From the
The thrombin receptor agonist peptide SFLLRN was less effective
than thrombin in eliciting the liberation of arachidonic acid and the
generation of thromboxane A
Thrombin, a serine protease, is a potent agonist for platelets,
eliciting shape change, secretion of granular contents, and
aggregation. Thrombin also evokes the release of arachidonic acid from
platelet membrane phospholipids and induces the generation of
prostaglandin endoperoxides, TXA
We have shown
recently that the Ca
Our study shows that in human platelets, the thrombin
receptor agonist peptide SFLLRN causes only partial phosphorylation and
activation of cPLA
The structure
determination of the thrombin receptor by cloning has provided a new
insight into the mechanism by which this receptor is
activated(2) . The peptide SFLLRN was found to be an agonist for
the thrombin receptor and to mimic many cellular effects of
thrombin(2, 3, 4, 28, 29) .
However, in human platelets, SFLLRN fails to match the production of
TXA
As previously demonstrated, increasing
[Ca
Studies on the
involvement of MAP kinases in functional properties of platelets, a
terminally differentiated and highly specialized cell, have only
recently been initiated. In agreement with our studies, Nozawa and
co-workers (41) observed that thrombin stimulates the MAP
kinases ERK1/2 in human platelets with optimal activation occurring
after 2 min. On the other hand, Papkoff et al.(42) reported that in human platelets, thrombin activates
the MAP kinase ERK2 but not ERK1 since they could not detect either
enzymatic activity or a shift in the electrophoretic mobility of ERK1.
Clearly, in the present study, thrombin induced the activation of both
ERK1 and ERK2 as new bands with altered reduced mobilities appeared
with the concomitant disappearance of the faster migrating forms.
Conversely, SFLLRN failed to activate the MAP kinases ERK1 and ERK2 as
demonstrated by gel shift analysis (Fig. 7B) and assay
of Thr
The MAP kinases ERK1/2 are currently
thought to be responsible for the activating phosphorylation of
cPLA
In summary, the differential activation of cPLA
Aliquots (8.3 µl; derived from
We gratefully acknowledge the synthesis of SFLLRN by
Eddie Angleton and thank Larry Froelich, Chi Lin, and Bob Shuman for
contributions to this work. Our special thanks go to Beth Strifler,
Todd Pickard, and John Sharp for providing purified cPLA
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
by human platelets. We found
that while SFLLRN evokes an initial transient increase in cytosolic
free calcium concentration
([Ca
]
) of similar
magnitude as that caused by thrombin, the SFLLRN-induced elevation of
[Ca
]
declines more
rapidly to near resting levels than that evoked by thrombin, suggesting
that disparate levels of [Ca
]
may contribute to the attenuated arachidonic acid release.
Furthermore, we observed that SFLLRN is less effective than thrombin in
mediating the ``activating'' phosphorylation of cytosolic
phospholipase A
(cPLA
). Both thrombin and
SFLLRN rapidly and transiently activated kinases that phosphorylate the
21-residue synthetic peptide Thr
derived from the
epidermal growth factor receptor, but the maximal activation of
proline-directed kinases by SFLLRN was less pronounced than that by
thrombin. MonoQ chromatography and immunoblot analysis of extracts from
stimulated platelets revealed that while thrombin induced a prominent
activation of the mitogen-activated protein kinases ERK1 and ERK2,
SFLLRN completely failed to do so. On the other hand, SFLLRN, like
thrombin, stimulated the activity of a proline-directed kinase distinct
from ERK1/2, but the activation of this kinase was less pronounced
following stimulation of platelets with SFLLRN compared with thrombin.
We conclude 1) that the partial activation of cPLA
and the
subsequent attenuated mobilization of arachidonic acid in response to
SFLLRN may be the consequence of a less prolonged elevation of
[Ca
]
and insufficient
activation of proline-directed kinase(s) by SFLLRN and 2) that the
ability of SFLLRN to mediate the activating phosphorylation of
cPLA
in the absence of ERK1/2 stimulation suggests that, at
least in human platelets, proline-directed kinases other than ERK1/2
may phosphorylate and activate cPLA
.
,
(
)and other metabolites of arachidonic acid(1) .
These thrombin-induced platelet responses are mediated by cell-surface
receptors that belong to the family of seven-transmembrane domain
receptors coupling to G proteins, but that are activated by a novel
mechanism(2) . Thus, receptor activation occurs when thrombin
cleaves the extracellular amino-terminal portion of the thrombin
receptor, exposing a new amino terminus that functions as a tethered
peptide ligand for the receptor(2) . SFLLRN peptides, containing
the first six residues of the new amino terminus, have been shown to
mimic the effect of thrombin, causing platelets to secrete and
aggregate(2, 3, 4) . Like thrombin, they
activate phospholipase C, phosphatidylinositol 3-kinase, and protein
kinase C(5, 6, 7) ; inhibit adenylate
cyclase(6, 8) ; and induce phosphorylation on tyrosine
residues of multiple platelet proteins(7, 9) . However,
there is increasing evidence that thrombin receptor peptides are not
full agonists for activation of platelets (10, 11) and
other cells(12, 13, 14) .
