Stimulation of platelet thrombin receptors
or protein kinase C causes fibrinogen-dependent aggregation
that is a function of integrin
IIb
3
activation. Such platelets rapidly and transiently form
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and a small amount of phosphatidylinositol 3,4-bisphosphate
(PtdIns(3,4)P2). After aggregation, a larger amount of
PtdIns(3,4)P2 is generated. We report that this latter
PtdIns(3,4)P2 arises largely through wortmannin-inhibitable
generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase
(PtdIns3P 4-K), a novel pathway apparently contingent upon the
activation of the Ca2+-dependent protease
calpain. Elevation of cytosolic Ca2+ by ionophore, without
integrin/ligand binding, is insufficient to activate the pathway.
PtdIns3P 4-K is not the recently described "PIP5KII
."
Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P
4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can
be regulated negatively by the 
subunit of heterotrimeric
GTP-binding protein. After aggregation, PtdIns3P 4-K
calpain-dependently loses its susceptibility to G
and is, in addition, activated. Both PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 have been shown to stimulate PKB
/Akt
phosphorylation and activation by
phosphoinositide-dependent kinase 1. We find that
activation of PKB
/Akt in platelets is
phosphorylation-dependent and biphasic; the initial phase
is PtdIns(3,4,5)P3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P2
generated after aggregation. There is thus potential for both pre- and
post-aggregation-dependent signaling by PKB
/Akt.
 |
INTRODUCTION |
The activation of platelets via the thrombin receptor
(THR-R)1 has been found to cause the rapid
(within 60 s) stimulation of two phosphoinositide 3-kinases:
PI3K
and p85/PI3K (1). Each of these enzyme activities results
in the generation of PtdIns(3,4,5)P3 (PtdInsP3)
by phosphorylation of PtdIns(4,5)P2; shortly thereafter, a
small amount of PtdIns(3,4)P2 is produced, either by
hydrolysis of PtdInsP3 or phosphorylation of PtdIns4P (2).
After 5-10 min, however, a large increase in PtdIns(3,4)P2
can be observed with THR-R agonists, as described below. Both
PtdInsP3 and PtdIns(3,4)P2 have the
potential to act as second messengers, stimulating in vitro
the activities of some protein kinase C species (3-6), the
protooncogene product PKB/Akt via PDK1 (7-9), and, in the case of
PtdIns(3,4)P2, direct activation of PKB/Akt (10-12).
Hence, elucidation of the routes by which these second messengers are synthesized and metabolized has potentially important implications for
cell signaling.
PI3K
is stimulated by G
, whereas activation of p85/PI3K
appears to be down-stream of a protein kinase C (stimulated after THR-R-dependent activation of phospholipase C). Activation
of p85/PI3K, but not of PI3K
, can be achieved by the addition to platelets of protein kinase C-activating PMA (13). Apparently, it is
p85/PI3K, rather than PI3K
, whose activity contributes to the
re-organization of integrin
IIb
3 (13),
allowing it to bind fibrinogen (FIB), which thereby facilitates
platelet aggregation and subsequent signaling events. After platelets
aggregate, the substantial burst of PtdIns(3,4)P2
accumulation that occurs is prevented by the integrin antagonist RGDS,
removal of Ca2+, or the absence of integrin
IIb
3 (14, 15). This increase has been
found to be blocked by cell-permeable inhibitors of the Ca2+-dependent protease, calpain (16-18), an
enzyme that is activated when platelets aggregate in an integrin
IIb
3-dependent manner (19).
Since one of several proteins cleaved by calpain is
PtdIns(3,4)P2 4-phosphatase, leading to decreased activity,
it had been suggested that inhibition of 4-phosphatase is responsible
for the aggregation/calpain-dependent rise in
PtdIns(3,4)P2 (17). Our recent data, however, point to
another explanation (18). Integrin
IIb
3
can be activated directly by LIBS (antibody Fab fragment to
3; Ref. 20), an event that causes
FIB-dependent aggregation and PtdIns(3,4)P2 accumulation (21). We have found that LIBS+FIB-induced accumulation of
PtdIns(3,4)P2, which is inhibited by calpain inhibitors
such as calpeptin, cannot be accounted for by inhibition of
4-phosphatase (18). Instead, a new synthetic route is triggered, in
which no PtdInsP3 is formed, but PtdIns3P is generated
transiently by PtdIns 3-K in a calpeptin- and wortmannin-sensitive
manner, and PtdIns (3,4)P2 arises through
phosphorylation of PtdIns3P by Ptd Ins3P 4-K (18). The
PtdIns(3,4)P2 that is formed after aggregation is generated
primarily by this pathway and the increase in the activity of PtdIns3P
4-K is also inhibited by calpeptin. Moreover, the
PtdInsP3-independent generation of
PtdIns(3,4)P2 by this route leads to PKB/Akt
activation in vivo. The purposes of the present study are to
determine whether the new pathway is triggered in PMA+FIB and
THR-R+FIB-activated platelets, whether PKB/Akt is activated only by
PtdIns(3,4)P2 or by both PtdInsP3 and
PtdIns(3,4)P2 in these cells, whether such activation(s)
is/are dependent upon PKB/Akt phosphorylation, which PKB isoforms are
stimulated, and to begin to address how PtdIns3P 4-K is regulated.
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MATERIALS AND METHODS |
All reagents and preparations of platelets were as described
(18). Synthetic phosphoinositides were also purchased from Echelon
Research Laboratories (Salt Lake City, UT). Antibodies to PKB (
,
, and
isoforms) and PDK1 were generously contributed by Dr.
Dario Alessi (University of Dundee, Dundee, United Kingdom). Drs. Kath
Hinchliffe and Robin Irvine kindly provided an affinity-purified antibody to "PIP5KII
" that is immunoprecipitating and recognizes recombinant type II
PIP kinase on Western blots. G
subunits of
heterotrimeric GTP-binding proteins were isolated from bovine brain, as
reported (22), and kindly provided by Dr. Xiu-wen Tang (University of
Dundee). Protein phosphatase PP1
(human recombinant) and phosphatase
inhibitor microcystin-LR were the generous gifts of Drs. P. T. W. Cohen and C. MacKintosh (University of Dundee).
ARK-PH
was expressed and purified as described (1).
Metabolic Studies with 32P-Labeled
Platelets--
For studies with platelets labeled to equilibrium,
washed and aspirin-treated platelets were prepared and labeled as
described (18, 23), and incubated (2 × 109/ml) in the
presence of Ca2+ and apyrase (18), with or without SFLLRN
(25 µM) in the presence of RGDS (400 µM) or
FIB (400 µg/ml), while stirring at 37 °C for up to 12 min. In
other experiments,
PMA (200 nM) with or without FIB or
Ca2+ ionophore A23187 (2 µM) with RGDS was
substituted for SFLLRN. For some 2-min and 10-min incubations, 20 nM wortmannin , 0-300 µM calpeptin, or
0-400 µg/ml FIB was included in incubations. All incubations were
terminated with CHCl3/MeOH/HCl, and lipids extracted,
resolved, and quantitated as described in Ref. 18.
For non-equilibrium labeling, platelets were exposed to
[32P]Pi for 2 min with or without FIB prior
to the addition of SFLLRN or PMA for 7 min, while stirring (20). When
FIB was omitted, RGDS was included to block effects of secreted FIB
caused by SFLLRN. Incubations were terminated as above, and lipids were
extracted and incorporation of 32P into phosphoinositides
assessed. [32P]PtdIns(3,4)P2 was digested for
positional analysis of label as described (2, 18).
Platelet Fractions--
Triton X-100-insoluble CSK (1, 24) was
isolated from platelets treated with SFLLRN+FIB or PMA+FIB
(±calpeptin) after various periods of stirring. EGTA (20 mM) and calpeptin were included in Triton buffer for lysis.
Washed CSK (50 µg) was incubated with 1 mg/ml mixtures (23) of
diC16PtdIns3P, PtdIns, PtdIns4P, diC16PtdIns4P, PtdIns(4,5)P2, or
diC16PtdIns(3,4)P2, and phosphatidylserine, for
assays of lipid kinase activity (13, 18). In some cases,
ARK-PH (5 µM; Ref. 1) or G
(0.5 µM) was
included in assays. PKB was immunoprecipitated from Triton-soluble
fractions using antibodies to
,
, or
isoforms, and its kinase
activity was assayed with "Crosstide" peptide as described (18). In
some experiments, PKB immunoprecipitates were incubated for 30 min at
room temperature in Pi-free PKB assay buffer ± 50 milliunits/ml PP1
phosphatase (which does not hydrolyze
PtdIns(3,4)P2 or PtdInsP3), followed by excess
(2 µM) phosphatase inhibitor microcystin-LR, before assay
of PKB activities. The presence of PKB and PDK1 in CSK and
Triton-soluble fractions was also determined by Western blot (18).
Cytosol was prepared from unstimulated platelets (1). An antibody to
PIP5KII
(25-28) was used for immunoprecipitations from cytosol.
After immunoprecipitation or after "mock" immunoprecipitation (resulting from incubation with antibody buffer and protein
G-Sepharose), supernatants were assayed as above for PtdInsP kinase
activities, and in some cases, synthetic
diC16PtdIns5P or diC16PtdIns4P was used as
a substrate. For separations of glycerophospho-Ins(3,5)P2 (derived from PtdIns(3,5)P2) and
glycerophospho-Ins(3,4)P2 (derived from
PtdIns(3,4)P2), HPLC fractions were collected every
10 s and monitored by scintillation spectrophotometry,
rather than by Flo-One Beta detection (23).
 |
RESULTS |
Generation of Labeled 3-Phosphorylated Phosphoinositides in Intact
Platelets--
As shown in Figs. 1 and
2, SFLLRN and PMA were each able to
induce transient accumulations of labeled PtdInsP3,
PtdIns3P, and sustained accumulations of PtdIns(3,4)P2 in
32P-labeled platelets. Only the increases in PtdIns3P and
PtdIns(3,4)P2, however, were dependent upon the presence of
FIB (Figs. 1 (B and C) and 2 (B and
C)), the former totally dependent, and the latter largely
FIB-dependent. The effects of FIB were linear up to 200 µg/ml (data not shown). These effects were very similar to those resulting from exposure of platelets to LIBS (which directly activates
IIb
3)+FIB, under aggregating conditions
(18). That we were indeed measuring changes in
PtdIns(3,4)P2, and not in recently described
PtdIns(3,5)P2 (29, 30), is illustrated in Fig.
3, which shows the results for a mixture
of platelet PtdInsP2s that had been deacylated, resolved by
HPLC, and counted. Separation to base line was achieved, and we
observed that PtdIns(3,5)P2 contained a very minor fraction
of the amount of 32P seen for PtdIns(3,4)P2.
After exposure of platelets to PMA±FIB for 10 min, 32P in
PtdIns(3,5)P2 increased 2-3-fold with FIB
versus FIB-free controls, whereas
[32P]PtdIns(3,4)P2 increased over 20-fold
(Fig. 3). The addition of RGDS with PMA had no effect in comparison
with PMA alone. Accumulations of PtdInsP3, which do not
occur with LIBS+FIB (18), were rapid and unaffected by FIB (Figs.
1A and 2A), nor were they altered by inclusion of
RGDS (13). All increases in 3-phosphorylated phosphoinositides were
inhibited more than 90% by wortmannin (data not shown). After
platelets were incubated with Ca2+ ionophore with stirring,
but aggregation was prevented by omitting FIB and including RGDS to
prevent binding of secreted FIB, a rapid increase in
PtdInsP3 and a similarly rapid, but modest, increase in
PtdIns(3,4)P2 levels occurred (Fig.
4). There was no increase, however, in
PtdIns3P.

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Fig. 1.
Effects with time of SFLLRN±FIB on
accumulations of 3-phosphorylated phosphoinositides in platelets.
Platelets, labeled to equilibrium with 32P, were incubated
with SFLLRN+RGDS (open symbols) or SFLLRN+FIB (filled
symbols) with stirring for various periods. Lipids were extracted,
digested, and resolved by HPLC with in-line isotopic detection.
Data are shown for radiolabeled PtdInsP3
(A), PtdIns3P (B), and PtdIns(3,4)P2
(C) and are representative of two experiments.
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Fig. 2.
Effects with time of PMA±FIB on
accumulations of 3-phosphorylated phosphoinositides in platelets.
32P-Labeled platelets, as in Fig. 1, were incubated
for various periods with PMA (open symbols) or PMA+FIB
(filled symbols). PtdInsP3 (A),
PtdIns3P (B), and PtdIns(3,4)P2
(C).
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Fig. 3.
Resolution by HPLC of PtdInsP2
species from platelets. 32P-Labeled platelets were
incubated for 10 min as in Fig. 2 with PMA (open symbols) or
PMA+FIB (filled symbols). PtdInsP2 was digested
and resolved on HPLC, and fractions were collected every 10 s and
counted. The fraction designated PtdIns(3,5)P2 had the same
retention time (consistent with those published; Ref. 28) as
phosphoinositide products derived from in vitro incubations
of cytosol with PtdIns3P and PtdIns5P (contributions of
PtdIns(3,4)P2 and PtdIns(4,5)P2
excluded).
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Fig. 4.
Effects with time of cytosolic
Ca2+ elevation on accumulations of 3-phosphorylated
phosphoinositides in platelets. As in Fig. 1,
32P-labeled platelets were incubated with A23187+RGDS (RGDS
alone had no effects) for various periods. Filled circles,
PtdInsP3; open circles,
PtdIns(3,4)P2; filled squares, PtdIns3P.
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When the relative specific activities of 32P at the 1-, 3-, and 4-positions of the inositol ring of PtdIns(3,4)P2 were
analyzed after non-equilibrium labeling of platelets and exposure of
stirred platelets to either SFLLRN±FIB or PMA±FIB for 7 min, it was
found that the presence of FIB affected the relative positional
labeling (Fig. 5). As was true for
LIBS+FIB-stimulated platelets, position 4 was hotter than position 3, and labeling of position 1 was negligible after platelets were
aggregated in response to either SFLLRN or PMA. This indicated that
32P was added to position 4 after 3, i.e. that
the predominant synthetic pathway for PtdIns(3,4)P2 is
PtdIns3P
PtdIns(3,4)P2. In contrast, as is true for
platelets exposed briefly (20 s or 60 s) to thrombin or SFLLRN (2,
18), platelets incubated for 7 min with PMA or SFLLRN+RGDS in the
absence of FIB, and therefore without aggregation, contained
"hotter" position 3 than 4 in PtdIns(3,4)P2. Under
these conditions, the predominant pathway for PtdIns(3,4)P2
synthesis could be either PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns(3,4)P2 or PtdIns4P
PtdIns(3,4)P2.

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Fig. 5.
Distribution of 32P on the
inositol ring of PtdIns(3,4)P2. Non-equilibrium
32P-labeled, stirred platelets were exposed to PMA or
SFLLRN for 7 min under conditions which permitted (filled
bars) or prevented (open bars) binding of FIB. The
amount of 32P at each position of the inositol ring was
analyzed and shown as a percent of the total. Data are the means ± S.D. of two or three experiments.
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Activation of PKB/Akt in Stimulated
Platelets--
Immunoprecipitations with antibodies specific for
PKB
, -
, or -
revealed that only PKB
had significant
activity, and only this activity was stimulated when platelets were
activated. PKB
and PDK1 were found to be present in both
Triton-soluble and CSK fractions, as detected by Western blotting;
however, when the same amount of CSK protein from resting and activated
platelets was compared, detectable PKB
and PDK1 did not increase.
Total CSK protein did increase, however, in activated platelets, rising 2-3-fold; therefore, the amounts of PKB
and PDK1 increased as CSK-associated proteins. The majority of PKB
and PDK1 was present in
the Triton-soluble fraction of platelets (data not shown).
In contrast to the case for LIBS+FIB (18), in which PKB
activity
increased (3-fold) rather late (maximally at 10-12 min), following
platelet aggregation and paralleling PtdIns(3,4)P2 levels (20-30-fold), PKB
activity in response to SFLLRN±FIB increased much more rapidly, and to a greater degree (8-9-fold), following the
increases in PtdInsP3 (8-fold) after a 15-s lag (Fig.
6). This was totally inhibited by
wortmannin (data not shown). After 10 min, however, when
PtdInsP3 had decreased to control (agonist-free) levels,
PKB
activity had also returned to base-line levels, except where FIB
was present (Fig. 7A). The
presence of FIB had no effect on accumulations of PtdInsP3
(shown as well in Fig. 1A), including the return to
base-line levels, but had a marked enhancing effect on
PtdIns(3,4)P2 accumulations (26-fold; Figs. 1C
and Fig. 7A) and PKB
activity (3-fold; Fig.
7A). Calpeptin (IC50 1 µM) did not
inhibit increases in PtdInsP3 or early activation of PKB
(Fig. 7A; 2 min), but totally inhibited the rises in
PtdIns(3,4)P2 and PKB
activity associated with later
incubations in the presence of FIB (Fig. 7A; 10 min). The
same phenomena were observed when PMA was used as an agonist, in place
of SFLLRN (Fig. 7B). "Second phase" stimulations of
PKB
and accumulations of PtdIns(3,4)P2 in response to
either SFLLRN+FIB or PMA+FIB were thus similar to the increases
observed with LIBS+FIB (18).

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Fig. 6.
Initial formation of PtdInsP3 and
activation of PKB in platelets exposed to SFLLRN. As in Fig. 1,
32P-labeled platelets, or non-labeled platelets, were
incubated with SFLLRN for short periods. Incubations were terminated
with organic solvents (32P-labeled platelets) or ice-cold
Triton lysis buffer (unlabeled platelets). Lipids were resolved and
[32P]PtdInsP3 (filled circles)
quantitated, or PKB was immunoprecipitated from Triton-soluble
fractions, and its activity assayed (open circles). There
was negligible production of PtdIns(3,4)P2 within the first
30 s.
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Fig. 7.
Effects of FIB binding or calpeptin exposure
on platelet production of PtdInsP3,
PtdIns(3,4)P2, and PKB activation in response to
agonists. As in Fig. 6, formation of
[32P]PtdIns(3,4)P2 and
[32P]PtdInsP3 were quantitated after
platelets were exposed to SFLLRN (A) or PMA (B).
Two incubation periods were examined and conditions varied with respect
to the presence of FIB, RGDS, and prior exposure of platelets to
calpeptin. The data with calpeptin are for its maximum effective
concentration. The IC50 for calpeptin was 1 µM. Calpeptin, FIB, and RGDS had no effect on basal
activity of PKB or radiolabeling of phosphoinositides. Data shown
are presented as a multiple of agonist-free (basal) values and are
representative of three or four experiments.
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In order to determine whether phosphorylation of PKB
, presumably by
PDK1, was required for its early stage and late stage activations,
immunoprecipitated PKB
from platelets incubated with or without
SFLLRN (2 min) or with LIBS+FIB (stirring, 10 min) were incubated with
or without protein phosphatase and then phosphatase inhibitor prior to
assay of PKB
activity. We observed (Table
I) that incubation with phosphatase
inhibited SFLLRN-activated PKB
by 100% and LIBS+FIB-activated
PKB
by 95%.
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Table I
Inhibition of activated PKB by dephosphorylation
PKB was immunoprecipitated after incubations of platelets with
buffer or SFLLRN+RGDS or LIBS+FIB+stirring. Immunoprecipitates were
incubated with or without protein phosphatase; incubations were
terminated with excess phosphatase inhibitor, and PKB activities
were assayed. Background dpm (assays without PKB immunoprecipitates)
are subtracted from all results.
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Regulation of CSK-associated PtdIns3P 4-K and PtdIns4P 3-K from
Activated Platelets--
Exposure of platelets to SFLLRN+FIB led to an
initial (within 3 min) inhibition of CSK-associated PtdIns3P 4-K
activity, followed (5-10 min) by increased activity (Fig.
8A). The initial inhibition was overcome by the addition to the assay incubation mixture of
ARK-PH, which binds G
, known to be present in CSK of
SFLLRN/THR-activated platelets (1). Incubation of platelets with
calpeptin had no effect on the early PtdIns3P 4-K activities in CSK,
whether or not
ARK-PH was added to assay mixtures. At late times,
however, the stimulated PtdIns3P 4-K activity could not be increased
further by
ARK-PH, but it was blocked by exposure of platelets to
calpeptin prior to SFLLRN+FIB. That G
can indeed inhibit PtdIns3P
4-K activity at early time points, and this is the likeliest
explanation for the effects of
ARK-PH shown in Fig. 8A,
is illustrated in Fig. 9A.
There, G
added to assay mixtures is shown to have inhibited PtdIns3P 4-K in CSK of resting platelets or of platelets exposed for 2 min to PMA+FIB, whereas
ARK-PH was without effect (PMA does not
cause liberation of G
to platelet CSK; Ref. 13). After 10 min,
however, PtdIns3P 4-K lost its sensitivity to G
. Prior treatment
of platelets with calpeptin, however, allowed PtdIns3P 4-K to remain
susceptible to inhibition by G
, even after 10 min. It is known
that CSK of SFLLRN/THR-activated platelets contains increased
activities of both p85/PI3K and PI3K
, the latter due to G
, and
that this G
-dependent activation is blocked by
ARK-PH added to isolated CSK (1). We have confirmed this in Fig.
8B, for 0-3 min. Early activation of PtdIns4P 3-K (which utilized either commercial PtdIns4P or synthetic
diC16PtdIns4P with similar results) was inhibited about
50% by
ARK-PH, but not by calpeptin treatment of platelets. The
sensitivity to
ARK-PH also decreased with time, but this was not
improved by calpeptin treatment. Calpeptin was inhibitory at late time
points, and the combination of
ARK-PH + calpeptin was more
inhibitory than either alone, implying the presence of both a
calpain-activated PtdIns4P 3-K and some remaining G
-sensitive
PtdIns4P 3-K in CSK of "late stage" SFLLRN-activated platelets. In
Fig. 9B, the results for PtdIns4P 3-K activity in CSK of
platelets activated for 10 min with PMA+FIB (where released G
is
not a significant factor, and thus
ARK-PH has no effect) show that
calpeptin treatment was inhibitory here, as well. In contrast,
activating effects of G
were preserved by calpeptin treatment.
Thus, there appears to be a calpain-promoted PtdIns4P 3-K activity in
the CSK of both SFLLRN- and PMA-stimulated platelets, as well as
stimulated PtdIns3P 4-K activity. Regulation by G
of both
PtdIns4P 3-K (activating) and PtdIns3P 4-K (inhibiting) activities,
however, appears to be decreased by calpain action.

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Fig. 8.
Effects of calpeptin and ARK-PH on
CSK-associated phosphoinositide kinase activities of SFLLRN-activated
platelets. Unlabeled platelets were incubated as in Fig. 7, were
incubated with (open or filled diamonds) or
without calpeptin (circles) prior to exposure to buffer or
SFLLRN+FIB for varied periods. Incubations were terminated with
ice-cold Triton lysis buffer. Washed, Triton-insoluble CSKs at the same
concentration were incubated with kinase assay buffer, using PtdIns3P
(for 4-K; A) or PtdIns4P (for 3-K; B) substrates
in the presence (open circles, open diamonds) or
absence (filled circles, filled diamonds) of
ARK-PH. [32P]PtdIns(3,4)P2 was
quantitated, and the results expressed as a multiple of control
(unstimulated platelet) values. Data are representative of two
incubations, in duplicate.
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Fig. 9.
Effects of calpeptin, G , and ARK-PH
on CSK-associated phosphoinositide kinase activities of PMA-activated
platelets. Platelets were incubated as in Fig. 8, substituting PMA
for SFLLRN. In some cases, G was added to kinase assay mixtures.
Substrates were PtdIns3P (for 4-K; A) and PtdIns4P (for 3-K;
B). calp, calpeptin; ARK,
ARK-PH. Results are expressed as a multiple of control (no agonist
or inhibitors) values as means ± S.D. for two or three
experiments.
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Depletion of PtdInsP Kinase Activities with Antibody to
PIP5KII
--
Most PtdIns3P 4-K activity in unstimulated platelets
was cytosolic, and inhibitable by G
, as above, but unaffected by
GTP
S (data not shown). In order to gain additional information about the identity of this platelet PtdIns3P 4-K, immunodepletion experiments were performed. Immunodepletions of platelet cytosol, using an antibody
to PIP5KII
(which has been cloned and expressed; Refs. 25 and 26),
led to several findings (Fig. 10). 1)
When PtdIns4P (either synthetic or natural) was used as a substrate,
PtdIns 4P 5-K (i.e. "type I" activity), yielding
PtdIns(4,5)P2, was depleted by 80%, indicating
cross-reactivity of the antibody with type I 5-K. 2) When PtdIns5P was
used, PtdIns5P 4-K (erroneously, according to recent literature,
referred to as type II "5-K"; Ref. 28), which also yielded
PtdIns(4,5)P2, was depleted by 65%. 3) No significant depletion was seen for 3-K activities, i.e. PtdIns4P 3-K or
PtdIns5P 3-K. 4) PtdIns3P 5-K activity was depleted 50%. 5) Very
importantly, no significant depletion was seen for platelet
PtdIns3P 4-K activity (and no PtdInsP3 was formed in these
assays). The finding for PtdIns3P 4-K was rather surprising, in that it
has been reported (28) that type II "5-K" (actually PtdIns5P 4-K)
also acts on PtdIns3P, albeit less efficiently, forming
PtdIns(3,4)P2. Platelet PtdIns3P 4-K, by these
immuno-criteria, therefore, qualifies as neither type I nor type II
"5-K."

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Fig. 10.
Effect of depletion with PIP5KII antibody
on platelet cytosolic PtdInsP kinase activities. Platelet
cytosolic fractions were incubated with or without antibody and then
protein G-Sepharose. After immunoprecipitation (or after mock
immunoprecipitation), supernatants were assayed for kinase activity
with synthetic or natural PtdIns4P, synthetic PtdIns3P, and synthetic
PtdIns5P substrate, in the linear range of the time course, and the
PtdInsP2 products resolved, after digestion, by HPLC.
Glycerophospho-Ins(3,5)P2 was separated from
glycerophospho-Ins(3,4)P2 as in Fig. 3. Results are the
means ± S.D. of two to four experiments, in duplicate.
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DISCUSSION |
We have demonstrated (Figs. 6 and 7 (A and
B)) that stimulation of platelets by either SFLLRN or PMA,
in the absence of FIB, leads to monophasic activation of PKB
/Akt due
to the generation of PtdInsP3 (see also Figs. 1A
and 2A). This is the first indication, to our knowledge, of
PtdInsP3-dependent activation in
vivo of PKB
/Akt in the absence of a stimulated increase in
PtdIns(3,4)P2. When FIB (which binds to activated integrin
IIb
3) is present, however, a second phase
of PKB
activation occurs. This phase is dependent upon the
accumulation of PtdIns(3,4)P2 (see also Figs. 1C
and 2C), consistent with findings that we have obtained in
studies in which
IIb
3 was activated
directly by LIBS (18). The
PtdIns(3,4)P2-dependent activation of PKB
can occur in the absence of PtdInsP3 (Fig. 7, A
and B; Ref. 18), and is not due to potentiation by
PtdIns(3,4)P2 of residual effects of PtdInsP3 that had accumulated earlier since, in LIBS+FIB-activated platelets, no
generation of PtdInsP3 occurs (18). It appears that
PtdInsP3 is more potent than is PtdIns(3,4)P2
as an activator of PKB
, which might be predicted from studies (8, 9)
that have demonstrated that PtdInsP3 regulates PDK1 more
efficiently than does PtdIns(3,4)P2. Since PDK1 is present
in platelets, and is required (via its phosphoinositide-stimulated phosphorylation of PKB
) as the intermediate stage in the activation of PKB
by PtdInsP3, this is the likely route by which
PKB
is regulated in platelets. Indeed, the exposure of
immunoprecipitated, activated PKB
to protein phosphatase prior to
assay of activity was found to decrease early
(PtdInsP3-dependent) or late
(PtdIns(3,4)P2dependent) stage PKB
activities by
95-100%, whereas basal PKB
activity was minimally affected (Table
I). Although this argues in favor of regulation of PKB
by
phosphorylation, presumably by PDK1, it cannot rule out additional
direct regulation of PKB
by PtdIns(3,4)P2 (10-12)
in vivo, which association might not have been preserved after immunoprecipitation of PKB
from Triton lysates. Nonetheless, the early and late phase activations of PKB
, the latter dependent on
a post-aggregatory event, point to possible roles for PKB
in both
pre- and post-aggregatory signaling.
Our data (Figs. 1B, 2B, and 5) indicate that the
majority of post-aggregatory PtdIns(3,4)P2 is synthesized
by a route that involves the generation of PtdIns3P, and its
phosphorylation by PtdIns3P 4-K. In the absence of FIB, however,
PtdIns(3,4)P2 is formed primarily from the hydrolysis of
PtdIns(3,4,5)P3 or by PtdIns4P 3-K. In the presence of FIB,
the large accumulation of PtdIns(3,4)P2 is dependent upon
the activation of
IIb
3, the binding of
FIB to this integrin (as indicated above), aggregation (18), and the
activation of the Ca2+-dependent protease,
calpain (Fig. 7; Ref. 18). The binding of FIB to integrin
IIb
3 has been reported to cause an influx of extracellular Ca2+ (31, 32), but in the absence of FIB
binding to integrin, the elevation of cytosolic Ca2+ is
insufficient to activate this pathway (Fig. 4) or to activate calpain
(19). It may be sufficient, however, to activate calmodulin, and
thereby p85/PI3K (33), which would account for the increase in
PtdInsP3 and the small increase in
PtdIns(3,4)P2 that we observed, in the absence of a rise in
PtdIns3P.
Data that we present here for CSK fractions point to a role for calpain
in the activation of PtdIns3P 4-K (Figs. 8A and
9A; Ref. 18), as is also true for PtdIns 3-K (18). Both of
these increased activities are necessary for integrin signaling leading to maximum PtdIns(3,4)P2 generation and PKB
activation
(18). Our data further indicate that PtdIns3P 4-K is negatively
regulated by G
, which can be released when THR-R is stimulated.
Presumably, this might minimize the formation of
PtdIns(3,4)P2 from endogenous PtdIns3P during early phases
of platelet activation by THR-R. It seems likely that some common
calpain target is involved in the negative regulation of PtdIns3P 4-K
and positive regulation of PtdIns4P 3-K (Fig. 9B) by G
since, once calpain is activated, the ability of G
to regulate
both of these activities is diminished. Therefore, calpain appears to
be important not only for eliminating regulatability of PtdIns3P 4-K
and PtdIns4P 3-K by G
, but also for activating these
phosphoinositide kinases by another mechanism. Our "positional
labeling" experiments (Fig. 5) do not rule out some participation of
PtdIns4P 3-K in integrin-linked production of
PtdIns(3,4)P2. Rather, they indicate that this is not the
predominant pathway. The data for CSK may thus have some bearing on
post-integrin generation of a minor portion of
PtdIns(3,4)P2 via PtdIns4P 3-K, as they do for pre-integrin
activation of PI3K
and/or p85/PI3K when SFLLRN/THR or PMA is the
agonist (1). Further studies are needed to address this issue.
In hopes of elucidating the nature of the PtdIns3P 4-K activity that we
have observed in the cytosol and in the CSK of platelets, we have
utilized an antibody that can immunoprecipitate and recognize on
Western blots PIP5KII
(25-28). This is an enzyme that has been expressed and found to phosphorylate synthetic PtdIns5P, as well as
PtdIns5P found as an impurity in PtdIns4P isolated from natural sources
(28). To a much lesser degree, it can also phosphorylate PtdIns3P, and
all phosphorylations occur at the 4-OH position of the inositol ring,
to produce PtdIns(4,5)P2 and PtdIns(3,4)P2; PtdIns(4,5)P2 is not produced from pure (synthesized)
PtdIns4P (28) by this enzyme. Technically, therefore, the type II
"5-K" is not a "5-K", but a "4-K." We have found that the
antibody not only recognizes and removes PtdIns5P 4-K from platelet
cytosol, but also PtdIns4P 5-K (utilizing either natural or synthetic
PtdIns4P substrate) and PtdIns3P 5-K (Fig. 10). Thus, the antibody
apparently can recognize true 5-K activities, as well. Despite removing
50-80% of these activities, however, the immunoprecipitation
procedure does not remove PtdIns3P 4-K or 3-K (PtdIns4P 3-K or PtdIns5P 3-K) activities. This observation with respect to PtdIns3P 4-K activity
is unexpected, since it has been reported recently that type II kinases
display PtdIns3P 4-K (27, 28) and concerted PtdIns3P
PtdIns(3,4)P2
PtdIns(3,4,5)P3 activities
(27). The PtdIns3P 4-K activity that we observe in platelet lysates is
thus not the type II or PtdIns5P 4-K enzyme described in the literature. Only purification and sequencing of the platelet enzyme will provide information on how these families of 4-kinases are related.
Our findings with respect to the activation of
IIb
3 integrin, PI3Ks, and PKB
/Akt are
summarized in Fig. 11. As indicated, the targets for PKB
/Akt (pre- and post-integrin) are as yet unknown. Additional targets for post-integrin-generated
PtdIns(3,4)P2 may also exist. It is possible, for example,
that such PtdIns(3,4)P2 may be involved in late
(post-aggregation) filopod formation (34). The involvement of
3-phosphorylated phosphoinositides in both early and late phases of
agonist-induced platelet signaling, and the nature of the
phosphoinositide kinases that are involved, should be important topics
for future exploration.

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Fig. 11.
Summary of pre-integrin and post-integrin
pathways leading to activation of PKB /Akt. For clarity,
activation of PI3K via THR-R has been omitted. Whereas PI3K does
not contribute to the activation of IIb 3
(13), it may, however, affect PKB through its contribution to
PtdInsP3.
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We thank Drs. Dario Alessi, Kath Hinchliffe,
and Robin Irvine for the generous contribution of antibodies, and Drew
Likens of the Cardeza Foundation and Michelle Levinski for artwork.