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Blood, 15 December 2000, Vol. 96, No. 13, pp. 4246-4253
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Fyn and Lyn phosphorylate the Fc receptor  chain downstream of
glycoprotein VI in murine platelets, and Lyn regulates a novel
feedback pathway
Lynn S. Quek,
Jean-Max Pasquet,
Ingeborg Hers,
Richard Cornall,
Graham Knight,
Michael Barnes,
Margaret L. Hibbs,
Ashley
R. Dunn,
Clifford A. Lowell, and
Steve P. Watson
From the Department of Pharmacology, University of
Oxford; the Nuffield Department of Medicine, John Radcliffe Hospital,
Headington, Oxford; the Department of Biochemistry, University of
Cambridge, Cambridge, United Kingdom; the Ludwig Institute for Cancer
Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital,
Victoria, Australia; and the Department of Laboratory Medicine,
University of California at San Francisco, San Francisco, CA.
 |
Abstract |
Activation of platelets by collagen is mediated by the complex
glycoprotein VI (GPVI)/Fc receptor  (FcR chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling
has been examined using murine platelets deficient in one or both
kinases. In the fyn / platelets, tyrosine
phosphorylation of FcR chain, phopholipase C (PLC) activity,
aggregation, and secretion are reduced, though the time of onset of
response is unchanged. In the lyn/
platelets, there is a delay of up to 30 seconds in the onset of
tyrosine phosphorylation and functional responses, followed by recovery
of phosphorylation and potentiation of aggregation and  -granule
secretion. Tyrosine phosphorylation and aggregation in response to
stimulation by collagen-related peptide is further attenuated
and delayed in fyn/lyn/
double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune
receptor tyrosine-based activation motif phosphorylation and PLC2
activation after the ligation of GPVI. Lyn plays an additional role in
inhibiting platelet activation through an uncharacterized inhibitory pathway.
(Blood. 2000;96:4246-4253)
© 2000 by The American Society of Hematology.
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Introduction |
Platelets interact with subendothelial
collagen through several receptors, including glycoprotein VI (GPVI)
and the integrin 2 1. Although
21 is important for platelet adhesion to
collagen, major changes in tyrosine phosphorylation, including
activation of phospholipase C 2 (PLC2), are thought to be
mediated predominantly by the ligation of GPVI.1,2 A
collagen-related peptide (CRP), based on a repeated GPP* sequence
(single-letter amino acid code: P*, hydroxyproline), activates
GPVI without binding to
21.3,4 More recently, the
major constituent of the venom of the tropical rattlesnake
Crotalus durissus terrificus, convulxin, has been described
as a specific ligand for GPVI.5,6 GPVI is a member of IgG
superfamily7 and is associated with the Fc receptor (FcR) chain, a low-molecular-weight (12-14 kd) homodimer that contains an immune receptor tyrosine-based activation motif (ITAM) in
its cytoplasmic tail.7 Tyrosine phosphorylation of the
ITAM recruits the cytoplasmic tyrosine kinase p72syk (Syk)
and initiates a signaling cascade, leading to the activation of
phosphoinositide 3'kinase (PI 3'kinase)8 and
PLC2.9 This sequence of events closely parallels those
for many immune receptors, indicating a divergence of function of this
signal transduction pathway in hematopoietic cells.
The Src family of tyrosine kinases is believed to phosphorylate
the ITAM after the activation of immune receptors. Available evidence
suggests that this is also the case for GPVI. Structurally distinct
Src-family kinase inhibitors PP1 and PD173956 inhibit platelet
activation by collagen, CRP, and convulxin.10,11 In contrast, they have a weak inhibitory effect against activation induced
by the G-protein-coupled receptor agonist thrombin.10,11 Two independent studies in human platelets have proposed a role for Fyn
and Lyn in mediating phosphorylation of the FcR chain, including the
observation that both kinases coimmunoprecipitate with FcR
chain.10,11 In contrast, similar interactions with the
FcR chain were not observed for other platelet Src-family kinases,
namely Src, Yes, Fgr, Lck, or Hck.10,11 Fyn and Lyn were
also found to associate with a number of other tyrosine phosphorylated proteins in GPVI-stimulated platelets,10 several of which
have since been characterized, including the adapter
SLP-76,12 the tyrosine kinase Btk,13 and
PLC2.12 However, little is known of the functional
roles of Fyn and Lyn in collagen-mediated platelet activation.
