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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1313-1318
A Critical Role for N-ethylmaleimide-Sensitive Fusion Protein
(NSF) in Platelet Granule Secretion
By
János Polgár and
Guy L. Reed
From the Harvard School of Public Health, Cardiovascular Biology
Laboratory, Boston, MA.
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ABSTRACT |
The molecular mechanisms that regulate membrane targeting/fusion
during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF
attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements
of a conserved molecular machinery for membrane targeting/fusion that
have been detected in platelets. We examined whether NSF, an ATPase
that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule
secretion. Peptides that mimic NSF sequence motifs inhibited both
 -granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar
amino acids in a scrambled sequence inhibited platelet secretion. The
peptides that inhibited platelet granule secretion also inhibited the
human recombinant -SNAP-stimulated ATPase activity of recombinant
NSF. It was also found that anti-NSF antibodies, which inhibited
recombinant -SNAP-stimulated ATPase activity of NSF, inhibited
platelet granule secretion in permeabilized cells. The inhibition by
anti-NSF antibodies was abolished by the addition of recombinant NSF.
These data provide the first functional evidence that NSF plays an
important role in platelet granule secretion.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DURING PLATELET GRANULE secretion, a
large number of biologically active substances are released that have
important roles in platelet activation, aggregation, and thrombus
formation.1-3 Still, the mechanisms through which platelet
activation is coupled to granule secretion are poorly understood. There
is little known about the membrane targeting/fusion events related to
platelet degranulation.
In many cells, N-ethylmaleimide-sensitive fusion protein
(NSF),4 soluble NSF attachment proteins
(SNAPs),5 and SNAREs (SNAP receptors) have been found to
mediate intracellular membrane targeting/fusion events. According to
the SNARE-hypothesis,6 vesicular (v-) and target (t-)
membrane SNAREs form stoichiometric complexes that pair biological
membranes in preparation for fusion. The v-SNARE synaptobrevin (also
known as vesicle-associated membrane protein [VAMP]) and the t-SNAREs
syntaxin and SNAP-25 form a stable ternary complex that docks the
vesicle with its target membrane. This complex then binds SNAPs
followed by NSF to form a 20S complex. In this model, the specificity
of membrane targeting relies on specific pairing of SNAREs, whereas the
highly conserved NSF, together with SNAPs, are the general elements of
the machinery. NSF and SNAPs dissociate the stable v-/t-SNARE complexes
using energy provided by the hydrolysis of ATP.7,8 The
ATPase NSF has been shown to play a critical role in membrane
targeting/fusion in many cells.
Recently, NSF, SNAPs, and SNAREs have been detected in
platelets.9,10 Their presence in platelets suggests that
membrane targeting/fusion events related to platelet degranulation are mediated by NSF and the SNARE machinery. However, the demonstration of
these secretory molecules does not prove that platelet secretion requires an NSF-mediated mechanism. In addition to the numerous examples in which NSF was required in intracellular membrane
targeting/fusion, NSF-independent mechanisms were also reported in
yeast and mammals as well.11-14 Both NSF-dependent and
NSF-independent, but SNARE-dependent, membrane targeting/fusion
processes may coexist in the same cell type.14 Furthermore,
it has been found recently that v- and t-SNAREs alone, when
reconstituted into separate vesicles, are sufficient for assembling a
fusion complex and for mediating the spontaneous fusion of the docked
membranes.15 This argues that the SNAREs are both necessary
and sufficient for membrane fusion and that NSF and the SNAPs are not
part of the minimal fusion machinery. So, without functional evidence,
the involvement of NSF in membrane targeting/fusion events associated
with platelet granule secretion remains speculative.
The goal of this study was to determine whether the ATPase NSF plays a
role in platelet granule exocytosis. We found that peptides that mimic
NSF sequences and inhibit  -SNAP-stimulated ATPase activity of NSF
in vitro inhibited both dense-granule and -granule secretion in
permeabilized human platelets. Anti-NSF antibodies, which inhibited
-SNAP-stimulated ATPase activity of NSF in vitro, also inhibited
platelet granule secretion in permeabilized cells, and this inhibition
could be abolished by the addition of recombinant NSF. These data
provide the first functional evidence that NSF is important for
platelet granule secretion.
