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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 921-929
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Molecular mechanisms of platelet exocytosis: role of SNAP-23
and syntaxin 2 in dense core granule release
Dong Chen,
Audrey M. Bernstein,
Paula P. Lemons, and
Sidney W. Whiteheart
From the Department of Biochemistry, University of Kentucky College
of Medicine, Lexington, KY.
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Abstract |
To characterize the molecular mechanisms of platelet secretion, we
focused on the calcium-induced exocytosis of dense core granules.
Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as
syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated
protein 23). By using an in vitro exocytosis assay, we have been able
to assign roles for some of these t-SNAREs in dense core granule
release. This calcium-induced secretion relies on the SNARE proteins
because it is stimulated by the addition of recombinant  -SNAP and
inhibited by a dominant negative -SNAP-L294A mutant or by
anti--SNAP and anti-NSF antibodies. SNAP-23 antibodies and an
inhibitory C-terminal SNAP-23 peptide both blocked dense core granule
release, demonstrating a role for SNAP-23. Unlike other cell types,
platelets contain a significant pool of soluble SNAP-23, which does not
partition into Triton X-114. Of the anti-syntaxin antibodies tested,
only anti-syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and
SNAP-23 do form a complex in vivo. These data clearly show that SNAPs,
NSF, and specific t-SNAREs are used for dense core granule release;
these data provide a greater understanding of regulated exocytosis in platelets.
(Blood. 2000;95:921-929)
© 2000 by The American Society of Hematology.
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Introduction |
Platelet activation and the release reaction can be
summarized as a progression through 3 steps: (1) an activating event
caused by contact with an agonist such as thromboxane, platelet
activating factor, collagen, or thrombin; (2) the generation of
intracellular signals through such molecules as G proteins,
phospholipase C, phospholipase A, and protein kinase C, and calcium
efflux from the dense tubular system or influx from the outside; and
(3) a set of cellular responses that include cytoskeletal rearrangement and exocytosis of storage granules (reviewed in
1). Platelets contain 3 types of granules:
dense core granules, containing such small molecules as ADP,
serotonin, calcium, and pyrophosphate;  -granules, containing such
proteins as von Willebrand factor, thromboglobulin, and
platelet-derived growth factor (PDGF); and lysosomes, containing acid
hydrolases (reviewed in 1). After platelet activation, the
contents of these granules are released into the extraplatelet space.
The exocytotic pathway in platelets is unique. As the platelet changes
shape upon stimulation, the secretory granules become increasingly
centralized within a constricting microtubular coil. These granules
then either fuse directly with the invaginations of the plasma
membrane, called the open canalicular system, or fuse with one another
(compound fusion) and then with the open canalicular
system.2-4 Although the molecular mechanism of signal
transduction has been studied extensively, the mechanism of granule to
plasma membrane fusion is still unclear.
The molecular mechanisms of membrane fusion events in other systems
have been studied intensively over the past few years.5-7 A
growing body of data supports the concept that membrane proteins from
both the transport vesicle and target membrane are, at least in part,
responsible for the specific fusion of the 2 lipid bilayers. As
originally stated,8 the SNARE (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptor) hypothesis proposed that a vesicle membrane protein from the synaptobrevin/VAMP (vesicle associated membrane protein) family (v-SNARE)
binds specifically to a heterodimeric complex in the target membrane
(t-SNARE) made up of 1 member of the syntaxin family and 1 from the
synaptosomal associated protein (SNAP)-23/25 family. The resulting
heterotrimeric, intermembrane complex is the core complex that is
minimally required for membrane fusion.9 It is also clear
from this body of work that there are numerous accessory proteins, such
as SNAPs and NSF, that "activate" the SNARE proteins so that they
attain fusion-competent configurations.10,11 Although the
specific details of the SNARE hypothesis continue to
evolve,12-14 it has served as a useful guide to dissecting
the mechanisms of exocytosis events.
In our initial studies of the molecular machinery of platelet
exocytosis, we reported that platelets contain the general accessory proteins -SNAP,  -SNAP, and NSF, as well as 2 specific plasma membrane t-SNAREs, syntaxin 2 and syntaxin 4.15 Subsequent
work by others using antibody inhibition experiments has shown that syntaxin 4 mediates -granule release.16 As to the
heterodimeric partner t-SNARE, SNAP-25 was undetectable in platelets,
so SNAP-23 became a good candidate. Several studies have indicated that
SNAP-23 can heterodimerize with each of the plasma membrane syntaxins (i.e., 1, 2, 3, and 4).17-20 SNAP-23 is expressed in
numerous tissues, where it has been localized to the plasma
membrane,21-24 mast cell granules,25 and
endosome.26 It has been detected in platelets and was
suggested to play a role in -granule release.16 As for
v-SNAREs, only 1 has been described in platelets,
VAMP-3/cellubrevin.27 Although it is clear
from botulinum toxin-based studies that at least 1 v-SNARE is required
for -granule exocytosis,16 it is unclear whether it is
VAMP-3/cellubrevin. Taken together, these data show that
many new insights into the mechanisms of platelet exocytosis are being
made, especially regarding -granule release, yet it is clear that
there are other, yet to be identified, components that play a role in
dense core granule and lysosome exocytosis.
In this report, we demonstrate that dense core granule secretion is
mediated by the general membrane-fusion components such as -SNAP and
NSF. Using an in vitro exocytosis assay, we also provide evidence that
the t-SNAREs SNAP-23 and syntaxin 2, but not syntaxin 4 or 7, are
involved in dense core granule exocytosis from platelets.
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Materials and methods |
Antibodies and reagents
Polyclonal anti--SNAP, anti-SNAP-23, and anti-syntaxin 2 and 4 antibodies were generated by immunizing rabbits with appropriate recombinant proteins.26,28 The lack of cross-reactivity
with SNAP-25 by our anti-SNAP-23 antibody is demonstrated in Figure 1A. Anti-syntaxin 7 antibodies were also
produced in our laboratory using a recombinant protein generated from a
human expressed sequence tag (Accession N31 042; Genome Systems Inc,
St Louis, MO). All antibodies were affinity purified with the
appropriate recombinant protein or protein G Sepharose
(Pharmacia, Piscataway, NJ). Fab fragments of ab23 (Fab23)
were prepared using the ImmunoPure Fab preparation kit (Pierce,
Rockford, IL). It should be noted that the platelet forms of the 3 syntaxins differ in apparent molecular weight (Figure 1B). These data
demonstrate the specificity of the antibody reagents used in this study
because none of our syntaxin-specific antibodies cross-reacted with an
inappropriate syntaxin. The anti-NSF monoclonal antibody
2E529,30 was prepared from ascites and purified on protein G Sepharose (Pharmacia).
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| Fig 1.
t-SNAREs in the platelet.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blotting analyses were used to demonstrate the presence of
SNAP-23 (A) and syntaxins 2, 4, and 7 (B) in platelets. One hundred
micrograms of whole platelet lysate was separated by SDS-PAGE and then
transferred to nitrocellulose. The resulting blots were probed with
anti-syntaxin 2 (syntaxin 2), anti-syntaxin 4 (syntaxin 4), and
anti-syntaxin 7 (syntaxin 7), and the immunodecorated proteins were
detected by ECL. For A, the anti-SNAP-23 antibody was preincubated
with 500 µg of either recombinant SNAP-23 or SNAP-25, as indicated.
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Reduced streptolysin O (SLO) was purchased from Murex (Dartford, UK).
The SNAP-23 C-terminal peptide (ANARAKKLIDS) was a generous gift from
Dr. David Castle (University of Virginia, Charlottesville, VA).
Thrombin, apyrase VII, heparin, prostaglandin I2, sodium pyruvate, P-nitrophenyl-N-acetyl- -D-glucosaminide, and NADH
were purchased from Sigma (St. Louis, MO).
[1,2-3H(N)]-hydroxytryptamine ([3H]5-HT)
was purchased from NEN (Boston, MA). All other chemicals were of reagent grade.
Complementary DNAs encoding -SNAP and -SNAP mutant (L294A) were
inserted into the vector pQE-9 (Qiagen, Chatsworth, CA) using the
HindIII and BamHI restriction sites. Constructs were transfected into Escherichia coli M15 pREP4 cells. The
transfected cells were selected with ampicillin (100 µg/mL) and
kanamycin (50 µg/mL). The constructs were confirmed by dideoxyribose
nucleic acid sequencing. Production of recombinant
proteins was performed as described previously.31
Freshly banked platelets were procured as units from the Central
Kentucky Blood Center (Lexington, KY).
Immunoprecipitation and western blotting
For co-immunoprecipitation experiments, ab23 was covalently coupled
to protein G Superose using dimethyl
pimelimidate.8 Fifty micrograms of detergent-solubilized
platelet extract was incubated with the antibody beads at 4°C for 2 hours. Supernatants were collected, the beads were washed 5 times with
phosphate-buffered saline (PBS) and 1% Triton X-100, and the bound
material was eluted with 100 mmol/L glycine, pH 2.5, 1% Triton X-100.
The supernatants and bound material were subjected to Western blotting
analysis with the indicated antibodies. For all Western blotting
experiments, the Enhanced Chemiluminescence (ECL) detection system
(Pierce, Rockford, IL) was used with secondary antibodies covalently
coupled to horseradish peroxidase to visualize the immunodecorated
proteins. Protein concentrations were determined using the
bicinchoninic acid assay (Pierce).
Subcellular fractionation of platelets
One unit of platelets was sedimented (700g) and then
resuspended in 50 mL of platelet wash buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 3 mmol/L NaH2PO4 [pH 7.4], 5.5 mmol/L D-glucose, 1 mmol/L MgCl2, and 0.35% bovine serum
albumin [BSA]). The platelets were resedimented and resuspended in 5 mL of homogenizing buffer, 25 mmol/L HEPES (pH 7.0, 38 mmol/L KCl, 108 mmol/L NaCl, 1 mmol/L dithiothreitol [DTT], and
1 × magic mix containing 1 mmol/L o-phenanthroline, 10 mmol/L EGTA, 1 mmol/L leupeptin, 40 µg/mL antipain, 0.12 U/mL aprotinin, 1 µmol/L pepstatin, 1 mmol/L benzamidine, and 40 µg/mL chymostatin). The platelets were disrupted by 5 freeze-thaw cycles and
then clarified by centrifugation at 100 000g for 1 hour. The supernatant (cytosol) was collected. The pellet was washed with homogenizing buffer plus 1 mol/L KCl and 1 mmol/L DTT and
recentrifuged. The resulting pellet was resuspended in either 100 mmol/L Na2CO3, pH 11.5, or in 1% Triton PBS
and incubated on ice for 30 minutes, then subjected to centrifugation
at 100 000g for 1 hour. The supernatants (soluble fractions)
and pellets (insoluble fractions) were analyzed by western blotting.
Triton X-114 partitioning was performed as described by
Bordier.32 The cytosol and membrane fractions (the
fractions after 5 freeze-thaw cycles) were diluted (1:10 v:v) into
homogenization buffer containing 1 × magic mix and 1% Triton
X-114, and incubated on ice for 1 hour. The Triton X-114-insoluble
material was sedimented. The supernatants were warmed at 37°C for 5 minutes and then subjected to centrifugation for 3 minutes. The aqueous
phase and detergent phase were collected, precipitated with 12%
trichloroacetic acid, and subjected to Western blotting using ab23 and
anti-syntaxin 4 antibody.
Preparation of [3H]5-HT-labeled platelets
One unit of freshly banked platelets was incubated at room
temperature for 5 minutes in the presence of 10 ng/mL prostaglandin I2 and then sedimented at 700g for 15 minutes at
room temperature. The platelets were resuspended in 2 to 5 mL of
the platelet-poor plasma, and the concentration of platelets was
measured. The concentration of platelets was adjusted to 109
to 5 × 1010 platelets/mL by the addition of
extra platelet-poor plasma. For [3H]5-HT labeling,
platelets were incubated at 37°C for 40 minutes in the presence of
0.2 µCi/mL [3H]5-HT. The labeled platelets were washed
twice in Ca++-free Tyrode's solution (154 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 5.6 mmol/L D-glucose, 7 mmol/L
NaHCO3, 0.6 mmol/L NaH2PO4, 5 mmol/L sodium PIPES [pH 6.5], 0.35% BSA, 5 mmol/L EGTA [pH 6.5 adjusted with KOH], 0.03 mg/mL apyrase, and 50 U/mL heparin). The
platelets were washed 1 more time with the same medium without heparin
(but with apyrase). Finally, the platelets were resuspended in assay
buffer (120 mmol/L sodium glutamate, 5 mmol/L potassium glutamate, 20 mmol/L HEPES/NaOH, pH 7.4, 2.5 mmol/L EDTA, 2.5 mmol/L EGTA, 3.15 mmol/L MgCl2, and 1 mmol/L DTT), and the concentration of
the platelets was adjusted to 109/mL. Note that the
inclusion of DTT in this buffer did not affect the efficiency of the
subsequent release reactions and could be eliminated if the
streptolysin O used was fully reduced (data not shown).
Permeabilization of platelets with SLO and assay of 5-HT,
hexosaminidase, lactate dehydrogenase (LDH), and PDGF release
Fifty microliters of platelets (107-108
platelets) in assay buffer was mixed with 50 µL of assay buffer
containing 8 mmol/L ATP, 1.6 U/mL SLO, and antibodies or recombinant
proteins for 10 minutes at room temperature. The reactions were further
incubated on ice for 30 minutes. After the samples had been warmed to
25°C for 5 minutes, CaCl2 was added to give the desired
final concentration,33 and the reactions were incubated at
25°C for another 5 minutes. The reactions were stopped by placing
the samples on ice for 4 minutes, followed by centrifugation at
13 000g for 1 minute. The supernatants were collected and
assayed as described later (see Figure 4B).
[3H]5-HT release was measured by scintillation counter.
Hexosaminidase was measured as described by Holmsen and
Dangelmaier.34 Five milliliters of citrate-phosphate
buffer, pH 4.5, and 2.5 mL of 10 mmol/L substrate
(P-nitrophenyl-N-acetyl--D-glucosaminide) were mixed and
aliquoted (100 µL) into 96-well plates, and 5 µL of the reaction
supernatant was added. After incubation at 37°C for 18 hours, 60 µL of 0.08N NaOH was added to stop the reaction. The absorbance was
read in a Titertek Multiscan Plus ELISA plate reader (Labsystems,
Stockholm, Sweden) with a 405-nm filter. In these assays,
the no-enzyme background was subtracted (OD405 = 0.040).
For the LDH assay, 700 µL of 0.2 mol/L Tris-HCl, pH 7.4, was mixed
with 100 µL of 3 mmol/L NADH and 100 µL of 10 mmol/L pyruvate.
Forty-five microliters of the supernatant from the SLO experiment was
added into the prewarmed reaction buffer, and the decrease in
absorbance at 340 nm versus time was recorded and converted to enzyme activity.
Release of -granules was measured by quantitative enzyme-linked
immunosorbent assay (ELISA) for the -granule protein PDGF. Supernatants from the SLO-permeabilized platelets were added in triplicate to wells of a microtiter high-protein-binding ELISA plate
(Costar, Cambridge, MA) containing 200 µL of 15 mmol/L
Na2CO3 and 35 mmol/L NaHCO3, pH
9.6. The samples were dried onto the plate by overnight incubation at
37°C. The wells were washed twice with ELISA wash buffer (3.5 mmol/L NaH2PO4, 31 mmol/L
Na2HPO4, 15.4 mmol/L NaCl, and 0.5% Tween-20).
Blocking was done for 1 hour at room temperature with 3% BSA (Sigma)
in 1 × PBS. Anti-PDGF-BB (R&D Systems, Minneapolis, MN)
primary antibody was diluted to 1 µg/mL in blocking solution, added
to the wells, and incubated for 2 hours at room temperature. The wells
were washed 4 times, and anti-goat secondary antibody (coupled to
horseradish peroxidase), diluted in blocking solution, was added and
allowed to incubate for 1 hour at room temperature. The wells were
again washed 4 times, and 200 µL of 1 mmol/L ABTS
(2,2'-azino-di-[3-ethylbenzthiazoline sulfonate) was added.
Samples were quantified spectrophotometrically at 405 nm.
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Results |
Biochemical characterization of platelet SNAP-23
SNAP-25 and SNAP-23 are homologous proteins sharing 59% amino acid
identity.17 Both appear to be anchored to the cytosolic side of the plasma membrane by thioester-linked acyl
groups.35-39 SNAP-23 was shown in numerous cases to be
membrane associated.21-26 To test whether SNAP-23 behaves
as a peripheral or integral membrane protein in platelets, we performed
a subcellular fractionation experiment (Figure
2A). Platelets were disrupted and the
membranes were pelleted by centrifugation at 100 000g for 30 minutes. The supernatant (S fraction) was collected, and the membrane
pellet was washed with 1 mol/L KCl. The supernatant was collected (S2 fraction), and the salt-washed pellet was resuspended in either 1%
Triton X-100 in PBS or in 0.1 mol/L sodium carbonate, pH 11.5, and
subjected to further centrifugation. The supernatants (Stx, supernatant
of Triton X-100 solubilization; Sc, supernatant of carbonate wash) were
collected, and the pellets (Ptx, Triton-insoluble pellet; Pc, carbonate
wash-insoluble pellet) were resuspended in 0.2% sodium dodecyl
sulfate (SDS) in PBS. SNAP-23 is present in both cytosolic and membrane
fractions. The membrane-bound SNAP-23 is 1 mol/L KCl and sodium
carbonate-resistant but can be partially solubilized by Triton X-100.
Syntaxin 4 served as an integral membrane protein control for this
fractionation experiment; it was enriched in the membrane fraction, was
not released by sodium carbonate, and was soluble in Triton X-100. The
presence of soluble SNAP-23 was also demonstrated using SLO-treated
platelets (Figure 2C). A portion of the SNAP-23, but none of the
syntaxin 4, diffused out of the permeabilized cells and was detected in
the supernatant.
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| Fig 2.
Distribution of SNAP-23 in platelets.
(A) Platelets were disrupted by freeze-thaw cycles and fractionated
into a soluble fraction (S1) and pellet by ultracentrifugation. The
pellet was incubated with 1 mol/L KCl on ice for 30 minutes. The
supernatant (S2) was collected, and the resulting pellet was either
solubilized with 1% Triton X-100 in PBS or washed again with 200 mmol/L Na2CO3 for 30 minutes. The supernatants
(Triton-soluble, Stx; and carbonate-released, Sc) were collected and
the pellets (Ptx and Pc) were resuspended in SDS-loading buffer. All of
the above fractions were subjected to western blotting using ab23 and
anti-syntaxin 4 antibody. Based on comparison with the starting
material, approximately 38.2%, 16.5%, 33.5%, 31.1%, 14.2%, and
8.5% of the total platelet protein was in S1, S2, Sc, Stx, Ptx, and
Pc, respectively. (B) The initial soluble and membrane fractions from A
were incubated with 1% Triton X-114 for 30 minutes on ice. The aqueous
and detergent phases were separated by warming the samples to 37°C,
followed by centrifugation. The aqueous and detergent phases of both
the soluble fraction and membrane pellet as well as the Triton
X-114-insoluble pellet were analyzed by western blotting using ab23
and anti-syntaxin 4 antibody. His6-SNAP-23 was also
subjected to Triton X-114 partitioning, and the aqueous and detergent
phases were analyzed by western blotting using ab23. (C) Soluble
SNAP-23 is released from platelets after the treatment with 0.8 U/mL
SLO. Platelets (108) were treated with 0.8 U/mL SLO for 10 minutes and then pelleted by centrifugation. The supernatant (S) and
the pellet (P) were subjected to western blotting with ab23 and
anti-syntaxin 4.
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Because this was the first evidence of cytosolic SNAP-23, we sought to
determine whether this was due to a difference in the biochemical
properties of platelet SNAP-23. Platelets were fractionated into
soluble and membrane fractions, and each fraction was subjected to
Triton X-114 partitioning to determine whether SNAP-23 retained the
hydrophobicity of a membrane protein (Figure 2B). The aqueous phase and
the detergent phase were collected and subjected to Western blotting
using ab23 and anti-syntaxin 4 antibody. As a control, 90% of the
recombinant, unacylated, His6-SNAP-23 partitioned into the
aqueous phase, as expected for a hydrophilic protein. Soluble platelet
SNAP-23 was enriched in the aqueous phase, whereas most of the
membrane-bound SNAP-23 was in the detergent phase or in the
detergent-insoluble pellet. The soluble SNAP-23 and membrane-bound
SNAP-23 appeared to have a difference in hydrophobicity. As an internal
control, the true integral membrane protein, syntaxin 4, was enriched
in the detergent phase of the membrane fraction.
To determine whether the soluble pool of SNAP-23 changes during
platelet activation, we performed a similar sequential fractionation of
thrombin-treated (1 U/mL) and resting platelets (Figure
3). The membrane pellets were washed
sequentially with high salt and sodium carbonate, and each time the
supernatants were collected. Finally, the pellets were
resuspended in Triton X-100 and divided into Triton-soluble and
-insoluble fractions. All of the fractions were subjected to Western
blotting using ab23 and anti-syntaxin 4 antibody. After stimulation of
the platelets, the soluble fraction of SNAP-23 decreased (from 60% to
17% of total), with a subsequent increase (from 11% to 41% of total)
in the Triton X-100-insoluble fraction (Figure 3B). Although this
could be due to simple trapping, it was not seen for syntaxin 4 from the same samples.
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| Fig 3.
Distribution of SNAP-23 in resting and activated
platelets.
(A) Platelets were resuspended in Ca++-free Tyrode's
buffer or Tyrode's (1 mmol/L Ca++) buffer containing 1 U/mL thrombin for 5 minutes. The resting and activated platelets were
disrupted by freeze-thaw and fractionated by centrifugation. The
supernatant (S1) was collected, and the pellets were washed
sequentially with 1 mol/L KCl, 200 mmol/L
Na2CO3, and 1% Triton X-100, as in Figure 2
(S2, Sc, and Stx). The supernatants and Triton X-100-insoluble pellet
(Ptx) were analyzed by Western blotting using ab23 and anti-syntaxin 4 antibody. (B) The Western blotting image was scanned and digitized by
using NIH 1.6 program (available at rsb.info.nih.gov/nih.image).
The pixel number of each band was normalized as a
percentage of total pixel number in all lanes of the treatment group.
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Description of a permeabilized platelet exocytosis assay
To study the roles of the fusion machinery proteins in platelet
exocytosis, it was first necessary to develop an in vitro exocytosis
assay using permeabilized platelets. The initial step was to determine
the conditions and concentration of SLO needed to permeabilize platelet
plasma membrane without affecting granule integrity. SLO was incubated
at various concentrations with platelets for 10 minutes at 25°C or
37°C, followed by chilling on ice for 30 minutes and then
reincubation at 25°C or 37°C for another 10 minutes.
Permeabilization of the plasma membrane was measured by the appearance
of LDH in the media. The integrity of the dense core granules,
lysosomes, and -granules was followed by measuring the appearance of
[3H]5-HT, hexosaminidase, and PDGF, respectively (Figure
4). LDH appeared in the supernatant at SLO
concentrations of 0.6 to 0.8 U/mL at both 25°C and 37°C.
However, at 37°C, [3H]5-HT was released when the
concentration of SLO exceeded 0.4 U/mL. This leakage was delayed at
25°C, so the optimal permeabilization condition appeared to be 0.8 U/mL of SLO at 25°C. The integrity of lysosome and -granule
membrane was also maintained under these conditions (Figure 4).
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| Fig 4.
Streptolysin O permeabilization of platelets.
(A) Increasing amounts of SLO (0-1.2 U/mL final concentration) were
added to the assay buffer containing 108 platelets.
Platelets were incubated at 25°C or 37°C for 10 minutes,
chilled on ice for 30 minutes, and further warmed to 25°C or
37°C for 10 minutes. The platelets were sedimented and the
supernatants were collected. The activity of lactate dehydrogenase
(LDH) and hexosaminidase and the amounts of [3H]5-HT and
PDGF in the supernatant were measured (see "Materials and
Methods") and compared with the total activity of Triton
X-100-solubilized platelets (n = 4). (B) The time line represents
the standard reaction scheme used in the permeabilized platelet
exocytosis assay.
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The next step was to optimize the Ca++ concentration needed
to stimulate exocytosis (Figure 5A).
[3H]5-HT-labeled platelets were first treated with 0.8 U/mL SLO and then chilled on ice for 30 minutes. This 4°C step is
required to allow equilibration of potential activators and inhibitors into the platelets without further permeabilization of granule membranes. SLO is inactive at 4°C.40 When this step was
performed at room temperature, there was an increase in the
Ca++-independent release of granule stores (data not
shown), suggesting that the granules were permeabilized during the
incubation. After rewarming to 25°C for 5 minutes, the platelets
were treated with increasing concentrations of Ca++ for 5 minutes. Exocytosis was stopped by chilling the samples on ice for 4 minutes, and then the platelets were removed by centrifugation at
13 000g for 1 minute. The supernatants were analyzed for
[3H]5-HT release. As shown in Figure 5A, dense core
granule secretion did not occur until the calcium concentration reached
10 µmol/L. This is similar to the intracellular calcium concentration
reached upon stimulation of intact platelets with
thrombin.41 This calcium concentration curve was also
similar to that reported from other permeabilized platelet exocytosis
assays.42,43 As the calcium concentration increased,
exocytosis reached a plateau but the standard error between samples
increased. This suggests that at the high calcium concentrations,
artifactual membrane fusion events were randomly occurring. For this
reason and for agreement with previous studies,41-43 we
chose to use 10 µmol/L as the calcium concentration to stimulate
exocytosis. The calcium-stimulated exocytosis requires energy. When
apyrase was added without the addition of ATP (30 µg/mL of apyrase,
0.5 U per assay point, enough to degrade 0.5 µmol of ATP in 15 minutes), the release of [3H]5-HT was eliminated (Figure
5A).
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| Fig 5.
The effects of Ca++, -SNAP, and NSF on
[3H]5-HT release.
(A) Increasing Ca++
(10 8-103 mol/L final) was used to
induce exocytosis in the presence of 50 µg/mL of wild-type -SNAP
or mutant -SNAP-L294A. The released [3H]5-HT was
measured as in Figure 4. In an additional titration, 30 µg/mL of
apyrase was added to deplete ATP from the reaction (n = 6). (B) In a
separate, summary experiment, the effects of 50 µg/mL bovine
wild-type -SNAP, 60 µg/mL anti--SNAP antibody, and 60 µg/mL
rabbit IgG on the 10 µmol/L Ca++-triggered
[3H]5-HT secretion were compared with the control (100%)
(n = 6). (C) [3H]5-HT-labeled and SLO-permeabilized
platelets were incubated with increasing amounts of the 2E5 monoclonal
antibody (anti-NSF; 0-0.32 mg/mL). The release of
[3H]5-HT was measured as before and normalized to the
control (no addition) (n = 5). (D) The 2E5 inhibitory effect can be
reversed by the addition of recombinant NSF. Radiolabeled and
SLO-permeabilized platelets were incubated on ice for 30 minutes with
buffer (control), 80 µg/mL 2E5, 80 µg/mL plus 0.75 mg/mL
recombinant NSF, or 0.75 mg/mL recombinant NSF alone. Platelets were
activated by 10 µmol/L Ca++, and the release of
[3H]5-HT was measured and normalized to the control group
(n = 5).
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To optimize the release time, we analyzed the time course of the dense
core granule secretion using 10 µmol/L Ca++ to activate
secretion. The release of [3H]5-HT started immediately
after the addition of Ca++ and was complete by 3 minutes
(data not shown). All of our later secretion assays were stopped after
5 minutes of Ca++ treatment, when the release of
[3H]5-HT should be complete. For all subsequent assays,
0.8 U/mL of SLO was used to permeabilize the platelets following the
scheme outlined in Figure 4B, and incubation with 10 µmol/L
Ca++ for 5 minutes was used to stimulate exocytosis. These
same assay conditions were used to test the level of exocytosis in
fresh platelets as compared with the freshly banked platelets reported here. In 3 separate preparations, fresh platelets released
49% ± 2.3% (n = 9 data points) of the total cellular
[3H]5-HT, and freshly banked platelets released
45% ± 0.9% of the total [3H]5-HT (n = 20 data
points). Because there was no apparent difference between fresh and
freshly banked platelets, the remaining experiments were performed with
freshly banked cells. Also under these assay conditions, it is possible
to measure Ca++ and GTP--S-stimulated release from all
3 platelet granule stores, as well as a Ca++-induced
increase in fibrinogen receptor affinity and centralization of platelet
granules caused by cytoskeletal rearrangements (Chen, Lemmons, and
Whiteheart, unpublished data).
Platelet dense core granule secretion is facilitated by SNAPs and
NSF
Platelets contain -SNAP, -SNAP, and NSF.15 To test
whether SNAPs are involved in platelet secretion, we added 50 µg/mL of wild-type -SNAP and dominant negative -SNAP-L294A
mutant44 to the Ca++ titration assay during the
SLO incubation step (Figure 5A). After 30 minutes of incubation on ice,
increasing amounts of Ca++ were added to the reaction, and
the release of [3H]5-HT was measured (Figure 5A).
Wild-type -SNAP appeared to increase the extent of
[3H]5-HT secretion from 43% to 68% of the total. The
mutant -SNAP decreased [3H]5-HT secretion to 27% of
the total. The effects of wild-type and mutant -SNAP were most
apparent at 10 µmol/L calcium and less so at higher calcium
concentrations. Research has shown that -SNAP also stimulates
membrane trafficking events, although not as effectively as
-SNAP.45,46 Addition of -SNAP (50 µg/mL) did
increase [3H]5-HT release, but only by 20% when compared
with control (data not shown). Anti--SNAP antibodies were also
tested for their effect on dense core granule release. In Figure 5B,
antibody to -SNAP almost completely inhibited [3H]5-HT
release, whereas the nonspecific antibody control had no effect.
Because -SNAP has been shown to serve as an adapter for NSF
binding,45 we next examined the role of NSF in
[3H]5-HT release. A monoclonal anti-NSF antibody (2E5)
inhibited dense core granule release (Figure 5C), and this inhibition
was reversed by preincubation of the 2E5 antibody with recombinant NSF
(Figure 5D). Not unexpectedly, the recombinant NSF alone showed little
enhancement of secretion because its size (approximately 480 kDa) makes
it unlikely to enter the SLO-induced pores in the platelet membrane. To
further confirm this result, we tested the effect of 2E5 on fresh
platelets that had been permeabilized with SLO. The anti-NSF antibody
reduced calcium-stimulated release from 49% ± 2.3% of total
[3H]5-HT to 9% of total in these cells.
SNAP-23 mediates dense core granule secretion
From the above data, it appears that platelet exocytosis uses SNAPs
and NSF; therefore, it is likely that SNARE proteins are involved. To
address this, we first focused on the t-SNARE SNAP-23. Fab fragments
made from ab23 (Fab23) appeared to significantly inhibit
Ca++-induced release of [3H]5-HT from dense
core granules (Figure 6). Fab23 almost
completely inhibited [3H]5-HT release at a concentration
of 0.16 mg/mL (Figure 6A). This inhibition of [3H]5-HT
release by Fab23 was partially reversed by the addition of recombinant
His6-SNAP-23, confirming the specificity of the Fab
fragment (Figure 6B). Recombinant His6-SNAP-23 had no
effect on [3H]5-HT secretion (Figure 6B), nor did a
preimmune IgG fraction. The C-terminal peptides of SNAP-23 and -25 have
proved to be good inhibitors of GLUT4 translocation and
neurotransmitter release.47,48 The C-terminal peptide of
human SNAP-23 inhibits [3H]5-HT secretion by 43%
compared with control. On the basis of our data, SNAP-23 is involved in
dense core granule secretion.
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| Fig 6.
Anti-SNAP-23 antibody inhibits [3H]5-HT
release.
(A) The permeabilized platelet exocytosis was performed in the presence
of increasing amounts of Fab23 (0-0.16 mg/mL). After 5 minutes of
Ca++ stimulation (10 µmol/L), [3H]5-HT
release was measured (n = 3). (B) Secretion triggered by 10 µmol/L
Ca++ was analyzed in the presence of Fab23 (0.12 mg/mL),
Fab23 (0.12 mg/mL) preincubated with His6-SNAP-23 (0.45 mg/mL), His6-SNAP-23 (0.45 mg/mL), human SNAP-23
C-terminal peptide (0.15 mg/mL), and rabbit IgG purified from preimmune
sera (0.15 mg/mL) (n = 6).
|
|
Syntaxin 2 mediates dense core granule secretion
We next focused on the syntaxin family of t-SNARE. Syntaxins 2, 4, and 7 are present in platelets (Figure 1).15 To determine which syntaxin is involved in dense core granule secretion, we tested
the antibodies against syntaxins 2, 4, and 7 in the in vitro secretion
assay (Figure 7A). Syntaxin 2 antibody
dramatically inhibited dense core granule release, reaching 90%
inhibition by 0.18 mg/mL. This inhibition was reversed by
His6-syntaxin 2 recombinant protein but not by
His6-syntaxin 4 (Figure 7B). His6-syntaxin 2, a cytoplasmic domain of syntaxin 2, by itself had no effect on
secretion. Neither anti-syntaxin 4 nor anti-syntaxin 7 antibodies affected dense core granule secretion when comparable concentrations were used, nor did a preimmune IgG fraction. An additional monoclonal anti-syntaxin 4 antibody, shown to inhibit -granule
release,16 also had no effect on [3H]5-HT
release (data not shown). These data indicate that syntaxin 2 is
involved in dense core granule release in platelets.
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| Fig 7.
Anti-syntaxin 2 antibody inhibits [3H]5-HT
release.
(A) The permeabilized platelet exocytosis assay was performed in the
presence of increasing amounts of anti-syntaxin 2 ( ), 4 ( ), and
7 ( ) antibodies and rabbit IgG ( (0-0.18 mg/mL). After
stimulation with Ca++ (10 µmol/L), [3H]5-HT
release was measured and normalized as a percentage of control release.
(B) [3H]5-HT secretion stimulated by 10 µmol/L
Ca++ was analyzed in the presence of anti-syntaxin 2 antibody (20 µg/mL), anti-syntaxin 2 antibody (20 µg/mL)
preincubated with His6-syntaxin 2 (0.24 mg/mL),
His6-syntaxin 2 alone (0.24 mg/mL), anti-syntaxin 2 antibody (0.02 mg/mL) preincubated with His6-syntaxin 4 (0.24 mg/mL), and IgG purified from preimmune sera (20 µg/mL). The
[3H]5-HT release was measured and normalized as a
percentage of control release (n = 9).
|
|
SNAP-23 and syntaxin 2 interact in platelets
Because both SNAP-23 and syntaxin 2 are involved in dense core
granule secretion, according to the SNARE hypothesis, they should form
a complex in the platelet. To test this, we performed an
immunoprecipitation experiment to trap this complex. SNAP-23 has been
shown to bind to each of the plasma membrane syntaxins in
vitro17; however, co-immunoprecipitation from dilute
detergent-solubilized extracts seems to be the only method available to
demonstrate that a SNAP-23-containing complex exists in vivo.
Thrombin-activated and resting platelets were solubilized with 1%
Triton X-100 PBS and subjected to immunoprecipitation by ab23
covalently coupled to protein G beads. The bound and free materials
were recovered and subjected to Western blot with ab23 and
anti-syntaxin 2 antibody. In both activated and resting platelets,
weak but detectable complexes were formed; however, there was no
obvious change in the amount of complex recovered either before or
after thrombin stimulation (Figure 8).
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| Fig 8.
SNAP-23 and syntaxin 2 can form a complex.
Fifty micrograms of Triton X-100-solubilized platelet extracts from
resting and thrombin-activated (1 U/mL for 5 minutes) platelets was
subjected to immunoprecipitation (IP) with ab23 coupled to protein G
beads. The precipitated material (P) and the unbound supernatant
material (S) were analyzed by Western blotting with ab23 and
anti-syntaxin 2 antibody. The immunodecorated proteins were detected
by ECL as described earlier. Hexosaminidase release was measured to
confirm the activation of platelets.
|
|
| |
Discussion |
In our previous studies of platelet exocytosis, we identified many
of the putative components of the platelet secretory machine, such as
-SNAP, -SNAP, NSF, p115/TAP, VAMP-3/cellubrevin,
and 2 of the known syntaxins, syntaxins 2 and
4.15,27 In this report, we increase this list by
demonstrating the presence of 2 additional t-SNAREs, SNAP-23 and
syntaxin 7. As our focus turns from identification to functional
studies, we have adapted a permeabilized platelet exocytosis assay to
dissect the roles of these secretory machinery proteins. Using this
assay, we have shown that -SNAP and NSF play a role in the
Ca++-induced secretion of dense core granules. Using
t-SNARE-specific antibodies, we have further demonstrated that
syntaxin 2 and SNAP-23 are involved in dense core granule release.
These data confirm the fact that platelet exocytosis is mediated by
SNARE proteins and offer new insight into the proteins that facilitate
the exocytosis events of the platelet.
SNAP-23, like its homologue SNAP-25, behaves as an integral membrane
protein in all of the cell types examined.21,22,24,25,47,49 This is presumably because the protein is bound to the membrane through
a series of thioester-linked palmitoyl groups, attached to a conserved
cluster of cysteine residues in the center of both SNAP-23 and
SNAP-25.35-39 The distribution of SNAP-23 in platelets is
unique in that not all of it is associated with membranes. Subcellular
fractionation and SLO permeabilization experiments show that a portion
of platelet SNAP-23 is soluble. This soluble SNAP-23 no longer
partitions into the detergent phase of a Triton X-114 extraction,
suggesting that it has lost its hydrophobicity. This loss of
hydrophobicity could result from a difference in the acyl groups
attached to platelet SNAP-23, or it could result from a loss of or
incomplete addition of palmitoyl groups. Given the inherent instability
of thioester bonds, it seems possible that SNAP-23 irreversibly
deacylates as platelets age. It is interesting that the pool of soluble
SNAP-23 decreases upon platelet activation. This change in distribution
could indicate some functional relocation of the molecule, as has been
proposed for SNAP-23 in 3T3-L1 and mast cells,25,50 or
simple trapping in the aggregated cytoskeleton.51 At this
stage, the functional relevance of the formation and redistribution of
soluble SNAP-23 is unknown; however, soluble SNAP-23 is not found in
the megakaryoblastic leukemia cell line, Meg-O1s (Chen and Whiteheart, unpublished data). Further detailed experimentation will be required to determine the significance of this observation.
To gain insight into the machinery of platelet secretion, we adapted a
previous technology42,43 in which the pore-forming bacterial toxin SLO was used to permeabilize platelets. Based on the
titration presented in Figure 4, it is clear that permeabilization at
25°C with 0.8 U/mL SLO allows access to the cytosol without significantly damaging the granule membranes. Under these conditions, cells can be permeabilized and then incubated on ice with activators or
inhibitors to equilibrate these reagents into the cells. This cold step
decreases the activity of SLO and therefore lessens the
permeabilization of granule membranes.40 When the
equilibration step is performed at room temperature, granule contents
leak, thereby decreasing the calcium-dependent secretion signal.
Chilling, however, can stimulate changes in platelet shape and
-granule secretion,52,53 presumably through a
cold-induced leakage of calcium from intraplatelet
stores.54-56 The inclusion of EGTA in the permeabilization
buffer appears to eliminate this effect because no release of
[3H]5-HT is seen in the absence of added calcium (See
Figures 4A and 5A), nor is there any apparent cytoskeletal
rearrangement as detected by electron microscopy (Lemons and
Whiteheart, unpublished data). Once the permeabilized platelets are
rewarmed to 25°C, they can be quickly stimulated with
Ca++ and secretion can be measured. Calcium
(108-103 mol/L) was titrated into
the permeabilized platelet assay and, as shown in Figure 5A, secretion
occurred only when the Ca++ concentration reached 10 µmol/L. As Ca++ was increased, release of granule
contents did not increase but became more erratic, as suggested by the
increase in the standard errors. For this reason as well as the fact
that stimulation of platelets in vivo usually results in an increase in
intracellular Ca++ to greater than 1 µmol/L,57 we chose to use 10 µmol/L Ca++ to
stimulate exocytosis from the permeabilized platelets. In all cases,
content release was dependent on ATP (Figure 5A) and temperature (data
not shown). In the presence of apyrase (0.5 U), no
Ca++-induced release of granule contents occurred. In
addition, granule release occurs rapidly after the addition of
Ca++ and appears to plateau by 3 minutes (data not shown).
This is very similar to the kinetics of thrombin-induced secretion in vivo.58 For these experiments, we chose to use freshly
banked platelets because there was no apparent difference in the degree of calcium-stimulated release between these cells and freshly prepared
platelets (see "Results").
The major difference between the properties of the permeabilized cell
system and intact platelets is the extent of content release. The
extent of release in the permeabilized cells is approximately half of
that seen for thrombin-induced intact platelets or for cells that have
been permeabilized for only a short time (approximately 2 minutes43). However, it is similar to the efficiency of the calcium-dependent release (approximately 52%) when the cells were permeabilized for 30 minutes.43 The difference in
efficiency between intact and permeabilized cells is probably due to
the dilution of important cytosolic components as they diffuse from the
platelets. Padfield et al43 indicated that this was a
likely explanation for their apparent inability to recapitulate
GTP--S-stimulated release in their permeabilized platelet system.
From the data presented in this manuscript, it is clear that increasing
the total concentration of at least 1 cytosolic component, -SNAP, by
adding recombinant protein to the assay can increase the efficiency of
[3H]5-HT release (Figure 5A). This is consistent with the
explanation that proteins (at least of a certain molecular weight) can
freely diffuse both in and out of the permeabilized cells and affect secretion efficiency. Additionally, it should be noted that in our
system, exocytosis was measured at room temperature rather than
37°C, which also might account for some decrease in release efficiency.
Using the above assay, our first task was to demonstrate a role for
SNAPs in platelet exocytosis. In other systems, such as intercisternal
Golgi transport45 and neurotransmission in
squid,46 increasing -SNAP over endogenous levels leads
to an increase in membrane fusion events. In platelets, the addition of
-SNAP caused an almost 2-fold increase in the extent of
[3H]5-HT release. The importance of -SNAP was further
demonstrated by the fact that, at the concentration used, the
dominant-negative -SNAP mutant (-SNAP-L294A44) and
the anti--SNAP antibody both inhibited dense core release. In it
interesting that in the Ca++ titration of Figure 5A, the
effects of the wild-type and mutant SNAPs are lost as the
Ca++ level is increased above 10 µmol/L, suggesting that
other factors (such as calpain activation) affect granule release. NSF
was also shown to play a role in [3H]5-HT release because
anti-NSF antibodies almost completely blocked secretion, and inhibition
was reversed by the addition of recombinant NSF (Figures 5C and 5D).
These data indicate that these 2 general membrane fusion machinery
components, SNAP and NSF, are involved in exocytosis, thus confirming
the similarity between the platelet release reaction and other
regulated secretion events.
Given these data, we next attempted to determine which t-SNAREs play a
role in dense core granule release. Figures 6A and 6B show that SNAP-23
antibodies inhibit dense core granule release. The inhibition was
reversed by the addition of recombinant SNAP-23, supporting the
specificity of the effect. To determine which syntaxin is involved in
dense core granule release, we used syntaxin-specific antibodies as
inhibitors. Only anti-syntaxin 2 antibodies inhibited dense core
granule release (Figure 7A). This inhibition was reversed by
competition with syntaxin 2 protein but not with syntaxin 4 protein
(Figure 7B), supporting the antibody specificity. Syntaxin 4 has been
shown to be involved in -granule exocytosis16 (and demonstrated in our laboratory; Chen et al, in preparation), but neither the syntaxin 4 antibody used by Flaumenhaft et al16 nor the one produced by our group had any inhibitory effect on dense
core granule release. It is interesting that dense core granule and
-granule as well as lysosome release use SNAP-23 (Chen, Lemons, and
Whiteheart, unpublished data), and this suggests that the specificity
of the 3 platelet exocytosis events is controlled by the syntaxin
component of the t-SNARE heterodimer. This also suggests that SNAP-23
is, in fact, a general t-SNARE that pairs with either syntaxin 2 or 4. As for the v-SNAREs in platelets, it is clear that some
synaptobrevin/VAMP is required for -granule release,16
but it remains to be determined which v-SNARE is involved. At present,
only 2 potential v-SNAREs have been identified in
platelets27 (also Rutledge et al, in preparation).
Further experimentation will be required to clarify which SNAREs are
involved in which platelet exocytosis events. However, from the data
presented here and in other reports,16 it appears that
syntaxin 2 controls dense core release, syntaxin 4 contributes to
-granule release, and SNAP-23 plays a role in both secretion events
(Lemons and Whiteheart, unpublished data). As we get closer to a
complete mapping of the secretory machinery of platelets, the next
series of questions will focus on how these proteins and their
interactions are regulated. In this respect, SNARE binding proteins
such as Munc18's19 and pantophysin59,60
and additional regulatory proteins such as the
rabs61 and p115/TAP62 will become important
subjects for study. The ultimate goal is to determine how extracellular
signals impinge on the secretory machinery to mediate granule release.
Note: During the revision of this manuscript, a report by Poiger and
Reed63 appeared that suggested a role for NSF in platelet exocytosis.
| |
Acknowledgments |
The authors would like to thank Dr David Castle for his generous gift
of the SNAP-23 peptide. They also thank the staff of Central Kentucky
Blood Center for their assistance and the members of the Whiteheart lab
for their helpful discussions. They would especially like to thank Dr
Susan A. Buhrow for her expert editing of this manuscript.
| |
Footnotes |
Submitted June 10, 1999; accepted September 24, 1999.
Supported by National Institutes of Health Grant HL56652 to
S.W.W.
Reprints: Sidney W. Whiteheart, Department of
Biochemistry, University of Kentucky College of Medicine, 800 Rose
Street, Lexington, KY 40536; e-mail: whitehe{at}pop.uky.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction