From the
NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced
protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa
proteins in human platelets, whereas F(ab`)
A number of antibodies directed against antigens on the platelet
membrane activate platelets (Horsewood et al., 1991). Whereas
the antigens involved are diverse, the known antibodies with
stimulatory properties are predominantly against CD9, a 24-kDa cell
surface glycoprotein (Higashihara et al., 1985; Rendu et
al., 1987; Jennings et al., 1990; Carroll et
al., 1990), which is confirmed by the fact that most of the
monoclonal antibodies (MoAbs)
There is an increasing body of
evidence for an important role of protein tyrosine phosphorylation
facilitated by tyrosine kinases in the regulation of cell functions,
especially those related to cell growth and oncogenesis. While
platelets lack the ability to proliferate, they possess a high level of
tyrosine kinase activity. All platelet tyrosine kinases reported to
date are non-receptor types, p60
Recently, we found
that anti-CD9 MoAb induces protein tyrosine phosphorylation and that
there is an increased level of 3`-phosphorylated polyphosphoinositides,
the production of which is physiologically related to tyrosine kinase
activity (Yatomi et al., 1993). In this study, we investigated
the changes in p72
We then evaluated the effect of
PGI
Previous studies have suggested that the platelet activation
induced by anti-CD9 MoAb is largely dependent upon Fc
An increase in the
p72
With intact
IgG of anti-CD9 MoAb, which induces a full picture of platelet
activation, an increase in the p72
Alternately, the process of aggregation may deactivate
p72
Involvement of
p72
We gratefully acknowledge the kind donation of PMA2,
an anti-CD9 MoAb, by Dr. Takaaki Hato (Ehime University, Japan).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
fragments of
NNKY 1-19 did not, suggesting that the stimulation of Fc
II
receptors is required for the induction of protein tyrosine
phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa
were associated with aggregation, while NNKY 1-19-induced protein
tyrosine phosphorylation was completely inhibited by prostaglandin
I
(PGI
). The activity of p72
was assessed in immunoprecipitation kinase assays to
determine at which step the signal transduction pathway leading to
protein tyrosine phosphorylation was suspended. NNKY 1-19 induced
a rapid and transient increase in the
p72
-associated tyrosine kinase activity that
peaked at 10 s and subsided to the original level 2 min after
stimulation. Coinciding with this time course,
p60
transiently associated with
p72
. In platelets preexposed to GRGDS peptides
or PGI
, NNKY 1-19 also increased the
p72
-associated tyrosine kinase activity and led
to the association of p60
with
p72
. However, in contrast to the control without
any inhibitor, the elevated tyrosine kinase activity and the associated
state of the two tyrosine kinases persisted as long as 5 min after
stimulation. F(ab`)
fragments of NNKY 1-19 induced
changes similar to those observed with the effects of GRGDS peptides or
PGI
treatment on intact IgG NNKY 1-19 stimulation.
F(ab`)
fragments of another CD9 MoAb, PMA2, had effects on
p72
essentially similar to those of NNKY
1-19. These findings suggest that the binding of anti-CD9 MoAb to
CD9 on the platelet membrane per se induces an increase in the
p72
-associated tyrosine kinase activity but that
Fc
II receptor-mediated signal(s) is required for the full
activation of platelets and the appearance of tyrosine-phosphorylated
proteins. The elevated intracellular cAMP level induced by PGI
acts at a step distal to the activation of p72
and inhibited the signal transduction pathway leading to
protein tyrosine phosphorylation and aggregation. p72
activation occurs in the absence of aggregation, but aggregation
appears to reduce the elevated p72
activity
induced by anti-CD9 MoAb.
(
)that induced
rapid aggregation were directed against CD9, as presented at the
platelet workshop of the IV Leucocyte Typing Conference in Vienna
(Powling et al., 1989). Anti-CD9 MoAb is a potent activator of
platelet function comparable to thrombin and induces the entire range
of functions, including aggregation, granule secretion, protein
phosphorylation, phosphoinositide hydrolysis with Ca
mobilization, and arachidonic acid metabolism. These findings
suggested that CD9 is unique among platelet membrane glycoproteins and
that it is a signal transducer. Molecular cloning of CD9 revealed that
it is a multiply inserted membrane protein containing four putative
transmembrane domains, suggesting that it could indeed be involved in
signal transduction pathways (Boucheix et al., 1991) Various
attempts have been made to clarify the mechanism by which anti-CD9
MoAbs activate platelets. Phospholipase A
activation plays
an important role but is not a prerequisite (Ozaki et al.,
1991). CD9 physically associates with GPIIb/IIIa in the course of
anti-CD9 MoAb-induced platelet activation (Slupsky et al.,
1989). These studies were based upon the concept that the effects of
anti-CD9 MoAbs are exerted through CD9. However, this concept was
challenged by recent findings that F(ab`)
fragments of
anti-CD9 MoAbs lack the ability of platelet activation and that the
blocking of the Fc
II receptor by corresponding monoclonal
antibodies inhibited aggregation and Ca
mobilization
induced by anti-CD9 MoAbs (Slupsky et al., 1989; Worthington et al., 1990). Moreover, stimulation of the Fc
II receptor
by cross-linkage or by aggregated IgG presented a picture of platelet
activation similar to that of MoAbs to CD9 (Henson and Spiegelberg,
1973; Anderson and Anderson, 1990). These findings imply that the
signal transduction pathway is mediated entirely through the Fc
II
receptor, independent of CD9.
being the most abundant (Rendu et al., 1989). In
addition to p60
, several tyrosine
kinases including p59
,
p62
, p54/58
,
p72
, and p125
have been identified
(Huang et al., 1991; Lipfert et al., 1992). Upon
platelet activation induced by various stimulators, a set of proteins
undergo tyrosine phosphorylation (Nakamura and Yamamura, 1989; Salari et al., 1990). Although it is not yet clear which tyrosine
kinase is responsible for a particular tyrosine-phosphorylated protein,
several kinases change their activities upon platelet stimulation. The
activity of p72
is rapidly increased by 10-fold
upon thrombin activation, reaching a maximum at 10 s (Taniguchi et
al., 1993). Platelet activation also elevates the
p60
activity, albeit to a lesser
degree and with a slower time course (Wong et al., 1992). The
activity of p125
appears to be modified even later by
fibrinogen binding to glycoprotein IIb/IIIa on the platelet membrane
(Lipfert et al., 1992). These lines of evidence suggest that
protein tyrosine phosphorylation and tyrosine kinases are actively
engaged in the regulation of platelet functions from the initial phase
of activation to the late stage of aggregation.
activity induced by anti-CD9
MoAb with reference to protein tyrosine phosphorylation.
Materials and Chemicals
MoAb against
p72, p59
, or
p54/58
was obtained from Wako Chemicals (Tokyo).
MoAbs against p60
(GD11) and
phosphotyrosine (4G10) were obtained from Upstate Biotechnology.
Acetoxymethyl ester of fura 2 were from Dojin Chemicals (Kumamoto,
Japan). GRGDS peptides were obtained from Peptide Institute (Osaka,
Japan). EGTA, enolase, prostaglandin I
(PGI
),
leupeptin, phenylmethylsulfonyl fluoride, and sodium orthovanadate were
purchased from Sigma. Protein A-Sepharose and CNBr-activated Sepharose
were from Pharmacia Japan (Tokyo). Hepes buffer containing 138 mM NaCl, 2.8 mM KCl, 0.8 mM
KH
PO
, 0.8 mM MgCl
, 10
mM Hepes (pH 7.2), and 5.5 mM glucose was sterilized
by filtration and stored at 4 °C until use. NNKY 1-19,
anti-CD9 MoAb, was raised by immunizing mice with human platelets
(Nagata et al., 1990). Another anti-CD9 MoAb, PMA2, was a
generous gift from Dr. Takaaki Hato (Ehime University, Japan) (Hato et al., 1990). F(ab`)
fragments of NNKY 1-19
or PMA2 were prepared by pepsin digestion (Lamoyi and Nisonoff, 1983).
Briefly, NNKY 1-19 was dialyzed overnight at 4 °C against 100
mM acetate buffer, pH 7.0. Pepsin (3% w/w) was added, and the
mixture was incubated in acetate buffer, pH 4.2, for 12 h at 37 °C.
The solution was fractionated on a Sephadex G150 column to obtain
F(ab`)
fragments, and the purity of the F(ab`)
was confirmed by SDS-PAGE.
Platelet Separation
Citrated,
anti-coagulated venous blood was obtained from healthy human donors who
had not received any medication for a minimum of two weeks prior to the
experiment. The blood was centrifuged at 160 g for 15
min to obtain platelet-rich plasma. Platelets were isolated by
differential centrifugation as described (Golden and Brugge, 1989) and
finally resuspended at a concentration of 10
cells/ml in
Hepes buffer containing 1 mM Ca
unless
otherwise stated.
Preparation of Fura 2-loaded Platelets and Measurement of
[Ca
To platelet-rich
plasma obtained as described above, fura 2-AM at a final concentration
of 3 µM was added, and the mixture was incubated at 37
°C for 30 min. The platelets were then washed twice, and
resuspended at a concentration of 1 ]
10
cells/µl. Fura 2 fluorescence was measured with a Hitachi
F-2000 fluorescence spectrophotometer, with an excitation wavelength
alternating every 0.5 s from 340 to 380 nm, and the emission wavelength
was set at 510 nm. The [Ca
]
values were determined from the ratio of fura 2 fluorescence
intensity at 340 and 380 nm excitation (Grynkiewicz et al.,
1985).
Immunoprecipitation Kinase Assay
The
platelets were activated with anti-CD9 MoAb or its F(ab`) fragments with constant stirring unless otherwise stated. After
the indicated periods of time, the reaction was terminated with an
equal volume of ice-cold lysis buffer (2% Triton X-100, 100 mM
Tris/HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 2 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, and 100
µg/ml leupeptin). The lysate was sonicated and separated by
centrifugation at 16,000
g for 5 min. The supernatant
was precleared with Sepharose beads twice and then mixed with
anti-p72
antibody bound to protein A-Sepharose
or CNBr-activated Sepharose. The mixture was rotated for 2 h at 4
°C. The Sepharose beads were washed three times with lysis buffer.
The sample was then split into two portions. One was used for
immunoblotting, described elsewhere, and the other was processed
further for in vitro kinase assay. In vitro kinase
assay was performed essentially as described (Clark and Brugge, 1993).
The beads were washed once with low salt buffer (100 mM NaCl,
5 mM MnCl
, 10 mM Tris, pH 7.4) incubated
with 25 µl of kinase reaction buffer (20 mM Tris, pH 7.5,
10 mM MnCl
) with or without 10 µg of
acid-treated enolase. The reaction was initiated by the addition of 10
µCi of [
-
P]ATP and 2 µM ATP. After 10 min at 20 °C, the reaction was stopped by the
addition of Laemmli buffer and then subjected to boiling for 3 min. The
proteins were separated under reducing conditions by 8 or 12% SDS-PAGE
and electrically transferred onto Clear Blot Membrane P (Atto, Tokyo).
The membrane was treated with 1 M KOH for 60 min, dried, and
quantified with a BAS-2000 Phosphorimager (Fuji Film, Japan).
Protein Analysis by
Immunoblotting
Laemmli sample buffer was added to platelets
activated with anti-CD9 MoAb for the indicated periods, and then the
mixture was boiled for 3 min. In some experiments, proteins separated
for immunoprecipitation kinase assay were similarly processed. Platelet
proteins were separated by 8% SDS-PAGE and electroblotted onto Clear
Blot Membrane P (Atto, Tokyo). The immunoblots were incubated with 1
µg/ml MoAb directed to phosphotyrosine or
p60 for 3 h. Antibody binding was
detected using peroxidase-conjugated goat anti-mouse IgG (Cappel, PA)
and visualized with ECL detection reagents (Amersham, UK).
PTP Induced by NNKY 1-19, Anti-CD9
MoAb
In the presence of 1 mM extracellular
Ca and without aspirin, NNKY 1-19 at
concentrations as low as 1 µg/ml induced platelet aggregation,
[Ca
]
elevation, and
serotonin release (Nagata et al., 1990). The optimal
concentration of NNKY 1-19 varied among individuals tested but
usually fell in the range of 3 to 10 µg/ml. In experiments
thereafter, NNKY 1-19 was used at a concentration of 10
µg/ml. Upon NNKY 1-19 stimulation, a set of
tyrosine-phosphorylated proteins appeared with different profiles in
the time course (Fig. 1A). A 75-kDa band appeared as
early as 30 s after stimulation and tended to disappear after several
minutes. The appearance of 97- and 125-kDa bands was a late event and
probably corresponded to those alleged to be related to aggregation in
thrombin-induced platelet activation (Golden et al., 1990),
since these bands of PTP did not appear when platelets were treated
with GRGDS (data not shown). F(ab`)
fragments of NNKY
1-19 up to a concentration of 30 µg/ml had no effects on
platelets in terms of the appearance of PTP (Fig. 1B),
aggregation, or [Ca
]
elevation, confirming that stimulation of Fc
II
receptors is required for the full picture of platelet activation
(Worthington et al., 1990).
Figure 1:
Protein tyrosine phosphorylation
induced by anti-CD9 MoAb. Platelets were suspended in Hepes buffer
containing 1 mM Ca. Platelets were activated
either with 10 µg/ml of intact IgG NNKY 1-19, an anti-CD9
MoAb, or with 10 µg/ml F(ab`)
fragments of NNKY
1-19 with constant stirring for the indicated periods, and
reactions were terminated with Laemmli sample buffer. Platelet proteins
were applied to SDS-PAGE, and tyrosine-phosphorylated proteins were
detected by Western blotting using 4G10, anti-phosphotyrosine MoAb. A, intact IgG of NNKY 1-19; B, F(ab`)
fragments.
Effects of Various Inhibitors on PTP Induced by NNKY
1-19
We previously found that extracellular Ca and the production of thromboxane A
greatly
facilitate platelet activation induced by a MoAb to CD9 (Ozaki et
al., 1991). Thus, the effects of chelating extracellular
Ca
with EGTA and of aspirin were evaluated on NNKY
1-19-induced [Ca
]
elevation and PTP. Platelets in platelet-rich plasma were
treated with 0.5 mM aspirin for 30 min and then washed and
resuspended in Hepes buffer containing 200 µM EGTA and no
Ca
. Chelation of extracellular Ca
and aspirin pretreatment markedly reduced
[Ca
]
elevation induced
by NNKY 1-19 (0.3 ± 0.2 versus 4.8 ± 1.1
in terms of Fura 2 fluorescence ratio). The appearance of 75-kDa PTP
band was delayed, but in contrast to the control sample with
extracellular Ca
and no aspirin, the intensity of PTP
persisted for up to 3 min (data not shown). The 97- and 125-kDa bands
were barely, if at all, detectable.
on NNKY 1-19-induced PTP. PGI
raises
the intracellular cAMP level, which attenuates PTP induced by thrombin
(Pumiglia et al., 1990). Incubating platelets with 0.4
µM PGI
for 5 min completely abrogated the
appearance of PTP, aggregation, and
[Ca
]
elevation induced
by NNKY 1-19 (data not shown).
NNKY 1-19 Stimulation Induces a Transient Rise in
p72
NNKY
1-19 stimulation increased the level of p72-associated Tyrosine Kinase Activity with
Concomitant Association of p60
autophosphorylation, which peaked 10-60 s after
stimulation and subsided to lower level thereafter (Fig. 2, upperpanel). Densitometry revealed a
1.5-3-fold increase, which was substantially lower than that for
thrombin activation (Taniguchi et al., 1993). The
autophosphorylated amino acid of p72
was
exclusively tyrosine (Ohta et al., 1992 and data not shown).
Concomitant with the change in autophosphorylation, in vitro kinase assays revealed that the tyrosine kinase activity for
exogenous substrates was increased transiently along with the faint
band of a 60-kDa tyrosine-phosphorylated protein (Fig. 2, lowerpanel). Western blotting using
anti-p60
MoAb revealed that the
60-kDa band was p60
(Fig. 3),
suggesting that p60
transiently
associates with p72
upon NNKY 1-19-induced
platelet activation. Unlike anti-p72
,
anti-p59
MoAb or anti-p54/58
MoAb did not coprecipitate
p60
, suggesting that
p60
specifically associates with
p72
during platelet activation induced by
anti-CD9 MoAb (data not shown).
Figure 2:
p72-associated tyrosine kinase activity
induced by anti-CD9 MoAb. Platelets suspended in a buffer containing 1
mM Ca were activated with 10 µg/ml NNKY
1-19 for the indicated periods. The reaction was terminated with
lysis buffer, and p72 was isolated by immunoprecipitation with anti-p72
MoAb. Immunoprecipitates were either directly subjected to Western
blotting using anti-phosphotyrosine MoAb (upper), or to in
vitro kinase assay using enolase as exogenous substrate (lower). Arrowheads represent the bands presumably
derived from IgG.
Figure 3:
Association of p60 with p72
induced by anti-CD9 MoAb stimulation. Platelets were activated with
NNKY 1-19, and p72-associated proteins were isolated by
immunoprecipitation with anti- p72 MoAb. The sample was applied to
SDS-PAGE, and Western blotting was performed using anti-p60
MoAb.
F(ab`)
The F(ab`)Fragment of NNKY
1-19 Induces a Persistent Increase in the
p72
-associated Tyrosine Kinase Activity and the
Persistent Association of p60
with
p72
fragment of NNKY
1-19 at a concentration of 10 µg/ml also increased the level
of p72
autophosphorylation and the association
of tyrosine-phosphorylated 60-kDa protein with
p72
. However, in contrast to an early decay in
the p72
activity and the early dissociation of
the 60-kDa band from p72
, the
F(ab`)
-induced process was persistent up to 5 min after
stimulation (Fig. 4). At this time, the autophosphorylated level
of p72
and the associated 60-kDa PTP was often
greater than the maximum level attained by intact IgG of NNKY
1-19. Western blotting with
anti-p60
MoAb revealed the
persistent p60
association with
p72
(data not shown). That the preparation of
F(ab`)
fragments was not contaminated with intact antibody
was confirmed by SDS-PAGE under non-reducing conditions (data not
shown). Furthermore, IV.3, an anti-Fc
II receptor MoAb, was used to
block Fc receptor activation induced by residual, if any, intact
antibody. The absence of inhibitory effects of IV.3 on the increased
tyrosine kinase activity induced by F(ab`)
fragments of
NNKY1-19 suggests that F(ab`)
fragments of
NNKY1-19 are capable of activating p72
(Fig. 5). To reinforce the notion that F(ab`)
fragments of anti-CD9 MoAb induce p72
activation in platelets, the effects of another CD9 MoAb,
PMA2 (Hato et al., 1990), were evaluated on the changes in
p72
-associated tyrosine kinase activity and the
association between p72
and
p60
. Fig. 6shows that the
effects of intact IgG PMA2 or the F(ab`)
fragments of PMA2
were essentially similar to those of NNKY 1-19. A transient
increase in the autophosphorylated level of p72
and the association between p72
and
p60
was observed with intact
antibody, and these processes were persistent with the F(ab`)
fragments.
Figure 4:
The p72-associated tyrosine kinase
activity induced by F(ab`) fragments of anti-CD9 MoAb
compared with that by intact IgG. Platelets suspended in a buffer
containing 1 mM Ca
were activated with 10
µg/ml intact IgG or F(ab`)
fragments of NNKY 1-19
for the indicated periods. The reaction was terminated with lysis
buffer, and p72 was isolated by immunoprecipitation with anti-p72 MoAb. In vitro kinase assay was performed on the isolated sample as
described under ``Experimental Procedures.'' A,
intact IgG; B, F(ab`)
fragments.
Figure 5:
Effects of IV.3, anti-FcII receptor
MoAb, on p72-associated tyrosine kinase activity induced by anti-CD9
MoAb F(ab`)
fragments. Platelets were first incubated with
10 µg/ml IV.3 for 3 min, and the changes in p72-associated tyrosine
kinase assay was assessed (A) at the indicated point of time.
F(ab`)
fragments, 10 µg/ml of NNKY1-19, were then
added, and the changes in tyrosine kinase activity were evaluated for
another 3 min (B).
Figure 6:
p72-associated tyrosine kinase activity
and p60 association with p72 induced by intact antibody or
F(ab`)
fragments of PMA2, another anti-CD9 MoAb. Platelets
suspended in a buffer containing 1 mM Ca
were activated with 10 µg/ml intact antibody or F(ab`)
fragments of PMA2, an anti-CD9 MoAb for the indicated periods.
The reaction was terminated with lysis buffer, and p72 was isolated by
immunoprecipitation with anti-p72 MoAb. Immunoprecipitates were either
directly subjected to Western blotting using anti-phosphotyrosine MoAb
or to in vitro kinase assay using enolase as exogenous
substrate. Arrowheads represent the bands presumably derived
from IgG. A and C, in vitro kinase activity; B and D, Western blotting using anti-p60
MoAb. A and B, the changes induced by intact
antibody; C and D, the changes induced by
F(ab`)
fragments of PMA2.
Effects of Various Inhibitors on
p72
Aspirin slightly prolonged the elevated level
of tyrosine kinase activity induced by NNKY 1-19. The overall
tyrosine kinase activity associated with p72-associated Tyrosine Kinase Activity Induced
by NNKY 1-19
was
still detectable at 60 s after stimulation albeit to a lesser degree
than that at 10 s (Fig. 7A). Western blotting using
anti-p60
MoAb showed that the
association of p60
with
p72
was also slightly extended (Fig. 7B). However, the general profile of changes in
p72
-associated tyrosine kinase including a
transient increase in p72
autophosphorylation
and the transient association of p60
with p72
, followed by a marked
decrease in kinase activity and the complete dissociation of
p60
, was essentially similar to
that of the control sample without aspirin.
Figure 7:
The effects of aspirin pretreatment on
p72-associated tyrosine kinase activity and association between p72 and
p60 induced by anti-CD9 MoAb. Platelet-rich plasma was
incubated with or without 0.5 mM aspirin for 30 min. Platelets
were then washed and resuspended in a buffer containing 200 µM EGTA. Platelets were activated with 10 µg/ml NNKY 1-19
for the indicated periods, and the reaction was terminated with lysis
buffer. p72 was isolated by immunoprecipitation with anti-p72 MoAb. The
sample was analyzed either by in vitro kinase assay or to
SDS-PAGE followed by Western blotting using anti-p60
MoAb. A, in vitro kinase assay; B, detection of
p60
with Western blotting. Arrowheads represent
the band presumably derived from IgG.
We then asked whether
aggregation modified the changes in p72 activation induced by anti-CD9 MoAb. Chelation of
extracellular Ca
with 2 mM EGTA and 200
µM GRGDS peptide to inhibit platelet aggregation did not
suppress p72
activation in terms of its
autophosphorylation and the in vitro kinase activity (Fig. 8, A and B). However, a decrease in the
tyrosine kinase activity, which appears 3-5 min after
stimulation, was not observed in the absence of aggregation.
Figure 8:
Effects of GRGDS peptides on p72
autophosphorylation and p72-associated tyrosine kinase activity induced
by anti-CD9 MoAb. Extracellular Ca was chelated with
2 mM EGTA, and 200 µM GRGDS peptides were added
to inhibit platelet aggregation. Platelets were activated with 10
µg/ml NNKY1-19 for the indicated periods. The reaction was
terminated with lysis buffer, and p72 was isolated by
immunoprecipitation with anti-p72 MoAb. Immunoprecipitates were either
directly subjected to Western blotting using anti-phosphotyrosine MoAb
or to in vitro kinase assay using enolase as exogenous
substrate. Arrowheads represent the bands presumably derived
from IgG. A, Western blotting using anti-phosphotyrosine MoAb; B, in vitro kinase assay.
When
platelets were incubated with 0.4 µM PGI for 5
min, the profile of changes in the
p72
-associated tyrosine kinase activity was
similar to that observed without aggregation. The elevated level of
tyrosine kinase activity persisted even at 5 min after activation, and
the association of p60
with
p72
as assessed by Western blotting showed no
phase of dissociation (Figs. 9 and 10).
II receptor
stimulation. F(ab`)
fragments that lack the ability to
stimulate the Fc
II receptor did not induce
[Ca
]
elevation or
release of intracellular granule contents (Worthington et al.,
1990). PTP induced by anti-CD9 MoAb was inhibited by preincubation of
the platelets with anti-FcRII MoAb (Huang et al., 1992). Based
upon the fact that direct Fc
II receptor stimulation, such as that
caused by the cross-linkage of the receptors, is sufficient to induce
platelet activation, it has been doubted whether the binding of
anti-CD9 MoAb to CD9 molecules on the platelet membrane by itself
elicits any signal that leads to platelet activation. It could only
serve to prompt the binding of the Fc portion of MoAb to the Fc
II
receptor. This study confirmed, using NNKY1-19, an anti-CD9 MoAb
(Nagata et al., 1990), the previous findings that the
binding of MoAb to CD9 in the absence of Fc
II receptor stimulation
did not induce protein tyrosine phosphorylation or platelet aggregation
but also showed that F(ab`)
fragments of NNKY1-19 per se elicited intracellular signals in terms of
p72
activation and the association of
p60
with
p72
. The effect of residual intact antibody was
ruled out by the use of IV.3, anti-Fc
II receptor MoAb.
Furthermore, that the F(ab`)
fragments of another anti-CD9
MoAb, PMA2 (Hato et al., 1990), are capable of
activating p72
reinforces the notion that
antibody binding to CD9 alone can elicit a signal(s) leading to
p72
activation. Griffith et al.(1991)
have proposed that CD9 molecules induce an activation signal in
platelets, based upon their study with immobilized anti-CD9 MoAb F(ab`)
fragments. The association between CD9 and glycoprotein IIb/IIIa in
platelets (Slupsky et al., 1989) and that with CD9 and the
diphtheria toxin receptor in Vero cells (Mitamura et al.,
1992) implies that CD9 molecules exert a regulatory function for
various cell surface receptors. Our findings may provide a biochemical
basis for the proximal signal that CD9 molecules generate within
platelets. Several non-receptor tyrosine kinases have been found to
associate with cell surface molecules (Samelson et al., 1990;
Hatakeyama et al., 1991). A more recent finding provides
evidence for the close association between B-cell antigen receptor and
p72
(Yamada et al., 1993). In
platelets, p59
, p62
, and
p54/58
associate with CD36 (Huang et
al., 1991). We have found that p72
specifically associates with p60
during platelet activation induced by CD9 MoAb. By analogy
to other cell types, it is tempting to postulate that CD9 or some other
related membrane protein generates a signal(s) to recruit
p72
and p60
.
Whether CD9 actually localizes these tyrosine kinases awaits further
investigation.
-associated tyrosine kinase activity along
with association between p72
and
p60
induced by F(ab`)
fragments, however, does not suffice to induce protein tyrosine
phosphorylation or other parameters of platelet activation. These
findings suggest that an additional signal(s) mediated by Fc
II
receptor occupancy is required for the full picture of platelet
activation. PGI
pretreatment, which elevates the
intracellular cAMP content, completely inhibited the appearance of PTP
and aggregation induced by intact IgG of anti-CD9 MoAb, whereas
p72
activation and association between
p72
and p60
was preserved. The reduction in
p72
-associated tyrosine kinase activity and the
apparent dissociation of p60
from
p72
, which occur 1-3 min after activation
with intact IgG of anti-CD9 MoAb, was suppressed by PGI
treatment. Thus, PGI
reverted the overall profile of
intact IgG anti-CD9 MoAb-induced activation to one similar to that with
F(ab`)
fragments. Taken together, these findings suggest
that cAMP acts at a step distal to the activation of p72
and association between p72
and
p60
and that intracellular cAMP
accumulation facilitated by PGI
must act at a particular
step of the signal transduction pathway that links Fc
II receptor
activation to PTP and the dissociation of p72
and p60
.
-associated
tyrosine kinase activity and association between p72
and p60
are transient,
lasting 30-60 s at most. F(ab`)
fragments lacking the
Fc portion led to the persistent activation of p72
and association between the two tyrosine kinases. These
findings suggest that Fc
II receptor stimulation induces the
apparent dissociation between p72
and
p60
and the reduction in
p72
-associated tyrosine kinase activity. Calcium
influx or the thromboxane pathway do not play a decisive role in these
processes, although they may support them to some extent; the exclusion
of either or both of these pathways did not suppress p72
activation or the association between P72
and p60
but slightly
suspended the dissociation and the decay of
p72
-associated tyrosine kinase activity. On the
other hand, aggregation appears to play a key role on the apparent
dissociation between p72
and
p60
and the reduction in
p72
-associated tyrosine kinase activity. A
number of kinases including p60
and
substrates colocalize to the cytoskeleton in an aggregation-dependent
manner (Grondin et al., 1991; Clark and Brugge, 1993) and
therein transmit signals to the target proteins. p72
also translocates to the cytoskeletal fractions upon
platelet activation (Yanagi et al., 1994). It is plausible
that the associated form of p60
and
p72
translocates to the cytoskeletal fraction,
thus reducing the recovery of p60
associated with p72
, although, to
date, we are not able to demonstrate that the associated form of
p72
and p60
colocalizes with the cytoskeleton (data not shown).
and terminate the association between
p72
and p60
.
The recovery of p72
in anti-p72
MoAb immunoprecipitates after NNKY 1-19 stimulation
was virtually the same as that of the resting state (data not shown). A
portion of activated p72
associated with the
cytoskeleton cannot totally explain the magnitude of the reduction in
p72
-associated tyrosine kinase activity,
although we lack accurate stoichiometry. The rapid activation of
p72
and subsequent deactivation within 60 s
after stimulation has been noted with platelet activation induced by
thrombin and thromboxane A
mimetics (Taniguchi et
al., 1993; Maeda et al., 1993). It is likely that
p72
is actually deactivated during the course of
NNKY 1-19 activation, which is mediated by Fc
II receptor
stimulation and dependent upon aggregation.
has been suggested for Fc receptor signaling
with HL60 and macrophages (Agarwal et al., 1993; Greenberg et al., 1994) and for Fc
receptor stimulation with a mast
cell line (Benhamou et al., 1993). Direct association between
p72
and subunits of Fc receptors may play an
important role in signal transduction mediated by Fc
I receptors
(Shuie et al., 1995). With platelets, cross-linking of
Fc
II receptors or aggregated IgG induces PTP and other parameters
of platelet activation (Anderson and Anderson, 1990; Huang et
al., 1992; Kang et al., 1993). We have found that
cross-linking of Fc
II receptors induces tyrosine phosphorylation
of p72
and elevates the
p72
in vitro kinase activity, which
suggests that p72
is also involved in the direct
stimulation of the Fc
II receptor. However, we have also found that
a tyrosine kinase distinct from p72
copurifies
with Fc
II receptors, which does not take place in platelet
activation induced by anti-CD9 MoAb.
(
)Thus, we
are inclined to believe that Fc
II receptor cross-linking differs
from anti-CD9 MoAb in the mode of platelet activation. Cross-linking of
Fc
II receptors may provide a signal(s) other than simple Fc
receptor occupancy by restricting the free movement of bound receptors
and initiating cytoskeletal reorganization. Based upon these findings,
we suggest that platelet activation induced by anti-CD9 MoAb involves
two separate signals, one generated by the binding of anti-CD9 MoAb to
CD9 molecules leading to the activation of p72
and the association between p72
and
p60
, and another that is mediated
by the Fc
II receptor. The former alone results in persistent
elevation of p72
activation and association
between p72
and p60
but does not lead to the full picture of platelet
activation. The latter generates a signal(s) that is inhibitable by
intracellular cAMP elevation, and in synergy with CD9 activation it
leads to platelet activation in terms of
[Ca
]
elevation and
aggregation. Whether the latter also involves p72
activation awaits to be clarified.
,
prostaglandin I
; PTP, protein tyrosine phosphorylation;
PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.