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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4156-4165
A Key Role of Adenosine Diphosphate in the Irreversible Platelet
Aggregation Induced by the PAR1-Activating Peptide Through the Late
Activation of Phosphoinositide 3-Kinase
By
Catherine Trumel,
Bernard Payrastre,
Monique Plantavid,
Béatrice Hechler,
Cécile Viala,
Peter Presek,
Elizabeth A. Martinson,
Jean-Pierre Cazenave,
Hugues Chap, and
Christian Gachet
From Institut Fédératif de Recherche en Immunologie
Cellulaire et Moléculaire, INSERM U 326, Hôpital Purpan,
Toulouse, France; INSERM U 311, ETS, Strasbourg, France; and
Rudolf-Buchheim-Institut für Pharmakologie, Giessen, Germany.
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ABSTRACT |
Although adenosine diphosphate (ADP), per se, is a weak platelet
agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The
molecular basis of the ADP-induced platelet activation is starting to
be understood since the discovery that 2 separate P2 purinergic
receptors may be involved simultaneously in the activation process.
However, little is known about how ADP plays its role as a cofactor in
platelet activation and which signaling pathway initiated by a specific
agonist can be modulated by the released ADP. To investigate these
points, we took advantage of a model of platelet activation through the
thrombin receptor PAR1 in which both ADP scavengers and
phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to
transform the classical irreversible aggregation into a reversible one.
We have observed that, among the different PI 3-kinase products, the
accumulation of phosphatidylinositol 3,4-bisphosphate
[PtdIns(3,4)P2] was dramatically and specifically
attenuated when ADP was removed by apyrase treatment. A comparison
between the effects of PI 3-kinase inhibitors and apyrase strongly
suggest that the late, ADP-dependent, PtdIns(3,4)P2 accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to
the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we
found that both ADP and PI 3-kinase play an important role in
PAR1-dependent reorganization of the cytoskeleton through a control of
myosin heavy chain translocation and the stable association of
signaling complexes with the actin cytoskeleton.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBIN, A POTENT agonist for platelets,
is known to induce the synthesis of both phosphatidylinositol
3,4-bisphosphate [PtdIns(3,4)P2] and
phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P3], 2 phosphoinositides phosphorylated at
the D3 position of the inositol ring by phosphoinositide 3-kinases (PI
3-kinases). The D3-phosphoinositides generated by the various PI
3-kinases are now considered as second messengers capable of binding
protein modules, including src homology region 2 (SH2) or pleckstrin
homology (PH) domains, present in their targets,1 thus
regulating specific signaling pathways. In platelets stimulated by
thrombin, the synthesis of PtdIns(3,4,5)P3 is rapid and
transient,2,3 whereas PtdIns(3,4)P2 accumulates upon increasing stimulation times.4-6 Using platelets from
thrombasthenic patients or Arg-Gly-Asp-Ser (RGDS)-treated platelets, we
have demonstrated that the synthesis of a major part of
PtdIns(3,4)P2 is dependent on the engagement of
 IIb 3 integrin.7 It is
important to note that fibrinogen binding to its receptor
IIb3 is not sufficient per se to induce a
full activation of this pathway, because aggregation is also required,
as demonstrated by using thrombin-treated platelets in the absence of
stirring.3 Furthermore, platelets adhering to a fibrinogen
matrix require adenosine diphosphate (ADP) for spreading,
phosphorylation of focal adhesion kinase (p125FAK),
and synthesis of PtdIns(3,4)P2.8,9 These
results suggest that the level of PtdIns(3,4)P2 is
regulated by complex mechanisms involving cross-talk between different
signaling pathways. Although the accumulation of
PtdIns(3,4)P2 in stimulated platelets is thought to play an
important role,3,7,9 its function and target remain
unknown. It has been suggested that PI 3-kinase and its products may
play a role in irreversible aggregation, possibly by maintaining the
IIb3 integrin in its activated
state.10 These lipids may also be involved in mediating
actin filament uncapping and specific filopodial actin
assembly.11
We have recently shown that washed human platelets stimulated by ADP in
the presence of fibrinogen were unable to accumulate significant
amounts of PtdIns(3,4)P2.12 Indeed, as compared with thrombin, ADP is a weak platelet agonist inducing only reversible aggregation with partial and reversible cytoskeleton
reorganization.12-14 Again, this observation indicates that
fibrinogen binding to IIb3 is not
sufficient per se to induce the accumulation of
PtdIns(3,4)P2 and that the absence of accumulation of this
lipid correlates with a reversible aggregation. However, although ADP
is a weak agonist per se, the use of ADP receptor antagonists or of
enzymes capable of degrading ADP leads to clear attenuation of platelet responses to low thrombin concentrations or other agonist-like collagen.13-15 Present at very high concentrations in the
platelet dense granules,13,14 ADP is secreted when
platelets are stimulated by other aggregating agents. Among all
platelet-released substances, ADP has been shown to be selectively
responsible for the stabilization of thrombin-induced platelet
aggregates.16,17
The molecular basis of ADP-induced platelet activation is only
beginning to be understood with the finding that 2 separate P2
receptors could be involved simultaneously in the activation process.18-22 The P2 family of receptors is composed of 2 classes of receptors, namely the P2X receptors, which are ligand-gated ion channels, and the P2Y receptors, which belong to the serpentine G
protein-coupled receptor family.23 In the case of
platelets, the P2Y1 receptor is coupled to calcium
mobilization and has been shown to be responsible for ADP-induced shape
change. In addition, a not yet identified P2 receptor negatively
coupled to adenylyl cyclase seems to be necessary for the completion of
the aggregation response.18-20 Selective antagonists and
inhibitors have recently been developed that allow specific
discrimination between P2Y1 and P2/adenylyl
cyclase-dependent responses. Adenosine 2'-phosphate 5'-phosphate (A2P5P) is a selective P2Y1
antagonist,18-20,24 whereas AR-C66096 has been found to
selectively block the inhibitory effect of ADP on adenylyl
cyclase.18 The pharmacology of AR-C66096 is strikingly
similar to that of the well-known antiplatelet drug clopidogrel, which
inhibits selectively ADP-induced platelet aggregation by blocking the
effect of ADP on adenylyl cyclase.25
We have investigated here how ADP plays its role as a cofactor in
platelet activation and whether a specific signaling pathway, PI
3-kinase activation, is modulated by released ADP. We took advantage of
2 models of reversible aggregation previously
described10,26 in which the PAR1 thrombin receptor is
stimulated by the peptide SFLLRNP (TRAP) in the presence of an
ADP-scavenger26 or in the presence of PI 3-kinase
inhibitors.10 We demonstrate that ADP plays a key and
specific role in the late accumulation of PtdIns(3,4)P2 induced by TRAP through its receptor coupled to inhibition of adenylyl
cyclase. The late and sustained activation of a PI 3-kinase, which is
responsible for the ADP-dependent production of
PtdIns(3,4)P2, was found to be necessary for the
irreversible aggregation of platelets stimulated by TRAP. Finally, we
show that the cytoskeletons from TRAP-stimulated platelets display
major differences in their myosin heavy chain and RhoA content,
depending on the presence of ADP and synthesis of
PtdIns(3,4)P2.
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MATERIALS AND METHODS |
Reagents.
Apyrase was purified from potatoes as previously
described.27 Its specific ADPase activity was 12.5 U/mg.
TRAP was purchased from Bachem (Budendorf, Switzerland),
rabbit anti-p85 antibody from Upstate Biotechnology Inc (Lake
Placid, NY), rabbit anti-p125FAK, and
anti-RhoA antibodies from Santa Cruz Biotechnology Inc (Santa Cruz,
CA). LY294002 was purchased from Biomol Research Laboratories, Inc
(Plymouth, PA). Enhanced chemiluminescence (ECL) Western blotting reagents and [32P]-orthophosphate were obtained from
Amersham International (Buckinghamshire, UK). AR-C66096, formerly known
as FLP66096 or ARL66096, was a generous gift from ASTRA Charnwood
(Loughborough, UK). All other reagents were from Sigma (St Louis,
MO) unless otherwise indicated.
Preparation of washed human platelets.
Human blood was collected from a forearm vein, and platelet suspensions
were prepared as previously described.27 In some experiments, platelets were labeled with sodium
[32P]-orthophosphate (400 µCi/mL) for 1 hour at
37°C during a first washing step in Tyrode's buffer lacking
phosphate. The final resuspending medium (pH 7.35) was a Tyrode's
buffer containing 2 mmol/L Ca2+, 1 mmol/L Mg2+,
0.35% human serum albumin (Etablissement de Transfusion Sanguine, Strasbourg, France), and apyrase (0.02 U/mL) to prevent desensitization of platelet responses to ADP. Platelets were stored at 37°C
throughout the experiments, and the cell count was adjusted in the
final suspension to 7.5 × 105/µL using a Sysmex 100 particle counter (Merck Clevenot, Nogent-sur-Marne, France).
Platelet aggregation studies.
Aggregation was measured at 37°C by a turbidimetric method using a
dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). A 1.45-mL aliquot of nonlabeled or
32P-labeled platelet suspension was stirred at 1,100 rpm
and activated by the addition of TRAP in the absence or presence of 1 U/mL apyrase or one of the following selective ADP receptor
antagonists: 100 µmol/L ATPS, an antagonist of both
P2Y1 receptor and the unidentified P2 receptor coupled to
adenylyl cyclase; 100 µmol/L A2P5P, a selective P2Y1
antagonist20,24; or 1 µmol/L of the recently described ATP analogue AR-C66096, a strong and selective antagonist of the P2
receptor coupled to inhibition of adenylyl cyclase.18,28
Measurement of adenylyl cyclase activity.
A 450-µL aliquot of washed platelets was stirred at 1,100 rpm in an
aggregometer cuvette and the following reagents were added at 30-second
intervals: prostaglandin E1 (PGE1); A2P5P,
AR-C66096, or ATPS; and TRAP at indicated concentrations. One minute
later, the reaction was stopped by addition of 50 µL of ice-cold 6.6 mol/L perchloric acid. Perchloric acid extracts were centrifuged at
11,000g for 5 minutes to eliminate protein precipitate, and cyclic adenosine monophosphate (cAMP) was isolated from the
supernatants as described.20 The upper aqueous phase was
freeze-dried, and the dry residue was dissolved in the buffer provided
with the commercial radioimmunoassay kit for cAMP measurement (Amersham).
Cytoskeleton extraction.
Reactions were stopped and the cytoskeleton was immediately isolated as
described previously.3 Cytoskeletal material was collected
by centrifugation (12,000g for 10 minutes at 4°C), washed once with Csk buffer (50 mmol/L Tris-HCl, pH 7.4, 10 mmol/L EGTA, 1 mmol/L Na3VO4, 2 µg/mL aprotinin, 2 µg/mL
leupeptin, and 1 mmol/L phenylmethylsulfonylfluoride) containing
0.5% (vol/vol) Triton X-100 and then twice with Csk buffer
without Triton X-100.
Gel electrophoresis and immunoblotting.
Cytoskeletal proteins were solubilized in the electrophoresis sample
buffer, boiled for 5 minutes, separated on 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized
by Coomassie Blue staining or transferred onto a nitrocellulose
membrane (Gelman Sciences, Ann Arbor, MI) for immunoblotting as
reported previously.3 Immunodetections were performed with
relevant antibodies and reactions were visualized using the ECL
chemiluminescent system.
Lipid extraction and analysis.
Reactions were stopped by the addition of 2 vol of chloroform/methanol
and lipids were extracted, separated, deacylated, and finally analyzed
by high-performance liquid chromatography (HPLC) on a
partisphere SAX column (Whatman International Ltd, Maidstone, UK) as reported previously.29
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RESULTS |
The apyrase-induced reversible aggregation of TRAP-stimulated human
platelets is associated with a lack of PtdIns(3,4)P2
accumulation.
As previously shown by Lau et al,26 when platelets were
stimulated with 40 µmol/L TRAP, the addition of apyrase caused a reversion of aggregation, starting at 60 to 80 seconds
(Fig 1A). Figure 1B indicates that
treatment of platelets with the ADP scavenger led to a dramatic change
in [32P]PtdIns(3,4)P2 accumulation induced by
TRAP. Interestingly, the responses were quite similar until 40 seconds,
and then a large decrease in the intensity of labeling was observed in
the presence of apyrase, followed by a rapid disappearance of this
lipid, which was complete at 3 minutes. This strongly suggests a lack
of sustained PI 3-kinase activation and points out a degradation
pathway occurring under these conditions. These results clearly
indicate a major role of ADP in the late increase in
PtdIns(3,4)P2 occurring after 1 minute in TRAP-stimulated
platelets. In contrast, the production of
[32P]PtdIns(3,4,5)P3 evoked by TRAP was rapid
(maximal at 20 seconds), transient, and not significantly affected by
apyrase (Fig 1C). The level of [32P]PtdIns(3)P was low
under resting conditions and did not change significantly upon TRAP
stimulation either in the presence or in the absence of apyrase (not
shown).
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| Fig 1.
ADP is involved in the accumulation of
[32P]PtdIns(3,4)P2 induced by TRAP.
