(Received for publication, July 5, 1995; and in revised form, January 4, 1996)
From the
Platelets exposed to thrombin or thrombin receptor agonist
peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC),
and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function
of the activation and relocalization of two cytoskeletally-associated
phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K. We now
report that exposure of platelets to PKC-activating
-phorbol
myristate acetate (
PMA) does not stimulate PI 3-K
, but rather
stimulates p85/PI 3-K, which associates with the cytoskeleton.
Wortmannin is an inhibitor of both PI 3-Ks, known to act with more
potency on p85/PI 3-K.
PMA-stimulated 3-PPI accumulation is more
sensitive to wortmannin (IC
= 1.3 nM) than
is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC
= 10 nM). The activity of p85/PI 3-K in
immunoprecipitates or in cytoskeletal fractions is inhibited more
potently by exposure of platelets to wortmannin than is the activity of
PI 3-K
.
PMA or SFLLRN promotes the conversion of platelet
integrin
into a fibrinogen-binding
form required for platelet aggregation. Activation of
in response to
PMA or SFLLRN
is inhibited by wortmannin with an IC
of 1 nM in
each case. Wortmannin inhibits neither activation of
by ligand-induced binding site
antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet
aggregation in the presence of fibrinogen, indicating that this type of
``outside-in'' signaling by
is largely PI 3-K-independent. We conclude that p85/PI 3-K, in
preference to PI 3-K
, contributes to activation of
when the thrombin receptor or PKC
is stimulated.
We have reported that activation of the thrombin receptor on
human platelets leads to the accumulation of PtdIns(3,4,5)P(
)and PtdIns(3,4)P
as a function of the
stimulation of two phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and
PI 3-K
, both of which are dependent upon GTP-binding
proteins(1, 2) . Each PI 3-K contributes to the
production of both 3-PPIs. The former PI 3-K, a heterodimer containing
a regulatory noncatalytic p85 subunit, is activated via the small
G-protein Rho(1, 3) , whereas the latter, lacking p85,
is activated via the
subunit of dissociated heterotrimeric
G-proteins(1, 4) . Phospholipase C (PLC) and protein
kinase C (PKC) are also activated under the same conditions, and
inhibition of PKC in permeabilized platelets by a pseudosubstrate
peptide partially inhibits the accumulation of 3-PPI in platelets
exposed to thrombin or the direct G-protein agonist
GTP
S(5) . Tumor-promoting
-phorbol esters are known
to activate PKC in platelets(6, 7) , as in other
cells, and earlier studies with such a phorbol ester (2) indicated that 3-PPI accumulation could be stimulated,
although not as strongly as by thrombin.
In view of findings about
the two species of PI 3-K that are activated by thrombin(1) ,
we have now re-examined the activation of PI 3-K by phorbol esters. We
reasoned initially that PMA, which has been shown in some cells to
stabilize some heterotrimeric G-proteins(8) , would be unlikely
to promote the release of activating G
, and therefore be
unable to activate PI 3-K
. This would perhaps account for some of
the weaker effects of
PMA versus thrombin in stimulating
3-PPI accumulation. It has been reported more recently, however, that,
when added to platelets,
PMA causes phosphorylation of the
-subunit of G
(9) , and that such
phosphorylation inhibits association of
with
(10) . The possibility of a release of
from G
provided us with an additional reason to evaluate
the activation of PI 3-K
in response to
PMA. Wortmannin, a
fungal metabolite with hemorrhagic effects in animals, is an
irreversible inhibitor of PI 3-K(11) , and p85/PI 3-K in
neutrophils is more sensitive to wortmannin than is PI
3-K
(12) . Therefore, in this study we examined the
relative susceptibility to inhibition by wortmannin of
PMA- and
thrombin receptor-stimulated 3-PPI production as well as the effects of
wortmannin on p85/PI 3-K and PI 3-K
activities in platelets.
