From the Division of Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, September 7, 2000, and in revised form, April 6, 2001
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ABSTRACT |
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To understand the molecular basis of
granule release from platelets, we examined the role of
phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)
in Phospholipid metabolism has long been regarded as an essential
component of regulated secretion in platelets. Initial studies performed in intact (19) and electropermeabilized (20) platelets indicated a role for the products of phosphatidylinositol-specific PLC,
diacylglycerol and inositol 1,4,5-trisphosphate, in stimulating granule
secretion (21). However, both dense and Phosphoinositide phosphorylation has been invoked in vesicle fusion
models and shown to mediate granule secretion. Initial observations in
chromaffin cells demonstrated that the maintenance of
polyphosphoinositides is crucial for vesicle secretion (26). Subsequent
studies revealed a PtdIns 4-kinase activity associated with granules in
chromaffin, mast, and pancreatic Although the role of PtdIns(4,5)P2 itself in platelet
granule secretion has not previously been evaluated,
PtdIns(4,5)P2 metabolism has been studied in platelets, and
PtdIns(4,5)P2 has been shown to mediate several other
platelet processes. PtdIns(4,5)P2 is involved in uncapping
of F-actin barbed ends, thereby contributing to platelet shape change
(33). PtdIns(4,5)P2 is also thought to mediate platelet
spreading (34). Upon platelet activation with thrombin (35, 36) or
thrombin receptor agonist peptides (33), PtdIns(4,5)P2
increases 10-40% above base-line levels. The generation of
PtdIns(4,5)P2 is presumed to be mediated by up-regulation
of PtdIns kinases. However, the synthetic pathway by which
PtdIns(4,5)P2 is synthesized in platelets is not well established. Synthesis of PtdIns(4,5)P2 via progressive
phosphorylation of PtdIns by PtdIns 4-kinase followed by
PtdIns-4-phosphate 5-kinase represents the dominant pathway in most
cell types. However, platelet PtdIns 5-phosphate levels increase upon
exposure to thrombin (37). In addition, type II PIPK is abundant in
platelets (38) and undergoes aggregation-dependent
translocation to the platelet cytoskeleton (39). These observations
raise the possibility that PtdIns(4,5)P2 can be
synthesized via a PtdIns-5-phosphate 4-kinase-dependent
pathway. In this study, we demonstrate that PtdIns(4,5)P2
mediates platelet Chemicals and Reagents--
All buffer constituents, solvents,
ATP, CaCl2, PtdIns, PtdIns 4-phosphate,
PtdIns(4,5)P2, and PtdIns-specific PLC (from Bacillus cereus) were purchased from Sigma. Sepharose 2B was obtained from Amersham Pharmacia Biotech. Reduced SL-O was purchased from Corgenix (Peterborough, England). Wortmannin and LY294002 were purchased from
Calbiochem. Phycoerythrin-conjugated anti-P-selectin antibody AC1.2 was
purchased from Becton Dickinson (San Jose, CA).
[32P]Orthophosphoric acid was obtained from PerkinElmer
Life Sciences. Silica Gel G TLC plates were obtained from Whatman Ltd.
(Kent, England). GST-tagged type II PIPK was generously provided by Dr. Lucia Rameh. All solutions were prepared using water purified by
reverse-phase osmosis on a Millipore Milli-Q purification water system.
Antibodies--
Affinity-purified mouse monoclonal antibodies to
PtdIns 3-kinase p85 Platelet Preparation--
Blood from healthy donors who had not
ingested aspirin in the 2 weeks prior to donation was collected by
venipuncture into 0.4% sodium citrate. Citrate-anticoagulated blood
was centrifuged at 200 × g for 20 min to prepare
platelet-rich plasma. Platelets were then purified from platelet-rich
plasma by gel filtration using a Sepharose 2B column equilibrated in
PIPES/EGTA buffer (25 mM PIPES, 2 mM EGTA, 137 mM KCl, 4 mM NaCl, and 0.1% glucose, pH 6.4).
Final gel-filtered platelet concentrations were 1-2 × 108 platelets/ml.
Permeabilization of Platelets--
Platelets were permeabilized
using reduced SL-O. The ability of each batch of SL-O to permeabilize
platelets was tested by analyzing for incorporation of fluorescein
isothiocyanate-dextran sulfates by flow cytometry as described
previously (12, 40). Briefly, gel-filtered platelets (20 µl) were
incubated with 25 µM fluorescein isothiocyanate-dextran
sulfate of various molecular masses in the presence or absence of SL-O
for 15 min. Platelets were subsequently analyzed for fluorescence by
flow cytometry. An increase in fluorescence in the samples exposed to
SL-O compared with non-permeabilized samples confirmed permeabilization.
Analysis of P-selectin Surface Expression--
For analysis of
P-selectin surface expression from permeabilized platelets, 20 µl of
gel-filtered platelets (1-2 × 108/ml) in the
indicated concentration of MgATP were incubated with the concentrations
of reduced SL-O indicated in the figure legends. Samples were adjusted
to pH 6.9 immediately following permeabilization. The timing of
addition of inhibitors varied according to the inhibitor that was being
evaluated and is indicated in the figure legends. Following the
incubation with inhibitor, CaCl2 was added to the reaction
mixture. The amount of CaCl2 required to give a free Ca2+ concentration of 10 µM in the presence
of 2 mM EGTA at pH 6.9 was calculated for each condition
using a computer program (gift from Dr. P. J. Padfield) based on
the algorithms described by Fabiato and Fabiato (41). Following an
additional incubation after the addition of Ca2+, 10 µl
of reaction mixture were transferred to 5 µl of
phycoerythrin-conjugated anti-P-selectin antibody AC1.2.
Phosphate-buffered saline (500 µl) was added to the sample after a
20-min incubation, and the platelets were analyzed immediately by flow
cytometry as described below.
Immunoblot Analysis--
Gel-filtered platelets (1-2 × 107/ml) or GST-tagged PIPK was diluted in sample buffer
(62.5 mM Tris-HCl, 2% SDS, 0.5% Analysis of Platelet Phosphoinositides--
Gel-filtered
platelets (350 µl/sample) were incubated in the presence of 2 mCi/ml
[32P]orthophosphoric acid at 37 °C for 2 h.
Unbound 32P was separated from platelets by sequential
centrifugation. Platelets were then permeabilized with the indicated
concentrations of SL-O in the presence or absence of ATP. Samples were
adjusted to pH 6.9 immediately following permeabilization. The timing
of addition of inhibitors varied according to the inhibitor and is
indicated in the figure legends. Platelets were subsequently
solubilized in 250 µl of a 20:40:1 solution of chloroform, methanol,
and 12 N HCl and mixed vigorously. Chloroform (75 µl) was
added to this solution. The solution was mixed vigorously, and its
phases were separated at 1300 × g for 10 min at
4 °C. The upper phase was discarded, and the lower phase was washed
three times with 625 µl of the 20:40:1 solution of chloroform,
methanol, and 12 N HCl. Each extract (5 µl) was applied
to one lane of a 20 × 20-cm Silica Gel G plate previously soaked
in 1% potassium oxalate for 30 min and then baked for 5 min at
100 °C. Phospholipids were separated by chromatography in 1-propanol
and 2 M acetic acid (65:35). The location of PtdIns
4-phosphate and PtdIns(4,5)P2 was determined by applying 5 µg of known standards to each lane. Standards were detected by
staining in a saturated iodine tank. The radioactivity on the plates
was detected using a Bio-Rad GS-525 molecular imaging system.
Phosphoinositide levels were quantified both by calculating the pixel
density of bands comigrating with PtdIns standards using Molecular
Analyst software (Bio-Rad) and by extracting the radioactivity comigrating with the PtdIns standards from the plate and quantifying radioactivity using a Tri-Carb 2100TR liquid scintillation analyzer (Packard Instrument Co.). Under the conditions of our assay, a pixel
density of 1 corresponds to 10 cpm.
Flow Cytometry--
Flow cytometry was performed on gel-filtered
platelet samples using a Becton Dickinson FACSCalibur flow
cytometer. Fluorescent channels were set at logarithmic gain. 10,000 particles were acquired for each sample. A 530/30-nm band-pass
filter was used for FL-2 fluorescence. Phycoerythrin was measured in
the FL-2 channel. Data were analyzed using CellQuest
software on a Macintosh Power PC.
SL-O-permeabilized Platelets Synthesize
PtdIns(4,5)P2--
To study the molecular mechanisms of
platelet
The observation that ATP, but not platelet cytosol, is required for
Ca2+-induced Effect of PtdIns-specific PLC on Ca2+-induced
Effect of Exogenous PtdIns(4,5)P2 on
Ca2+-induced Effect of Wortmannin, LY294002, and Anti-PtdIns 3-Kinase Antibody
on Ca2+-induced
Experiments performed in the presence of increasing concentrations of
wortmannin demonstrated that wortmannin inhibited
Ca2+-induced Effect of Anti-type II PIPK Antibodies on Ca2+-induced
To assess the possibility that wortmannin and type II
PIPK inhibit Ca2+-induced One advantage of permeabilized secretory systems is that
target-specific, cell-impermeable inhibitors such as antibodies and enzymes can be introduced intracellularly. This characteristic permits
for more precise identification of molecules directly involved in the
secretory process. The fact that Ca2+-induced A second advantage of permeabilized secretory systems is that they
allow for the dissection of the secretory process into component parts.
Like other permeabilized cells, platelets will undergo
Ca2+-mediated secretion only following exposure to ATP (42,
43, 46). SL-O-permeabilized platelet systems differ from many
permeabilized cell systems, however, in that Ca2+-induced
granule secretion from platelet systems does not exhibit a requirement
for exogenous cytosolic proteins (12, 45, 52). The observation that
PtdIns(4,5)P2 synthesis occurs in SL-O-permeabilized platelets in the absence of exogenous cytosol suggests that some PtdIns(4,5)P2 synthetic machinery remains cell-associated
in this permeabilized secretory system. In our studies, inhibitors of PtdIns(4,5)P2 synthesis inhibited Previous studies of hematopoietic cells have implicated PtdIns
3-kinase in the process of regulated granule exocytosis. For example,
nanomolar concentrations of wortmannin inhibit granule secretion from a
basophilic leukemia cell line (RBL-2H3) (49) and from natural killer
cells (50). However, the fact that nanomolar concentrations of
wortmannin, 33.3 µM LY294002, and antibodies directed at
PtdIns 3-kinase p85 In addition to demonstrating that PtdIns(4,5)P2 is
synthesized in SL-O-permeabilized platelets, these studies demonstrate that the type II PIPK-dependent pathway is involved in the
synthesis of PtdIns(4,5)P2 required for
Ca2+-induced The fact that exposure of SL-O-permeabilized platelets to micromolar
concentrations of wortmannin results in a 50% reduction of both
PtdIns(4,5)P2 synthesis and Ca2+-induced,
ATP-dependent How might PtdIns(4,5)P2 mediate -granule secretion. Streptolysin O-permeabilized platelets
synthesized PtdIns(4,5)P2 when incubated in the presence of
ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced
PtdIns(4,5)P2 levels and resulted in a dose- and
time-dependent inhibition of Ca2+-induced
-granule secretion. Exogenously added PtdIns(4,5)P2 inhibited
-granule secretion, with 80% inhibition at 50 µM PtdIns(4,5)P2. Nanomolar concentrations of
wortmannin, 33.3 µM LY294002, and antibodies directed
against PtdIns 3-kinase did not inhibit Ca2+-induced
-granule secretion, suggesting that PtdIns 3-kinase is not involved
in
-granule secretion. However, micromolar concentrations of
wortmannin inhibited both PtdIns(4,5)P2 synthesis and
-granule secretion by ~50%. Antibodies directed against type II
phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate
4-kinase) also inhibited both PtdIns(4,5)P2 synthesis and
Ca2+-induced
-granule secretion by ~50%. These
antibodies inhibited
-granule secretion only when added prior to ATP
exposure and not when added following ATP exposure, prior to
Ca2+-mediated triggering. The inhibitory effects of
micromolar wortmannin and anti-type II phosphatidylinositol-phosphate
kinase antibodies were additive. These results show that
PtdIns(4,5)P2 mediates platelet
-granule secretion and
that PtdIns(4,5)P2 synthesis required for
Ca2+-induced
-granule secretion involves the type II
phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Granules are the dominant platelet secretory granules. They
contain many components that have been implicated in thrombosis and
atherosclerosis, including adhesion molecules, coagulation factors,
soluble mediators of inflammation, and growth factors (1). The
molecular mechanisms that mediate the release of these components are
not well defined. This secretory process is tightly controlled to
prevent unregulated release of thrombogenic factors. In many respects,
platelets represent a unique secretory system. They are anucleate,
contain an open canalicular system that undergoes eversion following
platelet activation (2), and demonstrate homotypic granule fusion (3).
Ultrastructural studies have shown that the
-granules of platelets
are secreted primarily via fusion with the surface-connected open
canalicular system (1) following apparent centralization of granules
and microtubule reorganization. Such observations have lead to
speculation that cytoskeletal reorganization is responsible for
-granule release. However, inhibition of actin polymerization (4) or
microtubule organization (5, 6) does not inhibit granule secretion. Furthermore, granule secretion and shape change can be dissociated under several experimental conditions (7-9). More recently,
SNARE1 proteins have been
demonstrated in platelets (10-12) and shown to mediate platelet
-granule secretion (12-15). Purified SNARE proteins are capable of
fusing lipid membranes in vitro (16, 17). However,
permeabilized cell systems require molecules other than SNARE proteins
to secrete granules. Furthermore, regulated secretion necessitates that
the SNARE protein apparatus respond to activation-dependent
signals (18). Thus, an understanding of the molecular mechanisms of
platelet granule secretion requires the identification of molecules
that are both modified upon platelet activation and capable of
interacting directly with the secretory machinery.
-granule secretion can be
stimulated in the absence of PLC activation, suggesting that PLC
activity is not essential to platelet granule secretion (22-24).
Subsequent studies in Ca2+-independent permeabilized
secretory systems showed that the generation of phosphatidic acid by
phospholipase D correlates closely with secretion (23, 24). Inhibition
of phospholipase D, however, fails to block
Ca2+-independent dense granule secretion, and phosphatidic
acid itself cannot stimulate dense granule secretion (25). These
observations support a modulatory role of phospholipase D in platelet
granule secretion. Thus, no product of phospholipid metabolism has been demonstrated to be fusogenic in platelets (25), and the role of
phospholipid metabolism in platelet membrane fusion remains to be defined.
-cells (27). In chromaffin cells,
this PtdIns 4-kinase activity correlates with granule secretion (28).
In PC12 cells, phosphatidylinositol transfer protein was determined to
be one of three cytosolic factors required to restore
Ca2+-induced secretion from semi-intact PC12 cells (29).
Type I PIPK is a second cytosolic factor necessary to reconstitute
secretion (30). Subsequent to these studies, phosphatidylinositol
transfer protein was found to reconstitute Ca2+-induced,
GTP
S-dependent granule secretion from SL-O-permeabilized HL-60 cells (31). In RBL-2H3 mast cells, ADP-ribosylation
factor-1 was demonstrated to stimulate exocytosis via
PtdIns(4,5)P2 synthesis (32). These data suggest a central
role for PtdIns(4,5)P2 in regulated secretion.
-granule secretion and that
PtdIns(4,5)P2 synthesis required for
-granule secretion
involves the type II PtdIns 5-phosphate 4-kinase-dependent pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(clone 8-2D-4D) were obtained from Lab Vision
(Fremont, CA). Affinity-purified rabbit anti-peptide polyclonal
antibodies to type II PIPK were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The anti-amino-terminal end antibody was raised
against a peptide consisting of the 19 amino-terminal amino acids of
type II
PIPK. The anti-carboxyl-terminal end antibody was raised
against a peptide consisting of the 18 carboxyl-terminal amino acids of type II
PIPK.
-mercaptoethanol, 10%
glycerol, and 0.01% bromphenol blue) at 95 °C for 5 min. Proteins were then separated by SDS-polyacrylamide gel electrophoresis on 12%
gels. Immunoblotting was performed using anti-type II PIPK antibodies
directed against the amino- and carboxyl-terminal ends of the
-isoform of human type II PIPK and visualized using enhanced chemiluminescence.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-granule secretion, we have developed and characterized an
SL-O-permeabilized, Ca2+-induced
-granule secretory
system (12). In this system, exposure of platelets to SL-O permits the
entry of molecules as large as 260 kDa (12). In the presence of 5 mM ATP, 96 ± 1% of platelets exposed to SL-O
expressed P-selectin on their surface in response to 10 µM Ca2+. In contrast, non-permeabilized
platelets expressed almost no P-selectin on their surface in response
to ATP and Ca2+ (Fig. 1)
(12). These observations demonstrate that >95% of platelets were
permeabilized following incubation with SL-O. Multiple Ca2+-induced permeabilized secretory systems, including
permeabilized platelet models (42, 43), have demonstrated a requirement for ATP exposure prior to Ca2+-induced triggering of
granule secretion. Many permeabilized secretory systems also require
cytosol to reconstitute significant secretion (44). A role for
PtdIns(4,5)P2 synthesis in PC12 cell granule secretion was
demonstrated when type I PIPK and PtdIns transfer protein were
identified as proteins within cytosol responsible for reconstituting
ATP-mediated priming of granule secretion (29, 30). In contrast,
cytosol is not required for Ca2+-mediated secretion of
-granules from SL-O-permeabilized platelets (12). However,
-granule secretion from SL-O-permeabilized platelets remained
ATP-dependent (Fig. 1). ATP did not support
-granule secretion from non-permeabilized platelets or under conditions in which
the free Ca2+ concentration was maintained at
pCa2+ < 8 with 2 mM EGTA (Fig.
1B). The ability of ATP to support
-granule secretion was
dose-dependent with an EC50 of ~1
mM. ADP neither supported
-granule secretion from
SL-O-permeabilized platelets nor augmented Ca2+-induced
-granule secretion in the presence of submaximal concentrations of
ATP (Fig. 1C). These data demonstrate that
Ca2+-induced
-granule secretion in this system requires
permeabilization with SL-O and exposure to ATP.
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Fig. 1.
Effect of ATP on
-granule secretion from SL-O-permeabilized
platelets. A, gel-filtered platelets (20 µl/sample)
in PIPES/EGTA buffer supplemented with 5 mM
MgCl2 were permeabilized with 4 units/ml SL-O in the
presence or absence of 5 mM ATP at pH 6.9 for 10 min.
Platelets were then exposed to 10 µM Ca2+ for
10 min. P-selectin surface expression was assayed by incubating the
sample (10 µl) with phycoerythrin-conjugated anti-P-selectin antibody
AC1.2 (5 µl) for 20 min. Phosphate-buffered saline (500 µl) was
added to the sample, and the platelets were analyzed immediately by
flow cytometry as described under "Experiment Procedures."
Histograms represent the distribution of the relative fluorescence of
10,000 platelets. B, gel-filtered platelets in PIPES/EGTA
buffer supplemented with 5 mM MgCl2 were
incubated in the presence or absence of 5 mM ATP as
indicated. Platelets were then incubated in the presence or absence of
4 units/ml SL-O at pH 6.9 for 10 min and subsequently exposed to buffer
or 10 µM Ca2+ for 10 min. Platelets were
assayed for P-selectin expression by flow cytometry. Error
bars represent the S.E. of three to seven independent experiments.
C, gel-filtered platelets in PIPES/EGTA buffer supplemented
with 5 mM MgCl2 were permeabilized with 4 units/ml SL-O in the presence of the indicated concentrations of ATP at
pH 6.9 for 10 min in the presence (
) or absence (
) of 500 µM ADP. Platelets were then exposed to 10 µM Ca2+ for 10 min and assayed for P-selectin
expression by flow cytometry. Error bars represent the S.E.
of six independent experiments.
-granule secretion raises the question of
whether SL-O-permeabilized platelets are able to synthesize
PtdIns(4,5)P2 upon exposure to ATP in the absence of added
cytosol. To address this question, platelets were radiolabeled with
[32P]orthophosphate, incubated in the presence or absence
of ATP, and analyzed for PtdIns(4,5)P2 levels by TLC.