[32P]-labeled human platelets (7.5 × 108/mL) were stimulated with 40 µmol/L TRAP in the
absence or the presence of 1 U/mL apyrase ( and + in [A],  and  in [B] and [C]) for various periods of time. (A)
Aggregation was measured as described in Materials and Methods. (B)
Time course of PtdIns(3,4)P2 accumulation. (C) Time course
of PtdIns(3,4,5)P3 accumulation. Lipids were analyzed using
an HPLC technique as indicated in Materials and Methods. Data are from
1 representative experiment of 3 independent experiments that gave very
similar results.
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Effect of apyrase on PtdIns, PtdIns(4)P, PtdIns(4,5)P2,
and phosphatidic acid metabolism induced by TRAP.
The specificity of ADP action as a cofactor in the accumulation of
PtdIns(3,4)P2 induced by TRAP was further assessed by
measuring the level of the other classes of phosphoinositides and of
phosphatidic acid (PtdOH). As shown in Fig
2, when TRAP was used as an agonist, PtdIns, PtdIns 4P, and
PtdIns(4,5)P2 displayed a characteristic pattern of
labeling not significantly modified by suppression of ADP. As
previously observed by Hartwig et al,30 we could measure a
net and steady increase in PtdIns(4)P labeling in TRAP-treated platelets. This increase was much more obvious than upon thrombin addition5 and probably subsequent, beside an increased
synthesis, to an absence of persistent phospholipase C activation. The
activation of PtdIns 4-kinase was not dependent on the presence of ADP.
The increase in PtdIns(4,5)P2 labeling (Fig 2) presented
some fluctuations from one experiment to another, as is often the case
in platelet phosphoinositide metabolic studies, yet apyrase effects
were always negligible. Apyrase did not modify the overall kinetics of
PtdOH formation (Fig 2), although removal of ADP did lead to a more rapid decrease in PtdOH levels. It is noteworthy that, in contrast to
the sustained increase of [32P]PtdOH synthesis observed
upon thrombin addition, even at low thrombin concentrations
(unpublished results), 40 µmol/L TRAP induced a
transient increase in the labeling of PtdOH. This time course of PtdOH
production might be correlated with the more transient cytosolic free
Ca2+ increase measured in the presence of TRAP in
comparison with thrombin.31 Together, the results from Figs
1B and 2 indicate that ADP has a specific effect as a cofactor of TRAP
on PtdIns(3,4)P2 metabolism, because TRAP alone (Fig 1B) or
ADP alone12 were unable to induce the late accumulation of
this phosphoinositide.
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| Fig 2.
Time courses of [32P]PtdIns,
[32P]PtdIns(4)P,
[32P]PtdIns(4,5)P2, and
[32P]PtdOH production and effects of released ADP in
TRAP-stimulated platelets. Time courses of [32P]-labeling
of PtdIns, PtdIns(4)P, PtdIns(4,5)P2, and PtdOH in
[32P]-labeled platelets activated by 40 µmol/L TRAP in
the absence () or the presence () of 1 U/mL apyrase.
Radioactivity incorporated into various phospholipids was quantified
using HPLC as in Fig 1. Results are representative of 3 experiments
with similar results.
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The late and sustained activation of PI 3-kinase is required for
PtdIns(3,4)P2 accumulation and irreversible aggregation.
As already reported,10 when platelets were pretreated with
PI 3-kinase inhibitors, the aggregation induced by TRAP became reversible (not shown), as in the absence of ADP (Fig 1). However, under these conditions, the synthesis of all PI 3-kinase products was
fully inhibited (not shown). Interestingly, addition of the 2 unrelated
PI 3-kinase inhibitors after 2 minutes of stimulation, when aggregation
and PtdIns(3,4)P2 production were at their maximum, induced
the reversion of aggregation in a dose-dependent manner (Fig 3A and B). At this stage of
stimulation, the rapid and transient PtdIns(3,4,5)P3
synthesis (Fig 1) has already occurred and was not affected (not
shown). In contrast, Fig 3C shows that the level of
PtdIns(3,4)P2 dramatically decreased immediately upon
addition of the PI 3-kinase inhibitors, demonstrating a very active
turnover of this phosphoinositide and a sustained activation of PI
3-kinase between 2 and 4 minutes. Interestingly, the decrease in
PtdIns(3,4)P2 was rapidly followed by the disaggregation
mechanism. Moreover, the addition of PI 3-kinase inhibitors at any time
up to 2 minutes of stimulation was without effect on the aggregation
response until 2 minutes, but, once at its maximum, the aggregation
became reversible (not shown). These results demonstrate a key role of a PI 3-kinase active after 2 minutes of stimulation in the accumulation of PtdIns(3,4)P2 and the stabilization of aggregation
induced by TRAP. Results from Fig 3 also strongly suggest that the late formation of PtdIns(3,4)P2 was actually the cause of
irreversible aggregation.