-sensitive cytosolic
phospholipase A
(cPLA
) in human platelets is
responsible for thrombin-stimulated mobilization of arachidonic acid
for the synthesis of TXA
. Incubation of platelets with
thrombin induced the phosphorylation of cPLA
, thereby
increasing its intrinsic catalytic activity(15) . In the present
study, we have examined the effect of the thrombin receptor agonist
peptide SFLLRN on the phosphorylation and activation of
cPLA
. We found that SFLLRN does not match the substantial
mobilization of arachidonic acid and TXA
generation induced
by thrombin and activates cPLA
only partially compared with
thrombin. We further discovered that SFLLRN is unable to evoke a
sustained increase in [Ca
]
or to mediate activation of the MAP kinases ERK1 and ERK2.
These results demonstrate that in human platelets, both
[Ca
]
transients and
activation of proline-directed kinases may play a critical role in
thrombin-induced activation of cPLA
.
Platelet Isolation and Incubation
Blood from
healthy volunteers was drawn into 0.16 volume of acid/citrate/dextrose
(85 mM trisodium citrate, 64 mM citric acid, and 111
mM glucose) containing a 10M concentration of a stable prostacyclin
analog(15, 16) . Platelets were isolated as described
previously (15) and suspended in 140 mM NaCl, 27 mM KCl, 1 mM MgCl
, 5.5 mM glucose, 0.5
mM EGTA, 10 mM Hepes, pH 7.4. After the addition of
CaCl
and the glycoprotein IIb-IIIa antagonist
cyclo-(S,S)-Mpr(Har)GDWPPen-NH
(kindly
provided by Dr. Robert Scarborough, COR Therapeutics) (17) to
final concentrations of 3 mM and 30 µM,
respectively, platelet suspensions (final concentration of 0.5
10
/ml or as indicated) were incubated at 37 °C with
-thrombin (
3500 NIH units/mg; Enzyme Research Laboratories)
or SFLLRN (C-terminal amide) synthesized as detailed before(3) .
Incubations were terminated, and platelet sonicates were prepared as
detailed previously(15) . Labeling of Platelet Phospholipids with
[
H]Arachidonic Acid and Agonist-induced
Release of [
H]Arachidonic
Acid-Platelet-rich plasma (20 ml) was prepared from blood of
donors who had taken 325 mg of aspirin the night before and again on
the morning of phlebotomy and incubated for 1 h at 37 °C with 10
µCi of [5,6,8,9,11,12,14,15-
H]arachidonic
acid (
60 Ci/mmol; DuPont NEN) added in 1 ml of 5 mM Tris/HCl, pH 8, bound to fatty acid-free bovine serum albumin (100
mg/ml). Free [
H]arachidonic acid was removed by
washing platelets as detailed above. The incorporation of
[
H]arachidonic acid into platelet phospholipids
was
25%, and the content of esterified
[
H]arachidonic acid was
2
10
dpm/10
platelets.
[
H]Arachidonic acid-labeled platelets suspended
at 0.5
10
/ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl
, 3 mM CaCl
, 5.5 mM glucose, 0.5 mM EGTA, 10
mM Hepes, pH 7.4, were incubated with thrombin (5 units/ml) or
SFLLRN (80 µM). Reactions were stopped by adding 2 ml of
Dole's reagent (2-propanol, heptane, 0.5 M sulfuric acid
(40:10:1)), and the released [
H]arachidonic acid
plus 12-lipoxygenase metabolites were extracted and quantified as
described(18) .
Intracellular Ca
[CaMobilization
]
was measured using the fluorescent Ca
indicator indo-1/AM (Molecular Probes, Inc.). Platelet-rich
plasma was incubated for 45 min at room temperature with 3.3 µM indo-1/AM. The platelets were then pelleted and washed as detailed
above; suspended at 0.5
10
/ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl
, 3 mM CaCl
, 5.5 mM glucose, 0.5 mM EGTA,
10 mM Hepes, pH 7.4, containing 30 µM glycoprotein IIb-IIIa antagonist; and transferred to a glass
cuvette, which was placed in a thermostatically controlled chamber at
37 °C in an SLM-AMINCO Model 48000 spectrofluorometer and stirred
magnetically. After 2 min, thrombin or SFLLRN was added directly to the
cuvette at the indicated concentrations. Indo-1 was excited at 355 nm,
and emission was detected at 405 and 485 nm, the fluorescence maxima
for Ca
-bound and Ca
-free indo-1,
respectively. Changes of the fluorescence intensities at these two
emission wavelengths were continuously monitored during stimulation.
The platelet suspension was then centrifuged, and the fluorescence of
the supernatants was assessed and subtracted from the suspension
readings to correct for extracellular probe. The relative changes in
the ratio of the fluorescence at 405 nm to that at 485 nm (i.e. ratio = 405 nm/485 nm) were used to quantitate the changes
in [Ca
]
upon
stimulation with thrombin or SFLLRN.
[Ca
]
was calculated
assuming a K
of 250 nM for
indo-1(19) .
MonoQ Chromatography
Soluble extracts were diluted
5-fold with 1 mM EGTA, 1 mM DTT, 100 µM NaVO
, 50 mM
-glycerophosphate, pH 7.2, and then applied to a MonoQ HR 5/5
column equilibrated in 25 mM NaCl, 1 mM EGTA, 1
mM DTT, 100 µM Na
VO
, 50
mM
-glycerophosphate, pH 7.2, using a Pharmacia Biotech
fast protein liquid chromatography system. After extensive washing, the
column was eluted with a 40-ml linear salt gradient from 25 to 500
mM NaCl at a flow rate of 1.5 ml/min. Fractions of 1 ml were
collected and assayed for kinase activity.
Assay for Proline-directed Kinases
Platelets were
prepared and exposed to thrombin or SFLLRN as detailed above. The
reaction was stopped by adding (final concentrations) 1% Triton X-100,
10 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.2
mM NaVO
, 1 µM
microcystin, 100 µM leupeptin, 0.2 mg/ml aprotinin, 10
µM pepstatin A, 1 mM phenylmethylsulfonyl
fluoride, 50 mM
-glycerophosphate, pH 7.5. The suspension
was centrifuged at 10,000
g for 15 min at 4 °C
(for kinase assays in lysates) or at 100,000
g for 60
min at 4 °C (for MonoQ chromatography). Kinase assays were
performed as described by Ahn et al.(20) with
modifications as follows. The final assay volume was 25 µl and
contained (final concentrations) 2 mM synthetic peptide
Thr
or Ala
(KRELVEP/ALTPSGEAPNQALLR;
Macromolecular Resources, Colorado State University), 0.3 mg/ml myelin
basic protein (Sigma), 1.5 mg/ml human cPLA
or S505A
cPLA
(modified by site-directed mutagenesis and produced
and purified from a baculovirus/insect cell expression system as
described previously (21)), and 8.33 µl of platelet extract (20
µg of protein) or MonoQ column fraction. Reactions were initiated
by the addition of 8.33 µl of 3
ATP reaction buffer to
provide (final concentrations) 0.1 mM Na
VO
, 1 mM DTT, 10 mM
MgCl
, 2 µM protein kinase A inhibitor peptide
(Sigma), 1 mM EGTA, 50 mM
-glycerophosphate, pH
7.5, 0.1 mM ATP (containing 40 µCi/ml
[
-
P]ATP,
2000 Ci/mmol; DuPont NEN) and
were allowed to proceed for 20 min at 30 °C. The reactions were
stopped by the addition of 10 µl of 90% formic acid containing 50
mM ATP. Aliquots of 25 µl were applied to phosphocellulose
membranes in SpinZyme separation units (Pierce). The membranes were
washed four times with 0.5 ml of 1% phosphoric acid using
centrifugation, retrieved from the unit, and transferred to
scintillation vials containing 1 ml of 1% SDS. After the addition of 10
ml of Ready Protein
(Beckman Instruments), the
radioactivity was counted by liquid scintillation counting. Nonspecific
incorporation of radioactivity was determined in the absence of
peptide/protein substrate.
SDS-PAGE and Immunoblotting
After the addition of
SDS sample buffer, platelet lysates were incubated at 60 °C for 15
min and electrophoresed at 4 °C on 10% Tris/glycine gels at 35 mA
for 3.5 h (for cPLA detection) or at 30 mA for 2.5 h (for
MAP kinase detection) using the Novex system. Standard proteins
included rainbow markers (range of 14.3-200 kDa; Amersham Corp.),
human cPLA
, and MAP kinase ERK1 (Upstate Biotechnology,
Inc.). After electroblotting, the nitrocellulose sheets were probed
with mouse M3-1/horseradish peroxidase conjugate or rabbit anti-MAP
kinase ERK1/2 antibodies (erk1-CT (Upstate Biotechnology,
Inc.) and ERK 2 (C-14) (Santa Cruz Biotechnology)) and developed using
the ECL detection system (Amersham Corp.) as detailed
before(15) .
Human
platelets from donors that had taken aspirin were labeled with
carrier-free [P
Labeling,
Immunoprecipitation, and Phosphoamino Acid Analysis
P]H
PO
as
described(15) . Platelets were incubated at 1
10
/ml for 5 min at 37 °C without or with 5 units/ml
thrombin as detailed above. The reaction was stopped by adding EDTA to
a final concentration of 10 mM and by pelleting platelets for
1 min at 8500
g at 4 °C. The platelets were
solubilized, and cPLA
was immunoprecipitated with rabbit
anti-cPLA
IgG as described(15) . Immunoprecipitated
cPLA
(
200 ng) derived from 4
10
platelets was subjected to SDS-PAGE (10% gel, 1.5 mm thick, 10
wells), electroblotted onto Immobilon-P (Millipore Corp.), and
visualized by Coomassie Blue staining. The band containing the
P-labeled cPLA
was excised and subjected to
acid hydrolysis with 5.7 N HCl for 1 h at 110 °C as
described(22) . The samples were dried in a Speed Vac (Savant
Instruments, Inc.); suspended in 5 µl of water containing 10 µg
each of phosphoserine, phosphothreonine, and phosphotyrosine; and
subjected to ascending thin-layer chromatography on cellulose plates
(Merck) with butanol/acetic acid/ethanol/H
O (1:1:1:1) as
the mobile phase. The plates were dried, sprayed with
EN
HANCE (DuPont NEN), and exposed to x-ray film. The
positions of standard phosphoamino acids were identified by staining
with ninhydrin.
Assay of PLA
PLAActivity
activity was assayed using
sonicated liposomes containing
1-palmitoyl-2-[
C]arachidonoyl-sn-glycero-3-phosphocholine
(
50 mCi/mmol; DuPont NEN) and sn-1,2-dioleoylglycerol
(Avanti Polar Lipids, Inc.) at a molar ratio of 2:1 as described
previously (23) with modifications as follows. The assay buffer
consisted of 1 mM DTT, 150 mM NaCl, 50 mM Hepes, pH 7.5, containing 1 mg/ml bovine serum albumin, 1 mM CaCl
, 2 µM
[
C]arachidonoylphosphatidylcholine (50,000 dpm),
1 µM dioleoylglycerol, and incubations were carried out at
37 °C for 15 min. The results are presented as
disintegrations/minute [
C]arachidonic acid
released.
Other Methods
Protein measurements were made in
the presence of 0.05% SDS using Coomassie Plus protein assay reagent
(Pierce) with bovine serum albumin as a reference standard. The
concentration of thromboxane B (reflecting TXA
generation) was determined by radioimmunoassay as described
previously(24) . Platelet aggregation and ATP release were
assayed as described previously(25) .
Functional Responses of Platelets
In agreement
with previous studies, we found that SFLLRN, like thrombin, induced
platelet aggregation and ATP
secretion(2, 3, 9) . We determined that the
concentrations of thrombin and SFLLRN for half-maximal release of ATP
were 0.33 ± 0.12 units/ml and 3.8 ± 1.7 µM
(mean ± S.E. of three independent experiments using different
donors), respectively. The concentrations causing half-maximal
aggregation of platelets induced by thrombin and SFLLRN were 0.15
± 0.05 units/ml and 2.6 ± 2.0 µM (mean
± S.E. of three independent experiments using different donors),
respectively. In agreement with previous work, the molar concentration
of peptide required to produce a response equivalent to that of
thrombin was 1000 times that of thrombin (1 unit/ml thrombin taken
to be 10 nM).
Release of Arachidonic Acid and TXA
We observed that human platelets stimulated
with the peptide SFLLRN produce less TXAProduction
than platelets
stimulated with thrombin (Fig. 1). As shown with a dose
dependence and time course study, the generation of TXA
by
platelets was maximal with 25 µM SFLLRN and stopped after
1 min of activation. In contrast, thrombin-induced TXA
formation continued to increase up to 10 min. We next examined
whether the lower amount of TXA
formed in response to
SFLLRN was the result of decreased mobilization of arachidonic acid. We
prelabeled aspirinized platelets with
[
H]arachidonic acid and measured the release of
radiolabeled arachidonic acid (including 12-lipoxygenase metabolites)
from platelet phospholipids after stimulation with thrombin or SFLLRN.
As shown in Fig. 2, the liberation of arachidonic acid was
significantly lower when platelets were stimulated with SFLLRN compared
with thrombin. These findings suggest that the enzyme responsible for
thrombin-induced arachidonic acid release, cPLA
, may not be
fully activated by SFLLRN. Kinetics of Elevation of [Ca
]
by Thrombin and SFLLRN-Previous studies using
cell-free systems and purified cPLA
have demonstrated that
Ca
plays a critical role in the activation of
cPLA
as it promotes association of cPLA
with
its membrane phospholipid substrate(26, 27) . We
therefore questioned whether dissimilar
[Ca
]
responses may be
the reason for the differential mobilization of arachidonic acid in
platelets stimulated with SFLLRN and thrombin. SFLLRN peptides were
reported to stimulate a transient increase in
[Ca
]
similar to
thrombin(2, 28, 29) . To verify that this was
also true for human platelets, we loaded platelets with indo-1 and
compared SFLLRN- and thrombin-induced changes in
[Ca
]
by
spectrofluorometry. As shown in Fig. 3, SFLLRN and thrombin
elevated [Ca
]
from
resting levels of
50 nM to 1 and 0.6 µM,
respectively, with two apparent differences in the time course of the
[Ca
]
transients.
First, the maximal increase in
[Ca
]
induced by SFLLRN
was greater than and preceded that induced by thrombin. Second, the
[Ca
]
transient evoked
by SFLLRN declined more rapidly with time than that induced by
thrombin. These results demonstrate that although SFLLRN reproduces the
thrombin-induced initial rise of
[Ca
]
to
600
nM, it is unable to maintain the prolonged elevation of
[Ca
]
at
300
nM caused by thrombin. Hence, the differential release and
metabolism of arachidonic acid observed with SFLLRN and thrombin could
be a consequence of dissimilar
[Ca
]
responses.
Figure 1:
Thrombin- and SFLLRN-induced TXA production in human platelets. A, dose dependence.
Platelets at 0.5
10
/ml were incubated for 5 min at
37 °C with thrombin and SFLLRN as indicated. B, time
course. Platelets were incubated at 37 °C with thrombin (5
units/ml) or SFLLRN (80 µM) for various times as
indicated. The reaction was stopped by adding indomethacin to a final
concentration of 10 µM; platelets were pelleted; and
supernatants were assayed for thromboxane B
(the stable
metabolite of TXA
) as described under ``Experimental
Procedures.'' The data are representative of two different
experiments, and values shown are the means ± range from two
duplicate incubations.
Figure 2:
Time course of thrombin- and
SFLLRN-induced release of [H]arachidonic acid in
platelets. Aspirinized platelets prelabeled with
[
H]arachidonic acid were suspended at 0.5
10
/ml and incubated with thrombin (5 units/ml) or SFLLRN
(80 µM) at 37 °C. The reaction was stopped, and
released [
H]arachidonic acid ([
H]AA; including
12-lipoxygenase products) was determined as described under
``Experimental Procedures.'' The data are representative of
two different experiments, and values shown are the means ±
range of two separate incubations.
Figure 3:
Effect of thrombin and SFLLRN on changes
in [Ca] in human platelets. Indo-1-loaded
human platelets (0.5
10
/ml) were exposed to
thrombin (2 units/ml) or SFLLRN (80 µM) for 5 min. The
change in indo-1 fluorescence was continuously monitored at 37 °C
in the fluorometer, and Ca
transients were
quantitated as described under ``Experimental Procedures.''
After 5 min, the [Ca
] in thrombin- and
SFLLRN-stimulated platelets in this experiment was 211 and 73
nM, respectively. The data are representative of three
separate experiments with platelets obtained from different
donors.
Phosphorylation and Activation of
cPLA
Thrombin induces phosphorylation of platelet
cPLA, improving its catalytic activity
2-3-fold(15) . Phosphoamino acid analysis of
P-labeled cPLA
immunoprecipitated from
platelets revealed the presence of phosphoserine after thrombin
stimulation (Fig. 4), indicating that a serine protein kinase may
be responsible for the activating phosphorylation of cPLA
in platelets. Thrombin-mediated phosphorylation converts
cPLA
to a form with decreased electrophoretic mobility,
thereby providing a convenient means to monitor stimulus-induced
phosphorylation and activation of cPLA
(15, 30). As
demonstrated with a dose dependence experiment (Fig. 5) and a
time course study (Fig. 6), SFLLRN induced an incomplete gel
shift and hence only partial activating phosphorylation of cPLA
compared with thrombin (Fig. 5A and 6A).
Accordingly, the SFLLRN-induced enhancement of cPLA
activity in lysates derived from stimulated platelets was lower
than that observed with thrombin (Fig. 5B and
6B). These results suggest that the liberation of arachidonic
acid and the production of TXA
in platelets may not be
sustained in response to SFLLRN compared with thrombin due to
incomplete phosphorylation and activation of cPLA
.
Figure 4:
Phosphoamino acid analysis of cPLA from control and thrombin-stimulated platelets.
P-Labeled human platelets (10
/ml) were
incubated for 5 min without (-THR) or with (+THR) 5 units/ml thrombin as detailed under
``Experimental Procedures.'' cPLA
was
immunoprecipitated with anti-cPLA
IgG, subjected to
SDS-PAGE, and electroblotted onto Immobilon-P. The strips containing
cPLA
were subjected to acid hydrolysis and phosphoamino
acid analysis by thin-layer chromatography on cellulose plates. Shown
here is the fluorogram of the thin-layer plate on which phosphoamino
acids were resolved by ascending chromatography as detailed under
``Experimental Procedures.'' The positions of the
phosphoamino acids were determined by ninhydrin staining of standards
added to each extract. P, free phosphate; P-Y,
phosphotyrosine; P-T, phosphothreonine; P-S,
phosphoserine.
Figure 5:
Dose dependence of the effect of thrombin
and SFLLRN on cPLA activity in lysates from human
platelets. Platelets (0.5
10
/ml) were incubated at
37 °C for 5 min in the presence of thrombin or SFLLRN as indicated
and lysed as detailed under ``Experimental Procedures.'' A, SDS-PAGE/immunoblot probing with mouse M3-1/horseradish
peroxidase conjugate. Solubilized lysates (20 µg of protein) from
platelets stimulated with SFLLRN (lanes 1-5) or thrombin (lanes 6-10) were electrophoresed on a 10% gel. B, determination of cPLA
activity in lysates (20
µg) from platelets stimulated with thrombin or SFLLRN. The data are
representative of two different experiments; values shown for
cPLA
activity are the means ± range of two separate
platelet incubations.
Figure 6:
Time course of activation of cPLA in lysates from thrombin- and SFLLRN-stimulated platelets.
Platelets (0.5
10
/ml) were incubated at 37 °C
in the presence of thrombin (1 unit/ml), SFLLRN (30 µM),
or buffer for the indicated times and lysed as detailed under
``Experimental Procedures.'' A, SDS-PAGE/immunoblot
probing with mouse M3-1/horseradish peroxidase conjugate. Solubilized
lysates (20 µg of protein) from platelets incubated for 10 min with
buffer (lane1) or stimulated for 1-10 min with
SFLLRN (lanes2-5) or thrombin (lanes
6-9) were electrophoresed on a 10% gel. B,
determination of cPLA
activity in lysates (20 µg) from
platelets stimulated with thrombin or SFLLRN. The data are
representative of two different experiments; values shown for
cPLA
activity are the means ± range of two separate
platelet incubations.
Activation of Proline-directed
Kinases
Phosphorylation by MAP kinases is thought to be a
prerequisite for activation of cPLA and subsequent
mobilization of arachidonic acid in Chinese hamster ovary cells that
overexpress cPLA
(30) . We compared the kinetics of
thrombin and SFLLRN-induced activation of these kinases using as
substrate the 21-residue synthetic epidermal growth factor receptor
peptide Thr
known to contain the consensus sequence
Pro-Leu-Thr
-Pro for MAP kinases(31) . As shown in Fig. 7A, thrombin induced the activation of kinases that
phosphorylate Thr
in a time-dependent manner, reaching
peak levels at 2 min and remaining significantly elevated after 10 min.
In contrast, SFLLRN caused an early transient kinase activation within
1 min of stimulation that rapidly declined thereafter and remained only
slightly elevated over basal levels. On average, the maximal activation
of kinases that phosphorylate Thr
by SFLLRN (after 1 min)
was reduced significantly (53 ± 13%, mean ± S.D. of five
independent experiments) compared with that achieved by thrombin (after
2 min). Immunoblot analysis using antibodies directed against ERK1 or
ERK2 (Fig. 7B) revealed that after treatment of
platelets with thrombin, two new immunoreactive bands of lower
electrophoretic mobility became visible with the concomitant
disappearance of the corresponding 42- and 44-kDa bands. This shift in
electrophoretic mobility was most pronounced after 2-5 min. In
contrast, no such mobility shift of the ERK1/2 bands indicative of
activation (32) could be detected when platelets were stimulated
with SFLLRN. Upon MonoQ chromatography of soluble platelet extracts,
the Thr
kinase activity eluted as two peaks after
exposure of platelets to thrombin and as one peak after stimulation
with SFLLRN (Fig. 8B). No kinase activity could be
detected in MonoQ fractions from control platelets (data not shown).
The first peak of kinase activity eluting with 150 mM NaCl
(Peak I) was only observed after stimulation with thrombin and
coincided with the elution of ERK1 and ERK2 as detected by
immunoblotting (Fig. 8C). The second peak of Thr
kinase activity eluting with 350 mM NaCl (Peak II) was
apparent after both thrombin and SFLLRN treatment of platelets and
comprised two-thirds and all of the Thr
kinase activity
induced by thrombin and SFLLRN, respectively. Preliminary
characterization of the Peak II kinase activity is summarized in , showing that it readily phosphorylated myelin basic
protein and cPLA
. Furthermore, the kinase in Peak II, like
ERK1/2, exhibited reduced activity with the synthetic peptide
Ala
, indicating that the amino-terminal proline is
necessary for maximal kinase activity. More important, S505A cPLA
was a poor substrate, suggesting that the Peak II kinase, like
ERK1/2, targets Ser
within the MAP kinase phosphorylation
site of cPLA
. In summary, the above findings demonstrate
that the magnitude and time course of proline-directed kinase
activation by SFLLRN are distinct from those observed with thrombin.
Furthermore, they show that while SFLLRN fails to stimulate the MAP
kinases ERK1/2, it activates, like thrombin, another proline-directed
kinase that may phosphorylate cPLA
at Ser
.
Figure 7:
Activation of proline-directed kinases in
human platelets stimulated by thrombin and SFLLRN. A, time
course of thrombin- and SFLLRN-stimulated kinase activities. Platelets
(0.5 10
/ml) were incubated at 37 °C with
thrombin (5 units/ml), SFLLRN (80 µM), or buffer and
solubilized with 1% Triton X-100, and kinase activities were determined
using the Thr
(EGFR
)
peptide substrate as detailed under ``Experimental
Procedures.'' B, immunoblot analysis of platelet lysates,
probing with anti-ERK1/2 antibodies (erk1-CT, anti-ERK1; or
ERK 2 (C-14), anti-ERK2). Platelets (0.5
10
/ml)
were incubated at 37 °C with buffer (lanes1 and 2) or were stimulated with thrombin (5 units/ml; lanes
3-8) or SFLLRN (80 µM; lanes
9-14) for 0-10 min. Cell lysates (2 µg) were
analyzed by SDS-PAGE/immunoblotting as detailed under
``Experimental Procedures.'' Agonist-induced phosphorylation
and activation of the MAP kinases ERK1/2 are reflected by an upward
mobility shift on SDS-PAGE. The data are representative of three
independent experiments yielding similar results, and values shown are
the means ± range of duplicate
determinations.
Figure 8:
MonoQ chromatography of thrombin- and
SFLLRN-stimulated Thr kinase activity. Soluble extracts
derived from 2
10
platelets stimulated at 1
10
/ml with thrombin (5 units/ml) for 2 min or with SFLLRN
(80 µM) for 1 min were subjected to chromatography on
MonoQ as detailed under ``Experimental Procedures.'' The
column was developed with a 40-ml linear gradient of 25-500
mM NaCl, and 1-ml fractions were collected at a flow rate of
1.5 ml/min. A, elution of proteins monitored by absorbance at
280 nm. B, determination of kinase activity in 8.3-µl
aliquots of fractions as indicated using the synthetic peptide
substrate Thr
(EGFR
).
The data shown are the means ± range of two separate assays.
Incorporation of radioactive phosphate was <250 dpm for flow-through
and high-salt (0.5-1 M NaCl) fractions. Likewise, assays
using MonoQ fractions from control platelets or assays performed in the
absence of the Thr
peptide yielded <250 dpm
P radioactivity. C, 25-µl aliquots of
fractions containing kinase activity (fractions 12-17 and
26-30) as well as 10 µg of control platelet lysate (laneC) and 20 ng of purified MAP kinase (laneErk1) were electrophoresed on 10% gels and immunoblotted
with anti-ERK1/2 antibodies (erk1-CT). The migration position
of the molecular mass marker ovalbumin is indicated on the right. The
data are representative of three independent
experiments.
and is less effective than thrombin in
promoting the release of arachidonic acid and production of
TXA
. We present evidence to indicate that this differential
activation may be attributed to differences in
[Ca
]
signaling and
attenuated activation of proline-directed kinases by SFLLRN compared
with thrombin. Our results further demonstrate that in
SFLLRN-stimulated platelets, the activating phosphorylation of
cPLA
is mediated by a proline-directed kinase distinct from
the MAP kinases ERK1/2, suggesting that phosphorylation and activation
of cPLA
may occur independent of ERK1/2.
induced by thrombin, as also reported by others (10, 11) while this present work was in progress. Our
studies indicate that this can be attributed to the inability of SFLLRN
to sustain the activation of cPLA
, the enzyme responsible
for supplying free arachidonic acid for the synthesis of TXA
in human platelets. It is of interest to note that SFLLRN
peptides failed to reproduce other characteristic cellular effects of
thrombin. Thus, in endothelial cells, thrombin receptor agonist
peptides were unable to induce expression of the intracellular adhesion
molecule ICAM-1(12) , to increase the mRNA levels of
thrombomodulin(33) , and to promote secretion of
platelet-derived growth factor(34) . The extent to which
thrombin receptor agonist peptides fail to mediate mitogenesis in
hamster fibroblast cells remains controversial(35, 36) .
]
from levels of
resting cells (
80 nM) to levels typically found in
stimulated cells (
300 nM) is necessary to promote
association of cPLA
with its membrane phospholipid
substrate via the CaLB domain(27) . In oocytes expressing
functional human thrombin receptor(2) , HEL cells(28) ,
and endothelial cells(13, 29) , SFLLRN peptides promoted
a transient increase in [Ca
]
that was similar to the
[Ca
]
response elicited
by thrombin. It is noteworthy that in endothelial cells, thrombin and
SFLLRN peptides achieve equivalent formation of lysophosphatidylcholine
and/or production of prostacyclin, suggesting a comparable activation
of PLA
(29, 37, 38) . On the other
hand, it was reported that in endothelial cells, thrombin receptor
agonist peptides failed to induce the translocation of protein kinase C
from the cytosol to the plasma membrane, indicative of protein kinase C
activation(13) . We observed that in human platelets, the
decrease of elevated [Ca
]
was accelerated with SFLLRN compared with thrombin. Thus,
the SFLLRN-induced increase in
[Ca
]
declined to near
resting levels within 1-2 min of stimulation, while the rise of
[Ca
]
after thrombin
treatment remained elevated around 300 nM for at least 3 min.
As the prolonged elevation of
[Ca
]
after the
addition of thrombin in platelets was found to be due to continuing
influx of external Ca
(39) , it appears that
SFLLRN does not mediate the later phase of external Ca
entry in stimulated platelets. In this regard, the SFLLRN-induced
[Ca
]
transient
resembles the one induced by thrombin in the absence of external
Ca
. It is well known that thrombin-induced
mobilization of arachidonic acid in human platelets is diminished in
the absence of extracellular Ca
(40) . Given
our previous observation that the activating phosphorylation of
cPLA
in human platelets occurs independent of external
Ca
(15) , we conclude that the discrepancy in
the late Ca
signaling induced by SFLLRN compared with
thrombin is not likely to be responsible for the differential
activation of proline-directed kinases, but may compromise the
association of cPLA
with its membrane phospholipid
substrate in the later phase of platelet activation.
kinase activity after MonoQ chromatography (Fig. 8B).
in stimulated cells. First, it was shown that purified
ERK2 phosphorylates cPLA
in vitro, increasing its
activity and altering its electrophoretic mobility, thereby mimicking
the agonist-induced activating phosphorylation of cPLA
observed in many cell systems(30, 43) . Second,
phosphorylation by the MAP kinase ERK2 in vitro was identical
to the phorbol ester-stimulated phosphorylation of cPLA
overexpressed in Chinese hamster ovary cells occurring at the
consensus phosphorylation site for MAP kinase
(Pro-Leu-Ser
-Pro). Third, studies with macrophages
stimulated with physiological agents demonstrated that the activation
of ERK1/2 correlated closely with the activation of cPLA
cells, suggesting that ERK1/2 may not only be responsible for the
activation of overexpressed, but also endogenous
cPLA
(44, 45) . On the other hand,
phosphorylation and activation of cPLA
can take place
independent of the MAP kinases ERK1/2. First, in macrophages treated
with ionophore A23187, the activity of cPLA
in lysates was
increased despite the fact that A23187 was unable to promote
stimulation of ERK1/2(44) . Second, it was recently reported
that heparin suppresses endothelin-induced stimulation of the MAP
kinases ERK1/2 in glomerular mesangial cells without affecting the
activation of cPLA
(46). Third, we show here that in
SFLLRN-stimulated platelets, cPLA
exhibits an
electrophoretic mobility shift indicative of the activating
phosphorylation despite the fact that both ERK1 and ERK2 are inactive.
However, SFLLRN, like thrombin, activates another proline-directed
kinase (Peak II in Fig. 8B) that phosphorylates
cPLA
. The maximal activation of this kinase by SFLLRN is
approximately half of that induced by thrombin and thus correlates with
the half-maximal activation of cPLA
by SFLLRN compared with
thrombin.
by thrombin and the thrombin receptor agonist peptide SFLLRN
appears to be the result of a difference in the prolonged elevation of
[Ca
]
that may affect
the association of cPLA
with its membrane phospholipid
substrate and a distinction in the stimulation of proline-directed
kinases that may activate cPLA
by phosphorylation. SFLLRN
fails to stimulate the MAP kinases ERK1/2, but induces, at least
partly, the activating phosphorylation of cPLA
, suggesting
that a proline-directed kinase distinct from ERK1/2 may activate
cPLA
by phosphorylation. Hence, the thrombin receptor
agonist peptide provides a useful tool to dissect and elucidate the
biochemical events associated with thrombin-induced activation of
cPLA
. It will be of great interest to further define the
signaling pathway(s) connecting the thrombin receptor to cPLA
and to identify the proline-directed kinase(s) responsible for
cPLA
activation in human platelets.
Table: Comparison of the substrate specificity of
thrombin-activated proline-directed kinases resolved by MonoQ
chromatography
2
10
platelets) of MonoQ fractions from thrombin-stimulated
platelets (as in Fig. 8) comprising Peaks I and II (eluting with 150
and 350 mM NaCl, respectively) were incubated with substrates
as detailed under ``Experimental Procedures.'' Incorporation
of radioactive phosphate into various substrates was normalized with
respect to phosphorylation of the Thr
peptide (
7000
and
12,000 dpm for Peak I and II fractions, respectively). The
values represent the average of two separate MonoQ chromatographies,
each assayed in duplicate. Identical results were obtained with Peak II
fractions from SFLLRN-stimulated platelets. In control assays performed
in the absence of substrate, the
P radioactivity recovered
on phosphocellulose membranes was <2%.
, thromboxane A
;
cPLA
, cytosolic phospholipase A
;
[Ca
], cytosolic free calcium concentration;
MAP, mitogen-activated protein; DTT, dithiothreitol; PAGE,
polyacrylamide gel electrophoresis.
and S505A cPLA
and to Natalie Ahn for helpful advice
on the MAP kinase assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.