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MATERIALS AND METHODS |
Materials.
Peptides acetylated at the NH2-terminus and amidated at the
COOH-terminus were synthesized, purified to greater than 95% by high-performance liquid chromatography, and analyzed by
Mass Spectral Analysis (MALDI/TOF) by Research Genetics Inc
(Huntsville, AL) and Advanced ChemTech (Louisville, KY).
 -Thromboglobulin enzyme-linked immunosorbent assay (ELISA) kit was
from American Bioproducts (Parsippany, NJ). 5-Hydroxy
[2-14C] tryptamine creatinine sulphate (50 µCi/mL at 56 mCi/mmol; 14C-serotonin) was from Amersham
(Chicago, IL). Colorimetric lactate dehydrogenase (LDH) assay kit,
saponin (catalogue no. S4521) and bovine thrombin were from Sigma
Chemical Co (St Louis, MO). Escherichia coli XL-1 Blue was
obtained from Stratagene (La Jolla, CA). PQE-30 vectors and
Nickel-nitrilotriacetic acid-agarose (Ni-NTA-agarose) were from Qiagen
Inc (Valencia, CA). Protein A Sepharose was from Pierce (Rockford, IL).
Centrifugal concentrators were from Amicon (Beverly, MA).
Double-stranded DNA sequencing kit was from US Biochemical (Cleveland,
OH). Proteinase K was from Boehringer Mannheim Co (Indianapolis, IN).
Enhanced chemiluminescence detection reagents were from Amersham
(Piscataway, NJ).
Preparation of 14C-serotonin labelled platelet
suspensions.
Human blood was drawn by venipuncture through 19-gauge needles from
apparently healthy volunteers who had not taken any medication for at
least 10 days. This protocol was approved by the Human Subject
Committee at the Harvard School of Public Health. The blood (6 vol) was
collected into 1 vol 85 mmol/L trisodium citrate, 65 mmol/L citric
acid, and 110 mmol/L glucose, pH 4.5, and the sample was mixed gently
by inversion. Platelet-rich plasma (PRP) was prepared by
centrifugation. PRP was preincubated with 14C-serotonin (3 µL/mL PRP) for 60 minutes at 37°C. Platelets were pelleted by
centrifugation at 1,500g for 10 minutes, the supernatant was
completely removed, and the cells were resuspended in a modified Ca2+-buffering media16 containing 20 mmol/L
PIPES, pH 7.4, 150 mmol/L potassium glutamate, 5 mmol/L glucose, 5 mmol/L ATP, 12.5 mmol/L magnesium diacetate, 2.5 mmol/L EDTA, 2.5 mmol/L EGTA, and 0.05% bovine serum albumin (BSA) (buffer A). The
platelet count was adjusted to 1 × 109/mL and the
sample was stored at 25°C and used within 3 hours.
Measurement of granule secretion in saponin-permeabilized platelets.
To 60 µL of buffer A, containing 10 to 16 µg/mL saponin and various
additions, was added 20 µL platelet suspensions in 1.5-mL plastic
tubes. Samples were incubated at 25°C for 10 minutes (experiments with peptides) or for 15 minutes (experiments with antibodies). Granule
secretion was induced by increasing the free
Ca2+-concentration to 10 µmol/L16 by adding
2.3 µL 100 mmol/L CaCl2 with or without 1.7 µL 50 U/mL
thrombin. The samples were incubated for 5 minutes and centrifuged at
3,000g for 2.5 minutes, and 70 µL of supernatants was saved
for scintillation counting of released 14C-serotonin and
measurement of -thromboglobulin and LDH. Under these experimental
conditions, maximal granule secretion by 10 µmol/L free
Ca2+ was typically 40% to 50% of total cell content both
for 14C-serotonin and -thromboglobulin. All sets of
experiments contained these controls: samples containing 0.2% Triton
X-100 for the measurement of total cellular 14C-serotonin,
-thromboglobulin, and LDH; samples with no CaCl2 addition for the measurement of nonspecific leakage of the granule contents during the procedure. This nonspecific leakage was typically less than 10% to 15% of total secretion both for
14C-serotonin and -thromboglobulin. In agreement with
earlier reports,17 the concentration of saponin required to
produce the same degree of permeabilization estimated by LDH leakage
had to be titrated for each individual experiment. For some
experiments, peptides were pretreated with proteinase K at a peptide to
enzyme ratio of 80 to 1 (wt/wt) at 37°C for 60 to 120 minutes.
Assay of stimulation of the ATPase activity of recombinant NSF by
recombinant -SNAP.
The assay was essentially performed as described by Schweizer et
al.18 The released phosphate was quantified by the
colorimetric assay of Lanzetta et al.19
Preparation of recombinant proteins.
cDNA encoding mouse NSF, SKD2 clone in pYES2 vector, was generously
provided by Dr Carol A. Vandenberg20 (University of California, Santa Barbara, CA). NSF cDNA was genetically
engineered into the BamHI/Sal I site of pQE-30
expression vector, resulting in a construct comprising a
His6 tag, the entire open reading frame, and the portion of
the cDNA downstream of the stop codon. The NSF was expressed in XL-1
Blue Escherichia coli and purified as described by Whiteheart
et al,8 except that gel filtration on a Superose 12 column
was used in place of Superose 6 in the final purification step. Human
-SNAP was cloned from human platelet cDNA by the polymerase chain
reaction using standard techniques. The primers corresponded to the 5'
and 3' coding sequence and contained sites (BamHI and
HindIII) that permitted ligation in frame to the pQE-30 vector.
The sequence was verified by double-stranded DNA sequencing. Expression
in XL-1 Blue E coli and purification of
His6--SNAP was performed essentially as described by
Whiteheart et al.7
Production and purification of anti-NSF antibodies.
Polyclonal rabbit anti-NSF antibodies were generated by immunizing a
rabbit with purified His6-NSF as described by Morgan and
Burgoyne.21 The IgG were purified from preimmune and immune rabbit sera by affinity chromatography on protein A Sepharose, dialyzed
against Tris-buffered saline, pH 7.4, and concentrated to 12 mg/mL
using Centricon-30 centrifugal concentrators.
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RESULTS |
Peptides that mimic NSF sequence motifs inhibit platelet granule
secretion.
Figure 1 shows that NSF is present in human
platelets. This prompted us to examine whether SNAP-triggered NSF
activity is necessary for platelet secretion. In a recent report,
Schweizer et al18 used peptides that correspond to
potential sites of NSF-SNAP interaction to inhibit NSF function. Two of
6 peptides, NSF-2 and NF-3, inhibited neurotransmitter release by more
than 50% when injected into the giant presynaptic terminal of
squid.18 We used a similar approach to study whether NSF is
necessary for platelet granule secretion. Although the small size of
platelet makes microinjection difficult, substances can be introduced
into the platelet cytosol by permeabilizing the cell membrane.
Saponin-permeabilized human platelets were used to study the effect of
various peptides on platelet granule secretion. The peptides used in
this study as well as the corresponding sequence motifs in NSF are
shown in Fig 2. The NSF-2 peptide inhibited
platelet dense-granule secretion in a concentration-dependent manner
(Fig 3). The inhibition reached a plateau
at 1 to 1.5 mmol/L, a peptide concentration that was also effective in
the squid model.18 To verify the specificity of this
inhibitory effect, we used a scrambled peptide, scr-NSF-2 peptide,
containing a similar amino acid composition to NSF-2, but with a
different order. The scr-NSF-2 peptide had no effect on granule
secretion (Fig 4), even when used in
concentrations as high as 2 mmol/L (not shown), indicating that the
inhibition with NSF-2 peptide was sequence-specific. Pretreatment of
NSF-2 peptide with proteinase K, which digested the peptide, also
abolished the inhibitory effect of NSF-2 (not shown), providing further evidence for the sequence specificity of inhibition by NSF-2. In
contrast, NSF-3, which was equally effective as NSF-2 for inhibiting neurotransmission in the squid,18 did not inhibit
dense-granule secretion in the human platelet (Fig 4). However,
hu-NSF-3, a 19-mer peptide synthesized to mimic the human sequence for
NSF-3, inhibited granule secretion in the human platelet (Fig 4) in a concentration-dependent manner (not shown).
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| Fig 1.
Anti-NSF antibodies recognize NSF in human platelets.
Platelet lysate (50 µg; lane 1) and recombinant NSF (50 ng; lane 2)
were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by Western blotting. The positions
of prestained molecular mass markers are shown on the left.
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| Fig 2.
Amino acid sequence of the peptides used in this study,
as well as the corresponding sequence motifs in human and squid NSF.
Residue numbers are indicated on the top of the human NSF sequences.
Boxes indicate residues that are identical between the human and squid
sequences. Arrows show amino acid differences between the NSF-3 and
hu-NSF-3 peptides. The 19-mer hu-NSF-3 peptide is identical to the
human NSF sequence from 265 to 283.
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| Fig 3.
Concentration-dependent inhibition of platelet
dense-granule secretion by NSF-2 peptide in permeabilized human
platelets. Platelets with 14C-serotonin-loaded dense
granules were preincubated with saponin and various concentrations of
NSF-2 peptides or with no peptide addition. Granule secretion was
induced by increasing free Ca2+-concentration to 10 µmol/L. Secretion was measured by counting radioactivity in the cell
supernatants (see Materials and Methods; mean ± SD, n = 2).
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| Fig 4.
Comparison of the effect of NSF-2, scr-NSF-2, NSF-3, and
hu-NSF-3 peptides on platelet dense-granule secretion. Platelets with
14C-serotonin-loaded dense granules were preincubated with
saponin and different peptides (1 mmol/L) before granule secretion was
induced by increasing free Ca2+ concentration to 10 µmol/L. Secretion was measured by counting radioactivity in the cell
supernatants. The figure shows the percentage of inhibition of
secretion by various peptides when granule secretion with no peptide
additive is considered as 100% secretion (see Materials and Methods;
mean ± SD, n = 4 to 8; platelet samples from 4 different
donors).
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To examine the effect of these peptides on -granule secretion, the
release of -thromboglobulin was measured by ELISA. NSF-2, which
almost completely inhibited dense-granule secretion (Figs 3 and 4),
practically abolished -granule secretion as well, whereas scr-NSF-2
did not inhibit (Fig 5).
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| Fig 5.
NSF-2 peptide inhibited -granule secretion in
permeabilized human platelets. Platelets were preincubated with saponin
and NSF-2 or scr-NSF-2 peptides or with no peptide. Granule secretion
was induced by increasing free Ca2+-concentration to 10 µmol/L. -Granule secretion was monitored by ELISA measuring
-thromboglobulin in the cell supernatants (see Materials and
Methods; mean ± SD, n = 2).
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The previous data showed that peptides that mimic NSF sequence motifs
inhibit platelet granule secretion elicited by Ca2+, which
mimics the increased intracellular Ca2+ induced by cellular
activation provoked by extracellular agonists. We examined whether the
peptides could also inhibit agonist-induced granule secretion in
platelet. This question is complicated by the need to permeabilize the
platelets (in a Ca2+-buffering buffer system) to permit the
entry of the peptides. However, CaCl2 (pCa = 5) and
thrombin (1 U/mL) induced 30% to 40% more granule secretion in
permeabilized cells compared with the effect of pCa = 5 alone
(Fig 6). This indicated that the signaling pathways remained (at least partially) intact under the experimental conditions used in this study and made it possible to examine the
effect of the peptides on agonist-induced granule secretion. Under
these conditions, NSF-2 blocked secretion both induced by pCa = 5 alone
or pCa = 5 and thrombin (Fig 6). This strongly suggests that
these peptides inhibit both Ca2+-induced and
thrombin-induced platelet granule secretion.
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| Fig 6.
NSF-2 peptides inhibit both Ca2+-induced
and agonist-induced platelet granule secretion. Platelets with
14C-serotonin-loaded dense granules were preincubated with
saponin and with or without NSF-2 peptides (1.5 mmol/L). Granule
secretion was induced by increasing the free
Ca2+-concentration to 10 µmol/L (Ca2+),
with or without 1 U/mL thrombin (thr). Secretion was measured by
counting radioactivity in the cell supernatants (see Materials and
Methods; mean ± SD, n = 2; representative of 2 independent
experiments with similar results).
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Peptides that inhibit platelet secretion also inhibit
-SNAP-stimulated ATPase activity of NSF.
Experiments were performed to determine whether the effects of the
NSF-2 and hu-NSF-3 peptides were related to their ability to interfere
with SNAP-NSF interaction. In these assays, the effect of the peptides
on the -SNAP-stimulated ATPase activity of NSF was examined using
recombinant proteins. It was found that both peptides inhibited human
recombinant -SNAP-stimulated ATPase activity of NSF
(Fig 7). As seen with platelet secretion,
NSF-2 was the most potent inhibitor, followed by hu-NSF3. NSF-3, which did not inhibit platelet granule secretion (Fig 4), also had no inhibitory effect on the SNAP-stimulated ATPase activity of NSF (Fig
7).
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| Fig 7.
Inhibition of human recombinant -SNAP-stimulated
ATPase activity of recombinant NSF by peptides that mimic NSF sequence
motifs. Peptide sequences are shown in Fig 2. Data are expressed as
nanomoles of liberated phosphate per hour per microgram of NSF (see
Materials and Methods; mean ± SD, n = 3).
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Anti-NSF antibodies inhibit granule secretion in platelet.
We used anti-NSF antibodies (see Fig 1) as an additional tool to verify
the involvement of NSF in the process of platelet granule secretion. It
was found that rabbit polyclonal anti-NSF antibodies inhibited
-SNAP-stimulated ATPase activity of NSF using recombinant proteins
(not shown). These anti-NSF antibodies also significantly inhibited
platelet granule secretion compared with samples with no antibodies or
with preimmune rabbit IgG (Fig 8). The
inhibition by anti-NSF antibodies was abolished by recombinant NSF,
which itself did not affect granule secretion in these assays (Fig 8).
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| Fig 8.
Anti-NSF antibodies inhibited platelet dense-granule
secretion in permeabilized human platelets. Platelets with
14C-serotonin-loaded dense granules were preincubated with
saponin and 300 µg/mL rabbit IgG purified from preimmune serum
(preimm. IgG), anti-NSF IgG from immune serum (anti-NSF) with or
without 20 µg/mL recombinant NSF (rNSF). Granule secretion was
induced by increasing free Ca2+-concentration to 10 µmol/L. Secretion was measured by counting radioactivity in the
cell supernatants (see Materials and Methods; mean ± SD, n = 4)
The P values were calculated using the paired Student's
t-test.
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DISCUSSION |
The accumulating evidence that the SNAREs are the core components of
most intracellular membrane trafficking/fusion events suggests that
SNAREs may play a critical role in the membrane targeting/fusion that
occurs with platelet granule secretion. The assembly of v-/t-SNAREs
appears to be under the kinetic control of various regulatory
mechanisms, involving the members of Rab GTPase family and Sec1
family.21 NSF and SNAPs dissociate the stable v-/t-SNARE
complexes using energy provided by the hydrolysis of
ATP.22,23 Because of this, NSF and SNAPs may also have a role in controlling the kinetics of membrane targeting/fusion, acting
either prefusion22,23 or postfusion.9,22
However, because NSF-independent intracellular membrane
targeting/fusion mechanisms exist,11-14 the involvement of
NSF in a particular process, eg, in platelet granule secretion, cannot
be assumed without functional evidence.
Consequently, we examined whether NSF plays a role in platelet granule
secretion, a process in which membrane targeting/fusion is crucial. A
saponin permeabilization assay was established to study the effects of
various peptides and anti-NSF antibodies on platelet secretion. Saponin
permeabilization has been successfully used to introduce molecules, up
to the size of antibodies, into platelets.17,24-27 It was
found that peptides that mimic human NSF sequence motifs inhibited
granule secretion in permeabilized human platelets. In these studies,
we have taken several steps to verify that the inhibitory effect was
mediated through NSF. We first established that the inhibitory effects
were sequence-specific because (1) digestion of the peptides by
proteinase K abolished the inhibition; (2) peptides with a similar
amino acid composition in a scrambled sequence did not inhibit; and (3)
NSF-3, a 20-mer that differs from the corresponding human NSF sequence,
did not inhibit, but an hu-NSF-3 peptide, which was identical with the human NSF sequence motif, did inhibit secretion. In addition, the
inhibitory effects of the peptides on platelet secretion were directly
related to their ability to block functional NSF-SNAP interactions in
vitro: NSF-2 and hu-NSF-3, which inhibited platelet secretion, also
inhibited human recombinant -SNAP-stimulated ATPase activity of
recombinant NSF. The NSF-3 peptide, which did not inhibit platelet
secretion, did not inhibit ATPase activity. Finally, anti-NSF
antibodies were used as an independent approach to verify the role of
NSF in platelet secretion. Anti-NSF antibodies inhibited granule
secretion in permeabilized platelets. The specificity of inhibition by
anti-NSF antibodies was demonstrated by showing that (1) these
antibodies recognize only 1 major band in platelet lysate and that this
band comigrates with recombinant NSF; (2) they inhibited
-SNAP-stimulated ATPase activity of NSF with recombinant proteins;
(3) recombinant NSF abolished the inhibitory effects of the anti-NSF
antibodies both in vitro and in platelet secretion; and (4) preimmune
IgG, purified from preimmune serum, did not inhibit. Taken together,
these findings indicate that NSF is important for platelet secretion.
The fact that dense-granule and -granule secretion were almost
completely inhibited by the NSF-2 peptide, the most potent inhibitor of
NSF-ATPase activity in vitro, strongly suggests that platelet NSF plays
a critical role in granule secretion that probably cannot be bypassed
by other mechanisms.
Recent studies have indicated that platelets contain NSF, SNAPs, and a
full complement of interacting SNARE machinery-related proteins.9,10 Indeed, during the review of our manuscript, Flaumenhaft et al28 published functional evidence for the
involvement of SNARE proteins in Ca2+-induced -granule
secretion in streptolysin O-permeabilized platelets. Our study provides
the first functional evidence for the involvement of NSF in platelet
granule secretion and provides additional independent evidence for the
hypothesis that the SNARE-machinery plays a pivotal role in regulated
exocytosis in platelets.
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ACKNOWLEDGMENT |
The authors are grateful to Prof Carol A. Vandenberg for providing cDNA
encoding mouse NSF. We thank Lin Liu, Michael L. Fitzgerald, Aiilyan
Houng, and Brian Robinson for their help in cloning human -SNAP and
raising antibodies.
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FOOTNOTES |
Submitted November 6, 1998; accepted April 12, 1999.
Supported in part by Grant No. HL-57314 from the National Institutes of
Health and by a grant to Harvard School of Public Health from
Bristol-Myers Squibb.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part in abstract form at the 1998 national meeting of the
American Heart Association in Dallas, TX.
Address reprint requests to Guy L. Reed, MD, Cardiovascular Biology
Laboratory, Harvard School of Public Health, 665 Huntington Ave,
Boston, MA 02115.
| |
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