Furthermore, there have been no comparative studies of the roles of Fyn
and Lyn in platelet signaling.
Our results show that lack of Fyn or Lyn results in reduced or
delayed phosphorylation of the FcR chain, respectively, in response
to CRP. Further, in lyn/ platelets, the
initial delay is followed by a recovery of FcR chain phosphorylation
and potentiation of activation. A double deficiency of both Fyn and Lyn
causes a greater impairment of tyrosine phosphorylation and functional
responses of platelets to CRP, and recovery of potentiation is lost.
| |
Materials and methods |
Materials
Source of mice.
Fyn/ breeding pairs, engineered on an Sv129
background,14 were from Jackson Laboratories (Bar Harbor,
ME). Controls for fyn/ experiments were
fyn+/ mice raised from crosses between
fyn/ and C57Bl6J mice and wild-type C57Bl6J
and CD1 mice. Lyn/ mice15 were
initially bred from a population of heterozygotes kindly supplied by Dr
V. Tybulewicz (Institute of Medical Research, London, England).
Controls were either wild-type littermates or CD1 mice.
Fyn/lyn/ double-knockout
mice were bred on a mixed-strain background. These mice were raised
from mixed breedings of fyn/
background14 and lyn/
mice.16 Controls for the double-knockout mice were
double-positive wild-type mice from a mixed background and C57Bl6J
mice. Fgr/ mice were as
described.17 Mice were genotyped by PCR amplification of
mouse tail genomic DNA, and lack of expression of protein was confirmed
by Western blot platelet lysates. There were no significant differences
in platelet responses between littermates and the various strains
of mice.
Materials for functional assays.
CRP (GCP*[GPP*)]10GCP*G; single-letter amino acid code
where P* represents hydroxyproline) was cross-linked as
described.4 PP1 was dissolved in dimethyl sulfoxide
(DMSO). The final concentration of DMSO in platelet samples did not
exceed 0.1%. Flow cytometric analysis (FACS) was performed using
FACScalibur apparatus (Becton Dickinson, Oxfordshire, United
Kingdom), and the results were analyzed using Cell Quest
software (Becton Dickinson). Materials for FACS analysis of P-selectin
surface expression were purchased from sources described
previously.18 Fura-2-AM, a cell-permeant ratiometric calcium reporter, was purchased from Molecular Probes (Leiden, The Netherlands). The fluorometer used was supplied by PerkinElmer (Norwalk, CT). Data were analyzed using FWinlab
software (PerkinElmer). [32P]-Orthophosphate and
Hyperfilm (Amersham Pharmacia Biotech, Amersham, United
Kingdom) used for autoradiography were obtained as described previously.3 Anti-Btk (BL7), anti-Syk (BR15), and
anti-PLC2 (DN84) rabbit polyclonal antiserum were the kind gifts
of Drs M. G. Tomlinson and J. Bolen (DNAX Research Institute,
Palo Alto, CA). Anti-SLP-76 (H3) monoclonal antibody was the kind
gift of Dr G. Koretzky (Abramson Family Cancer Research Institute,
Philadelphia, PA). Other antibodies and reagents were obtained
from sources previously described.8,13
Methods
Isolation of murine platelets.
Blood was drawn from terminally CO2-anesthetized mice by
cardiac puncture using acid citrate dextrose as an anticoagulant (1:9
vol/vol). Preparation of murine platelets has been previously described.18 Platelets were resuspended to the appropriate
platelet concentration in acidified HEPES-Tyrode's buffer (20 mmol/L
HEPES, 135 mmol/L NaCl, 3 mmol/L KCl, 0.35 mmol/L
Na2HPO4, 12 mmol/L NaHCO3, and 1 mmol/L MgCl2, pH 7.30). Typically, 1.0 mL whole blood from an adult male wild-type mouse yields 5 to
8 × 108 platelets.
Stimulation of murine platelets.
Aggregation and flow cytometric analysis studies were carried out at a
concentration of 1 × 108 platelets/mL. Aggregation was
measured using a 300-µL sample in a Born aggregometer (Chronolog,
Havertown, PA) with high-speed stirring (1200 rpm) at
37°C.
FACS analysis of P-selectin surface expression in murine
platelets.
For measurements of P-selectin cell surface expression, murine
platelets were stimulated at a concentration of
8 × 107/mL in aliquots of 50 µL, with mixing but
without stirring. Platelets were stimulated by various agonists for 10 minutes in the presence of 10 µmol/L indomethacin and 1 mmol/L
CaCl2 unless stated otherwise. Two microliters primary
antimouse P-selectin (CD62P) IgG and 5 µL secondary fluorescein
isothiocyanate-conjugated rat antimouse IgG were used per platelet
sample to detect exposed P-selectin. Samples were incubated at room
temperature for 10 minutes with primary and secondary antibodies and
then diluted by the addition of 500 µL HEPES-Tyrode's buffer and
analyzed immediately.
Calcium fluorometry.
Platelets isolated from PRP were resuspended in HEPES-Tyrode's buffer
(pH 7.3) to a concentration of 5 × 108/mL. Murine
platelets were loaded with the calcium reporter Fura-2 by incubation
with 10 µmol/L Fura-2-AM for 1 hour at 30°C. Platelets were stimulated with fast stirring at 37°C in a fluorometer with an
excitation wavelength of 530 nm and emission wavelengths at 330 and 380 nm. The platelet concentration used was 8 × 107/mL. The
ratio of Fura-2 emissions were measured and analyzed using
phycoerythrin FWinlab software. Ratiometric analysis was converted to a
concentration of Ca++ by taking measurements of
Rmax (maximal fluorescence in lysed, labeled platelets in
the presence of 5 mmol/L CaCl2) and Rmin (minimum fluorescence in lysed, labeled platelet suspension in the
presence of 5 mmol/L EGTA).
[32P]-Labeling of murine platelets and
preparation of lysates for SDS-PAGE and for thin-layer chromatography
of phospholipids.
Platelets were labeled with 500 µCi
[32P]-orthophosphate per milliliter platelet
suspension for 60 minutes at 37°C. Washed, labeled platelets were
stimulated at a concentration of 2 × 108/mL in the
presence of 10 µmol/L indomethacin and 1 mmol/L EGTA in 100 µL
sample volumes in an aggregometer, as described above. Twenty-microliter aliquots of platelets were removed and added to equal
volume 2× sample buffer at various time points of stimulation and
resolved by 10% SDS-PAGE. Gels were stained with Coomassie blue before
drying to check for equal loading of protein. Pleckstrin was detected
as a phosphorylated band at 47 kd. Autoradiographs were taken using
Hyperfilm. The remaining 80-µL sample was lysed using 4 × volume of lipid extraction buffer (CHCl3:methanol, 1:1 vol/vol) and treated with 1 mmol/L EDTA and 0.4 mol/L HCl.
Each lysed sample was vortexed at high speeds for 30 seconds to ensure thorough mixing. Phases were separated by fast centrifugation. Lower-phase phospholipids were dried under N2 with gentle
heating (30°C) and resuspended in 30 µL CHCl3:methanol
(1:1, vol/vol). Silica thin-layer chromatography plates were pretreated
with a priming solution (0.5 mol/L oxalic acid in 70% methanol and
30% water) before the separation of phospholipids using a solution of
acidified CHCl3:methanol (87:13; vol/vol) as running
buffer. Phosphatidic acid appears as the major separated band using
pretreated plates and is visualized by autoradiography using Hyperfilm.
The phosphatidic acid band of silica was then scraped off the
thin-layer chromatography plate into scintillant for
scintillation counting.
Preparation of murine platelet lysates.
Platelets were stimulated in the presence of 10 µmol/L indomethacin
and 1 mmol/L EGTA, at 37°C under stirring conditions in an
aggregometer. Whole-platelet lysates and platelet lysates for immunoprecipitation were carried out at a concentration of
1 × 108 platelets/mL, using no more that 100 µL
platelet suspension per sample. Immunoprecipitation was carried out as
previously described.10,13
Analysis of data.
Results are shown as mean ± SEM. Statistical significance was tested
using the Student t test. All experiments were performed at
least 3 times.
| |
Results |
Selective inhibitor of Src-family kinases PP1 inhibits activation
of murine platelets by CRP
PP1 (10 µmol/L) completely inhibits shape change and aggregation
of murine platelets stimulated by an intermediate concentration of CRP
(1.0 µg/mL) (Figure 1A). Shape change
represents the transition from a resting discoid morphology to an
optically more dense spherical form. Aggregation of platelets is
detected as a decrease in optical density. The EC50 value
for inhibition by PP1 is approximately 3 µmol/L. Increasing the
concentration of CRP to a maximally effective concentration of 10 µg/mL does not overcome the inhibition (not shown). In contrast, PP1
(10 µmol/L) has no significant effect on thrombin (0.1 U/mL)-induced
shape change or the early phase of aggregation, though it inhibits
late-phase aggregation by up to 50% (Figure 1B). This suggests that,
as reported in human platelets, Src-family kinases are involved in
GPVI-mediated signaling and have a minor role in regulating later
phases of the thrombin pathway in murine platelets.
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| Figure 1.
Effect of PP1 on CRP and thrombin-stimulated
aggregation.
Platelets were incubated with 0.1% DMSO or PP1 for 5 minutes before
the addition of the agonist. (A) Shape change and aggregation of
wild-type murine platelets was measured in a Born aggregometer after
stimulation with CRP (1.0 µg/mL) in the absence and presence of PP1
(1-10 µmol/L). (B) Aggregation of wild-type murine platelets to
thrombin (0.1 U/mL) was measured in the absence or presence of PP1
(1-10 µmol/L). Arrowhead indicates the time of agonist addition. The
experiment is representative of 3 experiments with similar
results.
|
|
Independent expression of Fyn and Lyn in murine platelets
Immunoblots of whole-cell lysates from fyn/
platelets show that though there is no detectable Fyn expression,
Lyn is expressed at levels equivalent to those in wild-type platelets
(Figure 2A). A complementary set of
observations was made in lyn/ platelets.
Immunoblotting of platelet lysates from mice deficient in both kinases
confirms their lack of expression, whereas the expression of Tec family
kinase Bruton tyrosine kinase (Btk) was normal (Figure 2C). Therefore,
we found no evidence of compensatory changes in the expression of
Lyn and Fyn in fyn/ and
lyn/ mice.
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| Figure 2.
Immunoblots of lysates from
fyn/,
lyn/, and
fyn/lyn/
platelets.
Murine whole-platelet lysates were immunoblotted with anti-Fyn,
anti-Lyn, and anti-Btk antisera after 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (A)
Fyn/ platelets express no detectable Fyn but
normal levels of Lyn. (B) Conversely, lyn/
platelets express normal levels of Fyn but not Lyn. (C)
Fyn/lyn/ express neither Fyn
nor Lyn but normal amounts of Btk. The traces are representative of 5 to 20 experiments.
|
|
Tyrosine phosphorylation in fyn/
platelets
The profile of tyrosine phosphorylation in whole-cell lysates in
fyn/ platelets stimulated by collagen, CRP,
or thrombin was not significantly different from that of control cells
(not shown). However, FcR chain precipitated from CRP-stimulated
fyn/ platelet lysates using a GST fusion
protein of the tandem SH2 domains of Syk (GST-(SH2)2-Syk)
is less phosphorylated than in control platelets (Figure
3A). FcR chain appears as several
bands in the region of 12 to 14 kd, which are likely to represent
different tyrosine-phosphorylated forms.19 Tyrosine
phosphorylation of immunoprecipitated Syk and the adapters LAT and
SLP-76 from CRP-stimulated fyn/ platelets
were also similarly reduced compared to control platelets (Figure
3B-C). However, CRP-induced tyrosine phosphorylation of PLC2 was not
decreased in fyn/ platelets (Figure 3D).
Wild-type and fyn/ samples were prepared in
parallel and resolved on the same gel. The separate panels shown for
each protein precipitation in the figure were from the same exposure. A
similar set of phosphorylation results was obtained in
collagen-stimulated platelets (not shown).
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| Figure 3.
Tyrosine phosphorylation in CRP-stimulated wild-type and
fyn/ platelets.
Murine platelets were treated with 1 mmol/L EGTA and 10 µmol/L
indomethacin for these experiments. Antiphosphotyrosine immunoblots
show that CRP (1.0 µg/mL)-induced tyrosine phosphorylation of FcR
chain (A), Syk (B), LAT (C), and PLC2 (D) in wild-type and
fyn/ platelets. FcR chain was
precipitated using GST fusion protein of the tandem SH2 domains of Syk.
We were unable to obtain a reprobe for LAT from LAT
immunoprecipitations because the antibody could not be readily used for
Western blotting for murine platelets. The results are representative
of 3 to 5 experiments. Wild-type and knockout samples were obtained
from experiments run in parallel. Results shown in each panel in A are
from samples resolved on the same gel and are from the same exposure on
the same film. This also applies to B-D.
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Parameters of PLC activity are reduced in
fyn/ platelets
The degree of tyrosine phosphorylation of PLC2 does not
necessarily correlate with its activity because other pathways are also
involved in the regulation of the phospholipase for example, the PI
3'kinase pathway.8,20 It was, therefore, important to
measure PLC activity. Although inositol 1,4,5-triphosphate (InsP3) is a direct product of PLC, it cannot be measured
in the small number of platelets obtained from mice using standard
techniques. As an alternative, we measured levels of phosphatidic acid
(PA), a metabolite of 1,2-diacylglycerol, in
[32P]-labeled platelets and the formation of
[32P]-pleckstrin, a substrate of protein kinase
C.8,18 Activation of phospholipase D does not contribute
significantly to the increase in [32P]-PA because of the
low turnover of the inositol head group, which means that the phosphate
group in the 1-position is minimally radiolabeled during short
incubations. A reduction in PA formation of approximately 50% was seen
in response to CRP in fyn/ platelets
compared to controls (Figure 4A). The
basal level of [32P]phosphatidic acid was similar in both
wild-type and fyn/ platelets. In contrast,
thrombin-induced [32P]phosphatidic acid production was
slightly increased in fyn/ platelets (Figure
4A), though this was not statistically significant. A shorter exposure
of the autoradiogram is shown for the more powerful agonist, thrombin,
compared with CRP in the figure to enable differentiation between PA
levels induced by different concentrations of thrombin. Pleckstrin
phosphorylation was also reduced in fyn/
platelets stimulated by low concentrations of CRP but reached normal levels after stimulation with higher concentrations (Figure 4B).
This recovery is in contrast to the results for PA and can be explained
by the difference in concentration-response relations for these 2 responses in that only submaximal concentrations of CRP are
required for the maximal phosphorylation of pleckstrin. In contrast,
pleckstrin phosphorylation was not significantly altered in
fyn/ platelets in response to thrombin
compared to controls (Figure 4B).
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| Figure 4.
PLC activation in fyn/
platelets.
Pleckstrin phosphorylation (an indicator of PKC activation) and
phosphatidic acid (PA) production (a metabolite of diacylglycerol) were
measured in [32P]-labeled platelets in the presence of 1 mmol/L EGTA and 10 µmol/L indomethacin. (A) The autoradiographs show
pleckstrin phosphorylation stimulated by a range of concentrations of
CRP (0.3-3.0 µg/mL) and thrombin (0.1-1.0 U/mL) in wild-type and
fyn/ platelets. The results are
representative of 3 to 5 experiments. (B) Autoradiographs show
phosphatidic acid production stimulated by a range of concentrations of
CRP (0.3-3.0 µ/mL) and thrombin (0.1-1.0 U/mL) in wild-type and
fyn/ platelets. The results are
representative of 3 to 5 experiments. Wild-type and
fyn/ samples were run in parallel, and the
same exposure times are shown. A lighter exposure of the autoradiogram
from representative samples of the more powerful agonist thrombin is
shown to enable differentiation between PA levels stimulated by
different concentrations of thrombin. (C) Ca2+ mobilization
was measured in Fura-2-labeled murine platelets in the presence of 1 mmol/L EGTA. Wild-type and fyn/ platelets
(i) and wild-type and lyn/ platelets (ii)
were stimulated by 0.3 µg/mL CRP over 200 seconds. The arrow
indicates the point of addition of an agonist. Results are
representative of 5 experiments.
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Ca++ mobilization from intracellular stores was measured in
Fura-2-labeled platelets in the presence of 1 mmol/L EGTA and 10 µmol/L indomethacin. CRP (0.3 µg/mL) stimulated a rapid
Ca++ peak that declined to a plateau by 300 seconds in
control platelets. In fyn/ platelets, there
was a marked reduction in the initial peak, though the plateau was not
altered (Figure 4Ci). Similar results were seen for other
concentrations of CRP (not shown). In contrast, there was no change in
peak response to thrombin, though the duration of the plateau was
slightly increased in the fyn/ platelets
(not shown).
