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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1663-1670
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Rapid tyrosine phosphorylation and activation of Bruton's
tyrosine/Tec kinases in platelets induced by
collagen binding or CD32 cross-linking
Atsushi Oda,
Yasuo Ikeda,
Hans D. Ochs,
Brian J. Druker,
Katsutsohi Ozaki,
Makoto Handa,
Tadashi Ariga,
Yukio Sakiyama,
Owen N. Witte, and
Matthew I. Wahl
From the Division of Hematology, Department of Internal Medicine,
and the Blood Center, Keio University, Keio, Japan; the Department of
Microbiology, Immunology, and Molecular Genetics, University of
California at Los Angeles, Los Angeles, CA; the Howard Hughes Medical
Institute; the Department of Pediatrics, University of Washington
School of Medicine, Seattle, WA; the Division of Hematology and Medical
Oncology, Oregon Health Sciences University, Portland, OR; and the
Department of Human Gene Therapy, Hokkaido University School of
Medicine, Sapporo, Japan.
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Abstract |
Stimulation of the platelet nonintegrin collagen receptor,
glycoprotein VI, evokes a signaling response similar to that induced by
antigen receptor activation in B and T lymphocytes. A key transducer of
the lymphocyte signaling pathways is the Bruton's tyrosine kinase
(Btk)/Tec kinase family, which connects receptors to the elevation of
intracellular-free calcium levels. An important signaling function for
Btk in collagen-induced platelet activation in vitro was recently
demonstrated by other researchers using Btk-deficient platelets from
patients with X-linked agammaglobulinemia (XLA). Since Btk-deficiency
does not induce an overt platelet-based bleeding disorder in vivo,
collagen receptor responses may include other Btk/Tec kinase family
members in normal platelets. Both Btk and Tec had increased tyrosine
following stimulation of collagen receptors or CD32 cross-linking. Data
from kinetic analyses and inhibitor studies and the use of
phosphopeptide-specific antibodies recognizing 2 Btk regulatory
phosphorylated tyrosine residues suggest a mechanism for coordinate
recruitment of Btk and Tec through the immunoreceptor tyrosine-based
activation motif, Src family kinases, and phosphatidylinositol 3-kinase. In XLA platelets, collagen treatment increased tyrosine phosphorylation of Tec and several other signaling proteins, including Lyn, Fyb, Slp-76, and the Wiskott-Aldrich syndrome protein. This indicates that important elements of the collagen signaling pathway proximal and distal to Btk and Tec are preserved despite the lack of
functional Btk. The results are consistent with the conclusion that
activation of Tec may sustain XLA platelet function in vivo, while some
in vitro assays of nonintegrin collagen receptor signaling through the
Btk/Tec kinase family reflect the additive dosage of the transducers.
(Blood. 2000;95:1663-1670)
© 2000 by The American Society of Hematology.
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Introduction |
Disruption of vessel walls through injury or rupture of
atherosclerotic plaques exposes subendothelial collagen to blood
platelets and results in formation of a hemostatic
thrombus.1-3 Impairment of the platelet-collagen binding
interaction due to absence or blocking of platelet receptors is
associated with bleeding diathesis, thus illustrating the essential
role for this interaction in hemostasis.1-3 Platelets bind
to collagen through several specific membrane receptor types. Collagen
engagement of these receptors induces a series of biochemical and
morphological events as the platelets are incorporated into a forming
thrombus. The platelet membrane protein integrin  2 1 binds collagen directly,
whereas platelet glycoprotein Ib binds indirectly to collagen through
the von Willebrand factor. After initial adhesion to collagen, other
nonintegrin receptors, such as glycoprotein VI (GPVI), are engaged, and
the platelet proceeds through an irreversible activation sequence.
Collagen-induced platelet intracellular signaling through GPVI
resembles the functional response of B- and T-cell antigen receptors
(BCR and TCR, respectively) to cross-linking.3-8 Activated GPVI signals the platelets by physical association with a critical immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptor (FcR) chain, which is a docking site for proximal transducers of receptor signaling pathways.3
Dimerization and aggregation of receptor chains after ligand
binding or antibody cross-linking activates the proximal
transducers initiating an intracellular signal. In platelets, loss of
the FcR chain function abrogates collagen-induced platelet
activation and aggregation through a decrease in GPVI-mediated
intracellular signal transduction.7 Collagen receptor
signaling through the FcR chain results in activation of multiple
transducers including the Src family kinases, syk tyrosine kinase,
phosphatidylinositol 3-kinase (PI 3-kinase), Btk/Tec kinase
family, and phospholipase C-2 (PLC-2).3-9 These proximal mediators of signal transduction function in a variety of
hematopoietic cells to link lineage-specific surface receptors to
common downstream responses such as release of intracellular calcium,
redistribution of cytoskeletal proteins, and secretion of
granules.3
The Btk/Tec tyrosine kinase family comprises structurally
homologous, functionally interchangeable, dosage-sensitive signal transducing proteins.10 Multiple intracellular signaling
pathways, including the Src family kinases, PI 3-kinase, protein kinase C, and G proteins,11-21 appear to converge in the
modulation of Btk/Tec kinase activity. By influencing PLC-2 activity
and other targets, Btk/Tec kinases are important components of receptor pathways that lead to increased intracellular calcium
levels.9,13,22,23 Btk/Tec kinases may also function in
platelets and lymphoid cells by binding to and tyrosine phosphorylating
the Wiskott-Aldrich syndrome protein (WASP).24-30 Genetic
deficiency of WASP causes abnormal platelet morphology, number, and
functional responses (reviewed in Ochs31 and in Snapper and
Rosen32). Tyrosine phosphorylation of WASP by the Btk/Tec
kinases may be an important mechanism to regulate WASP function in
platelet signaling pathways.
Btk deficiency dramatically influences BCR signaling pathways in human
and murine B lineage cells, and more subtle defects have been
identified in murine mast cell Fc R function.10,29 A role
for Btk/Tec kinases in platelets was recently demonstrated by the
observation of decreased in vitro collagen-induced aggregation of
platelets from XLA patients.9 This influence on platelet function correlated with decreased collagen-induced tyrosine
phosphorylation of PLC-2, delayed intracellular calcium release, and
decreased secretion from dense granules. In contrast to the profound
influence of Btk deficiency on the B-cell lineage, XLA patients do not
exhibit a platelet-dependent bleeding disorder. The presence of a
functional defect of XLA platelets in vitro, without a corresponding
clinical phenotype of bleeding diathesis, suggests that expression of
alternative members of the Btk/Tec kinase family may permit
normal collagen receptor signaling in vivo. To test the hypothesis that
multiple Btk/Tec kinases contribute to collagen receptor signaling, we tested platelets for expression of these kinases and their tyrosine phosphorylation in response to receptor stimulation. The data were
consistent with the coordinated activation of Btk and Tec in receptor
pathways signaling through the ITAM, PI 3-kinase, and Src
kinases. The parallel activation of Btk and Tec suggests that both
contribute to functional responses in platelets.
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Materials and methods |
The following products were purchased for this study: prostaglandin
E1 (PGE1), Arg-Gly-Asp-Ser (arginine, glycine,
aspartic acid, and serine; RGDS) peptide, dimethylsulfoxide (DMSO),
aspirin, apyrase (type VIII),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes),
sodium dodecyl sulfate (SDS), 2-mercaptoethanol, sodium orthovanadate,
bovine serum albumin (BSA), protein A-Sepharose, Triton X-100, and
tris(hydroxymethyl) aminomethane (Tris) (all from Sigma, St. Louis,
MO); nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA);
SDS-PAGE (polyacrylamide gel electrophoresis) molecular standards,
[-32P]ATP (adenosine triphosphate), and enhanced
chemiluminesence (ECL) reagents including secondary antibodies
(Amersham, Arlington Heights, IL); PI 3-kinase inhibitors LY294002 and
wortmannin (Calbiochem, La Jolla, CA); anti-CD32 monoclonal antibody
(mAb) IV.3 (MEDAREX, Annandale, NJ); F(ab')2 fragment of goat
antimouse immunoglobulin G (IgG) (ICN Pharmaceutical, Aurora, OH);
anti-Btk goat serum (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-Fyb/Slp-130 mAb (Transduction Laboratories, Lexington, KY);
anti-Tec and anti-Shc rabbit polyclonal antibody (pAb) (Upstate
Biotechnology, Lake Placid, NY); and horse tendon collagen (Hormon
Chemie, Munich, Germany).
Antisera against the N-terminal of Btk, phosphotyrosine
551, and phosphotyrosine 223 have been described.13 An
antiphosphotyrosine antibody, 4G10, was used as described. We also
received the following gifts: purified human thrombin (Green Cross,
Osaka, Japan) and recombinant thrombopoietin (TPO) (Kirin Brewery,
Tokyo, Japan).
Platelet preparation
Human blood from XLA patients and healthy volunteers was drawn by
venipuncture into 1/10 volume of 3.8% (wt/vol) trisodium citrate and
gently mixed. Before drawing blood, informed consent was obtained.
Genetic studies were performed in different laboratories for XLA patients A and B (Dr S. Tuskada, Osaka University School of
Medicine, Osaka, Japan) and XLA patients C-F (H.D.O., University of
Washington School of Medicine, Seattle, WA). Patients A and B are
brothers, and the genetic study revealed that the Btk allele in both
patients has a T558 G(TATTAG) mutation
resulting in the appearance of a premature stop codon. Patients C and D
are brothers, and both have a Btk allele containing an
A1898G(Glu589Gly) missense mutation. The Btk allele
in patient E has a C403 T(Glu91STOP) mutation. The Btk alleles in the unrelated patients F and G have missense mutations: A1223C(His364Pro) mutation for
patient F and G1970A(Gly613Asp) mutation for
patient G. Western blot analysis and/or flow cytometry for detection of
intracellular Btk revealed that platelets from patients A, B, and E do
not express even truncated Btk, while platelets from the other patients
may express a trace amount of mutated Btk.
Platelet rich plasma (PRP) was prepared by centrifugation of whole
blood at 200g for 20 minutes. PRP was aspirated and incubated with 2 µmol/L aspirin for 30 minutes at room temperature.
Following addition of 1 µmol/L PGE1 from a
stock solution in 1 mmol/L absolute ethanol, the PRP was spun at
800g to form a soft platelet pellet. The pellet was resuspended
in 1 mL modified Hepes-Tyrode buffer containing 129 mmol/L sodium chloride (NaCl), 8.9 mmol/L sodium hydrogen carbonate
(NaHCO3), 0.8 mmol/L monopotassium phosphate (KH2PO4), 0.8 mmol/L magnesium dichloride
(MgCl2), 5.6 mmol/L dextrose, and 10 mmol/L Hepes (pH 7.4);
supplemented with 0.6 units/mL apyrase; and washed twice. Platelets
(3 × 108 cells/mL) were resuspended for experiments
in the modified Hepes-Tyrode buffer supplemented with 0.6 units/mL
apyrase and 200 µg/mL RGDS peptide.
Platelet stimulation and protein analysis
Aliquots of platelets (0.5 mL at a concentration of
3 × 108 cells/mL) were incubated in modified
Hepes-Tyrode buffer supplemented with 0.6 units/mL apyrase and 200 µg/mL RGDS peptide. Platelet stimulation by agonists was terminated
by the addition of an equal volume of lysis buffer containing 15 mmol/L
Hepes (pH 7.4), 150 mmol/L NaCl, 1 mmol/L phenyl methyl sulfonyl
fluoride (PMSF), 10 mmol/L EGTA (ethylenegylcotetraacetic acid), 1 mmol/L sodium orthovanadate, 0.8 µg/mL leupeptin, and 2% Triton
X-100 (wt/vol). In some experiments, 2 mmol/L MgCl2 was
added to ensure the presence of Mg2+ during precipitation.
After 20 minutes on ice, the lysates were centrifuged at
10 000g at 4°C for 20 minutes. The lysate soluble supernatant was recovered, incubated with protein A-Sepharose (40 µL
of 50% slurry) for 1 hour, and centrifuged to obtain the cleared
supernatant. Immunoprecipitating antibody (1-5 µg/sample) was added
to the cleared supernatants for 2-3 hours on ice, then immune complexes
were bound by the addition of protein A-Sepharose (40 µL of 50%
slurry/mL supernatant). The immunoprecipitates were washed 3 times with
1 mL cold buffer (the same as the lysis buffer except using 1% Triton
X-100 [wt/vol]). Proteins were denatured by heating at 95°C for 5 minutes in modified Laemmli's sample buffer (10% glycerol, 1% SDS,
5% 2-mercaptoethanol, 50 mmol/L Tris-HCl [pH 6.8], 0.002%
bromophenol blue, 10 mmol/L EGTA, and 1 mmol/L sodium orthovanadate),
then separated by 1-dimensional SDS-electrophoresis with 10% or
7.5%-15% polyacrylamide gels.
Proteins were transferred from the gel onto nitrocellulose membranes in
transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20%
methanol). To block residual protein binding sites, membranes were
incubated in Tris-buffered saline-Tween (TBST; 10 mmol/L Tris [pH
7.6], 150 mmol/L NaCl, and 0.1% Tween 20) with 10% BSA. The
membranes were washed with TBST, then incubated overnight with primary
antibodies diluted in TBST as indicated: 1.0 µg/mL each of 4G10
antibody, anti-Btk goat antibody, and 1.0 µg/mL anti-Tec pAb; 1:1000
dilution anti-Btk rabbit antibody; and 0.5 µg/mL each of antibodies
223PY and 551PY. The membranes were washed with TBST then incubated
with horseradish peroxidase-conjugated second antibody diluted in TBST
at a concentration of 1:3000. The antibody binding to proteins was
visualized with ECL according to the manufacturer's instructions.
In vitro kinase assays
Immunoprecipitated Btk was washed twice with kinase buffer (150 mmol/L NaCl, 5 mmol/L MgCl2, 5 mmol/L manganese dichloride (MnCl2), 1 mmol/L sodium orthovanadate
(Na3VO4), and 10 mmol/L Hepes [pH 7.4]), then
incubated for 10 minutes at room temperature in 30 µL kinase buffer
containing 10 µmol/L ATP and 9.25 MBq (0.25 mCi/mL)
[-32P]ATP. Reactions were terminated with an equal
volume of modified Laemmli sample buffer, the proteins were separated
with SDS-PAGE, and the incorporation of 32P into Btk was
visualized by autoradiography.
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Results |
Btk and Tec in human platelets
The Btk/Tec kinase family plays important roles during development
and function of hematopoietic lineages. Each family member displays a
distinct pattern of expression in these lineages. To evaluate the
pattern of expression of Btk/Tec kinases in platelets, immunoblot
analysis was performed after separation of platelet extract proteins by
gel electrophoresis. Approximately 77-kd Btk proteins and approximately
65-kd Tec proteins were detected, but few Itk or Bmx proteins were
detected (data not shown).
Tyrosine phosphorylation of Btk/Tec kinases specifically induced by
nonintegrin collagen receptors
To test for the tyrosine phosphorylation of Btk and Tec, these
proteins were immunopurified from extracts of control and
collagen-treated platelets. Increases in the phosphorylation of Btk and
Tec were detected using antiphosphotyrosine immunoblot in samples with similar amounts of either protein (Figure
1A). Treatment of platelets with collagen
for increasing lengths of time induced a detectable increase in Btk
phosphorylation within 1 minute, with a peak at 5 minutes after
collagen stimulation (Figure 1B). Phosphorylation at 10 minutes
remained similar to the elevated levels at 5 minutes. To evaluate
the role of integrin and nonintegrin collagen receptors in Btk
activation, platelets were stimulated with collagen in the presence or
absence of extracellular magnesium ion with 10 mmol/L EDTA added. The
absence of magnesium ion blocks collagen binding to and stimulation of
platelet 21.1-3 With or
without magnesium, Btk tyrosine phosphorylation was retained in
collagen-treated platelets, which indicates that nonintegrin receptors
are sufficient for activation (Figure 1C). A kinetic evaluation of Tec
phosphorylation after collagen stimulation of nonintegrin receptors was
performed to compare it with the Btk response. Magnesium-independent
increased Tec phosphorylation was detectable at 1 minute and was
maximal at 5 minutes (Figure 1D). The similar kinetics of activation
support the conclusion that both kinases participate in this pathway.
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| Fig 1.
Increased tyrosine phosphorylation of Btk and Tec in
collagen-stimulated platelets.
(A) Aliquots of platelets were treated with 50 µg/mL of
either collagen or buffer for 5 minutes, then were lysed in detergent
buffer as described in "Materials and methods." Btk or Tec was
purified from the soluble extracts by immunoprecipitation, and the
denatured samples were divided into 3 equal aliquots. Replicate pairs
of the samples (without or with collagen treatment) were separated by
7.5%-15% SDS-PAGE, transferred to a nitrocellulose membrane, then
immunoblotted with either 4G10 total phosphotyrosine antibody (top
panel), anti-Btk antibody (middle panel), or anti-Tec antibody (bottom
panel). (B) Platelets were treated with 50 µg/mL of either
collagen or buffer for 10 minutes or less. Btk was purified from the
soluble extracts by immunoprecipitation, as described above, then
immunoblotted with either 4G10 total phosphotyrosine antibody (top
panel) or anti-Btk antibody (bottom panel). (C) Platelets were
incubated in nominally Ca++- and Mg2+-free
modified Hepes-Tyrode buffer and treated with 50 µg/mL of either
collagen or buffer for 5 minutes, followed by the addition of 10 mmol/L
EDTA. Btk was purified from the soluble extracts by immunoprecipitation
as described above, then immunoblotted with either 4G10 total
phosphotyrosine antibody (top panel) or anti-Btk antibody (bottom
panel). (D) Aliquots of platelets, as described in (C), were treated
with 50 µg/mL of either collagen or buffer for 0 to 10 minutes
following the addition of 10 mmol/L EDTA. Tec was purified from the
soluble extracts by immunoprecipitation as described above and
immunoblotted with either 4G10 total phosphotyrosine antibody (top
panel) or anti-Tec antibody (bottom panel).
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Collagen-induced Btk activation through the Src family kinases
An important mechanism for activation of the Btk/Tec kinases in
receptor signaling pathways occurs through phosphorylation of 2 regulatory tyrosine residues.11-13 Receptor stimulation
activates the Src family kinases, which then transphosphorylates the
Btk Y551 residue. This site is within the kinase domain activation loop, and phosphorylation increases the catalytic activity of the
enzyme. An early substrate for the activated kinase is the Btk Y223
residue. This phosphorylation site, within the SH3 domain, probably
alters intramolecular and intermolecular binding interactions.
To test for evidence of an Src family kinase-mediated activation of
Btk in the collagen signaling pathway, the phosphorylation of the 2 regulatory Btk tyrosine residues was measured for increasing lengths of
time after platelet stimulation. The immunopurified Btk from each
sample was immunoblotted with phosphopeptide site-specific antibodies
that recognize either phosphorylated Y551 or Y223 residue, and the
kinetics of phosphorylation of each site were compared (Figure
2A). A maximal increase in phosphorylation
of the activating residue Y551 was observed within 1 minute. In
contrast to other B-cell and mast-cell receptor responses, in which the
Y551 phosphorylation rapidly decreases, Btk in platelets retained a
high level of phosphorylation for at least 10 minutes.13
The Y223 residue was detectably phosphorylated in unstimulated
platelets, similar to its phosphorylation in other systems.
Phosphorylation of the Y223 residue increased slowly relative to the
Y551 residue, with a maximal increase noted at approximately 5 minutes.
To confirm that collagen-induced phosphorylation enhanced kinase
activity, Btk was immunopurified from control and stimulated platelets,
then incubated in the presence of radiolabeled [-32P]ATP. Autoradiography of the Btk protein
(Figure 2B) demonstrated significantly higher in vitro
autophosphorylation in the platelets treated with collagen for 1 minute. This strongly suggests that collagen treatment of platelets
activates Btk through an Src family kinase-dependent mechanism
similar to that described in other cell types.
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| Fig 2.
Src-mediated activation of Btk in collagen-treated
platelets.
(A) Platelets were treated with collagen as in Figure 1A for the
time indicated. Btk was purified from the soluble extracts by
immunoprecipitation as described above, then immunoblotted with Btk
phosphopeptide site-specific antibodies 551PY (top panel) and 223PY
(middle panel) or anti-Btk antibody (bottom panel). (B) Platelets were
treated with either collagen or buffer for 1 minute. Btk was purified
from the soluble extracts by immunoprecipitation as described above.
The immune complexes were incubated in a kinase buffer containing
[-32P]ATP for 5 minutes as described in
"Materials and methods." Incorporation of 32P
into Btk was visualized by autoradiography.
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PI 3-kinase in the platelet Btk activation pathway
In B cells, Btk activation and function are critically influenced by
the PI 3-kinase signaling pathway. A variety of structural and genetic
data indicate that the Btk pleckstrin homology domain directly binds to membrane phospholipid products of PI
3-kinase.14 Modulation of this interaction regulates the
capacity of receptor-associated Src family kinases to activate Btk. In
platelets, PI 3-kinase is an important mediator of platelet
aggregation,33-37 and inhibitors can prevent
collagen-induced phosphorylation of the downstream target protein
WASP.24 To test the hypothesis that the PI 3-kinase signaling pathway is required for Btk and Tec activation in platelets, collagen stimulation of platelets was performed in the absence or
presence of a specific inhibitor of PI 3-kinase activity (LY294002). After immunopurification from platelet extracts, the phosphorylation of
Btk (Figure 3A) and Tec (Figure 3B) was
measured by antiphosphotyrosine immunoblot. The presence of the
LY294002 inhibitor strongly attenuated the collagen-induced
phosphorylation of Btk and Tec. The role of PI 3-kinase in
collagen-stimulated Btk phosphorylation was further tested by using
either a lower dose of inhibitor LY294002 (5 µmol/L) or using inhibitor wortmannin (Figures 3C and 3D,
respectively).
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| Fig 3.
Influence of PI 3-kinase activity on Btk and Tec
phosphorylation.
Aliquots of platelets were treated with 0.1% DMSO (vehicle for
LY294002) or 50 µmol/L LY294002 for 10 minutes. Then 50 µg/mL collagen was added for 5 minutes, as indicated. (A) Btk or (B)
Tec were purified from the soluble extracts by immunoprecipitation as
described above. The proteins were analyzed by immunoblot with 4G10
total antiphosphotyrosine (top panels) and anti-Btk or anti-Tec
antibody (bottom panels). Collagen-stimulated Btk phosphorylation and
coprecipitation of phosphoproteins was inhibited by low-dose (5 µmol/L) LY294 002, (C) 10 nmol/L wortmannin, and (D) 100 nmol/L
wortmannin. MgCl2 was added to the platelet extracts in (C)
and (D).
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As divalent cations can be essential to maintaining protein
conformation and interactions, Mg2+ was added to the
platelet extracts during immunoprecipitation to enhance the association
of Btk with possible ligands or substrates. Interestingly, the presence
of Mg2+ enhanced coprecipitation of several tyrosine
phosphorylated proteins (apparent masses in approximate units of 150, 80, 70, and 40 kd) with Btk from extracts of collagen-stimulated
platelets (Figure 3C). PI 3-kinase inhibition not only decreased Btk
tyrosine phosphorylation, but it also blocked the apparent
coprecipitation of the other phosphoproteins. Consistent with the
influence of the PI-3 kinase inhibitor LY294002, treatment of platelets
with inhibitor wortmannin resulted in a dose-dependent block of
collagen-induced Btk phosphorylation and coprecipitation of
phosphoproteins (Figure 3D). These results are consistent with the
recent observations of Laffargue et al,37 who found that
Btk tyrosine phosphorylation is enhanced by platelet aggregation and
that this response is inhibited in a similar dosage range by
wortmannin. These results confirm the importance of PI 3-kinase as an
upstream regulator of Btk activation and association with other
signaling proteins during collagen receptor signaling platelets. They
also strengthen the similarity of the platelet mechanism to receptor
pathways previously described in other hematopoietic lineages.
Capacity of alternative platelet receptors to activate Btk and
Tec
To further define the specificity of the activation of the Btk/Tec
kinase family in platelet signaling responses, important regulators of
platelet function were assessed for their capacity to induce tyrosine
phosphorylation of Btk or Tec. TPO and thrombin bind to specific
platelet membrane receptors that elicit tyrosine phosphorylation of
multiple platelet proteins by mechanisms different from collagen.
Thrombin is known to activate PLC in platelets through receptors
coupled to trimeric G-proteins like Gq and Gi.38-40 Interestingly, a role for Gq in activation of Btk has been postulated in other cells.20 On the other hand, TPO is known to induce activation of Jak2 and Tyk2 without activation of PLC.41-48
To test whether Btk is recruited into these signaling pathways,
platelets were treated with either TPO (Figure
4A) or thrombin (Figure 4B) for increasing
lengths of time, then Btk was immunopurified and analyzed for
phosphotyrosine content. In comparison with collagen treatment, there
was little or no increase of Btk phosphorylation induced by these
agonists. This indicates that a robust Btk activation is not a general
response to platelet activation, which is in agreement with the above
cited report by Laffargue et al.37
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| Fig 4.
Influence of TPO and thrombin on Btk phosphorylation.
Platelets were treated with 50 µg/mL collagen, (A) 100 ng/mL TPO, (B) 1 unit/mL thrombin for 0 to 10 minutes, or (C) 1 unit/mL
thrombin for 0 to 5 minutes, as indicated. (A,B) Btk or (C) Tec was
purified from soluble extracts by immunoprecipitation as described
above and immunoblotted with either 4G10 antibody (top panels) or
anti-Btk or anti-Tec antibody (bottom panels).
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Since XLA platelets have normal responses to thrombin treatment,9
it is formally possible that thrombin may selectively signal
through Tec, independently of Btk. To test the influence of thrombin
directly, Tec was immunopurified from control and thrombin-treated
platelets and immunoblotted with antiphosphotyrosine. A small increase
in Tec tyrosine phosphorylation was observed (Figure 4C), which is
consistent with a previous report that maximal thrombin-induced Tec
phosphorylation is highly dependent on platelet aggregation.49 Hamazaki et al50 also found that
Tec phosphorylation is significantly dependent on platelet aggregation,
although to a lesser degree. The differences may be due to technical
nuances. For example, Laffargue et al39 observed an
inhibitory influence of cytochalasins D, whereas Hamazaki et
al50 did not. Thus, collagen induces stronger Btk and Tec
tyrosine phosphorylation than that provoked through other platelet
receptor signaling pathways in our experimental conditions.
Recent observations indicate that the platelet surface FcRIIA (CD32)
is similar to the collagen nonintegrin receptor in its use of the
ITAM-mediated signaling mechanisms.3 To test whether platelet receptor signaling through the ITAM-mediated signaling is
sufficient for Btk and Tec activation, the CD32 protein was stimulated
by cross-linking with the mAb IV.3. Btk (Figure
5A) and Tec (Figure 5B) were immunopurified
from platelet extracts and analyzed by antiphosphotyrosine immunoblot.
Cross-linking of CD32 is sufficient to increase the tyrosine
phosphorylation of both proteins. The similarity of CD32 signaling to
the collagen receptor pathway was further tested by treating platelets
with the inhibitor LY294002 prior to CD32 cross-linking. The capacity of CD32 cross-linking to increase the tyrosine phosphorylation of both
Btk and Tec was blocked by inhibition of PI 3-kinase activity. This
suggests that following ligation of CD32, PI 3-kinase activity is also
necessary for tyrosine phosphorylation of Btk and Tec. These results
demonstrate that the capacity to recruit the Btk/Tec kinase family in
platelet signaling pathways is critically dependent upon the proximal
mediators of the receptor activation.
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| Fig 5.
Influence of CD32 cross-linking on Btk and Tec
phosphorylation modulated by PI 3-kinase.
Platelets were incubated with 0.1% DMSO (vehicle for LY294002) or 50 µmol/L LY294002 for 10 minutes. We added 3 µg/mL anti-CD32 for 10 minutes, followed by 30 µg/mL (Fab')2 of goat antimouse IgG for
5 minutes as indicated. (A) Btk or (B) Tec was purified from the
soluble extracts by immunoprecipitation as described above, then
immunoblotted with 4G10 antibody (top panels) and anti-Btk or anti-Tec
antibody (bottom panels).
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Collagen-induced Tec tyrosine phosphorylation in XLA
platelets
The evaluation of Btk and Tec tyrosine phosphorylation in platelets
from normal subjects indicates that these signaling proteins are
coordinately activated in response to collagen binding to nonintegrin
receptors. Platelets from 7 classical XLA patients (5 families) were
evaluated to determine whether the level of Tec expression or the
activation of Tec in response to collagen is dependent on the presence
of functional Btk protein. Immunoblot analysis of platelet extracts
from XLA patients (A, B, and E) demonstrated an absence of the
full-length Btk protein compared with normal subjects, as detailed in
"Materials and methods" (Figure 6A;
data not shown). In contrast, Tec protein, at levels similar to normal
subjects, was present in platelet extracts from all 7 XLA patients,
thereby indicating that the expression of Tec protein is not regulated
by functional Btk (Figure 6A; data not shown).
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| Fig 6.
Analysis of platelets from XLA patients.
Soluble extracts were prepared from patient A or a normal control
(3 × 106 platelets/sample) and were then subjected
to immunoblot analysis with (A) Btk or Tec antiserum.
Collagen-stimulated tyrosine phosphorylation of Tec was analyzed as
described in the legend to Figure 1D, and analyses are
given for (B) patient A and (C) patient B. Tyrosine phosphorylation of
(D) WASP or (E) Slp-76 in XLA platelets prior to or after 50 µg/mL
collagen treatment for 5 minutes was examined by immunoblot
analysis with 4G10 antibody following immunoprecipitation (anti-WASP or
anti-Slp-76, patient B). The upper and lower portions of the membrane
from the (E) anti-Slp76 immunoprecipitate was immunoblotted with
anti-Fyb/Slp-130 or anti-Slp-76 as indicated. Tec and WASP
phosphorylation studies were also performed using platelets from
patients C-G, and similar results were obtained.
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|
Collagen treatment of XLA platelets increased the level of Tec tyrosine
phosphorylation maximally within 1 minute after stimulation (Figures 6B
and 6C; data not shown). Increased Tec phosphorylation was observed in
platelets from all XLA patients (7/7) tested. Thus, Tec recruitment
into the collagen receptor signaling pathway does not require
functional Btk. Increased tyrosine phosphorylation of Tec in XLA
platelets is consistent with the hypothesis that Tec function can
rescue platelet responses in the absence of Btk function, and by this
mechanism, Tec function can prevent overt clinical platelet-dependent
bleeding diathesis in XLA patients. We examined the platelet extracts
from XLA patients for evidence of the recruitment of other signaling
proteins in collagen receptor pathways. WASP is essential for normal
platelet morphology and function, and it is tyrosine phosphorylated in
normal collagen-stimulated platelets in a PI 3-kinase dependent
manner.24 Recent reports indicate that WASP is a direct
substrate of Btk in heterologous expression systems.28,30
This suggests that the Btk/Tec kinase family may be important upstream
regulators of WASP function in platelet receptor pathways. We tested
the hypothesis that WASP recruitment into the platelet collagen
nonintegrin receptor pathway occurs in the absence of Btk. XLA platelet
extracts were prepared prior to and after collagen treatment, then WASP
protein was immunopurified and immunoblotted with either
antiphosphotyrosine or anti-WASP (Figure 6D). XLA platelet extracts
were prepared prior to and after collagen treatment, then WASP protein
was immunopurified and immunoblotted with either antiphosphotyrosine or
anti-WASP (Figure 6D; data not shown). Collagen induced a clear
increase in WASP tyrosine phosphorylation in the XLA platelets from 7/7 patients tested, suggesting that Tec activation is sufficient to
preserve the WASP recruitment in this signaling pathway.
We investigated whether other collagen-dependent signaling events
appear to be preserved in the XLA platelets. Previous studies have
shown that Slp-76 is essential for activation of PLC-2 in collagen
or collagen-related peptide-induced platelet
activation.51,52 In XLA platelets, Slp-76 becomes tyrosine
phosphorylated following collagen treatment and is immunoprecipitated
in association with 130-, 55-, and 38-kd phosphoproteins (Figure 6E).
The 130-kd protein in the anti-Slp-76 immunoprecipitate reacted with
an anti-Fyb mAb (Figure 6E, upper panel). The 130-kd and 55-kd proteins
were previously identified as Fyb/Slp-130 and Lyn,
respectively.51,52 These observations indicate that the
platelet collagen receptor signaling pathways are largely intact
despite the absence of Btk and that phosphorylation of a downstream
substrate requires only the presence of Tec.
| |
Discussion |
Platelet function in hemostasis can be promoted or inhibited by
multiple extracellular factors binding to surface
receptors.38 Alternative receptors use specific, sometimes
overlapping, intracellular signaling pathways to regulate platelet
incorporation into a forming thrombus. One of the strongest promoters
of thrombogenesis, subendothelial collagen, plays a critical role both
in normal hemostasis and in pathophysiological events such as vascular
infarction subsequent to rupture of atherosclerotic
plaques.1-3
Collagen receptor regulation of intracellular calcium levels in
platelet functional responses is an important element of the irreversible engagement into a thrombus.1-3,38 The Btk/Tec
kinase family provides critical elements for the regulation of
receptor-dependent intracellular calcium responses in a variety of
hematopoietic cell lineages. The important role of the Btk/Tec kinase
family in the receptor signaling pathways has been confirmed by the
phenotypic analysis of cells harboring either naturally occurring
mutations, such as XLA and xid, or targeted-deletion of other Btk/Tec
family members. In the B-cell antigen receptor signaling pathway, PI 3-kinase and Src kinases function upstream of Btk, while Btk, in
conjunction with Syk, modulates phosphorylation of PLC-2 and intracellular calcium levels.15,22
Several observations suggest a similar functional role for the Btk/Tec
kinase family in the platelet nonintegrin collagen receptor signaling
pathway. Importantly, collagen-induced responses, such as PLC-2
phosphorylation, calcium mobilization, and granule release, are
decreased in Btk-deficient platelets compared with those from normal
donors.9 In our experiments, phosphorylation of Btk and Tec
was more strongly activated by receptors that signal through an
ITAM-containing subunit (collagen nonintegrin receptors and CD32) than
in response to platelet agonists that signal by other mechanisms
(thrombin or thrombopoietin). The role of Src family kinases in Btk
activation was demonstrated by detection of enhanced phosphorylation of
Btk tyrosine 551 (Tyr551), the Src family kinase substrate site within
the Btk kinase domain activation loop, using phosphopeptide-specific
antibodies.11-13 The increased phosphorylation of Btk
coincided with increased Btk enzymatic activity in an in vitro kinase
assay. We also observed that PI 3-kinase activity was required for
collagen- and CD32-induced tyrosine phosphorylation of both Btk and Tec
in platelets. Taken together, these results confirm that 2 of the major
regulatory mechanisms controlling recruitment of the Btk/Tec kinase
family into antigen receptor pathways are conserved in platelet
receptor responses.
The evaluation of collagen regulation of platelet function is
complicated by heterogeneity in responses among healthy volunteers, due
in part to variation in the expression of the integrin
21, a major collagen
receptor.53 Whereas collagen and CD32 were strong
activators of Btk and Tec tyrosine phosphorylation, thrombin treatment
of platelets in our experiments had only a weak influence. Thrombin-stimulated activation of Tec and Btk in platelets is largely
dependent on platelet integrins and
aggregation.37,49,50 In agreement with this,
PI 3-kinase activity is essential for platelet integrin
IIb3 function and cytoskeletal
reorganization.33 Thus, the small influence of thrombin on
Btk and Tec phosphorylation in our experiments probably reflects RGDS
inhibition of the integrin-mediated platelet aggregation.
Several important collagen-dependent platelet responses are diminished
in Btk-deficient platelets derived from XLA patients.9 Despite abnormalities in XLA platelet responses to collagen in vitro,
XLA patients do not demonstrate a platelet-dependent abnormal bleeding
tendency. These results suggest either that the Btk/Tec family kinase
activity is dispensable to platelet function or that there is
sufficient Btk/Tec kinase functional redundancy to rescue signaling in
specific receptor pathways. Our comparison of the kinetics and
mechanisms of Btk and Tec activation in a normal agonist-stimulated
environment indicates that these signaling proteins are coordinately
recruited into specific platelet receptor pathways. Furthermore, Tec
protein is expressed in Btk-deficient platelets at levels similar to
normal platelets, and Tec is rapidly phosphorylated in response to
collagen stimulation of XLA platelets. These results favor the
interpretation that functional redundancy of the Btk/Tec kinase family
rescues XLA platelet responses to collagen in vivo.
In platelets, one important signaling function of the Btk/Tec kinase
family may be to link specific receptors to the WASP signaling protein,
a critical regulator of platelet morphology and
function.31,32 This relationship is suggested by
similarities in the recruitment of the Btk/Tec kinase family and the
WASP signaling protein into platelet receptor signaling pathways. Both
CD32 and collagen signal to WASP via a PI 3-kinase dependent
mechanism.24-27,54 WASP becomes strongly tyrosine
phosphorylated in collagen-stimulated platelets, and this response is
blocked by wortmannin, whereas thrombin induces a much weaker increase
in WASP phosphorylation.24 WASP binds directly to Btk and
appears to be a direct substrate of Btk.28,30 However, in
WASP-null mice generated by gene targeting, functional deficits were
noted in T cells but not in B cells.55 Because T cells
express Btk/Tec kinases but not Btk, and B cells express predominantly
Btk, the physiologic significance of the Btk-induced WASP
phosphorylation in B-cell receptor pathways is unclear. The robust
increase in WASP phosphorylation observed in collagen-treated XLA
platelets indicates the role of another kinase in the phosphorylation
of WASP in this platelet receptor pathway. The current study reveals
that Tec is recruited by mechanisms similar to those used to recruit
WASP and Btk and that Tec expression is preserved in XLA platelets.
Thus, Tec is a prime candidate as the WASP kinase in XLA platelets, and
this role would support the hypothesis of functional redundancy in
signaling through Btk/Tec kinases.
The capacity of Tec to partially rescue XLA platelet function would
concur with the previous demonstration of functional similarity in
signal transduction using Itk and Tec to reconstitute BCR-induced calcium responses in Btk-deficient B cells.15 As Tec is not overexpressed in the platelets from XLA patients, Tec may only partially compensate for the loss of Btk. Decreased XLA platelet responses detectable in vitro may indicate that Btk/Tec signaling function in the collagen receptor pathway is dosage dependent, which is
similar to the graded reconstitution of B-cell function in a Btk
transgenic murine model system.56 In contrast to the data
regarding the collagen receptor pathway, Tec expression does not
readily account for the normal response of XLA platelets to thrombin9 because thrombin has only a small influence on
Tec tyrosine phosphorylation in our experimental conditions. This pathway may require Btk/Tec kinase function only distal to platelet aggregation.37,49,50
The emerging role of the Btk/Tec kinase family in the collagen
signaling pathway helps to clarify the molecular mechanism of action of
a major physiological regulator of platelet function. The observation
that certain aspects of the nonintegrin collagen receptor signaling
pathway are impaired in XLA platelets,9 coupled with an
extensive catalog of Btk mutations resulting in XLA, suggests that
platelets may provide an interesting system for the detailed
genotypic/phenotypic evaluation of Btk signaling function.
| |
Footnotes |
Submitted December 29, 1998; accepted October 26, 1999.
Supported in part by grants in aid from The Ministry of Education,
Science and Technology of Japan, Japan (Y.I. and A.O.), and The Ryoichi
Naito Foundation for Medical Research, Japan (A.O.); Research Grants
for Life Sciences and Medicine, Keio University Medical Science Fund,
Keio, Japan (A.O.); grant HD17427 from the National Institutes of
Health, Bethesda, MD (H.D.O.); grant 6-F496-0330 from the March of
Dimes Birth Defects Foundation, (H.D.O.); the Jeffery Module
Foundation; and the DeJoria Wiskott-Aldrich Research Fund (H.D.O.)
O.N.W. is an Investigator of the Howard Hughes Medical Institute, and
M.I.W. is supported by fellowship DRG-086 from the Cancer Research Fund
of the Damon Runyon Walter Winchell Foundation Fellowship.
Reprints: Atsushi Oda, Hokkaido Red Cross Blood Center,
Yamanote 2-2, Nishi-ku, Sapporo 063-0002, Japan; e-mail:
aoda{at}hokkaido.bc.jrc.or.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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R. Bobe, J. I. Wilde, P. Maschberger, K. Venkateswarlu, P. J. Cullen, W. Siess, and S. P. Watson
Phosphatidylinositol 3-kinase-dependent translocation of phospholipase C{gamma}2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation
Blood,
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678 - 684.
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C. Mao, M. Zhou, and F. M. Uckun
Crystal Structure of Bruton's Tyrosine Kinase Domain Suggests a Novel Pathway for Activation and Provides Insights into the Molecular Basis of X-linked Agammaglobulinemia
J. Biol. Chem.,
October 26, 2001;
276(44):
41435 - 41443.
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Abstract
Introduction