From the Department of Clinical and Laboratory
Medicine, Yamanashi Medical University, Yamanashi 409-3898, ¶ Pharmaceutical Research Laboratories, Toray Industries, Inc.,
1111 Tebiro, Kamakura, Kanagawa 248-8555, and the
Department
of Biochemistry, Meiji Pharmaceutical University,
Tokyo 204-8588, Japan
Received for publication, July 13, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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Although glycoprotein Ia/IIa (GPIa/IIa, integrin
Platelets adhere to collagen fibers exposed at sites of damage to
the endothelial lining and become activated through specific membrane
receptors for collagen. Many candidates have been proposed for putative
collagen receptors on platelet membranes. Of these, GP
Ia/IIa1 and GPVI have now established
their roles as collagen receptors (1). It
has been reported that human blood platelets that showed no response to
collagen lacked the surface expression of GPIa/IIa or GPVI, indicating
that GPIa/IIa and GPVI are collagen receptors (2-8). Collagen-related
peptide (CRP) containing repeats of the Gly-Pro-Hyp sequence (9-11),
and convulxin, which is a C-type lectin obtained from a tropical
rattlesnake venom, are reported to be platelet agonists acting on GPVI
(12, 13). These proteins have been very useful for investigating the
signal transduction pathways mediated by GPVI. Cross-linking of GPVI
results in phosphorylation of the Fc receptor On the other hand, relatively little has been elucidated regarding the
GPIa/IIa-mediated signal transduction system. Although the role of
GPIa/IIa as a collagen receptor is well established, whether GPIa/IIa
also mediates activation signals has remained to be elucidated. Only
recently, Kehrel et al. (10) has demonstrated that
GPVI-deficient platelets showed increased fibrinogen binding in
response to collagen but not to CRP. Since collagen but not CRP
contains the binding sites for GPIa/IIa, it is suggested that GPIa/IIa
also mediates certain activation signals leading to GPIIb/IIIa activation (10). Studies with inhibitors have also suggested that
GPIa/IIa can transduce activation signals in platelets; tyrosine phosphorylation of Syk or PLC In contrast to GPVI-mediated platelet activation, there have been few
GPIa/IIa agonists appropriate for investigating the GPIa/IIa-related
signal transduction pathway. Aggretin, purified from snake venom, is
reported to induce platelet aggregation acting as a GPIa/IIa agonist,
but its functional property has not been fully elucidated (22). JBS2,
an anti- Recently, we have isolated and characterized a functionally novel
platelet agonist, designated as rhodocytin, from the Calloselasma rhodostoma venom (25). We have suggested that rhodocytin belongs to the heterodimeric C-type lectin-like proteins and appears to induce
platelet aggregation by interacting with GPIa/IIa (25, 26). We found
that the GPIa/IIa-mediated signal induces Syk and PLC In this study, we present several lines of evidence to suggest that
rhodocytin directly and specifically binds to GPIa/IIa and that this
association is independent of divalent cations distinct from other
integrin-ligand bindings. Our findings suggest that, upon rhodocytin
stimulation, Src that constitutively associate with GPIa/IIa become
activated, and several signaling molecules including Cas, Syk, and
PLC Materials--
Rhodocytin was purified from the venom of
C. rhodostoma as described previously (25). Anti-GPIa mAbs,
7E10B and 3C8A, were generated by one of us. Briefly, human platelets
were used for immunizing 5-week-old Balb/c mice. The hybridomas
obtained were screened by the reactivity to human GPIa-transfected
Chinese hamster ovarian cells and human osteosarcoma-derived cell line,
MG63, which expresses GPIa. Immunoprecipitation study using MG63
confirmed that clones 7E10B and 3C8A react with GPIa. Clone 7E10B is an inhibitory antibody, and clone 3C8A is a nonfunctional antibody, as
determined by adhesion assays of platelets to collagen. Liposomes carrying recombinant fragments of GPIa/IIa (rGPIa/IIa liposome) were
made by the methods of Staatz et al. (5). Sepharose
beads conjugated with 7E10B were used to purify the GPIa/IIa
incorporated liposome. We have confirmed that rGPIa/IIa liposome has
more than 90% purity by SDS-PAGE. Anti-GPIa mAb (6F1), anti-CD9 mAb,
anti-GPIb mAb (WGA3), and collagen-related peptide (CRP) were kindly
donated by Dr. B. S. Coller (Mt. Sinai Medical Center, New York),
Dr. S. Nomura (Kansai Medical University, Osaka, Japan), Dr. M. Handa (Keio University, Tolyo, Japan) and Drs. M. Moroi and Y. Miura (Institute of Life Science, Kurume University, Fukuoka, Japan), respectively. The FcR
The following materials were obtained from the indicated suppliers:
anti-GPIa mAb (Gi9), Immnotech, Marseille, France; anti-Syk mAb and
leupeptin, Wako Pure Chemical Industries, Ltd., Tokyo, Japan;
anti-PLC Platelet Preparation--
Venous blood from healthy drug-free
volunteers was collected into a tube containing acid/citrate/dextrose
(ACD). Platelet-rich plasma (PRP) was obtained after centrifugation of
whole blood at 160 × g for 10 min. When indicated, PRP
was incubated with 1 mM ASA for 30 min to exclude the
secondary effects of TXA. The platelets were washed twice with 15% ACD
and 100 nM PGI2 and resuspended in HEPES buffer
containing 138 mM NaCl, 3.3 mM
NaHPO4, 2.9 mM KCl, 1.0 mM MgCl, 1 mg/ml glucose, and 20 mM HEPES, pH 7.4, at a concentration
of 109 cells/ml. Thirty minutes before experiments, the
platelet suspension was supplemented with 1 mM CaCl.
FcR Platelet Aggregation--
Washed human platelets at a
concentration of 2.0 × 108/ml or murine platelets at
a concentration of 1.3 × 108 cells/ml were activated
by the indicated concentrations of agonists under continuous stirring
at 1,000 rpm in a PA-100 platelet aggregation analyzer (Kowa, Tokyo,
Japan). When indicated, platelets were pretreated with the indicated
concentration of anti-GPIa mAbs, 1 µM PGE1, 5 µM PP1 or 5 µM PP2 at 37 °C for 5 min.
The instrument was calibrated with a platelet suspension for zero light
transmission and with buffer for 100% transmission.
Platelet Proteins That Bind to Rhodocytin--
Thirty five µg
of purified rhodocytin was covalently coupled to 50 mg of
CNBr-activated Sepharose 4B beads according to the manufacturer's
instructions. One ml of platelet lysates (3.5 × 109
cells) lysed by an equal volume of 2× ice-cold lysis buffer(2% Triton
X-100, 100 mM Tris/HCl, pH 7.5, 5 mM EGTA, 2 mM vanadate, 1 mM phenylmethylsulfonyl
fluoride, and 100 µg/ml of leupeptin) was clarified by centrifugation
at 16,000 × g for 5 min. The supernatant was
precleared by 100 µl of Sepharose 4B (50% slurry) for 1 h and
then was split into two portions. One-half was incubated with 80 µl
of rhodocytin-bound Sepharose 4B, and the other half was incubated as
the control with 80 µl of Sepharose 4B for overnight. The beads were
washed 5 times in 1× lysis buffer, and proteins were eluted from the
beads with 40 µl of SDS reducing buffer (19) and were boiled for 5 min.