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| Fig 3.
The late and sustained activation of PI 3-kinase is
required for PtdIns(3,4)P2 accumulation and irreversible
aggregation induced by TRAP in the presence of ADP. Platelet
aggregation was measured as described in Materials and Methods. Various
concentrations of the 2 unrelated PI 3-kinase inhibitors, wortmannin
(A) or LY294002 (B), were added 2 minutes after stimulation with 40 µmol/L TRAP, as indicated by the arrow. Data are representative of 3 independent experiments with very similar results. (C)
[32P]-labeled human platelets (7.5 × 108/mL) were stimulated with 40 µmol/L TRAP. After 2 minutes, 50 nmol/L wortmannin (), 25 µmol/L LY 294002 (), or
0.1% Me2SO ( ) was added. The reactions were stopped at
different times by addition of chloroform/methanol and the level of
[32P]PtdIns(3,4)P2 was measured by HPLC as
indicated in Materials and Methods.
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Effect of selective ADP receptor antagonists on TRAP-induced platelet
aggregation and PtdIns(3,4)P2 accumulation.
To assess which ADP receptor may be involved in the contribution to
PtdIns(3,4)P2 formation by TRAP, we used selective
antagonists that are known to discriminate between them.
Figure 4A shows that 100 µmol/L ATPS
and 1 µmol/L AR-C66096 could reverse TRAP-induced platelet
aggregation in a manner similar to that of apyrase, whereas 100 µmol/L of the selective P2Y1 antagonist A2P5P did not. In relation to this, ATPS or AR-C66096 inhibited TRAP-induced
accumulation of PtdIns(3,4)P2, whereas A2P5P was
ineffective (Fig 4B). Again, this effect was selective for this lipid,
because the other phosphoinositides were not affected by these
compounds (data not shown). These results strongly suggest that the
cofactor role of ADP in PtdIns(3,4)P2 accumulation and
irreversible aggregation may be due to its P2 receptor coupled to
inhibition of adenylyl cyclase.
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| Fig 4.
Effect of selective ADP receptor antagonists on
TRAP-induced platelet aggregation and PtIns(3,4)P2
accumulation. [32P]-labeled human platelets (7.5 × 108/mL) were stimulated with 40 µmol/L TRAP in the
absence or in the presence of the selective P2Y1 antagonist
A2P5P (100 µmol/L), the selective antagonist of the P2 receptor
coupled to adenylyl cyclase AR-C66096 (1 µmol/L), or an antagonist of
both ADP receptors ATP S (100 µmol/L). (A) Aggregation was measured
as described in Materials and Methods. (B) Time course of
PtdIns(3,4)P2 accumulation. Lipids were immediately
extracted and [32P]PtdIns(3,4)P2 was
separated and quantified as in Fig 1. Results are expressed as the
percentage of PtdIns(3,4)P2 produced, with 100% being the
maximal production observed upon TRAP stimulation, and are the means ± SD from 2 independent experiments.
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TRAP-induced reversion of cAMP formation by PGE1 involves
released ADP.
To assess further the role of this signaling pathway, apyrase and the
various ADP receptor antagonists were tested for their ability to
inhibit the effect of TRAP on cAMP accumulation. For this purpose,
adenylyl cyclase was stimulated by incubation of platelets with 1 µmol/L PGE1 in the presence of ADP receptor antagonists and, 1 minute later, the addition of ADP or TRAP. As shown in Fig 5A, apyrase, ATPS, and AR-C66096
were able to block the inhibitory effect of ADP on adenylyl cyclase,
whereas A2P5P did not, which confirmed previous work.18,20
Under these experimental conditions, reversion of cAMP formation by a
high concentration of TRAP (100 µmol/L) was due to released ADP,
because this effect was inhibited in a dose-dependent manner by ATPS
and AR-C66096, with a complete inhibition at 10 4
mol/L and 106 mol/L, respectively (Fig 5B and C).
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| Fig 5.
TRAP-induced reversion of cAMP formation by
PGE1 involves released ADP. The effects of apyrase and
various ADP receptor antagonists on cAMP accumulation were measured in
intact platelets stimulated by 5 µmol/L ADP (A). The effects of
various concentrations of AR-C 66096 and ATPS on cAMP accumulation
were measured in intact platelets stimulated by a high concentration of
TRAP (100 µmol/L) (B) as described in Materials and Methods. Results
are the means of triplicates ± SEM from 1 representative experiment
of 3 independent experiments that gave very similar results.
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ADP scavengers and PI 3-kinase inhibitors alter similarly the
cytoskeleton reorganization induced by TRAP.
The dramatic reorganization of the actin cytoskeleton observed during
platelet aggregation is thought to play an important role in the
stabilization of aggregation and in the formation of functional
signaling complexes. Based on the results described above, we decided
to investigate the potential changes that may occur in TRAP-induced
actin cytoskeleton reorganization in the absence of ADP or when PI
3-kinase is inhibited. Interestingly, Fig 6A clearly indicates
that, among the major proteins found in the actin cytoskeleton from
TRAP-aggregated platelets, the content of 200-kD myosin heavy chain was
strongly decreased when ADP was absent or when PI 3-kinase was
inhibited. In contrast, these inhibitors did not induce significant
changes in the content of F-actin, actinin, and actin-binding
protein (ABP). To evaluate the formation of signaling complexes linked
to the actin cytoskeleton, we compared the time courses of relocation
to the cytoskeleton of the p85 subunit of PI 3-kinase and of its
potential regulator, RhoA. Figure 6B indicates that TRAP induced a
significant association of p85 with the cytoskeleton, reaching a
plateau at approximately 2 minutes. In contrast, in the presence of
apyrase or PI 3-kinase inhibitors, after 2 minutes, a reversion of the
association of p85 with the cytoskeleton was observed. An even more
dramatic difference was found in the translocation of RhoA upon
addition of apyrase or PI 3-kinase inhibitors (Fig 6B) that paralleled effects of these agents on p85.
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| Fig 6.
Both apyrase and the PI 3-kinase inhibitor wortmannin
specifically affect the association of myosin heavy chain with the
cytoskeleton and the organization of signaling complexes. (A)
Cytoskeletons were isolated from 40 µmol/L TRAP-treated platelets at
various time points in the absence () or in the presence of 1 U/mL
apyrase () or 100 nmol/L wortmannin ( ). Cytoskeletal proteins
were separated by a 7.5% SDS-PAGE and visualized by Coomassie Blue
staining. Actin and the major actin-binding proteins were quantified by
densitometric analysis (ScanMaker IIHR, Microtek, Germany). The
relocation of p85 (B) and RhoA (C) to the cytoskeleton was analyzed
by Western blotting with specific antibodies. Data shown are
representative of 3 independent experiments with similar results.
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| |
DISCUSSION |
In a recent study dealing with ADP stimulation of washed platelets, in
the presence of exogenous fibrinogen to allow
IIb3 engagement, we have observed a
relationship between reversible aggregation, absence of
PtdIns(3,4)P2 accumulation, and a large reduction of the
amount of myosin heavy chain and RhoA in the cytoskeleton.12 These data, together with our previous
report of a parallelism between aggregation extent and
PtdIns(3,4)P2 labeling in thrombin-stimulated
platelets,3 suggest that irreversible aggregation may be
linked to the late accumulation of PtdIns(3,4)P2 in human
platelets. The present study shows that, in another model of reversible
aggregation, ie, activation of human platelets by TRAP in the presence
of the ADP scavenger apyrase,26 PtdIns(3,4)P2 is synthesized in the very early stages of activation but then quickly
disappears. In contrast, in the presence of released ADP, TRAP leads to
an irreversible aggregation and to a large increase in the production
of PtdIns(3,4)P2 up to 3 minutes of stimulation. The
metabolism of the other phosphoinositides, including
PtdIns(3,4,5)P3 and PtdIns(3)P, is not significantly
modified by apyrase, indicating the specificity of this mechanism.
However, based on these results alone, it is difficult to know whether
the accumulation of this lipid is a cause or consequence of the
irreversible aggregation. We show here that PI 3-kinase inhibitors,
added when aggregation is at its maximum after 2 minutes of
stimulation, are able to induce a very rapid and dramatic decrease in
the level of PtdIns(3,4)P2 followed by a disaggregation of platelets. Although a potential role of PI 3-kinase as a protein kinase
cannot be totally excluded, these results strongly suggest a role for
the late accumulation of PtdIns(3,4)P2 in strengthening aggregation. The particularly active turnover of this phosphoinositide indicates that its accumulation results from a sustained PI 3-kinase activation rather than the inhibition of PtdIns(3,4)P2
hydrolysis. Moreover, the fact that PtdIns(3,4,5)P3 does
not accumulate in the presence of ADP scavengers suggests that a
5-phosphatase32 does not play a critical role in the late
and large increase of PtdIns(3,4)P2. Therefore, we propose
that ADP is playing its essential role as cofactor of TRAP via,
notably, the late activation of a PI 3-kinase and the production of
PtdIns(3,4)P2. A C2 domain-containing PI 3-kinase,
activated by IIb3 engagement, has
recently been described in platelets.33 This enzyme
produces PtdIns(3)P, which is then phosphorylated to
PtdIns(3,4)P2 by a PtdIns(3)P 4-kinase. This new route
could be compatible with our results; however, in our model, the
binding of fibrinogen to its receptor
IIb3 is not sufficient per se to lead to
PtdIns(3,4)P2 production, because ADP-dependent signaling
is clearly necessary.
Because we have shown that TRAP (Fig 1) or ADP alone12 are
not sufficient per se to induce the accumulation of
PtdIns(3,4)P2, an important question is how a combination
of these agents can induce a full activation. One explanation might be
that IIb3 exposure to its ligand must
reach a certain level, obtained upon addition of 2 weak agonists (ie,
TRAP and ADP),24 so that the formation of strong focal
complexes might occur. The outside/in signaling of
IIb3 is thought to be linked to the
recruitment, around the 3 cytoplasmic tail, of signaling
complexes and cytoskeletal proteins.34 These complexes may
be different according to the degree of
IIb3 activation and the mechanical
strengths acting through this integrin.
Another possibility, based on the role of ADP in enhancing the
secretion response induced by other agonists,35 could be that other adhesive receptors have to cooperate with
IIb3 for full signaling through the
integrin. Consistent with this idea, a recent study suggests a
modulation of IIb3 function by
thrombospondin,36 which is released upon platelet activation.
The results obtained using selective ADP receptor antagonists strongly
suggest that the P2 receptor negatively coupled to adenylyl cyclase is
the ADP receptor required for the reinforcing role of ADP. This
observation is of consequence in terms of antithrombotic pharmacology,37-39 because the antiplatelet drug
clopidogrel, acting through this ADP receptor, inhibits thrombosis in
humans.25 The intracellular machinery involved in these
processes is currently under investigation. One can speculate that,
beside the inhibition of cAMP formation, the release of /
subunits from heterotrimeric G-proteins may be critical.
Surprisingly, the purinergic antagonists were able to totally reverse
the inhibition of cAMP formation caused by TRAP (Fig 5). At first
glance, this is in apparent contradiction with previously published
work showing that PAR1 stimulation leads to ADP-independent inhibition
of adenylyl cyclase by a Gi coupling mechanism in isolated platelet
membranes40,41 or intact cells.42 This
discrepancy may come from the fact that inhibition of adenylyl cyclase
is not measurable without stimulation of cAMP production by
PGE1 or PGI2 and that, in contrast to Giesberts
et al,42 who first stimulated platelets by TRAP or thrombin
and then, 5 minutes later, added PGI2, we increased the
cAMP levels by PGE1 1 minute before stimulation by TRAP.
This increase in cAMP level may result in a decrease of efficiency of
TRAP to directly activate Gi, probably via a partial desensitization of
the receptor,43 and thus may exacerbate the effect of
secreted ADP. This is consistent with the fact that, in similar
experiments, we observed that thrombin at high concentration (>0.05
U/mL) did inhibit cAMP production by PGE1 in an
ADP-independent manner, whereas, at lower concentrations, the role of
ADP was clear (not shown). Moreover, we show here that PAR1 does not
require ADP to induce the reversible phase of platelet aggregation. In
contrast, ADP plays a pivotal role to get an irreversible aggregation,
indicating its involvement in a rather late phase of platelet
activation induced by TRAP. Based on recent results demonstrating the
necessity of both Gi and Gq pathways to obtain ADP-induced reversible
aggregation,21,22 it is conceivable that PAR1-mediated
reversible aggregation by itself also requires these 2 pathways.
The impact of released ADP and the importance of newly synthesized
PtdIns(3,4)P2 for reorganization of the cytoskeleton and translocation of signaling enzymes were also evaluated in this study.
The cytoskeletal content of actin binding protein, -actinin, and
F-actin increases upon TRAP stimulation, with no effect of apyrase or
wortmannin. In contrast, these treatments markedly reduce the amount of
myosin associated with the cytoskeletal fraction and affect the
stability of the signaling complexes associated with the actin
cytoskeleton. It is likely that the translocation of myosin to the
cytoskeleton results from its binding to actin filaments,44
a process regulated by phosphorylation of myosin light chain (MLC).
Beside the role of the Ca2+/calmodulin-dependent MLC kinase
in the initial responses induced by thrombin, the RhoA-dependent
regulation of MLC phosphorylation might also occur.45 This
is supported by the striking parallelism observed here between the low
cytoskeletal content of myosin and RhoA and the reversible aggregation.
Because PI 3-kinase inhibitors and apyrase had similar effects, it is
tempting to suggest that ADP may play its role in the reorganization of
the cytoskeleton via potentiating PtdIns(3,4)P2 synthesis.
We are currently investigating whether this lipid could contribute to
the stabilization of focal contacts and, in turn, to the irreversible
ligand binding to IIb346 in
TRAP-stimulated platelets.
Together, our results emphasize the role of ADP as a cofactor and may
explain its involvement in stabilizing platelet aggregates induced by
other agonists.16,17 It is also noteworthy that ADP is
involved in platelet spreading,8,9 a mechanism that requires PI 3-kinase activity.47 A better understanding of
the molecular mechanisms involved in ADP-dependent regulation of the accumulation of PtdIns(3,4)P2 in TRAP-stimulated platelets,
as well as the identification of the targets of this phosphoinositide, may lead to new pharmacological strategies to modulate platelet aggregation or spreading in vivo.
| |
ACKNOWLEDGMENT |
The authors thank Dr G. Mauco for helpful discussions.
| |
FOOTNOTES |
Submitted February 3, 1999; accepted August 10, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Bernard Payrastre, PhD, INSERM
U 326, Hôpital Purpan, 31059 Toulouse, France.
| |
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M. J. Cho, T. I. Pestina, S. A. Steward, C. A. Lowell, C. W. Jackson, and T. K. Gartner
Role of the Src family kinase Lyn in TxA2 production, adenosine diphosphate secretion, Akt phosphorylation, and irreversible aggregation in platelets stimulated with gamma -thrombin
Blood,
April 1, 2002;
99(7):
2442 - 2447.
[Abstract]
[Full Text]
[PDF]
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P. Lova, S. Paganini, F. Sinigaglia, C. Balduini, and M. Torti
A Gi-dependent Pathway Is Required for Activation of the Small GTPase Rap1B in Human Platelets
J. Biol. Chem.,
March 29, 2002;
277(14):
12009 - 12015.
[Abstract]
[Full Text]
[PDF]
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J. Graff, U. Klinkhardt, V. B. Schini-Kerth, S. Harder, N. Franz, S. Bassus, and C. M. Kirchmaier
Close Relationship between the Platelet Activation Marker CD62 and the Granular Release of Platelet-Derived Growth Factor
J. Pharmacol. Exp. Ther.,
March 1, 2002;
300(3):
952 - 957.
[Abstract]
[Full Text]
[PDF]
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C. L. Yap, K. E. Anderson, S. C. Hughan, S. M. Dopheide, H. H. Salem, and S. P. Jackson
Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha IIbbeta 3
Blood,
January 1, 2002;
99(1):
151 - 158.
[Abstract]
[Full Text]
[PDF]
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N. J. Greco, G. Tonon, W. Chen, X. Luo, R. Dalal, and G. A. Jamieson
Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells
Blood,
July 1, 2001;
98(1):
100 - 107.
[Abstract]
[Full Text]
[PDF]
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A. R. L. Gear, S. Suttitanamongkol, D. Viisoreanu, R. K. Polanowska-Grabowska, S. Raha, and D. Camerini
Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function
Blood,
February 15, 2001;
97(4):
937 - 945.
[Abstract]
[Full Text]
[PDF]
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C. Leon, M. Freund, C. Ravanat, A. Baurand, J.-P. Cazenave, and C. Gachet
Key Role of the P2Y1 Receptor in Tissue Factor-Induced Thrombin-Dependent Acute Thromboembolism : Studies in P2Y1-Knockout Mice and Mice Treated With a P2Y1 Antagonist
Circulation,
February 6, 2001;
103(5):
718 - 723.
[Abstract]
[Full Text]
[PDF]
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M.-P. Gratacap, J.-P. Herault, C. Viala, A. Ragab, P. Savi, J.-M. Herbert, H. Chap, M. Plantavid, and B. Payrastre
Fcgamma RIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation
Blood,
November 15, 2000;
96(10):
3439 - 3446.
[Abstract]
[Full Text]
[PDF]
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P. Ohlmann, A. Eckly, M. Freund, J.-P. Cazenave, S. Offermanns, and C. Gachet
ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galpha q
Blood,
September 15, 2000;
96(6):
2134 - 2139.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION