|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1295-1303
Human Signaling Protein 14-3-3 Interacts With Platelet
Glycoprotein Ib Subunits Ib and Ib
By
David C. Calverley,
Terrance J. Kavanagh, and
Gerald J. Roth
From the Medical and Research Services, Seattle Veterans'
Affairs Medical Center, Seattle; and the Division of
Hematology, Department of Medicine, and Department of Environmental
Health, University of Washington, Seattle, WA.
 |
ABSTRACT |
The initiation of primary hemostasis is mediated by interaction of
the platelet glycoprotein Ib (GPIb) surface receptor and its arterial
subendothelial von Willebrand factor (vWF) ligand. The intracellular
signaling immediately following GPIb receptor occupancy connecting the
adhesive event to platelet activation and aggregation has not been well
characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous
protein family with diverse biologic roles, including functional
modulation of several prominent signaling proteins. We used the yeast
two-hybrid system and confocal microscopy to characterize the recently
described interaction between GPIb and platelet 14-3-3 , and provide
evidence for the potential signaling role of this protein. Two-hybrid
interactions suggest that platelet 14-3-3 associates with the
cytoplasmic domain of GPIb subunits Ib and Ib in transformed
yeast cells. The 14-3-3 interaction with GPIb may be partly mediated
through the latter's phosphorylated serine 166 residue as its
mutagenesis results in 20% to 40% reduced interaction. There was 51%
to 59% reduced interaction between GPIb and three 14-3-3 deletion
mutants compared with full-length 14-3-3, suggesting that either the
N-terminal dimerization or membrane-binding domains or more
than one noncontiguous 14-3-3 element may be required for optimal
GPIb interaction. Confocal studies of platelets and a megakaryocyte
cell line provided additional evidence for interaction of 14-3-3
with GPIb and GPIb. We also found that, similar to the signaling
mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal
14-3-3 content is increased following vWF and ristocetin
stimulation. We suggest that platelet 14-3-3 interacts with GPIb
and Ib, that this interaction may be partly mediated through
phosphoserine recognition, and that 14-3-3 cytoskeletal
translocation may serve as a GPIb post-receptor occupancy signaling
event.
| |
INTRODUCTION |
PLATELET ADHESION TO arterial
subendothelium is an integral component of thrombus formation and is
mediated by a shear-dependent interaction between the platelet
glycoprotein (GP) Ib/V/IX receptor on the platelet surface and its
ligand, von Willebrand factor (vWF). Following this interaction, the
platelet undergoes characteristic morphologic and biochemical changes
associated with its transition from a resting state to an activated
state. This includes a conformational change of the platelet surface
GPIIb-IIIa integrin that facilitates its interaction with ligands,
including plasma fibrinogen, that lead to platelet aggregation.
The platelet GPIb receptor consists of four polypeptide chains with
certain features in common.1 First, they are all
transmembrane proteins with extracellular and cytoplasmic domains. The
GPIb subunit is associated covalently with the smaller GPIb
subunit, while GPIX and GPV are noncovalently associated with the
complex. Surface expression of GPIb subunits is significantly reduced
or absent in the congenital bleeding disorder Bernard-Soulier syndrome, in which mutations involving three subunits have been
described.2,3 Each GPIb subunit contains one or more
24-amino acid tandem repeats rich in leucine that have been
demonstrated to participate in protein-protein interactions in other
cells.1
Tyrosine phosphorylation of multiple platelet proteins is demonstrable
upon GPIb stimulation with vWF and ristocetin, but the precise
molecular signaling events leading to platelet activation remain to be
elucidated. Previous investigation has demonstrated the translocation
of platelet Src and phosphatidylinositol 3-kinase to cytoskeletal
elements in response to vWF stimulation, suggesting these enzymes play
a role in the cytoskeletal reorganization associated with platelet
activation following vWF-GPIb interaction.4
The 14-3-3 proteins are a 27- to 30-kD family originally isolated from
the brain, with at least 10 homologous mammalian isoforms now
described.5-7 Current knowledge regarding the functional domains of the 14-3-3 family is shown in Fig
1. They exist as homodimers and
heterodimers in vivo and have been found to exhibit a wide array of
biologic functions in many mammalian cell types, plants, and
yeast.7-13 The 14-3-3 family has recently been
implicated in the regulation of intracellular signaling pathways
through interaction with several oncogene and proto-oncogene products. Association with and regulation of proteins with key signaling roles
such as Raf-1, protein kinase C, and phosphatidylinositol 3-kinase by
14-3-3 has been described.12,14,15 This group may have
their kinase activity altered by this binding depending on the
experimental conditions used.
View larger version (12K):
[in this window]
[in a new window]
| Fig 1.
14-3-3 structural features and deletion mutants used
in yeast 2-hybrid cotransformations with platelet GPIb subunits. Known and putative domains include a 26-residue N-terminal
dimerization domain (DD),8,9 a domain with homology to the
C terminus of the annexin protein family that acts as a protein kinase
C (PKC) inhibitor (AHD),10 and a putative C-terminal
PKC-inhibitory domain that a recent mutagenesis investigation was
unable to more precisely localize (PKC ID).8 A C-terminal
phosphoserine (residue 185*) has also been
characterized.11
|
|
Recent affinity chromatography experiments using a monoclonal antibody
to GPIb led to the coelution of a 29-kD protein with GPIb.16 Amino acid sequencing of the protein led to its
identification as the isoform of 14-3-3. In view of the association
of 14-3-3 with mediators that participate in known early signaling
events,14,15,17 and the protein's membrane-binding
domain8,18 and phosphoserine site,11 one may
speculate that this protein participates in GPIb signaling following
vWF stimulation. The current study uses the yeast two-hybrid expression
system, confocal microscopy, and platelet compartment immunoblot
studies to document and quantify the interaction of
14-3-3 with cytoplasmic domains of the GPIb subunits,
and begins to characterize its potential role in GPIb signaling.
| |
MATERIALS AND METHODS |
Yeast two-hybrid constructs and transformation.
The cytoplasmic domains of platelet GPIb and GPIb were each
polymerase chain reaction (PCR)-amplified from platelet cDNA generated
by platelet RNA reverse transcription. Platelet RNA was isolated from
the therapeutic pheresis material of a patient with essential
thrombocythemia. The sense primers each contained EcoRI sites
and the antisense primers contained SalI sites to facilitate
directional cloning of the restriction enzyme-digested PCR products
into the yeast expression vectors pGBT9 and pGAD424 (Clontech
Laboratories Inc, Palo Alto, CA). This resulted in the in-frame fusion
of each cytoplasmic domain to the 3 end of either the GAL4 (1-147)
DNA-binding domain (pGBT9) or the GAL4 (768-881) activation domain.
Similar PCR and cloning procedures led to construction of yeast
expression vectors containing full-length platelet
14-3-3.19
Construction of the GPIb (ser166ala) substitution mutant was
achieved with the site-directed mutagenesis technique of
Michael.20 PCR amplification of the Ib cytoplasmic
domain used the original two wild-type primers along with a third sense
primer incorporating the ser166ala mutation and an HpaI site.
The latter facilitated colony screening following transformation into
Escherichia coli without altering the amino acid sequence of
the resultant protein other than the desired substitution. PCR was
performed in the presence of a thermostable DNA ligase (Taq ligase;
Sigma Inc, St Louis, MO) to permit 5 to 3 ligation of the wild-type
sense strand to the first base of the mutagenic sense primer. The
14-3-3 deletion constructs (Fig 1) were prepared similarly to the
wild-type constructs using primers with EcoRI (sense) and
SalI (antisense) restriction site adaptors. All wild-type and
mutant DNA sequences were confirmed by automated sequencing technology.
Transformations of pairwise vector combinations into yeast strain
SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,
112, canr, gal4-542, gal80-538, URA3 :: GAL1-lacZ; Clontech) were made
as described by Bartel et al.21 After verification of the
strain's nutritional phenotype (auxotrophic for tryptophan and
leucine), early log-phase (OD600 = 0.2 to 0.3) cultures
were grown for 3 hours at 30°C in YPD medium before transformation of
100 µL with 100 ng of each construct. Following agitation in 40%
polyethylene glycol/100-mmol/L lithium acetate at 30°C for 30 minutes, addition of 10% dimethyl sulfoxide, and heat shock at 42°C
for 15 minutes, the transformants were plated on selective media at
30°C until 2-mm colonies were visible.
Liquid culture and colony filter-lift -galactosidase
assays.
For quantitative -galactosidase assays, a single yeast colony was
innoculated into 5 mL SD medium without tryptophan and leucine and
agitated overnight at 30°C, and 2 mL was added to 8 mL YPD liquid and
grown until the OD600 was 0.5 to 0.7. Then, 1.5 mL was
processed for quantitation of -galactosidase activity expressed in
units using ONPG substrate and taken to represent the strength of
interaction between the two fusion proteins.21-24 Colony
filter-lifts were performed on transformants by submersion of
nitrocellulose transfer membranes lifted from each colony plate into
liquid nitrogen (Clontech). This was followed by incubation at 30°C
for up to 8 hours over Whatman (Maidstone, UK) filter paper presoaked in Z buffer (60 mmol/L Na2
HPO4 · 7H2O, 40 mmol/L NaH2PO4H2O, 10 mmol/L KCl, and 1 mmol/L MgSO4, pH 7.0) with 0.27% -mercaptoethanol and
1.67% X-gal in 100-mm petri dishes.
Platelet and human erythroleukemia cell confocal microscopy studies.
Human erythroleukemia (HEL) cells were grown at 37°C in a 5%
CO2 atmosphere in RPMI 1640 medium (Bio-Whittaker Inc,
Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine
serum, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 100 U/mL
penicillin, 0.1 mg/mL streptomycin, 2.5 µg/mL amphotericin B, and
2.05 µg/mL desoxycholate (Fungizone; Life Technologies Inc, Grand
Island, NY). To separate the platelets, platelet-rich
plasma was centrifuged twice (1,000g for 10 minutes) followed by suspension of the platelet pellet in 35 mL washing
buffer (10 mmol/L Tris, pH 7.0, 150 mmol/L NaCl, 1 mmol/L EDTA, and 7 mmol/L theophylline). Following centrifugation at 100g for 10 minutes, the suspended platelets were pelleted at 1,000g for 10 minutes and the concentration was determined in 2 mL Tyrode's
buffer.25
Rabbit anti-human GPIb polyclonal antibody,26 mouse
monoclonal antibody Beb1 (anti-human GPIX), and mouse monoclonal
antibody C-34 (anti-human Ib) were used in confocal microscopy
studies. The latter two were characterized by our laboratory and others in association with the Fifth International Workshop on Leukocyte Differentiation Antigens.27 Mouse anti-CD62P was obtained
from Becton Dickinson Immunocytometry Systems (San Jose, CA). Gi27 (mouse anti-human GPIb) was a gift from Dr Sentot Santoso of Universitat Giessen (Giessen, Germany). Rabbit polyclonal anti-human 14-3-3 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Fluorescein-isothiocyanate (FITC)-conjugated goat F(ab)2 anti-rabbit
antibody was obtained from Southern Biotechnology Associates Inc
(Birmingham, AL), and CY3-conjugated goat anti-mouse antibody was obtained from Jackson Laboratories (West
Grove, PA).
Platelets and HEL cells assessed for 14-3-3 and GPIb colocalization
were aliquotted to 30 × 106 platelets/mL or 5 × 106 cells/mL phosphate-buffered saline (PBS) or PBS with
1% BME if subsequently labeled with anti-Ib. After 15 minutes, each
platelet sample was cytospun onto a slide for the rest of the labeling procedure while HEL cells were centrifuged (130g for 10 minutes) following each of the remaining steps and then resuspended for the next step. The PBS incubation step was followed by suspension of
platelets and HEL cells in 90% ethanol in PBS for 45 minutes.28 After washing in PBS, they were incubated for 30 minutes with 1:10 to 1:100 dilutions of two primary antibodies. After
washing twice, they were then incubated for 30 minutes with 1:10 to
1:100 dilutions of fluorochrome-conjugated secondary antibodies.
