(Received for publication, May 25, 1996, and in revised form, November 14, 1996)
From the INSERM U.311, The aim of our study was to evaluate the effect
of ADP and the role of cytoskeleton reorganization during reversible
and irreversible platelet aggregation induced by ADP and thrombin,
respectively, on the heterodimeric (p85 Phosphoinositide 3-kinase (PI
3-kinases)1 (1) are enzymes involved in
growth factor signal transduction through association with receptor and
nonreceptor tyrosine kinases and with G-protein-coupled receptors such
as the fMet-Leu-Phe receptor in neutrophils or the thrombin receptor in
platelets (1-3). Phosphoinositide kinases and their products have been
implicated in the reorganization of the cytoskeleton, and PI 3-kinase
is known to be directly involved in platelet-derived growth factor,
insulin-like growth factor-1, and insulin-induced membrane ruffling (4,
5). Blood platelets also undergo morphological changes in response to
stimulation, in particular shape change, extension of pseudopods,
secretion of granule contents, aggregation, and contraction, all of
which are linked to cytoskeletal modifications. Thrombin activation of
human platelets leads to cytoskeletal translocation of the heterodimeric (p85 Integrins are transmembrane heterodimers mediating cell-matrix and
cell-cell interactions. The platelet Depending on the cell type studied, the heterodimeric PI 3-kinase A close relationship between integrin
Platelet aggregation by ADP plays a key role in the development and
extension of arterial thrombosis (28). Specific inhibitors of the ADP
activation pathway such as the anti-aggregatory thienopyridine compounds ticlopidine and clopidogrel (29) markedly prolong the
bleeding time and are used clinically as antithrombotic drugs. Furthermore, a rare congenital bleeding disorder with impairment of
ADP-induced platelet aggregation (30, 31) strikingly resembles the
acquired thrombopathy resulting from ticlopidine or clopidogrel intake
(32). Contained at very high concentrations in the platelet-dense granules, ADP is released when platelets are stimulated by other aggregating agents such as thrombin or collagen and thus contributes to
and reinforces platelet aggregation. Low concentrations of ADP also
potentiate or amplify the effects of all other agents, even weak
agonists such as epinephrine or serotonin. Addition of ADP to washed
human platelet suspensions results in shape change, exposure of the
fibrinogen binding site on the The aim of the present study was to evaluate the direct effect of ADP
on PI 3-kinase activation and to compare PtdIns(3,4)P2 accumulation during reversible and irreversible aggregation. ADP was
found to induce a reversible modification of the cytoskeleton which
paralleled aggregation. The regulatory subunit p85 The rabbit anti-p85 Human blood was
collected from a forearm vein (6 blood volumes into 1 volume of
acid/citrate/dextrose anticoagulant), and twice-washed platelet
suspensions were prepared as described previously (39). In some
experiments, platelets were labeled with sodium [32P]orthophosphate (200 µCi/ml) for 1 h at
37 °C during a first washing step in Tyrode's buffer containing no
phosphate. The final resuspending medium, pH 7.35, was Tyrode's buffer
containing 2 mM Ca2+, 1 mM
Mg2+, 0.35% human serum albumin (Etablissement de
Transfusion Sanguine, Strasbourg, France), and apyrase (2 µg/ml, a
concentration which converted 0.25 µM ATP to AMP within 2 min at 37 °C). Platelets were stored at 37 °C throughout
experiments, and 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).
Aggregation was measured at
37 °C by a turbidimetric method in 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 addition of ADP in
the absence or presence of human fibrinogen (0.8 mg/ml), or of thrombin
in the absence of fibrinogen. The extent of aggregation was estimated
quantitatively by measuring the maximum curve height above base-line
level. At predetermined times, the reaction was stopped by addition of
1 ml of chloroform/methanol (v/v) for lipid extraction and analysis or
by addition of an equal volume of CSK buffer (50 mM
Tris-HCl, pH 7.4, 10 mM EGTA, 1 mM Na3VO4, 4 µg/ml aprotinin, 4 µg/ml
leupeptin, 100 µg/ml phenylmethylsulfonyl fluoride, 2% (v/v) Triton
X-100).
Lipids were extracted and
analyzed as described previously (2).
Unlabeled platelets, activated or
nonactivated, were mixed with 1 volume of CSK buffer and incubated
successively for 5 min at room temperature and for 10 min at 4 °C
under shaking. Cytoskeletal material was collected by centrifugation
(12,000 × g, 10 min, 4 °C), washed once with 2 volumes of CSK buffer containing 1% (v/v) Triton X-100, and then
washed five times with 2 volumes of CSK buffer containing no
Triton.
Cytoskeletal
proteins were solubilized in electrophoresis buffer (10 mM
Tris-HCl, pH 6.8, 15% (v/v) glycerol, 25 mM
dithiothreitol, 3% SDS), boiled for 5 min, and separated on 7.5%
SDS-PAGE gels. The protein bands were blotted onto nitrocellulose, and
immunodetection was performed with relevant antibodies as
described.
Washed human
platelets were stimulated with 10 µM ADP in the presence
of fibrinogen or with 1 unit/ml thrombin in the absence of added
fibrinogen. Typical aggregation curves were obtained (Fig.
1A), ADP inducing reversible and thrombin
irreversible aggregation. In some experiments, the reaction was stopped
at predetermined time points, the cytoskeleton was extracted and
cytoskeletal proteins were solubilized, separated by SDS-PAGE, and
stained with Coomassie Blue. Small amounts of actin binding protein
(250 kDa),
Using a polyclonal
antibody, we found an increase in amounts of the regulatory subunit
p85
ADP-induced translocation of PI 3-kinase
Washed human platelets were stimulated with 10 µM ADP in the presence of fibrinogen or with 1 unit/ml
thrombin in the absence of added fibrinogen; the cytoskeleton was
extracted at indicated time points and analyzed by Western blotting.
The tyrosine kinases pp60c-src and FAK were
translocated to the cytoskeleton in a similar manner to the PI 3-kinase
regulatory subunit p85
As is now well established (7, 16), thrombin activation of washed
human platelets resulted in translocation of the heterodimeric PI
3-kinase Surprisingly, PtdIns(3,4)P2 did not accumulate even at the
maximum amplitude of aggregation, although PI 3-kinase Inhibition of PI 3-kinase by wortmannin or LY294002 has been reported
to reverse the platelet aggregation induced by agonists such as TRAP
(46). These authors suggested that PI 3-kinase activation may be
necessary for prolonged Alternatively, the results presented here are also consistent with the
hypothesis that the typical reversibility of ADP-induced platelet
aggregation finds its origin in the lack of PI 3-kinase activation and
hence of PtdIns(3,4)P2 formation. Recent data on the
effects of wortmannin on platelet aggregation induced by TRAP (46) may
indicate that PI 3-kinase products are necessary to stabilize platelet
aggregates. Moreover, it has been demonstrated that
PtdIns(3,4,5)P3 is able to bind to the SH2 domains of
several proteins including p85 We thank C. Viala for technical assistance,
J. N. Mulvihill for reviewing the English of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-p110) phosphoinositide
3-kinase translocation to the cytoskeleton and its activation.
Reversible ADP-induced aggregation was accompanied by a reversible
reorganization of the cytoskeleton and an increase in levels of the
regulatory subunit p85
in this cytoskeleton similar to the increase
observed in thrombin-activated platelets. This translocation followed a
course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3,4)-bisphosphate
(PtdIns(3,4)P2) and
phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase
translocation. Moreover, in contrast to the situation for thrombin stimulation, the
GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of
pp60c-src and focal adhesion kinase did occur.
These results suggest (i) translocation of signaling enzymes does not
necessarily imply their activation, (ii) the reversibility of
ADP-induced platelet aggregation may be the cause or the result of a
lack of PI 3-kinase activation and hence of PtdIns(3,4)P2
production, and (iii) RhoA does not seem to be involved in the ADP
activation pathway of platelets. Whether PtdIns(3,4)P2 or
RhoA may contribute to the stabilization of platelet aggregates remains
to be established.
-p110) PI 3-kinase (PI 3-kinase
) and
accumulation of PtdIns(3,4)P2 in an integrin
IIb
3-dependent manner (6, 7).
IIb
3
integrin serves as an activation-dependent receptor for the
adhesive proteins fibrinogen, fibronectin, and von Willebrand factor.
In patients with Glanzmann's thrombasthenia (8), an inherited
hemorrhagic disorder where the
IIb
3
integrin is absent, reduced, or abnormal, platelets are unable to bind
fibrinogen upon activation and consequently do not aggregate. In
platelets, as in other cells, integrin ligation triggers the assembly
of specific cytoskeletal proteins and enzymes into structures termed
focal adhesion sites (9, 10). These focal adhesion structures comprise
proteins such as vinculin, talin, and the integrin
IIb
3 itself, enzymes such as PI 3-kinase
, phospholipase C, protein kinase C, the tyrosine kinases
pp60c-src, pp72SYK, and focal adhesion
kinase (FAK) or the small GTP-binding proteins, RhoA and Cdc42Hs
(11-16). Rho, Rac, and Cdc42Hs are all members of the Ras superfamily
of small GTP-binding proteins. These molecules are important regulators
of the cytoskeleton, and evidence is now accumulating that Rho promotes
the formation of focal adhesions and their anchoring to stress fibers
(17-19).
has been found to be activated by several pathways including interactions of the p85
regulatory subunit with phosphorylated receptor tyrosine kinases, tyrosine kinases of the src
family (20), p21ras (21, 22), RhoA (23, 24), Cdc42Hs (25), or
FAK (16). In platelets stimulated by thrombin, both RhoA and FAK could
participate to the activation of PI 3-kinase
, the former presumably
by an indirect mechanism (24) and the latter by a direct interaction with the SH3 domain of the p85
subunit (16). In
addition, heterotrimeric G-protein
complexes may be involved in
the stimulation of a second isoform of PI 3-kinase present in
platelets. This form is immunologically related to a recently cloned
monomeric PI 3-kinase, which was designated PI 3-kinase
and found
to be activated in vitro by
subunits (24, 26,
27).
IIb
3-dependent p85
translocation to the cytoskeleton and PtdIns(3,4)P2
accumulation has been demonstrated in thrombin-stimulated platelets
(16), suggestive of a role of the heterodimeric PI 3-kinase at this stage of 3-phosphoinositide synthesis. However, true assessment of the
direct action of thrombin on platelets is difficult due to the presence
of several mediators, in particular ADP and serotonin, released from
the platelet-dense granules and of mediators such as thromboxane
A2 resulting from activation of the arachidonic pathway,
all of which interact with their own specific receptors on the platelet
membrane.
IIb
3
integrin, and in contrast to other agonists such as thrombin,
reversible aggregation in the presence of fibrinogen and physiological
concentrations of Ca2+. At the intracellular level,
platelet activation following ADP binding to its receptor leads to a
transient rise in free cytoplasmic Ca2+, resulting from
both Ca2+ influx and mobilization of internal stores,
without apparent activation of phospholipase C or
D-myo-inositol 1,4,5-trisphosphate (33, 34). ADP
also inhibits stimulated adenylyl cyclase (35). On the basis of its
agonist selectivity and signaling properties, the platelet receptor for
ADP has been classified as a P2T receptor of the
P2 purinoceptor family (36). Although the biochemical structure of this receptor remains unknown, it may belong to the seven
transmembrane domain G-protein-coupled receptor family since ADP has
been found to activate the Gi2 subtype of the
heterotrimeric G-protein family (37, 38).
of PI 3-kinase
and FAK reversibly translocated to the cytoskeleton, and this
effect was dependent on the presence of
IIb
3 and on the binding of fibrinogen to
its receptor. However, significant accumulation of
PtdIns(3,4)P2 did not occur, indicating that although translocation of the heterodimeric PI 3-kinase
occurred, activation did not. Moreover, in contrast to thrombin stimulation, the small GTP-binding protein RhoA was not significantly translocated to the
cytoskeleton when platelets were stimulated with ADP, adding further
support to a functional relationship between RhoA and PI 3-kinase.
Materials
antibody was from Upstate
Biotechnology Inc. (Lake Placid, NY), and rabbit anti-FAK and anti-RhoA
antibodies were from Tebu (Santa Cruz Biotechnology Inc., Santa Cruz,
CA), and an affinity purified sheep polyclonal antibody against
pp60c-src was from Cambridge Research Biochemicals
Inc. (Cambridge, UK).
Reversible Modification of the Cytoskeleton
-actinin (100 kDa), and F-actin (45 kDa) were found in
the cytoskeleton of resting platelets. After stimulation with ADP,
actin binding protein, myosin (200 kDa), and actin were reversibly
translocated to the cytoskeleton, maximum incorporation corresponding
to the maximum amplitude of platelet aggregation (Fig. 1B, left
panel). Myosin was only weakly incorporated into the cytoskeleton
of ADP-stimulated platelets. When platelets were stimulated with
thrombin, translocation of actin binding protein, myosin, and actin to
the cytoskeleton was not reversible (Fig. 1B, right panel).
Actin polymerization induced by ADP was reversible and followed a
course parallel to the amplitude of aggregation (Fig.
1C).
Fig. 1.
Differential aggregation and cytoskeletal
reorganization induced by ADP and thrombin. A, platelet
aggregation was induced by ADP (10 µM) in the presence of
fibrinogen or by thrombin (1 unit/ml) in the absence of added
fibrinogen and followed as described under "Experimental
Procedures." Curves are representative of five independent
experiments giving very similar results. B, in parallel,
cytoskeletons were isolated from ADP- (10 µM + fibrinogen) or thrombin (1 unit/ml) -stimulated platelets at the
indicated times. Cytoskeletal proteins (corresponding to 7.5 × 107 platelets) were separated by SDS-PAGE (7.5%) and
revealed by Coomassie Blue staining, actin, and the major actin-binding
proteins being identified on the right side of the gel. Data are
representative of two independent experiments giving very similar
results. C, the F-actin content of ADP- or
thrombin-stimulated platelets was quantified by densitometric analysis
(ScanMaker IIHR, Microtek, Germany) of the Coomassie Blue-stained
gel.
[View Larger Version of this Image (37K GIF file)]
Subunit of PI
3-Kinase
without [32P]PtdIns(3,4)P2
Accumulation during Platelet Aggregation
in the cytoskeleton during both ADP- and thrombin-induced
platelet aggregation (Fig. 2A). In the case
of ADP-stimulated platelets, translocation was reversible and followed
the amplitude of aggregation. However, 20 and 40 s after
stimulation, levels of p85
in the cytoskeleton were identical using
either ADP or thrombin, and PI 3-kinase activity measured in the
cytoskeleton followed the same time course (not shown). In order to
measure [32P]PtdIns(3,4)P2 accumulation
during platelet aggregation, 32P-labeled washed human
platelets were stimulated with 10 µM ADP in the absence
or presence of fibrinogen or with 1 unit/ml thrombin in the absence of
added fibrinogen. Thrombin stimulation gave rise to the expected
time-dependent accumulation of
[32P]PtdIns(3,4)P2 (Fig. 2B, right
panel), whereas ADP did not induce any significant synthesis of
[32P]PtdIns(3,4)P2 (Fig. 2B, left
panel). In 5 of 7 experiments, where 32P incorporation
into lipids was especially high, we could detect only transient trace
amounts of radioactivity in both PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 at short times (20 s) following ADP
stimulation. Addition of fibrinogen did not increase this labeling.
Similar results were obtained using 100 µM ADP (data not
shown).
Fig. 2.
Reversible translocation of p85 to the
cytoskeleton without PtdIns(3,4)P2 accumulation in
ADP-stimulated platelets as compared with time-dependent
PtdIns(3,4)P2 accumulation after thrombin stimulation.
A, cytoskeletons were extracted from ADP- (10 µM + fibrinogen) or thrombin (1 unit/ml) -stimulated platelets at the indicated times. Proteins of the cytoskeleton (corresponding to 7.5 × 107 platelets) were separated
by SDS-PAGE (7.5%), blotted onto nitrocellulose, and tested for
reactivity with an anti-p85
antibody using enhanced chemiluminescence. B, the time course of
PtdIns(3,4)P2 accumulation in washed platelets stimulated
by ADP (10 µM + fibrinogen) or thrombin (1 unit/ml) was
followed as indicated under "Experimental Procedures."
[View Larger Version of this Image (23K GIF file)]
Translocation Requires Integrin
IIb
3 and Fibrinogen
Binding
to the
cytoskeleton was dependent on the presence of the integrin
IIb
3, since this effect was not
detectable using platelets from a type I Glanzmann's thrombasthenia
patient (40) (Fig. 3, right panels).
Translocation was also dependent on the binding of fibrinogen to its
receptor and was clearly reduced when fibrinogen was omitted (Fig. 3,
left panels). The residual translocation observed is
probably due to secreted fibrinogen.
Fig. 3.
Integrin IIb
3
and fibrinogen binding dependence of p85
translocation. Control
and type I Glanzmann's thrombasthenia (GT) platelets were
stimulated with ADP (10 µM) in the presence or absence of
added fibrinogen. At the indicated time points, the reaction was
stopped and translocation of p85
was evaluated as in Fig. 2.
[View Larger Version of this Image (17K GIF file)]
(Fig. 4). In contrast, although thrombin induced clearly detectable translocation of RhoA to
the cytoskeleton (Fig. 4, right panel), ADP did not (Fig. 4,
left panel), and we could distinguish only a faint band at 20 s.
Fig. 4.
Reversible translocation of
pp60c-src and FAK but not RhoA to the cytoskeleton
of ADP (10 µM + fibrinogen) -stimulated platelets, as
compared with irreversible translocation of all three proteins after
thrombin stimulation. Platelets were stimulated with ADP (10 µM + fibrinogen) or thrombin (1 unit/ml) for increasing periods as indicated on the figure. Reactions were then stopped; the
cytoskeletons were immediately extracted, and cytoskeletal proteins
(corresponding to 7.5 × 107 platelets) were separated
by SDS-PAGE (7.5% for p125FAK and
pp60c-src or 12% for RhoA), blotted onto
nitrocellulose, and examined for reactivity with the indicated
antibodies. Alkaline phosphatase detection was used for
pp60c-src analysis and the enhanced
chemiluminescence system for p125FAK and RhoA.
[View Larger Version of this Image (48K GIF file)]
to the actin-rich cytoskeleton, together with production and accumulation of PtdIns(3,4)P2 (Fig. 2B, right
panel). These effects of thrombin are dependent on the presence of
functional
IIb
3 integrin (6, 41). In
general terms, coordinated signaling through agonist receptors and
integrins results in reorganization of the cytoskeleton and formation
of focal adhesion structures with translocation and activation of
signaling proteins and enzymes (9). The aim of our study was to assess
the specific role of ADP in these events and to investigate PI 3-kinase
activation during reversible aggregation. So far, the molecular
mechanisms leading to platelet aggregation by ADP and its typical
feature of reversibility are not well understood. Our results showed an increase in levels of the regulatory subunit p85
in the cytoskeleton of ADP-stimulated platelets equivalent to the increase observed in
thrombin-stimulated platelets (Fig. 2A). This translocation was reversible and followed a course parallel to the amplitude of
aggregation. Furthermore, this effect of ADP was dependent on the
presence of the integrin
IIb
3 and on the
binding of fibrinogen to its receptor (Fig. 3), clearly indicating an
"outside-in" signaling event involving the "ADP-activated"
integrin. Reversibility of the association of transduction proteins
with the cytoskeleton of platelets activated by a thrombin receptor
agonist peptide (TRAP) has already been reported to occur 15 min after
stimulation in a Ca2+-dependent but
aggregation-independent manner (42). Our results demonstrate the
rapidly reversible association of these proteins in a manner differing
according to the agonist used and the aggregation response.
was
translocated to the cytoskeleton in amounts comparable with those found
in thrombin-aggregated platelets. This result demonstrates that
translocation of the enzyme is an aggregation-dependent
event but is not sufficient for activation of PI 3-kinase. At least
three pathways of activation of PI 3-kinases have been reported in
platelets, involving the small GTP-binding protein RhoA (23, 24) and
the tyrosine kinase FAK (16) for the (p85
-p110) enzyme, or the
subunit complex of heterotrimeric G-proteins for the p110 PI
3-kinase
(24, 26, 27). In the case of ADP-induced platelet
aggregation, we found pp60c-src and FAK to be
reversibly translocated to the cytoskeleton in a manner similar to
p85
, whereas RhoA was not. These observations deserve several
comments. (i) Gi2 proteins involved in the ADP signaling
pathway do not seem to provide PI 3-kinase activating
subunits
which would otherwise have activated such an enzyme. (ii) The clear-cut
difference between ADP and thrombin in inducing translocation of RhoA
suggests that PI 3-kinase
could be regulated by this small
G-protein as previously reported (23, 24). Interestingly, using
lysophosphatidic acid as an agonist of a G-protein-coupled receptor,
evidence has been provided that Rho-dependent assembly of
an actin-based signaling complex linked to integrins was stimulated downstream of Gq (43, 44). In contrast to thrombin, ADP
activates only Gi2 with no effect on Gq (38).
Our data would fit with this scheme, underlying a possible role of RhoA
in regulating PI 3-kinase activity and stabilization of the
cytoskeleton. (iii) One cannot exclude a direct role of FAK, since its
translocation to the cytoskeleton does not necessarily imply
activation. This point could be clarified by looking at tyrosine
phosphorylation of FAK during ADP-induced platelet aggregation.
Nevertheless, the ADP scavenger apyrase has been shown to prevent
tyrosine phosphorylation of FAK and the spreading of platelets on
immobilized fibrinogen, which suggests that ADP in fact induces this
phosphorylation (45).
IIb
3 activation
and irreversible platelet aggregation. However, using wortmannin up to
100 nM, we were unable to reverse the irreversible
aggregation induced by thrombin, even though the aggregates were
smaller (data not shown). Aggregation was also found to be necessary
for the late accumulation of PtdIns(3,4)P2 measured 3-5
min after thrombin addition (16). Furthermore, previous studies have
clearly established that thrombin-induced translocation to the
cytoskeleton of several signaling proteins including
IIb
3, pp60c-src, PI
3-kinase
(12, 16), as well as FAK tyrosine phosphorylation (47)
require platelet to platelet contacts. Hence, the current data suggest
that irreversible aggregation is necessary to initiate a
"mechanical" transduction pathway leading to PI 3-kinase
activation, whereas reversible aggregation would appear to be
insufficient. Thrombospondin, a large trimeric adhesive molecule
released from the
granules of thrombin- but not ADP-stimulated
platelets, could contribute to such a late signaling event by binding
to the plasma membrane through its receptor CD 36, which has been shown
to be linked to tyrosine kinases of the src family (48). On
the other hand, when platelets were stimulated with lysophosphatidic acid (49) or concanavalin A (50), aggregation did not appear to be
necessary for the synthesis of PtdIns(3,4)P2.
Identification of the PI 3-kinase isoforms involved in these processes
would contribute to a better understanding of the different pathways and stages of 3-phosphoinositide synthesis in platelets. Nevertheless, the physiological significance of the synthesis of
PtdIns(3,4)P2 depending on irreversible platelet
aggregation remains to be established. The microvesiculation and clot
retraction occurring at this stage of platelet activation are
controlled by mechanisms involving reorganization of the membrane and
cytoskeleton, which could be regulated by 3-phosphoinositide
synthesis.
(51), thereby blocking the binding of PI 3-kinase to tyrosine phosphorylated proteins, which would suggest direct involvement of these D3-phosphorylated phosphoinositides. Whether PtdIns(3,4)P2, among other components of assembled
signaling complexes, contributes directly to the stability of platelet
aggregates is not known. The differential effects of ADP and thrombin
on platelet activation and aggregation may thus provide a physiological model leading to the improvement of our understanding of PI 3-kinase pathways, at least in platelets.
*
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: INSERM U.311, Biologie
et Pharmacologie des Interactions du Sang avec les Vaisseaux et les
Biomatériaux, Etablissement de Transfusion Sanguine, B.P. no. 36, 10, rue Spielmann, 67065 Strasbourg Cédex, France. Tel.: (33) 3 88 21 25 25; Fax: (33) 3 88 21 25 21; E-mail:
christian.gachet{at}etss.u-strasbg.fr.
1
The abbreviations used are: PI 3-kinase,
phosphoinositide 3-kinase; PtdIns, phosphatidylinositol;
PtdIns(3,4)P2, phosphatidylinositol (3,4)-bisphosphate;
FAK, focal adhesion kinase; TRAP, thrombin receptor agonist peptide;
PAGE, polyacrylamide gel electrophoresis.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.