A
crucial question for investigators studying signal transduction is
``what role(s) do PI 3-Ks (and 3-PPIs) play in the functions of a
cell?'' Prior to its recognition as a PI 3-K inhibitor, wortmannin
was described as an inhibitor of PMA-induced platelet
aggregation(13) . Aggregation is the major hemostatic function
of the platelet in vivo. Integrin
mediates the aggregation process
by binding fibrinogen after platelet activation by
agonists(15, 16) . Accordingly, we have been
interested in the role of the two PI 3-Ks in regulating the conversion
of platelet integrin
during
platelet activation to a form that is competent to bind fibrinogen, a
process often referred to as ``inside-out''
signaling(1, 14) . Therefore, in the present study we
investigated the relative inhibitory effects of wortmannin on
activation induced by a peptide
stimulator of the thrombin receptor or by
PMA. As a control, we
studied the effects of wortmannin on induction of
binding function by an antibody
Fab fragment (anti-LIBS6 Fab) that binds to
, thereby
activating
(17) . This
antibody also afforded us the opportunity to study PI 3-K activation in
direct response to fibrinogen binding and platelet aggregation
(``outside-in'' signaling).
Blood was obtained from normal donors and
anti-coagulated with 0.15 volumes of NIH formula A acid-citrate
dextrose solution supplemented with 1 µM prostaglandin
E and 1 unit/ml apyrase. Platelet-rich plasma was obtained (20) and incubated for 10 min at 37 °C with either 100
nM wortmannin or 0.5% Me
SO. Platelets were then
gel-filtered into an incubation buffer containing 137 mM NaCl,
2.7 mM KCl, 1 mM MgCl
, 5.6 mM glucose, 1 mg/ml bovine serum albumin, 3.3 mM NaH
PO
, and 20 mM HEPES, pH 6.5,
adjusted to 2
10
/ml, and supplemented with 10
µM indomethacin, a cyclooxygenase inhibitor. Aliquots of
platelets (5 µl) were added to incubation buffer (45 µl), pH
7.4, such that the final mixture contained 10 µM indomethacin, 40 µg/ml FITC-PAC1, and agonist (0-10
µM ADP, 0.2 µM
PMA, 50 µM SFLLRN, or 150 µg/ml anti-LIBS6 Fab). After 5 min in the dark
at room temperature, samples were diluted with 200 µl of incubation
buffer and FITC-PAC1 binding to 10
platelets/sample was
analyzed by flow cytometry (18) . In some experiments, 10
µg/ml FITC-SSA6 was present as well as 40 µg/ml biotin-PAC1 (in
place of FITC-PAC1) and phycoerythrin-streptavidin (1:100). After
incubation and dilution as above, binding was quantitated by dual color
flow cytometry.
In experiments to study wortmannin dose-dependence,
platelet-rich plasma was incubated with 1 mM ASA for 20 min,
and no wortmannin or MeSO was added. Platelets were
concentrated, applied to a Sepharose column (20) in buffer at
pH 7.4, and diluted to 6
10
/ml in buffer lacking
bovine serum albumin (final [bovine serum albumin] =
0.04%). Platelets (45 µl) were added to 5 µl of wortmannin
(0-100 nM, final) and incubated for 5 min at 37 °C,
followed by addition of 5 µl of buffer, SFLLRN (10 µM,
final) or
PMA (200 nM, final), and incubation at 37
°C for 2 min. Incubations with SFLLRN alternated with those for
PMA at each concentration of wortmannin, 60 s apart, and were
terminated by the addition of 500 µl of 0.3% bovine serum
albumin/buffer at room temperature. Of this, 20 µl were mixed with
20 µl of 80 µg/ml FITC-PAC1, and kept in the dark. After 5 min,
160 µl of phosphate-buffered saline was added, and the fluorescence
signal read.
Figure 1:
Accumulation of P-labeled 3-PPI and PtdOH in response to SFLLRN or PMA.
Washed ASA-treated platelets were labeled with
P
as described under ``Experimental Procedures'' and
incubated for 120 s with 0-1000 nM
PMA (A), 1000 nM
PMA (A*), or up to 10
µM SFLLRN (B). Other incubations (C)
were for up to 5 min with SFLLRN (10 µM; solid
symbols) or
PMA (200 nM; open symbols).
Incubations were terminated with acidic chloroform/methanol, and
extracted lipids were resolved and quantitated. Results are expressed
as % of basal (agonist-free) values ± range of duplicates
(contained within symbols). Basal values: A and B,
respectively, PtdIns(3,4,5)P
(
) = 1738 ±
67 dpm, 2870 ± 240 dpm; PtdIns(3,4)P
(
)
= 2068 ± 26 dpm, 4187 ± 93 dpm; PtdOH (
)
= 12,382 ± 132 dpm, 22,731 ± 221
dpm.
Figure 2:
PI 3-K
activity in cell fractions derived from platelets activated with PMA or
SFLLRN. ASA-treated platelets were incubated for 120 s with buffer,
PMA (200 nM), or
PMA (200 nM). Incubations
were terminated with cold Triton lysis buffer and spun at 15,000
g
4 min. A, equal amounts of washed
cytoskeletal protein were assayed for PI 3-K activity, using
PtdIns(4,5)P
as substrate, in the absence (open
bars) or presence of
ARK-PH (5 µM; cross-hatched bars) or G
(1 µM; diagonally striped bars). In other studies (B),
p85/PI 3-K was removed (>94%) by immunoprecipitation (with
antibodies to p85(
,
)) of p85/PI 3-K, followed by removal of
Triton X-100 from the post-immunoprecipitation supernatant(1) .
PI 3-K activity in the post-immunoprecipitation supernatant was assayed
in the absence (open bars) or presence of
ARK-PH (cross-hatched bars) or G
(diagonally striped
bars) as above. Results are the mean ± range of a
representative experiment of two performed in duplicate. Similar
results were observed when thrombin replaced SFLLRN as agonist (60 s),
or when platelets were exposed to SFLLRN for 60
s.
In contrast to the findings for PI 3-K, a specific
increase (increase/µg of cytoskeletal protein) in the
immunologically detectable p85 subunits of PI 3-K (p85
and
p85
) was observed in cytoskeletal fractions when either SFLLRN (or
thrombin) or
PMA was the agonist. Whereas
PMA did not alter
the amount of p85 in cytoskeleton, exposure to
PMA for 60 s
increased p85
2.0-fold and p85
2.1-fold. Similarly, exposure
to SFLLRN increased p85
2.0-fold and p85
2.2-fold. The ratio
of p85
/p85
= 6/1. There was also an increase in the
presence of the small GTP-binding proteins Rho and CDC42Hs (2-fold),
which are known to promote p85/PI 3-K
activity(1, 3, 25) , in SFLLRN- and
PMA-stimulated platelet cytoskeletal fractions. In keeping with
activation of p85/PI 3-K already reported (1) for platelets
exposed to thrombin, ADP-ribosylation of Rho was found to inhibit the
accumulation of 3-PPI in permeabilized platelets stimulated with either
SFLLRN or
PMA, although the degree of inhibition of SFLLRN-induced
3-PPI (63-75%) was greater than that of
PMA-induced 3-PPI
(40-44%). A role for additional factors is implied by the smaller
inhibition observed when
PMA, a sustained, non-localized agonist
for PKC, was used.
Figure 3:
Sensitivity to wortmannin of 3-PPI
accumulation in platelets exposed to PMA or SFLLRN.
P-Labeled platelets (as in Fig. 1) were incubated
for 5 min at 37 °C with various concentrations of wortmannin,
followed by 120 s with buffer,
PMA (200 nM;
), or
SFLLRN (10 µM;
). After termination of the
incubation, resolved lipids were quantitated as described in the legend
to Fig. 1. [
P]PtdOH levels were
unaffected by wortmannin. Basal values are subtracted from stimulated
values. Data are expressed in terms of stimulated values, as a % of
wortmannin-free controls, and are the mean ± S.D. of the results
of three experiments in duplicate. Results for thrombin (5 units/ml;
one experiment in duplicate, not shown) yielded the same IC
(10-15 nM) as did those for SFLLRN. A,
PtdIns(3,4)P
; B, PtdIns(3,4,5)P
. In a
representative experiment, stimulated PtdIns(3,4,5)P
values
(dpm) were 9293 ± 838(SFLLRN) and 4686 ± 52(PMA) and
stimulated PtdIns(3,4)P
values (dpm) were 27,029 ±
1754(SFLLRN) and 6567 ± 144(PMA).
Figure 4:
Effects of low-dose wortmannin on p85/PI
3-K and PI 3-K activities in platelet fractions. A,
preparations of ASA-treated platelets were exposed to
Me
SO/buffer (open bars) or 2 nM wortmannin (diagonally striped bars) for 5 min in
duplicate, followed directly (no SFLLRN added) by ice-cold Triton lysis
and centrifugation at 100,000
g
60 min at 4
°C. The Triton-soluble fraction (which contained >99% of p85/PI
3-K and PI 3-K
and the same amount of protein ± wortmannin)
was exposed to immunoprecipitating antibodies to p85/PI 3-K for 2 h at
4 °C. Triton was removed from the post-immunoprecipitation
supernatant, containing virtually all of PI 3-K
(1) .
Washed p85/PI 3-K immunoprecipitates and post-immunoprecipitation
supernatants (PI 3-K
) were assayed in duplicate for PI 3-K
activity in the absence or presence of GTP
S (10 µM,
former) or G
(1 µM, latter). Basal activities
were subtracted from stimulated activities. Results are the mean
activity ± S.D. for two experiments in duplicate. B,
platelets, as in A, were incubated ± wortmannin
followed, however, by incubation with SFLLRN (10 µM) or
buffer for 120 s. Cytoskeletal fractions (15,000
g
4 min at 4 °C = ``CSK'')
were obtained rapidly after Triton lysis. The yields of cytoskeletal
protein were the same ± wortmannin, but increased 26% +
SFLLRN. Equal amounts of washed cytoskeletal protein preparations
(containing the majority of activated p85/PI 3-K and PI
3-K
, post-SFLLRN; 1) were assayed with or without
ARK-PH (5
µM) for PI 3-K activity. Results were then adjusted for
total cytoskeletal protein yield. Activities were calculated for
samples with (cross-hatched bars) and without (open
bars) wortmannin treatment. Total activated PI 3-K activity
= total activity
-total
activity
. Total activated PI 3-K
activity
= total activity
- total
activity
. Total activated
p85/PI 3-K activity = total activated PI 3-K activity -
total activated PI 3-K
activity. Results are the mean activity
± S.D. for two duplicate preparations. Numbers in
parentheses, -fold increase in specific p85
/PI 3-K; numbers in brackets, -fold increase in specific p85
/PI
3-K (activated/resting cytoskeleton) quantitated after Western
blotting, where the same amount of protein was applied to resolving
gels. The ratio of
/
-p85 was
6.2/1.
Interestingly, when the effects of wortmannin on
exposure of activated (as measured
by binding of PAC1 antibody) in response to the two agonists were
compared (Fig. 5A), the IC
values were
found to be equal. Furthermore, they were the same as for inhibition of
PMA-induced PtdIns(3,4,5)P
accumulation, i.e.
1 nM. A comparison of the effects of low-dose
wortmannin (i.e. 1 and 2 nM, in the range giving
close to maximal inhibition of PAC-1 binding) on 3-PPI accumulation in
response to
PMA or SFLLRN (Fig. 5B), revealed two
points: 1) whereas the % inhibition by wortmannin of
SFLLRN-induced 3-PPI accumulation was much less than that of
PMA-induced accumulation (see also Fig. 3), the total
decrease in labeled 3-PPI was very similar for the two agonists,
and 2) when (at 2 nM wortmannin) 3-PPI formation in response
to SFLLRN had decreased only 20%, the amount of 3-PPI remaining was
greater than the amount of 3-PPI produced by
PMA in the absence of wortmannin; yet, this amount of 3-PPI was
apparently unable to ``support'' activation of
(Fig. 5A). The
degree of inhibition by 2 nM wortmannin of 3-PPI accumulation
in response to SFLLRN did not vary appreciably over the course of a
5-min incubation with agonist (not shown).
Figure 5:
Sensitivity to wortmannin of
reorganization and 3-PPI
accumulation for platelets activated by
PMA or SFLLRN.
Preparations of ASA-treated platelets were incubated for 5 min at 37
°C with 0-100 nM wortmannin (A) prior to
exposure for 120 s to buffer,
PMA (200 nM,
) or
SFLLRN (10 µM,
), alternating at each concentration
of wortmannin between
PMA and SFLLRN, as was true for labeled
platelets (Fig. 3). FITC-PAC1 binding was assayed as described
under ``Experimental Procedures.'' Results are representative
of two experiments (each comparing platelet responses to SFLLRN or
PMA using the same preparation of platelets) performed in
duplicate, and are shown as arbitrary fluorescence units±range.
Basal fluorescence is indicated by
*. In both cases,
wortmannin's IC
for
PMA as agonist =
IC
for SFLLRN as agonist, i.e. <2 nM.
In studies with
P-labeled platelets (B), platelet
suspensions were incubated as in Fig. 3with 0-2 nM wortmannin for 5 min prior to exposure to buffer, SFLLRN (10
µM, open bars), or
PMA (200 nM, diagonally striped bars) for 90 s and labeled 3-PPI were
quantitated. The same preparation of labeled platelets was incubated
with agonists or buffer in order to assure common responsiveness and
common baselines values, so that absolute changes could be compared.
Results are for one of two experiments performed in duplicate and a
third experiment (±2 nM wortmannin), presented as the
average % of unstimulated (``Basal'') values
± range. In all experiments, the amount of residual 3-PPI
generated by platelets exposed to SFLLRN + 2 nM wortmannin exceeded that produced by platelets exposed to
PMA-wortmannin.
The magnitude of the
SFLLRN-induced PAC1-binding response was slightly less than that in
other (eg., Fig. 6) experiments because a lower
concentration of SFLLRN was used (in keeping with 3-PPI studies). The
inhibitory effect of wortmannin was limited to the PAC1-binding
conformation of , since the total
surface expression of this integrin, monitored with SSA6 antibody in
the same experiment, was unchanged by wortmannin (Fig. 6). More
specifically, the inhibition was limited to agonist-induced conversion of
, since
enhancement of PAC1 binding by anti-LIBS6-Fab, which promotes a direct
conversion of
to the activated
state, was not impaired by wortmannin (Fig. 7). For comparison,
the effects of wortmannin on PAC1 binding after platelets were exposed
to three other agonists for platelet aggregation are shown.
Figure 6:
Wortmannin affects the conformation, not
the total surface expression, of .
Platelets were incubated with wortmannin (100 nM) or 0.5%
Me
SO for 5 min at 37 °C prior to incubation with
buffer,
PMA, or SFLLRN. Binding of FITC-SSA6 (A, total
expression) or biotin-PAC1 (B, active conformation) was
assayed, as described, by dual color flow cytometry. Each data bar
represents the mean ± S.E. of three independent
experiments.
Figure 7:
Effect of wortmannin on agonist-induced
reorganization of fibrinogen
receptors. Platelets were incubated with 100 nM wortmannin or
Me
SO prior to gel filtration and incubation for 5 min in
the presence of the different agonists, indomethacin (10
µM; inhibiting thromboxane A
synthesis), and
FITC-PAC1 as described under ``Experimental Procedures.''
Each bar represents the mean ± S.D. of triplicate
values from a single experiment representative of two
performed.
As is
true for PMA and thrombin/SFLLRN, ADP, a known platelet agonist,
induces activation of
(Fig. 7) and fibrinogen-dependent aggregation, which are
also inhibited by wortmannin. This ADP response is of significance,
since washed and stirred platelets frequently contain and release
traces of ADP, which can complicate interpretations of aggregation
results. We confirmed, as reported before, that ADP (10
µM) stimulates the accumulation of
[
P]PtdOH in
P-labeled platelets (26) and further observed a modest increase in 3-PPI in
ASA-treated platelets (ASA included to block potentiating effects of
thromboxane A
; (27) ) in 5 min (increase in PtdOH
= 2.65 ± 0.05-fold; PtdIns(3,4,5)P
=
1.83 ± 0.04-fold; PtdIns(3,4)P
= 2.01
± 0.06-fold). RGDS, added to block the effects of any traces of
fibrinogen, was without effect on this response. Anti-LIBS6 Fab and
fibrinogen, added to ASA-treated platelets in the presence of an enzyme
system (CP-CPK) to decrease ADP, caused a small increase in labeled
PtdIns(4,5)P
(1.39 ± 0.01-fold). An increase in
PtdIns(3,4,5)P
(1.14 ± 0.03-fold) was barely
detectable. PtdIns(3,4)P
increased 1.99 ± 0.04-fold.
Basal values for PtdIns(4,5)P
, PtdIns(3,4,5)P
,
and PtdIns(3,4)P
were 5.31 ± 0.01
10
dpm, 4.78 ± 0.09
10
dpm, and 1.54
± 0.11
10
dpm, respectively. It thus appears
that, in the absence of other receptor-directed agonists, fibrinogen
binding leads to an ``outside-in'' stimulation of more than
one phosphoinositide-directed kinase. These changes are all quite
small, however, in comparison with the effects of thrombin/SFLLRN or
PMA. When the effects of wortmannin were evaluated with respect to
anti-LIBS6-Fab/fibrinogen-induced aggregation of ASA- and
CP-CPK-treated platelets, no inhibition was observed (not shown).
Our results point to two major conclusions: 1) whereas the
thrombin receptor activates two different PI 3-Ks in platelets, i.e. p85/PI 3-K and PI 3-K, stimulation of PKC leads to
the activation of only p85/PI 3-K, and 2) the activation of p85/PI 3-K,
rather than of PI 3-K
, is involved in inside-out signaling
affecting the conformational change in
that is a prerequisite for fibrinogen binding and platelet
aggregation.
Thrombin or SFLLRN, in activating heterotrimeric
GTP-binding proteins, stimulates PLC and, via generated diacylglycerol,
PKC. Apparently, this, in turn, promotes the activation of p85/PI 3-K
in a Rho-dependent manner(1) . Liberated G binds to
and activates PI 3-K
, (
)which is a Rho-insensitive
event(1) .
PMA does not activate PLC/diacylglycerol kinase (Fig. 1A), or free PLC- or PI 3-K
-activating
G
(Fig. 2). Indeed, exposure of platelets to
PMA
may even be inhibitory for PI 3-K
(Fig. 2B). (
)Rather, in activating PKC,
PMA stimulates a partially
Rho-dependent p85/PI 3-K and an increased association of Rho and
CDC42Hs (a direct agonist for p85/PI 3-K; (25) ), as well as
p85/PI 3-K (both
- and
-forms), with a cytoskeletal fraction.
The activation of p85/PI 3-K that we have observed in response to
PMA is not inhibited by RGDS, and is therefore independent of
fibrinogen binding. This would be expected, since fibrinogen was not
added, and
PMA, unlike SFLLRN/thrombin, is a poor secretogogue and
would not be expected to release stored fibrinogen. Consequently, we
are focussing here purely on inside-out signaling responses.
It has
been noted that p85/PI 3-K in neutrophils is more susceptible to
inhibition by wortmannin than is PI 3-K(12) . This appears
to be the case for platelets, as well (Fig. 4). Wortmannin is
also an irreversible inhibitor of PI
3-Ks(12, 28, 29) . Therefore, it was
important that short incubation periods be employed to optimize
differences in sensitivities between the two enzymes. Presumably, if
wortmannin, even at low concentrations, were stoichiometrically in
excess of PI 3-K targets, long periods of incubation would eventually
lead to complete inhibition at such concentrations were other factors
equal. The permeability barrier of the intact cell lowers effective
concentrations, and it is also possible that the arrangement of the
enzymes in the intact platelet also permits differences in
susceptibility to wortmannin to be detected more readily than in
cytosolic fractions incubated directly. (
)By incubating
platelets with different wortmannin concentrations under the same
conditions for studies of both 3-PPI generation and PAC1 binding (a
more sensitive measure of activated
exposure than is aggregation), we were able to compare the
susceptibilities of these responses. It is clear from Fig. 3and Fig. 5that, although there is a wortmannin-attributable
decrease of only 28% in the level of PtdIns(3,4,5)P
generated in response to the more potent agonist SFLLRN, the
inhibitory effect of wortmannin on activated exposure of PAC1-binding
is 80% of maximum. In contrast,
the inhibitory effects of varied wortmannin concentrations on
PMA-stimulated PtdIns(3,4,5)P
accumulation and
activated
exposure correlate well.
Surprisingly, the total amount of 3-PPI formed in the presence of 2
nM wortmannin + SFLLRN is greater than that formed in the
presence of PMA, without wortmannin (Fig. 5B), yet
this remaining SFLLRN-induced 3-PPI apparently is unable to promote
conversion of
(Fig. 5A). Of further interest, the total
amount (rather than %) of decrease in 3-PPI generated in response
to SFLLRN or
PMA caused by 2 nM wortmannin is similar for
the two agonists (Fig. 5B). These observations point to
the importance, crucial for
activation, of not only the mass of agonist-generated 3-PPI, but
also of another factor, which probably involves localization. This
additional factor appears to be related to the type of PI 3-K that
generates 3-PPI. At the low concentrations of wortmannin that maximally
inhibit
activation in response to
SFLLRN or
PMA (Fig. 5A) and 3-PPI production in
response to
PMA ( Fig. 3and Fig. 5B),
p85/PI 3-K is inhibited preferentially (Fig. 4). We calculate
that 75% of the total wortmannin-induced decrease in SFLLRN-stimulated
PI 3-K activity in the cytoskeletal fraction, resulting in about 80% of
the maximum inhibited binding of PAC1 to
, is due to inhibition by
wortmannin of p85/PI 3-K, whereas 25% of the inhibition of stimulated
PI 3-K is due to inhibition of PI 3-K
(Fig. 4B).
Since 40% of the original activated cytoskeletal PI 3-K (without
wortmannin treatment) is due to p85/PI 3-K, total stimulated PI 3-K
activity is inhibited only about 30% by this concentration of
wortmannin. Thus, about 70% of stimulated PI 3-K activity (the great
majority now due to PI 3-K
) is unimpaired by 2 nM wortmannin, yet most of the wortmannin-inhibitable PAC1 binding
is, at this point, inhibited. These results point to a role for p85/PI
3-K, as opposed to PI 3-K
, in promoting the activation or
maintenance of active
. The
selectivity of the effect may involve differences in localization of
the two PI 3-Ks which we are unable to detect using crude
Triton-insoluble fractions, since both p85/PI 3-K and PI 3-K
are
recruited to the Triton-insoluble cytoskeleton of
thrombin/SFLLRN-activated platelets. Our findings also raise the issue
of what function PI 3-K
-generated 3-PPI might serve.
The route
by which PMA (and PKC) activates p85/PI 3-K and causes increased
association with the cytoskeletal fraction has not yet been elucidated,
although it seems likely to involve tyrosine phosphorylation. Our data
indicate that inhibition of p85/PI 3-K activity by low doses of
wortmannin does not inhibit recruitment of p85/PI 3-K to the
cytoskeletal fraction. PMA has been shown to stimulate tyrosine
phosphorylation in human platelets(30) . We have examined p85
immunoprecipitates from activated platelets and found no evidence of
tyrosine-phosphorylated p85, as has been noted by others (31) .
It is known, however, that binding of appropriate
tyrosine-phosphorylated peptides to the SH2 domains of p85 can activate
p85/PI 3-K(32, 33, 34) . Furthermore,
inhibition of tyrosine phosphatases in platelets promotes PAC1 binding,
aggregation, and production of PtdIns(3,4)P
(35) ,
and inhibition of tyrosine kinases curtails thrombin-induced
aggregation and PtdIns(3,4)P
accumulation(36) .
Part of this activation, however, may be dependent upon interactions
between p85 and p125
. A recent interesting study (31) has demonstrated that p85/PI 3-K can be activated directly
by interactions between the SH3 domain of p85 and the proline-rich
region of p125
, a tyrosine kinase that localizes with
integrins at the platelet cytoskeleton. At the early,
``pre-aggregation'' stage of platelet activation,
p125
does not become itself tyrosine-phosphorylated,
since such phosphorylation appears to be dependent upon both
-mediated platelet aggregation and
PKC and Ca
signals(37, 38) . It is
conceivable that activation of PKC can affect the accessibility of the
p125
proline domains to p85/PI 3-K.
While these
studies were underway, it was reported that two different PI 3-K
inhibitors, wortmannin and LY294002, inhibit both conversion of
to a fibrinogen-binding form and
the aggregation of platelets stimulated by thrombin receptor-activating
peptide(39) . Our results are in partial agreement with those
reported. In contrast to the previous report, however, we have found
that concentrations of wortmannin (100 nM, 5 min) that
completely inhibit PI 3-K activity (Fig. 3) do not impair
exposure of activated
in response
to the Fab portion of an antibody known as anti-LIBS6 (Fig. 7),
nor does it impair anti-LIBS6 Fab-induced platelet aggregation when
fibrinogen is present in excess. We suggest that part of the reported
inhibitory effects may relate to the presence of ADP and/or thromboxane
A
, each of which (present experiments; (2) ) can
activate PI 3-K in platelet preparations that have not been treated
with agents to remove or block synthesis of these
agonists(39) . Furthermore, we have observed a slight
stimulation of phosphoinositide kinase product accumulations by
anti-LIBS6 Fab plus fibrinogen-induced platelet aggregation, but not as
pronounced as reported. Thus, in the absence of an additional agonist,
outside-in signaling via
-mediated
fibrinogen binding and aggregation does not seem to be as strong as the
incremental PtdIns(3,4)P
accumulation observed when
platelets are exposed to agonists such as thrombin (when fibrinogen
binding and aggregation are not blocked; Refs. 24 and 40).
Granted
that 3-PPI accumulation is important for agonist-induced
conformational changes, one can
only speculate at this point about how PtdIns(3,4,5)P
and/or PtdIns(3,4)P
exert effects. The activation by
3-PPI of protein kinase activity would be the most likely route for
3-PPI action, since the amounts of 3-PPI (especially
PtdIns(3,4,5)P
) generated are rather small with respect to
cytoskeletal proteins(41) , although highly localized
stoichiometric effects cannot be excluded at present. We have observed
in other studies (42) that PtdIns(3,4,5)P
(2
µM), added to saponin-permeabilized platelets, stimulates
a kinase that phosphorylates pleckstrin and overcomes the inhibitory
effects of wortmannin on pleckstrin phosphorylation. It is possible
that a PtdIns(3,4,5)P
-activated protein kinase is also
involved in the conversion of
.
A portion of agonist-induced conformational changes in
is wortmannin-insensitive (Fig. 5Fig. 6Fig. 7), some of which may be
attributable to wortmannin-insensitive generation of PtdOH. PtdOH has
been shown to promote activation of
(43) . Nevertheless, the
wortmannin-sensitive component is a significant proportion of total
activation, and therefore p85/PI
3-K activation should now be regarded as an important signal leading to
functional responses in platelets.