Following this, the platelets and HEL cells were washed twice and then
suspended in 50 µL PBS. For confocal microscopy, 20 µL
antibody-labeled suspended HEL cells were mounted onto a glass slide to
which a glass cover slip was applied separated by an adhesive tape
spacer to prevent crush artifact. Platelet and HEL cell slides were
visualized using an ACAS Ultima Laser Cytometer (Meridian Instruments,
Okemos, MI). Slides were scanned in dual-color confocal mode
(pinhole = 225 µm for platelets and 400 µm for HEL cells) using a
100× oil-immersion objective (numeric aperture = 1.3), a step size
of 0.1 µm/pixel for platelets and 0.2 µm for HEL cells, and a field
size of 360 × 360 pixels (platelets) or 180 × 180 or 270 × 270 pixels (HEL cells). FITC fluorescence was detected in PMT1 with a
530/30-nm band-pass filter, and CY3 fluorescence was detected after
separation with a dichroic beam splitter (560-nm short-pass filter) in
PMT2 with a 580/30-nm band-pass filter. Dual-color digital images were
displayed on a DASY 9000 Image analysis workstation (Meridian) using
the manufacturer's software, and were saved as tag interchange file format (TIFF) files. Similarly, after using a threshold for background fluorescence, pixel histograms were generated from these images and
displayed using the manufacturer's software and printed with a
Sony Mavigraph video printer (Sony Corp, Tokyo, Japan).
Platelet 14-3-3 translocation studies.
To study the relative amount of 14-3-3 present in platelet
compartments during the resting state or following stimulation with
known agonists, fresh platelets were first isolated from whole blood as
already described. After suspension in Tyrode's buffer,
108 platelets were aggregated for 5 minutes at 37°C
following addition of either (1) 10 µg/mL vWF isolated from human
cryoprecipitate,29 (2) 1 mg/mL ristocetin (American
Biochemical and Pharmaceutical Corp, New York, NY), (3) both 10 µg/mL
vWF and 1 mg/mL ristocetin, (4) 1 U/mL thrombin (Sigma), (5) 20 µmol/L adenosine diphosphate (ADP), or (6) 500 µg/mL
arachidonic acid (Bio/Data Corp, Hatboro, PA). This was followed by
sample incubation in platelet lysis buffer (10 mmol/L EGTA, 2% Triton
X-100, 1 mmol/L PMSF, 100 mmol/L benzamidine, 4 mg/mL leupeptin, 100 mmol/L Tris hydrochloride, pH 7.4, and 2 mmol/L sodium orthovanadate)
on a rocker at 4°C for 1 hour. Samples were then fractionated into
Triton X-100-soluble and -insoluble (cytoskeletal) components by
10,000g centrifugation for 5 minutes. This supernatant was in
turn centrifuged at 100,000g for 3 hours at 4°C to separate
the membrane skeletal component from platelet cytosol.30
Pellet samples, after washing twice in PBS/0.05% Tween 20, along with
50 µL of each supernatant solution were then resuspended in sodium
dodecyl sulfate (SDS) protein loading buffer, boiled for 2 minutes,
electrophoresed on a 10% SDS-polyacrylamide gel,31
electrotransferred to a 0.45-µm nitrocellulose membrane (Millipore
Corp, Bedford, MA), and immunoblotted with anti-14-3-3 antibody C-16
or K-19 (Santa Cruz Biotechnology) followed by 32.5 µCi
125I-labeled Staphylococcal protein A (New England Nuclear,
Boston, MA). Autoradiograph bands were subsequently analyzed
quantitatively using area integration on an ImageQuant densitometer
(Molecular Dynamics, Sunnyvale, CA).
| |
RESULTS |
Yeast two-hybrid assays.
We were interested in characterizing the interaction of the signaling
protein 14-3-3 with the cytoplasmic domains of the GPIb and subunits. Our aims were to determine to which of the GPIb subunits
14-3-3 would bind; to determine what role, if any, was played by a
previously documented phosphoserine residue on GPIb32;
and to map the GPIb binding domain on 14-3-3.
PCR-amplified cDNA fragments encoding the entire cytoplasmic domains of
GPIb and GPIb along with a GPIb sequence in which serine 166 had been mutated to alanine were fused in-frame to either the
DNA-binding domain or activation domain of the yeast plasmids pGBT9 or
pGAD424, respectively. cDNAs encoding deletion mutants along with
wild-type 14-3-3 (Fig 1) were similarly cloned into the same yeast
expression vectors. Fusion protein expression of transformed unpaired
GPIb and 14-3-3 constructs by themselves did not cause transactivation
of the lacZ GAL4 reporter gene present in the yeast host strain SFY526,
as determined by colony filter-lift assays. These vectors were next
transformed into yeast in pairwise combinations along with three
pairwise positive and four negative control transformations as follows:
wild-type full-length GAL4 gene in pGBT9 alone; murine
p53(72-390)/SV40 large T antigen(84-708); GPIb/GPIb; pGBT9/pGAD424; murine p53(72-390)/pGAD424;
pGBT9/SV40 large T antigen(84-708); and human lamin
C(66-230)/SV40 large T antigen(84-708)
(Clontech). To further exclude nonspecific interactions, each vector
was also cotransformed into yeast with other two-hybrid vectors
encoding irrelevant proteins not expected to interact with the GPIb or
14-3-3 constructs (Figs 2 and 3).
View larger version (24K):
[in this window]
[in a new window]
| Fig 2.
14-3-3 binds to the cytoplasmic domains of GPIb and
GPIb in yeast. Yeast were cotransformed with fusion proteins
composed of GAL 4 modular domains hybridized to either GPIb, 14-3-3,
or control proteins. Data bars represent the mean ± SE of 5 separate experiments using analysis of 36 independent colonies and 3 spectrophotometric readings per colony at different concentrations.
Five representative negative control transformations among the 8 performed are shown.
|
|
View larger version (26K):
[in this window]
[in a new window]
| Fig 3.
GPIb binding to 14-3-3 deletion mutants is reduced
compared with the interaction with full-length 14-3-3. The binary
protein interactions along with the further controls shown in Fig 2 and 3 additional positive and 4 negative control transformations not shown
were studied, and quantitative -galactosidase assays were performed.
Deletion mutant activity was reduced 51% to 59% v full-length 14-3-3. Data bars represent the mean ± SE of 5 separate experiments using analysis of 37 independent colonies with 3 spectrophotometric readings per colony at different concentrations.
|
|
Following incubation at 30°C for 3 to 4 days, individual colonies
were grown overnight in selective SD medium, and -galactosidase activity was quantitatively determined using ONPG
substrate.21 In this system, the extent of expression of
the reporter gene lacZ can be interpreted as an indication of the
strength of interaction between the two fusion
proteins.21-24 Figure 2 shows
the wild-type 14-3-3/GPIb fusion protein quantitative interactions
along with five of eight negative control combinations. The findings
suggest 14-3-3 interacts with both the GPIb and cytoplasmic
domains, with significantly increased -gal activity evident in both
of these interactions versus the eight negative control combinations (Fig 2 and data not shown). Furthermore, when ser166 on GPIb is
replaced with alanine, its interaction is reduced 20% to 40% (mean, 27%) with 14-3-3.
Figure 3 shows results for
-galactosidase quantitative assays when the Ib cytoplasmic domain
is cotransformed into yeast with full-length 14-3-3 and the
14-3-3 deletion mutants outlined in Fig 1. These findings along with
the findings from qualitative X-gal assays (Fig
4) suggest that both Ib and Ib
interact optimally with full-length 14-3-3 compared with any of the
deletion mutants studied. Assays of Ib/14-3-3 colonies from each
of the three 14-3-3 deletion mutant transformations showed a
reduction in reporter gene activity of 51% to 59% compared with the
analysis of Ib/14-3-3 full-length colonies. Interactions between
the cytoplasmic domain of Ib and the 14-3-3 deletion mutants were too low to quantify using an ONPG substrate, and were barely
appreciable using the more sensitive X-gal colony filter-lift assay
(Fig 4). Thus, we were unable to map the GPIb binding domain on 14-3-3 using the yeast two-hybrid assay, perhaps due to the need for at least
two noncontiguous regions of 14-3-3 for optimal interaction with
GPIb and/or the need for 14-3-3 to be dimerized or
membrane-associated.
View larger version (40K):
[in this window]
[in a new window]
| Fig 4.
-Galactosidase colony filter-lift assays of
GPIb/14-3-3 yeast 2-hybrid transformations. Reporter gene activity
is reflected by expression of blue (dark) colonies, most notable with
the combination of GPIb and full-length 14-3-3, followed by
GPIb and deletion mutant 14-3-3 (1-123) and GPIb and
full-length 14-3-3. The X-gal substrate used in
these assays is considered more sensitive to -galactosidase activity
than the ONPG used in the quantitative assays in Figs 2 and 3.
|
|
Confocal microscopy studies.
After demonstrating the interaction of 14-3-3 with GPIb and
GPIb through the yeast two-hybrid system, we were next interested in
documenting these relationships in vivo using human platelets and HEL
cells, a tumor cell line that manifests several megakaryocyte and
platelet antigens including GPIb.33 Following membrane
permeabilization, we proceeded to colabel platelets and cells with
anti-14-3-3 and either anti-human GPIb or anti-GPIb
antibodies followed by fluorochrome-conjugated secondary antibody
combinations. Samples were also colabeled with primary and secondary
antibody combinations to either 14-3-3 and CD62P, Ib and CD62P
(negative controls), or GPIb and GPIX (positive control). Cytospun
slides (platelets) or HEL cells in suspension were then visualized by
confocal microscopy. Our objective was to determine if each of the
protein pairs colocalized and associated with each other to within the
limits of resolution of a single pixel (~200 nm). Colocalization of
FITC (green) and Cy3 (red) is evidenced by a yellow appearance (Fig 5C
to E) and by corresponding
pixel histograms that demonstrate a linear correlation in antibody
expression for each fluorescence intensity level34 (Fig 5G
to I). Absence of colocalization is suggested by a red and green
appearance (Fig 5A and B) and by a pixel histogram exhibiting a diffuse
pattern34 (Fig 5F). These data support the yeast two-hybrid evidence that 14-3-3 interacts with GPIb and GPIb present in platelets and HEL cells.
View larger version (132K):
[in this window]
[in a new window]
View larger version (57K):
[in this window]
[in a new window]
| Fig 5.
Colocalization of GPIb subunits and 14-3-3 in HEL
cells (A to E) and platelets (F to I). Permeabilized platelets and HEL cells were colabeled with antibody pairs. HEL cells were labeled with
either (A) anti-GPIb/FITC (green) and anti-CD62P/CY-3 (red) (negative control), (B) anti-14-3-3/FITC and anti-CD62P/CY-3 (negative control), (C) anti-GPIb/FITC and anti-GPIX/CY-3 (positive control), (D) anti-GPIb/CY-3 and anti-14-3-3/FITC, or (E)
anti-GPIb/CY-3 and anti-14-3-3/FITC. Inset: detector 1, FITC
fluorescence intensity; detector 2, CY-3 fluorescence intensity. Yellow
fluorescence suggests that the 2 antibodies colocalize to within
approximately 200 nm. A and C to E field size, 36 × 36 µm; B field
size, 54 × 54 µm. Representative platelet pixel histograms for each
antibody pair are shown.
(F to I) and reflect colocalization of
GPIX/Ib (G), GPIb/14-3-3 (H), and GPIb/14-3-3 (I). Note
the approximately equal contribution from each fluorochrome-conjugated
secondary antibody at different fluorescence intensities in G (positive
control), H, and I. This suggests that each computer pixel is detecting an equal amount of both antibodies present, which implies their respective epitopes are present in equal amounts (colocalized) in the
3-dimensional volume scanned by the microscope. This contrasts with F
(1 of 2 negative controls), in which a diffuse pattern reflecting
noncolocalization is seen. Three experiments with HEL cells and 2 with
platelets were performed.
|
|
To provide statistical evidence for these conclusions, we calculated
the degree to which colocalization occurred in each of the colabeled
samples. This was determined by calculating the coefficient of
variation ([CV] the standard deviation divided by the mean) for the
ratio of red to green pixels for individual cells. These CVs were
averaged (4 to 15 cells per sample) for each image and analyzed for
differences between them using a two-sample one-tailed t test
assuming unequal variances. Thus, for example, in one experiment the
colocalization between GPIb/GPIX was found not to significantly
differ versus GPIb/14-3-3 (P = .23), while that between
GPIb/14-3-3 and CD62P/14-3-3 did (P = .05). Lower average
HEL cell CVs from three experiments confirmed the increased colocalization of GPIb and GPIb with 14-3-3, compared with
significantly less colocalization exhibited by GPIb and 14-3-3 with
CD62P (data not shown). Hence, the confocal photomicrographs, pixel
histograms, and statistical analysis suggest that colocalization
between 14-3-3 and both GPIb and GPIb cytoplasmic domains
occurs in platelets and HEL cells as suggested by the yeast two-hybrid
experiments.
Platelet 14-3-3 translocation studies.