PtdIns(4,5)P2 levels were markedly increased in
SL-O-permeabilized platelets incubated in the presence of ATP compared
with levels in platelets incubated in the absence of ATP (Fig.
2A). The contribution of
radioactivity from the 4 ± 1% of platelets that were not
permeabilized upon exposure to SL-O was calculated to constitute ~1%
of the total radioactivity in the PtdIns(4,5)P2 band. We
next performed a time course study to determine whether the ability to
secrete
-granules in response to Ca2+ exposure is
temporally correlated with PtdIns(4,5)P2 synthesis. PtdIns(4,5)P2 synthesis and the degree of
-granule
secretion in response to Ca2+ increased in parallel in
SL-O-permeabilized platelets over the first 30 min following exposure
to ATP (Fig. 2B). Thus, SL-O-permeabilized platelets are
capable of synthesizing PtdIns(4,5)P2 in response to ATP
without the addition of cytosol, and PtdIns(4,5)P2
synthesis correlates temporally with the ability to secrete
-granules in response to Ca2+ exposure.
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Fig. 2.
Effect of ATP on
PtdIns(4,5)P2 synthesis in SL-O-permeabilized
platelets. A, gel-filtered platelets (350 µl/sample)
in PIPES/EGTA buffer supplemented with 5 mM
MgCl2 were labeled with 2 mCi/ml
[32P]orthophosphoric acid at 37 °C for 2 h.
32P-Labeled platelets were then permeabilized with 3 units/ml SL-O in the presence or absence of 5 mM ATP at pH
6.9 for 30 min. The lipids were extracted, and
PtdIns(4,5)P2 levels were analyzed by TLC as described
under "Experimental Procedures." Error bars represent
the S.E. of six samples. B, to assess the time course of
PtdIns(4,5)P2 synthesis, gel-filtered platelets were
labeled with [32P]orthophosphoric acid and permeabilized
with 2 units/ml SL-O in the presence of 5 mM MgATP at pH
6.9 for the indicated amounts of time. The lipids were then extracted,
and PtdIns(4,5)P2 levels were analyzed by TLC ( ). To
assess the time course of P-selectin surface expression, gel-filtered
platelets were permeabilized with 2 units/ml SL-O in the presence of 5 mM MgATP, 10 µM Ca2+, and
phycoerythrin-conjugated anti-P-selectin antibody (5 µl) at pH 6.9 for the indicated amounts of time. Samples were then diluted in
phosphate-buffered saline (500 µl) and analyzed immediately by flow
cytometry (
). Data are expressed as the percentage of P-selectin
expression compared with a sample permeabilized in the presence of 5 mM MgATP and 10 µM Ca2+ for 30 min and subsequently incubated with phycoerythrin-conjugated
anti-P-selectin antibody for the indicated amounts of time. Error
bars represent the S.E. of four independent experiments.
-Granule Secretion--
To determine whether
PtdIns(4,5)P2 mediates Ca2+-induced
-granule
secretion from SL-O-permeabilized platelets, we hydrolyzed endogenous
platelet PtdIns(4,5)P2 using PtdIns-specific PLC.
PtdIns-specific PLC reduced endogenous PtdIns(4,5)P2 levels
in the SL-O-permeabilized platelets by ~75% (Fig.
3A). However, the use of
PtdIns-specific PLC in secretory assays is complicated by the fact that
the by-products of PtdIns-specific PLC activity, diacylglycerol and
inositol 1,4,5-trisphosphate, are themselves capable of influencing
secretion from permeabilized platelets (19). Diacylglycerol acts
primarily via activation of protein kinase C (21). Sloan and Haslam
(45) have shown that protein kinase C diffuses out of platelets
following exposure to SL-O. Consistent with these observations, we
found that stimulation of P-selectin surface expression by phorbol
12-myristate 13-acetate was reduced by >90% in SL-O-permeabilized
platelets compared with non-permeabilized platelets (data not shown).
Thus, permeabilized platelets are relatively insensitive to
diacylglycerol because of protein kinase C leakage. Inositol
1,4,5-trisphosphate is not likely to stimulate secretion in this system
because Ca2+ chelation by EGTA blocks the increase in
[Ca2+]i that is elicited by inositol
1,4,5-trisphosphate in unchelated systems. In the secretion
experiments, platelets were permeabilized with SL-O for 15 min prior to
exposure to PtdIns-specific PLC. Platelets were then incubated in the
presence or absence of PtdIns-specific PLC for 15 min prior to
Ca2+ exposure. Ca2+-induced
-granule
secretion was inhibited in permeabilized platelets exposed to
PtdIns-specific PLC (Fig. 3B). Platelets incubated with
PtdIns-specific PLC in the absence of Ca2+ exposure
demonstrated a small degree of secretion under these conditions (8 ± 2% P-selectin expression compared with samples incubated with
buffer and subsequently exposed to Ca2+) that might result
from the generation of diacylglycerol. Heat-denatured PtdIns-specific
PLC had no effect on Ca2+-induced
-granule secretion.
PtdIns-specific PLC had no effect on intact platelets since the amount
of SFLLR-induced P-selectin surface expression from intact
platelets exposed to 5 units/ml PtdIns-specific PLC was 97 ± 3%
of that from intact platelets not exposed to PtdIns-specific PLC.
Similarly, PtdIns-specific PLC failed to inhibit
Ca2+-induced P-selectin expression from platelets
permeabilized with
-toxin.
-Toxin creates pores that restrict the
entry of molecules greater than ~4.4 kDa (46).
Ca2+-induced P-selectin expression from platelets
permeabilized with
-toxin and subsequently exposed to 5 units/ml
PtdIns-specific PLC was 101 ± 9% of that from
-toxin-permeabilized platelets not exposed to PtdIns-specific PLC.
Thus, the effects of PtdIns-specific PLC on
-granule secretion
require entry into the platelet cytosol. Inhibition of
Ca2+-induced
-granule secretion by PtdIns-specific PLC
occurred in a dose-dependent manner with an
IC50 of ~0.5 units/ml (Fig. 3C). Inhibition of
-granule secretion by PtdIns-specific PLC was also time-dependent, with the amount of inhibition increasing to
~75% over a 45-min incubation time (Fig. 3D). These data
demonstrate that incubation of SL-O-permeabilized platelets with
PtdIns-specific PLC interferes with Ca2+-induced
-granule secretion.
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Fig. 3.
Effect of PtdIns-specific PLC on
PtdIns(4,5)P2 synthesis in and
Ca2+-induced -granule secretion
from SL-O-permeabilized platelets. A,
32P-labeled gel-filtered platelets (350 µl/sample) in
PIPES/EGTA buffer were permeabilized with 3 units/ml SL-O in the
presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized
platelets were then incubated in the presence of buffer (No
Addition) or 5 units/ml PtdIns-specific PLC for 40 min. The lipids
were extracted, and PtdIns(4,5)P2 levels were analyzed by
TLC. Data are expressed as percent PtdIns(4,5)P2 levels
compared with samples exposed to buffer alone. Error bars
represent the S.E. of six samples. B, gel-filtered platelets
were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were
then incubated for 15 min with buffer (No Addition), 5 units/ml PtdIns-specific PLC, or 5 units/ml heat-denatured
PtdIns-specific PLC. Following this incubation, platelets were exposed
to either buffer (No Ca2+) or 10 µM
Ca2+ for 5 min. Platelets were then assayed for P-selectin
expression by flow cytometry as described under "Experiment
Procedures." Histograms represent the distribution of the relative
fluorescence of 10,000 platelets. C, gel-filtered platelets
were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were
then incubated with the indicated concentrations of PtdIns-specific PLC
for 15 min. Platelets were exposed to 10 µM
Ca2+ for 5 min and assayed for P-selectin expression. Data
are expressed as the percent inhibition of P-selectin expression in
samples exposed to PtdIns-specific PLC compared with samples exposed to
buffer alone. Error bars represent the S.E. of three
independent experiments. D, gel-filtered platelets were
permeabilized with 3 units/ml SL-O in the presence of 5 mM
MgATP at pH 6.9 for 15 min. Permeabilized platelets were then incubated
with 5 units/ml PtdIns-specific PLC for the indicated times. After
1 h following permeabilization, platelets were exposed to 10 µM Ca2+ for 5 min and assayed for P-selectin
expression. Data are expressed as the percent inhibition of P-selectin
expression in samples exposed to PtdIns-specific PLC compared with
samples exposed to buffer alone. Error bars represent the
S.E. of three independent experiments.
-Granule Secretion--
The proposed role
of PtdIns(4,5)P2 in granule secretion from neuroendocrine
cells is to localize PtdIns(4,5)P2-binding proteins that
mediate secretion to specific locations to permit membrane fusion (47).
Consistent with this hypothesis, exogenously added PtdIns(4,5)P2 inhibits granule secretion from permeabilized
PC12 cells (48). We therefore determined the effect of exogenously added PtdIns(4,5)P2 on platelet
-granule release.
Platelets were permeabilized with SL-O for 15 min prior to the addition
of either PtdIns(4,5)P2 or buffer. Following an additional
15-min incubation, platelets were exposed to buffer alone or
Ca2+ for 5 min and subsequently analyzed for P-selectin
surface expression by flow cytometry. Exogenously added
PtdIns(4,5)P2 inhibited Ca2+-induced platelet
-granule secretion by ~80% (Fig.
4). PtdIns(4,5)P2 itself had
little effect on
-granule secretion in the absence of exposure to
Ca2+. The amount of SFLLR-induced P-selectin surface
expression from intact platelets exposed to exogenously added
PtdIns(4,5)P2 (50 µM) was 91 ± 2% of
that from intact platelets not exposed to exogenously added
PtdIns(4,5)P2. Similarly, Ca2+-induced
P-selectin expression from platelets permeabilized with
-toxin and
subsequently exposed to exogenously added PtdIns(4,5)P2 (50 µM) was 99 ± 6% of that from
-toxin-permeabilized platelets not exposed to exogenously added
PtdIns(4,5)P2. Thus, the effects of exogenously added
PtdIns(4,5)P2 require entry into the platelet cytosol.
These data demonstrate that incubation of SL-O-permeabilized platelets
with exogenous PtdIns(4,5)P2 inhibits
Ca2+-induced
-granule secretion.
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Fig. 4.
Effect of exogenous PtdIns(4,5)P2
on Ca2+-induced -granule secretion
from SL-O-permeabilized platelets. A, gel-filtered
platelets were permeabilized with 3 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 15 min. Permeabilized platelets were
then incubated with buffer (No Addition) or 50 µM PtdIns(4,5)P2 for 15 min. Following this
incubation, platelets were exposed to either buffer (No
Ca2+) or 10 µM Ca2+ for 5 min. Platelets were then assayed for P-selectin expression by flow
cytometry as described under "Experimental Procedures." Histograms
represent the distribution of the relative fluorescence of 10,000 platelets. B, data are expressed as percent P-selectin
surface expression compared with samples exposed to buffer alone.
Error bars represent the S.E. of three independent
experiments.
-Granule Secretion--
PtdIns 3-kinase
has previously been implicated in the process of regulated granule
exocytosis from hematopoietic cells (49, 50). We therefore sought to
determine whether PtdIns 3-kinase is involved in
Ca2+-induced
-granule secretion from SL-O-permeabilized
platelets. To this end, experiments using inhibitors of PtdIns
3-kinase, wortmannin and LY294002, were performed. In these
experiments, neither 250 nM wortmannin nor 33.3 µM LY294002 inhibited Ca2+-induced
-granule secretion from SL-O-permeabilized platelets (Fig.
5A). The activity of 250 nM wortmannin and 33.3 µM LY294002 was
confirmed in platelet aggregation studies. Reversible platelet aggregation, which is dependent on PtdIns 3-kinase activity (51), was
inhibited to 36 ± 3% of base-line levels by 250 nM
wortmannin and to 29 ± 6% of base-line levels by 33.3 µM LY294002. Thus, the concentrations of wortmannin and
LY294002 used to assess the role of PtdIns 3-kinase in
Ca2+-induced
-granule secretion are sufficient to
inhibit PtdIns 3-kinase-dependent platelet functions.
Antibodies directed at PtdIns 3-kinase p85
also failed to inhibit
Ca2+-induced
-granule secretion from SL-O-permeabilized
platelets (Fig. 5B). These results demonstrate that PtdIns
3-kinase is not involved in Ca2+-induced,
ATP-dependent
-granule secretion.
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Fig. 5.
Effect of wortmannin, LY294002, and
anti-PtdIns 3-kinase p85 antibody on
Ca2+-induced
-granule secretion
from SL-O-permeabilized platelets. A, gel-filtered
platelets were incubated with 0.3% dimethyl sulfoxide
(DMSO), 250 nM wortmannin, or 33.3 µM LY294002 for 30 min. Platelets were then permeabilized
with 2 units/ml SL-O for 5 min and subsequently incubated with 5 mM MgATP at pH 6.9 for 25 min. Platelets were exposed to 10 µM Ca2+ for 5 min and analyzed for P-selectin
expression by flow cytometry as described under "Experimental
Procedures." Data are expressed as percent P-selectin surface
expression compared with samples exposed to 0.3% dimethyl sulfoxide
alone. B, gel-filtered platelets were incubated with MgATP
in the presence of buffer (No addition), 40 µg/ml
anti-PtdIns 3-kinase p85
monoclonal antibody, or 40 µg/ml
nonimmune mouse IgG (nonimmune antibody). Platelets were
subsequently permeabilized with 2 units/ml SL-O at pH 6.9 for 25 min.
Platelets were then exposed to 10 µM Ca2+ for
5 min and analyzed for P-selectin expression by flow cytometry. Data
are expressed as percent P-selectin surface expression compared with
samples exposed to buffer alone.
-granule secretion to a maximum of ~50%
at 3.125 µM (Fig.
6A). Similarly, 3.125 µM wortmannin inhibited the synthesis of
PtdIns(4,5)P2 in SL-O-permeabilized platelets by ~50%
(Fig. 6B). To determine whether exposure to wortmannin
inhibits ATP- or Ca2+-dependent processes,
SL-O-permeabilized platelets were exposed to wortmannin either 1) prior
to ATP exposure or 2) after ATP exposure, but prior to Ca2+
exposure. Incubation of platelets with 3.125 µM
wortmannin for 45 min inhibited Ca2+-induced
-granule
secretion from SL-O-permeabilized platelets when wortmannin was added
prior to ATP exposure (Fig. 6C). However,
-granule
secretion was not inhibited in SL-O-permeabilized platelets exposed to
ATP for 15 min and subsequently incubated with 3.125 µM
wortmannin for 45 min (Fig. 6C). Thus, wortmannin inhibits ATP-dependent events, but not Ca2+-mediated
triggering of
-granule secretion. The correlation between the
reduction of PtdIns(4,5)P2 synthesis by micromolar
concentrations of wortmannin and the inhibition of an
ATP-dependent step of Ca2+-induced
-granule
secretion is consistent with a role for PtdIns(4,5)P2 synthesis in this process.
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Fig. 6.
Effect of micromolar concentrations of
wortmannin on Ca2+-induced
-granule secretion from and
PtdIns(4,5)P2 synthesis in SL-O-permeabilized
platelets. A, gel-filtered platelets were incubated
with the indicated concentrations of wortmannin for 30 min. Platelets
were then permeabilized with 2 units/ml SL-O for 5 min and subsequently
incubated with 5 mM MgATP at pH 6.9 for 25 min. Platelets
were exposed to 10 µM Ca2+ for 5 min and
analyzed for P-selectin expression by flow cytometry as described under
"Experimental Procedures." Data are expressed as percent P-selectin
surface expression compared with samples exposed to dimethyl sulfoxide
alone. Error bars represent the S.E. of three to seven
independent experiments. B, 32P-labeled
gel-filtered platelets (350 µl/sample) in PIPES/EGTA buffer were
incubated with either 0.3% dimethyl sulfoxide (vehicle) or
3.125 µM wortmannin for 30 min. Labeled platelets were
then permeabilized with 2 units/ml SL-O in the presence of 5 mM MgATP at pH 6.9 for 30 min. The lipids were extracted,
and PtdIns(4,5)P2 levels were analyzed by TLC. Data are
expressed as percent PtdIns(4,5)P2 levels compared with
samples exposed to vehicle alone. Error bars represent the
S.E. of six samples. C, one set of platelets was incubated
with either vehicle (white bars) or 3.125 µM
wortmannin (black bars) for 30 min. Platelets were
subsequently permeabilized with 2 units/ml SL-O for 5 min and incubated
with 5 mM MgATP at pH 6.9 for 15 min. A second set of
platelets was permeabilized with 2 units/ml SL-O for 5 min and
incubated with 5 mM MgATP at pH 6.9 for 15 min. Following
incubation with MgATP, these samples were incubated with vehicle or
3.125 µM wortmannin for 45 min. Following incubation with
MgATP, all samples were exposed to 10 µM Ca2+
for 5 min and analyzed for P-selectin surface expression. Data are
expressed as percent P-selectin surface expression compared with
samples exposed to vehicle. Error bars represent the S.E. of
three independent experiments.
-Granule Secretion--
PtdIns 5-phosphate levels increase in
platelets following stimulation with thrombin (37). Furthermore, type
II PIPK has previously been demonstrated to be abundant in platelets
(38). We therefore determined the effect of anti-type II PIPK
antibodies on PtdIns(4,5)P2 synthesis in and
-granule
secretion from SL-O-permeabilized platelets. Anti-type II PIPK
antibodies directed against the amino- and carboxyl-terminal ends of
the
-isoform of human type II PIPK were used in these studies. The
peptides used to generate these antibodies have no homology to type I
PIPK. Each antibody recognized a single band of 53 kDa in platelet
lysates (Fig. 7A). In
contrast, nonimmune IgG failed to recognize any bands in platelet
lysates (data not shown). In addition, both antibodies recognized a
recombinant GST-tagged type II PIPK fusion protein of 79 kDa in
immunoblot analysis (Fig. 7B). When incubated with
MgATP-exposed platelets prior to permeabilization with SL-O, both
anti-amino- and anti-carboxyl-terminal type II PIPK antibodies
inhibited PtdIns(4,5)P2 levels by ~50% compared with
permeabilized platelets incubated with nonimmune antibody (Fig.
7C). In contrast, neither anti-type II PIPK antibody inhibited PtdIns(4,5)P2 levels when incubated with
platelets for 15 min following permeabilization with SL-O. We next
determined the effect of anti-type II PIPK antibodies on
Ca2+-induced
-granule secretion from SL-O-permeabilized
platelets. In these experiments, platelets were incubated in the
presence of nonimmune antibody or anti-type II PIPK antibody directed
against either the amino- or carboxyl-terminal end of type II PIPK.
Platelets were permeabilized with SL-O and then exposed to 10 µM Ca2+. Both antibodies significantly
inhibited Ca2+-induced
-granule secretion from
SL-O-permeabilized platelets (Fig. 7D). In contrast,
nonimmune antibody had no effect on Ca2+-induced
-granule secretion. As observed with wortmannin, anti-type II PIPK
antibodies inhibited Ca2+-induced
-granule secretion
when added prior to, but not after, ATP (Fig. 7D). The
amount of SFLLR-induced P-selectin surface expression from intact
platelets exposed to anti-amino- or anti-carboxyl-terminal type II PIPK
antibody was 104 ± 24 and 93 ± 18%, respectively, of that
from intact platelets not exposed to anti-type II PIPK antibodies.
Similarly, Ca2+-induced P-selectin expression from
platelets permeabilized with
-toxin in the presence of anti-amino-
or anti-carboxyl-terminal type II PIPK antibody was 100 ± 6 and
91 ± 22%, respectively, of that from
-toxin-permeabilized
platelets not exposed to anti-type II PIPK antibodies. Thus, the
effects of exogenously added anti-type II PIPK antibodies require entry
into the platelet cytosol. We conclude that type II PIPK mediates
-granule secretion from Ca2+-induced SL-O-permeabilized
platelets and acts prior to Ca2+ exposure.
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Fig. 7.
Effect of anti-type II PIPK antibodies on
PtdIns(4,5)P2 synthesis in and
Ca2+-induced -granule secretion
from SL-O-permeabilized platelets. A, proteins from
human platelets were solubilized at 95 °C in sample buffer,
separated by SDS-polyacrylamide gel electrophoresis on a 12% gel, and
electrophoretically transferred to polyvinylidene difluoride membranes.
Immunoblotting was performed with antibodies directed against the
amino- and carboxyl-terminal peptides of type II PIPK as indicated.
Bands were visualized using enhanced chemiluminescence for detection.
The positions of the molecular mass standards used are indicated on the
left. B, recombinant GST-tagged type II PIPK was solubilized
at 95 °C in sample buffer, separated by SDS-polyacrylamide gel
electrophoresis on a 12% gel, and electrophoretically transferred to
polyvinylidene difluoride membranes. Immunoblotting was performed with
antibodies directed against the amino- and carboxyl-terminal peptides
as described for A. C, one set of
32P-labeled gel-filtered platelets (black bars)
was permeabilized with 4 units/ml SL-O in the presence of 40 µg/ml
nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK
antibody (N-term), or 40 µg/ml anti-carboxyl-terminal type
II PIPK antibody (C-term) with 5 mM MgATP at pH
6.9 for 90 min. A second set of 32P-labeled platelets
(white bars) was permeabilized with 4 units/ml SL-O in the
presence of 5 mM MgATP alone at pH 6.9 for 15 min. This set
of platelets was then exposed to 40 µg/ml nonimmune antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody, or 40 µg/ml
anti-carboxyl-terminal type II PIPK antibody for 75 min. The lipids
were extracted, and PtdIns(4,5)P2 levels were analyzed by
TLC. Data are expressed as percent PtdIns(4,5)P2 levels
compared with samples exposed to nonimmune antibody. Error
bars represent the S.E. of three to six samples. D, one
set of gel-filtered platelets (black bars) was incubated in
the presence of 40 µg/ml nonimmune antibody, 40 µg/ml
anti-amino-terminal type II PIPK antibody, or 40 µg/µl
anti-carboxyl-terminal type II PIPK antibody. Samples were then exposed
to 10 mM MgATP and permeabilized with 5 units/ml SL-O.
Following permeabilization, all samples were adjusted to pH 6.9 and
subsequently exposed to 10 µM Ca2+. A second
set of gel-filtered platelets (white bars) was incubated in
the presence of 10 mM MgATP at pH 6.9 with no addition.
Samples were then perme abilized with 5 units/ml SL-O. Following this MgATP exposure,
samples from the second set were incubated with 40 µg/ml nonimmune
antibody, 40 µg/ml anti-amino-terminal type II PIPK antibody, or 40 µg/µl of anti-carboxyl-terminal type II PIPK antibody, followed by
10 µM Ca2+. Following a 15-min incubation in
the presence of Ca2+, all samples were assayed for
P-selectin expression by flow cytometry as described under
"Experimental Procedures." Data are expressed as percent P-selectin
expression compared with samples incubated in the presence of buffer
alone. Error bars represent the S.E. of four to eight
independent experiments.
-granule secretion by distinct
mechanisms, we performed experiments using both inhibitors. In these
experiments, we incubated platelets in the presence of 3.125 µM wortmannin or vehicle for 30 min and exposed them to
nonimmune, anti-amino-terminal end, or anti-carboxyl-terminal end
antibodies prior to ATP exposure and SL-O permeabilization. Under these
conditions, ~40% inhibition of Ca2+-induced
-granule
secretion was observed in platelets exposed to only wortmannin or
either of the anti-type II PIPK antibodies compared with platelets
treated with vehicle and nonimmune antibody (Fig.
8). Increasing the concentration of
anti-type II PIPK antibodies to 200 µg/ml failed to increase the
degree of inhibition of
-granule secretion. However,
-granule
secretion from SL-O-permeabilized platelets exposed to both wortmannin
and anti-type II PIPK antibodies was inhibited by ~70% compared with
platelets exposed to vehicle and nonimmune antibody (Fig. 8). Thus,
inhibition by wortmannin and anti-type II PIPK antibodies is
additive.
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Fig. 8.
Effect of wortmannin and type II PIPK
antibodies on Ca2+-induced
-granule secretion from SL-O-permeabilized
platelets. Gel-filtered platelets were incubated in the presence
(black bars) or absence (white bars) of 3.125 µM wortmannin for 30 min. Platelets were then exposed to
40 µg/ml nonimmune antibody or 40 or 200 µg/ml antibody directed
against an amino-terminal (N-term) or carboxyl-terminal
(C-term) peptide of type II PIPK as indicated and
permeabilized with 4 units/ml SL-O in the presence of 5 mM
MgATP. Following permeabilization, all samples were adjusted to pH 6.9. After a 30-min incubation, samples were exposed to 10 µM
Ca2+ for an additional 10-min incubation. Samples were then
analyzed for P-selectin expression by flow cytometry as described under
"Experimental Procedures." Data are expressed as percent P-selectin
expression compared with samples incubated in the presence of vehicle
and nonimmune antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-granule
secretion from SL-O-permeabilized platelets is inhibited by
PtdIns-specific PLC (which cleaves PtdIns(4,5)P2), by
exogenously added PtdIns(4,5)P2 (which may act by
displacing proteins bound to endogenous PtdIns(4,5)P2), and
by two different anti-type II PIPK antibodies (which inhibit
PtdIns(4,5)P2 synthesis) provides strong evidence that
PtdIns(4,5)P2 mediates
-granule secretion in this
system. Since none of these inhibitors have inhibitory activity in
intact platelets or in
-toxin-permeabilized platelets, access to
the platelet cytosol is required for their inhibitory activity. The
assertion that PtdIns(4,5)P2 mediates
-granule secretion
is also supported by the observations that the ability to secrete
-granules upon Ca2+ exposure is temporally correlated
with PtdIns(4,5)P2 synthesis and that micromolar
concentrations of wortmannin inhibit both PtdIns(4,5)P2
synthesis and Ca2+-induced
-granule secretion.
-granule secretion
when added prior to ATP exposure, but not when added following ATP
exposure, but prior to Ca2+-mediated triggering of
secretion. This finding raises the possibility that
activation-dependent changes in localized
PtdIns(4,5)P2 concentrations mediate granule secretion.
Indeed, activation of platelets is accompanied by both an increase in
PtdIns kinase activity (33, 35, 36) and relocalization of PtdIns
kinases (53). Future studies will determine whether
activation-dependent increases in PtdIns(4,5)P2
at zones of granule secretion within the platelet are necessary for
-granule secretion.
did not inhibit secretion from SL-O-permeabilized platelets (Fig. 5) demonstrates that D3
phosphoinositides are not involved in
-granule secretion from
platelets. The observation that wortmannin failed to inhibit
-granule secretion at concentrations that inhibit PtdIns 3-kinase is
consistent with the data of Kovacsovics et al. (51). Their
studies using intact platelets demonstrated that although wortmannin at
nanomolar concentrations inhibits thrombin receptor agonist
peptide-induced activation of glycoprotein IIb-IIIa, it fails to affect
-granule secretion from platelets. Similarly, wortmannin at
nanomolar concentrations is a relatively poor inhibitor of neutrophil
granule secretion, even though it inhibits the neutrophil oxidative
burst with an IC50 of <5 nM (54). Thus,
granule secretion from various cells of hematopoietic origin differs in
requirements for phosphoinositides, and platelet
-granule secretion
demonstrates a requirement for D4, but not D3, phosphoinositides.
-granule secretion. In PC12 cells, type I,
but not type II, PIPK reconstitutes Ca2+-mediated secretion
(30). To our knowledge, type II PIPK has not previously been
demonstrated to function in a cellular process (55). However, PtdIns
5-kinase levels are increased upon exposure of intact platelets to
thrombin (37). In this study, we show that anti-type II PIPK antibodies
partially inhibit Ca2+-induced
-granule secretion from
SL-O-permeabilized platelets. Three isoforms of type II PIPK
(
,
, and
) have been identified. The antibodies used in this
study are directed to the amino- and carboxyl-terminal ends of the
-isoform. However, these antibodies cross-react with the
-isoform
(data not shown); and thus, we are not able to determine which
isoform(s) of type II PIPK is active in platelets. Anti-type II PIPK
antibodies do not cause complete inhibition of Ca2+-induced
-granule secretion from SL-O-permeabilized platelets. The degree of
inhibition of secretion was no greater when the concentration of
antibody was increased from 40 to 200 µg/ml (Fig. 8). Neither the
anti-amino- nor anti-carboxyl-terminal type II PIPK antibody is
directed against the proposed active site of the kinase. Thus, it is
possible that the antibodies cause partial inhibition of type II PIPK
function by disrupting its interactions with other molecules
(e.g. PtdIns kinases) required for PtdIns(4,5)P2 production (56). Another possibility is that partial
PtdIns(4,5)P2 production in the presence of anti-type II
PIPK antibodies is secondary to the activity of type I PIPK isoforms,
which are also found in
platelets.2
-granule secretion when added prior to, but not after, ATP exposure raises the possibility that the PtdIns 4-kinase pathway is also involved in
-granule secretion. Three isoforms of PtdIns 4-kinase have been identified and can be
distinguished based on their sensitivity to wortmannin. A 55-kDa PtdIns
4-kinase is resistant to wortmannin (57). A 230-kDa PtdIns 4-kinase
with a 97-kDa splice variant is inhibited by wortmannin with an IC50 of ~1 µM and is nearly completely
inhibited at 10 µM (58). PtdIns 4-kinase
is inhibited
by wortmannin with an IC50 of ~120 nM and is
completely inhibited at 1 µM. However, wortmannin at micromolar concentrations is not specific for PtdIns 4-kinases. Inhibition of the ATP-dependent step of
Ca2+-induced
-granule secretion by micromolar
concentrations of wortmannin may occur by interfering with molecules
not involved in PtdIns(4,5)P2 synthesis. The fact that
PtdIns(4,5)P2 synthesis is also inhibited by wortmannin may
be secondary to inhibition of a PtdIns 5-kinase. Although the PtdIns
5-kinases in platelets have not been well characterized, a 235-kDa
mammalian PtdIns kinase originally discovered in insulin-sensitive
cells (59) was subsequently found to synthesize PtdIns 5-phosphate
(60). Wortmannin inhibits this PtdIns 5-kinase with an IC50
of ~600 nM and completely inhibits kinase activity at 4 µM wortmannin (60). Thus, it is possible that the effect of wortmannin on PtdIns(4,5)P2 synthesis and
Ca2+-induced
-granule secretion is via inhibition of a
PtdIns 5-kinase pathway rather than through a PtdIns 4-kinase pathway.
However, the identification of the complete set of PtdIns kinase and
PIPK isoforms used in the synthetic pathway of
PtdIns(4,5)P2 required for
-granule secretion remains to
be determined.
-granule secretion?
Platelet
-granule secretion is known to require the SNARE protein
machinery (12-15). In other secretory systems, it has been postulated
that PtdIns(4,5)P2 might act by interacting with proteins,
such as synaptotagmin and Munc-18-interacting protein, that both bind PtdIns(4,5)P2 and interact with SNARE proteins (61). In
addition, PtdIns(4,5)P2 interacts with ADP-ribosylation
factors and GTPase-activating proteins to localize these molecules to
sites of membrane fusion (61). However, the presence of
ADP-ribosylation factors in platelets and the role of GTPase-activating
proteins and ADP-ribosylation factors in platelet secretion remain to
be explored. PtdIns(4,5)P2 also stimulates phospholipase D
(62), which generates phosphatidic acid and is thought to influence
platelet granule secretion (24). Phosphatidic acid, in turn, stimulates
type I PIPK activity (63). Thus, a feed-forward mechanism is
established whereby cell activation leads to increased synthesis of
phosphatidic acid and PtdIns(4,5)P2. Future studies will
provide a more detailed understanding of how PtdIns(4,5)P2
mediates interactions between platelet signaling events and SNARE
protein rearrangements that result in membrane fusion.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Christopher Carpenter for helpful discussion, advice, and critical reading of the manuscript and Bruce Furie for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
Supported by National Institutes of Health Grant HL63250,
Burroughs Wellcome Fund Career Awardee, and participant in the Beth Israel Deaconess Medical Center-Harvard/Massachusetts Institute of
Technology Health Sciences and Technology Clinical Investigator Training Program in collaboration with Pfizer. To whom
correspondence should be addressed: RE 319, Research East, P. O. Box
15732, Boston, MA 02215. Tel: 617-667-0627; Fax: 617-975-5505; E-mail:
rflaumen@caregroup.harvard.edu.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M008184200
2 C. L. Carpenter, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SNARE, soluble N-ethylmaleimide-sensitive fusion protein
(NSF) attachment protein receptor;
PLC, phospholipase C;
PtdIns, phosphatidylinositol;
PtdIns(4, 5)P2, phosphatidylinositol
4,5-bisphosphate;
PIPK, phosphatidylinositol-phosphate kinase;
GTPS, guanosine 5'-O-(3-thiotriphosphate);
SL-O, streptolysin O;
GST, glutathione S-transferase;
PIPES, 1,4-piperazinediethanesulfonic acid.
![]() |
REFERENCES |
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