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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2645-2652
Genetic and Pharmacological Analyses of Syk Function in
 IIb 3 Signaling in Platelets
By
Debbie A. Law,
Lisa Nannizzi-Alaimo,
Kathleen Ministri,
Paul E. Hughes,
Jane Forsyth,
Martin Turner,
Sanford J. Shattil,
Mark H. Ginsberg,
Victor L.J. Tybulewicz, and
David R. Phillips
From COR Therapeutics, Inc, South San Francisco, CA; Scripps Research
Institute, La Jolla, CA; and National Institute Medical Research, Mill
Hill, London, UK.
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ABSTRACT |
Agonists induce inside-out  IIb 3
signaling resulting in fibrinogen binding and platelet
aggregation. These in turn trigger outside-in signaling resulting in
further platelet stimulation. Because the Syk tyrosine kinase is
activated during both phases of integrin signaling, we evaluated its
role in IIb3 function in murine platelets
rendered null for Syk by gene targeting and in human platelets
incubated with piceatannol, a tyrosine kinase inhibitor reportedly
selective for Syk. Both Syk null murine platelets and
piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of
IIb3 by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets
adhered normally to immobilized fibrinogen, and mice with these
platelets exhibited normal tail bleeding times. In contrast,
piceatannol treatment of human platelets completely inhibited platelet
adhesion to immobilized fibrinogen. The discrepancy in extent of
integrin dysfunction between murine and human platelet models may be
due to lack of specificity of piceatannol, because this compound
inhibited the activity of Src and FAK as well as Syk and also reduced
tyrosine phosphorylation of multiple platelet proteins. These results
provide genetic evidence that Syk plays a role in
IIb3 signaling in platelets and
pharmacological evidence that, although piceatannol also inhibits
IIb3 signaling, it does so by inhibtion
of multiple protein tyrosine kinases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE MOST ABUNDANT integrin on platelets,
 IIb3 (glycoprotein [GP]
IIb-IIIa), participates in signaling events that are critical for
successful platelet aggregation, consolidation of the platelet
aggregate, and hemostasis. Upon receiving an inside-out signal during
the stimulation of platelets by agonists such as adenosine diphosphate
(ADP), epinephrine, and thrombin, the receptor function of
IIb3 is activated, resulting in the
binding of soluble fibrinogen or von Willebrand factor. Ligand binding,
together with subsequent platelet-platelet interactions during
aggregation, trigger the IIb3-mediated
outside-in signaling processes that generate stable platelet
aggregates.1 Although a variety of signaling events occur
in activated platelets, including phosphoinositide turnover, calcium
mobilization, arachadonic acid metabolism, activation of MAP kinases,
and phosphorylation of numerous proteins on serine/threonine and
tyrosine residues,2,3 a major unsolved problem is the identification of the pathways used for the signal transduction to and
from IIb3.
A preponderance of circumstantial evidence supports a role for Syk, a
72-kD protein tyrosine kinase, in both inside-out and outside-in
IIb3 signaling. For example, Syk is
phosphorylated early in response to stimulation of platelets by
thrombin, ADP, or collagen, regardless of the activation and/or ligand
binding status of the IIb3
integrin.4,5 Because each of these agonists is capable of
inducing IIb3 activation, Syk has become
a candidate for involvement in inside-out
IIb3 signal transduction. Additionally, Syk is thought to have a proximal position in the outside-in
IIb3 signal transduction cascade, because
tyrosine phosphorylation and activation of Syk occurs rapidly after
platelet aggregation mediated by fibrinogen binding and signaling
through IIb3. Syk is the only tyrosine
kinase in platelets that has been shown to be activated directly in
response to IIb3 ligation by soluble ligand.2,4 In addition to
IIb3 signaling, Syk has recently been
implicated in collagen-induced platelet signaling. Syk becomes tyrosine
phosphorylated upon collagen-induced platelet activation and associates
with GP VI through the intermediary immune receptor tyrosine-based
activation motif (ITAM)-containing Fc signaling subunit
and indeed Syk-deficient murine platelets fail to respond to
collagen.6 Further evidence to support a role of Syk in platelet function comes from studies using piceatannol, a tyrosine kinase inhibitor reported to exhibit selectivity toward Syk.
Piceatannol has been shown to inhibit platelet aggregation
induced by collagen, thrombin, or the thromboxane analogue
U46619.5
The present study was designed to determine the role of Syk in
IIb3 signal transduction using two
complementary approaches. First, Syk-deficient murine platelets,
generated using gene targeting methodology, were subjected to
functional analysis. Second, a pharmacologic model of Syk inhibition
was used involving pretreatment of human platelets with the tyrosine
kinase inhibitor, piceatannol. Interestingly, although piceatannol was
found to inhibit both IIb3 activation by
platelet agonists and IIb3-dependent
platelet adhesion and tyrosine phosphorylation, Syk deficiency was
found to have an effect only in one of the events studied herein,
namely the activation-induced binding of soluble fibrinogen to
IIb3 in platelets stimulated with ADP ± epinephrine. These observations establish a contributory role for
Syk in IIb3 signaling. Furthermore, they
demonstrate that the discrepancy between the effects of piceatannol and
Syk deficiency can be explained by a heretofore unappreciated lack of
selectivity of this inhibitor.
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MATERIALS AND METHODS |
Reagents.
Antibodies to ZAP-70 (M-20), Syk (C-20 or 4D10), and Fyn
(FYN3) were obtained from Santa Cruz Biotechnology, Inc
(Santa Cruz, CA). Antibody AB-1 to v-Src and purified Src enzyme were
from Oncogene Sciences (Cambridge, MA). The antiphosphotyrosine
antibody 4G10 and the purified enzymes Fyn and Lyn were from UBI (Lake Placid, NY). PY-20 was purchased from Transduction Laboratories (Lexington, KY). The rabbit antimouse Syk polyclonal, #2131; the IIb3 monoclonal antibody, A2A9; and the
rabbit anti-FAK polyclonal have been previously
described.7-9 Rabbit antihuman
IIb3 (#41), which cross-reacts with mouse
IIb3, was obtained by immunizing a rabbit
with purified IIb3 protein. PAC-1
antibody specific for the activated conformation of
IIb3 was fluorescein isothiocyanate (FITC) conjugated as described.10 Horseradish peroxidase
(HRP)-conjugated antirabbit IgG and antigoat IgG were from
Jackson Immunoresearch Laboratories (West Grove, PA). HRP-conjugated
antimouse IgG, ECL detection kits, and radiolabeled 33P
-adenosine triphosphate (ATP) were from Amersham Life
Science Inc (Arlington Heights, IL). Baculo-virus produced murine Syk was from S. Harmer and A. L. DeFranco (University of California San
Francisco, San Francisco, CA). Piceatannol was the kind gift of Mark
Cushman (Purdue University, West Lafayette, IN) and had a purity
greater than 98% by high-performance liquid chromatography (HPLC). Additional piceatannol was purchased from
Boehringer Mannheim Corp (Indianapolis, IN). ADP, epinephrine, phorbol
myristate acetate (PMA), and enolase were from Sigma
Chemical Co (St Louis, MO). Fibrinogen was purified from human
plasma.11 The cyclic RGD peptide
Mpr-RGDWP-Pen-NH2 was previously described.12
Generation of Syk-deficient chimeric mice.
Radiation chimeras were generated as previously described.6
Briefly, 8- to 10-week-old BALB/c mice received two doses of irradiation from a 60Co source (each of 500 rads at 3 hours
apart). The mice were then reconstituted with an intravenous injection
of 1.5 × 106 fetal liver cells obtained from 16.5-day
Syk /,
Syk+/, or Syk+/+ mouse
fetuses. These were generated by intercrossing mice heterozygous for
the Syktm1Tyb mutation
(Syk+/) backcrossed for at least five
generations onto a B10.D2 background.8 Reconstituted mice
received neomycin sulfate (0.16%) in their drinking water for 4 weeks
after irradiation and were used for experiments between weeks 5 and 6 after irradiation. The genotype of the reconstituting liver cells was
confirmed in each case by Southern blotting.8
Platelet preparation and pretreatment with piceatannol.
To obtain human platelets, blood was collected from healthy donors and
resting platelets were prepared as in Law et al.13 For
pretreatment with piceatannol, the rested platelets (at 4 to 5 × 108/mL) were incubated with the desired concentration of
piceatannol, or control dimethyl sulfoxide (DMSO)
vehicle, for 10 to 15 minutes.
In the case of murine platelets, blood was collected by cardiac
puncture from anesthetized mice. For fluorescence-activated cell
sorting (FACS) experiments, 700 µL of blood was drawn
into a syringe containing 1/10th volume of 3.8% trisodium citrate
(TSC). The blood was transfered to a 1.5-mL eppendorf tube, 700 µL of saline was added, and the sample was centrifuged at approximately 90g for 10 minutes. The platelet-rich plasma (PRP) layer was
carefully removed and used in the FACS experiments detailed below. To
obtain washed platelets, 700 µL of blood was drawn into a syringe
containing 200 µL ACD (2.5% trisodium citrate, 2% dextrose, 1.5%
citric acid [monohydrate]), 500 µL saline, and prostaglandin
E1 (PGE1; to give a final
concentration of 50 ng/mL). The mixture was transfered into a 1.5-mL microcentrifuge tube and centrifuged at 90g for 10 minutes, and the PRP layer was removed to a new tube. For maximal recovery of platelets, the red blood cell pellet from the first spin
was diluted to 1.4 mL with CGS (0.038% trisodium citrate, 0.6%
dextrose, 0.72% NaCl, pH to 7.0) containing PGE1 at 25 ng/mL and recentrifuged, and the supernatant was collected. The pooled supernatant samples were made up to a volume of 1.4 mL with CGS and
after gentle mixing were spun at 16,000g for 7 seconds. The pelleted platelets were resuspended in Ca2+- and
Mg2+-free Tyrodes buffer, counted, and diluted to 1.2 × 108/mL. MgCl2 was then added to a final
concentration of 1 mmol/L.
Adhesion to immobilized fibrinogen.
For assay with human platelets, the platelets were resuspended (at
109/mL) in phosphate-buffered saline (PBS), pH 7.4, and
labeled with 6 µmol/L BCECF-AM
[2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; Molecular Probes Inc, Eugene, OR] for 30 minutes
at 37°C. They were then pelleted and resuspended at 4 × 108/mL in Tyrodes buffer supplemented with 1 mmol/L
Ca2+. Human fibrinogen in PBS, pH 8.0, was plated onto
Immulon-2 microtiter plates at concentrations ranging from 1 ng to 2 µg/well and incubated overnight at 4°C. Plates were washed two
times with PBS, pH 7.4, and blocked for 2 hours at room temperature
with 20 mg/mL radioimmunoassay (RIA) grade bovine serum albumin (BSA;
Sigma Chemical Co) in PBS. Fifty microliters of labeled
platelets (2 × 108 total) was added per well. After 1 hour of incubation, nonadherent platelets were removed by aspiration
and the wells were washed twice with 150 µL Tyrodes buffer
supplemented with 1 mmol/L Ca2+. Adherent platelets were
then quantitated on a Fluorescence Concentration Analyser (Pandex,
Mundelein, IL) at excitation/emmission wavelengths of 485/535 nm.
Adherence of murine platelets to immobilized fibrinogen was determined
using BSA-blocked fibrinogen microtiter wells plates prepared as
described above, followed by the addition of 50 µL of platelets at
1.2 × 108/mL. After 1 hour of incubation,
nonadherent platelets were removed and the wells were washed twice with
150 µL Tyrodes buffer supplemented with 1 mmol/L Mg2+.
One hundred fifty microliters of pNpp buffer (0.1 mol/L citrate, pH
5.4, 0.1% Triton X-100, 5 mmol/L para-nitropheylphosphate) was added
for 1 hour at room temperature. Then, 100 µL of 2 mol/L NaOH was
added and adherent platelets were quantitated in a microplate reader
(Molecular Devices, Menlo Park, CA) at 405 nm. In assays using
piceatannol, the murine platelets were pretreated with the indictated
concentration of inhibitor for 10 minutes before addition to the
fibrinogen-coated microtiter wells.
The percentage of platelets adhering was determined by calculating the
ratio of bound/maximal signal at 405 nm, where maximal reading was
obtained from a microtiter well containing 2 × 108
platelets that was not subjected to washing procedures.
FACS analysis of FITC-PAC-1 and FITC-fibrinogen binding to
platelets.
The analysis of PAC-1 binding to agonist-activated human platelets was
performed as described.10 To assess the binding of fibrinogen to murine platelets, human fibrinogen was labeled with FITC
using a similar method to that described for PAC-1
labeling.10 Basically, human fibrinogen (0.4 mL at 5mg/mL)
was mixed with 60 µL 1 mol/L HaHCO3, pH 9.3, and 120 µL
of FITC-cellite at 20 mg/mL in PBS for 1 hour at room temperature. The
mix was then loaded onto a PD10 column, fractions were collected from
PBS washing, and the fluorophore-labeled fibrinogen fractions were
pooled. On the day of an experiment mixes were made up consisting of
the desired stimuli and a 1/6 dilution of FITC-fibrinogen in Walsh buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L
MgCl2.6H2O, 3.3 mmol/L
NaH2PO4.H2O, 3.8 mmol/L HEPES, pH 7.4)
containing 0.1% BSA and 0.1% dextrose. Twenty microliters of PRP was
added to 30 µL of these mixes, and samples were incubated for 30 minutes at room temperature in the dark. Samples were then diluted with 0.5 mL Tyrodes buffer and analyzed on a FACScan (Becton Dickinson, Mountain View, CA).
Syk expression levels in murine platelets.
A 100-µL sample of PRP from each mouse was washed with CGS
(16,000g for 2 minutes) and lysed in RIPA buffer (1% Triton
X-100, 1% deoxycholate acid [sodium form; DOC], 0.1% sodium dodecyl
sulfate [SDS], 20 mmol/L Tris, pH 7.5, 5 mmol/L EDTA) containing 1 mmol/L phenylmethylsulfonyl fluoride, 20 µmol/L leupeptin, and 0.15 U/mL aprotinin. Samples were separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE; 7.5% gel), transfered to nitrocellulose,
and immunoblotted with either rabbit anti-Syk antiserum (1/1,000) to
determine whether the Syk protein was expressed in the platelets or an
anti-IIb3 polyclonal antibody (#41 at 2 µg/mL) to determine expression levels of
IIb3. After detection with enhanced
chemiluminescence (ECL) reagent, immunoblots were
stripped (according to the manufacturer's recommendation; Amersham)
and reprobed with antiserum to ZAP-70 (2 µg/mL).
Determination of protein tyrosine phosphorylation in
piceatannol-treated human platelets.
Platelets pretreated with piceatannol, as described above, were
incubated at 5 × 108/mL with PBS, 0.1 U/mL thrombin,
or 0.1 U/mL thrombin with stirring for 2 minutes before lysis in RIPA
lysis buffer. For antiphosphotyrosine analysis of total protein, the
proteins were separated on gels, transferred to nitrocellulose, and
immunoblotted with 4G10 and PY-20. For assessment of FAK
phosphorylation, FAK was immunoprecipitated from platelet lysates by
incubating the lysates with anti-FAK antiserum and protein A/G
sepharose. The sepharose-bound material was washed twice with RIPA
buffer and the samples were boiled before separation by SDS-PAGE. After
transfering to nitrocellulose the blots were subjected to
antiphosphotyrosine immunoblotting.
Determination of kinase activity of piceatannol-treated enzymes.
Either purified enzymes or enzymes obtained by immunoprecipitation with
relevant antibodies from platelet lysates were incubated with the
desired concentration of piceatannol or DMSO (0.5% vol/vol final) for
10 minutes. In some experiments, platelets were pretreated with
piceatannol before immunoprecipitating the kinases from lysates. The
samples were then subjected to an in vitro kinase assay by the addition
of 0.5 µCi 33P -ATP, 10 µmol/L ATP with or without
exogenous substrate (enolase at 1.5 µmol/L). After 15 minutes,
reactions were stopped by the addition of laemmli sample buffer and
boiled. Proteins were separated by SDS-PAGE and bands were visualized
by autoradiography. Densitometry was performed using a Bio-Rad Imager
(Bio-Rad, Hercules, CA) with Molecular Analyst software
(Bio-Rad).
Bleeding times.
Mice were anesthetized with SQ ketamine cocktail (Ketamine, Xylazine,
AcePromazine) and 6 minutes later the tail was completely transected
0.5 cm from the tip with a scalpel. Blood was blotted onto SurgiCut
blotting paper (International Technidyne Corp, Edison, NJ) every 30 seconds and the bleeding time was defined by the time
required for cessation of blood flow. Gentle blotting every 30 seconds
was continued for 1 minute to detemine whether stable hemostasis had
been achieved. If bleeding continued for 30 minutes it was stopped
manually to prevent loss of life.
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RESULTS |
Genetic analysis of the role of Syk in murine platelet function.
Although recent data have indicated a role for Syk in the signaling
pathways activated by the binding of collagen to platelet GP
VI,6 support for the role of Syk in
IIb3-mediated signaling events remains
circumstantial. To address this issue in a more definitive fashion, we
sought a genetic approach where studies could be performed on murine
platelets in which the Syk protein was not expressed due to gene
targetting. Because genetically engineered mice that lack Syk
either die perinatally or within 1 to 5 days of birth,8,14
a radiation chimera system was used in which all hematopoietic cells,
including platelets, were Syk deficient (see Materials and Methods).
Syk expression in platelets from individual chimeras was evaluated by
Western blotting and, as can be seen from the examples in
Fig 1, platelets from animals repopulated
with Syk/ liver cells did not express
detectable levels of Syk, whereas appreciable levels of Syk were
present in mice repopulated with liver cells genotyped as
Syk+/ or Syk+/+. It was
determined that 3% contamination of Syk-deficient platelets by
Syk-expressing platelets could be detected in these Western blots (data
not shown). Similar levels of IIb3 were
present in the platelets from all reconstitutions (top panel, Fig 1), indicating that the lack of Syk had no deleterious effect on
IIb3 expression. Two of the 38 BALB/c
mice repopulated with Syk/ liver
cells were rejected for further study because they had detectable
levels of Syk protein in the platelet lysates, whereas samples from 4 other mice were eliminated due to inadequate sample size.
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| Fig 1.
Determination of IIb3
protein expression in murine platelets using immunoblotting. This
figure shows the analysis of platelets from 8 different radiation
chimeras that were repopulated with liver cells of the indicated Syk
genotype. One hundred microliters of PRP was washed with CGS and then
solubilized in RIPA buffer. The proteins were separated by SDS-PAGE on
a 7.5% gel and transferred to nitrocellulose. The resulting blot was
cut in two at approximately the 80-kD point. The upper portion of the
blot was immunoblotted with the anti-IIb3
polyclonal #41. The lower portion of the blot was probed with the
anti-Syk antiserum #2131. As little as 3% contamination of Syk null
platelets with Syk-expressing platelets could be detected by this
method.
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Effect of lack of Syk on murine tail bleed times.
A bleeding time model was used to assess the effect of lack of Syk
expression on platelet-dependent hemostasis. Prolonged bleeding time
can be a result of low platelet count or defective platelet function.
Indeed, mice deficient in 3, as a result of gene
targetting, have prolonged bleeding times similar to that observed in
humans lacking IIb3
expression.15 Treating mice with aspirin or a low molecular
weight IIb3-inhibitor also results in
prolonged bleeding times (Ministri and Hollenbach, manuscript in
preparation). As can be seen in
Fig 2, there was no difference in bleeding
times when chimeric mice lacking the Syk tyrosine kinase were compared
with those expressing Syk (either heterozygotes or homozygotes).
However, 2 of the 5 Syk/ mice rebled
within the 1 minute after the primary endpoint. The result given above
indicates that Syk is not required for cessation of bleeding after tail
transection in the mouse but suggests that Syk deficiency may decrease
the stability of the hemostatic plug.
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| Fig 2.
Bleeding times generated from
Syk/, Syk+/, and
Syk+/+ mice. Radiation chimera mice repopulated
with either ( ) Syk/, ( )
Syk+/, or ( ) Syk+/+
fetal liver cells were anesthetized and 6 minutes later
had 0.5 cm from the tip of the tail removed with a scalpel blade. Blood
was gently blotted onto surgical blotting paper every 30 seconds until cessation of bleeding. The mean and standard deviations
obtained for the bleeding times were 5.6 ± 3.4 (for
Syk/, where n = 5), 5.9 ± 3.1 (for
Syk+/, where n = 7), and 9.8 ± 8.2 (for
Syk+/+, where n = 3).
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Inside-out signaling in Syk null murine platelets.
The Syk null platelets were then used to address the role that Syk
might play in specific IIb3-mediated
signaling events. In the last decade there has been a surge of work
using gene targetted mice; however, relatively little has been
published on examining the function of murine platelets. Thus, we
developed and adapted two assays to examine platelet function that took
into account the relatively small number of platelets obtained per
mouse. The first assay examined whether the absence of Syk expression
had any effect on inside-out IIb3
signaling and used a FACS-based assay. The
IIb3 present on murine platelets, like
its human counterpart, needs to be activated to bind soluble
fibrinogen. The binding of FITC-labeled fibrinogen to murine platelets
stimulated by either 1 µmol/L ADP alone or a combination of 10 µmol/L ADP plus 25 µmol/L epinephrine was used as a measure of
inside-out signaling. Preliminary studies showed that the binding of
FITC-fibrinogen was saturable and inhibitable by EDTA and inhibited by
immunoblocking of IIb3, characteristics
typical of unmodified fibrinogen.
As demonstrated in Fig 3, Syk null
platelets activated by ADP plus epinephrine bound 32% less fibrinogen
than control. This functional defect, although partial, was
statistically significant (P = .0029, Student's
t-test) and was observed in the platelets from two independent
batches of Syk null radiation chimeras. Similar results were observed
when Syk null platelets were stimulated with ADP alone, with 28.6%
less fibrinogen being bound compared with control platelets (P = .08), although this experiment was limited to a single batch of mice
(data not shown). In contrast, Syk null platelets demonstrated the same
fibrinogen binding as control platelets in response to direct
activation of protein kinase C by PMA. This indicates that the
defective activation of IIb3 observed
with ADP ± epinephrine was not due to some defect in
IIb3 per se. We conclude from these
results that Syk does play a role in inside-out
IIb3 signaling when ADP ± epinephrine are used as agonists.
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| Fig 3.
FITC-fibrinogen binding to Syk null and Syk positive
platelets. PRP from ( ) Syk positive or ( ) Syk null mice was
treated with the indicated platelet agonists, PBS (control), ADP (10 µmol/L) plus epinephrine (25 µmol/L), or PMA (20 µmol/L).
FITC-labeled fibrinogen was added with the agonists. After 30 minutes
of incubation in the dark at room temperature, samples were diluted in
0.5 mL Tyrodes buffer and analyzed on a FACScan. Bars represent the
geometric mean ± standard error fluorescent channel for
PAC-1 binding and were 2.75 ± 0.26 (control), 28.94 ± 2.75 (ADP + epi), and 38.31 ± 4.33 (PMA) for the Syk null mice
(where n = 7), and 2.85 ± 0.22 (control), 42.4 ± 2.49 (ADP + epi), and 39.5 ± 2.89 (PMA) for Syk positive mice (where n = 12, including 10 mice repopulated with Syk+/ liver
cells and 2 repopulated with Syk +/+ liver
cells). *P = .0029 (Student's t-test). Similar
results were obtained with a different batch of radiation chimeras.
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Binding of murine Syk null platelets to immobilized fibrinogen.
The second assay to measure IIb3 function
in Syk-deficient platelets assessed the role of Syk in the binding of
platelets to immobilized fibrinogen. Unstimulated platelets can bind to immobilized fibrinogen and the initial recognition of immobilized fibrinogen by unstimulated platelets is possible because of the activation of fibrinogen by immobilization.16 We developed
an assay that, using an acid phosphoatase detection
system,17 was sufficiently sensitive to generate
fibrinogen-dependent adhesion curves with platelets from a single
mouse. When Syk/ platelets were
compared with control, either Syk+/ or
Syk+/+, for their ability to adhere to immobilized
fibrinogen, no differences were observed
(Fig 4). The concentration of fibrinogen
required to induce half-maximal binding was similar for Syk null (0.26 ± 0.05 µg/well) and control (0.22 ± 0.07 µg/well)
platelets. Approximately 28% to 32% of Syk null or control platelets
adhered to the immobilized fibrinogen at optimal fibrinogen
concentrations. These results suggest that
IIb3-adherence to immobilized fibrinogen
under static conditions does not require Syk.
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| Fig 4.
Adherence of Syk null and Syk positive platelets to
immobilized fibrinogen. Murine platelets (6 × 106),
either () Syk null or () Syk positive, were added to microtiter
wells coated with fibrinogen (at a range of concentrations from 1 ng to
2 µg per well). After 1 hour of incubation at room temperature, wells
were washed twice and 150 µL of pNpp buffer was added for a further 1 hour at room temperature. One hundred microliters of 2 N NaOH was added
and adherent platelets were quantitated in a microplate reader at 405 nm. Platelets from single mice where used to generate duplicate points
for platelet adherence to each fibrinogen concentration. The graph
depicts the mean ± standard error at each fibrinogen concentration,
where for Syk null mice n = 10 and for Syk positive mice n = 18.
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Previous studies have shown that members of the same family of tyrosine
kinases can compensate for a deleted kinase.18 The kinase
related to Syk is ZAP-70, a 70-kD protein tyrosine kinase with 73%
homology to Syk.19 To explore the possibility that ZAP-70
was compensating for the lack of Syk in the Syk null platelets, the PRP
lysates were immunoblotted with an anti-ZAP-70 antibody. A very low
level of ZAP-70 protein was detected in the PRP lysates, possibly due
to contaminating T cells. The expression levels were comparable in both
the Syk null and control samples.
Effect of piceatannol on platelet intergin signaling and protein
tyrosine kinases.
Piceatannol is a tyrosine kinase inhibitor with a reported selectivity
for Syk.20 Previous studies showing that this inhibitor blocks the platelet aggregation induced by several different agonists have been used to infer a role for Syk in platelet
function.5,21 However, on the basis of the results obtained
with Syk null murine platelets, we decided to re-examine the effect of
piceatannol on a number of platelet functions.
Consistent with results obtained with Syk null murine platelets,
piceatannol treatment of human platelets resulted in a partial inhibition of IIb3 activation. Inhibition
was dose-dependent such that PAC-1 binding to ADP-stimulated platelets
was reduced by approximately 50% at a piceatannol concentration of 30 µg/mL, and consistent inhibition was observed even at 10 µg/mL
(Fig 5). Similar results were obtained when
TRAP, an agonist peptide to the PAR-1 thrombin receptor, was used as
the agonist (data not shown). Piceatannol had no effect on the level of
PAC-1 binding to platelets stimulated with 0.2 µmol/L PMA. Thus, the
data obtained with both the Syk null platelets and with piceatannol
suggest a role for Syk in the activation of
IIb3 through ADP receptors but indicate
that Syk is independent of, or proximal to, agonist-induced activation
of protein kinase C.
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| Fig 5.
Effect of piceatannol on PAC-1 binding to platelets.
Human platelets were pretreated for 10 minutes at room temperature with
control DMSO vehicle or piceatannol at 1, 10, or 30 µg/mL. They were
then stimulated with the desired agonist ([] PBS control, []
0.1 µmol/L ADP, [] 1 µmol/L ADP, [] 10 µmol/L ADP,
[ ] 0.2 µmol/L PMA) and incubated with FITC-labeled PAC-1
antibody. The level of PAC-1 bound is expressed as a percentage of
maximal PAC-1 binding obtained when the DMSO-pretreated platelets were
stimulated with the highest concentration of agonist (either 10 µmol/L ADP for the ADP-treated samples or 0.2 µmol/L PMA for the
PMA-treated samples). Results shown are the mean ± SE values from
three separate experiments.
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In stark contrast to the data obtained with Syk null murine platelets,
in which lack of Syk had no effect on platelet adhesion to fibrinogen,
piceatannol inhibited the binding of human platelets to immobilized
fibrinogen in a dose-dependent manner, with a 30 µg/mL piceatannol
pretreatment leading to complete abrogation of platelet adherence to
fibrinogen (data not shown). The
IIb3-dependency of the platelet adherence
was confirmed using either the IIb3 blocking antibody, A2A9, or an RGD inhibitory peptide, both of which
abolished platelet adherence to the fibrinogen. Cytochalasin E (4 µmol/L), which disrupts the actin cytoskeleton, also blocked platelet
adherence to fibrinogen when added to the isolated platelets (not shown).
This result suggested that the effects of piceatannol might not be
limited to an inhibition of Syk function. To further address this
issue, we assessed the effect of piceatannol on protein tyrosine phosphorylation in platelets. The effect of piceatannol on both total
protein tyrosine phosphorylation, as well as on the phosphorylation of
the specific substrate, FAK, was examined. Piceatannol pretreated platelets were stimulated by the addition of thrombin (with or without
stirring). Surprisingly, pretreatment of platelets with 40 µg/mL
piceatannol had a profound effect on the extent of protein tyrosine
phosphorylation, with the level of phosphorylation of most proteins
rendered less than basal (Fig 6A).
Pretreating platelets with various concentrations of piceatannol
established that doses greater than 5 µg/mL decreased the tyrosine
phosphorylation of a number of proteins that are induced by
thrombin-induced aggregation, including FAK (Fig 6B). It is unlikely
that Syk is responsible for maintaining the tyrosine phosphorylation
state of proteins in the unstimulated platelets or for all the tyrosine
phosphorylations induced by thrombin stimulation. Thus, the ablation of
tyrosine phosphorylation of almost all proteins in control, activated, or aggregated platelets again suggests that the effects of the inhibitor are not specific to Syk.
View larger version (24K):
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[in a new window]
| Fig 6.
Piceatannol inhibition of protein tyrosine
phosphorylation in platelets. Human platelets were pretreated with DMSO
or piceatannol (40 µg/mL) for 10 minutes at room temperature before
the addition of PBS ( ), 0.1 U/mL thrombin, or 0.1 U/mL thrombin plus
stirring. After 2 minutes, the platelets were solubilized in RIPA
buffer and the proteins were separated by SDS-PAGE on an 8% gel and
transferred to nitrocellulose. (A) Total protein lysates. (B) FAK
immunoprecipitated from platelet lysates. In each case the blots were
probed with the antiphosphotyrosine antibodies 4G10 and PY-20. (B) The
blot stripped and reprobed with the anti-FAK antibody to confirm equal
loading of protein. Proteins were visualized using ECL detection
methods. The molecular weight standards are indicated to the left.
|
|
To assess whether piceatannol might be exerting an inhibitory effect
through some kinases in addition to Syk, the effect of piceatannol on
the activity of other tyrosine kinases known to be present in platelets
was assessed. Previously, investigators have suggested that piceatannol
selectively inhibits Syk, because doses in the 30 to 50 µg/mL range,
which totally abrogated Syk kinase activity, did not inhibit the
activity of the Src-family tyrosine kinase Lyn in in vitro kinase
assays.20 We repeated this experiment and found that
piceatannol at 40 µg/mL profoundly inhibited Syk kinase activity by
greater than 90%, as assessed by autophosphorylation, whereas Lyn
activity was unaffected. Furthermore, the kinase activity of Fyn,
another member of the Src-family of tyrosine kinases, was inhibited by
only approximately 20% at 40 µg/mL of piceatannol. However, the
kinase activity of Src was inhibited by greater than 75% at 40 µg/mL
of piceatannol and by 50% at 10 µg/mL
(Fig 7).
View larger version (21K):
[in this window]
[in a new window]
| Fig 7.
Effect of piceatannol on tyrosine kinase activity. ()
Src, () Fyn, () Syk, or () FAK were incubated with the
indicated concentration of piceatannol for 10 minutes at room
temperature. The enzymes were then incubated with 0.5 µCi
33P -ATP for 15 minutes at room temperature. Reactions
were stopped by the addition of Laemmli sample buffer and the proteins
were separated by SDS-PAGE. Gels were dried down and the radioactive
bands were visualized by autoradiography. Densitometry was preformed
using a Bio-Rad Imager equipped with Molecular Analyst software. The
graph shows the levels of autophosphorylation of relevant enzymes
expressed as a percentage of control, where control was the level of
autophosphorylation obtained in the absence of piceatannol but with
DMSO vehicle present. Similar results were obtained in at least two
separate experiments with each enzyme and also when enolase was used as
an exogenous substrate.
|
|
These data were in contrast to previous reports concluding that Src was
not inhibited by piceatannol based on observations showing that the
kinase activity of Src immunoprecipitated from platelets was not
affected by piceatannol pretreatment of the cells.5 To
determine whether different assay protocols accounted for the different
results obtained in these studies because our experiments used purified
enzymes, Src was immunoprecipitated from lysates made from platelets
pretreated with various concentrations of piceatannol. Significant
piceatannol inhibition of Src was still observed in these experiments,
although the dose-response curve was shifted to right, with
approximately 50% inhibition of Src activity seen at 20 µg/mL
piceatannol. Under the same conditions, the dose-response curve for Syk
was also shifted with at least 5 µg/mL piceatannol being required to
inhibit 90% of Syk activity compared with approximately 1 µg/mL when
purified enzyme was incubated directly with the inhibitor (data not shown).
The effect of piceatannol on FAK, another prominent platelet tyrosine
kinase, was also examined. FAK was immunoprecipitated from platelet
lysates and then incubated with piceatannol before performing an
immune-complex in vitro kinase assay. Both FAK autophosphorylation and
the phosphorylation of exogenous substrate were dramatically decreased
by 5 µg/mL piceatannol (Fig 7). Similar results were obtained with
two different sources of piceatannol. These results show that
piceatannnol can significantly inhibit FAK and Src at the 30 µg/mL
dose often used to determine the role of Syk in various cellular
function; indeed, even at doses of piceatannol lower than 10 µg/mL,
FAK kinase activity is still significantly impaired.
| |
DISCUSSION |
The purpose of this study was to determine the functional importance of
Syk in the IIb3-mediated signaling
processes that occur upon platelet activation. Using a genetic
approach, we found that the specific deficiency of this tyrosine kinase
led to a partial defect in the activation of
IIb3 in murine platelets. However, lack
of Syk did not affect the ability of platelets to adhere to immobilized
fibrinogen and neither did it have an effect on
IIb3-mediated primary hemostasis, as
assessed by tail bleeding times. These data were surprising in light of
previous reports, based on the kinetics of Syk phosphorylation and
activation and on the effects of piceatannol, that concluded that Syk
was important for all IIb3-mediated
signaling events.2,5 However, the present results indicate
that discrepancies in IIb3 dysfunction observed between the genetic and pharmacological approaches are due, at
least in part, to a lack of specificity of piceatannol. Regardless,
both experimental approaches yielded results consistent with Syk
playing an essential role in achieving maximal
IIb3 activation.
Our data draw into question the validity of using piceatannol to infer
specific roles for Syk in cell function. Piceatannol has been widely
used to study Syk and results obtained with this inhibtor have been
used to predict the role of Syk in a number of cell signaling
events.5,20,22,23 Experiments examining the activity of
tyrosine kinases immunoprecipitated from piceatannol-pretreated platelet lysates and the activity of kinases directly treated with
piceatannol both demonstrated that this inhibitor is not selective for
Syk and can inhibit other kinases, in particular FAK, at the
concentrations often used to infer the role of Syk in a particular
system. Such results highlight the problem of using piceatannol, even
at relatively low concentrations (5 to 10 µg/mL), as a reagent to
infer crucial roles for Syk in cell function and suggest that
alternative experimental approaches need to be considered. One such
approach is the study of platelets from genetically engineered mice.
In the present study we show that murine platelets lacking Syk are
defective in IIb3 signaling. This
defective activation of IIb3 was observed
when ADP ± epinephrine were used as agonists. This combination of
agonists can induce several signaling pathways, including calcium
mobilization and phosphoinositide hydrolysis, and results in platelet
shape change and IIb3
activation.24 It remains unclear whether Syk plays a role
in initiating one of the aforementioned signaling pathways, is involved
in a secondary signaling pathway, or takes part in an event directly
involving the IIb3 integrin. In other
systems in which Syk plays a critical role in signaling, such as via
the B-cell and Fc receptors, an interaction between Syk and proteins
containing a tyrosine-based motif sequence (ITAM) has always been
documented.25-28 Syk binds to and is activated by the
phosphorylated ITAMs via its tandem SH2 domains, allowing for its
localization to signaling complexes at the cell membrane.29
Indeed, in the one pathway in platelets in which Syk clearly plays a
role, namely collagen-induced signaling, an ITAM-containing protein,
Fc, is thought to be involved.6 Thus, whereas Syk is
required for maximal IIb3 activation, the partial phenotype displayed by the Syk null platelets suggests that
other pathways, and presumably other protein tyrosine kinases, must
also be involved in integrin activation. Further studies will be
necessary to characterize these pathways in more detail.
Our experiments with Syk-deficient platelets failed to demonstrate a
role for Syk in other IIb3-mediated
events, such as adherence to immobilized fibrinogen. In addition, work
by Poole et al6 demonstrated the ability of Syk-deficient
platelets to aggregate and release arachidonic acid in response to 10 U/mL thrombin stimulation. It is conceivable that some other kinase can
compensate for the lack of Syk in these platelets. Indeed, it has been
observed that the thymocytes from people who genetically lack the
tyrosine kinase ZAP-70 express Syk at higher than normal levels.18 However, no increased expression of ZAP-70, the
most likely candidate for a compensating kinase because it belongs to
the same family as Syk, was observed in the Syk-deficient platelets. This does not rule out the possibility of some unrelated kinase being
able to take over Syk's function in an ADP plus epinephrine-induced signaling pathway. However, there are data that suggest that in some
cases loss of Syk is not compensated for by any other tyrosine kinase.
For example, Syk-deficient B cells give the expected phenotype based on
loss of Syk, ie, they fail to progress past the pre-B-cell stage, in
keeping with the importance of Syk for signaling via the pre-B-cell
receptor complex.8,14 In addition, the complete loss of
response towards collagen in Syk null platelets also indicates that Syk
cannot be compensated for in this particular signaling reaction.6
Further evidence indicating a lack of importance of Syk in certain
IIb3-mediated processes comes from
bleeding time data. It is clear that in the mouse, as in the human,
prolonged bleeding times can correlate with defective
IIb3 function or defective platelet
signaling. Mice genetically engineered to lack 3 have increased bleeding times.15 In addition, mice that lack
Gq have a defect in platelet activation and also have
prolonged bleeding times.30 No increase in tail bleeding
time was observed when Syk/ animals
were compared with Syk+/ or
Syk+/+ controls, as had previously been
observed.6 Whereas Syk-deficiency had no effect on bleeding
times here, it is possible that this is due to a limitation of the
model, because it is known that these Syk-deficient platelets fail to
mount responses to collagen and, thus, by analogy to humans with
defective collagen-induced platelet responses,31,32 might
be expected to have prolonged bleeding times on that basis alone.
However, Syk deficiency was assocaited with rebleeding after primary
hemostasis in 2 of 5 animals. It is not known whether this indicates
unstable hemostatic plug formation in these animals, and neither is it
known whether this is a contributing factor in death due to hemorrhage
that happens to most Syk knockout mice shortly after
birth.8,14
The discrepancies between the pharmacological and genetic analysis of
the role of Syk in IIb3-mediated platelet
processes reiterate the need for caution when interpreting results
obtained with inhibitors. They also highlight the strength of a genetic approach that, as gene targeting technology becomes more commonplace, is an increasingly viable strategy for determining protein function. This method, incorporating the micro-assays of platelet function used
herein, may be particularly useful for studying the role of proteins in
platelet function, because platelets are not amenable to more
traditional genetic manipulation such as transfection.
| |
ACKNOWLEDGMENT |
The authors thank Mark Smyth (Cor medicinal chemistry group) for
checking the purity of piceatannol and all the members of the
Tybulewicz laboratory for their help during this work.
| |
FOOTNOTES |
Submitted July 28, 1998; accepted December 9, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David R. Phillips, PhD, COR
Therapeutics, Inc, 256 E Grand Ave, South San Francisco, CA 94080;
e-mail: david_phillips{at}corr.com.
| |
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G.A. Stouffer and S.S. Smyth
Effects of Thrombin on Interactions Between {beta}3-Integrins and Extracellular Matrix in Platelets and Vascular Cells
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[Abstract]
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K. S. S. Prasad, P. Andre, M. He, M. Bao, J. Manganello, and D. R. Phillips
Soluble CD40 ligand induces {beta}3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling
PNAS,
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[Abstract]
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Z. Wang, T. M. Leisner, and L. V. Parise
Platelet {alpha}2{beta}1 integrin activation: contribution of ligand internalization and the {alpha}2-cytoplasmic domain
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[Abstract]
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T. Willeke, J. Schymeinsky, P. Prange, S. Zahler, and B. Walzog
A role for Syk-kinase in the control of the binding cycle of the {beta}2 integrins (CD11/CD18) in human polymorphonuclear neutrophils
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August 1, 2003;
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[Abstract]
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H. W. Sohn, H. Gu, and S. K. Pierce
Cbl-b Negatively Regulates B Cell Antigen Receptor Signaling in Mature B Cells through Ubiquitination of the Tyrosine Kinase Syk
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T. R. Kyriakides, P. Rojnuckarin, M. A. Reidy, K. D. Hankenson, T. Papayannopoulou, K. Kaushansky, and P. Bornstein
Megakaryocytes require thrombospondin-2 for normal platelet formation and function
Blood,
May 15, 2003;
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[Abstract]
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A. Mocsai, H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C. A. Lowell
G-protein-coupled receptor signaling in Syk-deficient neutrophils and mast cells
Blood,
May 15, 2003;
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[Abstract]
[Full Text]
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J. C. Resendiz, S. Feng, G. Ji, K. A. Francis, M. C. Berndt, and M. H. Kroll
Purinergic P2Y12 Receptor Blockade Inhibits Shear-Induced Platelet Phosphatidylinositol 3-Kinase Activation
Mol. Pharmacol.,
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[Abstract]
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A. Obergfell, K. Eto, A. Mocsai, C. Buensuceso, S. L. Moores, J. S. Brugge, C. A. Lowell, and S. J. Shattil
Coordinate interactions of Csk, Src, and Syk kinases with {alpha}IIb{beta}3 initiate integrin signaling to the cytoskeleton
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April 15, 2002;
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[Abstract]
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W.-M. Yu, T. S. Hawley, R. G. Hawley, and C.-K. Qu
Role of the docking protein Gab2 in beta 1-integrin signaling pathway-mediated hematopoietic cell adhesion and migration
Blood,
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[Abstract]
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F. Wolter, A. Clausnitzer, B. Akoglu, and J. Stein
Piceatannol, a Natural Analog of Resveratrol, Inhibits Progression through the S Phase of the Cell Cycle in Colorectal Cancer Cell Lines
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[Abstract]
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E. Bulanova, V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus
The IL-15R{alpha} Chain Signals Through Association with Syk in Human B Cells
J. Immunol.,
December 1, 2001;
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[Abstract]
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M. Majeed, E. Caveggion, C. A. Lowell, and G. Berton
Role of Src kinases and Syk in Fc{gamma} receptor-mediated phagocytosis and phagosome-lysosome fusion
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S. Yanagi, R. Inatome, J. Ding, H. Kitaguchi, V. L. J. Tybulewicz, and H. Yamamura
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[Abstract]
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P. Desaulniers, M. Fernandes, C. Gilbert, S. G. Bourgoin, and P. H. Naccache
Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals
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October 1, 2001;
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A. Oda, H. D. Ochs, L. A. Lasky, S. Spencer, K. Ozaki, M. Fujihara, M. Handa, K. Ikebuchi, and H. Ikeda
CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk
Blood,
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[Abstract]
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V. Bertagnolo, M. Marchisio, F. Brugnoli, A. Bavelloni, L. Boccafogli, M. L. Colamussi, and S. Capitani
Requirement of Tyrosine-phosphorylated Vav for Morphological Differentiation of All-trans-Retinoic Acid-treated HL-60 Cells
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April 1, 2001;
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[Abstract]
[Full Text]
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Y. Kuno, A. Abe, N. Emi, M. Iida, T. Yokozawa, M. Towatari, M. Tanimoto, and H. Saito
Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12)(q22;p12)
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[Abstract]
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K. J. Clemetson, J. M. Clemetson, A. E. I. Proudfoot, C. A. Power, M. Baggiolini, and T. N. C. Wells
Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets
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[Abstract]
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B. A. Judd, P. S. Myung, L. Leng, A. Obergfell, W. S. Pear, S. J. Shattil, and G. A. Koretzky
Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIbbeta 3 and collagen receptors
PNAS,
October 24, 2000;
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[Abstract]
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M. T. Santos, A. Moscardo, J. Valles, M. Martinez, M. Pinon, J. Aznar, M. J. Broekman, and A. J. Marcus
Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, {alpha}IIb{beta}3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets
Circulation,
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[Abstract]
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A. Mocsai, Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, and E. Ligeti
Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases
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M. Shiraga, A. Ritchie, S. Aidoudi, V. Baron, D. Wilcox, G. White, B. Ybarrondo, G. Murphy, A. Leavitt, and S. Shattil
Primary Megakaryocytes Reveal a Role for Transcription Factor NF-E2 in Integrin {alpha}IIb{beta}3 Signaling
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V. Ramakrishnan, P. S. Reeves, F. DeGuzman, U. Deshpande, K. Ministri-Madrid, R. B. DuBridge, and D. R. Phillips
Increased thrombin responsiveness in platelets from mice lacking glycoprotein V
PNAS,
November 9, 1999;
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[Abstract]
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K. J. Cowan, D. A. Law, and D. R. Phillips
Identification of Shc as the Primary Protein Binding to the Tyrosine-phosphorylated beta 3 Subunit of alpha IIbbeta 3 during Outside-in Integrin Platelet Signaling
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[Abstract]
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I. Canobbio, A. Bertoni, P. Lova, S. Paganini, E. Hirsch, F. Sinigaglia, C. Balduini, and M. Torti
Platelet Activation by von Willebrand Factor Requires Coordinated Signaling through Thromboxane A2 and Fcgamma IIA Receptor
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[Abstract]
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T. R. Faruqi, E. J. Weiss, M. J. Shapiro, W. Huang, and S. R. Coughlin
Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides. DETERMINANTS OF SPECIFICITY AND UTILITY IN ASSAYS OF RECEPTOR FUNCTION
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P. Maschberger, M. Bauer, J. Baumann-Siemons, K. J. Zangl, E. V. Negrescu, A. J. Reininger, and W. Siess
Mildly Oxidized Low Density Lipoprotein Rapidly Stimulates via Activation of the Lysophosphatidic Acid Receptor Src Family and Syk Tyrosine Kinases and Ca2+ Influx in Human Platelets
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[Abstract]
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A. Obergfell, K. Eto, A. Mocsai, C. Buensuceso, S. L. Moores, J. S. Brugge, C. A. Lowell, and S. J. Shattil
Coordinate interactions of Csk, Src, and Syk kinases with {alpha}IIb{beta}3 initiate integrin signaling to the cytoskeleton
J. Cell Biol.,
April 15, 2002;
157(2):
265 - 275.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION