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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3429-3434
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Thrombopoietin potentiates collagen receptor signaling in
platelets through a phosphatidylinositol 3-kinase-dependent
pathway
Jean-max Pasquet,
Barbara S. Gross,
Marie-Pierre Gratacap,
Lynn Quek,
Sophie Pasquet,
Bernard Payrastre,
Gijsbert van Willigen,
Joanne C. Mountford, and
Steve P. Watson
From the Department of Pharmacology, University of Oxford, Oxford,
UK; the Department of Haematology (G03.647), University Hospital,
Utrecht, The Netherlands; and U326 INSERM, Hopital Purpan, Toulouse,
France.
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Abstract |
Collagen activates platelets through a tyrosine kinase-dependent
pathway, involving phospholipase C 2. Functional responses such as
aggregation and secretion induced by collagen are potentiated by
preincubation with thrombopoietin (TPO). In this study, we show that
collagen and thrombopoietin activate the phosphatidylinositol 3-kinase
(PI 3-kinase) pathway and that this contributes to their respective
actions. The structurally distinct inhibitors of PI 3-kinase,
wortmannin, and LY294002, completely inhibit formation of
phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a
substantial reduction in the formation of inositol phosphates and
phosphatidic acid, 2 indices of PLC activity, and the consequent
inhibition of intracellular Ca++
[Ca++]i, aggregation and secretion.
Potentiation of the collagen response by TPO is prevented in the
presence of wortmannin and LY294002. However, when the 2 PI 3-kinase
inhibitors are given after the addition of TPO but before the collagen,
recovery of potentiation is observed. This suggests that potentiation
is mediated through activation of PI 3-kinase. TPO stimulates
aggregation of platelets from a low percentage of donors and this is
also blocked by wortmannin. These results suggest that the PI 3-kinase
pathway plays an important role in signaling by collagen and in the
priming action of TPO.
(Blood. 2000;95:3429-3434)
© 2000 by The American Society of Hematology.
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Introduction |
Thrombopoietin (TPO) was described in 1994 as a growth
and developmental factor for the platelet precursor cell, the
megakaryocyte.1 The cellular receptor for TPO is c-mpl, a
transmembrane receptor with close homology to the erythropoietin
receptor. Expression of c-mpl has been found restricted to
hematopoietic progenitor cells, megakaryocytes, and platelets. However,
recent studies suggest c-mpl expression in neutrophils and endothelial
cells.2,3 TPO potentiates (or primes) platelet activation
by a variety of stimuli, including thrombin, adenosine diphosphate
(ADP), collagen, and adrenaline; whereas it has no
effect on its own in the majority of donors.4-9 The most
thoroughly characterized signaling cascade from the TPO receptor is the
JAK/STAT pathway.5,10 In addition, TPO stimulates tyrosine
phosphorylation of a number of proteins in platelets, including Cbl,
p85 subunit of phosphatidylinositide 3-kinase (PI 3-kinase), Shc, Vav,
and cortactin.11-13 The mechanism of platelet priming by
TPO is not established.
Over the last few years, we and others14,15 have shown that
the major collagen receptor underlying platelet activation is a
multimeric assembly consisting of glycoprotein VI (GPVI) and the Fc
receptor -chain (FcR -chain). Activation by collagen leads to
tyrosine phosphorylation of the FcR -chain on 2 conserved tyrosines
in an immunoreceptor tyosine-based activation motif (ITAM), enabling
binding of the tyrosine kinase Syk.14 Activation of Syk in
turn leads to tyrosine phosphorylation and activation of a number of
signaling proteins, including the adapters LAT and SLP-76, and
phospholipase C2 (PLC2).16-18 The importance of this
pathway in platelet activation by collagen is illustrated by the
complete inhibition of secretion and aggregation to collagen in
platelets from mice deficient in the FcR -chain, Syk, LAT, and
SLP-76.16-18 The platelet low-affinity immune receptor
FcRIIA is thought to induce activation through a similar pathway.
Recently, a collagen-related peptide (CRP), based on a
glycine-proline-hydroxyproline repeat motif, was reported to stimulate formation of 3-phosphorylated inositol lipids in platelets through the
PI 3-kinase pathway.19 CRP is a powerful agonist at the collagen receptor GPVI but does not recognize the integrin  2 1 (GPIa-IIa).20 The structurally distinct inhibitors of the
PI 3-kinase pathway, wortmannin, and LY294002, partially inhibit activation of phospholipase C (PLC) and aggregation by CRP, suggesting that this pathway has a critical role in signaling by GPVI. This is
supported by the observation that chelation of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3) in megakaryocytes by
microinjection of the pleckstrin homology (PH) domain to Bruton's
tyrosine kinase (Btk), which binds selectively to this lipid species,
inhibits CRP-induced elevation of Ca++. The PI 3-kinase
pathway also plays a critical role in platelet activation by
FcRIIA.21 PI3,4,5P3 is believed to be the
active component in this pathway because it restores full activation in
response to cross-linking of the immune receptor in permeabilized platelets.21
In this study, we have investigated the role of PI 3-kinase in the
regulation of platelets by TPO and collagen. Our results provide
evidence for an important role for PI 3-kinase in the activation of
PLC2 by collagen and show that priming of the collagen response by
TPO is mediated through a PI 3-kinase-dependent pathway.
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Materials and methods |
Materials
Recombinant human TPO was from Genzyme Diagnostics (West Malling,
Kent, UK). The antiphosphotyrosine monoclonal antibody (mAb), 4G10, and
the anti-PI 3-kinase p85 serum were from Upstate Biotechnology Inc
(TCS Biologicals, Bucks, UK). The mAb IV.3, which is selective for
FcRIIA, was purchased from Medarex Inc (Annandale, NJ). Anti-PLC2 polyclonal antibody (Q-20) was from Santa Cruz (Autogen Bioclear, Wiltshire, UK) and anti-PLC2 serum was kindly provided by Dr Y. H. Lee (Pohang University of Science and Technology, Kyung-Buk, South
Korea). Collagen fibers, as Horm collagen, a suspension of type I
fibers from equine tendon, were obtained from Nycomed (Munich,
Germany). Bovine thrombin, wortmannin, phosphatidylinositol, and
F(ab')2 antimouse IgG fragments were from Sigma
(Poole, Dorset, UK). LY294002 was purchased from Calbiochem-Novabiochem
(Nottingham, UK). Nonidet P-40 (NP40) was from BDH (Poole, Dorset, UK).
Horseradish peroxidase-conjugated sheep antimouse IgG (NA931),
myo-[3H]inositol and
[32P]orthophosphate were from Amersham International
(Cardiff, UK). Other reagents were from previously described
sources.22,23
Fura2-AM, wortmannin, and LY294002 were dissolved in dimethyl sulfoxide
(DMSO) with other reagents made up in saline. The concentration of DMSO
in the incubation never exceeded 1:1000 final dilution and an
equivalent volume of DMSO was present in control samples.
Platelet isolation and measurement of aggregation, secretion, and
[Ca++]i
Human-washed platelets were prepared from donors who had not taken
aspirin in the previous 10 days.23 Platelets were separated from plasma by centrifugation in the presence of prostacyclin (100 ng/mL), resuspended in a modified Tyrodes-HEPES buffer without added
Ca++ and recentrifuged in the presence of prostacyclin
before final suspension in the Tyrodes-HEPES buffer at a density of 2 to 4 × 108 cells per milliliter. Platelets were
left for 30 minutes to allow recovery from exposure to the
prostacyclin. Indomethacin (10 µmol/L), EGTA (1 mmol/L), and apyrase
(5 U/mL) were included as required. In a limited number of aggregation
studies, the platelets were separated from plasma by gel
filtration.24
Platelet suspensions (450 µL) were prewarmed to 37°C for 5 minutes before incubation with TPO or inhibitors of PI
3-kinase. Platelets were transferred to a
Born-aggregometer (Chronolog; Labmedics, Cheshire, UK)
and stirred at 1200 rpm for 60 seconds before addition of agonist. The
5-HT secretion was measured as previously described.22 For
measurement of [Ca++]i, the platelets were
loaded with Fura2-AM in platelet-rich plasma.25 [Ca++]i was measured in a spectofluorimeter
at 37°C under continuous agitation, with excitation at 340 and 380 nm and emission at 510 nm.
Measurement of [3H]-inositol phosphates
Washed platelets were labeled with 1.85 MBq/mL (50 µCi/mL) of [3H]myo-inositol for
3 hours at 30°C, washed, and resuspended at 4 × 108 cells per milliliter.26 LiCl
(10 mmol/L) was added to inhibit conversion of inositol phosphates into
free inositol. Reactions were performed in a final volume of 250 µL
and stopped after 5 minutes of stimulation with 0.94 mL
chloroform/methanol/HCl (50:100:1, by vol). Water (0.31 mL) and
chloroform (0.31 mL) were then added. After the separation of phases,
total [3H]inositol phosphates (mono-, bis-,
trisphosphates) were eluted by Dowex anion-exchange chromatography as
described.26
Measurement of [32P]3-phosphoinositides and
[32P] phosphatidic acid
Platelets were labeled with [32P]orthophosphate (22.2 MBq/mL [0.6 mCi/mL]) for 60 minutes, washed, and
resuspended at 1 × 109 cells per milliliter.
Reactions were stopped at the indicated time by addition of
chloroform/methanol (1:1 v/v) containing 0.4 mol/L HCl, and lipids were
immediately extracted and analyzed by high-performance liquid
chromatography (HPLC) as described.21 Phosphatidic acid was
separated by thin-layer chromatography (TLC), detected by
autoradiography, and analyzed by densitometry.
Measurement of PI 3-kinase activity
p85 or 4G10 immunoprecipitates (see below) were washed and
preincubated in 0.1 mol/L NaCl, 20 mmol/L Tris (pH 7.6), and 10 µg
phosphatidylinositol (final volume 50 µL) for 10 minutes. The reaction was initiated by addition of 10 mmol/L MgCl2, 0.02 mmol/L ATP, and 0.37 MBq (10 µCi)
[32P]ATP for 30 minutes at 30°C. Labeled
phosphoinositides were extracted, separated by TLC, and visualized by
autoradiography.27 Silica was scraped from TLC plates and
radioactivity was measured in a Beckmann scintillation counter
(Beckmann Instruments, High Wycombe, UK).
Immunoblotting and immunoprecipitation studies
For measurement of whole-cell tyrosine phosphorylation, platelet
suspensions (4 × 108/mL) were stopped with an equal
volume of Laemmli sample buffer, and the mixture was heated for 5 minutes at 100°C.23 For immunoprecipitation studies,
platelet suspensions (4 × 108/mL) were stopped by
addition of an equal volume of a nondenaturing extraction buffer (2%
NP-40, 300 mmol/L NaCl, 20 mmol/L Tris, 1 mml/L PMSF, 10 mmol/L EDTA, 2 mmol/L Na3VO4, 20 µg/mL leupeptin, 20 µg/mL
aprotinin, and 5 µg/mL pepstatin; for final concentrations divide by
2). Immunoprecipitations were carried out with 5 µL/mL anti-p85 serum
or 5 µL/mL anti-PLC2 per sample. Proteins were separated
by 10% SDS/PAGE and transferred to polyvinylidene difluoride (PVDF)
blotting. Membranes were blotted for tyrosine phosphorylation using 1 µg/mL mAb 4G10, or 1:1000 dilution of the anti-PI 3-kinase p85 or
anti-PLC2 as described. Horseradish peroxidase-conjugated sheep
antimouse IgG (NA931) or sheep antirabbit IgG (NA934) was applied as a
secondary antibody and detected by enhanced chemiluminescence (ECL).
The membranes were stripped of bound antibody by washing in TBS-T
containing 2% SDS for 30 minutes at 80°C. After verifying stripping by the use of the secondary antibody, blots were reprobed with a different primary antibody as appropriate.
Analysis of data
Each experiment was performed at least 3 times. Results are shown as
mean ± SE mean of 1 experiment performed in triplicate, or as pooled results where n represents the number of
experiments. Statistical testing was by Student t test with
P < .05 taken to indicate significance.
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Results |
Collagen and thrombopoietin stimulate formation of PI 3,4,5 P3
The ability of collagen and TPO to stimulate formation of
3-phosphorylated lipids was measured in platelets labeled with
[32P]orthophosphate. Collagen (5 µg/mL) stimulated a
significant increase in formation of
[32P]PI3,4,5P3 and
[32P]PI3,4P2 (phosphatidylinositol
3,4-bisphosphate) by 2 minutes (Table 1),
at which time, aggregation and secretion responses were more than 80%
complete. The increase in [32P]PI3,4,5P3 was
approximately 1 order of magnitude lower than the response to
CRP,19 but greater than that to the G protein-coupled receptor agonist ADP.28 TPO stimulated a lower increase in
formation of [32P]PI3,4,5P3 and
[32P]PI3,4P2 (Table 1). Formation of
PI3,4,5P3 and PI3,4P2 by both agonists was
completely inhibited by wortmannin (100 nmol/L) (Table 1) and LY294002
(50 µmol/L; not shown). Responses to collagen and TPO were maintained
in the presence of cyclooxygenase inhibitor indomethacin, GPIIb-IIIa
antagonist, RGDS, and apyrase (Table 1 and not shown).
We investigated whether tyrosine phosphorylation plays a role in the
regulation of the heterodimeric p85/110 form of PI 3-kinase by TPO and
collagen. TPO has been reported to stimulate tyrosine phosphorylation
of the p85 subunit of PI 3-kinase in platelets.12 This
observation was confirmed in this study (Figure
1A). In comparison, collagen (10 µg/mL)
induced negligible tyrosine phosphorylation of the p85 subunit of PI
3-kinase and had no effect on phosphorylation induced by TPO (Figure
1A). The consequence of phosphorylation of the p85 subunit by TPO was
investigated by in vitro measurement of PI 3-kinase activity after
immunoprecipitation, using the antiphosphotyrosine mAb 4G10, or the
anti-p85 serum, using phosphatidylinositol as the substrate lipid. TPO
stimulated an increase in PI 3-kinase activity in both sets of
immunoprecipitates that was completely inhibited by 100 nmol/L
wortmannin (Figure 1B and not shown). Collagen had no effect on the
activity of PI 3-kinase in the p85 or 4G10 immunoprecipitates (not
shown).
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| Fig 1.
TPO treatment of platelets results in tyrosine
phosphorylation and activation of PI 3-kinase.
(A) Phosphorylation of p85 subunit of PI 3-kinase. Platelet samples
were treated with 10 µg/mL collagen (2 minutes), 50 ng/mL TPO (3 minutes), or the combination of both agents (TPO for 3 minutes,
followed by collagen for 2 minutes). The platelets were then lysed
under nondenaturing conditions in 1% NP-40 and protein
immunoprecipitated with p85 antiserum. The immunoprecipitates were
submitted to SDS-PAGE and, after transfer, the membrane was blotted
with the antiphosphotyrosine monoclonal antibody (mAb) 4G10 and exposed
by ECL (upper panel). The same membrane was stripped and reprobed with
anti-p85 serum (lower panel). Results are from 1 experiment
representative of 5; the extent of phosphorylation varied widely
between donors. (B) TPO stimulates an increase in PI 3-kinase in p85
immunoprecipitates. Activity was measured by monitoring formation of
phosphatidylinositol 3-monophosphate as described in "Materials and
methods." Results are performed in duplicate and are representative
of 2 experiments.
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These results demonstrate that TPO and collagen stimulate the PI
3-kinase second messenger pathway through distinct mechanisms. Activation of PI 3-kinase by TPO is associated with tyrosine
phosphorylation of the p85 subunit of PI 3-kinase. In contrast,
collagen does not stimulate tyrosine phosphorylation of p85, but most
likely activates the p85p/110 heterodimer through recruitment to the membrane via association with the adapters LAT29 and
Cbl.30
The effect of PI 3-kinase inhibitors on PLC2 activation
A role for the PI 3-kinase pathway in the activation of PLC2 by
CRP and FcRIIA has been proposed.19,21 We were therefore interested to determine whether the PI 3-kinase pathway is also required for activation of PLC by collagen. PLC activity was assessed by measurement of [3H]inositol phosphates and
[32P]phosphatidic acid, a metabolite of
1,2-diacylglycerol (Figure 2). LY294002 and
wortmannin inhibited the response to collagen (10 µg/mL) over the
effective concentration range for inhibition of PI 3-kinase. Maximally
effective concentrations of wortmannin (100 nmol/L) and LY294002 (50 µmol/L) reduced the formation of inositol phosphates and phosphatidic
acid to collagen by up to 75%. In contrast, they had no significant
effect on the formation of inositol phosphate by the G protein-coupled
receptor agonist, thrombin (Figure 2A and 2B).
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| Fig 2.
Wortmannin and LY294002 partially inhibit
collagen-induced formation of inositol phosphates and phosphatidic
acid.
(A) [3H]Inositol labeled platelets resuspended at
4 × 108/mL were incubated with different
concentrations of either LY294002 (i) or wortmannin (ii) for 3 to 15 minutes before stimulation with 10 µg/mL collagen (s) or 0.1 U/mL
thrombin (l) for 5 minutes. Results are expressed as percentage (%) of
the response to collagen or thrombin (= 100% in each case) from 1 experiment made in quadruplicate, which is representative of 4 experiments. The mean ± SEM basal dpm was 101 ± 12.4, and
maximum stimulations over basal were 1019 ± 133.2 (collagen) and
954 ± 8.4 (thrombin). (B)
[32P]orthophosphate-labeled platelets were stimulated
with collagen (10 µg/mL) for 2 minutes and phosphatidic acid
extracted as described in "Materials and methods." LY294002 (LY)
or wortmannin (Wt) for 3 to 15 minutes before stimulation with
collagen. Results are shown as mean ± SEM percentage of the
increase in phosphatidic acid by collagen over basal from 3 experiments.
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The effect of PI 3-kinase inhibitors on aggregation and
dense granule secretion
Experiments were designed to investigate whether the inhibitory
effect of PI 3-kinase inhibitors on PLC activation by collagen is
associated with inhibition of aggregation and dense granule secretion.
Aggregation induced by a low concentration of collagen is converted to
shape change in the presence of the PI 3-kinase inhibitors LY294002 and
wortmannin (Figure 3A). Full inhibition of
aggregation by LY294002 and wortmannin occurs at 20 µmol/L and 100 nmol/L, respectively (Figure 3A). These concentrations correspond to
those required for full inhibition of PI 3-kinase activity in platelets
stimulated with collagen. Aggregation induced by higher concentrations
of collagen (more than 3 µg/mL) was slowed and partially inhibited by
wortmannin and LY294002 (Figure 3B). The 2 PI 3-kinase inhibitors also
inhibited secretion of [3H]5-HT from dense granules,
producing a rightward shift in the concentration response curve to
collagen (Figure 3C).
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| Fig 3.
Effect of PI 3-kinase inhibitors on platelet aggregation
and secretion of 5-hydroxytryptamine (5-HT).
Platelets were isolated as described in "Materials and methods"
and resuspended at 2 to 4 × 108/mL in a modified
Tyrode's buffer. Platelets were treated with LY294002 (LY, i) or
wortmannin (Wt, ii) for 3 minutes at 37°C before stimulation. All
experiments were performed in a Born aggregometer. (A) and (B) An
increase in optical density (OD) represents shape change that precedes
the decrease, due to aggregation. The traces are representative of at
least 10 experiments. (C) [3H]5-HT secretion was analyzed
as described in "Material and methods," the concentrations of
LY294002 and Wt were 50 µmol/L and 100 nmol/L, respectively.
Platelets were incubated with collagen for 2 minutes. Results are shown
as the mean ± SEM of 3 experiments.
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TPO (50 ng/mL) stimulated aggregation in approximately 20% of donors
after isolation of platelets by gel filtration, which could be
reproduced for the same donor over time (Figure
4). A lower frequency of response (less
than 5%) was seen in platelets isolated by centrifugation. Aggregation
induced by TPO was not preceded by shape change and was inhibited
completely by the PI 3-kinase inhibitors, wortmannin, and LY294002, ADP
scavengers, and by the protein kinase C antagonist, Ro 31-8220 (Figure
4 and not shown).
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| Fig 4.
TPO stimulates aggregation of platelets in a
subpopulation of donors.
Aggregation of gel-filtered platelets was monitored by light
transmission where a decrease in optical density (OD) reflects
aggregation. (i) Platelets from approximately 1 in 5 donors were found
to undergo aggregation in response to TPO (50 ng/mL). (ii)
Wortmannin (Wt) preincubated 15 minutes before the addition of TPO
was found to completely block aggregation. The aggregation trace is
representative of 4 experiments.
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Potentiation of collagen-induced platelet activation by
thrombopoietin abrogated by inhibitors of PI 3-kinase
Potentiation of the collagen response by TPO is illustrated in
Figure 5, in which the response to a
threshold concentration of the matrix protein is converted to maximal
aggregation after treatment with the cytokine. We sought to determine
whether activation of the PI 3-kinase pathway by TPO underlies this
potentiation. Wortmannin and LY294002 inhibited aggregation induced by
the combination of TPO and the threshold concentration of collagen
(Figure 5A). However, it is unclear whether this inhibitory action is
due solely to an effect against collagen or because of an additional
action against TPO. To address this, the platelets were treated with TPO before the addition of wortmannin or LY294002 and then challenged with collagen. Recovery of potentiation was observed in both cases (Figure 5A).
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| Fig 5.
Priming of platelet responses to collagen by TPO is
mediated through PI 3-kinase.
(A) Platelets were preincubated with (i) 50 µmol/L LY294002 (LY) or
(ii) 100 nmol/L wortmannin (Wt) given either 3 minutes before or 2 minutes after TPO (50 ng/mL). In both sets of experimental paradigms,
platelets were left for 5 minutes after the addition of TPO before
stimulation by collagen (1 µg/mL). (B) Platelets were preincubated 3 minutes with TPO (100 ng/mL) and/or apyrase (1 U/mL) as indicated and
then stimulated by collagen. Results are shown from 1 experiment that
is representative of at least 5.
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Secretion of ADP serves as a positive feedback signal in the activation
of platelets by collagen. It was therefore important to establish
whether the ability of TPO to induce potentiation was maintained in the
presence of the ADP scavenger, apyrase. Apyrase causes a shift to the
right in the collagen concentration response curve to collagen for
aggregation, as illustrated in Figure 5B. Potentiation by TPO was
retained in the presence of apyrase, demonstrating that it is not
dependent on release of ADP (Figure 5B).
Experiments were designed to investigate whether the priming action of
TPO on collagen is associated with elevation of
[Ca++]i. TPO potentiated the rate and
magnitude of the Ca++ response to collagen (Figure
6A, i). When wortmannin was added before
TPO and collagen, the increase in Ca++ was reduced to a
lower level than for collagen alone (Figure 6A, ii). In contrast, when
wortmannin was added after TPO but before collagen, potentiation of the
Ca++ response was restored (Figure 6A, ii). Potentiation of
the collagen Ca++ response by TPO was also observed in the
presence of apyrase, confirming that it is not dependent on the release
of ADP (Figure 6B).
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| Fig 6.
Priming of platelet responses to collagen by TPO is
accompanied by a PI 3-kinase-dependent Ca++ increase.
Fura-2-loaded platelets (108/mL) were stimulated in a
plastic cuvette with stirring at 37°C in the presence of 1 mmol/L
extracellular Ca++. Fluorescence emission was recorded at
510 nm for an excitation ratio 340/380 nm. In (A) experiments were
performed as follows: (i) collagen (2 µg/mL) or TPO (100 ng/mL) for 3 minutes and then collagen (2 µg/mL); (ii)
wortmannin (Wt) (100 nmol/L) for 3 minutes, followed by TPO for 3 minutes and then collagen (2 µg/mL), or TPO (100 ng/mL) for 3 minutes, followed by Wt (100 nmol/L) for 3 minutes and then collagen (2 µg/mL). In (B) experiments were performed as follows: (i) collagen (5 µg/mL), or TPO (100 ng/mL) for 3 minutes, followed by collagen (5 µg/mL); (ii) same as (i) but in the presence of apyrase (1 U/mL). The
addition of collagen is indicated by an arrow. Results are from 1 experiment made in triplicate representative of 4.
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We investigated whether the effect of the 2 PI 3-kinase inhibitors and
TPO on the response to collagen is mediated by alteration of
phosphorylation of PLC2. A submaximal concentration of collagen (10 µg/mL) stimulated tyrosine phosphorylation of PLC2 (Figure 7), whereas TPO (50 ng/mL) had only a
negligible effect (not shown). Neither wortmannin nor LY294002 had a
significant effect on the collagen-stimulated
tyrosinephosphorylation of PLC2 (Figure 7), although a small
reduction in phosphorylation was observed in some experiments
(not shown). A similar observation was reported in platelets stimulated
by CRP.18 There was no significant change in the
tyrosine phosphorylation of PLC2 in response to collagen in the
presence of TPO (Figure 7).
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| Fig 7.
Phosphorylation of PLC2 by collagen in the
presence of TPO.
PLC2 tyrosine phosphorylation induced by TPO and collagen. Platelets
were stimulated with stirring by collagen (10 µg/mL) for 90 seconds;
TPO (50 ng/mL) for 300 seconds; TPO (50 ng/mL) for 300 seconds,
followed by collagen for 90 seconds. Wortmannin (100 nmol/L) and
LY294002 (50 µmol/L) are preincubated 3 minutes before the addition
of TPO or collagen. PLC2 was immunoprecipitated and probed for
tyrosine phosphorylation using the monoclonal antibody 4G10 (upper
panel). Membranes are stripped and reprobed for PLC2 (lower panel).
Results are from 1 experiment representative of 3.
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Discussion |
We have previously reported that collagen stimulates tyrosine
phosphorylation and activation of PLC2 in platelets. In this study,
we show that collagen also activates the PI 3-kinase pathway in
platelets, leading to the formation of PI3,4,5P3 and
PI3,4P2. The PI 3-kinase pathway is required for full
activation of PLC activity because 2 structurally distinct inhibitors
of PI 3-kinase, LY294002 and wortmannin, partially inhibit formation of
inositol phosphates and phosphatidic acid by collagen at concentrations that result in complete inhibition of PI 3-kinase activity. This is
associated with inhibition of aggregation, secretion, and
Ca++ elevation. In contrast, neither inhibitor has a major
effect on the activation of PLC by the G protein-coupled receptor
agonist thrombin. These observations confirm our previous study using CRP,19 which provided evidence for a partial role for the
PI 3-kinase pathway in the regulation of PLC2 by the GPVI-selective peptide, CRP. A similar conclusion has also been made for the regulation of PLC2 by FcRIIA.21 It is notable that
collagen induces a much lower increase in the formation of
PI3,4,5P3, compared with CRP, but that wortmannin and
LY294002 cause a similar reduction in the formation of inositol
phosphates by both stimuli. This suggests that the level of the newly
generated 3-phosphorylated lipids is not rate limiting in the
regulation of PLC2. In support of this, we have shown that the
elevation of PI3,4,5P3 in murine platelets deficient in the
5' lipid phosphatase, SHIP, in response to GPVI cross-linking,
does not lead to increased activation of phospholipase C (J. Pasquet
and S. Watson, unpublished data).
Our study also provides evidence that the cytokine TPO enhances
platelet secretion and aggregation by collagen through a PI 3-kinase-dependent pathway. TPO stimulates formation of
PI3,4,5P3 and its metabolite PI3,4P2. The
treatment of platelets with either LY294002 or wortmannin before the
addition of TPO prevents potentiation of the response to collagen,
although this could be due solely to inhibition of the response to
collagen. However, when either inhibitor is given after TPO but before
the addition of collagen, potentiation is restored. This potentiation
is maintained for several minutes in the presence of wortmannin and LY
294002, suggesting that the liberated 3-phosphorylated lipids are
protected against metabolism, presumably as a consequence of
interaction with the PH domains.
There are several mechanisms whereby elevation in PI3,4,5P3
and/or PI3,4P2 in response to TPO could lead to
potentiation of the response to collagen. These include enhanced
recruitment and activation of the tyrosine kinase Btk and/or PLC2 to
the plasma membrane, and potentiation of Ca++ entry.
PI3,4,5P3 has been shown to support the translocation of
Btk to the plasma membrane, where it is able to phosphorylate PLC2.31 PI3,4,5P3 has also been shown to be
required for translocation of PLC1 to the membrane through
interaction with its src homology 2 (SH2) or PH
domains.32,33 PI 3-kinase is also required for the
recruitment of PLC2 to the membrane by GPVI in megakaryocytes because translocation is inhibited by the pretreatment with wortmannin (R. Bobe and S. Watson, unpublished data).
Additionally, elevation of PI3,4,5P3 in murine mast
cells deficient in the 5' lipid phosphatase, SHIP, leads to
enhanced Ca++ entry34; a similar result has
been observed in platelets (J. Pasquet and S. Watson, unpublished data).
Platelets from a limited number of donors aggregate in response to TPO
through a pathway that is exquisitely sensitive to wortmannin, and
which may therefore be related to the mechanism that underlies
potentiation of the response to collagen. Additionally, we show that
the activation of protein kinase C and the release of ADP are required
for aggregation by TPO in these donors. Although the relationship
between these events is not established, it is notable that
3-phosphorylated lipids support activation of atypical forms of protein
kinase C, and that this pathway contributes to platelet
aggregation.35,36 Further, the requirement for ADP in
aggregation suggests that this might be the result of potentiation of
the ADP response by TPO, possibly through the same pathway as described
in this study.
In summary, we have shown that PI 3-kinase has an important role in
platelet activation by collagen through the regulation of PLC2, and
that the priming effect of TPO on collagen is mediated through a PI
3-kinase-dependent pathway, leading to potentiation of PLC activity
and increased intracellular Ca++ concentration.
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Footnotes |
Submitted July 7, 1999; accepted January 27, 2000.
Supported by funding from The Wellcome Trust and The British Heart
Foundation. S.P.W. is a British Heart Foundation Research Fellow.
B.S.G. is a Wellcome Trust Prize Student. L.Q. holds a BHF Studentship.
G.V.W. is a Fellow of the Catharijne Foundation. This project was
initiated by J.C.M., who laid much of the foundation for the work.
J.P., B.S.G., and M.-P.G. contributed equally to this work.
Reprints: Steve P. Watson, Department of Pharmacology,
University of Oxford, Mansfield Rd, Oxford Ox1 3QT, UK; e-mail: stevewatson{at}pharm.ox.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction