(Received for publication, June 29, 1995; and in revised form, August 14, 1995)
From the
Human platelets pretreated with indomethacin release arachidonic
acid predominantly through the activity of cytosolic phospholipase
A (cPLA
), an 85-kDa protein. This enzyme is
regulated by an increase in intracellular Ca
, a
necessary condition for arachidonic acid liberation, and by
phosphorylation. Phosphorylation of cPLA
enhanced the
Ca
-induced arachidonic acid release in platelets
stimulated by the ionophore A23187 and phorbol ester (phorbol
12,13-dibutyrate (PDBu)). In thrombin-stimulated platelets, however,
phosphorylation appeared not to be necessary for arachidonic acid
release since the latter response was not impaired in the presence of
staurosporine, which inhibited phosphorylation. Collagen, thrombin, and
PDBu induced phosphorylation of platelet cPLA
as well as
activation of mitogen-activated protein kinase (MAPK; p42
and p44
). cPLA
activation was not dependent on protein kinase C (PKC) in
thrombin- and collagen-stimulated platelets, as preincubation with the
PKC inhibitor Ro 31-8220 neither interfered with cPLA
phosphorylation nor reduced arachidonic acid release. However,
collagen- and thrombin-induced activation of MAPK was inhibited by Ro
31-8220, indicating that PKC is necessary for MAPK stimulation in
platelets. Although MAPK may underlie phosphorylation of cPLA
in PDBu-activated human platelets, our results provide evidence
for PKC- and MAPK-independent phosphorylation of cPLA
in
platelets stimulated by the physiological activators collagen and
thrombin.
Upon stimulation, human platelets release eicosanoids as
mediators in blood clotting events. Arachidonic acid is cleaved from
the sn-2 position of phospholipids through the activity of
phospholipase A (PLA
) (
)and
metabolized by cyclooxygenase and lipoxygenase enzymes. Two different
forms of PLA
exist in platelets: a 14-kDa PLA
,
which is secreted into the plasma and depends on millimolar
Ca
for activation(1) , and the recently
discovered cytosolic PLA
(cPLA
), which is an
85-kDa enzyme requiring submicromolar Ca
for
activation(2) . cPLA
has been purified (3, 4) and cloned(5, 6) . The enzyme
is regulated by intracellular Ca
, which induces
translocation to membranes (5) through a
Ca
-dependent lipid-binding motif in its N
terminus(7) . Phosphorylation on serine residues leads to an
increase in enzymatic activity(2, 8, 9) . A
third structural feature of cPLA
, serine 228, is involved
in the catalytic mechanism(10) .
The signaling events
leading to phosphorylation and activation of cPLA are not
established. Phorbol 12-myristate 13-acetate potentiates release of
arachidonic acid by the Ca
ionophore A23187 in
platelets suggesting a role for protein kinase C (PKC; (11) ).
Nemenoff et al.(12) reported that purified
cPLA
can be phosphorylated by either purified PKC or
mitogen-activated protein kinase (MAPK). The site of phosphorylation of
cPLA
by MAPK has been identified as serine 505 in
transfected Chinese hamster ovary cells and lies in a consensus
sequence for this kinase(9) . MAPK activity is regulated
through MAPK kinase(13, 14, 15) , which is
itself activated downstream of PKC-dependent and -independent
pathways(16, 17) .
In human platelets, cPLA is phosphorylated after thrombin stimulation, and this is
associated with an increase in its specific activity(2) . Both
p42
and p44
are present
in platelets(18, 19, 20) , but only
p42
has been reported to undergo activation in
thrombin-stimulated human platelets (19) .
In this study, we
have investigated the regulation of cPLA in human platelets
stimulated by collagen and thrombin. Collagen binds to glycoprotein
receptors on the platelet surface, which results in an increase in
phospholipase C (PLC) activity through phosphorylation of PLC
-2 on
tyrosine residues(21, 22) . In contrast, thrombin
activates PLC
isoforms via a G protein-dependent
pathway(23) . To investigate the importance of PKC and MAPK in
the regulation of cPLA
, we used kinase inhibitors that
block the intracellular signaling pathways elicited by collagen and
thrombin. Staurosporine is a strong inhibitor of tyrosine and
serine/threonine kinases, including PKC, but its lack of specificity
limits its use for studying the role of PKC(24, 25) .
We therefore included the staurosporine analogue Ro 31-8220 in
our study, which is a potent and more selective inhibitor of
PKC(26, 27) . The role of Ca
has
also been investigated using the divalent cation ionophore A23187 and
the intracellular Ca
chelator BAPTA-AM. The results
demonstrate that cPLA
undergoes phosphorylation in
collagen- and thrombin-stimulated platelets independent of PKC and MAPK
and that Ca
has a major role in its regulation
independent of phosphorylation.
Electrophoretic mobility analyses to distinguish
phosphorylated from nonphosphorylated cPLA was performed on
10% SDS-PAGE as described previously(2) .
The Ca dependence of arachidonic acid formation in collagen-stimulated
platelets has previously been demonstrated by Smith et al.(28) using the Ca
chelator BAPTA-AM (100
µM), which permeates the plasma membrane and becomes
trapped as a tetraanion inside the cell after cleavage by nonspecific
cytosolic esterases. Since BAPTA-AM markedly suppressed PLC activation
at concentrations higher than 15 µM(30) , we used
BAPTA-AM at a concentration of 10 µM. This blocked the
thrombin- and collagen-stimulated Ca
rise in
platelets (Fig. 1) and inhibited over 90% of the
[
H]arachidonic acid release after stimulation by
collagen, thrombin, or A23187 (Table 1). To check that BAPTA-AM
did not inhibit PLC/PKC activation, we monitored phosphorylation of
pleckstrin, the major PKC substrate in
platelets(31, 32) . BAPTA-AM had no effect on
[
P]phosphorylation of pleckstrin induced by
collagen (106.8 ± 5.1%, n = 3, relative to
collagen, 100%) or by thrombin (108.7 ± 6.4%, n = 4, relative to thrombin, 100%), indicating that the
observed inhibition of [
H]arachidonic acid
release is due to the Ca
-chelating properties of
BAPTA-AM. A rise in intracellular Ca
is therefore a
necessary condition for the formation of arachidonic acid in platelets.
Figure 1:
Inhibition of collagen- and
thrombin-induced Ca elevation in human platelets by
BAPTA-AM. Platelets were loaded with fura2-AM and treated with
indomethacin (10 µM). They were incubated with either
Me
SO (1%) or BAPTA-AM (10 µM) at room
temperature for 15 min. Samples were transferred to a
spectrofluorimeter where the fluorescence ratio (excitation at 340 and
380 nm; emission at 510 nm) was measured. After 60 s, platelets were
stimulated with collagen (100 µg/ml) or with thrombin (1 unit/ml)
in a stirred solution at room temperature.
Figure 2:
Concentration-response curve of the
[H]arachidonic acid release from platelets after
ionophore-stimulation. [
H]Arachidonic
acid-labeled platelets (4
10
/ml) were treated with
indomethacin and BW4AC and then incubated with Me
SO
(
, 1%), PDBu (
, 1 µM), Ro 31-8220
(
, 10 µM), or PDBu and Ro 31-8220 (
)
for 5 min. They were stimulated with A23187 for 2 min at 37 °C.
After adding an equal volume of 6% glutaraldehyde, the radioactivity of
the supernatant was counted. Points show mean ± S.E. from
quadruplicate determination.
Further experiments were therefore designed to investigate
whether PKC is involved in the formation of
[H]arachidonic acid after activation by
physiological stimuli, such as collagen and thrombin. Both Ro
31-8220 and staurosporine inhibited phosphorylation of the PKC
substrate pleckstrin in collagen- and thrombin-treated platelets (Fig. 3). Ro 31-8220 did not alter the collagen-induced
[
H]arachidonic acid release significantly,
whereas staurosporine blocked it (Fig. 4A). This is in
agreement with results reported by Daniel et al.(21) and
parallels the effect of each inhibitor on PLC activation(35) .
The inhibitory effect of staurosporine is consistent with a role for
tyrosine phosphorylation in early signaling events in
collagen-stimulated platelets(21, 22, 36) .
Since Ro 31-8220 did not decrease the formation of
[
H]arachidonic acid after collagen stimulation,
PKC activity does not appear to be necessary for arachidonic acid
liberation.
Figure 3:
Phosphorylation of pleckstrin (p47).
Platelets were labeled with P
, pretreated with
indomethacin and were incubated with Me
SO (D, 1%),
Ro 31-8220 (Ro, 10 µM), or staurosporine (st, 10 µM) for 5 min at 37 °C. They were
stimulated with collagen (100 µg/ml) for 5 min in a stirred
solution (1200 rpm) or with thrombin (1 unit/ml) for 2 min. Total
tissue samples were resolved on 10% SDS-PAGE, and the dried gel was
autoradiographed.
Figure 4:
Effect of the protein kinase C inhibitors
Ro 31-8220 and staurosporine on the release of
[H]arachidonic acid and on the intracellular
Ca
rise in platelets stimulated with collagen and
thrombin. A, [
H]arachidonic acid-labeled
platelets (4
10
/ml) were treated with
indomethacin/BW4AC. They were incubated with Me
SO (DMSO, 1%), Ro 31-8220 (Ro 31, 10
µM), or staurosporine (stauro, 10
µM) for 5 min at 37 °C and stimulated with collagen
(100 µg/ml) for 5 min in a stirred solution (1200 rpm).
Radioactivity released into the buffer was determined as described
under ``Materials and Methods.'' 100% stimulation correspond
to 4135 dpm, 0% (basal) corresponds to 2290 dpm. B,
concentration-response curve of the
[
H]arachidonic acid release from
indomethacin/BW4AC pretreated platelets stimulated with thrombin.
[
H]Arachidonic acid-labeled platelets were
incubated with Me
SO (
), staurosporine (
, 10
µM), or Ro 31-8220 (
, 10 µM) for 5
min at 37 °C and stimulated with thrombin for 2 min. In A and B, each point is the mean ± S.E. from
quadruplicate determinations. The results shown are representative of
at least three other similar experiments. C, intracellular
Ca
was measured in fura2-AM-loaded platelets as
described for Fig. 1. Platelets were incubated with
Me
SO (1%), Ro 31-8220 (10 µM) or
staurosporine (10 µM) for 5 min at room temperature and
stimulated with collagen (100 µg/ml) or thrombin (1 unit/ml) as
indicated.
[H]Arachidonic acid was generated
in a concentration-dependent manner following stimulation of platelets
with thrombin (Fig. 4B). At submaximal concentrations
of thrombin (0.2-1 units/ml), both staurosporine and Ro
31-8220 enhanced [
H]arachidonic acid
release without having any significant effect on their own (for
example, compare release of [
H]arachidonic acid
at 0.01 units/ml thrombin). Activation of PLC by thrombin is not
altered in the presence of staurosporine but is potentiated
significantly by Ro 31-8220(24, 37) ; the effect
of Ro 31-8220 can be explained by inhibition of the negative
feedback action of PKC on PLC(38) . This is in good agreement
with the effect of Ro 31-8220 on
[
H]arachidonic acid release, suggesting that
thrombin-induced formation of [
H]arachidonic acid
is an event downstream of PLC and Ca
.
To further
investigate this hypothesis, intracellular Ca elevation was measured in the presence of Ro 31-8220 and
staurosporine. As shown in Fig. 4C, Ro 31-8220
had no effect on the intracellular Ca
rise in
collagen-stimulated platelets, whereas staurosporine blocked it. Both
inhibitors increased and prolonged the intracellular Ca
elevation after thrombin stimulation. These data correlate with
the release of [
H]arachidonic acid (Fig. 4, A and B) and provide further evidence
that arachidonic acid is generated downstream of Ca
in human platelets.
Figure 5:
Phosphorylation of cPLA. A, indomethacin-treated and
P
-labeled
platelets (1
10
/sample) were incubated with
Me
SO (D, 1%), Ro 31-8220 (Ro, 10
µM), or staurosporine (st, 10 µM)
for 5 min. Platelets were stimulated with collagen (100 µg/ml) for
5 min in a stirred solution (1200 rpm) or with thrombin (1 unit/ml) for
2 min at 37 °C. cPLA
was immunoprecipitated and
resolved by SDS-PAGE (10%). After autoradiography (upper
panels), the membranes were Western blotted for cPLA
(lower panels). Unspecific bands represent IgG bands detected
by the secondary antibody. Phosphorylation was quantified by
densitometry analysis, collagen 100%, + Ro 31-8220 107%,
+ staurosporine below basal levels; thrombin 100%, + Ro
31-8220 99%, + staurosporine 5%. Similar results were
obtained with platelets isolated from different donors (n = 3). B, indomethacin-treated platelets were
incubated with Me
SO (1%), Ro 31-8220 (Ro, 10
µM), or staurosporine (stauro, 10
µM) and stimulated as in A. Stimulation was
stopped by adding 2
Laemmli sample buffer after 0 s, 1, 2, or 5
min. Samples were boiled for 5 min and subjected to 10% SDS-PAGE. Gels
were transferred to polyvinylidene difluoride and Western blotted for
cPLA
.
Both isoforms of MAPK were
activated in collagen- and thrombin-stimulated platelets, whereas there
was little p42 activity under basal conditions (Fig. 6A). p42
was the predominantly
active form, but in contrast to results obtained by Papkoff et
al.(19) , p44
was also activated. The
collagen-induced MAPK activation peaked after 2 min of stimulation (Fig. 6A) and declined slightly by 5 min to 13.4
± 3.0-fold (n = 5) over basal levels. Thrombin
stimulated maximal MAPK activity between 1 and 2 min (at 2 min, 94.2
± 31.9-fold, n = 5), which declined to
half-maximal levels after 5 min. Preincubation of platelets with Ro
31-8220 or staurosporine blocked MAPK activation by either
stimulus at all time points measured (Fig. 6A). All
samples contained similar levels of p42/p44
as analyzed
by Western blotting of a small portion of each immunoprecipitate (Fig. 6B). We have confirmed these results by using a
monoclonal antibody to immunoprecipitate p42
prior to
the in-gel renaturation kinase assay (not shown), and by Western
blotting MAPK immunoprecipitates for phosphotyrosine residues.
Thrombin-stimulated p42
tyrosine phosphorylation peaked
at 2 min (Fig. 6C); faint p44
tyrosine
phosphorylation was detectable at longer exposure times (not shown).
Tyrosine phosphorylation was blocked in the presence of Ro
31-8220 or staurosporine (Fig. 6C).
Collagen-induced p42
tyrosine phosphorylation was much
weaker but could be detected after 2 and 5 min of stimulation (not
shown); it was also inhibited by Ro 31-8220 and staurosporine.
These results suggest that p42/p44
is regulated
downstream of PKC in platelets stimulated by collagen or thrombin.
Figure 6:
Activation of p42 and
p44
is inhibited by staurosporine and Ro 31-8220.
Indomethacin-treated platelets (1
10
/sample) were
incubated with Me
SO (1%), Ro 31-8220 (10
µM), or staurosporine (10 µM) for 5 min at 37
°C. A, platelets were stimulated with collagen (100
µg/ml) for 5 min in a stirred solution (1200 rpm) or with thrombin
(1 unit/ml) for 2 min. p42
and p44
were immunoprecipitated under denaturing conditions using a
polyclonal antibody recognizing both isoforms and were resolved on
SDS-PAGE (10%, copolymerized with 0.5 mg/ml MBP). Gels were renatured
as described under ``Materials and Methods'' and were
incubated with 50 µM ATP and 20 µCi/ml
[
-
P]ATP in kinase buffer. Autoradiographs
were taken from dried gels. Under the conditions used during the kinase
assay, no autophosphorylation was detectable (not shown). B,
small portions of the immunoprecipitates were resolved on 10% SDS-PAGE,
transferred to polyvinylidene difluoride and Western blotted for MAPK
using a polyclonal anti-p42/p44
antibody. C, membranes obtained as in B were Western blotted
for phosphotyrosine residues using the monoclonal anti-phosphotyrosine
antibody 4G10. Bands around 50 kDa (B and C)
represent IgG heavy chains and were detected by secondary antibody
alone. The results are representative of three similar
experiments.
Figure 7:
Effect of PDBu and A23187 on PKC and
p42 activities and on cPLA
phosphorylation.
Platelets were treated with indomethacin and stimulated with
Me
SO (1%, basal), PDBu (1 µM), A23187 (2
µM), or both drugs together at 37 °C for 2 min. A, phosphorylation of pleckstrin as indicator of PKC activity. Total
tissue samples from
P
-labeled platelets were
resolved on 10% SDS-PAGE, and the dried gel was autoradiographed. The
position of the PKC substrate pleckstrin is indicated. B,
activity of p42
(in-gel renaturation kinase
assay). p42
was immunoprecipitated using a
monoclonal anti-p42
antibody and renatured in
MBP (0.5 mg/ml) containing 10% SDS-PAGE gels. The autoradiograph shows
bands of renatured p42
activity. C,
autoradiograph of immunoprecipitated cPLA
from
P
-labeled platelets. After stimulation,
platelets were lysed, and cPLA
was immunoprecipitated as
described under ``Materials and Methods.'' As analyzed by
densitometry, cPLA
phosphorylation induced by A23187
corresponded to 40% of the response to PDBu 100%, and both agents
together caused 130% of the PDBu response. An equal amount of
cPLA
was immunoprecipitated in each sample as controlled by
Western blotting (not shown).
The abundance of cPLA in human platelets over
secretory PLA
(2) and the millimolar Ca
requirement of the latter (1) suggest that the bulk of
arachidonic acid is generated through the activity of cPLA
.
Consistent with this, inhibitors of secretory PLA
do not
reduce the release of arachidonic acid from human
platelets(39) , whereas arachidonyl trifluoromethyl ketone, an
inhibitor of cPLA
(40) and
Ca
-independent PLA
(41) , induces
substantial inhibition after stimulation with thrombin (39) and
A23187(42) .
Evidence against a major role for
diacylglycerol lipase in the liberation of arachidonic acid is provided
by Halenda et al.(11, 33) , demonstrating
that Ca ionophores induce substantial arachidonic
acid release in the absence of PLC activation, and by the present work
with BAPTA-AM. BAPTA-AM markedly inhibited thrombin- and
collagen-stimulated [
H]arachidonic acid release (Table 1) but had little effect on pleckstrin phosphorylation, an
indicator of PKC activation in human
platelets(32, 43, 44) . This shows indirectly
that PLC activation (and therefore formation of diacylglycerol, the
substrate for diacylglycerol lipase) was not impaired (see also (30) ). Our results support the model of cPLA
activation whereby a Ca
-dependent lipid-binding
domain is responsible for the translocation of cPLA
to
phospholipid membranes(5, 7) .
To address the
question as to whether PKC and kinases regulated downstream of PKC are
involved in the activation of cPLA, experiments were
carried out using agents that either increase (PDBu) or inhibit (Ro
31-8220, staurosporine) PKC activity. Human platelets contain the
PKC isoenzymes
,
,
, and
of which PKC-
,
-
and -
translocate to the membrane fraction after
stimulation by thrombin, which correlated with the first phase of
diacylglycerol formation and pleckstrin phosphorylation(45) .
Ro 31-8220 inhibits purified PKC-
, -
, -
, and
-
isoforms as well as the PKC activity of a partially purified rat
brain PKC preparation (which presumably also contains PKC-
and
-
isoforms) with an IC
of less than 30
nM(27) . We would therefore expect Ro 31-8220
(10 µM) to completely inhibit all PKC activity in
platelets, and consistent with this, Ro 31-8220 inhibited
pleckstrin phosphorylation (Fig. 3). Ro 31-8220 also
blocks PKC-dependent processes such as secretion of 5-hydroxytryptamine
and slows down the rate of aggregation, whereas it does not affect
Ca
-dependent shape change in platelets, which is
mediated via myosin light chain kinase (34) , or tyrosine
phosphorylation(35) .
When applied on their own, phorbol
esters do not stimulate arachidonic acid release from platelets or
elevate intracellular Ca(46) . However, the
synergy of phorbol esters and A23187 on arachidonic acid formation, and
the inhibition of this by staurosporine and Ro 31-8220, provides
evidence for a role of PKC in the regulation of PLA
activity (present results and (33) ). The absence of any
effect of Ro 31-8220 on A23187-induced release of
[
H]arachidonic acid (Fig. 2) is consistent
with the observation that A23187 does not stimulate significant PKC
activity (Fig. 7A). Similar results have been described
by Qiu and Leslie (47) in macrophages where inhibition of PKC
either by GF109203X (25) or through down-regulation did not
interfere with A23187-stimulated release of
[
H]arachidonic acid. The molecular basis of the
synergy between phorbol esters and Ca
-ionophore is
probably mediated through PKC/MAPK dependent phosphorylation of
cPLA
(Fig. 7B and C) which, despite increasing the
specific activity of cPLA
, does not induce activation of
cPLA
in vivo in the absence of
Ca
-dependent translocation(7) .
In
contrast to the results observed with PDBu and ionophore, PKC appears
not to directly regulate collagen- or thrombin-induced arachidonic acid
formation. Ro 31-8220 did not block
[H]arachidonic acid release, nor did it inhibit
formation of inositol phosphates (35, 37) and the
intracellular Ca
rise by collagen and thrombin.
Moreover, staurosporine, which neither inhibited PLC activity in
thrombin-stimulated platelets (24) nor intracellular
Ca
elevation, did not inhibit formation of
[
H]arachidonic acid (Fig. 4, also
mentioned in (33) ). Additional evidence for the regulation of
arachidonic acid formation downstream of PLC but not downstream of PKC
comes from the effect of PDBu on thrombin-stimulated platelets; PDBu
decreased [
H]arachidonic acid release by 50% (not
shown) in parallel with its inhibiting effect on PLC(24) .
These results are in contrast with reports on zymosan-stimulated
macrophages where inhibition of PKC partially decreased the release of
[
H]arachidonic acid(47) , and on
thrombin- and ATP-stimulated Chinese hamster ovary cells where phorbol
esters enhanced [
H]arachidonic acid
release(48) . The present results indicate a major role of PLC
and Ca
in the stimulation of arachidonic acid
formation by both collagen and thrombin but provide evidence against a
direct role for PKC in this response.
Activation of cPLA in intact cells is associated with phosphorylation of the
enzyme(2, 49, 50, 51, 52, 53) ,
which increases cPLA
activity in a reconstitution
assay(2, 9, 12) . Experiments using phorbol
ester indicate that phosphorylation is not sufficient for cPLA
activation (present results and (49) ). In contrast to
results by Lin et al.(8) , who observed an inhibitory
effect of staurosporine on A23187- and ATP-induced arachidonic acid
release in Chinese hamster ovary cells overexpressing cPLA
,
we show that phosphorylation of cPLA
is not necessary for
arachidonic acid formation. In platelets, staurosporine abolished
cPLA
phosphorylation after thrombin stimulation but did not
reduce formation of [
H]arachidonic acid (Fig. 4B and Fig. 5). It might be possible that
a decrease in cPLA
activity due to the inhibition of
phosphorylation by staurosporine was counterbalanced by the enhancing
effect of the drug on Ca
. However, it is interesting
to see that despite the different effects of staurosporine and Ro
31-8220 regarding cPLA
phosphorylation, both agents
increased thrombin-induced Ca
elevation and
[
H]arachidonic acid release in a similar manner.
Furthermore, A23187 was a powerful stimulus of
[
H]arachidonic acid release within 2 min but
caused minimal phosphorylation of cPLA
during this
stimulation time.
Both p42 and p44
are
present in platelets (18, 19, 20) , and we
found that not only p42
but also p44
is
activated upon stimulation with collagen, thrombin, and PDBu. In
agreement with a report by Qiu and Leslie(47) , where A23187
did not activate MAPK in macrophages, a rise in intracellular
Ca
was not sufficient to activate this enzyme in
platelets. However, Ca
-dependent activation of MAPK
has been described previously(54, 55) .
Lin et
al.(9) demonstrated a causal link between MAPK activation
and cPLA phosphorylation by cotransfection experiments and
mutation of Ser-505, the MAPK phosphorylation site of cPLA
.
Subsequent studies in several cell types showed strong correlation
between MAPK activation and cPLA
phosphorylation under
physiological
conditions(12, 47, 51, 56, 57) .
Since the correlation between MAPK activation and cPLA
phosphorylation does not on its own establish a causal link
between these two events, we investigated the PKC dependence of the two
signaling pathways. PKC stimulates MAPK in a number of cell types
through a pathway that may involve direct activation of
Raf(58, 59) . In agreement with this, we observed that
activation of PKC by phorbol ester was sufficient to stimulate platelet
MAPK. Moreover, PKC was necessary for MAPK activation since Ro
31-8220 inhibited p42/p44
when platelets were
stimulated with the agonists tested in this study (Fig. 6). In
contrast, phosphorylation of cPLA
was maintained in the
presence of Ro 31-8220, which is consistent with the absence of
an inhibitory action of Ro 31-8220 on collagen- and
thrombin-stimulated release of [
H]arachidonic
acid. cPLA
phosphorylation does therefore not appear to be
mediated downstream of PKC, which contrasts with the regulatory pathway
of MAPK in platelets stimulated by collagen and thrombin. This suggests
that platelets must contain enzymes different from PKC and MAPK that
are capable of phosphorylating cPLA
. In agreement with
this, two reports dissociating MAPK activation and cPLA
phosphorylation were recently published(60, 61) .
In conclusion, the present study demonstrates that activation of
MAPK and phosphorylation of cPLA are regulated by distinct
pathways in collagen- and thrombin-stimulated platelets. p42
and p44
seem to be regulated downstream of PKC but
do not contribute to the physiological regulation of cPLA
.
An unidentified kinase therefore mediates phosphorylation of cPLA
in collagen- and thrombin-stimulated human platelets.