Having used the yeast two-hybrid system and confocal microscopy studies
to obtain structural data concerning the interaction of 14-3-3 and
platelet GPIb, we were interested in determining whether 14-3-3
translocated between the platelet cytosol and either the platelet
cytoskeleton or membrane skeleton in response to stimulation with known
agonists.30 Translocation to the cytoskeletal component in
response to surface receptor occupancy is a property of many signaling
proteins; these events typically lead to cytoskeletal reorganization,
which in turn promotes events such as shape change, locomotion, and
activation.4,35
Following isolation from whole blood, platelets were stimulated with
either vWF alone, ristocetin alone (controls), both vWF and ristocetin,
thrombin, ADP, or arachidonic acid. They were then subjected to
aggregometry, lysed, and separated by low- and high-speed
centrifugation into cytosolic, membrane skeletal, and cytoskeletal
components, which were in turn subjected to SDS-polyacrylamide gel
electrophoresis and immunoblotted with one of two anti-14-3-3 antibodies. Immunoblot band densitometry determinations from these experiments showed a 4.9-fold increase of cytoskeletal 14-3-3 in
vWF/ristocetin-stimulated platelets compared with unstimulated platelets, and a 2.9-fold increase compared with thrombin-stimulated platelets (Table 1). Additional studies
showed a similar or reduced amount of cytoskeletal 14-3-3 in
response to ADP and arachidonic acid stimulation, respectively,
compared with thrombin stimulation (data not shown).
View this table:
[in this window]
[in a new window]
|
Table 1.
Anti-14-3-3 Immunoblot Densitometry: Fold Changes in
Platelet Lysate 14-3-3 Binding Over Resting State in Same
Compartment
|
|
| |
DISCUSSION |
Intracellular signaling that takes place immediately following GPIb
receptor occupancy connecting the adhesive event to platelet activation
and aggregation has not been well characterized. In the present study,
we used yeast two-hybrid experiments and confocal microscopy
to further characterize the interaction of platelet GPIb with an
isoform of the 14-3-3 protein family. While their exact function
remains elusive, the 14-3-3 protein family shows a diverse array of
associations and biologic activities related to signaling and
cell-cycle progression.7,12
We have shown that 14-3-3 will interact in vivo with both the GP
Ib and Ib subunit cytoplasmic domains; we are unable to determine
the preference, if any, of 14-3-3 for either subunit with these
methods. This interaction is modestly reduced when serine 166 on
GPIb is replaced with alanine. We have also demonstrated that GPIb
interacts optimally with full-length 14-3-3, while showing an
approximately equally reduced interaction with each of three deletion
mutants. Confocal microscopy studies of platelets and HEL cells have
provided additional characterization of the 14-3-3 interaction with
GPIb receptor subunits. In this respect, the positive confocal data
support the yeast two-hybrid findings but do not conclusively prove
them, because of the imaging system's limits of resolution. Negative
confocal data, on the other hand, would have refuted the findings.
Finally, platelet 14-3-3 translocation studies in stimulated and
resting platelets have provided evidence that 14-3-3 may translocate
from the cytosolic component to the cytoskeleton in response to vWF and
ristocetin. A similar agonist-induced cytoskeletal relocalization of
platelet molecules with signaling roles such as Src, Syk, and FAK has
been observed.30,35 Our methods were unable to detect any
appreciable amount of agonist-induced translocation to or from the
membrane skeleton.
Using affinity chromatography, a 29-kD protein found to be 14-3-3
was recently coeluted with GPIb-IX by Du et al,16 who later
showed that the 14-3-3 binding site on GPIb resides within a 15 residue C-terminal sequence.36 Binding was not contingent upon the presence of actin-binding protein and was abolished by removing the C-terminal five residues of GPIb, suggesting their critical role in the association.16,36 Our results also
suggest that 14-3-3 will associate with the cytoplasmic domain of
GPIb in vivo along with GPIb (Figs 2, 4, and 5). Du's group
provided some evidence that 14-3-3 may not interact with
GPIb.36 Our study differs from theirs with respect to
the methods used to address this question (yeast two-hybrid studies and
confocal microscopy v peptide inhibition and synthetic peptide
binding studies). In addition, this group's synthetic peptides and
anti-GPIb immunogens did not incorporate the cytoplasmic domain
GPIb phosphoserine residue. Recent investigation has provided
evidence that phosphoserine recognition may play an important role in
14-3-3 interaction with other proteins.37,38
Since GPIb contains the receptor's ligand binding site and
interacts with the membrane skeleton and GPIb contains a potentially signal-transducing phosphoserine residue in its intracellular domain,
it is not unreasonable to speculate that 14-3-3 may indeed interact
with both subunits. In this respect, dimerization of 14-3-3 not only
may serve to potentially link receptors and signaling proteins but may
also bridge important functional elements within a multiple subunit
complex like GPIb.39
The reduction in interaction between 14-3-3 and GPIb when serine 166 is replaced by alanine is supported by the observation that
phosphoserine recognition may play an important role in the former's
interaction with other proteins.37,38,40,41 It is thus
reasonable to speculate that the platelet GPIb interaction with 14-3-3 may be mediated, in part, by the phosphoserine-containing cytoplasmic
domain of GPIb. Other groups have suggested that 14-3-3 may
recognize clusters of serine residues in an amphipathic helical
structure appropriate for its ligand-binding groove recently characterized through crystallography studies.36,42,43 Such a serine-rich cluster exists at the C-terminal region (residues 596 to
610) of GPIb where interaction with 14-3-3 has been recently characterized.36 Our yeast two-hybrid and confocal
microscopy data provide evidence for both of the 14-3-3 recognition
motif theories by demonstrating 14-3-3 interactions with GPIb and
GPIb.
Our yeast two-hybrid experiments suggest that GPIb interacts optimally
with full-length 14-3-3, with 51% to 59% reduced interaction demonstrable between GPIb and deletion mutants of 14-3-3 (Figs 1 and
3). Using these techniques, we were therefore unable to map a specific
GPIb binding site on 14-3-3. These results may be attributable to
the dimerization domain involving the N-terminal 26-amino acid
residues, suggesting that 14-3-3 may need to homodimerize or
heterodimerize for optimal activity.8,9,13,44,45 The 14-3-3 proteins have been found at high concentrations on synaptic plasma
membranes, and another study suggests the N-terminal region is
responsible for this membrane binding.8,18 Optimal
GPIb-14-3-3 interaction may thus be facilitated by binding of the
latter to the platelet membrane. A third possible contributing cause to these results rests with the fact that GPIb-14-3-3 interaction may be
mediated by at least two distinct and noncontiguous regions of 14-3-3. In this respect, the C-terminal phosphoserine 185 of 14-3-3 is
surrounded by a consensus sequence for cyclin-dependent kinases, and
amino acids 171 to 213 are required for interaction with phosphorylated
tryptophan hydroxylase, the rate-limiting enzyme of serotonin
synthesis.11,46 Therefore, for example, if both the
N-terminal dimerization domain and C-terminal residues 171 to
213 were required for optimal interaction, only the full-length 14-3-3 species would be capable of maximal interaction with GPIb, as
our findings suggest.
The translocation of 14-3-3 to the platelet cytoskeleton upon vWF
and ristocetin stimulation is suggestive evidence that this protein
participates in GPIb signaling (Table 1). Similar fractionation
procedures have been used to demonstrate the cytoskeletal translocation
of membrane skeleton and selective signaling molecules, such as the
tyrosine kinases Src and FAK, upon platelet surface receptor
binding.4,30,35,47 vWF stimulation and aggregation of
platelets is capable of inducing cytoskeletal translocation and
activation of both the lipid kinase phosphatidylinositol 3-kinase and
Src independently of other agonists.4 These events were not
observed in Bernard-Soulier platelets. Recent investigation has
demonstrated an association of 14-3-3 and phosphatidylinositol 3-kinase with additional evidence showing that it may act as a negative
regulator of this enzyme by directly binding to its catalytic subunit.15 It could be hypothesized that 14-3-3 may
interact with platelet GPIb under resting conditions and thereby exert a similar negative regulatory effect on phosphatidylinositol 3-kinase activity.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted September 29, 1997.
Supported by Grants No. HL39947 and ES07033 from the National
Institutes of Health, a Merit Review Award from the Department of
Veterans Affairs, and the Medical Research Council of Canada.
Presented in part at the 1996 38th Annual Meeting of the
American Society of Hematology, Orlando, FL (December 7-10, 1996).
Address reprint requests to Gerald J. Roth, MD, Medical Service,
Seattle VA Medical Center (111), 1660 S Columbian Way, Seattle, WA
98108.
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.
| |
ACKNOWLEDGMENT |
We are grateful to Chao-Yang Li and Kin Ritchie for helpful discussion,
to Susan Danner and Tanya Hill for excellent technical assistance, and
to Sally Swedine for help with the graphics.
| |
REFERENCES |
1.
Roth GJ:
Developing relationships: Arterial platelet adhesion, glycoprotein Ib and leucine-rich glycoproteins.
Blood
77:5,
1991[Free Full Text]
2.
Bernard J,
Soulier J-P:
Sur une nouvelle variete de dystrophie thrombocytaire-hemorragipare congenitale.
Semin Hop Paris
24:3217,
1948
3.
Nurden AT,
Dupuis D,
Kunicki TJ,
Caen JP:
Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
J Clin Invest
67:1431,
1981
4.
Jackson SP,
Schoenwaelder SM,
Yuan Y,
Rabinowitz I,
Salem HH,
Mitchell CA:
Adhesion receptor activation of phosphatidylinositol 3-kinase.
J Biol Chem
269:27093,
1994[Abstract/Free Full Text]
5. Moore BW, Perez VJ: Specific acidic proteins of the nervous
system, in Carson FD (ed): Physiological and Biochemical Aspects of
Nervous Integration. Englewood Cliffs, NJ, Prentice-Hall, 1967, p 343
6.
Burbelo PD,
Hall A:
14-3-3 Proteins. Hot numbers in signal transduction.
Curr Biol
5:95,
1995[Medline]
[Order article via Infotrieve]
7.
Aitken A:
14-3-3 Proteins on the MAP.
Trends Biomed Sci
20:95,
1995
8.
Jones DHA,
Martin H,
Madrazo J,
Robinson K,
Nielsen P,
Roseboom PH,
Patel Y,
Howell SA,
Aitken A:
Expression and structural analysis of 14-3-3 proteins.
J Mol Biol
245:375,
1995[Medline]
[Order article via Infotrieve]
9.
Luo Z-J,
Zhang X-F,
Rapp U,
Avruch J:
Identification of the 14-3-3 domains important for self-association and raf binding.
J Biol Chem
270:23681,
1995[Abstract/Free Full Text]
10.
Schlaepfer CC,
Jones J,
Haigler HT:
Inhibition of protein kinase C by annexin V.
Biochemistry
31:1886,
1992[Medline]
[Order article via Infotrieve]
11.
Aitken A,
Howell S,
Jones D,
Madrazo J,
Patel Y:
14-3-3 and  are the phosphorylated forms of raf-activating 14-3-3 and .
J Biol Chem
270:5706,
1995[Abstract/Free Full Text]
12.
Morrison D:
14-3-3: Modulators of signaling proteins?
Science
266:56,
1994[Free Full Text]
13.
Jones DH,
Leys S,
Aitken A:
Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: Implications for function as adapter proteins.
FEBS Lett
368:55,
1995[Medline]
[Order article via Infotrieve]
14.
Robinson K,
Jones D,
Patel Y,
Madrazo J,
Martin S,
Howell S,
Elmore M,
Finnen MJ,
Aitken A:
Mechanism of inhibition of protein kinase C by 14-3-3 isoforms.
Biochem J
299:853,
1994
15.
Bonnefoy-Berard N,
Liu Y-C,
von Willebrand M,
Sung A,
Elly C,
Mustelin T,
Yoshida H,
Ishizakza K,
Altman A:
Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.
Proc Natl Acad Sci USA
92:10142,
1995[Abstract/Free Full Text]
16.
Du X,
Harris SJ,
Tetaz TJ,
Berndt MC:
Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex.
J Biol Chem
269:18287,
1994[Abstract/Free Full Text]
17.
Irie K,
Gotoh Y,
Yashar BM,
Errede B,
Nishida E,
Matsumoto K:
Stimulatory effects of yeast and mammalian 14-3-3 proteins on the raf protein kinase.
Science
265:1716,
1994[Abstract/Free Full Text]
18.
Martin H,
Rostas J,
Patel Y,
Aitken A:
Analysis of subcellular distribution of 14-3-3 isoforms in rat brain using specific antibodies.
J Neurochem
63:2259,
1994[Medline]
[Order article via Infotrieve]
19.
Zupan LA,
Steffens DL,
Berry CA,
Landt M,
Gross RW:
Cloning and expression of a human 14-3-3 protein mediating phospholipolysis.
J Biol Chem
267:8707,
1992[Abstract/Free Full Text]
20.
Michael SF:
Mutagenesis by a phosphorylated oligo during PCR amplification.
Biotechniques
16:410,
1994[Medline]
[Order article via Infotrieve]
21. Bartel PL, Chien C-T, Sternglanz R, Fields S: Using the
two-hybrid system to detect protein-protein interactions, in Hartley DA
(ed): Cellular Interactions in Development. A Practical Approach.
Oxford, UK, IRL, 1993, p 153
22.
Yang M,
Wu Z,
Fields S:
Protein-peptide interactions analyzed with the yeast two-hybrid system.
Nucleic Acids Res
23:1152,
1995[Abstract/Free Full Text]
23.
Yan K,
Kalyanaraman V,
Gautam N:
Differential ability to form the G protein betagamma complex among members of the beta and gamma subunit families.
J Biol Chem
271:7141,
1996[Abstract/Free Full Text]
24.
Eiganthaler M,
Hofferer L,
Shattil SJ,
Ginsberg MH:
A conserved sequence motif in the integrin 3 cytoplasmic domain is required for its specific interaction with 3-endonexin.
J Biol Chem
272:7693,
1997[Abstract/Free Full Text]
25.
Fox JEB,
Berndt MC:
Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets.
J Biol Chem
264:9520,
1989[Abstract/Free Full Text]
26.
Canfield VA,
Ozols J,
Nugent D,
Roth GJ:
Isolation and characterization of the and chains of human platelet glycoprotein Ib.
Biochem Biophys Res Commun
147:526,
1987[Medline]
[Order article via Infotrieve]
27. Schlossman S, Boumsell L, Gilks W, Harlan JM, Kishimoto T,
Morimoto C, Ritz J, Shaw S, Silverstein RL, Springer TA (eds):
Leukocyte Typing, V. Oxford, UK, Oxford University Press, 1995
28.
Schimenti KJ,
Jacobberger JW:
Fixation of mammalian cells for flow cytometric evaluation of DNA content and nuclear immunofluorescence.
Cytometry
13:48,
1992[Medline]
[Order article via Infotrieve]
29.
Roth GJ,
Titani K,
Hoyer LW,
Hickey MJ:
Localization of binding sites within human von Willebrand factor for monomeric type III collagen.
Biochemistry
25:8357,
1986[Medline]
[Order article via Infotrieve]
30.
Fox JEB,
Lipfert L,
Clark EA,
Reynolds CC,
Austin CD,
Brugge JS:
On the role of the platelet membrane skeleton in mediating signal transduction.
J Biol Chem
268:25973,
1993[Abstract/Free Full Text]
31.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680,
1970[Medline]
[Order article via Infotrieve]
32.
Wardell MR,
Reynolds CC,
Berndt MC,
Wallace RW,
Fox JEB:
Platelet glycoprotein Ib is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase.
J Biol Chem
264:15656,
1989[Abstract/Free Full Text]
33. Papayannopoulou T, Yokochi T, Nakamoto B, Martin P: The surface
antigen profile of HEL cells, in Stamatoyannopoulous G, Neinhuis AW
(eds): Globin Gene Expression and Hematopoietic Differentiation. New
York, NY, Liss, 1983, p 277
34.
Calverley DC,
Yagi M,
Stray SM,
Roth GJ:
Human platelet glycoprotein V: Its role in enhancing expression of the glycoprotein Ib receptor.
Blood
86:1361,
1995[Abstract/Free Full Text]
35.
Clark EA,
Shattil SJ:
Regulation of protein tyrosine kinases in platelets.
Trends Biomed Sci
19:464-469,
1994
36.
Du X,
Fox JE,
Pei S:
Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib.
J Biol Chem
271:7362,
1996[Abstract/Free Full Text]
37.
Muslin AJ,
Tanner JW,
Allen PM,
Shaw AS:
Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.
Cell
84:889,
1996[Medline]
[Order article via Infotrieve]
38.
Liao J,
Omary MB:
14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor.
J Cell Biol
133:345-357,
1996[Abstract/Free Full Text]
39.
Vincenz C,
Dixit VM:
14-3-3 proteins associate with A20 in an isoform-specific manner and function both as chaperone and adapter molecules.
J Biol Chem
271:20029,
1996[Abstract/Free Full Text]
40.
Michaud NR,
Fabian JR,
Mathes KD,
Morrison DK:
14-3-3 is not essential for Raf-1 function: Identification of Raf-1 proteins that are biologically activated in a 14-3-3 and Ras-independent manner.
Mol Cell Biol
15:3390,
1995[Abstract]
41.
Furukawa Y,
Ikuta N,
Omata T,
Yamauchi T,
Isobe T,
Ichimura T:
Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein.
Biochem Biophys Res Commun
194:144,
1993[Medline]
[Order article via Infotrieve]
42.
Xiao B,
Smerdon SJ,
Jones DH,
Dodson GG,
Soneji Y,
Aitken A,
Gamblin SJ:
Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways.
Nature
376:188,
1995[Medline]
[Order article via Infotrieve]
43.
Liu D,
Bienkowska J,
Petrosa C,
Collier RJ,
Fu H,
Liddington R:
Crystal structure of the zeta isoform of the 14-3-3 protein.
Nature
376:191,
1995[Medline]
[Order article via Infotrieve]
44.
Loeb LA,
Gross RW:
Identification and purification of sheep platelet phospholipase A isoforms.
J Biol Chem
261:10467,
1986[Abstract/Free Full Text]
45.
Braselman S,
McCormick F:
Bcr and Raf form a complex in vivo via 14-3-3 proteins.
EMBO J
14:4839,
1995[Medline]
[Order article via Infotrieve]
46.
Ichimura T,
Uchiyama J,
Kunihiro O,
Ito M,
Horigome T,
Omata S,
Shinkai F,
Kaji H,
Isobe T:
Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase.
J Biol Chem
270:28515,
1995[Abstract/Free Full Text]
47.
Oda A,
Druker B,
Smith M,
Salzman EW:
Association of pp60src with Triton X-100 insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.
J Biol Chem
267:20075,
1992[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
F.-T. Mu, R. K. Andrews, J. F. Arthur, A. D. Munday, S. L. Cranmer, S. P. Jackson, F. C. Stomski, A. F. Lopez, and M. C. Berndt
A functional 14-3-3{zeta}-independent association of PI3-kinase with glycoprotein Ib{alpha}, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex
Blood,
May 1, 2008;
111(9):
4580 - 4587.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-Z. Luo, X. Mo, V. Afshar-Kharghan, S. Srinivasan, J. A. Lopez, and R. Li
Glycoprotein Ib{alpha} forms disulfide bonds with 2 glycoprotein Ib{beta} subunits in the resting platelet
Blood,
January 15, 2007;
109(2):
603 - 609.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Dai, R. Bodnar, M. C. Berndt, and X. Du
A critical role for 14-3-3{zeta} protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications
Blood,
September 15, 2005;
106(6):
1975 - 1981.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Mangin, T. David, V. Lavaud, S. L. Cranmer, I. Pikovski, S. P. Jackson, M. C. Berndt, J.-P. Cazenave, C. Gachet, and F. Lanza
Identification of a novel 14-3-3{zeta} binding site within the cytoplasmic tail of platelet glycoprotein Ib{alpha}
Blood,
July 15, 2004;
104(2):
420 - 427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Bialkowska, Y. Zaffran, S. C. Meyer, and J. E. B. Fox
14-3-3{zeta} Mediates Integrin-induced Activation of Cdc42 and Rac: PLATELET GLYCOPROTEIN Ib-IX REGULATES INTEGRIN-INDUCED SIGNALING BY SEQUESTERING 14-3-3{zeta}
J. Biol. Chem.,
August 29, 2003;
278(35):
33342 - 33350.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Perrault, P. Mangin, M. Santer, M.-J. Baas, S. Moog, S. L. Cranmer, I. Pikovski, D. Williamson, S. P. Jackson, J.-P. Cazenave, et al.
Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex
Blood,
May 1, 2003;
101(9):
3477 - 3484.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Bodnar, X. Xi, Z. Li, M. C. Berndt, and X. Du
Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibbeta
J. Biol. Chem.,
November 27, 2002;
277(49):
47080 - 47087.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kanaji, S. Russell, and J. Ware
Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome
Blood,
August 28, 2002;
100(6):
2102 - 2107.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kasirer-Friede, J. Ware, L. Leng, P. Marchese, Z. M. Ruggeri, and S. J. Shattil
Lateral Clustering of Platelet GP Ib-IX Complexes Leads to Up-regulation of the Adhesive Function of Integrin alpha IIbbeta 3
J. Biol. Chem.,
March 29, 2002;
277(14):
11949 - 11956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kuwahara, M. Sugimoto, S. Tsuji, H. Matsui, T. Mizuno, S. Miyata, and A. Yoshioka
Platelet Shape Changes and Adhesion Under High Shear Flow
Arterioscler Thromb Vasc Biol,
February 1, 2002;
22(2):
329 - 334.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. K. Andrews, A. D. Munday, C. A. Mitchell, and M. C. Berndt
Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex
Blood,
August 1, 2001;
98(3):
681 - 687.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Ni, V. Ramakrishnan, Z. M. Ruggeri, J. M. Papalia, D. R. Phillips, and D. D. Wagner
Increased thrombogenesis and embolus formation in mice lacking glycoprotein V
Blood,
July 15, 2001;
98(2):
368 - 373.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Ramakrishnan, F. DeGuzman, M. Bao, S. W. Hall, L. L. Leung, and D. R. Phillips
A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V
PNAS,
February 13, 2001;
98(4):
1823 - 1828.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Mistry, S. L. Cranmer, Y. Yuan, P. Mangin, S. M. Dopheide, I. Harper, S. Giuliano, D. E. Dunstan, F. Lanza, H. H. Salem, et al.
Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX-von Willebrand factor interaction
Blood,
November 15, 2000;
96(10):
3480 - 3489.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Butte, P. Tamayo, D. Slonim, T. R. Golub, and I. S. Kohane
Discovering functional relationships between RNA expression and chemotherapeutic susceptibility using relevance networks
PNAS,
October 4, 2000;
(2000)
220392197.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. D. Munday, M. C. Berndt, and C. A. Mitchell
Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3zeta
Blood,
July 15, 2000;
96(2):
577 - 584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zaffran, S. C. Meyer, E. Negrescu, K. B. Reddy, and J. E. B. Fox
Signaling across the Platelet Adhesion Receptor Glycoprotein Ib-IX Induces alpha IIbbeta 3 Activation Both in Platelets and a Transfected Chinese Hamster Ovary Cell System
J. Biol. Chem.,
May 26, 2000;
275(22):
16779 - 16787.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Feng, N. Christodoulides, J. C. Resendiz, M. C. Berndt, and M. H. Kroll
Cytoplasmic domains of GpIbalpha and GpIbbeta regulate 14-3-3zeta binding to GpIb/IX/V
Blood,
January 15, 2000;
95(2):
551 - 557.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Gu, X. Xi, G. D. Englund, M. C. Berndt, and X. Du
Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX-Mediated Activation of Integrin {alpha}IIb{beta}3 Using a Reconstituted Mammalian Cell Expression Model
J. Cell Biol.,
November 29, 1999;
147(5):
1085 - 1096.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Bodnar, M. Gu, Z. Li, G. D. Englund, and X. Du
The Cytoplasmic Domain of the Platelet Glycoprotein Ibalpha Is Phosphorylated at Serine 609
J. Biol. Chem.,
November 19, 1999;
274(47):
33474 - 33479.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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;
96(23):
13336 - 13341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Falati, C. E. Edmead, and A. W. Poole
Glycoprotein Ib-V-IX, a Receptor for von Willebrand Factor, Couples Physically and Functionally to the Fc Receptor gamma -Chain, Fyn, and Lyn to Activate Human Platelets
Blood,
September 1, 1999;
94(5):
1648 - 1656.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Torti, A. Bertoni, I. Canobbio, F. Sinigaglia, E. G. Lapetina, and C. Balduini
Rap1B and Rap2B Translocation to the Cytoskeleton by von Willebrand Factor Involves Fcgamma II Receptor-mediated Protein Tyrosine Phosphorylation
J. Biol. Chem.,
May 7, 1999;
274(19):
13690 - 13697.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Gu and X. Du
A Novel Ligand-binding Site in the zeta -Form 14-3-3 Protein Recognizing the Platelet Glycoprotein Ibalpha and Distinct from the c-Raf-binding Site
J. Biol. Chem.,
December 11, 1998;
273(50):
33465 - 33471.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Ware, S. Russell, and Z. M. Ruggeri
Generation and rescue of a murine model of platelet dysfunction: The Bernard-Soulier syndrome
PNAS,
March 14, 2000;
97(6):
2803 - 2808.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Butte, P. Tamayo, D. Slonim, T. R. Golub, and I. S. Kohane
Discovering functional relationships between RNA expression and chemotherapeutic susceptibility using relevance networks
PNAS,
October 24, 2000;
97(22):
12182 - 12186.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract