From the Center for Hemostasis and Thrombosis
Research, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts 02215, § Howard Hughes
Medical Institute, and
Department of Biochemistry, Tufts
University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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To understand the molecular basis of
granule release from platelets, we examined the role of
vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in
The study of the secretory machinery using cell-free systems and
permeabilized cells has demonstrated the existence of a class of
proteins, termed SNARE1
proteins, that mediate vesicle secretion (9, 10). These proteins have
been found to mediate vesicle secretion in essentially all organisms
investigated, from yeast to human (9). SNARE family proteins have been
found in numerous cell types including cells responsible for
maintaining vascular integrity such as platelets (11), leukocytes
(12-14), and endothelial cells (15). Three sets of SNARE proteins have
been identified (16). vSNAREs are type II integral membrane proteins
located on vesicles and oriented such that the majority of the protein
resides within the cytosol. tSNAREs are membrane proteins that are
associated with target membranes and are also oriented toward the
cytoplasm. Soluble SNAREs are cytoplasmic proteins that associate with
vSNARE-tSNARE complexes. Functional evaluation of these proteins has
led to the SNARE hypothesis that states that interactions between
vSNAREs and tSNAREs mediate vesicle fusion with target membranes (17). Certain vSNAREs and tSNARES contain coiled-coil structures that bind in
a parallel (18) manner to create a stable exocytotic core complex (19).
The soluble SNARE proteins that are subsequently recruited to this
complex then mediate membrane fusion via a process that involves ATP
hydrolysis (17).
Analysis of the components of the exocytotic core complex has
demonstrated a large number of vSNARE and tSNARE isoforms. Such diversity may provide the specificity required to target vesicles to
their appropriate destinations (20). vSNAREs include vesicle-associated membrane proteins (VAMPs) 1 and 2 (also known as synaptobrevins 1 and
2) as well as cellubrevin. Isoform-specific expression of VAMP-1 and
VAMP-2 has been demonstrated in a number of tissues (21-23).
Cellubrevin has been detected in both neuronal and nonneuronal tissue
(24, 25). SNAP-23 and SNAP-25 are tSNAREs that associate with the inner
leaflet of the plasma membrane via a palmitoylation anchor. SNAP-23 is
ubiquitously expressed, whereas SNAP-25 expression is somewhat more
restricted (26). Syntaxins are a family of tSNARES with multiple
isoforms (26). Specific tSNAREs will only bind to a limited number of
vSNAREs (19). Such restricted interaction of SNARE proteins is thought
to form the basis for specificity of vesicle targeting. VAMP, SNAP-25,
and syntaxin interact to form the heterotrimeric complex that serves as
a receptor for soluble SNARE proteins. Exocytosis proceeds through this complex.
Understanding of the role of SNARE proteins in mediating vesicle
secretion in mammalian cells is derived largely from studies in
neuronal and neuroendocrine cell systems. To what extent the principles
derived from these studies can be applied to platelet granule secretion
is uncertain. Several fundamental differences exist between granule
secretion by platelets and vesicle secretion by neurons and
neuroendocrine cells. Platelets are anucleate cytoplasmic fragments
derived from megakaryocytes. Although some Chemicals and Reagents--
All buffer constituents and dextran
sulfate-fluorescein isothiocyanate conjugates were purchased from
Sigma. Sepharose 2B was obtained from Amersham Pharmacia Biotech.
Reduced streptolysin O (SL-O) was purchased from Murex (Dartford, UK).
PE-conjugated AC1.2 anti-P-selectin antibody was purchased from Becton
Dickinson. Micro-BCA protein assay kit was purchased from Pierce and
used according to the manufacture's instructions.
Serine-phenylalanine-leucine-leucine-arginine (SFLLR) was
synthesized using solid phase Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied
Biosystems model 430A peptide synthesizer. Bovine brain homogenate was
obtained from StressGene (Victoria, Canada). Human endothelial cell
homogenate was obtained from Transduction Laboratories (Lexington,
Kentucky). All solutions were prepared using water purified by
reverse-phase osmosis on a Millipore Milli-Q purification Water System.
Antibodies and Toxins--
Rabbit polyclonal and mouse
monoclonal anti-VAMP-2 antibodies were obtained from StressGene
(Victoria, Canada). The polyclonal antibody is directed against a
21-amino acid peptide fragment of human VAMP-2. The monoclonal antibody
was raised against human synaptic vesicles and recognizes both VAMP-1
and VAMP-2. VAMP-1 antibody was a generous gift from Dr. S. W. Trimble. SNAP-23 antibody, obtained from Synaptic Systems (Gottingen,
Germany), is a polyclonal antibody raised against the C-terminal end of
SNAP-23. Anti-syntaxin 4 antibody, purchased from Transduction Labs
(Lexington, Kentucky), reacts specifically with syntaxin 4 and has no
cross-reactivity with syntaxin 2. Anti-P-selectin cytoplasmic tail
antibody, a gift from Dr. Michael Berndt, is a rabbit polyclonal
antibody directed against an 11-amino acid fragment of the P-selectin
cytoplasmic tail (30). Tetanus toxin was obtained from List Biologics,
Inc. (Campbell, CA).
