Direct Demonstration of Involvement of Protein Kinase C
in the Ca2+-induced Platelet Aggregation*
Arata Tabuchi,
Akira Yoshioka
,
Tomohito Higashi,
Ryutaro Shirakawa,
Hiroaki Nishioka
,
Toru Kita ¶ and
Hisanori Horiuchi ||
From the
Department of Geriatric Medicine and
¶Cardiovascular Medicine, Graduate School of
Medicine, Kyoto University, 606-8507 Kyoto, Japan
Received for publication, December 5, 2002
, and in revised form, March 31, 2003.
 |
ABSTRACT
|
---|
Platelets play critical roles in hemostasis and thrombosis through their
aggregation following activation of integrin
IIb
3. However, the molecular mechanism of
the integrin activation inside platelets remains largely unknown.
Pharmacological experiments have demonstrated that protein kinase C (PKC)
plays an important role in platelet aggregation. Because PKC inhibitors can
have multiple substrates and given that non-PKC-phorbol ester-binding
signaling molecules have been demonstrated to play important roles, the
precise involvement of PKC in cellular functions requires re-evaluation. Here,
we have established an assay for analyzing the
Ca2+-induced aggregation of permeabilized platelets. The
aggregation of platelets was inhibited by the addition of the
arginine-glycine-aspartate-serine peptide, an integrin-binding peptide
inhibitor of
IIb
3, suggesting that the
aggregation was mediated by the integrin. The aggregation was also dependent
on exogenous ATP and platelet cytosol, indicating the existence of essential
cytosolic factors required for the aggregation. To examine the role of PKC in
the aggregation assay, we immunodepleted PKC
and
from the
cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity,
which was recovered by the addition of purified PKC
. Furthermore, the
addition of purified PKC
in the absence of cytosol did not support the
aggregation, whereas the cytosol containing less PKC supported it efficiently,
suggesting that additional factors besides PKC would also be required. Thus,
we directly demonstrated that PKC
is involved in the regulation of
Ca2+-induced platelet aggregation.
 |
INTRODUCTION
|
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Platelets play critical roles in hemostasis and thrombosis through
aggregation following activation of integrin
IIb
3
(13).
Although
IIb
3 of platelets in the resting
stage does not bind fibrinogen or von Willebrand factors
(vWF),1 the activation
of platelets results in conformation changes, which allow
IIb
3 to bind these ligands
(13).
When fibrinogen or vWF binds to
IIb
3, the
ligand-occupied integrin signals downstream to stabilize platelet aggregation
through reorganization of the actin cytoskeletal network and the release of
bioactive substances stored in the granules
(13).
Thus, the process of platelet activation and aggregation consists of a series
of orchestrated responses
(13).
However, the molecular mechanisms that underlie this process remain unclear
because it is difficult to use molecular biology and biochemistry in platelets
that do not synthesize new proteins. To overcome this difficulty, semi-intact
assays using permeabilized platelets have been established for the study of
granule secretion
(48).
Although some semi-intact aggregation assays have now been developed, it has
been difficult to demonstrate a cytosol dependence in these experiments
(8).
Protein kinase C (PKC) family members are important signaling molecules
regulating many cellular functions
(9,
10). Among the family members,
conventional PKCs (cPKC), which include PKC
,
I,
II, and
, have regulatory Ca2+- and phorbol ester-binding
domains (9,
10). The involvement of cPKCs
in the cellular functions has been analyzed mainly pharmacologically using
cell-permeable small compounds of inhibitors and stimulators such as phorbol
esters. However, these experiments are somewhat indirect because 1) no
inhibitors have absolute specificity and 2) important signaling molecules
other than PKCs have also been demonstrated to contain phorbol ester-binding
C1-domains, which were first identified in cPKC
(9,
10). For example, Ras-guanyl
nucleotide-releasing protein
(11,
12) contains the C1 domain at
its C terminus and acts as a stimulator for small GTPase Ras involved in the
regulation of cell growth. Chimerin
(13,
14) also contains a C1 domain
and acts as a GTPase-activating protein of small GTPase Rac involved in the
regulation of cytoskeletal reorganization. Thus, the effects of phorbol esters
could be through multiple pathways. Therefore, it is important to re-evaluate
and demonstrate the involvement of PKCs in certain cellular functions in a
direct fashion (15).
In platelets, PKC has been considered to play important roles in
aggregation (16,
17) and granule secretion
(18). For the granule
secretion, we have recently demonstrated the direct involvement of PKC
(19). We have established a
semi-intact secretion assay
(19,
20) where the secretion does
not occur upon stimulation without adding exogenous cytosol, indicating the
existence of cytosolic essential factors. We purified an essential factor for
the secretion and identified it to be PKC
(19). On the other hand, it
has been so far difficult to demonstrate the involvement of PKC in platelet
aggregation without using small compounds of PKC inhibitors or phorbol esters.
Here, we directly demonstrate the involvement of PKC
in the regulation
of platelet aggregation by using a stable semi-intact aggregation assay with
cytosol dependence.
 |
EXPERIMENTAL PROCEDURES
|
---|
MaterialsAnti-PKC
and anti-pan-PKC
mouse monoclonal antibodies were purchased from Transduction Laboratories.
This anti-PKC
antibody, which was described in the manufacturer's
instruction to interact with PKC
but not with other PKCs, was used for
immunodepletion experiments. Another anti-PKC
mouse monoclonal antibody
purchased from Santa Cruz Biotechnology had no cross-reactivity with PKC
and was used for the immunoblotting. Anti-PKC
, anti-PKC
, and
anti-PKC
mouse monoclonal antibodies were from Transduction
Laboratories, and anti-PKC
I and anti-PKC
II rabbit polyclonal
antibodies were from Santa Cruz Biotechnology. A control mouse IgG used for
the immunodepletion experiment was from Zymed Laboratories Inc. Horseradish
peroxidase-labeled anti-mouse and anti-rabbit IgG polyclonal antibodies were
from Amersham Biosciences, which were used as secondary antibodies for
immunoblotting visualized by enhanced chemiluminescence method (Amersham
Biosciences). Unless otherwise specified, all of the chemicals including
peptides of arginine-glycine-aspartate-serine (RGDS) and
arginine-glycine-glutamate-serine (RGES) were purchased from Sigma with the
exception of SLO, which was from Dr. Bhakdi (Mainz University, Mainz,
Germany). Protein concentrations were determined by the Bradford method (Bio
Rad) (21) or densitometric
scanning of the Coomassie Blue-stained band in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis
(22) using bovine serum
albumin as a standard.
The Aggregation Assay with Permeabilized PlateletsThe
aggregation assay was established by modification of our previous assay
(23). Unless otherwise
specified, the standard assay method was used as follows. Washed human
platelets from healthy donors were prepared
(24), resuspended in ice-cold
Buffer A (50 mM Hepes/KOH, pH 7.2, 78 mM KCl, 4
mM MgCl2, 0.2 mM CaCl2, 2
mM EGTA, 1 mM dithiothreitol, and the calculated free
[Ca2+] was
20 nM
(25)) containing 4 mg/ml
bovine serum albumin and 20 ng/ml prostaglandin E1 and kept at 4
°C. The platelets were incubated with 0.6 µg/ml SLO at 4 °C for 10
min and washed once to remove unbound SLO
(19,
20,
26,
27), The treated platelets
were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin
at a density of 5 x 108/ml, quantified with a Coulter
Counter, and incubated at 30 °C for 5 min to make holes in their plasma
membrane (19,
20,
26,
27). The permeabilized
platelets were kept on ice for 1530 min with 2 mg of proteins/ml human
platelet cytosol (or rat brain cytosol), an ATP-regenerating system
(19,
20,
26,
27), and tested substances.
Because fibrinogen has been added in previously established aggregation assays
using washed platelets at 0.4 mg/ml by Kinlough-Rathbone et al.
(28) and 0.38 mg/ml by Santos
et al. (29), we also
added fibrinogen (Sigma) in our assay at the concentration of 0.4 mg/ml. The
platelets then were incubated at 37 °C for 3 min and stimulated by the
addition of calcium chloride to make final [Ca2+] at 200
µM (25). The
aggregation was measured by a light transmission aggregometer, MCM HEMA TRACER
212 (MC Medical). For the morphological analysis, permeabilized platelets were
incubated with 200 µM or 20 nM
Ca2+ for 20 min in the standard assay condition. The
samples then were immediately examined with a phase-contrast microscopy.
Cytosol PreparationFor generation of platelet cytosol,
platelets from healthy donors were washed and resuspended in ice-cold Buffer A
containing a protease inhibitor mixture (Sigma). The platelets then were
sonicated, and the supernatant after the low speed centrifugation at 1000
x g for 10 min was further centrifuged at 100,000 x
g for 30 min. The final supernatant was dialyzed extensively with
Buffer A and kept as platelet cytosol at 80 °C until use. The
cytosol of rat brain was prepared in a similar way except using a Potter-type
blender instead of sonication. For generating the PKC-depleted cytosol, the
human platelet cytosol (0.4 mg of proteins) was incubated with protein
A-agarose beads (Roche Diagnostics) coated with 25 µg of the
anti-PKC
antibody (Transduction Laboratories) or the control mouse IgG
for 6 h at 4 °C. After the beads were removed by centrifugation, the
supernatants were used as the PKC-depleted cytosol and the control cytosol,
respectively. PKC
was purified from rat brain cytosol as described
previously (19).
 |
RESULTS
|
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Establishment of an Assay Analyzing the
Ca2+-induced Aggregation of Permeabilized
PlateletsWe have established an aggregation assay with platelets
permeabilized by SLO, which has been shown to form pores of
30 nm in
diameter in the membrane (27).
In this method, we permeabilized only the plasma membrane judging from
observations that the intracellular membrane structures of the platelets
appeared to be intact morphologically
(30) and that vWF stored in
-granules did not leak out
(19,
20). Because the condition
used here with 0.6 µg/ml SLO induced leakage of 80% cytosolic lactate
dehydrogenase from platelets
(19,
20), it was presumed that most
of ATP and cytosol were also lost by diffusion through the pores in the plasma
membrane. Therefore, we exogenously added ATP and cytosol in the assay to
reconstitute the aggregation. We also added 0.4 mg/ml fibrinogen in our assay,
which would bridge the activated integrin
IIb
3 on both sides of platelets to be
aggregated (2,
3). Because calcium ionophore
has been demonstrated to induce platelet aggregation
(31,
32), we used calcium ions at a
calculated concentration at 200 µM
(25) as a stimulus.
We first examined morphologically whether aggregates of permeabilized
platelets were indeed generated in the assay. After confirming the
Ca2+-induced aggregation by the light-transmission
aggregometer (data not shown), the samples were subjected to observation with
a phase-contrast microscopy. A typical set of photographs showed that many
platelet aggregates were formed upon stimulation with 200 µM
Ca2+, whereas the permeabilized platelets incubated with
20 nM Ca2+ remained unaggregated
(Fig. 1A). The
quantification of the platelets in the images revealed that unaggregated
platelets were reduced upon Ca2+ stimulation
(Fig. 1B) and that the
aggregates consisting of >10 platelets were drastically increased
(Fig. 1C). Thus, the
formation of the aggregates in the assay was confirmed morphologically.

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FIG. 1. Morphological examination of the Ca2+-induced
aggregation of permeabilized platelets. A, the permeabilized
platelets were incubated with 20 nM Ca2+
(Ca2+ stimulation ()) or 200 µM
Ca2+ (Ca2+ stimulation (+)) at 37
°C for 20 min and examined by phase-contrast microscopy (x1000) as
described under "Experimental Procedures." An arrowhead
and an arrow indicate an unaggregated platelet and an aggregate
consisting of >10 platelets, respectively. B and C, 20
individual fields of the sample after the incubation were randomly selected,
and numbers of unaggregated platelets (B) and aggregates consisting
of >10 platelets (C) were counted. The data shown are expressed as
means + S.E. of four independent experiments.
|
|
We next examined the effects of Ca2+ concentrations
on the platelet aggregation. Although Ca2+ at 20 and 200
nM did not induce the platelet aggregation,
Ca2+ at 2200 µM efficiently induced
the aggregation (Fig. 2).
Because it has been shown that [Ca2+] in resting
platelet cytosol is around 10 nM and that it increases to
110 µM upon platelet activation
(33), the
Ca2+ sensitivity of the aggregation in the assay using
permeabilized platelets was similar to physiological conditions.

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FIG. 2. The Ca2+ concentration-dependent aggregation of
the permeabilized platelets. Permeabilized platelets were first incubated
for 30 min at 4 °C with 2 mg of proteins/ml rat brain cytosol followed by
stimulation with indicated concentrations of Ca2+
(25) as described under
"Experimental Procedures." The data shown are the representative
of four independent experiments with similar results.
|
|
Aggregation of Permeabilized Platelets Was Mediated by the
IntegrinIt has been well known that platelet aggregation is
mediated by activated integrin
IIb
3
(2,
3). We examined whether it was
the case for the aggregation of permeabilized platelets. RGD is the
integrin-binding sequence
(34), which is present in both
fibrinogen and vWF, and the aggregation of intact platelets has been shown to
be inhibited by the addition of the RGD-containing peptide
(35,
36). As shown in
Fig. 3, the RGDS peptide
inhibited the Ca2+-induced aggregation of permeabilized
platelets, whereas the control RGES peptide did not. Furthermore, when
fibrinogen was omitted from the assay, the aggregation was also inhibited
(data not shown). Taken together, the aggregation in the assay was mediated by
the integrin.
Platelet Aggregation Was Cytosol-dependentATP and cytosol
in the permeabilized platelets would be lost by diffusion through the pores.
Without the addition of ATP, the permeabilized platelets did not aggregate
upon the Ca2+ stimulation (data not shown), indicating
that ATP is essential for the aggregation. When the cytosol was not added
exogenously into the assay, the permeabilized platelets did not aggregate upon
the Ca2+ stimulation in the condition where ATP and
fibrinogen were sufficiently supplemented
(Fig. 4A). On the
other hand, the aggregation was reconstituted by the addition of platelet
cytosol in a concentration-dependent manner
(Fig. 4A). These
results indicated the existence of cytosolic essential factor(s). We next
tested rat brain cytosol for the reconstitution of the aggregation. The rat
brain cytosol also supported the aggregation as efficiently as the human
platelet cytosol (Fig.
4B). These results indicated that cytosolic essential
factor(s) were expressed ubiquitously.

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FIG. 4. The Ca2+-induced aggregation was
cytosol-dependent. A, the permeabilized platelets were first
incubated for 15 min at 4 °C with indicated concentrations of platelet
cytosol, and the Ca2+-induced aggregation was analyzed
as described under "Experimental Procedures." B, the
permeabilized platelets were first incubated for 15 min at 4 °C with rat
brain cytosol or human platelet cytosol at 1.5 mg of proteins/ml, and the
Ca2+-induced aggregation was analyzed as described under
"Experimental Procedures." The data shown are the representative
of four independent experiments with similar results.
|
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Involvement of PKC in the Regulation of Platelet
AggregationThe cytosol dependence of the aggregation indicates
that some cytosolic factors are required for the platelet aggregation.
Although the identity of these factors is unknown, one important factor could
be cPKC. As shown previously with intact platelets
(37), GF109203X, an inhibitor
of cPKCs, also affected the Ca2+-induced aggregation in
our semi-intact assay in a concentration-dependent manner
(Fig. 5).

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FIG. 5. The Ca2+-induced aggregation was inhibited by
GF109203X, a cPKC inhibitor. The permeabilized platelets were first
incubated for 30 min at 4 °C with indicated concentration of GF109203X,
and the Ca2+-induced aggregation was then analyzed as
described under "Experimental Procedures." The data shown are the
representative of four independent experiments with similar results.
|
|
To examine the involvement of PKC directly, we first prepared PKC-depleted
platelet cytosol. As shown in Fig.
6A, PKC
was completely depleted from the platelet
cytosol with the anti-PKC
antibody-coated beads while PKC
stayed
in the cytosol after the same procedure with control IgG-coated beads. Among
other cPKCs, PKC
I and PKC
II were detected in platelets, whereas
PKC
, a neuronal specific cPKC, was not (data not shown). By the
immunodepletion, PKC
was also completely depleted because of the
cross-reactivity of the antibody (Fig.
6A). When we used lower amounts of the anti-PKC
antibody for the immunodepletion, PKC
was completely depleted while
PKC
still remained in the cytosol (data not shown), suggesting that
platelet cytosol contained more PKC
than PKC
. As expected,
although PKC
and PKC
, both of which are classified as novel
PKCs, were detected in platelets, they were not affected by the
immunodepletion procedure either with anti-PKC
antibody or control IgG
(Fig. 6A).
The PKC-depleted cytosol lost the aggregation activity, whereas the
Ca2+-induced platelet aggregation was
efficiently reconstituted with the cytosol treated with the control IgG
(Fig. 6C). When
PKC
purified from rat brain (Fig.
6B) (19)
was supplemented to the PKC-depleted cytosol, the aggregation activity was
recovered (Fig. 6C),
indicating that cPKC, possibly PKC
, is an essential cytosolic factor
for the platelet aggregation. We next examined whether PKC
is a
sufficient cytosolic factor for the aggregation. As shown in
Fig. 7, purified PKC
(50
nM) alone was not sufficient to support the
Ca2+-induced platelet aggregation. On the other hand,
platelet cytosol at 0.6 mg of proteins/ml, which contained 15 nM
PKC
determined by Western blot with the anti-PKC
antibody using
purified PKC
as a control (data not shown), efficiently induced
platelet aggregation (Fig. 7).
Thus, PKC
is not a sufficient cytosolic factor for platelet
aggregation. Furthermore, the addition of purified PKC
to the low
concentration of cytosol (0.6 mg of proteins/ml) strongly enhanced the
platelet aggregation, suggesting that PKC
is a limiting factor for the
Ca2+-induced platelet aggregation. Thus,
PKC
is an essential but not sufficient cytosolic factor for the
Ca2+-induced platelet aggregation.
 |
DISCUSSION
|
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Here we established an aggregation assay system using permeabilized
platelets, and using this assay, we first directly demonstrated that PKC is an
essential but not sufficient factor in the cytosol for platelet
aggregation.
Because platelets lack the protein-producing activity, it is difficult to
apply molecular biology for investigating the molecular mechanism of
aggregation and granule secretion inside activated platelets. Therefore, the
research in these fields has been performed mainly pharmacologically. To
overcome this difficulty, much effort has been made to establish semi-intact
assay systems using permeabilized platelets. In the research of platelet
granule secretion, several semi-intact assays have been established
(48,
19,
20). However, for platelet
aggregation, only a few semi-intact aggregation assays have been established
(8), and as far as we know, no
stable assays with cytosol dependence have been established.
We have previously established an aggregation assay system with
SLO-permeabilized platelets and demonstrated that small GTPase Rho plays an
important role in thrombin-induced aggregation
(23). However, the assay did
not demonstrate cytosol dependence since a low concentration of SLO (0.1
µg/ml) was used for the permeabilization and the aggregation was induced
without adding exogenous cytosol
(23). Here, we have
established another semi-intact aggregation assay by modifying the previous
one (23). The aggregation of
the permeabilized platelets in our in vitro assay appears
physiological since the time course, the Ca2+
sensitivity and the involvement of the integrin are similar to those of intact
platelets. Since the cytosol was extensively depleted from the permeabilized
platelets in our semi-intact aggregation assay using a higher concentration of
SLO than that used previously
(23), permeabilized platelets
did not aggregate in response to calcium stimulation without adding exogenous
cytosol. This cytosol dependence would widen the application of the assay to
investigate the molecular mechanism of platelet aggregation.
The cytosol dependence also indicated the existence of essential cytosolic
factor(s) for aggregation. A cytosolic protein PKC has been shown to play an
important role in platelet aggregation by pharmacological experiments using
cell-permeable small compounds of inhibitors and stimulators such as phorbol
esters (16,
17). However, the results
obtained from such experiments appear somewhat indirect because the
specificity of inhibitors is not absolutely strict and important signaling
molecules containing the phorbol ester-binding C1 domain other than PKC have
been recently identified such as Ras-guanyl nucleotide-releasing protein
(11,
12) and chimerin
(13,
14). Munc13-1 present in the
presynapse also contains the C1 domain
(38), and it has very recently
been demonstrated that the effect of phorbol ester in the neurotransmitter
release is through Munc13-1
(39). Thus, at the moment, it
is ambiguous whether phorbol esters exert their functions through PKC or other
non-PKC-signaling molecules. Therefore, re-evaluation and direct demonstration
are required in various cell functions where PKCs have been suggested to be
involved (15,
43).
Using the semi-intact aggregation assay, we have directly demonstrated the
involvement of PKC
in the Ca2+-induced platelet
aggregation. First, an inhibitor of conventional PKC affected the aggregation.
Second, immunodepletion of PKC
and PKC
from the cytosol abolished
the Ca2+-induced aggregation. Third, the
aggregation-supporting activity of PKC
/
-depleted cytosol was
rescued by supplementation of purified PKC
. Supplementation of
PKC
alone to the PKC
/
-depleted cytosol was enough to
reconstitute the aggregation, suggesting that PKC
but not PKC
is
the essential factor or otherwise that the activity of cPKC, namely PKC
or PKC
, is essential. Because PKC
and PKC
show similar
substrate specificity in vitro
(40), we cannot exclude a
possibility that added PKC
covered the lack of PKC
activity in
the assay. Although cPKC is an essential factor for the aggregation, it is not
a sufficient cytosolic factor since the addition of purified PKC
alone
without exogenous cytosol did not support the aggregation. Furthermore,
platelet cytosol containing less PKC supported the aggregation efficiently,
and purified PKC
strongly enhanced the aggregation in the presence of
low concentration of cytosol, suggesting that PKC
is a limiting factor
in the cytosol for the aggregation and that additional factors besides PKC
would also be required for the aggregation.
PKC
and PKC
are known to be activated by
Ca2+, diacylglycerol, and phosphatidylserine
(9,
10). Although we did not add
these stimulators besides Ca2+, the purified PKC
added to the assay was indeed active since the purified PKC
phosphorylated a PKC substrate efficiently in the similar assay condition used
here (19). We speculate that
the components inside the platelets, possibly including phosphatidylserine,
help support the activity of PKC
(19). Furthermore, because PKC
increases the intracellular Ca2+ concentration by
modulating Ca2+ channels in the plasma membrane in
neurons (41,
42), it remains unclear
whether PKC acts upstream and/or downstream of increased
Ca2+. Because we used Ca2+ as a
stimulus, we could safely say that PKC
plays an important role at least
at the downstream of increased Ca2+. Further
investigation is required for elucidation of how PKC
activates the
integrin
IIb
3 and induces platelet
aggregation. The assay established here will be a powerful tool for future
experiments aimed at elucidating these mechanisms.
 |
FOOTNOTES
|
---|
* This work was supported by Research Grants from Ministry of Education,
Science, Sports, and Culture of Japan (Grants 15590740 (to H. H.) and 12CE2006
and 13307034 (to T. K.)), Research Grants from Ministry of Health, Labor, and
Welfare of Japan (Comprehensive Research on Aging and Health Grant
H14-choju-012 (to T. K. and H. H.)), and partially by grants from Takeda
Science Foundation, Suzuken Memorial Foundation, Study Group of Molecular
Cardiology and Novartis Foundation for Gerontrogical Research (to H. H.). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Present address: Dept. of Internal Medicine, Mitsubishi Kyoto Hospital,
615-8087 Kyoto, Japan. 
Present address: Sir William Dunn School of Pathology, University of
Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom. 
||
To whom correspondence should be addressed. E-mail:
horiuchi{at}kuhp.kyoto-u.ac.jp.
1 The abbreviations used are: vWF, von Willebrand factors; PKC, protein
kinase C; cPKC, conventional protein kinase C; SLO, streptolysin-O; RGDS,
arginine-glycine-aspartate-serine; RGES, arginine-glycineglutamate-serine. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. H. McBride and Dr. H. Arai for the critical reading
of the paper and to T. Matsubara for excellent technical assistance.
 |
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