From the
The synthesis of phosphatidylinositol 3`,4`-bisphosphate
(PtdIns(3,4)P
Human platelets accumulate the 3-phosphorylated
phosphoinositides phosphatidylinositol 3`,4`-bisphosphate
(PtdIns(3,4)P
The biochemical pathways and the regulatory mechanisms
leading to the production of the 3-phosphorylated phosphoinositides are
not completely understood. The enzyme mainly responsible for the
formation of these lipids is the phosphatidylinositol 3-kinase (PtdIns
3-kinase)
Synthesis of
PtdIns(3,4)P
The GP
IIb-IIIa belongs to the integrin family of receptors for adhesive
proteins (integrin
The understanding of the exact
role played by GP IIb-IIIa in regulating these events is complicated by
the fact that the classical experimental strategies used to block its
receptor activity (specific monoclonal antibodies, RGD-containing
peptides, thrombasthenic platelets, etc.) also result in the consequent
inhibition of platelet aggregation. Therefore, under these conditions,
it is not clear whether a certain event is directly controlled by GP
IIb-IIIa or is linked to other events occurring during platelet
aggregation. It is known that secondary events, following fibrinogen
binding, occur on the cell surface during platelet aggregation. These
events include clustering of GP IIb-IIIa
(23) , the stabilization
of the platelet aggregates by binding of secreted thrombospondin to its
surface receptor
(24, 25) , and the interaction of GP
IIb-IIIa and other glycoproteins, such as the platelet-endothelial cell
adhesion molecule 1 (PECAM-1), with the intracellular
cytoskeleton
(26, 27) .
Looking for an experimental
system able to discriminate between the influence of GP IIb-IIIa and
platelet aggregation on the production of PtdIns(3,4)P
Here, we show that ConA
stimulates the accumulation of PtdIns(3,4)P
In this study, we demonstrate that synthesis of
PtdIns(3,4)P
The most intriguing consideration about the aggregation-independent
synthesis of PtdIns(3,4)P
One of the membrane components
clustered during platelet aggregation and upon binding of ConA is the
GP IIb-IIIa
(23, 33) . We used platelets from patients
affected by Glanzmann thrombasthenia, which lack the GP IIb-IIIa, to
clarify the role of clustering of the fibrinogen receptor. It was
previously reported that thrombin displayed a reduced ability to induce
synthesis of PtdIns(3,4)P
We
propose here a working model to explain the GP IIb-IIIa-dependent
synthesis of PtdIns(3,4)P
The signaling pathway activated by the cluster of membrane
glycoproteins and leading to PtdIns(3,4)P
In conclusion, the aggregation-independent synthesis of
PtdIns(3,4)P
Platelets from healthy donors and from two
patients affected by Glanzmann thrombasthenia (C. S. and M. L.) were
labeled with
We thank Dr. M. Poggio (Transfusion Unit, Hospital L.
Sacco, Milan, Italy) for providing blood samples of thrombasthenic
patients and Drs. B. Payrastre and C. Racaud-Sultan (INSERM U326,
Toulouse, France) for helpful discussion.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) in
P-labeled human platelets
induced by the tetrameric lectin concanavalin A and the physiological
agonist thrombin were compared. Like thrombin, concanavalin A
stimulated a time-dependent accumulation of PtdIns(3,4)P
,
which reached maximal levels after 5 min of stimulation. However, while
synthesis of PtdIns(3,4)P
induced by thrombin was dependent
on platelet aggregation, the production of PtdIns(3,4)P
induced by concanavalin A was unchanged when aggregation was
prevented by the omission of stirring or when fibrinogen binding to
platelets was inhibited by the tetrapeptide RGDS. Accumulation of
PtdIns(3,4)P
was not observed in platelets stimulated with
succinyl-concanavalin A, a dimeric derivative of the lectin that binds
to the same receptors on the platelet surface but does not promote
clustering of membrane glycoproteins. The synthesis of
PtdIns(3,4)P
induced by concanavalin A was also independent
of the membrane glycoprotein IIb-IIIa, as normal accumulation of this
lipid was observed in platelets from two patients affected by Glanzmann
thrombasthenia. In contrast, thrombin showed a strongly reduced ability
to stimulate PtdIns(3,4)P
production in thrombasthenic
platelets. Although concanavalin A was able to induce association of
the regulatory subunit of the phosphatidylinositol 3-kinase with
tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor
tyrphostin AG-213 did not inhibit the lectin-induced synthesis of
PtdIns(3,4)P
. These results demonstrate the existence of a
novel mechanism of PtdIns(3,4)P
synthesis in human
platelets, which is independent of glycoprotein IIb-IIIa and
aggregation, but requires clustering of membrane glycoproteins. As
clustering events occur during platelet aggregation promoted by
physiological agonists, this new mechanism may also be involved in the
aggregation-dependent production of PtdIns(3,4)P
in
thrombin-stimulated platelets.
) and phosphatidylinositol
3`,4`,5`-trisphosphate (PtdIns(3,4,5)P
) in response to
strong agonists such as thrombin and the thromboxane analogue U46619
(1-3).
(
)(4) . The PtdIns 3-kinase is a
heterodimer composed of a 110-kDa catalytic subunit (p110) and an
85-kDa regulatory subunit (p85). In growth factor-stimulated cells, p85
is tyrosine phosphorylated and interacts, through its SH2 domains, with
receptor-associated and cytosolic protein-tyrosine kinases
(4) .
However, interaction between p85 and Src-like kinases could also be due
to the association of SH3 domain of the kinase and a proline-rich
sequence of p85
(5) . In human platelets stimulated with
thrombin, the PtdIns 3-kinase was found associated with the tyrosine
kinases pp60
, pp59
, and
pp120
(6, 7) , and the accumulation of
PtdIns(3,4)P
was reduced by preincubation of the cells with
the tyrosine kinase inhibitor tyrphostin
(8) . These evidences
indicate that tyrosine phosphorylation events may regulate the
synthesis of the 3-phosphorylated phosphoinositides. However, it has
been recently demonstrated that the PtdIns 3-kinase may be activated by
different mechanisms involving both
-subunits of
heterotrimeric G-proteins
(9) and the low molecular weight
G-proteins Ras and Rho
(10, 11) .
and PtdIns(3,4,5)P
are differently
regulated in human platelets stimulated with thrombin.
PtdIns(3,4,5)P
accumulates rapidly, while
PtdIns(3,4)P
synthesis occurs in a late phase of platelet
activation
(12) . The increase of PtdIns(3,4)P
is
enhanced by extracellular Ca
, which, in contrast, has
no effect on the accumulation of
PtdIns(3,4,5)P
(12) . It has been demonstrated that
in thrombin-stimulated platelets, synthesis of PtdIns(3,4)P
but not PtdIns(3,4,5)P
was inhibited by the
tetrapeptide RGDS, which blocks interaction of fibrinogen with its
membrane receptor, the glycoprotein IIb-IIIa (GP
IIb-IIIa)
(12, 13) . Moreover, a strongly reduced
accumulation of PtdIns(3,4)P
in response to thrombin was
observed in platelets from patients affected by Glanzmann
thrombasthenia, which lack GP IIb-IIIa and fail to aggregate (13).
These evidences indicate a regulatory role of GP IIb-IIIa and platelet
aggregation on the synthesis of PtdIns(3,4)P
.
) and plays an
essential role in platelet physiology
(14) . Platelet stimulation
with thrombin results in the activation of GP IIb-IIIa, which then
binds released fibrinogen and triggers platelet aggregation (15).
Recently, it has been demonstrated that GP IIb-IIIa may also act as a
transducer of signals into the platelets that can influence platelet
function. An increasing number of events observed in activated cells
have been found to be inhibited by blocking the receptor activity of GP
IIb-IIIa: activation of the Na
/H
exchanger
(16) , intracellular Ca
increase
(17) , tyrosine phosphorylation of several
proteins
(18, 19) , activation of protein phosphotyrosine
phosphatases
(20) , interaction of signaling molecules, such as
pp60
and the low molecular weight G-protein
rap2B, with the cytoskeleton
(21, 22) , and synthesis of
PtdIns(3,4)P
(13) .
, we
performed studies using the lectin concanavalin A (ConA). ConA is a
strong platelet agonist able to induce phospholipase C
activation
(28) , protein-tyrosine phosphorylation
(29) ,
and secretion and aggregation
(30) . Although ConA binds to
several mannose-containing glycoproteins, the main platelet receptor
for this lectin has been identified as GP
IIb-IIIa
(31, 32) . Unlike physiological agonists, ConA
can induce in nonaggregated platelets events that are usually observed
only in aggregated cells. For instance, ConA promotes clustering of
membrane glycoproteins, especially clustering of GP
IIb-IIIa
(33) , and induces the association of GP IIb-IIIa with
the intracellular cytoskeleton
(30) .
in human
platelets and that this effect does not require fibrinogen binding to
GP IIb-IIIa and platelet aggregation but is dependent on the
lectin-induced clustering of membrane glycoproteins.
Materials
Concanavalin A, thrombin, the
tetrapeptide RGDS, leupeptin, and aprotinin were obtained from Sigma.
Succinyl-ConA was from Vector Laboratories. Tyrphostin AG-213 was a
gift from Dr. A. Levitzki (Hebrew University, Jerusalem, Israel).
[P]Orthophosphate and enhanced chemiluminesce
reagents were from Amersham Corp. Protein A-Sepharose was from
Pharmacia Biotech Inc. Nitrocellulose was from Schleicher and Schuell.
Anti-p85 polyclonal antiserum and anti-phosphotyrosine monoclonal
antibodies were from UBI. Prestained molecular weight markers,
peroxidase- and alkaline phosphatase-conjugated antibodies were
obtained from Bio-Rad.
Platelet Preparation and Labeling
Human
platelets were prepared from platelet concentrates as previously
described
(3) . Platelets were resuspended at the concentration
of 10 cells/ml and labeled with
[
P]orthophosphate (0.3 mCi/ml) in 135
mM NaCl, 2.7 mM KCl, 12 mM
NaHCO
, 0.16 mM NaH
PO
, 2
mM MgCl
, 0.2 mM EGTA, 5.5 mM
glucose, 0.34% bovine serum albumin, pH 6.5 (buffer A), for 90 min at
room temperature. Platelets were then washed with buffer A without
glucose and EGTA (buffer B) and finally resuspended at the
concentration of 10
cells/ml in buffer B, pH 7.4,
containing 2.5 mM CaCl
. Thrombasthenic platelets
were prepared from freshly drawn blood and labeled as described above.
In parallel, platelets from healthy donors were prepared and used as
controls.
Platelet Incubation, Lipid Extraction, and HPLC
Analysis
Platelet samples (1 ml, 10 cells/ml)
were equilibrated at 37 °C. RGDS (1 mM, final
concentration), tyrphostin (100 µM, final concentration),
or buffer were added 2 min before stimulation with 100 µg/ml ConA
or 1 unit/ml thrombin. As tyrphostin was stored as a 100 mM
stock solution in dimethyl sulfoxide, an equal volume of dimethyl
sulfoxide was added to control samples. Stimulation with the agonists
was usually performed for 5 min at 37 °C with or without stirring
as indicated. Lipids were extracted, deacylated by methylamine
treatment, separated, and identified by HPLC on a Whatman Partisphere
strong anion exchange column as previously described
(3) .
Immunoprecipitation and
Immunoblotting
Platelet stimulation was stopped by addition
of 1 volume of 40 mM Tris/HCl, pH 7.4, 200 mM NaCl,
20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.2 mM
phenylmethylsulfonyl fluoride, 2 mM
NaVO
, 2% Triton X-100. Samples were vortexed,
placed on ice for 10 min, and then centrifuged at 13,000
g for 5 min. Lysates were incubated with 5 µl of anti-p85
polyclonal antiserum, anti-phosphotyrosine antibodies, or control serum
for 2 h at 4 °C. Immunocomplexes were recovered with protein
A-Sepharose (80 µl of a 50 mg/ml stock suspension), washed three
times with 1 ml of 20 mM Tris/HCl, pH 7.4, 100 mM
NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM
Na
VO
, 1% Triton X-100, and finally resuspended
in SDS-sample buffer (100 mM Tris/HCl, pH 8.0, 10% glycerol,
25 mM dithiothreitol, 3% SDS). Samples were boiled 10 min, and
proteins were separated by SDS-polyacrylamide gel electrophoresis on
7.5% polyacrylamide gels and then transferred to nitrocellulose.
Immunoblotting with anti-p85 polyclonal antiserum (1:500 dilution) or
anti-phosphotyrosine antibodies (1:1000 dilution) was performed as
previously described
(34) . Appropriated secondary antibodies
conjugated to alkaline phosphatase or horseradish peroxidase were used.
Immunoreactive proteins were visualized with 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium reaction or by the
chemiluminescent reaction.
Production of PtdIns(3,4)P
The deacylated lipids from
in
ConA-stimulated Platelets
P-labeled human platelets, unstimulated and aggregated for
5 min with 1 unit/ml thrombin or 100 µg/ml ConA under constant
stirring, were separated by strong anion exchange HPLC (Fig. 1).
As previously reported
(3) , thrombin induced a marked increase
of GroP, corresponding to the deacylated form of phosphatidic acid,
which is produced by the sequential action of phospholipase C and
diacylglycerol kinase, and the appearance of a new peak corresponding
to deacylated PtdIns(3,4)P
(GroPIns(3,4)P
),
which was not detected in resting platelets. The HPLC profile of the
deacylated lipids from
P-labeled platelets stimulated with
ConA was very similar to that from thrombin-stimulated platelets,
indicating that, like thrombin, ConA induced both phospholipase C
activation and accumulation of PtdIns(3,4)P
. We also
observed a small amount of GroPIns(3)P in resting platelets, which did
not significantly change upon treatment with the agonists. The
PtdIns(3,4,5)P
was not considered, since, in most of our
experiments, it was not efficiently separated from an unknown peak
eluting at about 68 min.
Figure 1:
HPLC profiles of the deacylated lipids
from resting, thrombin-, and ConA-stimulated platelets.
P-Labeled platelets (10
cells) were incubated
at 37 °C in the absence or in the presence of 1 unit/ml thrombin or
100 µg/ml ConA for 5 min under constant stirring. Lipids were
extracted, deacylated, and analyzed by strong anion exchange HPLC.
Profiles are representative of more than 10 separate experiments.
GroP, sn-glycero-3-phosphate; GroPIns(3)P,
sn-glycero-phosphoinositol-3`-phosphate; GroPIns(4)P,
sn-glycero-phosphoinositol-4`-phosphate;
GroPIns(3,4)P,
sn-glycero-phosphoinositol-3`,4`-bisphosphate;
GroPIns(4,5)P,
sn-glycero-phosphoinositol-4`,5`-bisphosphate.
Fig. 2
shows the time course of GroP
and GroPIns(3,4)P production in ConA-stimulated platelets.
In contrast to thrombin, which is known to induce activation of
phospholipase C within seconds
(35) , increase of GroP in
ConA-treated platelets was evident only after 2 min of stimulation.
This is in agreement with previously reported results measuring the
formation of soluble inositol phosphates in
[
H]inositol-labeled platelets
(28) . The
synthesis of GroPIns(3,4)P
showed a similar kinetic, being
detectable between 1 and 2 min after the addition of the agonist.
Maximal accumulation of GroPIns(3,4)P
was measured at 5
min.
Figure 2:
Time course of the formation of
phosphatidic acid and PtdIns(3,4)P in ConA-stimulated
platelets.
P-Labeled platelets were incubated at 37 °C
with 100 µg/ml ConA under constant stirring for the indicated
times. Lipids were extracted, deacylated, and separated by HPLC.
A, time course of GroP accumulation; B, time course
of sn-glycero-phosphoinositol-3`,4`-bisphosphate synthesis.
Data are representative of three similar
experiments.
Synthesis of PtdIns(3,4)P
To investigate the role of platelet aggregation in
the synthesis of PtdIns(3,4)P in
ConA-stimulated Platelets Did Not Require Platelet
Aggregation
,
P-labeled
platelets were stimulated at 37 °C with 1 unit/ml thrombin and 100
µg/ml ConA for 5 min with and without constant stirring. Both
thrombin and ConA-treated platelets did not aggregate when stirring was
omitted (data not shown). As shown in Fig. 3A, the
production of PtdIns(3,4)P
in thrombin-stimulated platelets
was dramatically decreased in the absence of aggregation (about 60%
inhibition). In contrast, a similar amount of PtdIns(3,4)P
was measured in ConA-stimulated platelets independently from the
occurrence of aggregation. The aggregation-independent synthesis of
PtdIns(3,4)P
in ConA-stimulated platelets was confirmed by
using the tetrapeptide RGDS. It was reported that preincubation of
platelets with 1 mM RGDS caused about 60% inhibition of
PtdIns(3,4)P
synthesis induced by thrombin
(13) .
However, as shown in Fig. 3B, treatment with 1
mM RGDS did not modify the ability of ConA to induce
accumulation of PtdIns(3,4)P
in human platelets.
Figure 3:
Effect of platelet aggregation on the
production of PtdIns(3,4)P. A,
P-Labeled platelets were stimulated with 1 unit/ml
thrombin or 100 µg/ml ConA at 37 °C for 5 min with or without
stirring as indicated. Formation of PtdIns(3,4)P
was
determined by HPLC analysis of the deacylated lipids. Synthesis of
PtdIns(3,4)P
in stimulated platelets under constant
stirring (4308 ± 1112 dpm in thrombin-stimulated platelets and
2357 ± 626 dpm in ConA-stimulated platelets) is reported as 100%
of production. Statistic significances by Student's t test are as follows: thrombin with stirring versus thrombin without stirring, 0.01 < p < 0.02; ConA
with stirring versus ConA without stirring, not significant
(p > 0.3). B,
P-labeled platelets
were incubated at 37 °C for 2 min with buffer or with 1 mM
RGDS before stimulation with 100 µg/ml ConA for 5 min under
constant stirring. Results are expressed as percentage of the
production of PtdIns(3,4)P
observed in ConA-stimulated
platelets. Student's t test indicates that the
difference between ConA and RGDS + ConA is not statistically
significant (0.1 < p < 0.2).
Accumulation of PtdIns(3,4)P
The lectin ConA is a
tetrameric ligand able to induce clustering of its membrane
receptor
(33) . We investigated the role of platelet
glycoproteins clustering in ConA-induced synthesis of
PtdIns(3,4)P in
ConA-treated Platelets Was Triggered by Clustering of Membrane
Glycoproteins Different from GP IIb-IIIa
by using a dimeric derivative of the lectin,
the succinyl-ConA (S-ConA). S-ConA maintains the same glycoprotein
specificity as the native lectin and can recognize the same components
on the platelet surface
(36) . However, unlike ConA, S-ConA does
not induce clustering of its receptor
(37) . Fig. 4shows
that S-ConA, unlike the tetrameric ConA, did not induce accumulation of
PtdIns(3,4)P
in human platelets and did not behave as a
platelet agonist. This indicates that the clustering of the ConA
receptor by the native lectin rather than the mere binding to the
membrane glycoproteins can activate the signaling pathways leading to
PtdIns(3,4)P
accumulation.
Figure 4:
Succinyl-ConA did not induce synthesis of
PtdIns(3,4)P.
P-Labeled platelets were
incubated with buffer (Resting), with 100 µg/ml ConA
(ConA) or 100 µg/ml S-ConA at 37 °C for 5 min with constant
stirring. Typical HPLC profiles, representative of several experiments,
are reported. Abbreviations are as in Fig.
1.
As GP IIb-IIIa is the main
platelet receptor for ConA, which becomes clustered upon binding of the
lectin
(33) , we analyzed the ConA-induced accumulation of
PtdIns(3,4)P in platelets from two patients affected by
type I Glanzmann thrombasthenia. Platelets from both patients did not
possess immunologically detectable GP IIb-IIIa (data not shown).
shows that the amount of PtdIns(3,4)P
observed
in ConA-treated thrombasthenic platelets was only slightly reduced when
compared to that observed in ConA-treated normal platelets. We also
tested the effect of thrombin on platelets from one thrombasthenic
patient. As previously described (13), we confirmed that synthesis of
PtdIns(3,4)P
in thrombin-stimulated thrombasthenic
platelets was greatly reduced when compared with normal platelets
().
Protein-tyrosine Phosphorylation Did Not Mediate
ConA-induced Synthesis of
PtdIns(3,4)P
It has been previously
shown that ConA induces tyrosine phosphorylation of several proteins in
human platelets
(29) . Since a proposed mechanism of PtdIns
3-kinase activation involves the tyrosine phosphorylation of the
regulatory subunit p85
(4) , we analyzed whether the regulatory
subunit of the PtdIns 3-kinase, p85, was tyrosine phosphorylated in
ConA-stimulated platelets. Lysates from resting and ConA-treated
platelets were immunoprecipitated either with anti-p85 antiserum or
with a preimmune serum. Immunoprecipitated proteins were separated by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and probed with anti-phosphotyrosine antibodies. As shown in
Fig. 5
, no tyrosine-phosphorylated proteins with molecular mass
corresponding to the regulatory subunit of the PtdIns 3-kinase were
detected in the anti-p85 immunoprecipitates. In contrast, p85 was
clearly detected in immunoprecipitates obtained with
anti-phosphotyrosine antibodies from ConA-treated platelets but not
from resting platelets (Fig. 5). This indicates that ConA induces
the association of nonphosphorylated p85 with tyrosine-phosphorylated
proteins. A similar behavior was also reported in thrombin-stimulated
platelets
(8) .
Figure 5:
ConA-induced association of p85 with
tyrosine-phosphorylated proteins. Lysates from resting (basal
(Bas)) or ConA-stimulated platelets (100 µg/ml ConA, 5 min
at 37 °C) were immunoprecipitated with anti-p85 polyclonal
antiserum, anti-phosphotyrosine antibodies, or preimmune serum as
indicated under ``Experimental Procedures.''
Immunoprecipitated proteins and a sample of total platelet lysate
(PLT) were transferred to nitrocellulose and probed with
anti-phosphotyrosine antibodies (A) or anti-p85 polyclonal
antiserum (B) as indicated. Molecular mass markers are
indicated on the left. The position of p85 is indicated on the
right.
The role of protein-tyrosine phosphorylation
events in the synthesis of PtdIns(3,4)P induced by ConA was
further investigated using the specific tyrosine kinase inhibitor
tyrphostin AG-213. This compound was known to inhibit both aggregation
and PtdIns(3,4)P
synthesis in thrombin-stimulated platelets
(8). We found that, when added to tyrphostin-treated platelets, ConA
was able to induce accumulation of PtdIns(3,4)P
similar to
that observed in control platelets (Fig. 6). On the other hand,
we confirmed that in our experimental condition tyrphostin was able to
strongly decrease both aggregation and accumulation of
PtdIns(3,4)P
in thrombin-stimulated platelets (data not
shown).
Figure 6:
Effect of tyrphostin AG-213 on the
production of PtdIns(3,4)P in ConA-stimulated platelets.
P-Labeled platelets were incubated for 2 min at 37 °C
with 100 µM tyrphostin AG-213 or an equal volume of
dimethyl sulfoxide, and then buffer or ConA (100 µg/ml, final
concentration) was added. After 5 min, lipids were extracted and
deacylated, and the production of PtdIns(3,4)P
was
determined by HPLC analysis. Data are expressed as percentage of the
PtdIns(3,4)P
production in ConA-stimulated
platelets.
in human platelets stimulated with the lectin
ConA does not require platelet aggregation. Prevention of aggregation
by the omission of stirring or by preincubation of platelets with the
tetrapeptide RGDS did not impair the ability of ConA to stimulate the
accumulation of PtdIns(3,4)P
. These evidences are
particularly interesting when compared with the results obtained using
thrombin as platelet agonist. Previous works demonstrated that
inhibition of fibrinogen binding by RGD-containing peptides resulted in
an inhibition of PtdIns(3,4)P
synthesis induced by
thrombin
(12, 13) , and a strong reduction of
accumulation of PtdIns(3,4)P
was observed in platelets from
patients affected by Glanzmann thrombasthenia, which lack the
fibrinogen receptor and fail to aggregate
(13) . This evidence
supported a role for GP IIb-IIIa in regulating the activation of PtdIns
3-kinase in human platelets. It should be noted, however, that the use
of RGD-containing peptides or thrombasthenic platelets actually
interferes with two closely related but clearly different events
occurring in activated cells: fibrinogen binding to GP IIb-IIIa and
platelet aggregation. Both events require a functional GP IIb-IIIa, as
binding of fibrinogen is strictly necessary to start aggregation.
However, while fibrinogen binding to GP IIb-IIIa fails to produce
aggregation if platelets are not stirred, when stimulated platelets are
simultaneously stirred, aggregation ensues, and secondary events on the
cell surface occur: the GP IIb-IIIa is clustered
(23) , several
glycoproteins, including the GP IIb-IIIa itself and the PECAM-1,
interact with the intracellular cytoskeleton
(26, 27) ,
and other adhesive proteins, such as released thrombospondin, bind to
their receptors on the cell surface to stabilize platelet
aggregates
(25, 26) . As inhibition of fibrinogen binding
necessarily results in the prevention of platelet aggregation and
related events, the available data actually do not clarify if the
synthesis of PtdIns(3,4)P
is directly regulated by GP
IIb-IIIa or by other events occurring during platelet aggregation. In
this paper, we show that stimulation of platelets with thrombin in the
absence of stirring, in such a way to allow fibrinogen binding to GP
IIb-IIIa, but not aggregation resulted in a marked decrease of
PtdIns(3,4)P
synthesis, which is similar to the decrease
previously described in thrombin-stimulated, RGDS-treated platelets
(12, 13). This indicates that the regulatory role of GP IIb-IIIa on
PtdIns(3,4)P
production is mainly played through platelet
aggregation. When ConA was used as platelet agonist, we observed
accumulation of PtdIns(3,4)P
independent of both fibrinogen
binding and platelet aggregation (i.e. in the absence of
stirring and in the presence of the peptide RGDS). Therefore, our
results identify an excellent simplified experimental system to study
the mechanisms regulating the PtdIns 3-kinase in human platelets.
in ConA-stimulated platelets is
that, unlike other lectins, ConA reproduces in nonaggregated platelet
events that usually occur only during aggregation when physiological
agonists are used. For instance, ConA induces clustering of membrane
glycoproteins including GP IIb-IIIa
(33) , which is normally
promoted by fibrinogen during aggregation
(23) . Moreover, ConA
promotes the association of the fibrinogen receptor with the
intracellular cytoskeleton
(30) , which, in thrombin-stimulated
platelets, is normally a consequence of aggregation
(26) . Here,
we demonstrate that the clustering of membrane glycoproteins is a
crucial event in ConA-induced synthesis of PtdIns(3,4)P
. In
fact, the dimeric derivative of the lectin, the succinyl-ConA, which
binds to the same surface components as the native lectin but does not
induce clustering of the receptors, is unable to induce the synthesis
of PtdIns(3,4)P
in human platelets. Although it cannot be
ruled out that ConA and thrombin activate the PtdIns 3-kinase through
different signaling pathways, these results may indicate that also in
thrombin-aggregated platelets the signal leading to accumulation of
PtdIns(3,4)P
is generated by the fibrinogen-promoted
clustering of membrane glycoproteins.
in thrombasthenic
platelets
(13) . Here, we confirm this observation. However, when
we compared the production of PtdIns(3,4)P
in ConA-treated
thrombasthenic platelets with that of ConA-treated normal platelets, we
only observed a slight reduction, which was not comparable with the
inhibition observed when thrombin was used as agonist. We conclude that
the reduced synthesis of PtdIns(3,4)P
in thrombin-treated
thrombasthenic platelets was related not to the absence of GP IIb-IIIa
but to the inability of these platelets to aggregate. The different
behavior of ConA indicates that, when distinguished from platelet
aggregation, GP IIb-IIIa is not an essential regulatory factor for
PtdIns 3-kinase activation. It is thus clear that the signal
transduction pathway initiated by the lectin ConA and leading to
accumulation of PtdIns(3,4)P
requires clustering of
membrane glycoproteins different from the fibrinogen receptor.
in thrombin-aggregated platelets.
When platelets are activated, fibrinogen binds to GP IIb-IIIa, and in
the presence of stirring, aggregation ensues. During aggregation,
fibrinogen triggers clustering of GP IIb-IIIa. As aggregation proceeds,
other membrane glycoproteins are included in these clusters, maybe
facilitated by the action of other adhesive proteins. In this regard,
it is interesting that a proposed model to explain the role of secreted
thrombospondin in the stabilization of platelet aggregates involved the
physical association between fibrinogen-bound GP IIb-IIIa and its
putative receptor, the glycoprotein IV
(25, 38) . The
formation of these heterogenic clusters finally generates the signal
leading to PtdIns(3,4)P
accumulation. ConA, by binding
directly to the membrane glycoproteins, may act bypassing the initial
steps and directly promoting the signal-generating clusters. As ConA
binds to several major and minor membrane glycoproteins, it is very
difficult to identify the receptor responsible for the activation of
the PtdIns 3-kinase. Moreover, we cannot rule out that the recruitment
of different glycoproteins rather than a single class of molecules into
the cluster is responsible for the activation of the PtdIns 3-kinase.
accumulation is
still unknown. We investigated the possible relationship between
protein-tyrosine phosphorylation and activation of PtdIns 3-kinase.
Although we found that ConA induced the association of the regulatory
subunit of the enzyme with tyrosine-phosphorylated proteins, we did not
observe any tyrosine phosphorylation of p85 under conditions producing
maximal accumulation of PtdIns(3,4)P
. Moreover, we found
that the tyrosine kinase inhibitor tyrphostin did not affect the
production of PtdIns(3,4)P
in ConA-stimulated platelets.
These evidences indicate that tyrosine phosphorylation events are not
essential for PtdIns 3-kinase activation. Inhibition of
PtdIns(3,4)P
synthesis by tyrphostin was described in
thrombin-stimulated platelets
(8) . However, treatment of
platelets with this tyrosine kinase inhibitor also resulted in the
inhibition of platelet aggregation
(8) . Therefore, it was
questionable whether the effect of tyrphostin on PtdIns(3,4)P
production was directly related to the inhibition of tyrosine
kinases or mediated by the inhibition of platelet aggregation. The data
presented in this work using an aggregation-independent model suggest
that the observed inhibition of PtdIns(3,4)P
synthesis by
tyrphostin in thrombin-activated platelets was most likely related to
the inhibition of platelet aggregation. Thus, the signaling pathway
leading to PtdIns(3,4)P
synthesis in human platelets seems
to be different from that operating in nucleated cells stimulated with
growth factors, where an important role is played by the
protein-tyrosine kinases
(4) . In a recent report, activation of
PtdIns 3-kinase by the low molecular weight GTP-binding protein Rho was
demonstrated in human platelets
(11) . Rho is known to play a
role in the cytoskeletal organization
(39) , and it is
interesting that the cytoskeleton of aggregated platelets interacts
with both membrane glycoproteins and activated PtdIns
3-kinase
(26, 40, 41) . The exact role of the
platelet cytoskeleton and associated proteins in the stimulation of the
synthesis of PtdIns(3,4)P
remains to be investigated, but
recent results by Guinebault et al.(7) suggest that
interactions between pp120
and PtdIns 3-kinase in
specific regions of the cytoskeleton may directly activate the PtdIns
3-kinase.
in ConA-stimulated platelets described in this
paper represents an excellent experimental model used to clarify the
role played by GP IIb-IIIa, membrane glycoproteins clustering, and
protein-tyrosine kinases in the stimulation of PtdIns(3,4)P
production in human platelets. This model can be usefully
exploited in future investigations to provide further insights into the
biochemical mechanisms regulating PtdIns(3,4)P
synthesis in
human platelets.
Table:
Synthesis of PtdIns(3,4)P in
thrombasthenic platelets
P and stimulated at 37 °C for 5 min with
1 unit/ml thrombin or 100 µg/ml ConA. Production of
PtdIns(3,4)P
was analyzed by HPLC. Data are expressed as
percentage of the production of PtdIns(3,4)P
observed in
stimulated normal platelets.
, phosphatidylinositol 3`,4`-bisphosphate;
PtdIns(3,4,5)P
, phosphatidylinositol
3`,4`,5`-trisphosphate; ConA, concanavalin A; S-ConA, succinyl-ConA;
GP, glycoprotein; HPLC, high performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.