Platelet Preparation--
Blood from healthy donors who had not
ingested aspirin in the two weeks before 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 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, 0.1% glucose, 0.1% bovine serum albumin, pH 6.4). Final gel-filtered platelet concentrations were 1-2 × 108 platelets/ml.
Platelet Permeabilization with SL-O--
Gel-filtered platelets
(20 µl) were incubated with 25 µM indicated dye in the
presence or absence of 2 units/ml SL-O reduced with 5 mM
dithiothreitol (1 µl). After a 15-min incubation, platelets were
diluted in phosphate-buffered saline (500 µl) and analyzed immediately for fluorescence. Results are expressed as the fluorescence of platelets exposed to SL-O divided by the fluorescence of unexposed platelets. A value of 1 represents no permeabilization.
Analysis of P-selectin Surface Expression--
Gel-filtered
platelets (20 µl) were exposed to 2 units/ml reduced SL-O. After the
indicated amount of time, 5 mM MgATP was added to the
reaction mixture at pH 6.4. We have previously demonstrated that
maximal Immunoblot Analysis--
In experiments evaluating the presence
of individual proteins in platelets, samples were heated to 100 °C
before electrophoresis. In experiments evaluating SNARE protein
complexes, 500 µl of resting platelets or platelets exposed to 100 µM SFLLR were pelleted and solubilized in 250 µl of
sample buffer (62.5 mM Tris-HCl, 0.2% SDS, 0.5%
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. Ten thousand
particles were acquired for each sample. A 530/30 band pass filter was
used for FL-1 fluorescence, and a 585/42 band pass filter was used for
FL-2 fluorescence. Platelet-associated fluorescein isothiocyanate was
measured in the FL-1 channel. PE was measured in the FL-2 channel. Data
were analyzed using CellQuest software on a MacIntosh PowerPC.
Platelet VAMP, SNAP-23, and Syntaxin 4--
To determine whether
an exocytotic core complex consisting of VAMP, SNAP-23, and syntaxin 4 exists in platelets, we assayed for the presence of these proteins in
platelet lysate. As illustrated in Fig.
1, all three proteins are detected in
platelet lysates subjected to immunoblotting. In these experiments,
solubilized lysates were heated at 100 °C before loading onto the
gel. The VAMP antibody used in this experiment was a polyclonal IgG
directed against a peptide fragment of VAMP-2. This sequence is located in the SNAP-23/syntaxin binding domain, a region conserved among vesicle-associated membrane proteins (35) (Fig. 1a). This
antibody recognized a single protein in bovine brain lysates and in
human platelet lysates. However, the VAMP species in bovine brain had a
molecular mass of 18 kDa, whereas the VAMP species in platelets migrated on the gel with an apparent molecular mass of 14 kDa. The VAMP
species in platelets was not recognized by a monoclonal antibody
specific to VAMP-2 (11) nor by an antibody specific to VAMP-1 (data not
shown). These results suggest the platelet VAMP is yet another VAMP
isoform. The antibody to SNAP-23 is a polyclonal antibody raised
against the C-terminal end of SNAP-23. This antibody recognized a
protein of approximately 23 kDa in both bovine brain and in human
platelet lysates. The syntaxin 4 antibody is a monoclonal antibody
raised against syntaxin 4. Proteins of approximately 34 kDa were
detected by this antibody in both human endothelial cell and human
platelet lysates.
VAMP, SNAP-23, and Syntaxin 4 Complex Formation in
Platelets--
An SDS-resistant, heat-sensitive complex consisting of
VAMP, SNAP-23, and syntaxin 4 has been described in neurons (36, 37).
To determine whether such a complex exists in platelets, platelet
lysate was solubilized in SDS-containing sample buffer at 37 °C for
30 min and analyzed by SDS-PAGE followed by immunoblotting using the
anti-VAMP-2, anti-SNAP-23, and anti-syntaxin 4 antibodies. A doublet of
approximately Mr 70,000 was recognized by
antibodies directed against VAMP (Fig.
2a) and SNAP-23 (Fig.
2b). A band of approximately Mr
70,000 was also recognized by the anti-syntaxin antibody (Fig.
2c). As has been shown in neuronal tissue, the high
molecular weight complex was not observed when platelet lysate was
subjected to 100 °C (36, 37). Nonimmune antibody did not detect any
bands in the platelet lysate.
To determine whether disassembly of this complex occurred with platelet
activation, platelets were exposed to SFLLR and subsequently assayed
for the presence of the 70-kDa SDS-resistant, heat-sensitive complex.
Disassembly of the complex was observed under these conditions upon
platelet activation with SFLLR (Fig. 3).
The amount of talin in the gels containing sample from resting and
activated platelets was determined to assure equal protein content. The
amount of talin present in each of the lanes did not vary by more than
10% (data not shown), indicating that differences in intensity of the
70-kDa band in resting samples compared with activated samples was not
secondary to differences in protein loading. Activation of platelets
upon exposure to SFLLR was confirmed by demonstrating increases in
P-selectin surface expression in the SFLLR-exposed platelets using flow
cytometry (data not shown).
Ca2+-induced
We next sought to determine whether platelets permeabilized with SL-O
would secrete
Many SL-O-permeabilized systems lose the ability to secrete
in response to Ca2+ as a function of time after
permeabilization (40). To determine whether
SL-O-permeabilized platelets lose their responsiveness to
Ca2+ over time, we permeabilized platelets with SL-O and
exposed them to Ca2+ at various times after
permeabilization. SL-O-permeabilized platelets express
P-selectin on their surface in response to Ca2+ up to
2 h after permeabilization (Fig. 6).
There is no detectable loss of responsiveness to Ca2+ over
this period. Furthermore, no spontaneous P-selectin expression was
observed in SL-O-permeabilized platelets that were not
exposed to Ca2+. Thus, SL-O-permeabilized platelets secrete
Effect of Anti-VAMP Antibodies and Tetanus Toxin on
Ca2+-induced
Tetanus toxin cleaves VAMP family proteins specifically (43). The next
series of experiments were performed to determine the sensitivity of
platelet VAMP to tetanus toxin. We exposed permeabilized platelets to
tetanus toxin and assayed for VAMP by immunoblot analysis using the
polyclonal anti-VAMP-2 peptide antibody. The VAMP family protein that
is recognized by the polyclonal anti-VAMP-2 peptide antibody is
sensitive to degradation by tetanus toxin (Fig.
8a). Tetanus toxin had no
effect on VAMP from nonpermeabilized platelets. Given the observation
that tetanus toxin degrades the VAMP family protein found in platelets,
we sought to determine whether tetanus toxin would inhibit
Ca2+-mediated Effect of Antibodies Directed against Syntaxin 4 on
Ca2+-induced An understanding of the mechanisms that mediate Of the exocytotic core components evaluated in this study, only
syntaxin 4 has been previously demonstrated in platelets. Lemons
et al. (11) did not find VAMP-2 in a preparation from outdated platelets enriched for SNARE proteins by immunoprecipitation of a 20 S complex that typically includes a VAMP species. They probed
for VAMP-2 using a monoclonal antibody (clone C1 41.1). In contrast, we
detected VAMP in platelets using a polyclonal antibody directed against
a peptide fragment of VAMP-2 and a different monoclonal antibody (clone
SP10) that recognizes both VAMP-1 and VAMP-2. Platelet VAMP has an
apparent molecular weight that is distinct from VAMP species found in
brain (Fig. 1). In addition, greater concentrations of tetanus toxin
are required to cleave and inactivate platelet VAMP (>0.5
µM) than are required to cleave neuronal VAMP (<50
nM) (Fig. 8) (49, 50). Furthermore, platelet VAMP is not
recognized by monoclonal antibodies specific to either VAMP-2 (Lemons
et al. (11)) or VAMP-1 (data not shown). For these reasons,
we believe that platelet VAMP represents a novel species of VAMP. VAMP
species with reduced or absent sensitivity to clostridial toxins have
been identified in pancreatic zymogen granules (21), enterochromaffin
cells (51), and adipocytes (52). However, the apparent molecular weight
of the VAMP species described in these studies was similar to those of
VAMP-1 and VAMP-2.
Lemons et al. (11) similarly did not find SNAP-25 in
platelets. However, we found SNAP-23, which has a wider tissue
distribution (53), in platelets (Fig. 1). Platelets contain both
syntaxin 2 and 4 but not syntaxins 1, 2, and 5 (11). The anti-syntaxin 4 antibody used in these studies to detect platelet syntaxin was raised
to syntaxin 4, which has less than 50% amino acid sequence homology
with syntaxin 2. Furthermore, this antibody does not cross-react with
purified syntaxin 2. Therefore, the syntaxin isoform relevant to these
studies is syntaxin 4.
The novel VAMP, SNAP-23, and syntaxin 4 assemble into a heterotrimeric
complex in platelets as evidenced by the presence of a SDS-resistant,
heat-sensitive complex in platelets. This complex contains platelet
VAMP, SNAP-23, and syntaxin 4 as demonstrated by the fact that bands of
approximately 70 kDa are detected by anti-VAMP, anti-SNAP-23, and
anti-syntaxin 4 antibodies. The fact that the anti-VAMP and
anti-SNAP-23 antibodies detect a doublet whereas the anti-syntaxin 4 antibody does not (Fig. 2) suggests that the doublet may represent
complexes containing syntaxin 2 and syntaxin 4, which differ in
molecular mass. A recently published study by Foster et al.
(29) using recombinant VAMP-2, SNAP-23, and syntaxin 4 in an vitro
binding assay did not demonstrate a SDS-resistant complex of these
three proteins (29). The discrepancy between these results and the
detection of a SDS-resistant complex containing a novel VAMP isoform,
SNAP-23, and syntaxin in platelets may result from the fact that only
this novel VAMP can participate in the complex. Alternatively, the
differences in experimental conditions between an in vitro
assay using recombinant proteins and detecting native complex in
vivo may account for the difference in results. The fact that this
complex disassembles upon platelet activation is consistent with
observations in neuronal and neuroendocrine systems that the exocytotic
core complex is disassembled upon membrane fusion. In these other
systems, soluble SNARE proteins including
N-ethylmaleimide-sensitive fusion protein (NSF) and SL-O-permeabilized cell models have been used to define the
molecular mechanisms of various secretory events in neuronal, neuroendocrine, and endocrine cells. For example, incubation of SL-O-permeabilized pancreatic In this study, we have used a SL-O-permeabilized platelet
model to demonstrate that both vSNAREs and tSNAREs mediate With regard to tSNAREs, we assessed the function of syntaxin 4 in
The presence of the exocytotic core complex in platelets (11) and the
demonstration that constituents of this complex mediate In summary, we have detected a heterotrimeric exocytotic core complex
in platelets and have demonstrated that a platelet VAMP and syntaxin 4 mediate -granule secretion. A vesicle-associated membrane protein, SNAP-23,
and syntaxin 4 were detected in platelet lysate. These proteins form a
SDS-resistant complex that disassembles upon platelet activation. To
determine whether these proteins are involved in
-granule secretion,
we developed a streptolysin O-permeabilized platelet model
of
-granule secretion. Streptolysin O-permeabilized
platelets released
-granules, as measured by surface expression of
P-selectin, in response to Ca2+ up to 120 min after
permeabilization. Incubation of streptolysin O-permeabilized platelets with an antibody directed against
vesicle-associated membrane protein completely inhibited
Ca2+-induced
-granule release. Tetanus toxin cleaved
platelet vesicle-associated membrane protein and inhibited
Ca2+-induced
-granule secretion from streptolysin
O-permeabilized platelets. An antibody to syntaxin 4 also
inhibited Ca2+-induced
-granule release by
approximately 75% in this system. These results show that
vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form
a heterotrimeric complex in platelets that disassembles with
activation and demonstrate that
-granule release is dependent on
vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions.
INTRODUCTION
Top
Abstract
Introduction
References
-Granules are the most abundant platelet secretory granule.
These granules contain many components that have been implicated in
thrombosis and atherosclerosis, including adhesion molecules, coagulation factors, soluble mediators of inflammation, and growth factors (1).
-Granule constituents are released from the platelet after platelet stimulation. Secretion of
-granules is tightly controlled to prevent unregulated release. Although there are many
known signal transduction pathways that are activated concurrently with
-granule secretion, little is known regarding the mechanisms that
lead directly to membrane fusion between granules and surface-connected membranes. Ultrastructural studies have shown that the
-granules of
platelets are secreted primarily via fusion with the surface-connected open canalicular system (2) after apparent centralization of granules
and microtubule reorganization. Such observations have led to
speculation that cytoskeletal reorganization is responsible for
-granule release. However, inhibition of actin polymerization (3) or
microtubule organization (4, 5) does not inhibit granule secretion.
Furthermore, granule secretion and shape change can be dissociated
under several experimental conditions (6-8). Thus,
-granule
secretion is not solely dependent on shape change, indicating that
other mechanisms need to be explored to understand the molecular
mechanisms of
-granule secretion.
-granule proteins are
acquired via both fluid phase and receptor-mediated endocytosis, the
majority of
-granular proteins are incorporated during
thrombopoiesis (27). In contrast, biogenesis of synaptic vesicles
occurs de novo from newly synthesized proteins and from assembly of recycled vesicles (28). Furthermore, platelets have a
unique secretory pathway. Morphologic studies have shown that platelet
granules are secreted primarily via fusion with the open canalicular
system (1). Such studies have also shown that platelet granules fuse
with one another before exocytosis. However, platelet granule fusion
with plasma membrane occurs only occasionally. In contrast, vesicles
from neurons and neuroendocrine cells are exocytosed via fusion with
plasma membrane exclusively. Whether the molecular mechanisms mediating
vesicle-membrane fusion in platelets and cells of neural crest origin
also differ is not well characterized. An exocytotic complex consisting
of VAMP 2, SNAP-23, and syntaxin 4 has been found in several
nonneuronal cell types. However, these individual components failed to
form a heterotrimeric complex when co-incubated in vitro
(29). Furthermore, a previously published study failed to demonstrate
some of the essential components of the exocytotic machinery
(e.g. SNAP-25 and VAMP-2) in platelets (11). In this report,
we demonstrate the presence of an exocytotic core complex consisting of
a VAMP, SNAP-23, and syntaxin 4 in platelet lysate and show that
platelet vSNARE-tSNARE interactions mediate
-granule secretion from platelets.
EXPERIMENTAL PROCEDURES
-granule secretion occurs in permeabilized platelets at pH
6.4.2 After a 15-min
incubation, Ca2+ was added to the reaction tube unless
otherwise indicated. The amount of CaCl2 required to give a
free Ca2+ concentration of 10 µM in the
presence of 2 mM EGTA was calculated for each condition
using a computer program (gift from Dr. P. J. Padfield) based on
the algorithms described by Fabiato and Fabiato (32). After an
additional 5-min incubation, PE-conjugated AC1.2 anti-P-selectin
antibody (10 µl) was transferred to the reaction tube.
Phosphate-buffered saline (500 µl) was added to the sample after a
15-min incubation, and the platelets were analyzed immediately by flow
cytometry as described below. In experiments designed to determine the
efficacy of various inhibitors, the inhibitor was added 45 min before
the addition of MgATP. Tetanus toxin was activated with the addition of
5 mM dithiothreitol.
-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue) at
37 °C. SDS-PAGE using either 10%, 12% gels, or 8%/15%
discontinuous gels, immunoblotting, and enhanced chemiluminescence were
carried out using standard protocols (33, 34). Densitometry was
performed using Gelbase/Gelblot-Pro software on a MacIntosh PowerPC.
RESULTS
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Fig. 1.
Detection of VAMP, SNAP-23, and syntaxin 4 in
platelet lysate. a, proteins from bovine brain (20 µg) and human platelets (50 µg) were solubilized at 100 °C in
sample buffer, separated by SDS-PAGE on a 12% gel, and
electrophoretically transferred to polyvinylidene difluoride membranes.
Immunoblotting was performed with anti-VAMP antibody. b,
proteins from bovine brain (20 µg) and human platelets (1 µg) were
prepared as in a, and immunoblotting was performed with
anti-SNAP-23 antibody. c, proteins from human endothelial
cells (20 µg) and human platelets (3 µg) were prepared as in
a, and immunoblotting was performed with anti-syntaxin 4 antibody. Bands were visualized using enhanced chemiluminescence for
detection. The position of the molecular mass standards used are
indicated on the right.
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Fig. 2.
Detection of a SDS-resistant, heat-sensitive
complex in platelets. Gel-filitered platelets were solubilized in
sample buffer at either 37 °C or 100 °C, as indicated. Platelet
proteins (50 µg) were then separated by SDS-PAGE and
electrophoretically transferred to polyvinylidene difluoride membranes.
Immunoblotting was performed using either anti-VAMP monoclonal antibody
(a), anti-SNAP-23 polyclonal peptide antibody
(b), or anti-syntaxin 4 monoclonal antibody (c).
Bands were visualized using enhanced chemiluminescence for detection.
The position of the molecular mass standards used are indicated on the
right.
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Fig. 3.
Disassembly of the heterotrimeric complex
upon exposure of intact platelets to SFLLR. Gel-filtered platelets
(50 µg), either resting or exposed to 100 µM SFLLR,
were solubilized in sample buffer at either 37 or 100 °C, as
indicated. Platelet proteins were then separated by SDS-PAGE and
electrophoretically transferred to polyvinylidene difluoride membranes.
a, monomers and complex were visualized using a mixture of
anti-VAMP-2, anti-SNAP-23, and anti-syntaxin 4 antibodies and enhanced
chemiluminescence for detection. b, bands detected in lysate
from resting and activated platelets solubilized at 37 °C were
quantitated by densitometry. Band intensities are expressed as percent
intensity in activated platelet samples compared with bands from
resting platelets.
-Granule Secretion in
SL-O-permeabilized Platelets--
We next sought to determine whether
the components of the exocytotic core complex serve a functional role
in
-granule secretion. To access the platelet cytoplasm in whole,
functional platelets with specific inhibitors of SNARE proteins such as
antibodies and clostridial toxins, we exposed platelets to SL-O. SL-O
was used for this purpose because it inserts preferentially into plasma membrane without permeabilizing granules; it creates stable, uniform pores, large enough to permit molecules of greater than 150 kDa into
the cytoplasm. The efficacy of permeabilization was assessed by
incubating SL-O-permeabilized platelets with fluorescent
compounds of different molecular masses and quantitating the
internalization of the compounds by flow cytometry (38). The results
depicted in Fig. 4 demonstrate that
although incorporation of fluorescent compounds into
SL-O-permeabilized platelets decreases with increasing molecular mass, incorporation of compounds as large as 260 kDa is
significantly increased in permeabilized versus
nonpermeabilized platelets. These results demonstrate that
permeabilization with SL-O will allow antibodies and clostridial toxins
access to the platelet cytoplasm.
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Fig. 4.
Permeabilization of platelets with SL-O.
Gel-filtered platelets (20 µl) were incubated with 2 units/ml SL-O or
buffer in the presence of fluorescein isothiocyanate-dextran sulfate of
the indicated molecular mass. Fluorescent compounds were at 25 µM. After 15 min, platelets were diluted in
phosphate-buffered saline (500 µl) and analyzed immediately by flow
cytometry. Incorporation of the indicated compounds into platelets is
expressed as the fluorescence of SL-O-exposed platelets relative to
nonpermeabilized platelets. Error bars represent the S.E. of
four independent experiments.
-granules in response to Ca2+. In these
experiments, platelets were permeabilized with SL-O and subsequently
exposed to MgATP. Incubation of permeabilized platelets with MgATP has
previously been shown to be necessary for Ca2+-stimulated
secretion (39). After 15 min, platelets were exposed to either buffer
alone, SFLLR, or Ca2+. Intact platelets were also exposed
to buffer alone, SFLLR, or Ca2+. Platelets were then
assessed for
-granule release by assaying for surface expression of
P-selectin by flow cytometry.2
SL-O-permeabilized platelets express P-selectin on their
surface in response to Ca2+ (Fig.
5). Ca2+-mediated
-granule
release from SL-O-permeabilized platelets was approximately equivalent
in degree to that elicited from intact platelets by exposure to SFLLR.
In contrast, SFLLR failed to stimulate
-granule release from
SL-O-permeabilized platelets (39). Similarly, Ca2+ failed to stimulate
-granule release from
nonpermeabilized platelets (Fig. 5).
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Fig. 5.
Ca2+ stimulates -granule
secretion from SL-O-permeabilized platelets.
Gel-filtered platelets (20 µl) were incubated with buffer
(nonpermeabilized) or 2 units/ml SL-O (permeabilized) for 5 min.
Permeabilized samples were then incubated for 15 min with 5 mM MgATP at pH 6.4. Buffer, 10 µM
Ca2+, or 100 µM SFLLR was then added to the
nonpermeabilized and permeabilized platelets as indicated, and the
reaction mixtures were allowed to incubate for 5 min. P-selectin
surface expression was assayed by incubating samples with a
PE-conjugated AC1.2 anti-P-selectin antibody (10 µl) for 15 min.
Phosphate-buffered saline (500 µl) was added to the sample after a
15-min incubation, and the platelets were analyzed immediately by flow
cytometry as described under "Experimental Procedures." P-selectin
expression is indicated by PE-AC1.2 fluorescence. Data represent the
distribution of the relative fluorescence of 10,000 platelets.
-granules in response to Ca2+, and this response is
stable for at least 2 h.
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Fig. 6.
SL-O-permeabilized platelets
secrete -granules in response to Ca2+ for 2 h after
permeabilization. Gel-filtered platelets (20 µl) were incubated
with 2 units/ml SL-O for 5 min. 5 mM MgATP was then added
to the reaction mixture at pH 6.4. After the indicated amount of time
after permeabilization, Ca2+ (
) or buffer (
) was
added to the reaction tube. After an additional 5-min incubation,
PE-conjugated AC1.2 anti-P-selectin antibody (10 µl) was transferred
to the reaction tube. Phosphate-buffered saline (500 µl) was added to
the sample after a 15-min incubation, and the platelets were analyzed
immediately by flow cytometry. P-selectin expression is indicated by
PE-AC1.2 fluorescence. Error bars represent the S.E. of
three independent experiments.
-Granule Secretion--
We next sought to
determine whether platelet VAMP plays a role in mediating
-granule
secretion. In these experiments, we incubated
SL-O-permeabilized platelets with polyclonal anti-VAMP-2 antibody for 1 h before exposure to Ca2+. Anti-VAMP-2
antibody inhibits Ca2+-mediated P-selectin surface
expression from SL-O-permeabilized platelets by greater than
95% (Fig. 7). In contrast, rabbit
nonimmune IgG had no effect on Ca2+-mediated P-selectin
surface expression. To determine whether an antibody directed at an
-granule membrane protein thought not to be involved in
-granule
secretion affects secretion (41), we exposed
SL-O-permeabilized platelets to a polyclonal antibody directed against the cytoplasmic tail of P-selectin (30). This antibody
had no effect on Ca2+-induced P-selectin surface
expression. This result argues that the P-selectin cytoplasmic tail is
not involved in secretion despite its phosphorylation during activation
(42). It also suggests that an antibody directed at a
-granule
surface protein will not nonspecifically interfere with secretion.
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Fig. 7.
Effect of anti-VAMP-2 antibody on P-selectin
surface expression in SL-O-permeabilized platelets.
A, gel-filtered platelets (20 µl) were incubated with 2 units/ml SL-O for 5 min. Platelets were then incubated for 45 min with
buffer (panels a and b), 50 µg/ml nonimmune IgG
(panel c), or 50 µg/ml anti-VAMP antibody (panel
d). MgATP (5 mM) was added to the reaction mixture at
pH 6.4, and the reaction mixtures were incubated for 15 min. To
stimulate platelet activation, either buffer (panel a) or
Ca2+ (panels b, c, and d)
was added to the reaction tube. Samples were assayed for P-selectin
surface expression as described previously. Histograms represent the
distribution of the relative fluorescence of 10,000 platelets.
P-selectin expression is indicated by PE-AC1.2 fluorescence.
B, data are expressed as percent P-selectin expression
compared with Ca2+-stimulated samples exposed to buffer
alone. Error bars represent the S.E. of four independent
experiments.
-granule secretion. In these experiments,
SL-O-permeabilized platelets were exposed to 2 µM tetanus
toxin for 1 h before exposure to Ca2+. Tetanus toxin
inhibited Ca2+-mediated surface P-selectin surface
expression by approximately 70% (Fig. 8B). Dithiothreitol,
which was used to reduce the tetanus toxin, had no significant effect
on Ca2+-induced P-selectin surface expression. Tetanus
toxin that had been inactivated by boiling for 10 min had no effect on
-granule secretion (data not shown). The effect of tetanus toxin was
dose-dependent (Fig. 8C).
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Fig. 8.
Effect of tetanus toxin on platelet VAMP and
on P-selectin surface expression in SL-O-permeabilized
platelets. A, gel-filtered platelets (250 µl) were
exposed to either 2 units/ml SL-O or buffer for 5 min. Platelets were
then incubated with 1 µM tetanus toxin or buffer for 45 min. Platelet proteins were separated by SDS-PAGE and transferred to a
polyvinylidene difluoride membrane. VAMP was visualized by
immunoblotting using an anti-VAMP peptide antibody and enhanced
chemiluminescence for detection. B, gel-filtered platelets
(20 µl) were incubated with 2 units/ml SL-O for 5 min. Platelets were
then incubated for 45 min with buffer (panels a and
b), 5 mM dithiothreitol (panel c), or
2 µM tetanus toxin (panel d). MgATP (5 mM) was added to the reaction mixture at pH 6.4, and the
reaction mixtures were incubated for 15 min. To stimulate platelet
activation, either buffer (panel a) or Ca2+
(panels b, c, and d) was added to the
reaction tube. Samples were assayed for P-selectin surface expression
as described previously. Histograms represent the
distribution of the relative fluorescence of 10,000 platelets.
P-selectin expression is indicated by PE-AC1.2 fluorescence.
C, SL-O-permeabilized platelets were exposed to
increasing concentrations of reduced tetanus toxin for 45 min, exposed
to MgATP at pH 6.4, and stimulated with Ca2+. -Granule
secretion was than assessed by assaying for P-selectin surface
expression. Data are expressed as percent P-selectin expression
compared with Ca2+-stimulated samples exposed to buffer
alone.
-Granule Secretion--
Syntaxin 4 is
found in platelets (11) and is thought to play a role in the regulated
secretion of granules from hematopoietic cells (14). To determine
whether this tSNARE mediates
-granule secretion, we incubated
SL-O-permeabilized platelets with an anti-syntaxin 4 monoclonal antibody for 1 h before exposure to Ca2+.
Anti-syntaxin 4 antibody inhibited Ca2+-induced P-selectin
surface expression by approximately 75% (Fig. 9). Nonimmune mouse IgG1 had
no effect.
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Fig. 9.
Effect of anti-syntaxin 4 antibody on
P-selectin surface expression in SL-O-permeabilized
platelets. A, gel-filtered platelets (20 µl) were
incubated with 2 units/ml SL-O for 5 min. Platelets were then incubated
for 45 min with buffer (panels a and b), 50 µg/ml nonimmune IgG (panel c), or 50 µg/ml anti-syntaxin
4 antibody (panel d). MgATP (5 mM) was added to
the reaction mixture at pH 6.4, and the reaction mixtures were
incubated for 15 min. To stimulate platelet activation, either buffer
(panel a) or Ca2+ (panels b,
c, and d) was added to the reaction tube. Samples
were assayed for P-selectin surface expression as described previously.
Histograms represent the distribution of the relative
fluorescence of 10,000 platelets. P-selectin expression is indicated by
PE-AC1.2 fluorescence. B, data are expressed as percent
P-selectin expression compared with Ca2+-stimulated samples
exposed to buffer alone. Error bars represent the S.E. of
four independent experiments.
DISCUSSION
-granule
release has evolved over the past two decades. Morphologic studies demonstrated that platelets undergo shape change upon activation that
results in the apparent centralization of granules and in microtubular
band reorganization (44-46). These observations have led investigators
to evaluate the roles of actin and microtubule reorganization in
granule secretion. However, experiments using cytochalasin E at
concentrations that inhibit actin polymerization and platelet shape
change have no effect on
-granule release (3). Similarly, although
pharmacological agents that affect microtubule organization inhibited
granule secretion in some studies, further investigation suggested that
these agents worked through mechanisms other than disrupting
microtubule reorganization (4, 5). Furthermore, granule secretion and
shape change can be dissociated under several experimental conditions
(6-8). Subsequent detailed ultrastructural studies demonstrated that
71% of dense granules are within 12.5 nm of the plasma membrane and
that this population is preferentially released upon platelet
activation (47, 48). Thus, it is not certain that granule movement is required for platelet granule secretion. Some of the protein
components, termed SNARE proteins, of the secretory machinery that
mediate vesicle secretion in other cell types have been shown to exist in platelets (11). However, several of the components thought to be
essential for granule release were not found. The present study
demonstrates that three SNARE proteins, VAMP, SNAP-23, and syntaxin 4, are found in platelets and form an exocytotic core complex that
disassembles upon platelet activation. This study also uses a
functional assay to show that vSNARE-tSNARE interactions mediate
-granule secretion.
-SNAP
are thought to mediate core complex disassembly via NSF hydrolysis of
ATP. This mechanism has yet to be evaluated in platelets. However, the
experiments performed in this study do demonstrate that constituents of
this core complex participate in
-granule secretion.
cells with an antibody
directed at synaptotagmin III inhibited insulin secretion (54).
Incubation of SL-O-permeabilized enterochromaffin cells and
islet
cell lines with tetanus toxin inhibited
Ca2+-evoked release of amylase (55) and insulin (56)
release, respectively. The finding that the Ca2+-mediated
secretory response of SL-O-permeabilized platelets is stable
over time has certain implications with regard to the organization of
the secretory machinery in platelets. For example, the SNARE hypothesis
maintains that after interactions between vSNAREs and tSNAREs,
cytoplasmic factors associate with these membrane proteins and mediate
fusion events (17). The concentration of such cytoplasmic factors is
decreased by several orders of magnitude upon permeabilization. In many
systems, cytosol must be included in the permeabilization buffer to
retain the secretory response to Ca2+ (40, 57, 58). The
fact that platelets retain the ability to secrete
-granules upon
exposure to Ca2+ after SL-O permeabilization without added
cytosol raises the possibility that either cytoplasmic factors are not
required for
-granule secretion or that such factors are already
bound to the vSNARE-tSNARE complexes before permeabilization. Further
studies will be required to differentiate between these two possibilities.
-granule secretion. Platelet VAMP mediates
-granule secretion, as evidenced by the fact that both an antibody directed against the protein and
tetanus toxin, which cleaves VAMP (31), inhibit
-granule secretion.
The concentration of tetanus toxin required to achieve 50% inhibition
of
-granule secretion in this system was approximately 900 nM. This concentration is more than 10-fold greater than
that required to inhibit catecholamine release from permeabilized
chromaffin cells (49) or acetylcholine release from synaptosomes (50). However, the concentrations of tetanus toxin required to inhibit
-granule release are similar to those required to inhibit
Ca2+-induced histamine release from permeabilized
enterochromaffin cells (51). These differences may be because platelets
and enterochromaffin cells contain VAMP species that are relatively
poor substrates for tetanus toxin compared with VAMP-1 and VAMP-2.
-granule secretion. Both syntaxins 2 and 4 are found in platelets
(11). Syntaxin 2 is widely expressed and is thought to participate in
constitutive secretion (20). In contrast, syntaxin 4 is more
specifically expressed. Other hematopoetic cells, including macrophages
and neutrophils, synthesize syntaxin 4 (13, 14). Syntaxin 4 may play a
role in the regulated secretion of lysosomes by neutrophils (14). For
these reasons, we choose to target syntaxin 4 to determine whether or
not syntaxin family proteins function in
-granule secretion. The
fact that this antibody inhibits
-granule secretion in
SL-O-permeabilized platelets implicates this syntaxin
isoform in
-granule secretion. However, this antibody does not
completely block
-granule secretion. This observation suggests that
either other molecules (e.g. syntaxin 2) can partially compensate for syntaxin 4 activity or that this particular monoclonal antibody is not capable of inhibiting syntaxin 4 activity completely.
-granule
secretion suggest a model for
-granule secretion from platelets.
According to this model, platelet VAMP, SNAP-23, and syntaxin 4 form a
complex that docks
-granules to surface-connected membranes,
primarily the open canalicular system. The heterotrimeric complex is
then decorated with soluble SNARE proteins such as NSF and
-SNAP,
both of which have been found in platelets (11). Binding of these
soluble factors to the heterotrimeric complex may occur before
activation, because permeabilization of platelets does not create a
requirement for exogenously added factors. With activation, hydrolysis
of ATP by NSF and release of Ca2+ into the cytosol cause
complex disassembly and membrane fusion. Further investigation will be
required to evaluate this model and define the relationship between
platelet signaling events and SNARE protein rearrangements leading to
membrane fusion.
-granule secretion. These observations suggest that despite
morphologic differences in granule secretion between platelets and
nucleated cells,
-granule secretion shares several fundamental
mechanisms with neuronal and neuroendocrine cell vesicle secretion at
the molecular level. An understanding of the molecular mechanisms of
-granule secretion is not only of interest in platelet biology but
is also of pharmacological interest given the possibility that proteins
mediating
-granule secretion may serve as appropriate targets for
anti-thrombogenic agents.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL51926 and HL57140.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.
¶ A Howard Hughes Medical Institute Physician Postdoctoral Fellow. To whom correspondence should be addressed: RE 318, Research East, P. O. Box 15732, Boston, MA 02215. Tel.: 617-667-0627; Fax: 617-975-5505; E-mail: rflaumen{at}bidmc.harvard.edu.
The abbreviations used are: SNARE, SNAP receptor; SNAP-23, soluble (N-ethylmaleimide-sensitive fusion protein) attachment protein 23; VAMP, vesicle-associated membrane protein; SL-O, streptolysin O; PE, phycoerythrin: PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; NSF, N-ethylmaleimide-sensitive fusion protein.
2 Flaumenhaft, R., Furie, B., Furie, B. C., J. Cell. Physiol., in press.
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