Fyn/ platelets exhibited a reduced
aggregation response over the length of the concentration response
curve to CRP (Figure 5Ai). This was
associated with an apparent prolongation of shape change to
intermediate concentrations of CRP, which was the consequence of the
reduction in aggregation. Similar results were obtained with collagen
(1.0-10.0 µg/mL, not shown). In contrast, shape change and
aggregation of fyn/ platelets in response to
submaximal concentrations of thrombin (0.1 U/mL) were not decreased at
early time points when compared to controls. However, there was a
reduction in the late-phase aggregation response to thrombin (Figure
5Aii). In comparison, there was no alteration in aggregation induced by
CRP in platelets deficient in the Src-family kinase, Fgr (not shown),
which is also expressed in platelets.17 Activation of
platelets by CRP is also associated with the exposure of P-selectin, a
constituent of -granule membrane. P-selectin expression in
fyn/ platelets was partially inhibited in
response to CRP (0.1-1.0 µg/mL) (Figure 5B), whereas the response to
thrombin was not significantly altered (not shown).
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| Figure 5.
Functional responses of control and kinase-deficient
platelets.
(A) Platelet shape change and aggregation stimulated with CRP or
thrombin. (i) Wild-type and fyn/ platelets
stimulated with CRP (0.3-3.0 µg/mL). (ii) Wild-type and
fyn/ platelets stimulated with thrombin (0.1 U/mL). Results are representative of 5 experiments. (B) Fibrinogen
binding and P-selectin exposure in fyn/
platelets stimulated by CRP in the presence of 1mmol/L
CaCl2. The results are mean ± standard error of mean
of 4 experiments.  indicates fyn+/+;  ,
fyn/; *, P < .05; **,
P < .01). (C) P-selectin exposure in
lyn/ platelets stimulated by CRP in the
presence of 1 mmol/L CaCl2 or 1 mmol/L EGTA. The results
are mean ± standard error of mean of 4 experiments. indicates
lyn+/+; , lyn/; *,
P < .05; **, P < .01.
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Protein tyrosine phosphorylation in lyn/
platelets
Protein tyrosine phosphorylation induced by CRP (1.0 µg/mL) was
significantly delayed in lyn/ platelets
compared to controls. A time-course of CRP-stimulated whole-platelet
phosphorylation was performed to pinpoint this delay in initiation of
the GPVI-dependent tyrosine phosphorylation. There was little
detectable increase in phosphorylation observed after 20 seconds of
stimulation by 1.0 µg/mL CRP (Figure 4A). Recovery of CRP-induced
protein tyrosine phosphorylation in the lyn/
platelets was seen in the whole-cell lysates by 45 sec and was complete by 90 seconds (Figure 6A).
FcR chain, Syk, LAT, and PLC2 were among the proteins that show
an initial reduction in phosphorylation at 30 seconds, with full
recovery at 180 seconds (Figure 6A-B). A similar set of results was
seen in platelets stimulated with collagen (not shown).
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| Figure 6.
Tyrosine phosphorylation in CRP-stimulated wild-type and
lyn/ platelets.
(A) Antiphosphotyrosine immunoblots show whole-platelet tyrosine
phosphorylation stimulated by CRP (1.0 µg/mL) from 20 to 180 seconds
in wild-type and lyn/ platelets. Regions
known to comigrate with Syk (70-80 kd), LAT (36-38 kd), and FcR
chain (14-16 kd) are indicated. (B) Immunoprecipitates of Syk (i) and
PLC2 (ii) immunoblotted with antiphosphotyrosine mAb in wild-type
and lyn/ platelets at 30 and 180 seconds.
Results are representative of 4 experiments. Wild-type and knockout
samples were obtained from experiments run in parallel. Results shown
in both panels are from samples resolved on the same gel and are from
the same exposure on the same film.
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Parameters of PLC activity are reduced in
lyn/ platelets
There was a significant impairment of CRP-stimulated pleckstrin
phosphorylation and production of [32P]PA in
lyn/ platelets at time points up to 90 seconds, which was restored to control levels by 180 seconds (Figure
7A-B). For example, the level of PA in
response to 1.0 µg/mL CRP was reduced by 54% ± 5% at 90 seconds
in lyn/ platelets (P < .05).
In contrast, thrombin-induced pleckstrin phosphorylation and PA
formation were similar in lyn/ and control
platelets at all time points (Figure 7A-B). Lyn/
platelets also show a delay of up to 45 seconds in
Ca++ mobilization in response to 1.0 µg/mL CRP (Figure
4Cii). The initial response was followed by a sustained plateau in
lyn/ platelets. It is notable that the
plateau response of wild-type platelets declined toward baseline levels
more rapidly than in lyn/ platelets
(Figure 4Cii).
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| Figure 7.
PLC activation in wild-type and
lyn/ platelets.
Phosphorylation of pleckstrin and formation of phosphatidic acid were
measured over time from 0 to 180 seconds after the addition of an
agonist in platelets treated with 1 mmol/L EGTA and 10 µmol/L
indomethacin. The autoradiographs show pleckstrin phosphorylation (A)
and phosphatidic acid production (B) stimulated by CRP (1.0 µg/mL)
and thrombin (0.1 U/mL) in wild-type and lyn/
platelets compared with wild type. Results are representative of 4 experiments.
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|
Lyn/ platelets exhibited a delay of up to 30 seconds in the onset of shape change in response to CRP, which was
followed by potentiation of the rate and magnitude of aggregation
(Figure 8B). Similar results were
obtained with collagen (1.0-10.0 µg/mL, not shown). In contrast, the
initial shape change response of lyn/
platelets to thrombin (0.1 U/mL) was not altered relative to controls, though aggregation was slightly enhanced in terms and rate
and magnitude (Figure 8B). Expression of P-selectin in response to CRP
was also significantly increased in lyn/
platelets compared with controls when measured after 120 seconds, either in the absence or the presence of extracellular Ca++
(Figure 5C). In contrast, the response to thrombin was not altered (not
shown).
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| Figure 8.
Aggregation responses in lyn/
compared with wild-type platelets.
Shape change and aggregation of wild-type and
lyn/ platelets in response to CRP (A,
0.3-3.0 µg/mL) and thrombin (B, 0.1 U/mL) were measured in a Born
aggregometer. Other conditions are as in the legend to Figure 1.
Results are representative of 3 experiments.
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GPVI-dependent tyrosine phosphorylation in
fyn/lyn/ platelets
The partial inhibition of ITAM tyrosine phosphorylation and
platelet activation in fyn/ and
lyn/ platelets suggests functional
redundancy within this pathway between Src-family kinases.
Additionally, the participation of Lyn in inhibitory pathways indicates
that Src-family kinases have distinct signaling roles. The responses in
fyn/lyn/ platelets were
investigated to characterize further the roles of the 2 Src family
kinases in GPVI signaling.
Whole-cell protein tyrosine phosphorylation was severely reduced in
platelets deficient in Fyn and Lyn in response to CRP. There was
minimal tyrosine phosphorylation of the 12- to 14-kd bands that
comigrated with FcR chain in the whole-cell lysates, and the
phosphorylation of Syk and of PLC2, measured after
immunoprecipitation, was severely attenuated at times up to 180 seconds
(Figure 9).
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| Figure 9.
CRP-stimulated tyrosine phosphorylation in wild-type and
fyn/lyn/
platelets.
Platelets in this experiment were treated with 1 mmol/L EGTA and 10 µmol/L indomethacin. (A) Antiphosphotyrosine immunoblots show
tyrosine phosphorylation at 0, 30, and 180 seconds in CRP (1.0 µg/mL)-stimulated wild-type and
fyn/lyn/ platelets. The 12- to 14-kd band corresponds to the FcR chain. Phosphorylation of Syk
(B) and PLC2 (C), measured after immunoprecipitation from platelets
stimulated as in panel A. Results are representative of 3 experiments.
Wild-type and knockout samples were obtained from experiments run in
parallel. Results shown in each panel of A are from samples
resolved on the same gel and are from the same exposure on the same
film. This also applies to B-D.
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Responses of fyn/lyn/
platelets to CRP are severely impaired
Shape change and aggregation of
fyn/lyn/ platelets in
response to CRP was severely delayed and, in the case of aggregation, markedly inhibited. Shape change was only elicited in response to
relatively high concentrations of CRP (1.0-3.0 µg/mL) after a delay
of 60 to 80 seconds (Figure 10Ai). A
weak aggregation response was only seen in response to a maximal
concentration of CRP (10.0 µg/mL), with no accelerated second phase
(Figure 10Aii). The residual shape change and aggregation responses in
the double-knockout mouse was inhibited by the Src-family kinase
inhibitor PP1 (10 µmol/L, not shown). Shape change and aggregation of
fyn/lyn/ platelets in
response to thrombin were similar to control responses (Figure
10B).
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| Figure 10.
Aggregation stimulated by CRP in wild-type and
fyn/lyn/ platelets.
(Ai) Shape change of wild-type and
fyn/lyn/ platelets was
stimulated by CRP (1.0-3.0 µg/mL). (Aii) Aggregation of wild-type and
fyn/lyn/ platelets was
induced by a higher concentration of CRP (10.0 µg/mL) in the presence
of added 1 mmol/L CaCl2. (B) Aggregation of wild-type and
fyn/lyn/ platelets was
stimulated with 0.1 U/mL thrombin. Results are representative of 3 experiments.
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Discussion |
Previous reports, based on a biochemical approach and use of
inhibitors, have suggested that the Src kinases Fyn and Lyn are involved in regulating collagen-induced platelet
activation.10,11 However, these studies were unable to
dissect the individual functions of Fyn and Lyn because of the lack of
selectivity of available inhibitors, such as PP1 and PD17036. The
current study has addressed this through a genetic approach using
fyn/ and lyn/
platelets. Our findings confirm that Fyn and Lyn regulate FcR chain tyrosine phosphorylation on ligation of GPVI by CRP.
Fyn/ platelets have reduced Fc chain
tyrosine phosphorylation, resulting in the partial loss of
phosphorylation of downstream proteins including the tyrosine kinase
Syk and the adapters SLP-76 and LAT. Although tyrosine phosphorylation
of PLC2 does not appear to be affected by Fyn deficiency, its
activity is significantly reduced. The mismatch between the tyrosine
phosphorylation of PLC2 and proteins involved in its regulation is
unclear. It could, for example, reflect a role of a Fyn-regulated
protein phosphatase in the regulation of PLC2 phosphorylation. The
inhibition of PLC2 activity, despite the normal level of
phosphorylation, is likely to be due to the decrease in phosphorylation
of adapter proteins such as LAT (thereby reducing the recruitment of
PLC2 to the membrane) or possibly phosphorylation at novel sites in the phospholipase. In response to CRP, lyn/
platelets have a delay 30 to 45 seconds in the onset of
aggregation, tyrosine phosphorylation, and PLC2 activation, followed
by either recovery or potentiation of response. The initial delay in
activation observed in the Lyn-deficient platelets, in comparison to
the reduction observed in the fyn/ cells,
suggests that Lyn may have the more important role in initiating
phosphorylation of the ITAM.
fyn/lyn/ platelets exhibit a
severe inhibition and a marked delay in activation. There is some
recovery in the response of
fyn/lyn/ platelets with
higher concentrations of CRP, but potentiation is not seen. Residual
activation of fyn/lyn/
platelets is blocked by the inhibitor of Src kinases, PP1,
suggesting that an additional Src-family kinase may underlie this
response. This kinase is likely to have a relatively minor role
relative to that of Fyn and Lyn in platelet activation by GPVI.
Fyn and Lyn have been shown to mediate phosphorylation of the ITAM in
the Fc chain on stimulation of other Fc receptors, including Fc RI
receptor.21 The role of Fyn and Lyn in mediating this
event has been attributed to their presence in glycolipid-enriched membrane domains (GEMs), an association that is dependent on
palmitoylation of both Src family kinases. In contrast, Src, which is
not palmitoylated and therefore is not present in GEMs, is unable to
induce phosphorylation of the Fc chain.21 This
mechanism is likely to account for the role of Fyn and Lyn in mediating
phosphorylation of the Fc chain in platelets rather than Src, which
is expressed at a much greater level.
The delayed potentiation in lyn/ platelets
suggests that Lyn is also involved in regulating a novel inhibitory
pathway. This pathway appears to be mediated at least partly through an
increase in PLC2 activity. In lyn/
platelets, there was an initial delay in the activation of PLC2 that recovered to control levels by 180 seconds, whereas in
fyn/ platelets, PLC2 activity was
inhibited at all times. The increase in PLC2 activity is consistent
with the sustained increase in Ca++ in the
lyn/ platelets. It seems likely, however,
that an additional mechanism contributes to the increase in aggregation
and -granule secretion observed in lyn |
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Abstract
Introduction