instance, platelet responsiveness to the
thromboxane A2 analog U46619, to protease-activated
receptor-1-activating peptide, or to cross-linking of Fc
RIIA is
strongly compromised when secretion is prevented by protein kinase C
inhibitors or when extracellular ADP is neutralized by specific
scavengers, such as apyrase or creatine phosphate/creatine phosphokinase (9-12). It has also been clearly shown that, although ADP can bind to two different G-protein-coupled receptors on the platelet surface (the P2Y1 receptor coupled to
Gq and the P2Y12 receptor coupled to
Gi), only the latter one is responsible for potentiation of
platelet activation induced by other agonists (10-12).
The exact mechanism for the Gi-mediated potentiation of
platelet activation is still unclear. The
-subunits of the
Gi family of heterotrimeric G-proteins are known to inhibit
adenylyl cyclase, but several findings indicate that reduction of basal
cAMP levels does not contribute to ADP-mediated potentiation of
platelet activation (13, 14). By contrast, several studies using
specific cell-permeable inhibitors have suggested a crucial role for
phosphatidylinositol 3-kinase (PI3K) in this event (11, 12, 14, 15).
The recent finding that stimulation of a Gi-coupled
receptor is sufficient to trigger activation of Rap1B also suggests
that this small GTPase could be involved in potentiation of platelet aggregation.
In this work, we have investigated the mechanism of Rap1B activation
downstream of stimulation of Gi-coupled receptors in an attempt to reveal a correlation between this small GTPase and the
potentiation of agonist-induced platelet activation. We have found that
inhibition of adenylyl cyclase is not sufficient to stimulate GTP
binding to Rap1B. By contrast, it is shown here that PI3K plays an
important role in the Gi-mediated activation of Rap1B. We
also provide evidence suggesting that activation of Rap1B downstream of
PI3K is associated with the Gi-mediated potentiation of
platelet activation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Epinephrine, ADP, thrombin, sheep anti-mouse
F(ab')2 fragments, apyrase, creatine phosphate, creatine
phosphokinase, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) were
from Sigma. Dideoxyadenosine, SQ22536, wortmannin, and LY294002 were
from Alexis. AR-C69931MX was a generous gift from AstraZeneca
(Charnwood, UK). Di-C16-PtdIns(3,4,5)P3 and Di-C16-PtdIns(3,4)P2 were from Matreya,
Inc. Monoclonal antibody (mAb) IV.3 against Fc
RIIA was obtained from
Medarex. Sepharose CL-2B, GSH-Sepharose 2B, and the enhanced
chemiluminescence substrate were from Amersham Biosciences. The rabbit
polyclonal antiserum against Rap1B was described previously (16).
Polyclonal and monoclonal (5G3) antibodies against Akt and phospho-Akt
Thr308 were from New England Biolabs, Inc. The cDNA for
the Rap-binding domain (RBD) of the Ral guanine nucleotide dissociation
stimulator was kindly provided by Dr. Johannes L. Bos (Department of
Physiological Chemistry, University of Utrecht, The Netherlands).
Peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad.
Human Platelet Isolation and Stimulation--
Human platelets
were isolated by gel filtration on Sepharose CL-2B and eluted with
HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.9 mM KCl, and 12 mM NaHCO3, pH 7.4)
as previously described (17). Platelet concentration was adjusted to
0.35 × 109 platelets/ml. Platelet samples (0.5 ml)
were incubated at 37 °C in an aggregometer under constant stirring
and typically stimulated with 10 µM ADP and 1 µM epinephrine or by cross-linking of Fc
RIIA through
addition of 2 µg/ml mAb IV.3 for 2 min, followed by 30 µg/ml sheep
anti-mouse F(ab')2 fragments. Platelet stimulation was
typically performed for 1 min. Where indicated, 1 unit/ml apyrase, 5 mM creatine phosphate, 40 units/ml creatine phosphokinase, 500 µM PAPS, or 100 nM AR-C69931MX was added
to the platelet samples 2 min before stimulation. Preincubation with
wortmannin or LY294002 was performed for 15 min at 37 °C. Inhibition
of adenylyl cyclase was achieved by incubation of platelets with 100 µM dideoxyadenosine or 300 µM SQ22536 for
30 min. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were
dissolved in Me2SO and added to the platelet suspension at a final concentration of 30 µM. Measurement of platelet
aggregation was performed under the same conditions indicated above,
and aggregation was monitored continuously over 10 min.
Rap1B Activation Assay--
Activation of Rap1B was evaluated
using GST-RBD immobilized on GSH-Sepharose, which is known to bind
specifically and selectively the GTP-bound form of Rap1B from a
platelet lysate. Platelet stimulation was stopped by addition of an
equal volume of ice-cold modified 2× RIPA buffer (100 mM
Tris-HCl, pH 7.4, 400 mM NaCl, 5 mM
MgCl2, 2% Nonidet P-40, 20% glycerol, 2 mM
phenylmethylsulfonyl fluoride, 2 µM leupeptin, 0.2 µM aprotinin, and 0.2 mM
Na3VO4). Cell lysis was performed on ice for 10 min, and the insoluble material was eliminated by centrifugation at
13,000 rpm for 10 min at 4 °C. Recombinant purified GST-RBD was
coupled to GSH-Sepharose by incubating 200 µg of the protein with 100 µl of GSH-Sepharose (75% slurry) for 2 h at room temperature
under constant tumbling and then added to the cleared platelet lysates
(20 µg of GST-RBD/sample). Precipitation of GTP-bound Rap1B was
performed by incubation at 4 °C for 45 min. The precipitates were
collected by brief centrifugation, washed three times with modified 1×
RIPA buffer, and finally resuspended in 25 µl of SDS sample buffer
(25 mM Tris, 192 mM glycine, pH 8.3, 4% SDS,
1% dithiothreitol, 20% glycerol, and 0.02% bromphenol blue).
Precipitated Rap1B was separated by SDS-PAGE on 10-20% acrylamide
gradient gels and transferred to nitrocellulose. The presence of active
Rap1B in precipitates with GST-RBD was evaluated by staining the
nitrocellulose filters with a specific polyclonal antiserum directed
against Rap1B, used at a final dilution of 1:1000. Reactive proteins
were detected by enhanced chemiluminescence reaction. Data in all
figures are representative of at least three separate experiments.
Measurement of Akt Phosphorylation--
Platelet samples (0.2 ml, 109 platelets/ml) were incubated at 37 °C and
stimulated as indicated in the figure legends for 1 min. Platelets were
lysed in 2% SDS in HEPES buffer, and protein concentration was
determined. Aliquots containing 80 µg of total platelet lysates were
heated at 96 °C for 5 min in SDS sample buffer, separated on 12%
acrylamide gel, and transferred to nitrocellulose. Blots were probed
with anti-phospho-Akt Thr308 antibody and then reprobed
with anti-Akt antibody. Analysis of Akt phosphorylation in platelets
stimulated by cross-linking of Fc
RIIA was performed with
immunoprecipitated Akt because preliminary experiments revealed a
cross-reactivity of the anti-phospho-Akt Thr308 antibody
with the heavy chains of mAb IV.3, used to activate Fc
RIIA, that
compromised the interpretation of the results (data not shown).
Platelet samples were lysed in ice-cold RIPA buffer and precleared with
protein A-Sepharose. The precleared lysates that were also devoid of
mAb IV.3 were used to immunoprecipitate Akt with mAb 5G3.
Immunoprecipitates were then analyzed by immunoblotting with
anti-phospho-Akt Thr308 antibody and reprobed with anti-Akt antibody.
Studies with Mouse Platelets--
PI3K
-deficient mice were
generated as previously described (18). Blood was collected from
anesthetized mice from the inferior vena cava into syringes containing
heparin solution (5 units/ml). Blood was centrifuged at 90 × g for 10 min, and the platelet-rich plasma was
collected. Aggregation studies were directly performed with the
platelet-rich plasma upon adjustment of the platelet count to 2 × 108 platelets/ml with autologous platelet-poor plasma. For
analysis of Rap1B activation, washed platelets were prepared by
centrifuging the platelet-rich plasma at 150 × g for
10 min. The platelet pellet was washed once with 0.038% trisodium
citrate, 0.6% glucose, and 0.72 NaCl, pH 7.0, containing 25 ng/ml
prostaglandin E1 and finally resuspended in HEPES buffer at
a final concentration of 3 × 108 platelets/ml.
Stimulation of platelet samples (0.4 ml), lysis, and pull-down assay
for Rap1B activation were performed as described above.
 |
RESULTS |
Gi-dependent Activation of Rap1B Does Not
Require Inhibition of Adenylyl Cyclase--
We have previously
demonstrated that stimulation of a Gi-dependent
pathway by epinephrine or by binding of ADP to the P2Y12 receptor is sufficient to trigger activation of the small GTPase Rap1B
and that Rap1B activation promoted by cross-linking of Fc
RIIA is
completely dependent on the stimulation of the Gi-coupled
P2Y12 receptor by secreted ADP (7). To investigate the
signaling pathway linking Gi to Rap1B, we first analyzed
the possible role of G
i-mediated inhibition of adenylyl
cyclase. Gel-filtered platelets were treated with two different
membrane-permeable inhibitors of adenylyl cyclase (SQ22536 and
dideoxyadenosine), and activation of Rap1B was evaluated upon
precipitation of the GTP-bound form of the protein with GST-RBD.
Although SQ22536 and dideoxyadenosine were used at concentrations
reported to maximally inhibit forskolin-stimulated adenylyl cyclase
(13), neither compound by itself caused detectable activation of Rap1B
(Fig. 1A). These results are
in agreement with those recently reported by Woulfe et al.
(8). However, we considered the possibility that, although not
sufficient by itself to induce Rap1B activation, inhibition of adenylyl
cyclase could contribute to this process in association with
co-stimulation of other signaling pathways. In platelets stimulated by
cross-linking of Fc
RIIA, activation of Rap1B is suppressed by the
ADP scavenger creatine phosphate/creatine phosphokinase, but is
restored by the simultaneous addition of epinephrine (7). By contrast, in the presence of creatine phosphate/creatine phosphokinase, neither
SQ22536 nor dideoxyadenosine was able to restore Rap1B activation upon
cross-linking of Fc
RIIA (Fig. 1A). This indicates that
the contribution of the Gi pathway to Rap1B activation does not require the inhibition of adenylyl cyclase by the G-protein
-subunit.

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Fig. 1.
Gi-dependent
activation of Rap1B is not regulated by cAMP. A,
gel-filtered platelets were preincubated at 37 °C with 100 µM dideoxyadenosine (DDA), 300 µM SQ22536, or 5 mM creatine phosphate plus
40 units/ml creatine phosphokinase (CP-CPK) as indicated and
then treated with buffer (none) or stimulated by clustering
of Fc RIIA with 2 µg/ml mAb IV.3 and 30 µg/ml sheep anti-mouse
F(ab')2 fragments for 1 min. After cell lysis, active
GTP-bound Rap1B was precipitated using GST-RBD and identified by
immunoblotting with a specific polyclonal antiserum. Aliquots (20 µl)
of each cell lysate were withdrawn before addition of GST-RBD and
immunoblotted with anti-Rap1B antiserum to evaluate the level of the
protein in the different samples. B, gel-filtered platelets
were preincubated with or without 10 µM prostaglandin
E1 (PGE1) for 30 min and then treated
with buffer (none) or stimulated by clustering
(clust.) of Fc RIIA with 2 µg/ml mAb IV.3 and 30 µg/ml
sheep anti-mouse F(ab')2 fragments, with 10 µM ADP, or with 1 µM epinephrine
(Epi). After 1 min, platelets were lysed, and GTP-bound
Rap1B was isolated with GST-RBD and visualized by immunoblotting. Total
Rap1B in cell lysates was monitored by immunoblotting of 20-µl
aliquots.
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We also considered that the very low intracellular levels of cAMP in
resting platelets may represent a permissive condition to allow
agonist-induced Rap1B activation. To verify this possibility, human
platelets were incubated with prostaglandin E1 to stimulate adenylyl cyclase and to increase cAMP levels and then treated with
agonists that activate Rap1B through stimulation of Gi.
Fig. 1B shows that, even when intracellular cAMP levels were
increased by prostaglandin E1, activation of Rap1B induced
by clustering of Fc
RIIA, epinephrine, or ADP occurred normally.
Taken together, these results indicate that the Gi-mediated
activation of Rap1B is completely independent of the modulation of
intracellular cAMP levels.
Gi-mediated Activation of Rap1B Is Regulated by
PI3K--
It is known that activation of Gi is necessary
to support full platelet secretion and aggregation as well as Rap1B
activation induced by U46619 or by cross-linking of Fc
RIIA (7, 10, 12). Moreover, it has been shown that PI3K plays a crucial role in the
Gi-mediated potentiation of platelet activation and is required for irreversible aggregation (11, 12, 14, 15). Therefore, we
investigated the role of PI3K in Fc
RIIA-mediated activation of Rap1B
using two structurally unrelated inhibitors of the enzyme, wortmannin
and LY294002. Fig. 2A shows
how activation of Rap1B induced by clustering of Fc
RIIA was totally
suppressed by both compounds. By contrast, Rap1B activation induced by
thrombin was not significantly affected by LY294002 (Fig.
2A) or by wortmannin (data not shown). We have previously
shown that Fc
RIIA-induced activation of Rap1B requires the binding
of secreted ADP to the P2Y12 receptor (7). To verify
whether, upon Fc
RIIA recruitment, PI3K signals to Rap1B downstream
of the Gi-coupled receptor for ADP, human platelets were
stimulated by cross-linking of Fc
RIIA in the presence of PAPS, a
selective antagonist of the P2Y1 receptor for ADP. Under
these conditions, secreted ADP can bind exclusively to the
Gi-coupled P2Y12 receptor. As shown in Fig.
2B, binding of secreted ADP to the P2Y12
receptor was sufficient to allow Rap1B activation in response to
Fc
RIIA cross-linking. Moreover, under these conditions, inhibition
of PI3K by wortmannin or LY294002 still completely suppressed
activation of Rap1B (Fig. 2B).

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Fig. 2.
PI3K inhibitors prevent Rap1B activation
induced by clustering of Fc RIIA. A,
platelet samples were preincubated at 37 °C with the indicated
concentrations of LY294002 or wortmannin or with an equivalent volume
of Me2SO for 15 min and then stimulated by clustering of
Fc RIIA with 2 µg/ml mAb IV.3 and 30 µg/ml sheep anti-mouse
F(ab')2 fragments or with 1 unit/ml thrombin. After 1 min,
GTP-bound Rap1B was precipitated from lysed platelets using GST-RBD and
immunoblotted with a specific polyclonal antiserum. B,
platelets preincubated with Me2SO, 25 µM
LY294002, or 50 nM wortmannin were stimulated by clustering
of Fc RIIA for 1 min in the absence or presence of 500 µM PAPS (A3P5PS) as indicated. The immunoblot
shows active Rap1B isolated by precipitation with GST-RBD.
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To further demonstrate that PI3K lies downstream of the
Gi-coupled P2Y12 receptor, we analyzed Rap1B
activation in response to exogenous ADP. Fig.
3A shows that, when exogenous
ADP was allowed to bind exclusively to the P2Y12 receptor,
i.e. in the presence of PAPS, activation of Rap1B was
prevented by the PI3K inhibitors wortmannin and LY294002. Finally, we
analyzed the role of PI3K in Rap1B activation induced by epinephrine,
which binds exclusively to the Gi-coupled
2A-adrenergic receptor on the platelet surface. Fig.
3B shows that both wortmannin and LY294002 almost completely suppressed epinephrine-induced activation of Rap1B. These results confirm and extend previously reported data (8) and indicate that PI3K
is a key element in the Gi-dependent pathway
for Rap1B activation.

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Fig. 3.
Rap1B activation by ADP and epinephrine is
prevented by PI3K inhibitors. PI3K was inhibited by incubation of
platelets with 25 µM LY294002 or 50 nM
wortmannin for 15 min. Control samples were preincubated with an equal
volume of Me2SO. Platelets were then stimulated with 10 µM ADP in the absence or presence of 500 µM
PAPS (A3P5PS) (A) or with 1 µM
epinephrine (B) for 1 min. Accumulation of GTP-bound Rap1B
was evaluated as described under "Experimental Procedures."
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PI3K
Is Not Involved in Either Rap1B Activation or Potentiation
of Platelet Aggregation Mediated by Gi--
It is known
that human platelets express different isoforms of PI3K (23). At least
two members of the class I PI3K family, viz. PI3K
and PI3K
, are activated by G-protein 
-dimers (24-26) and
therefore may represent the isoforms linking Gi to Rap1B. To investigate the possible role of PI3K
, we compared
agonist-induced activation of Rap1B in platelets from PI3K
-deficient
and wild-type mice. Because mouse platelets do not express Fc
RIIA,
these studies have been performed using ADP and epinephrine as Rap1B
activators. As shown in Fig. 4,
activation of Rap1B induced by ADP or epinephrine in PI3K
-deficient
platelets was almost identical to that observed in platelets from
wild-type mice. This finding argues against a role for PI3K
in
coupling activation of Gi to Rap1B. However, because
several intracellular messengers, including calcium, diacylglycerol, and tyrosine kinases, can mediate Rap1 activation (6), we considered that, in the absence of PI3K
, other signaling pathways may become predominant and result in an almost equally efficient activation of
Rap1B. By using a selective antagonist of the P2Y12
receptor, we found that, in both wild-type and PI3K
/
mice, ADP-induced activation of Rap1B was equally dependent on stimulation of the Gi-coupled receptor (Fig. 4). Moreover,
in both control and PI3K
-deficient platelets, ADP- or
epinephrine-induced activation of Rap1B was still prevented by the PI3K
inhibitors LY294002 (Fig. 4) and wortmannin (data not shown). These
results clearly indicate that a PI3K isoform different from PI3K
is
involved in the Gi-mediated activation of Rap1B.

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Fig. 4.
Activation of Rap1B in
PI3K -deficient mice. Platelets were
isolated from wild-type (PI3K+/+) or PI3K -deficient
(PI3K / ) mice and stimulated with buffer
(none), 10 µM epinephrine (Epi), or
10 µM ADP for 1 min. Preincubation with 25 µM LY294002 was performed for 15 min before stimulation,
whereas preincubation with 100 nM AR-C69931MX was for 2 min
before addition of ADP. Active Rap1B was precipitated from platelet
lysates with GST-RBD and revealed by immunoblotting with a specific
polyclonal antiserum. The amount of total Rap1B in cell lysates was
also monitored by immunoblotting.
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Both PI3K and Rap1B have been hypothesized to be involved in the
Gi-dependent potentiation of platelet
aggregation. Because activation of Rap1B downstream of Gi
occurs normally in PI3K
/
mice, these platelets
represent a good model to test the specific contribution of PI3K
versus Rap1B to epinephrine-induced potentiation of platelet
aggregation. In mouse platelets, ADP caused a reversible platelet
aggregation that required the concomitant stimulation of the
Gq-coupled P2Y1 and Gi-coupled
P2Y12 receptors. A small (but significant) reduction of
ADP-induced platelet aggregation in PI3K
/
mice was
reported in an earlier study (32). This effect was more evident when
low doses of ADP were used to stimulate washed platelets. In the
present work, such inhibition was negligible because we used high doses
of ADP to stimulate platelets in platelet-rich plasma. However, Fig.
5 shows that selective blockade of the
P2Y12 receptor strongly inhibited platelet aggregation
induced by high doses of ADP in both wild-type and
PI3K
/
mice. However, in the presence of the
P2Y12 receptor antagonist, addition of epinephrine, which
activates a Gi-dependent signaling pathway
leading to stimulation of Rap1B activation, strongly, although not
completely, restored ADP-induced platelet aggregation in both wild-type
and PI3K
/
platelets. This indicates that PI3K
is
involved neither in Rap1B activation nor in the
Gi-dependent potentiation of platelet
aggregation.

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Fig. 5.
Epinephrine restores ADP-induced platelet
aggregation blocked by a P2Y12 receptor antagonist in
wild-type and
PI3K /
mice. Platelets from PI3K / mice (lower
panel) or wild-type littermates (upper panel) were
placed in an aggregometer and preincubated with buffer (trace
A) or with 100 nM AR-C69931MX for 2 min (traces
B and C). Samples were then stimulated with 10 µM ADP (traces A and B) or with 10 µM ADP and 10 µM epinephrine (trace
C). Representative traces of platelet aggregation are
reported.
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PI3K-dependent Activation of Rap1B Is Mediated by the
Lipid Product PtdIns(3,4,5)P3, but Not by
PtdIns(3,4)P2--
Activation of PI3K leads to the
accumulation of the lipid products PtdIns(3,4,5)P3 and
PtdIns(3,4)P2, which, in turn, promote stimulation of Akt.
To investigate the role of 3-phosphorylated phosphoinositides in Rap1B
activation, we analyzed their ability to relieve the inhibition of
Rap1B activation in wortmannin-treated platelets. Preliminary
experiments with permeabilized platelets revealed that saponin
treatment caused an almost total loss of platelet responsiveness to the
analyzed agonists (data not shown). However,
PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been
shown to trigger biological responses even when added to whole cells
(19-21). Moreover, when dissolved in Me2SO and then added
to intact platelets, both lipids have been found to be rapidly
incorporated into the cell membrane (22). Fig.
6A shows that, in platelets
stimulated by cross-linking of Fc
RIIA, inhibition of Rap1B
activation by wortmannin was partially reversed by addition of
PtdIns(3,4,5)P3. By contrast, the related lipid
PtdIns(3,4)P2 was unable to relieve the wortmannin-induced
inhibition of Rap1B activation. Similarly, even when activation of
Rap1B was triggered by direct stimulation of Gi-coupled
receptors by ADP or epinephrine, PtdIns(3,4,5)P3 was able
to partially counteract the inhibitory effect of wortmannin (Fig. 6,
B and C). These results indicate that the lipid
product PtdIns(3,4,5)P3, but not PtdIns(3,4)P2,
is directly involved in the PI3K-dependent activation of
Rap1B downstream of stimulation of Gi.

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Fig. 6.
Wortmannin-induced inhibition of Rap1B
activation is reversed by PtdIns(3,4,5)P3. Platelets
were preincubated with 50 nM wortmannin (wort)
or an appropriated volume of Me2SO (DMSO) for 15 min and then treated with buffer (bas) or stimulated with 2 µg/ml mAb IV.3 and 30 µg/ml sheep anti-mouse F(ab')2
fragments to induce clustering of Fc RIIA (A), with 10 µM ADP (B), or with 1 µM
epinephrine (C) for 1 min. Where indicated,
PtdIns(3,4,5)P3 (30 µM) or
PtdIns(3,4)P2 (30 µM) was added together with
the agonists. Active Rap1B was precipitated and revealed by
immunoblotting with a specific polyclonal antiserum.
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We next investigated the ability of PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 to promote Akt phosphorylation using
phospho-specific antibodies against phospho-Akt Thr308. As
shown in Fig. 7A, platelet
activation by cross-linking of Fc
RIIA induced the phosphorylation of
Akt at Thr308, which was prevented by preincubation with
wortmannin. When analyzed using the same experimental approach,
phosphorylation of Akt induced by thrombin was found to be comparable
to that induced by stimulation of Fc
RIIA (Fig. 7A). Fig.
7B shows that addition of PtdIns(3,4,5)P3 or
PtdIns(3,4)P2 to intact platelets caused phosphorylation of Akt to a similar extent, as evaluated by immunoblotting of whole platelet lysates. The level of Akt phosphorylation induced by the
3-phosphorylated phosphoinositides was lower than that observed in
platelets stimulated with thrombin, but comparable to that induced by
ADP. Parallel analysis of Rap1B activation showed that addition of
PtdIns(3,4,5)P3 to platelets was sufficient to stimulate GTP binding to Rap1B (Fig. 7C). By contrast,
PtdIns(3,4)P2, although able to promote Akt
phosphorylation, did not stimulate activation of Rap1B. These results
indicate that exogenous PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 can mimic the effects of endogenous lipids
and that phosphorylation of Akt does not seem to be required for Rap1B activation.

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|
Fig. 7.
Phosphorylation of Akt by
PtdIns(3,4,5)P3 and PtdIns(3,4)P2.
A, gel-filtered platelets were stimulated by clustering
(clust.) of Fc RIIA with 2 µg/ml mAb IV.3 and 30 µg/ml
sheep anti-mouse F(ab')2 fragments for 1 (1') or
3 (3') min or with 1 unit/ml thrombin (THR) for 1 min. Preincubation with 50 nM wortmannin (wt)
was for 15 min before stimulation. Platelets were lysed in RIPA buffer,
and Akt was immunoprecipitated (Ip) from the precleared
lysates with mAb 5G3 and then analyzed by immunoblotting with
anti-phospho-Akt Thr308 antibody (p-Akt (Thr
308)). Blots were stripped and reprobed with anti-Akt antibody
(Akt). B, gel-filtered platelets were treated
with 30 µM PtdIns(3,4,5)P3, 30 µM PtdIns(3,4,5)P3, 10 µM ADP,
or 1 unit/ml thrombin at 37 °C for 1 min. Samples were lysed in 2%
SDS, and the levels of Akt phosphorylation (p-Akt) at
Thr308 were measured by immunoblotting with
phospho-specific antibodies. Blots were then stripped and reprobed with
anti-Akt antibody. C, similar samples were lysed in RIPA
buffer and processed for measurement of Rap1B activation.
|
|
In the presence of ADP scavengers, platelet aggregation induced by
cross-linking of Fc
RIIA is inhibited, but can be restored by the
simultaneous addition of epinephrine (12). Under the same conditions,
also activation of both PI3K and Rap1B is prevented and can be restored
by epinephrine (7, 12). Therefore, we wondered whether direct
activation of Rap1B by PtdIns(3,4,5)P3 could by-pass the
need for PI3K activation to induce platelet aggregation. Fig.
8 shows that Fc
RIIA-mediated platelet
aggregation was totally prevented when platelets where stimulated in
the presence of apyrase, but could be almost completely restored by
epinephrine. Addition of PtdIns(3,4,5)P3 at a concentration
shown to restore Rap1B activation also resulted in a limited (but
significant) recovery of platelet aggregation. At higher concentration
of PtdIns(3,4,5)P3, the recovery of platelet
aggregation was even more evident, although clearly less marked than
that induced by epinephrine. Taken together, these results indicate
that, when ADP secreted upon cross-linking of Fc
RIIA is neutralized,
exogenous PtdIns(3,4,5)P3 causes both activation of Rap1B
and restoration of platelet aggregation.

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|
Fig. 8.
Inhibition of
Fc RIIA-mediated platelet aggregation by
apyrase is reversed by PtdIns(3,4,5)P3. Gel-filtered
platelets were placed in an aggregometer, and clustering of Fc RIIA
was induced by addition of 2 µg/ml mAb IV.3 and 30 µg/ml sheep
anti-mouse F(ab')2 fragments as indicated by the
arrows. Aggregation was monitored continuously. The
representative traces shown refer to samples treated as follows.
trace A, no treatment; trace B, 1 unit/ml
apyrase; trace C, 1 unit/ml apyrase and 10 µM
epinephrine; trace D, 1 unit/ml apyrase and 30 µM PtdIns(3,4,5)P3; trace
E, 1 unit/ml apyrase and 60 µM
PtdIns(3,4,5)P3. Apyrase was added together with mAb IV.3,
whereas epinephrine or PtdIns(3,4,5)P3 was added together
with sheep anti-mouse F(ab')2 fragments.
|
|
 |
DISCUSSION |
During the last few years, a number of studies using scavenger
systems and receptor antagonists demonstrated the essential role played
by secreted ADP in potentiating platelet activation in response to many
different extracellular agonists (9-12). Although the biological
effects of ADP on human platelets require concomitant activation of two
different G-protein-coupled receptors, P2Y1 (coupled to
Gq) and P2Y12 (coupled to Gi),
potentiation of platelet activation induced by other agonists is
mediated mainly by activation of a Gi-dependent
pathway through the P2Y12 receptor (10, 12). In an attempt
to identify the intracellular messengers promoting Gi-dependent potentiation of platelet
activation, we (7) and others (8) have recently found that the small
GTPase Rap1B is indeed activated upon the sole binding of ADP to the
P2Y12 receptor or upon activation of the
Gi-coupled
2A-adrenergic receptor by
epinephrine. Moreover, in platelets from mice that lack
G
i2 or G
z, activation of Rap1B by ADP or
epinephrine is abolished (8). Rap1 is emerging as a modulator of cell
adhesion and integrin function (6), and a very recent study by Bertoni
et al. (27) demonstrates that active Rap1B regulates the
affinity state of integrin
IIb
3 in
mouse megakaryocytes. Therefore, Rap1B may represent a link
between activation of a Gi pathway and stabilization of
integrin-mediated platelet aggregation.
In this work, we investigated the biochemical mechanism of Rap1B
activation downstream of stimulation of a Gi signaling
pathway. By using inhibitors and activators of adenylyl cyclase, we
demonstrated that the reduction of the intracellular levels of cAMP
promoted by the Gi
-subunits is not required to induce
activation of Rap1B. These results confirm and extend those recently
reported by Woulfe et al. (8) and strengthen the idea that
G
i-mediated inhibition of adenylyl cyclase does not
directly contribute to the Gi-promoted potentiation of
platelet activation (13, 14).
In addition to the activated
-subunits, stimulation of a
Gi-coupled receptor generates free 
-dimers that are
able to stimulate different intracellular effectors, including
selective isoforms of the lipid-metabolizing enzyme PI3K, such as
PI3K
and PI3K
. In this work, we have actually demonstrated that
activation of PI3K is an essential step in the Gi-mediated
signaling pathway leading to Rap1B activation. In fact, the PI3K
inhibitors wortmannin and LY294002 have been found to totally
suppress Rap1B activation upon clustering of Fc
RIIA by acting
downstream of P2Y12 receptor stimulation by secreted ADP.
Moreover, as recently described (8), we also found that PI3K inhibitors
prevented activation of Rap1B induced by direct binding of ADP to the
P2Y12 receptor or of epinephrine to the
2A-adrenergic receptor. Therefore, PI3K appears to link Gi-coupled receptors to the small GTPase Rap1B.
How activation of Gi results in the stimulation of PI3K and
how PI3K promotes activation of Rap1B are still unclear. Free 
-dimers generated upon stimulation of a Gi-coupled
receptor may activate some class I PI3K isoforms, viz.
PI3K
and PI3K
(24-26). PI3K
is highly expressed in platelets,
and it has been reported to play an essential role in ADP-induced
platelet activation downstream of the Gi-coupled
P2Y12 receptor (32). Therefore, we hypothesized that this
isozyme could link Gi to activation of Rap1B. However,
studies with platelets from PI3K
knockout mice clearly showed that
the lack of expression of this isoform does not compromise the ability
of epinephrine or ADP to induce activation of Rap1B. A variable
decrease in agonist-induced Rap1B activation in
PI3K
/
mice has been recently reported (8). Although
some small differences were occasionally observed in our experiments
(see, for instance, Rap1B activation induced by epinephrine in the
representative experiment reported in Fig. 4), the analysis of several
determinations led us to conclude that these occasional differences
were not significant and that the contribution of PI3K
to
Gi-mediated Rap1B activation, if any, is negligible.
Our results also demonstrate that the Gi-mediated pathway
for platelet aggregation, which can be monitored by the ability of
epinephrine to replace ADP, is normally functional in
PI3K
/
mice. This is consistent with a role for
activated Rap1B, but not for PI3K
, in the signaling pathway from
Gi to platelet aggregation. We also have shown that, in
PI3K
/
mice, Gi-mediated activation of
Rap1B is still prevented by PI3K inhibitors. This clearly indicates
that a different isoform of the enzyme is mainly involved.
Interestingly, platelets express high levels of PI3K
(32), which is
also activated by 
-dimers (25, 26). Moreover, a recent report
demonstrates that PI3K
, but not PI3K
, represents a link between

-dimers released upon stimulation of the Gi-coupled
receptor for lysophosphatidic acid and activation of the small GTPase
Ras (33). Therefore, it is most likely that PI3K
represents the main
PI3K isoform involved in the regulation of Rap1B.
In this work, we have further strengthened the functional correlation
between PI3K and Rap1B activation by demonstrating that exogenous
PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, can
restore wortmannin-inhibited activation of Rap1B in platelets
stimulated with ADP or epinephrine or by cross-linking of Fc
RIIA.
This indicates that the action of PI3K on Rap1B activation is actually
mediated, at least in part, by the main lipid product of this enzyme.
It is interesting to note that, in stimulated platelets, accumulation of PtdIns(3,4,5)P3 is rapid and transient, whereas
synthesis of PtdIns(3,4)P2 occurs mainly as a consequence
of integrin
IIb
3-mediated platelet
aggregation (28, 29). Therefore, the ability of
PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, to
support Rap1B activation is consistent with a role for this GTPase in
an early phase of platelet stimulation that precedes integrin
IIb
3 activation and cell aggregation. Evaluation of Akt activation by exogenous PtdIns(3,4,5)P3
and PtdIns(3,4)P2 revealed that both lipids were able to
stimulate phosphorylation of Akt at Thr308. A previous
report suggested that, in contrast to PtdIns(3,4)P2, addition of PtdIns(3,4,5)P3 to intact platelets does not
activate Akt (20). Although we cannot provide a definitive explanation for this discrepancy, our results are clearly in line with the current
concept of PtdIns(3,4,5)P3 being a major regulator of Akt
(24). It is interesting to note that, although both 3-phosphorylated phosphoinositides were able to activate Akt, only
PtdIns(3,4,5)P3 caused activation of Rap1B. The finding
that PtdIns(3,4)P2 induces Akt phosphorylation, but not
Rap1B activation, suggests a dissociation between the two events. This
is also supported by the finding that ADP induced normal,
PI3K-dependent activation of Rap1B in PI3K
/
mouse platelets (Fig. 4) under conditions in
which phosphorylation of Akt has been reported to be totally suppressed
(32).
The data in Fig. 6 clearly show that the restoration of Rap1B
activation by PtdIns(3,4,5)P3 never returns the
amount of active Rap1B to that observed in control platelets stimulated
in the absence of wortmannin. This indicates either that generation of PtdIns(3,4,5)P3 is not the only mechanism by which PI3K
participates in Rap1B activation or that endogenously generated lipids
act more efficiently. In this regard, it should be noted that
stimulators of Gi, such as ADP and epinephrine, are really
weak stimulators of PI3K (12, 30). For instance, epinephrine induces
the accumulation of <20% of the amount of PtdIns(3,4,5)P3
measured in thrombin-stimulated platelets (12). Despite this, the
finding that PI3K inhibitors totally suppressed Rap1B activation
clearly indicates that such a small amount of 3-phosphoinositides plays
a crucial role. Thus, the endogenously generated
PtdIns(3,4,5)P3 seems to have a very high efficiency in
stimulating Rap1B activation. Although the reason for this effect is
not known, it may be hypothesized that a specific and optimal
localization of endogenously generated PtdIns(3,4,5)P3 may
improve its effect on Rap1B. In this regard, it is interesting to note
that a preferential distribution of the PI3K-generated lipids in
platelet membrane rafts has been recently reported (31). In this work,
we have also provided evidence that
PtdIns(3,4,5)P3-mediated activation of Rap1B is an
important step in the Gi-dependent pathway for
platelet aggregation. In fact, exogenous PtdIns(3,4,5)P3,
in addition to partially restore Rap1B activation, can overcome the
inhibition of Fc
RIIA-mediated platelet aggregation promoted by ADP
scavengers. In this context, the effect of PtdIns(3,4,5)P3
resembles that of epinephrine, which activates a truly
Gi-dependent pathway. Our data show that
PtdIns(3,4,5)P3 is much less efficient than epinephrine in
promoting restoration of Fc
RIIA-mediated platelet aggregation in
apyrase-treated platelets. However, this fits well with the reduced
ability of this lipid to restore Rap1B activation.
In conclusion, we have shown that stimulation of a Gi
signaling cascade leads to activation of the small GTPase Rap1B through the action of the PI3K lipid product PtdIns(3,4,5)P3,
rather than through inhibition of adenylyl cyclase. These results also
suggest that Rap1B represents a downstream effector for PI3K in the
Gi-dependent signaling pathway for potentiation
of platelet aggregation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Johannes L. Bos for providing
the RBD cDNA and Dr. Bob Humphries (AstraZeneca) for AR-C69931MX.
 |
FOOTNOTES |
*
This work was supported by grants from the Consiglio
Nazionale delle Ricerche (Target Project Biotechnology and Agenzia
2000), Ministero dell'Istruzione, Università e Ricerca
Scientifica (Progetti di Ricerca di Interesse Nazionale 2001),
Consorzio Interuniversitario Biotecnologie, and University of Pavia
(Progetto di Ateneo) and by European Union Fifth Framework Programme
QLG1-2001-02171 (to E. H. and M. P. W.).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. Tel.: 39-382-507238;
Fax: 39-382-507240; E-mail: mtorti@unipv.it.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M204821200
 |
ABBREVIATIONS |
The abbreviations used are:
Fc
RIIA, Fc
receptor IIA;
PI3K, phosphatidylinositol 3-kinase;
PAPS, adenosine
3'-phosphate 5'-phosphosulfate;
PtdIns(3, 4,5)P3,
phosphatidylinositol 3,4,5-trisphosphate;
PtdIns(3, 4)P2,
phosphatidylinositol 3,4-bisphosphate;
mAb, monoclonal antibody;
RBD, Rap-binding domain;
GST, glutathione S-transferase;
RIPA, radioimmune precipitation assay.
 |
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