(Received for publication, December 28, 1995; and in revised form, January 22, 1996)
From the
p38 mitogen-activated protein kinase (MAPK) was identified in
platelets on the basis of (a) its reactivity with antibodies
to C-terminal and N-terminal peptides, and (b) its ability to
activate MAPK-activated protein kinase-2, which phosphorylates the
small heat shock protein, hsp27. p38 MAPK was activated in platelets by
collagen fibers, a collagen-related cross-linked peptide, thrombin, or
the thromboxane analogue U46619. A highly specific inhibitor of p38
MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet
enzyme in vitro (IC
0.5 µM).
At similar concentrations it also inhibited agonist-stimulated
phosphorylation of hsp27 in platelets, and platelet aggregation and
secretion induced by minimal aggregatory concentrations of collagen or
U46619, but not thrombin. Inhibition of aggregation was overcome by
increasing agonist dose. SB203580 might act by inhibiting thromboxane
generation, but this was only inhibited by 10-20% at low agonist
concentrations.
p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.
p38 mitogen-activated protein kinase (MAPK) ()is a
member of a family of enzymes activated by dual phosphorylation upon
threonyl and tyrosyl residues separated by a single amino
acid(1, 2, 3, 4) . The first members
of this family to be discovered in mammalian cells were the
closely-related p42 and p44 MAPKs, which are activated by a wide
variety of growth factors and many other stimuli(5) .
Subsequently p54 MAPKs (
,
, and
) (6) and p38
MAPK (1, 2, 3, 4) were identified.
These latter types of MAPK were found to be activated only weakly by
mitogens, but very strongly by stressful stimuli, endotoxin, and the
inflammatory cytokines interleukin 1 (IL1) and tumor necrosis
factor(1, 2, 3, 4, 6, 7) .
p42 and p44 MAPK phosphorylate a variety of intracellular proteins including other kinases, transcription factors, and cytoplasmic and cytoskeletal proteins. They are important for proliferative and differentiative responses(5, 8) .
p54 MAP kinases strongly phosphorylate and activate the transcription factors c-Jun (they are also known as c-Jun N-terminal kinases or JNK) and activating transcription factor-2(9, 10, 11) . Their major function appears to be to control expression of c-Jun itself and other genes regulated by activator protein-1 complexes(12) .
The function of p38 MAPK is unclear. It is related to the yeast gene product HOG1, which lies on an osmosensing pathway that regulates glycerol synthesis enabling the micro-organism to withstand hyperosmolar conditions(13) . In mammalian cells p38 MAPK is a potent activator of MAPK-activated protein kinase-2 (MAPKAPK-2), which phosphorylates the small heat shock protein (hsp27)(2, 3) . The physiological significance of this is controversial, but it may help cells resist thermal stress(14, 15) .
A group of pyridinyl imidazole compounds have recently been found to be highly specific inhibitors of p38 MAPK(4, 16) . They were discovered serendipitously as inhibitors of endotoxin-stimulated cytokine production(17) . They strongly interacted with one particular 40-kDa cellular protein which was purified, cloned, and identified as the human p38 MAPK(4) . The inhibitors are potentially a powerful experimental probe to explore the role of p38 MAPK in physiology.
During platelet activation there is a strong increase in phosphorylation in hsp27(18) . We therefore examined the possibility that p38 MAPK may be causing this and that the enzyme plays a role in platelet responses. Use of a specific inhibitor in this relatively simple cell system could provide clues to the wider functions of p38 MAPK.
For studies in intact
cells, platelets were incubated with 100 µCi/ml
[P]orthophosphate for 60 min at 30 °C, then
washed and incubated with imidazole compound or vehicle
(Me
SO) for an additional 30 min. Platelets were lysed, and
hsp27 was immunoprecipitated(3) . Immunoprecipitates were run
on SDS-gel electrophoresis, and dried gels were autoradiographed. hsp27
phosphorylation was measured by scanning autoradiographs.
Aggregation was measured at 37 °C turbidimetrically (20) and agonists were used at the minimum dose needed to elicit a full aggregatory response. In some instances aggregation and secretion of ATP were measured concomitantly in a Chronolog Aggro-Meter (model 550, Coulter Electronics Ltd.) using a bioluminescence technique(20) .
Figure 1: Detection of p38 MAPK in platelets by Western blotting (A) and as immunoprecipitated kinase (B). A, platelet lysates were run on SDS-gel electrophoresis alongside samples of purified recombinant p38 MAPK and p42 MAPK. Proteins were transferred to nitrocellulose and reacted with the indicated antisera as described under ``Experimental Procedures.'' Bars indicate positions of standard protein molecular mass markers. B, assay of immunoprecipitated p38 MAPK on MAPKAPK-2. Platelets were stimulated with 20 µg/ml collagen-related peptide for the indicated time and lysed. p38 MAPK was immunosorbed and assayed for ability to activate MAPKAPK-2, as described under ``Experimental Procedures.'' Lanes 1-5 are an experiment with natural hsp27; for lanes 6-12, recombinant hsp27 was used. Lanes 1, 2, and 6 are material from unstimulated platelets; lanes 3-5 are from platelets stimulated for 1 min with collagen-related peptide. For lanes 1 and 3, lysates were treated with preimmune serum; for lanes 2 and 4-10, they were treated with antiserum to p38 MAPK C-terminal peptide. MAPKAPK-2 was omitted from the incubation for lane 5. For lanes 7-10, platelets were stimulated for 15, 45, 120, and 300 s, respectively. Lanes 11 and 12 are samples of MAPKAPK-2 after and before phosphatase treatment.
The activity of p38 MAPK immunoprecipitated from lysates was assayed by its ability to activate MAPKAPK-2 as judged by the latter enzyme's ability to phosphorylate hsp27. Lysates of platelets activated by a cross-linked collagen-related synthetic peptide, found previously to be highly aggregatory for platelets(20) , contained increased p38 MAPK activity when compared with lysates from unstimulated cells (Fig. 1B, lane 2 versus lane 4). hsp27 phosphorylation was not seen if preimmune serum was used (lanes 1 and 3), or MAPKAPK-2 omitted (lane 5). Natural hsp27 is partially cleaved and runs as two bands. Recombinant hsp27 was used for assay of immunoprecipitates of platelets stimulated by the collagen-related peptide for increasing times. These showed increasing activity (Fig. 1B, lanes 6-10). Recombinant hsp27 substrate was used for subsequent experiments. The immunological reactivity and substrate specificity (2, 3) of the enzyme identify it as p38 MAPK.
p38
MAPK was rapidly activated in platelets stimulated by thrombin,
collagen, collagen-related peptide or the thromboxane analogue U46619. Fig. 2shows the time course (a-d) and dose
dependence (e-h) for each agonist. All increased
activity within 45 s; that induced by thrombin, collagen, and U46619
declined after 2 min. The thrombin receptor-related peptide SFLLRNPND
also activated p38 MAPK (data not shown). Activation was not dependent
on aggregation of platelets, since it was unaffected by including the
peptide RGDS (which prevents aggregation by blocking
) in the suspension at 500
µM. The effects of optimal concentration of collagen and
thrombin were not secondary to thromboxane production since they were
unaffected by aspirin treatment (data not shown).
Figure 2: Activation of p38 MAPK by platelet agonists. Platelets were prepared and stimulated for indicated times and doses. p38 MAPK was immunoprecipitated from lysates and assayed for ability to reactivate MAPKAPK-2. Each ligand was tested on at least five different batches of platelets, and representative experiments are shown. Agonists were as follows: a and e, bovine thrombin (1 unit/ml in a); b and f, native type I collagen fibers (10 µg/ml in f); c and g, collagen-related peptide (20 µg/ml in c); d and h, U46619 (0.2 µM in d).
Figure 3:
Effect of pyridinyl imidazoles upon
activity of immunoprecipitated p38 MAPK (A) and hsp27
phosphorylation in intact platelets (B). A, p38 MAPK
was immunoprecipitated from collagen-activated platelets (10 µg/ml
for 1 min), preincubated with SB203580 () or SKF86002 (
)
for 20 min, and then assayed for ability to reactivate MAPKAPK-2. A
representative experiment is shown. B, platelets were
incubated with 100 µCi/ml [
P]orthophosphate
for 60 min, then washed, and SB203580, SKF86002 or vehicle were added
for an additional 30 min. Platelets were then stimulated (1 unit/ml
thrombin for 1 min) and lysed, and hsp27 was immunoprecipitated. hsp27
phosphorylation was measured by scanning autoradiographs of SDS-PAGE.
Thrombin caused a 3-fold increase (from 2600 to 7950 arbitrary units)
in phosphorylation; results are shown as percent inhibition of total
hsp27 phosphorylation.
We then
investigated the ability of these compounds to inhibit p38 MAPK in
intact platelets. Platelets were metabolically labeled with
[P]orthophosphate, pretreated with inhibitor,
and then stimulated with thrombin and lysed. hsp27 phosphorylation was
measured as an indicator of p38 MAPK activity. SB203580 inhibited the
increase in hsp27 phosphorylation in the range 1-10
µM. SKF86002 was also effective (Fig. 3B).
Similar results were obtained with collagen.
These results were
consistent with the phosphorylation of hsp27 being regulated by p38
MAPK. MAPKAPK-2 can be activated by p42 MAPK in
vitro(2) , and p42 MAPK may be activated in
platelets(22, 23) . However, p38 MAPK is a much
stronger activator of MAPKAPK-2 than p42 MAPK(2, 3) ,
so it was not surprising that its inhibition strongly affected hsp27
phosphorylation. We next tested the effect of SB203580 upon platelet
aggregation. Platelets were stimulated by the minimum concentration of
collagen fibers sufficient to cause full aggregation. Aggregation was
strongly inhibited by 1 µM SB203580 ( Fig. 4(A and B) shows platelets of two different individuals) with
concomitant inhibition of secretion (Fig. 4B). The
relationship between the concentration of inhibitor and the degree of
inhibition was similar to that observed for inhibition of hsp27
phosphorylation (Fig. 3B). IC for
platelets from 5 individuals varied within the range 0.2-1
µM SB203580. SKF86002 was also effective. A structurally
related compound (SKF105809), which is inactive on p38
MAPK(16) , did not inhibit aggregation. The inhibition of
aggregation could be overcome by increasing the agonist concentration
3-fold (Fig. 4C). Aggregation induced by a minimum
concentration of U46619 was also strongly inhibited. Inhibition was
overcome by doubling U46619 concentration (Fig. 4D).
However, aggregation induced by a minimum aggregatory concentration of
thrombin was not reproducibly inhibited (only one of five platelet
samples was inhibitable).
Figure 4: Effects of SB203580 on platelet aggregation and secretion. A, aggregation stimulated by native type I collagen fibers at 1 µg/ml (minimal concentration to elicit a full aggregatory response). Platelets were preincubated for 30 min at 37 °C with indicated concentrations of SB203580 or vehicle. Addition of agonist is indicated with an arrow. B, a separate experiment in which secretion was also measured in response to collagen (1 µg/ml). C, inhibition by SB203580 is overcome by increasing collagen fiber concentration. D, inhibition by SB203580 of aggregation induced by U46619 is overcome by increased agonist concentration.
Thromboxane production is necessary for
platelet aggregation and might be impaired by SB203580 interfering with
cyclooxygenase. Platelets were pretreated with SB203580 then stimulated
for 1 min with collagen (10 µg/ml) as described in Fig. 1,
in the presence of both EGTA and RGDS peptide (100 µM) to
prevent aggregation. Thromboxane released into the medium (measured as
thromboxane B) rose from 3.6 to 200 ng/ml upon stimulation
and was unaffected by concentrations of SB203580 ranging from 0.3 to 10
µM. SB203580 was not acting as an inhibitor of
cyclooxygenase.
p38 MAPK appeared to provide a signal necessary for
platelet aggregation caused by low concentrations of collagen or
U46619. Aggregation caused by low concentrations of collagen fibers is
dependent upon production of thromboxane A, which causes
positive feedback via its own receptor. It was possible that p38 MAPK
activation was needed for the generation of thromboxane by low
concentrations of collagen fibers. SB203580 had a small but significant
(10-20% inhibition) effect on the amounts of thromboxane produced
in response to low concentrations of collagen. Fig. 5shows an
experiment representative of five on platelets from different
individuals. We estimated that, since a minimum aggregatory
concentration of U46619 was 30-40 nM and basal
thromboxane levels were
10 nM, then a 3-4-fold
increase over the basal level may be needed to trigger aggregation.
SB203580 is therefore as likely to be causing inhibition by interfering
with secretion or other aspects of the response, as with thromboxane
generation.
Figure 5:
Effect of SB203580 on thromboxane B generation by collagen-stimulated platelets. Platelets
(10
/ml) were incubated in suspension buffer containing the
peptide RGDS (100 µg/ml) in the absence (
) or presence of
(
) 1 µM SB203580 for 15 min. They were then
stimulated with indicated concentrations of collagen fibers for 4 min.
Suspension buffer was assayed for thromboxane B
as
described under ``Experimental Procedures.'' Results are
means of duplicate experiments.
The results indicate that p38 MAPK plays a role in platelet aggregation. It enables platelets to respond to low concentrations of collagen or thromboxane. It is not necessary for full sensitivity to thrombin. It is likely that thrombin at low concentration and collagen or thromboxane at high concentrations activate pathways that bypass p38 MAPK, rendering it redundant.
Addendum-As this paper was being prepared for submission, Kramer et al. (24) reported the activation of a p38 MAPK-like enzyme in platelets by thrombin and thrombin receptor peptide.