Adhesion of rGPIa/IIa Liposomes or Platelets to Rhodocytin- or
Collagen-coated Plates--
For the preparation of microtiter plates
coated with rhodocytin or collagen, 100 µl of the indicated
concentrations of rhodocytin or collagen was added to each well of a
96-well flat-bottomed plate and was left to stand overnight at room
temperature. After two cycles of washing with PBS, each well was
blocked with 1% BSA in PBS for 30 min at room temperature. Before use,
1% BSA-containing PBS was heated at 80 °C for 10 min and sterilized
by filtration. One hundred µl of rGPIa/IIa liposomes (2 µg/ml) or
washed platelet (1 × 104 cells/ml) was then added to
each well and incubated at 30 °C for 3 h or 20 min,
respectively. With rGPIa/IIa liposome, wells were incubated with
biotinylated anti-GPIa mAb (3C8A) after removing unbound rGPIa/IIa
liposome. rGPIa/IIa liposome binding was detected by horseradish
peroxidase-conjugated avidin and visualized with o-phenylenediamine. The optical density was measured
by a microplate reader (NOVAPATH, Bio-Rad). When platelet adhesion was
measured, after removing unbound platelet, wells were incubated with
3% paraformaldehyde for 30 min at room temperature. After platelet membranes were lysed with 0.2% Triton X-100, they were stained by 0.1 µg/ml tetramethylrhodamine isothiocyanate-conjugated
phalloidin. Cells were viewed under a BH microscope (Olympus, Tokyo,
Japan) and photographed on Fujicolor super G400 (Fujifilm, Tokyo,
Japan). The number of adhered platelet was calculated on the photographs.
Immunoprecipitation and Western Blotting--
After platelets
were activated with rhodocytin for the indicated time intervals,
reactions were terminated with an equal volume of ice-cold lysis
buffer. The lysate was sonicated and centrifuged at 16,000 × g for 5 min. The supernatant was precleared with protein A-Sepharose beads for 30 min at 4 °C and then mixed with the
indicated antibodies. The mixture was rotated for 1-2 h at 4 °C
and, after the addition of protein A-Sepharose beads, was further
rotated for 1 h. The Sepharose beads were washed three times with
1× lysis buffer. SDS reducing buffer was then added to the beads,
followed by boiling for 3 min. The proteins were separated by 8%
SDS-PAGE and electrophoretically transferred onto Clear Blot
Membrane-P. The membranes were blocked with 1% BSA in PBS. After
extensive washing with PBS containing 0.1% Tween 80, the immunoblots
were incubated for 2 h with the indicated antibody. Antibody
binding was detected by using peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG and visualized with ECL chemiluminescence reaction reagents (Amersham Pharmacia Biotech) and Konica x-ray film (JX 8 × 10, Konica Co., Tokyo, Japan). Where indicated, levels of tyrosine
phosphorylation were quantified using PDI1400oe Scanner and Quantity
One 2.5a software for Macintosh.
Immunoprecipitation Kinase Assay--
After preclearing, the
sample was split into two portions. One-half was used for
immunoblotting as described elsewhere, and the other half was processed
further for an in vitro kinase assay. For in
vitro kinase assay, the beads were washed once with HEPES buffer
(10 mM HEPES/NaOH, 1 mM vanadate, pH 8.0) and
then incubated with 25 µl of kinase reaction buffer (300 mM HEPES/NaOH, 15 mM MnCl2, 150 mM MgCl2, pH 8.0) with 10 µl of acid-treated
enolase. The reaction was initiated by the addition of 2 µM ATP (10 µCi of [ Effects of Anti-GPIa mAbs and an Anti-Fc
In contrast, IV.3 (anti-Fc
We also examined the effects of an anti-GPIa antibody on the
interaction between platelets and immobilized rhodocytin. Washed platelets incubated with A2-IIE10 or control mouse IgG were added to
rhodocytin-coated wells. Platelet binding was detected by staining with
rhodamine-conjugated phalloidin. This binding was reduced to
12.5% of the control in the presence of 25 µg/ml A2-IIE10 (data not
shown). The fact that anti-GPIa antibody almost completely inhibits the
association between platelets and rhodocytin indicates that GPIa/IIa is
the major receptor responsible for the rhodocytin-platelet interaction.
Platelet Aggregation in FcR Precipitation of GPIa/IIa in Platelet Lysates with
Rhodocytin-coupled Beads--
Based on the effects of specific mAbs,
we have already suggested that rhodocytin induces platelet aggregation
by interacting with GPIa/IIa, independently of GPIb (25, 26). To
confirm this hypothesis, we sought to determine whether
rhodocytin-coupled beads bound to GPIa/IIa. Sepharose 4B beads were
covalently coupled with rhodocytin and added to platelet lysates.
Immunoblot analysis showed that rhodocytin-coupled Sepharose 4B beads
effectively precipitated GPIa, whereas there was no recovery of GPIa
with Sepharose 4B control beads (Fig.
3A). On the other hand,
neither rhodocytin-coupled Sepharose 4B beads nor Sepharose 4B beads as a control precipitated GPIb (Fig. 3B). We could not detect
coprecipitation of GPIIa in rhodocytin-coupled Sepharose 4B beads,
although an anti-GPIIa antibody detected the presence of GPIIa in whole
cell lysates of platelets. These findings suggest that rhodocytin binds to GPIa but not to GPIIa or GPIb.
GPIa/IIa or Platelet Adhesion to Immobilized Rhodocytin and the
Effects of EDTA--
To investigate whether rhodocytin binds directly
or indirectly to GPIa (for example, via an unidentified protein which
associates with GPIa/IIa), we evaluated the binding of liposomes
carrying recombinant fragments of GPIa/IIa (rGPIa/IIa liposomes) to
immobilized rhodocytin. As shown in Fig.
4, rGPIa/IIa liposomes adhered to immobilized rhodocytin or collagen. This binding was potently blocked
by 10 µg/ml anti-GPIa blocking mAbs, 7E10B and Gi9. It is well known
that the binding of integrins to their ligands requires the presence of
divalent cations. The adhesion of rGPIa/IIa liposomes to collagen was
completely inhibited by 10 mM EDTA. To our surprise, EDTA
did not inhibit the binding of rGPIa/IIa liposomes to rhodocytin at
all. These findings suggest that rhodocytin specifically and directly
interacts with GPIa/IIa, independently of divalent cations.
Platelet Aggregation, Intracellular Calcium Mobilization, Tyrosine
Phosphorylation of Syk, and PLC Rhodocytin Stimulation Induces Tyrosine Phosphorylation of an
Adapter Protein, Cas, Independent of PGE1
Treatment--
Recently, several groups reported that Cas
(Crk-associated protein), an adapter protein of 130 kDa, and FAK are
involved in cell activation induced by cross-linking of
Src Associates with Cas in a Manner Dependent upon Cas Tyrosine
Phosphorylation--
Several studies have suggested that Src, FAK,
PYK2, or Fyn play an important role in Cas tyrosine phosphorylation in
other cells (30, 32, 40-43, 46, 48-52). Immunoprecipitation with an
anti-Cas antibody, followed by Western blotting with an anti-Src mAb,
revealed that Src associates with Cas upon GPIa/IIa stimulation (Fig.
6). The association between Src and Cas appears to be related to the
tyrosine-phosphorylated level of Cas. In contrast, we were unable to
identify the co-presence of FAK, PYK2, Fyn, Lyn, or Syk in Cas
immunoprecipitates (data not shown). These findings suggest that Src
specifically associates with Cas and that Src but not FAK or PYK2 may
phosphorylate Cas in platelets. We did not detect Cas in the
immunoprecipitates of Src, probably because the total amount of Src in
platelets far exceeds that of Cas in platelets (data not shown).
Src and Lyn Constitutively Associate with GPIa/IIa and the
GPIa/IIa-associated Kinase Activity Rapidly Increases Upon GPIa/IIa
Stimulation--
Immunoprecipitation of GPIa/IIa with an anti-GPIa
antibody, followed by Western blotting with antibodies against FAK,
Fyn, phosphatidylinositol 3-kinase, Lck, Jak1, Jak2, PTP1D/SHP2, Cas, Cbl, 14-3-3, and Fc receptor PP1 Potently Inhibits Platelet Aggregation Induced by
Rhodocytin--
Since we found that GPIa/IIa-associated Src activity
is the most proximal step in the GPIa/IIa stimulation, we next sought to investigate the effects of PP1, a specific inhibitor of Src kinase
inhibitor, on platelet aggregation induced by rhodocytin. As shown in
Fig. 9, 5 µM PP1 completely
inhibited platelet aggregation induced by 10 nM rhodocytin,
and this inhibitory effect was not overcome by the higher concentration
of rhodocytin (100 nM). These findings suggest that Src is
activated after rhodocytin stimulation and plays a functionally
important role in rhodocytin-induced platelet aggregation.
Cytochalasin D Inhibits the Activation of GPIa/IIa-associated
Kinase, Tyrosine Phosphorylation of Cas--
Cytochalasin D, which
disrupts actin filament organization, inhibits tyrosine kinase-mediated
integrin signaling in several systems (44). We have already shown that
cytochalasin D inhibits platelet aggregation, intracellular calcium
mobilization, and Syk or PLC Rhodocytin Induces Platelet Aggregation by Interacting with
GPIa/IIa--
In the present study, we sought to identify the membrane
molecule that rhodocytin binds and the mechanism by which it elicits platelet activation. Several membrane glycoproteins have been identified, which induce activation signals upon ligand binding. They
include GPIb, Fc
Immunoprecipitation study using rhodocytin-coupled beads showed that
rhodocytin indeed interacts with GPIa/IIa. We could not detect the
association of GPIIa with rhodocytin-coupled beads, suggesting that
rhodocytin binds to GPIa but not to GPIIa. Failure of anti-GPIIa
antibody (100 µg/ml of DE9) to inhibit rhodocytin (10 nM)-induced platelet aggregation also supports this
hypothesis (data not shown). To determine whether rhodocytin directly
binds to GPIa or indirectly via certain proteins on the platelet
membrane, we performed binding studies of rGPIa/IIa liposomes to
immobilized rhodocytin. We found that GPIa/IIa liposomes bound to
immobilized rhodocytin in a dose-dependent manner and that
this binding was totally inhibited by anti-GPIa mAbs. These findings
clearly demonstrate that the rhodocytin binding to GPIa/IIa is direct
and specific. Furthermore, the fact that the anti-GPIa antibodies
inhibit rhodocytin-induced platelet aggregation suggests that the
binding of rhodocytin to GPIa/IIa is functionally relevant.
To our surprise, EDTA did not inhibit the binding of rhodocytin to
rGPIa/IIa liposomes at all. Most of the ligand-integrin interactions
are dependent upon divalent ions and thus can be inhibited by EDTA (5).
To the best of our knowledge, echovirus 1 binding to integrin
Relatively high concentrations of immobilized rhodocytin were required
for rGPIa/IIa liposomes binding, compared with those required for
eliciting platelet aggregation. This may be attributed to the possible
reduction in the binding ability of rhodocytin to GPIa/IIa during the
immobilization procedure. Alternatively, it is also possible that
rhodocytin induces conformational changes of GPIa/IIa on the platelet
membrane, as in the case of ligand binding to other integrins.
Conformational changes of GPIa/IIa on the platelet membrane may result
in the increased binding capacity of GPIa/IIa to rhodocytin, whereas
this apparently cannot happen with rGPIa/IIa liposomes. Further
experiments are needed to address this issue.
The Most Proximal Step of the Rhodocytin-mediated Signal
Transduction Pathway--
We then investigated the activation signals
most proximal to GPIa/IIa by the use of immunoprecipitation studies.
Immunoprecipitation of GPIa/IIa, followed by Western blotting with
anti-Src mAb or anti-Lyn pAb, revealed that Src and Lyn constitutively
associated with GPIa/IIa. GPIa/IIa-associated kinase activity increased
upon rhodocytin stimulation, which was only slightly modified by
PGE1 treatment. We assume that this kinase activity belongs
to that of Src, since its molecular mass, 60 kDa, is similar to that of Src, and no association was detected between GPIa/IIa and other major
kinases present in platelets including FAK, Fyn, phosphatidylinositol 3-kinase, Lck, Jak1, and Jak2.
Furthermore, we found that PP1, an inhibitor of Src family kinases,
completely inhibited rhodocytin-induced platelet aggregation at the
concentration of 5 µM. Higher concentrations of
rhodocytin (up to 100 nM) could not overcome the inhibitory
effect of PP1. We also examined effects of PP2, which is another
inhibitor of Src family kinase, and we obtained results similar to that
of PP1 (data not shown). Based on these findings, we assume that GPIa/IIa-associated Src is the most proximal step of the
GPIa/IIa-mediated signal transduction pathway, although we cannot
totally exclude the possibility that the Lyn kinase activity is also
involved in rhodocytin-induced platelet activation via GPIa/IIa. Since Triton X-100 treatment does not completely disrupt the noncovalent bond
between GPIa and GPIIa (data not shown), it is still premature to draw
a conclusion as to whether Src associates with GPIa or GPIIa and which
molecule plays a more important role in the GPIa/IIa-mediated signal
transduction pathway.
From these findings described above, we suggest that rhodocytin
mediates platelet activation signals through GPIa/IIa (integrin
Based upon these findings, we propose the following steps for
rhodocytin-induced platelet activation; the binding of rhodocytin to
GPIa/IIa results in activation of Src, which constitutively binds to
GPIa/IIa. Activated Src associated with GPIa/IIa then induces tyrosine
phosphorylation of Cas. Several activation signals including Syk and
PLC The Similarities and Differences between Rhodocytin-induced and
Collagen-induced Activation Signals--
We suggest that rhodocytin
induces platelet aggregation by interacting with GPIa/IIa, one of the
most important collagen receptors. We found several similarities and
differences between rhodocytin-induced and collagen-induced platelet
activation, which may give insights into the platelet activation
pathway related with collagen receptors.
1) Rhodocytin induces aggregation of Fc
It may be also argued that rhodocytin binds to yet unidentified
molecules other than GPIa/IIa, although the interaction between rhodocytin and GPIa/IIa is essential. In this study, we showed by
several lines of evidence that rhodocytin binds to GPIa/IIa but not to
GPVI or GPIb. However, we cannot totally exclude the possibility that
rhodocytin may have binding sites other than GPIa/IIa on the platelet
membrane, and this may contribute to the initiation of activation
signals. We need to address this issue in the near future.
2) It is reported that Fyn and Lyn but not Src are involved in the
GPVI-mediated signal transduction pathway (15, 17), whereas rhodocytin
activates GPIa/IIa-associated Src. Ichinohe et al. (60)
reported that collagen induces Src activation in GPVI-deficient
platelets, and an anti-
3) Cytochalasin D totally inhibited all the rhodocytin-elicited signals
we evaluated in a previous study and this one. GPIa/IIa-associated Src
activation, Cas tyrosine phosphorylation, Syk and PLC
Susceptibility to cytochalasin D suggests that GPIa/IIa clustering is
required to activate GPIa/IIa-associated Src. How does rhodocytin,
which is a heterodimer of 18- and 15-kDa units (25), induce GPIa/IIa
clustering? In a preliminary experiment, we estimated the molecular
mass of rhodocytin to be 61 kDa in water by gel filtrating
chromatography.2 Wang
et al. (55) also isolated and partially characterized a
protein whose structural and functional property is identical with
rhodocytin, although the complete sequence awaits to be determined. This protein probably has a quaternary structure consisting of at least
two disulfide-linked dimers since it has a native molecular mass of
about 66 kDa as determined by gel filtration. Thus, rhodocytin appears
to form a multimer in water. This multivalency in water may induce
platelet activation by clustering GPIa/IIa. Cytochalasin D is likely to
disturb GPIa/IIa clustering induced by rhodocytin stimulation,
resulting in inhibition of the GPIa/IIa-mediated signals from the most
proximal step.
Alternatively, cytochalasin D may directly inhibit the signal
transduction related to GPIa/IIa. It is known that GPIa/IIa is linked
to the membrane cytoskeleton through association with actin-binding
protein and short actin filaments. Therefore, disruption of actin
polymerization may inhibit transducing GPIa/IIa-mediated activation signals.
4) Although we found that rhodocytin-induced tyrosine phosphorylation
of Syk and PLC
5) Rhodocytin, which binds to GPIa/IIa and not to GPVI, induces faint
tyrosine phosphorylation of Syk, which typically peaks at 2 min after
stimulation, whereas collagen induces marked phosphorylation of Syk in
the early stage of activation. Others and we (26, 65) found that
CRP that binds to GPVI also induces marked phosphorylation of Syk in
the early stage of platelet activation, similar to collagen. Ichinohe
et al. (60) reported that collagen-induced Syk tyrosine phosphorylation was severely compromised in platelets lacking GPVI. Our
finding is in accordance with previous reports and confirms that GPVI
is a main receptor that induces Syk tyrosine phosphorylation in
collagen-induced platelet activation.
2
1) has established its role as a
collagen receptor, it remains unclear whether GPIa/IIa mediates
activation signals. In this study, we show that rhodocytin, purified
from the Calloselasma rhodostoma venom, induces platelet
aggregation, which can be blocked by anti-GPIa monoclonal antibodies.
Studies with rhodocytin-coupled beads and liposomes loaded with
recombinant GPIa/IIa demonstrated that rhodocytin directly binds to
GPIa/IIa independently of divalent cations. In vitro kinase
assays and Western blotting of GPIa immunoprecipitates revealed that
Src and Lyn constitutively associate with GPIa/IIa and that Src
activity increases transiently after rhodocytin stimulation. Src
specifically associates with p130 Crk-associated substrate (Cas) in a
manner dependent upon Cas phosphorylation, suggesting that Src is
responsible for Cas tyrosine phosphorylation. While all these phenomena
occur early after rhodocytin stimulation in a cAMP-resistant manner,
tyrosine phosphorylation of Syk and phospholipase C
2, intracellular
Ca2+ mobilization, and platelet aggregation occur later in
a cAMP-sensitive manner. Cytochalasin D, which interferes with actin
polymerization and blocks receptor clustering, inhibits all the
rhodocytin-mediated signals we examined in this study. We suggest that
rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated
Src, which then mediates downstream activation signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(FcR
)-chain by the
Src family kinases, Fyn and Lyn, then the binding of Syk to the
-chain, with resultant Syk activation (14-17). Syk activation
appears to lie upstream of the tyrosine phosphorylation of PLC
2,
which releases intracellular calcium (18).
2 induced by collagen was attenuated by
pretreatment of anti-GPIa/IIa inhibitory antibodies or integrin
1 (GPIIa)-cleaving metalloproteinase, Jararhagin
(19-21).
2 integrin monoclonal antibody (mAb) that
promotes collagen binding to T-cells, stimulated tyrosine
phosphorylation of PLC
2 in human platelets. However, when Fc
receptor II (Fc
RII) was blocked by an anti-Fc
RII mAb, IV.3, JBS2
did not induce PLC
2 phosphorylation, suggesting that platelet
activation induced by this anti-
2 mAb occurs mainly through Fc
RII
(20). Thus, although it has been well established that GPIa/IIa plays a
major role in platelet adhesion to collagen (1, 23, 24), it remains
unclear what signals GPIa/IIa mediates that finally lead to
platelet activation.
2 activation,
similar to GPVI-mediated signals. However, distinct from GPVI-mediated
signals, tyrosine phosphorylation of Syk and PLC
2 induced by
GPIa/IIa stimulation is potently inhibited by acetylsalicylic acid
(ASA) or cytochalasin D treatment. These findings suggest that Syk and
PLC
2 activation requires actin polymerization and that tyrosine
phosphorylation of Syk and PLC
2 is facilitated by the thromboxane
A2 (TXA2)-producing system in the
GPIa/IIa-mediated signal transduction pathway. Susceptibility to ASA or
cytochalasin D is reminiscent of collagen-induced platelet activation.
2 undergo tyrosine phosphorylation. It is suggested that
clustering of GPIa/IIa is necessary for the GPIa/IIa-mediated signal
transduction, since all of these phenomena related to tyrosine
phosphorylation are sensitive to cytochalasin D, which blocks actin polymerization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain-deficient C57BL/6 (B6) mice and control B6 mice were generously provided by Dr. T. Takai (Tohoku University, Sendai, Japan).
2 polyclonal antibody (pAb), Santa Cruz, CA; the hybridoma
of Fc
RII mAb (clone IV.3), American Type Culture Collection, Manassas, VA; anti-phosphotyrosine (PY20) mAb, anti-Cas mAb, and anti-GPIIa mAb (clone 18), Transduction Laboratories, Lexington, KY;
anti-GPIa mAb (A2-IIE10), anti-Src mAb (327), and anti-Lyn pAb, Upstate
Biotechnology, Lake Placid, NY; type I collagen, Nycomed Pharma GMBH,
Munich, Germany; the peptide Gly-Arg-Gly-Asp-Ser (GRGDS), Peptide
Institute, Osaka, Japan; bovine serum albumin (BSA), prostaglandin
I2 (PGI2), phenylmethylsulfonyl fluoride, sodium orthovanadate, Triton X-100, tetramethylrhodamine
isothiocyanate-conjugated phalloidin, and enolase, Sigma; protein
A-Sepharose and CNBr-activated Sepharose 4B beads, Amersham Pharmacia
Biotech; peroxidase-conjugated goat anti-mouse IgG, Cappel Organ
Teknika Co.; PGE1, Funakoshi, Tokyo, Japan; Fura2-AM,
Dojindo Laboratories, Kumamoto, Japan; PP1, Biomol Research
Laboratories, Inc., Plymouth Meeting, PA; PP2, Calbiochem;
[
-32P]ATP, PerkinElmer Life Sciences.
-Chain-deficient Mice and Preparation of Murine
Platelets--
From FcR
-chain-deficient C57BL/6 (B6) mice and
control B6 mice, blood (500-900 µl) was taken into a 1-ml plastic
syringe containing 0.1 ml of ACD by cardiac puncture immediately after the mice died from deep ethyl ether anesthesia. PRP was obtained after
centrifugation of whole blood at 160 × g for 10 min.
Residual blood after PRP had been removed was further diluted by adding 200 µl of Tyrodes-HEPES buffer (134 mM NaCl, 2.9 mM KCl, 0.34 mM
Na2HPO4, 12 mM NaHCO3,
20 mM HEPES, 1 mM MgCl2, pH 6.6).
The mixture was centrifuged again, and the supernatant was obtained for
the maximum recovery of platelets. After repeating this step several
times, PRP was centrifuged at 1,000 × g for 5 min in
the presence of 100 nM PGI2 and 15% ACD. The
platelets were resuspended in Tyrodes-HEPES buffer, pH 7.3, at a
concentration of 1.3 × 108 cells/ml.
-32P]ATP). After 10 min at 20 °C, reactions were stopped by the addition of Laemmli
buffer and then subjected to boiling for 3 min. The proteins were
separated under reducing conditions by 8% SDS-polyacrylamide gel
electrophoresis (PAGE) and electrophoretically transferred onto Clear
Blot Membrane-P (Atto, Tokyo, Japan). The membrane was treated with 1 M KOH for 60 min, dried, and visualized with a BAS-2000
PhosphorImager analyzer (Fuji Film, Japan). Where indicated, levels of
radioactivity were quantified using PDI1400oe Scanner and Quantity One
2.5a software for Macintosh.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RII mAb on
Rhodocytin-induced Platelet Aggregation--
We previously reported
that echicetin, which is GPIb-blocking snake venom, does not inhibit
rhodocytin-induced platelet aggregation, suggesting that rhodocytin
induces platelet aggregation independently of GPIb (25). We also showed
that rhodocytin induces platelet aggregation with a long lag time and
that rhodocytin-induced platelet aggregation is susceptible to ASA and
cytochalasin D treatment (26). Since all of these properties are
reminiscent of collagen-induced platelet aggregation, we investigated
the effect of antibodies directed against GPIa/IIa, which is one of the
most important collagen receptors in platelets, on rhodocytin-induced
platelet aggregation. A2-IIE10, a mAb directed against GPIa, potently
blocked collagen-induced platelet aggregation, whereas Gi9, another mAb directed against GPIa, only partially inhibited collagen-induced platelet aggregation even at a concentration of 100 µg/ml (Fig. 1A). Rhodocytin (10 nM)-induced platelet aggregation was inhibited by 50 µg/ml of Gi9 and was completely inhibited by A2-IIE10 at 25 µg/ml,
the concentration that also inhibits collagen-induced platelet
aggregation (Fig. 1A). Relatively high concentrations of
anti-GPIa mAbs required for inhibition of rhodocytin-induced platelet
aggregation imply that rhodocytin binds to GPIa/IIa with high affinity.
Performed as negative control experiments, none of these anti-GPIa
antibodies inhibited platelet aggregation induced by CRP or thrombin
(Fig. 1A). We confirmed that a blocking antibody against
integrin-associated protein (B6H12), one of the platelet membrane
proteins, had no effect on platelet aggregation induced by rhodocytin,
even at a concentration of 100 µg/ml (26).
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Fig. 1.
Effects of monoclonal antibodies against GPIa
and Fc RII on platelet aggregation induced by
rhodocytin or other agonists. A, platelets were
preincubated for 5 min with a vehicle solution (a), 25 µg/ml of A2-IIE10 (b), and 100 µg/ml of Gi9
(c). Platelets were then stimulated with 10 nM
rhodocytin, 10 µg/ml of collagen, 0.5 µg/ml of CRP, or 0.2 units/ml
of thrombin. Platelet aggregation was monitored by changes in light
transmission. B, platelets were preincubated for 5 min with
a vehicle solution (a and c) and 3 µg/ml
Fc
RII-blocking mAb (IV.3) (b and d). Platelets
were then stimulated with 10 nM rhodocytin (a
and b) and 100 µg/ml Fc
RII-activating mAb (NNKY1-19)
(c and d).
RII blocking mAb), at a concentration that
could completely inhibit platelet aggregation induced by a
Fc
RII-activating mAb (NNKY1-19), had virtually no effect on
rhodocytin-induced platelet aggregation (Fig. 1B). These
findings suggest that rhodocytin interacts with GPIa/IIa but not with
GPIb or Fc
RII.
-chain Knockout Mice Induced by
Rhodocytin--
We investigated whether rhodocytin-mediated platelet
aggregation is mediated by GPVI, one of the major collagen receptors. FcR
-chain, which associates with GPVI is necessary for GPVI-mediated signal transduction, and collagen-related peptide (CRP), which is a
specific GPVI agonist, fails to induce aggregation of platelets obtained from GPVI-deficient patients or FcR
-chain knockout mice (18). As shown in Fig. 2, 20 nM rhodocytin induced aggregation of platelets obtained
from Fc
/
mice, whereas CRP up to the concentration of 1 µg/ml
failed to do so. In contrast, both rhodocytin and CRP elicited platelet
aggregation with Fc
+/+ mice. These findings suggest that rhodocytin
induces platelet aggregation independently of GPVI.
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Fig. 2.
Effects of rhodocytin, collagen and CRP on
platelets obtained from FcR -deficient
mice. Platelets obtained from control mice (A) or
FcR
-deficient mice (B) were stimulated with 20 nM rhodocytin (a), 10 µg/ml collagen
(b), or 1 µg/ml of CRP (c). Platelet
aggregation was monitored by changes in light transmission. The
ordinate represents percent changes in light transmission. The data are
representative of three experiments.
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Fig. 3.
Affinity precipitation of GPIa with
rhodocytin-coupled beads from platelet lysates. Unstimulated
washed platelets were lysed directly Triton X-100 lysis buffer. Whole
cell lysates (Whole, lane 1) and proteins
precipitated by affinity with Sepharose 4B alone (Seph,
lane 2) or rhodocytin-coupled Sepharose 4B
(rhod-Seph, lane 3) were resolved by 8%
SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted
with anti-GPIa pAb (AB1936) (A), with anti-GPIb mAb (WGA3)
(B), or with anti-GPIIa mAb (clone 18) (C).
Molecular mass markers are indicated in kDa on the right
side of panels. WB, Western blot.
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Fig. 4.
Adhesion of rGPIa/IIa liposomes on the
rhodocytin-coated plate or the collagen-coated plate. Plastic
plates (96-well) were coated with the indicated concentrations of
rhodocytin (upper panel) or collagen (lower
panel) overnight at room temperature. Wells were blocked with 1%
BSA for 30 min and washed twice with PBS. One hundred µl of rGPIa/IIa
liposomes (2 µg/ml) was added to each well and incubated for 3 h
at 30 °C. The amount of bound rGPIa/IIa liposome was detected by
enzyme immunoassay as described under "Experimental Procedures."
The ordinate represents the optical density. The data are
representative of at least three experiments.
2 Mediated by GPIa/IIa Are Completely
Inhibited by PGE1--
We previously reported that
rhodocytin-induced GPIa/IIa stimulation elicits tyrosine
phosphorylation of Syk, subsequent PLC
2 activation, and
intracellular calcium mobilization, which finally leads to platelet
aggregation and that these activation signals are inhibited by ASA
treatment (26). We also confirmed that Syk and PLC
2 were activated
even when platelets were pretreated with GRGDS peptides and EGTA,
suggesting that this activation is aggregation-independent and that
this is not mediated through GPIIb/IIIa (data not shown). In this
study, we extended our study to evaluate the effects of
PGE1, which increases the intracellular cAMP concentration.
As shown in Fig. 5, platelet aggregation, intracellular calcium mobilization, and tyrosine phosphorylation of Syk
and PLC
2 were completely inhibited by 1 µM
PGE1. Since several parameters of collagen-induced platelet
activation are resistant to the elevation of intracellular cAMP
(27-29), we then sought to determine rhodocytin-induced activation
signals, which were resistant to PGE1.
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Fig. 5.
Platelet aggregation, intracellular calcium
mobilization, Syk, and PLC 2 tyrosine
phosphorylation induced by GPIa/IIa stimulation were inhibited by
PGE1. A, washed platelets were pretreated
with vehicle solution (upper panel) or 1 µM
PGE1 (lower panel) for 5 min and then activated
with 10 nM rhodocytin added at the time indicated by
arrows. Platelet aggregation was monitored by the changes in
light transmission. The ordinate represents percent changes
in light transmission. B, platelets were incubated with
vehicle solution (upper panel) or 1 µM
PGE1 (lower panel) for 5 min in buffer
containing 1 mM CaCl2 and then stimulated with
10 nM rhodocytin, added at the time indicated by
arrows. [Ca2+]i elevation was
monitored as changes in fura-2 fluorescence for 300 s. The
ordinate represents the ratio of fura-2 fluorescence.
C and D, platelets were incubated with a vehicle
solution or 1 µM PGE1 for 5 min and then
activated by 20 nM rhodocytin for the indicated times.
Reactions were terminated with lysis buffer, and platelet proteins
associated with p72Syk or PLC
2 were immunoprecipitated
with anti-p72Syk mAb (C) or anti-PLC
2 pAb
(D). The sample was then Western-blotted with
anti-phosphotyrosine mAbs (4G10 plus PY20), anti-Syk mAb, or
anti-PLC
2 pAb. The data are representative of at least three
experiments.
1 integrin or cell adhesion to extracellular matrix
including collagen in other cells (30, 31, 39). Recently, we identified
Cas in platelets (33) and found that it undergoes changes upon platelet
activation (34). Therefore, we investigated tyrosine phosphorylation of
Cas and FAK upon rhodocytin stimulation. FAK tyrosine phosphorylation occurred relatively late after stimulation and was dependent upon platelet aggregation (data not shown). On the other hand, Cas underwent
tyrosine phosphorylation 15 s after stimulation, and the
tyrosine-phosphorylated Cas was dephosphorylated after 1 min, as
aggregation proceeded further (Fig.
6A). Tyrosine-phosphorylated Cas may be dephosphorylated by the increased phosphatase activity mediated through GPIIb/IIIa (34). We also confirmed that Cas tyrosine
phosphorylation is independent of platelet aggregation, which is
distinct from FAK tyrosine phosphorylation (34). The onset of Cas
tyrosine phosphorylation was significantly earlier (15 s) than that of
Syk or PLC
2 (2 min after stimulation in both cases), which suggests
that Cas tyrosine phosphorylation lies more proximal to GPIa/IIa than
Syk or PLC
2. Quantification of Cas tyrosine phosphorylation relative
to Cas protein showed that PGE1 pretreatment did not
inhibit Cas tyrosine phosphorylation (Fig. 6B). Under
PGE1 pretreatment, which inhibited platelet aggregation, the level of tyrosine phosphorylation was kept elevated 1 min after
stimulation, whereas the tyrosine-phosphorylated Cas was dephosphorylated after 1 min without PGE1. This is in
strong contrast to Syk and PLC
2, the tyrosine phosphorylation of
which was completely abrogated by PGE1. Since Cas is an
adapter protein without kinase activity, a tyrosine kinase must be
activated before tyrosine phosphorylation of these adapter proteins
occurs.
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Fig. 6.
PGE1-independent tyrosine
phosphorylation of Cas induced by GPIa/IIa stimulation and Src
associates with Cas in a manner dependent upon Cas tyrosine
phosphorylation. A, washed platelets were pretreated
with vehicle solution or 1 µM PGE1 for 5 min
and then activated by 20 nM rhodocytin for the indicated
times. Reactions were terminated with lysis buffer, and platelet
proteins associated with Cas were immunoprecipitated (IP)
with anti-Cas pAb. The sample was then Western blotted
(Blot) with anti-phosphotyrosine mAbs, anti-Cas mAb and
anti-Src mAb. The data are representative of at least three
experiments. B, tyrosine phosphorylation of Cas was
quantified with the image analyzing software, Quantity One. Cas
tyrosine phosphorylation was quantified as relative intensity to Cas
protein.
-chain produced negative results, suggesting that these proteins do not associate with GPIa/IIa (data not
shown). However, immunoprecipitates with anti-Src mAb or anti-Lyn pAb
revealed that Src and Lyn constitutively associated with GPIa/IIa and
that Src but not Lyn dissociated from GPIa/IIa as platelets formed
concrete aggregates (Fig. 7A).
Src and Lyn did not co-precipitate with control IgG (data not shown).
In vitro kinase assays of GPIa/IIa immunoprecipitates
revealed that the 60-kDa kinase activity increased 10 s after
stimulation and completely disappeared 1 min after stimulation (Fig.
7B). Lyn constantly associated with GPIa/IIa, even when the
kinase activity was totally absent at 3 min. On the other hand, Src
almost completely dissociated from GPIa/IIa 3 min after stimulation.
Thus, the presence of Src correlates well with the GPIa/IIa-associated
kinase activity. Since the Western-blotted membrane was incubated with
KOH, to minimize serine/threonine phosphorylation, the kinase activity most probably belongs to a tyrosine kinase. The molecular mass of this autophosphorylated kinase, 60 kDa, is the same as that of Src
but distinct from that of Lyn (56/53 kDa). None of other tyrosine
kinases that exist in platelets were detected in GPIa/IIa immunoprecipitates. These findings strongly suggest that the
GPIa/IIa-associated kinase activity belongs to that of Src. When
platelets were pretreated with EGTA and GRGDS to inhibit platelet
aggregation, the association between GPIa/IIa and Src/Lyn remained
constant, regardless of GPIa/IIa stimulation (Fig.
8A, left panel). Although the
amount of GPIa/IIa-associated Src remained constant under GRGDS
treatment, in vitro kinase assays of GPIa/IIa
immunoprecipitates revealed that the 60-kDa kinase activity increased
10-20 s after stimulation and decreased after 1 min (Fig.
8A). We sometimes detected a yet unidentified 40-kDa
phosphorylated protein. The pattern of the 60-kDa kinase activity was
not greatly altered by PGE1; it increased 10-20 s after
stimulation and decreased after 1 min (Fig. 8A, right panel,
and Fig. 8B). Taken together, we suggest that a change in
the GPIa/IIa-associated Src activity is the most proximal step in the
GPIa/IIa-mediated signal transduction.
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Fig. 7.
Src and Lyn constitutively associate GPIa/IIa
and the kinase activity associated with GPIa/IIa is transiently
activated upon rhodocytin stimulation. Washed platelets were
activated by 20 nM rhodocytin for the indicated times.
Reactions were terminated with lysis buffer, and GPIa/IIa was isolated
by immunoprecipitation with anti-GPIa mAb, Gi9. The samples were
divided into two parts, one-half was used for in vitro
kinase assay with enolase as exogenous substrate, and the other half
was for Western blotting with anti-Src mAb (A, upper panel)
and anti-Lyn pAb (A, lower panel). Radioactivity of
GPIa/IIa-associated protein was visualized with a Bio-Imaging analyzer
BAS2000 (B).
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Fig. 8.
The GPIa/IIa-associated kinase is transiently
activated upon rhodocytin stimulation, independent of
PGE1. Washed platelets were pretreated with vehicle
solution or 1 µM PGE1 in buffer containing
500 µM GRGDS and 500 µM EGTA and then
activated by 20 nM rhodocytin for the indicated times.
Reactions were terminated with lysis buffer. GPIa/IIa was isolated by
immunoprecipitation (IP) with anti-GPIa mAb, 6F1. The samples were
divided into two parts. One-half was used for in vitro
kinase assay, with enolase as exogenous substrate; the other half is
for Western blotting (Blot) with anti-Src antibody (A,
lower panel). Radioactivity of GPIa/IIa-associated protein was
visualized with Bio-Imaging analyzer BAS2000 (A, upper
panel) and quantified with the image analyzing software, Quantity
One (B). Kinase activity toward enolase was
calculated.
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Fig. 9.
Inhibitory effects of PP1 on platelet
aggregation induced by rhodocytin. Washed platelets were
pretreated with vehicle solution (a) or 5 µM
PP1 (b) for 5 min, then activated with 10 nM
(A) or 100 nM (B) rhodocytin added at
the time indicated by arrows. Platelet aggregation was monitored by
changes in light transmission. The ordinate represents % changes in
light transmission.
2 tyrosine phosphorylation induced by
rhodocytin (26). In this study, we evaluated the effects of
cytochalasin D on the signaling molecules more proximal to GPIa/IIa,
which are the GPIa/IIa-associated kinase activity and Cas. As shown in
Fig. 10, cytochalasin D potently inhibited the activation of GPIa/IIa-associated kinase and tyrosine phosphorylation of Cas. Thus, cytochalasin D inhibited all the activation signals evaluated in this study, implying that cytochalasin D inhibits the GPIa/IIa-mediated signals from the most proximal step.
The potent inhibitory effect of cytochalasin D indicates that the
clustering of GPIa/IIa is important for eliciting the downstream
activation signals induced by rhodocytin.
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Fig. 10.
Inhibitory effects of cytochalasin D on
rhodocytin-induced signals. Washed platelets were pretreated with
a vehicle solution or 10 µg/ml of cytochalasin D for 5 min, then
activated by 20 nM rhodocytin for the indicated periods of
time. In the lower panel, 500 µM GRGDS was
also used to inhibit platelet aggregation. Reactions were terminated
with lysis buffer, and platelet proteins associated with Cas and
GPIa/IIa were immunoprecipitated with anti-Cas mAb (upper
panel) and anti-GPIa mAb (lower panel), respectively.
The samples were then Western blotted with the anti-phosphotyrosine
mAbs, anti-Cas mAb and anti-Src mAb. In the lower panel,
in vitro kinase assays were also performed, with enolase as
exogenous substrate. The data are representative of at least three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RII, GPIa/IIa, and GPVI. In a previous study, we
found that echicetin, GPIb-blocking snake venom, has no effect on
rhodocytin-induced platelet aggregation (25). We found in this study
that IV.3, an anti-Fc
RII antibody, did not inhibit platelet
aggregation induced by rhodocytin. It is well known that FcR
-chain
physically associates with GPVI and plays a crucial role in the
GPVI-mediated signaling pathway. It is reported that CRP, a GPVI
agonist, fails to induce aggregation of Fc
-deficient platelets (18).
In this study, aggregation of Fc
-deficient platelets was induced by
rhodocytin. These findings taken together suggest that rhodocytin
induces platelet aggregation independently of GPIb, Fc
RII, or GPVI.
On the other hand, three anti-GPIa mAbs potently inhibited platelet
aggregation induced by rhodocytin, suggesting that it interacts with
GPIa/IIa, a collagen receptor on the platelet membrane. Furthermore,
complete inhibition of platelet adhesion to rhodocytin by anti-GPIa
mAbs indicates that the major binding site of rhodocytin on the
platelet membrane is GPIa/IIa.
2
1 (GPIa/IIa) is the only case reported that is independent of divalent cations (45, 47). The inhibitory effects of anti-
2 (GPIa) or
1 (GPIIa)
antibodies on echovirus binding to integrin
2
1 also had a profile distinct from
collagen fiber binding to integrin
2
1
(47). Later, they determined the binding site of echovirus 1 on the
integrin
2 I domain, which is distinct from the metal
ion-dependent adhesion site residues essential for the
divalent cation-dependent interaction with collagen (53).
The binding site of rhodocytin on GPIa/IIa also appears to be different
from that of collagen, and this may partly explain the several
differences between rhodocytin-induced and collagen-induced signals.
The binding site of rhodocytin on GPIa/IIa needs to be addressed in the future.
2
1). By analogy to integrin-mediated
signaling pathways in other cells, in which FAK and Cas are involved,
we evaluated the changes in FAK and Cas in rhodocytin-induce platelet
activation. FAK tyrosine phosphorylation occurred relatively late and
was dependent upon platelet aggregation. On the other hand we found
that rhodocytin stimulation induced the level of Cas tyrosine
phosphorylation 15 s after stimulation. Src but not FAK associated
with Cas in a manner dependent upon the tyrosine phosphorylation of
Cas. These findings suggest that, unlike other cells in which FAK
phosphorylates Cas in an integrin-related activation, Src is
responsible for Cas tyrosine phosphorylation in GPIa/IIa-related
platelet activation. Since we previously found that ASA and
PGE1 potently inhibited platelet aggregation, intracellular
calcium mobilization, and tyrosine phosphorylation of Syk and PLC
2,
we then examined the effects of these inhibitors on Cas tyrosine
phosphorylation. Cas tyrosine phosphorylation was not inhibited by ASA
(data not shown) or PGE1. The GPIa/IIa-associated Src
activity enhanced upon rhodocytin stimulation was also resistant to ASA
(data not shown) or PGE1 treatment. This is in marked
contrast to Syk and PLC
2 tyrosine phosphorylation, Ca2+
mobilization, and platelet aggregation, which are almost completely inhibited by these agents, suggesting that GPIa/IIa-associated Src
activity and Cas tyrosine phosphorylation are located at the more
proximal steps in the rhodocytin-mediated signaling pathway.
2 are also involved in rhodocytin-induced platelet activation.
-deficient platelets, whereas
collagen does not. The "two-site, two-step" theory has been
proposed for collagen activation of platelets. Based on this notion,
collagen first interacts with one type of collagen receptors, and then
the second receptor is involved to induce full activation of platelets.
The major role of GPIa/IIa for platelet adhesion and the lack of
collagen-induced platelet aggregation in GPIa/IIa-deficient platelets
have suggested that GPIa/IIa is the first receptor to react with
collagen and plays the initial role in the collagen-platelet interaction. However, this notion was challenged by a recent report by
Jung and Moroi (35) who suggest that GPVI activates GPIa/IIa through
its inside-out activation pathway. Furthermore, CRP, which specifically
interacts with GPVI, is a potent agonist of platelet activation in
various aspects, whereas the peptides that specifically bind GPIa/IIa
appear to induce no activation signal in platelets (64). These findings
have provided evidence for the major role of GPVI in collagen-induced
platelet activation. In this aspect, it is of interest how rhodocytin
elicits platelet aggregation by interacting with GPIa/IIa but not with
GPVI. It is possible that the mode of binding between rhodocytin and
GPIa/IIa, which is distinct from that between collagen fibers and
GPIa/IIa, elicits intracellular activation signals that cannot be
observed with the binding between GPIa/IIa and its specific peptides.
Alternatively, it is possible that cross-linking of GPIa/IIa by
rhodocytin may suffice to produce intracellular activation signals
without the involvement of GPVI. Collagen fails to induce platelet
aggregation in GPIa/IIa-deficient platelets, suggesting that the
interaction between collagen fiber and GPVI does not lead to full
activation of platelets. On the other hand, CRP, which binds only to
GPVI potently, induces platelet aggregation. Thus extensive
cross-linking of one type of collagen receptors may suffice to activate
platelets, and the same mode of activation may be applied to that of rhodocytin.
2 blocking antibody inhibited this activation. The Src activation is most probably mediated through
GPIa/IIa, although the involvement of other collagen receptors cannot
be excluded. Our findings that rhodocytin induces the
GPIa/IIa-associated Src kinase activity appear to be in good agreement
with their study using GPVI-deficient platelets. Taken together, we
suggest that Src activation is involved in the GPIa/IIa-mediated signal transduction pathway, whereas Fyn and Lyn are involved in the GPVI-mediated signal transduction pathway.
2 activation, intracellular calcium mobilization, and platelet aggregation were completely blocked (26). It is reported that several collagen-induced activation signals are also susceptible to cytochalasins (29, 62, 63).
On the other hand, cytochalasin D does not inhibit platelet
aggregation, intracellular calcium mobilization, or tyrosine phosphorylation of Syk and PLC
2 induced by CRP as we have previously reported (26). Therefore, the susceptibility to cytochalasin D of
collagen-induced platelet activation is most likely derived from the
GPIa/IIa-mediated signaling pathway. There have been a number of
reports (54, 56-59) that suggest that integrin clustering, which is
facilitated by cytoskeletal reorganization, activates tyrosine kinases
associated with integrins. Since cytochalasin D interferes with
cytoskeletal assembly, cell activation mediated by integrins is
severely suppressed by cytochalasin D. Our findings that rhodocytin
mediates tyrosine phosphorylation of several proteins via GPIa/IIa,
which can be inhibited by cytochalasin D, fit well with this notion.
2 was potently inhibited by ASA or PGE1, several reports (27-29, 61) suggest that tyrosine phosphorylation of
several proteins induced by collagen is resistant to TXA2
blocking or cAMP-elevating agents. These reports used high dose
collagen (50-150 µg/ml) to prove the resistance of collagen-induced
protein tyrosine phosphorylation to these inhibitors. It is well
established that several parameters of platelet activation induced by
low dose collagen are highly dependent on TXA2 generation.
In our work, 1 mM ASA or 1 µM
PGE1 apparently inhibited Syk and PLC
2 tyrosine
phosphorylation induced by 10 µg/ml collagen. The maximum level of
PLC
2 tyrosine phosphorylation induced by 10 µg/ml collagen was
decreased to 60% of the control by ASA treatment, and the peak time
was delayed from 30 s to 5 min.3 Thus, the inhibitory
profile of these agents on low dose collagen stimulation is similar to
that of rhodocytin, which seems to activate platelets by binding to
GPIa/IIa. On the other hand, similar to high dose collagen, Syk and
PLC
2 tyrosine phosphorylation induced by GPVI agonists is not
dependent on TXA2 generation (26) or cAMP (27). Taken
together, we speculate that low concentrations of collagen mediate
activation signals through GPIa/IIa and GPVI, whereas at high
concentrations of collagen, signals from GPVI become predominant over
those mediated by GPIa/IIa.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. B. S. Coller at
the Mt. Sinai Medical Center; M. Moroi and Y. Miura at Kurume
University; T. Takai at Tohoku University; S. Nomura at Kansai Medical
University; and M. Handa at Keio University for providing 6F1, CRP,
FcR-chain deficient mice, NNKY1-19, and WGA3, respectively. We
thank H. Meguro at Toray Industries, Inc., for technical assistance
with generating 7E10B and 3C8A. We also thank Drs. H. Tezuka and K. Mukawa at Yamanashi Medical University for helping us with the management of knockout mice. We are grateful to Dr. S. P. Watson in University of Oxford for careful review of the manuscript and helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Clinical and Laboratory Medicine, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi 409-3898, Japan. Tel.: 81 55 273 9884; Fax: 81 55 273 6713; E-mail: yozaki@res.yamanashi-med.ac.jp.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M006191200
2 C-Q. Ma and T. Morita, unpublished observations.
3 K. Suzuki-Inoue, Y. Ozaki, Y. Shin, Y. Yatomi, K. Satoh, and T. Morita, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
GPIa/IIa, glycoprotein Ia/IIa;
Cas, p130 Crk-associated substrate;
FAK, focal
adhesion kinase;
ASA, acetylsalicylic acid;
PGE1, prostaglandin E1;
PLC2, phospholipase C
2;
CRP, Collagen-related peptide;
Fc
RII, Fc
receptor II;
mAb, monoclonal
antibody;
pAb, polyclonal antibody;
TXA2, thromboxane
A2;
PYK2, proline-rich tyrosine kinase 2;
PAGE, polyacrylamide gel electrophoresis;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
PGI2, prostaglandin
I2;
PRP, platelet-rich plasma.
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REFERENCES |
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