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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 571-579
Identification of a Cellubrevin/Vesicle Associated Membrane Protein 3 Homologue in Human Platelets
By
Audrey M. Bernstein and
Sidney W. Whiteheart
From the Department of Biochemistry, University of Kentucky College
of Medicine, Chandler Medical Center, Lexington, KY.
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ABSTRACT |
Several studies suggest membrane trafficking events are mediated by
integral, membrane proteins from both transport-vesicle and target
membranes, called v- and t-SNAREs (SNAp REceptors), respectively.
Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to
detect these v-SNAREs in human platelets, although membrane proteins
from these cells could support 20S complex formation. To
identify v-SNAREs in platelets, we used a polymerase chain reaction
(PCR) approach with degenerate primers to amplify potential VAMP-like
v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human
megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this
protein has greater than 93% identity with human VAMP 1, 2, and rat
cellubrevin over the conserved core region, but has a unique
N-terminal domain. Northern blot analysis showed that the 2.5-kB mRNA
encoding Hceb is expressed in every human tissue tested. Hceb from
detergent-solubilized platelet membranes, participated in
 -SNAP-dependent 20S complex formation and adenosine triphosphate
(ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in
platelets. Immunofluorescence microscopy, using an anti-Hceb antibody
showed a punctate, intracellular staining pattern in platelets,
megakaryocytes, and HEK-293 cells. This same pattern was observed in
surface-activated platelets even though all dense core and most
-granule contents had been released. These data suggest that Hceb
may reside on a platelet organelle that is not primarily involved in
the exocytic pathway.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ONCE ACTIVATED, PLATELETS secrete
components important for clot formation and wound repair. Of special
importance to their hemostatic function are their granules. It is the
alpha and dense core granules that contain the components necessary for
hemostasis. Binding to blood vessel lesions activates platelets causing
the granules to concentrate to the center of the cell. One theory
proposes that the concentrated granules fuse with specialized regions
of the plasma membrane called the open canalicular system (OCS),1 thereby releasing the granule contents into
channels that lead to the extracellular space. An alternative model
proposes that the OCS is first extruded into the plasma membrane, then the granules concentrate to the center of the cell where they undergo
compound fusion and subsequently fuse with the plasma membrane.2 Regardless of the mechanism, these fusion events result in the release of the contents of the alpha granules and dense
core granules into the extracellular space.3
Platelets also appear to undergo constitutive and regulated
endocytosis. Clathrin-coated vesicles budding from the OCS have been
observed by electron microscopy4 and electron-dense,
fluid-phase markers can be detected in platelet granules.4
To date, the most studied example of endocytic traffic involves the
receptor-mediated internalization of fibrinogen via the fibrinogen
receptor (gpII /III). Because megakaryocytes and platelets do not
synthesize fibrinogen,5,6 all of the fibrinogen in the
alpha granules must be endocytosed from the plasma.7
Kinetic studies of fibrinogen uptake into megakaryocytes, established
the multivesicular bodies (MVBs) as endocytic, precursors of the alpha
granules.8 Platelets also endocytose fibrinogen from the
plasma.7,9 Endocytosis in resting platelets occurs
constitutively at a slow rate,9 but the rate is increased
when platelets are partially stimulated with agonists such as adenine
diphosphate (ADP) or low concentrations of thrombin.10
Although platelets contain MVBs, the precursor relationship to alpha
granules has not been established.8
Studies of membrane trafficking in mammalian systems and in yeast have
suggested that docking and fusion of transport vesicles with target
membranes are mediated by soluble proteins (ie, N-ethylmaleimide sensitive factor (NSF), and  -soluble NSF attachment proteins (SNAPs)) and by integral membrane proteins, known as SNAp REceptors or
SNAREs.11-13 The synaptobrevin/vesicle associated membrane
protein (VAMP) family of SNARE proteins reside on the vesicle and are therefore termed, v-SNAREs. The v-SNAREs, VAMP 1 and 2 and rat cellubrevin, are small molecular weight ( 18 kD), type
II integral membrane proteins with a unique N-terminus and two highly
conserved coiled-coil domains, which are important for targeting VAMPs
to vesicles14 and for binding to t-SNAREs.15,16
The syntaxins and synaptosomal associated protein (SNAP)-23/25 family
members are thought to form a heterodimer on the target membrane and
are therefore called t-SNAREs.17 Since the discovery of the
SNARE proteins in neurons, homologues have been found in a number of different tissues, and importantly, on different membranes within a
cell.18 The original SNARE hypothesis proposed that for
each vesicular trafficking event there will be a specific v-SNARE that binds to its cognate t-SNARE heterodimer.12,19 Once the v- and t-SNAREs bind, they form a 7S complex, which can interact with
-SNAP and NSF. This series of binding interactions results in the
formation of the 20S complex.20 Adenosine triphosphate (ATP) hydrolysis by NSF mediates 20S complex disassembly and is a
required step for membrane trafficking events in
vitro.21,22 Recent studies have indicated that this complex
disassembly represents a priming step that is integral to the
activation of SNAREs for membrane fusion.23-26
To understand more about the membrane trafficking events in platelets,
we have continued to identify proteins that could mediate platelet
exocytosis or endocytosis. In the present study, we report the
identification of a novel VAMP/synaptobrevin family member. Biochemical
studies show that this v-SNARE is present in platelet membranes and can
participate in 20S complex assembly and ATP-dependent disassembly.
Originally cloned from a human megakaryocyte cDNA library, we find by
Northern blotting analysis that this v-SNARE is expressed in many human
tissues. Due to its high homology to other known VAMPs, its broad
tissue distribution, and its subcellular localization, this protein
appears to be the human equivalent of the rodent cellubrevin. In
platelets, our studies show that Hceb resides on a compartment that is
not mobilized to the plasma membrane on calcium or thrombin
stimulation. The functional significance of a platelet cellubrevin will
be discussed.
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MATERIALS AND METHODS |
Antibodies, cell lines, and reagents.
Oligonucleotide polymerase chain reaction (PCR) primers were from
GibcoBRL (Grand Island, NY). The cDNA library was constructed with mRNA
from CMK cells that were induced to differentiate with phorbol
12-myristate 13-acetate for 4 hours and was generously provided by Dr
Takeyuki Sato through Mochida Pharmaceutical Co, LTD, Tokyo,
Japan.27 The multiple tissue Northern blot was from Clonetech (Palo Alto, CA). Outdated platelets were procured as units
from the Central Kentucky Blood Center (Lexington, KY). HEK-293 cells
were a generous gift of Dr L.B. Hersh (University of Kentucky,
Lexington, KY). HepG2 cells were a generous gift of Dr D.J. Noonan
(University of Kentucky, Lexington, KY). Anti-VAMP 1 antibody
(Ab)28 and anti-VAMP 2 Ab (C1
69.1)29 were kindly provided by Dr Reinhard Jahn (Yale
University, New Haven, CT). Antirat cellubrevin Ab (MC16) was kindly
provided by Dr P. DeCamilli (Yale University, New Haven,
CT).28 Antitransferrin receptor Ab (MAB1451) was purchased
from Chemicon International, Inc (Temecula, CA). Anti-gpII/III Ab
(N77140M) was purchased from Biodesign (Kennebunkport, ME).
Antiserotonin Ab (18-0077) was purchased from Zymed (San Francisco,
CA). Anti-von Willibrand Factor Ab was purchased from Sigma (St Louis,
MO).
Cloning of cDNA encoding human cellubrevin.
The plasmid-based, CMK cDNA library was divided into 15 pools
containing 70,000 clones each (1.05 × 106 clones
total). Each pool was analyzed by PCR using the following degenerate
primers: 5 -GGAATTCC CA(AG) CA(AG) AC(TCA) CA(AG) GC(TCA) CA(AG)
G-3 and 5-CGGGATC CAT (TC)TT (AG)CA (AG)TT (TC)TT CCA C-3, based on conserved regions of human, bovine, and
Drosophila VAMP proteins (see Fig 1B).30 PCR was
run for 30 cycles using a 45°C annealing step and 1 µg of
template DNA from each of the library pools. One pool gave a positive
PCR product of the predicted size and on DNA sequencing proved to be
homologous to known VAMPs. Colonies from the positive pool were then
screened by filter hybridization with standard procedures using a
[32P]-cytidine triphosphate (CTP; NEN, Boston,
MA) labeled riboprobe (Promega, Madison, WI) made from the
initial PCR product.
Production and purification of antihuman cellubrevin antibody.
The N-terminal human cellubrevin (Hceb) peptide, MSTGPTAATGSN-(C),
was synthesized at the Macromolecular Structural Analysis Facility
(Lexington, KY) and coupled to Imject Activated BSA Super Carrier
(Pierce, Rockford, IL). Anti-Hceb Ab was produced by
injecting the conjugate into New Zealand White rabbits at the Division
of Laboratory Animal Resources facility (Lexington, KY). To affinity
purify the Hceb Ab, the cDNA encoding the cytosolic domain of Hceb
(a.a. 1-75) was subcloned into the pGEX-KG vector (Pharmacia,
Piscataway, NJ). The Hceb-glutathione-S-transferase (GST) fusion
protein was expressed in Escherichia coli (E. coli), bound to glutathione agarose beads (Sigma), and cross-linked to the
beads with Bis(Sulphosuccinimidyl)suberate (2 µg/mL)
(Pierce). The sera from the immunized rabbit were separated by a 50%
ammonium sulfate precipitation, brought up in 50% the initial volume
in phosphate-buffered saline (PBS), and dialyzed overnight against PBS
at 4°C. The dialyzate was passed over the GST-Hceb fusion protein
column and the bound antibody was eluted with 100 mmol/L glycine pH
2.5. The purified antibody was further incubated with GST-bound
glutathione agarose beads to adsorb any contaminating anti-GST
reactivity. To test the specificity of the anti-Hceb antibody, the
cDNAs encoding human VAMP 1 and 2 were amplified from a human QUICK
Clone spleen cDNA library (Clonetech, Palo Alto, CA) using PCR primers
made to their N and C terminus (Accession No. J05611). The correct
products encoding VAMP 1, 2 and Hceb were subcloned into a pQE-9 vector
(Qiagen, Valencia, CA) and expressed in E. coli.
SNARE activity assay.
Human platelet membrane extract (HPE) was prepared as described by
Lemons et al.31 Briefly, human platelet membranes are salt
washed to remove all of the peripheral proteins and then detergent
solubilized to produce a HPE. 20S Complexes were formed by incubating
HPE (5 mg) with recombinant -SNAP (100 µg), recombinant -SNAP
(10 µg), and recombinant C-terminal-myc-tagged NSF (50 µg) in
binding buffer (20 mmol/L Hepes/KOH pH 7.0, 100 mmol/L KCl, 1%
polyethyleneglycol, 1% glycerol, 1% Triton-X 100, 1 mmol/L dithiothreitol [DTT]) with 1 mmol/L ATPS and 1 mmol/L
EDTA for 30 minutes on ice. In these reactions, membrane extract must
constitute 50% or less of the total reaction volume to achieve
noninhibitory protein/detergent ratios. NSFmyc containing complexes
were then recovered by adding a saturating amount of anti-myc antibody
(9E10)32 coupled to Protein G Superose beads (Pharmacia),
and incubated for an additional 2 hours at 4°C. The beads were
washed four times at room temperature (RT) with 0.5 mL of binding
buffer with 1 mmol/L ATPS and 4 mmol/L MgCl2 (wash, see
Fig 5). The 20S complexes bound to beads were then incubated for 1 hour
at 4°C with 1 mL of binding buffer with 5 mmol/L ATP and 4 mmol/L
MgCl2 (release, see Fig 5). The beads were removed by
centrifugation and the supernatant was recovered. The beads were
further incubated with (100 mmol/L glycine pH 2.5, 1% Triton-X 100) to
remove any protein not released by ATP hydrolysis (beads, see Fig 5).
The proteins present in the various wash and elution supernatants were
recovered by precipitation with 10% trichloroacetic acid and analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot. Immunodecorated proteins were detected with an
antirabbit Ig-horseradish peroxidase conjugate and the Enhanced
Chemiluminscent System (Pierce).
Immunofluorescence studies.
Sterile coverslips were preincubated with 5 µg/mL laminin overnight
at 37°C. HEK-293 cells were seeded onto the laminin-coated coverslips and allowed to grow overnight. HEK-293 cells were washed once with PBS pH 7.0 containing 1.26 mmol/L CaCl2, 0.49 mmol/L MgCl2, 0.41 mmol/L MgSO4 (Ca/Mg PBS) and
fixed with methanol for 6 minutes on dry ice. The cells were blocked
with 5% fetal calf serum and 5% goat serum diluted in PBS (block
buffer). Primary antibodies were diluted in block buffer and incubated
with HEK-293 cells for 40 minutes. Cells were washed with the block
buffer five times and incubated with either fluorescein isothiocyanate (FITC)-conjugated or Texas Red conjugated secondary antibodies (Vector
Laboratories, Burlingame, CA) for 20 minutes. Cells were washed with
block buffer five times, then twice with PBS. Cells were mounted on
slides using Vectashield mounting medium (Vector Laboratories). Cells
were examined under appropriate illumination with an E-600
epifluorescence microscope (Nikon, Melville, NY) with a 100X oil
objective. Images were recorded with a U-III camera system (Nikon), and
overlapping of the FITC and Texas Red images was done with Photoshop
5.0 (Adobe, San Jose, CA).
Resting platelets were prepared by adding 300 nmol/L prostaglandin
I2 (Sigma). Platelets were separated from red blood cells by centrifugation at 150xg, collected by centrifugation at
900xg and resuspended in (140 mmol/L NaCl, 10 mmol/L
NaHCO3, 2.5 mmol/L KCl, 0.5 mmol/L
Na2HPO4, 1.0 mmol/L MgCl2, 22.0 mmol/L Na3 citrate, 0.55 mmol/L glucose, 0.35% bovine
serum albumin [BSA], pH 6.5) with 25 µg/mL apyrase (Sigma) and 500 µmol/L CaCl2 and incubated for 15 minutes at 37°C.
The platelets were seeded onto poly-lysine coated slides. Platelets
were fixed in 3.7% formaldehyde (Sigma) for 20 minutes at RT and
permeabilized with 100 µg/mL digitonin (Calbiochem, La Jolla, CA) for
5 minutes at RT. The remainder of the staining procedure was performed
as above.
Platelets for surface activation were prepared without prostaglandin
I2 or apyrase. Platelets were resuspended in PBS and seeded
onto poly-lysine-coated slides. Platelets were either incubated with
10 µmol/L CaCl2 in PBS for 15 minutes or 10 µmol/L
CaCl2 in PBS for 15 minutes followed by 0.1 U/mL thrombin
(Sigma), 10 µmol/L CaCl2 in PBS for 15 minutes at RT. The
remainder of the procedure was performed as above. Bone marrow samples
containing megakaryocytes were seeded onto poly-lysine-coated slides,
fixed in 3.7% formaldehyde, and permeabilized in 100 µmol/L
digitonin. Cells were processed as above.
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RESULTS |
Cloning and characterizing human cellubrevin cDNA.
Degenerate PCR primers based on homologous regions of six previously
cloned VAMPs were used to amplify new VAMP-encoding cDNAs from a
megakaryocyte cDNA library. Primary screening of pools from a
megakaryocyte-like CMK cell cDNA library yielded a 209-bp PCR product
that was 88%, 79%, and 82% identical to the nucleotide sequences of
rat cellubrevin, human VAMP 1, and 2, respectively. The one positive
pool was then screened by colony hybridization using a riboprobe made
from the initial PCR product. A single positive clone was isolated and
sequenced (Fig 1A). The clone, termed human
cellubrevin (Hceb), is 693 bp in length and contains: 24 bp of 5
untranslated sequence, including an upstream stop codon; a 300-bp
coding region; and 369 bp of 3 untranslated sequence that does
not include a poly A tract (Fig 1A). The nucleotide sequence
immediately adjacent to the proposed initiator ATG is noncanonical
(gccaaaATGt), yet it is identical to that found in rat
cellubrevin.30
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| Fig 1.
Nucleotide and predicted amino acid sequence of human
cellubrevin. (A) Nucleotide and amino acid sequence of Hceb. The
numbers correspond to the number of nucleotides and the underlined
regions correspond to the sequences for which
degenerate primers were designed. The sequence of the Hceb
clone was submitted to Genbank, accession number U64520.
(B) Comparison of the Hceb amino acid sequence to other human VAMPs and
rat cellubrevin. The dots represent identical amino acids. The
underlined region represents the peptide that was used to make a
specific antihuman cellubrevin Ab. The bold methionine (M) represents
the VAMP 2 vesicle targeting signal, which is also present in Hceb.
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Human cellubrevin protein has an overall 92% amino acid identity to
rat cellubrevin, 74% to human VAMP 1, and 83% to human VAMP 2 (alignment shown in Fig 1B). The homology is highest in the central
core region (a.a.11-77), which is predicted to contain the two
coiled-coil domains important for t-SNARE binding.15,16 In
addition, Hceb contains the methionine 46 of VAMP 2, shown to be
critical for synaptic vesicle targeting.14 This region also
contains the specific cleavage sites used by tetanus and botulinum B,
D, F, and G neurotoxins.33 Like other VAMPs, the N-terminal
domain of Hceb is unique and there is only limited homology in the
C-terminal transmembrane domain.
Human cellubrevin is widely distributed.
A Northern blot of poly A+ mRNA from the indicated human
tissues was incubated with a [32P]-labeled nick
translated probe made from 292 bp of the 3 untranslated region
of Hceb (bp 402-693 Fig 1A). mRNA load controls were confirmed with a
[32P] nick translated -actin DNA probe that will
hybridize to more than one isoform in some tissues (pancreas and
placenta) (Fig 2). The results of this
Northern blot show that the Hceb message is expressed in every human
tissue tested. The 2.5-kB message is similar to the size of VAMP 1 and
2 mRNAs34 and slightly larger than the message for rat
cellubrevin (1.8 kB).30 Homologous expressed sequence tags
(ESTs) derived from human lung, placenta, pancreas, pineal gland,
pituitary gland, brain, and heart are available, but as listed in the
database, none contain the entire coding region.
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| Fig 2.
Human cellubrevin is expressed in many human tissues. The
Northern blot containing 2 µg of poly A+ mRNA from the
indicated human tissues was incubated with a [32P]
nick-translated DNA probe made from the 292-bp of the 3
untranslated region of human cellubrevin (bp 402-693, Fig 1A). The blot
was washed and exposed to film for 18 hours at  80°C with an
intensifying screen. The positions of the size standards in kilobases
are shown at right. The same blot was also probed with a
[32P] nick-translated -actin DNA probe, washed, and
exposed to film at 80°C for 2 hours. Some tissues contain
two isoforms that will hybridize with the -actin probe (pancreas and
placenta).
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Antihuman cellubrevin antibody production and characterization.
A specific antipeptide human cellubrevin antibody was made against the
first 12 amino acids of the N-terminus of human cellubrevin. To show
that this antibody is specific for Hceb, its reactivity against VAMP 1 and 2 was tested. E. coli cells expressing
His6-tagged VAMP 1, 2, and Hceb constructs were induced
with 1 mmol/L isopropyl thio--D-galactoside (IPTG) for 4 hours to
produce each recombinant protein. The bacterial extracts were separated
on a SDS-PAGE gel and stained with Coommassie Brilliant Blue dye
(Fig 3A). The identical samples were
separated on a SDS-PAGE gel, transferred to nitrocellulose, and probed
with the anti-Hceb Ab (Fig 3B). As shown, the antipeptide antibody
while detecting Hceb does not cross-react with either human VAMP 1 or
2.
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| Fig 3.
Antihuman cellubrevin antibody does not cross-react with
VAMP 1 or VAMP 2. (A) E. coli cells expressing
His6-tagged VAMP 1, 2, and Hceb constructs were induced
with 1 mmol/L IPTG for 4 hours to produce each recombinant protein.
Bacterial extracts from the minus and plus IPTG-induced samples were
separated on a 12.5% SDS-PAGE gel and proteins were stained with
Coomassie Brilliant Blue. Lanes 1 and 2, VAMP 1 ± IPTG; lanes 3 and
4, VAMP 2 ± IPTG; lanes 5 and 6, Hceb ± IPTG. (B) The same samples
were separated by SDS-PAGE and then transferred to nitrocellulose and
blotted with a specific anti-Hceb Ab at a concentration of 1/1,000.
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Human cellubrevin colocalizes with the transferrin receptor in
HEK-293 cells.
To determine the subcellular distribution of Hceb, we performed
immunofluorescence microscopy with the anti-Hceb antibody on HEK-293
cells. HEK-293 cells showed a perinuclear punctate staining pattern
with anti-Hceb Abs that did not resemble Golgi complex or endoplasmic
reticulum staining (Fig 4A). This staining pattern appears similar to the antirat cellubrevin perinuclear, punctate staining of CHO cells.28 The transferrin receptor
is often used as a marker for recycling endosomal
compartments35 and was shown to colocalize with rodent
cellubrevin in Chinese hamster ovary (CHO) cells.
Double-labeling experiments in HEK-293 cells with anti-Hceb antibody
and the antitransferrin receptor antibody were performed to determine
if Hceb like rat cellubrevin, is localized to recycling endosomes. As
shown in Fig 4A through C, the transferrin receptor and Hceb share a
similar staining pattern, supporting the conclusion that Hceb is
endosomal. When compared directly, the staining of transferrin receptor
and Hceb showed a high degree of overlap as indicated in yellow (Fig
4C). A similar pattern of anti-Hceb staining and overlap with
transferrin receptor was observed in HepG2 cells (data not shown). The
staining pattern of anti-lysosome-associated membrane protein
(LAMP) 1 Ab, a late endosome and lysosomal marker, did
not overlap with Hceb staining in HEK-293 and HepG2 cells (data not
shown). These data are consistent with Hceb being the equivalent to
rodent cellubrevin.
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| Fig 4.
Human cellubrevin colocalizes with the transferrin
receptor in HEK-293 cells and is present in megakaryocytes. HEK-293
cells were fixed with methanol and incubated with (A) anti-Hceb
antibody (1/25), FITC-conjugated antirabbit antibody (1/150); (B)
antitransferrin receptor antibody (1/25), Texas Red-conjugated
antimouse antibody (1/150); (C) overlap of (A) and (B), which was
generated with Adobe Photoshop 5.0; (D) differential interference
contrast microscopy (DIC) image of the same cells. Original
magnification 600×. Megakaryocytes were fixed with 3.7%
formaldehyde, permeabilized with 100 µg/mL digitonin and incubated
with (E) anti-Hceb antibody (1/25), FITC-conjugated antirabbit antibody
(1/150); (F) anti-gpII/III antibody (1/100), Texas Red-conjugated
antimouse antibody (1/150). Original magnification 1,000×.
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Human cellubrevin acts as a SNARE in platelets.
Having shown that Hceb exhibits properties consistent with it being the
human homologue of rat cellubrevin, we next sought to determine if it
was in fact present in platelets. Anti-Hceb antibody was able to detect
a 16-kD protein in as little as 60 µg of salt-washed,
detergent-solubilized platelet membrane extracts (data not shown). To
show that Hceb in platelets has SNARE activity, we used the method of
Söllner et al.17 HPE (5 mg), which contains all of
the platelet SNARE molecules, was incubated with recombinant -SNAP,
-SNAP, and NSFmyc in the presence of EDTA and ATPS. The
recombinant proteins will interact with all of the platelet SNARE
proteins to form 20S complexes, which can then be isolated by
coimmunoprecipitation through the epitope-tagged NSF in the complex
(Fig 5). The 20S complexes are washed and
then incubated with ATP/Mg2+ to promote 20S complex
disassembly. The proteins from the ATPS/Mg2+ wash (wash,
lanes 1 and 4), the ATP/Mg2+ release supernatant (release,
lanes 2 and 5), and the glycine eluate (beads, lanes 3 and 6) were
analyzed by Western blotting using the anti-Hceb antibody. As shown in
Fig 5, Hceb participated in 20S complex formation, but only when the
adapter protein -SNAP was added (lanes 4 through 6). This
SNAP-dependent complex formation together with the
ATP/Mg2+-dependent release from NSF (lane 5) are
characteristic of a SNARE protein as originally
described.17,22 This data indicates that Hceb is an
integral membrane protein present in platelets. It further shows that
its t-SNARE partners must also be present because v-SNAREs alone will
not associate with -SNAP and NSF.36 As further proof of
Hceb's SNARE activity, when HPE was incubated with an -SNAP-GST
fusion protein, Hceb was precipitated with the -SNAP-GST containing
complexes (data not shown). To date, no proteins detectable by
antibodies to rat cellubrevin, VAMP 1, or VAMP 2 have been observed
using either purification technique31 (and data not shown).
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| Fig 5.
Human cellubrevin participates in 20S particle assembly
and ATP-dependent disassembly. Five milligrams of HPE were incubated
with NSFmyc and either with (+) or without (-) recombinant
-SNAP. 20S Complex was immunoprecipitated with an anti-myc
antibody cross-linked to agarose beads. The beads were harvested by
centrifugation, washed in the presence of ATPS (wash), and then
incubated with ATP/Mg. The resulting supernatant was harvested
(release), and the beads were then stripped with glycine (beads). The
proteins were recovered by trichloroacetic acid (TCA)
precipitation and analyzed by Western blotting with an anti-Hceb
antibody (1/100).
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Human cellubrevin exhibits punctate staining in platelets and
megakaryocytes.
Immunofluorescence microscopy studies of platelets using the anti-Hceb
antibody resulted in a punctate staining pattern in all platelets
(Fig 6A). This pattern suggests that Hceb
resides on an intracellular compartment, however, it did not overlap
with the staining pattern seen using antibodies to other platelet
granule markers (ie, LAMP-1, serotonin, von Willebrand factor [vWF];
data not shown). The anti-Hceb staining pattern was specific in that preincubation with recombinant Hceb eliminated immunoreactivity, whereas preincubation with recombinant VAMP 2 was without effect (Fig
6B and C).
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| Fig 6.
Immunofluorescence studies of human cellubrevin in
platelets. Platelets were fixed with 3.7% formaldehyde and
permeabilized with 100 µg/mL digitonin. The cells were then incubated
with (A) anti-Hceb antibody (1/5) followed by FITC-conjugated
antirabbit antibody (1/150); (B) anti-Hceb antibody that had been
preincubated with 200 µg of GST-Hceb recombinant protein followed by
FITC-conjugated antirabbit antibody (1/150); (C) anti-Hceb antibody
that had been preincubated with 200 µg of GST-VAMP 2 recombinant
protein followed by FITC-conjugated antirabbit antibody (1/150). A DIC
image of each panel is included below. Original magnification 2,800×.
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To determine if Hceb was also expressed in the megakaryocyte, we
performed double-labeling, immunofluorescence microscopy on bone marrow
megakaryocytes with a megakaryocytic marker antibody against
gpII/III,37 (Fig 4E) and the anti-Hceb Ab (Fig 4F). The double positive staining allows identification of the mature megakaryocyte and shows that Hceb is present not only in platelets, but
also in their bone marrow progenitor cell. Hceb shows punctate perinuclear staining that is similar to, but does not completely overlap with the fibrinogen receptor staining.
Hceb staining remains intracellular in surface-activated platelets.
To determine if Hceb was mobilized to the plasma membrane on
stimulation, platelets were seeded onto poly-lysine-coated slides and
either maintained in a resting state, surface-stimulated in the
presence of 10 µmol/L CaCl2, or stimulated with 0.1 U/mL
thrombin. The distribution of the fibrinogen receptor (gpII/III)
can be used as a marker to distinguish resting platelets from the
various states of platelet activation.38-40 In resting
platelets, the antifibrinogen receptor Ab shows an internal punctate
staining pattern (Fig 7, first row). When
surface-stimulated in the presence of CaCl2, the platelets
displayed a distinct ring-like structure (Fig 7, middle row). In
thrombin stimulated platelets, spikes or finger-like pseudopodia were
clearly delineated by the antifibrinogen receptor staining (Fig 7,
bottom row) indicating mobilization of gpII/III to the cell
surface. The final, completely spread stage of surface-activated platelets39 is not represented. In the left panel, the
platelets are doubled-stained with an anti-gpII/III Ab and an
anti-Hceb Ab. The Hceb staining is punctate in resting platelets and
remains internal, but centrally concentrated in stimulated platelets. In the middle panel of Fig 7, platelets were double-stained for serotonin and the fibrinogen receptor. The antiserotonin Ab staining is
internally punctate in resting platelets and is completely abolished in
stimulated platelets, suggesting that the dense core granules contents
have been released. In the right most panel, platelets were
double-stained for fibrinogen receptor and vWF. Again, resting
platelets exhibit a granular staining pattern, but the stimulated
platelets lost most, if not all, of the vWF staining. The Hceb positive
organelle remains detectable in activated platelets even after
serotonin and vWF immunoreactivity have all but disappeared.
Interestingly, Hceb, unlike the fibrinogen receptor, does not mobilize
to the cell surface after stimulation.
View larger version (45K):
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| Fig 7.
Human cellubrevin staining remains intracellular in
surface-activated platelets. Platelets were prepared as resting (see
Materials and Methods) or surface-stimulated in the presence of 10 µmol/L CaCl2 or 10 µmol/L CaCl2 and 0.1 U/mL thrombin. Platelets were fixed with 3.7% formaldehyde and
permeabilized with 100 µg/mL digitonin. Left panel,
anti-gpII/III antibody (1/100), Texas Red-conjugated antimouse
antibody (1/150) (gpII/III); anti-Hceb antibody (1/25),
FITC-conjugated antirabbit antibody (1/150) (Hceb). Middle panel,
anti-gpII/III antibody (1/100), Texas Red-conjugated antimouse
antibody (1/150) (gpII/III); antiserotonin antibody (1/10),
FITC-conjugated antirabbit antibody (1/150) (serotonin). Right panel,
anti-gpII/III antibody (1/100), Texas Red-conjugated antimouse
antibody (1/150) (gpII/III); anti-vWF antibody (1-500),
FITC-conjugated antirabbit antibody (1/500) (vWF). Original
magnification 2,800×.
|
|
| |
DISCUSSION |
In an attempt to identify VAMP-like molecules in platelets, we screened
a megakaryocyte cDNA library with degenerate PCR primers based on
conserved VAMP sequences. We isolated one clone, termed human
cellubrevin, which is highly homologous to other VAMP proteins except
for its divergent N-terminus. Hceb mRNA is widely expressed, and the
protein is present in each human cell type tested; HEK-293 cells
(kidney), HepG2 (liver), bone marrow megakaryocytes, and platelets. In
HEK-293 cells, Hceb has similar staining pattern to the transferrin
receptor, supporting the proposal that human cellubrevin is functioning
in an endocytic pathway. In platelets, Hceb is an integral membrane
protein that participates in 20S complex formation and ATP-dependent
disassembly demonstrating its function as a SNARE protein. This also
indicates that its t-SNARE partners are present, as v-SNAREs need
t-SNAREs to participate in 20S complex formation.36
Immunofluorescence studies with an anti-Hceb antibody showed a
punctate, intracellular staining pattern in resting, as well as in
stimulated platelets (Fig 7). In contrast, antibodies to serotonin
(dense core granule) and vWF (alpha granule) showed a punctate staining
pattern in resting cells that all but disappeared when the cells were
stimulated. Compared with the membrane protein gpII/III, Hceb
does not mobilize to the cell surface on stimulation. Two possible
scenarios could explain these results. Hceb could be present on a
granule that is involved in exocytosis, but is somehow trapped in the
granule membrane and prevented from laterally diffusing to the plasma
membrane after fusion. Alternatively, Hceb could be present on an
organelle that is not involved in exocytosis. This later possibility is
in keeping with preliminary immunofluorescence studies that show only
limited overlap between Hceb positive compartments and those that stain
with antibodies to serotonin, vWF, or LAMP-1 (Bernstein and Whiteheart
unpublished). Further, immunoelectron microscopy studies will be
required to more definitively define Hceb's intraplatelet locale.
Studies of the rodent cellubrevin in nucleated cells suggest that it
plays a role in endocytic pathways. Tetanus toxin cleaves and
inactivates cellubrevin.30 When added to streptolysin
O-permeabilized CHO cells, tetanus toxin causes a 30% decrease in the
recycling of preloaded [125I]-labeled
transferrin.28 Studies of the role that cellubrevin plays
in Glut 4 mobilization have been more equivocal and seem to depend on
the mode of introducing the toxin. In the best case, when adipocytes
were incubated overnight with toxin in isotonic, low ionic strength
media, there was a 64% decrease in the insulin-stimulated glucose
uptake.41 At this stage, it appears that cellubrevin does
play a role in the overall process of receptor recycling, yet it is
unclear at which transport step it functions.
Platelets and megakaryocytes undergo endocytosis.42
Clathrin-coated vesicles containing fibrinogen bud from the OCS and fuse with alpha granules, loading them with fibrinogen.43
Recently, endocytic compartments termed MVB I and II have been defined
in platelets and megakaryocytes.8 Kinetic uptake
experiments performed in megakaryocytes showed a sequential uptake of
BSA and fibrinogen into the MVBs and then into alpha granules. After
granule-granule fusion, the fibrinogen receptor (gpII/III) may be
recycled back to the plasma membrane.44 Consistent with its
proposed role in other cells, Hceb could function in this or other
endocytic pathways in megakaryocytes and platelets. If Hceb is involved in the endocytic process, it is likely most active in megakaryocytes when loading of alpha granules is most prevalent. However, based on the
apparent abundance of Hceb in platelets, its homogeneous expression in
all platelets examined and the fact that its t-SNARE partners are also
present in platelet membrane extracts, Hceb could still function in
platelets. Further studies will be required to determine the role of
this v-SNARE in platelets and megakaryocytes.
| |
NOTE ADDED IN PROOF |
During the revision of this manuscript, a publication by Galli et al
(Mol Biol Cell 9:1437,1998) appeared. In this report, the
investigators concurred with the conclusion that our clone represents
the human homologue of the rodent cellubrevin. The Genbank submission
(U64520) was referenced in their text.
| |
ACKNOWLEDGMENT |
We would like to thank Dr Takeyuki Sato for providing the CMK cDNA
plasmid library, Dr Reinhard Jahn for the anti-VAMP 1 and anti-VAMP 2 Ab (C1 69.1) antibodies, Dr P. DeCamilli for the antirat cellubrevin
antibody, Dr G. Vanzant for the bone marrow samples, and the staff of
the Kentucky Blood Center for their assistance. We would also like to
thank the members of the Whiteheart laboratory for their productive
discussions and help.
| |
FOOTNOTES |
Submitted April 7, 1998;
accepted September 9, 1998.
Supported by Grant No. CB-153 from the American Cancer Society (to
S.W.W.) and Grant No. HL56652 from the National Institutes of Health
(to S.W.W.).
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.
Address reprint requests to Sidney W. Whiteheart, PhD,
Department of Biochemistry, University of Kentucky College of Medicine,
Chandler Medical Center, 800 Rose St, Lexington, KY 40536-0084; e-mail:
whitehe{at}pop.uky.edu.
| |
REFERENCES |
1.
White JG, Escolar G:
The blood platelet open canalicular system: a two-way street.
Eur J Cell Biol
56:233, 1991[Medline]
[Order article via Infotrieve]
2.
Morgenstern E, Patscheke H, Mathieu G:
The origin of the membrane convolutions in degranulating platelets.
Blut
60:15, 1990[Medline]
[Order article via Infotrieve]
3.
Kaplan K, Nossel H, Drillings M, Lesznik G:
Radioimmunoassay of platelet factor 4 and beta-thromboglobulin: Development and application to studies of platelet release in relation to fibrinopeptide A generation.
Br J Haematol
39:129, 1978[Medline]
[Order article via Infotrieve]
4.
Behnke O:
Coated pits and vesicles transfer plasma components to platelet granules.
Thromb Haemost
62:718, 1989[Medline]
[Order article via Infotrieve]
5.
Cramer E, Debili N, Martin J, Gladwin A, Breton-Gorius J, Harrison P, Savidge G, Vainchecker W:
Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes.
Blood
73:1123, 1989[Abstract/Free Full Text]
6.
Louache F, Debili N, Cramer E, Breton-Gorius J, Vainchenker W:
Fibrinogen is not synthesized by human megakaryocytes.
Blood
77:311, 1991[Abstract/Free Full Text]
7.
Harrison P, Wilbourn B, Debili N, Vainchenker W, Breton-Gorius J, Lawrie A, Masse J, Savidge G, Cramer E:
Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets.
J Clin Invest
84:1320, 1989
8.
Heijnen HFG, Debili N, Vainchencker W, Breton-Gorius J, Geuze HJ, Sixma JJ:
Multivesicular bodies are an intermediate stage in the formation of platelet -granules.
Blood
91:2313, 1998[Abstract/Free Full Text]
9.
Morgenstern E, Ruf A, Patscheke H:
Transport of anti-glycoprotein ii/iii-antibodies into the alpha-granules of unstimulated human blood platelets.
Thromb Haemost
67:121, 1992[Medline]
[Order article via Infotrieve]
10.
Wencel-Drake J, Boudignon-Proudhon C, Dieter M, Criss A, Parise L:
Internalization of bound fibrinogen modulates platelet aggregation.
Blood
87:602, 1996[Abstract/Free Full Text]
11.
Bajjalieh SM, Scheller RH:
The biochemistry of neurotransmitter secretion.
J Biol Chem
270:1971, 1995[Free Full Text]
12.
Rothman JE:
Mechanisms of intracellular protein transport.
Nature
372:55, 1994[Medline]
[Order article via Infotrieve]
13.
Südhof TC:
The synaptic vesicle cycle: A cascade of protein-protein interactions.
Nature
375:645, 1995[Medline]
[Order article via Infotrieve]
14.
Grote E, Hao JC, Bennett MK, Kelly RB:
A targeting signal in VAMP regulating transport to synaptic vesicles.
Cell
81:581, 1995[Medline]
[Order article via Infotrieve]
15.
Calakos N, Bennett M, Peterson K, Scheller R:
Protein-protein intereactions contributing to the specificity of intracellular vesicular trafficking.
Science
263:1146, 1994[Abstract/Free Full Text]
16.
Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H:
Synaptic vesicle membrane fusion complex: Action of clostridial neurotoxins on assembly.
EMBO J
13:5051, 1994[Medline]
[Order article via Infotrieve]
17.
Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE:
SNAP receptors implicated in vesicle targetting and fusion.
Nature
362:318, 1993[Medline]
[Order article via Infotrieve]
18.
Bock J, Scheller R:
A fusion of new ideas.
Nature
387:133, 1997[Medline]
[Order article via Infotrieve]
19.
Rothman JE, Warren G:
Implications of the SNARE hypothesis for intracellular membrane topology and dynamics.
Curr Biol
4:220, 1994[Medline]
[Order article via Infotrieve]
20.
Söllner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE:
A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.
Cell
75:409, 1993[Medline]
[Order article via Infotrieve]
21.
Whiteheart SW, Rossnagel K, Buhrow SA, Brunner M, Jaenicke R, Rothman JE:
N-ethylmaleimide-sensitive fusion protein: A trimeric ATPase whose hydrolysis of ATP is required for membrane fusion.
J Cell Biol
126:945, 1994[Abstract/Free Full Text]
22.
Wilson DW, Whiteheart SW, Wiedmann M, Brunner M, Rothman JE:
A multisubunit particle implicated in membrane fusion.
J Cell Biol
117:531, 1992[Abstract/Free Full Text]
23.
Banerjee A, Barry V, DasGupta B, Martin T:
N-ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2+-activated exocytosis.
J Biol Chem
271:20223, 1996[Abstract/Free Full Text]
24.
Mayer A, Wickner W, Haas A:
Sec18p (NSF)-driven release of Sec17p (-SNAP) can precede docking and fusion of yeast vacuoles.
Cell
85:83, 1996[Medline]
[Order article via Infotrieve]
25.
Mayer A, Wickner W:
Docking of yeast vacuoles is catalyzed by the ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF).
J Cell Biol
136:307, 1997[Abstract/Free Full Text]
26.
Weber T, Zemelman B, McNew J, Westermann B, Gmachl M, Parlati F, Söllner T, Rothman J:
SNAREpins: Minimal machinery for membrane fusion.
Cell
92:759, 1998[Medline]
[Order article via Infotrieve]
27.
Sato T, Fuse A, Eguchi M, Hayashi Y, Ryo R, Adachi M, Kishimoto Y, Teramura M, Mizoguchi H, Shima Y, Komori I, Sunami S, Okimoto Y, Nakajima H:
Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukemia.
Br J Haematol
72:184, 1989[Medline]
[Order article via Infotrieve]
28.
Galli T, Chilcote T, Mundigl O, Binz T, Niemann H, DeCamilli P:
Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin recptor-containin vesicle in cho cells.
J Cell Biol
125:1015, 1994[Abstract/Free Full Text]
29.
Edelmann P, Hanson PI, Chapman ER, Jahn R:
Synaptobrevin binding to synaptophysin: A potential mechanism for controlling the exocytic fusion machine.
EMBO J
14:224, 1995[Medline]
[Order article via Infotrieve]
30.
McMahon HT, Ushkaryov YA, Edelmann L, Link E, Binz T, Niemann H, Jahn R, Sudhof TC:
Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein.
Nature
364:346, 1993[Medline]
[Order article via Infotrieve]
31.
Lemons P, Chen D, Bernstein A, Bennett M, Whiteheart S:
Regulated Secretion in platelets: Identification of elements of the platelet exocytosis machinery.
Blood
90:1490, 1997[Abstract/Free Full Text]
32.
Evan GI, Lewis GK, Ramsay G, Bishop MJ:
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol Cell Biol
5:3610, 1985[Abstract/Free Full Text]
33.
Montecucco C, Schiavo G:
Mechanism of action of tetanus and botulinum neurotoxins.
Mol Microbiol
13:1, 1994[Medline]
[Order article via Infotrieve]
34.
Trimble W, Gray T, Elferink L, Wilson M, Scheller R:
Distinct patterns of expression of two VAMP genes within the rat brain.
J Neurosci
10:1380, 1990[Abstract]
35.
Dautry-Varsat A, Ciechanover A, Lodish H:
pH and the recycling of transferrin during receptor-mediated endocytosis.
Proc Natl Acad Sci USA
80:2258, 1983[Abstract/Free Full Text]
36.
McMahon HT, Sudhof TC:
Synaptic core complex of synaptobrevin, syntaxin, and SNAP-25 forms high affinity -SNAP binding sites.
J Biol Chem
270:2213, 1995[Abstract/Free Full Text]
37.
Cramer EM, Debili N, Martin JF, Gladwin A, Breton-Gorius J, Harrison P, Savidge GF, Vainchenker W:
Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes.
Blood
73:1123, 1989
38.
Wencel-Drake JD, Frelinger AL III, Dieter MG, Lam S:
Arg-Gly-Asp-dependent occupancy of gpII/III by applaggin: Evidence for internalization and cycling of a platelet integrin.
Blood
81:62, 1993[Abstract/Free Full Text]
39.
Leistikow EA, Barnhart MI, Escolar G, White JG:
Receptor-ligand complexes are cleared to the open canalicular system of surface-activated platelets.
Br J Haematol
74:93, 1990[Medline]
[Order article via Infotrieve]
40.
Kieffer N, Guichard J, Breton-Gorius J:
Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading
Am J PathOL
140:57, 1992[Abstract]
41.
Chen F, Foran P, Shone C, Foster K, Melling J, Dolly J:
Botulinum neurotoxin b inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type a toxin which failed to proteolyze the SNAP-23 present.
Biochemistry
36:5719, 1997[Medline]
[Order article via Infotrieve]
42.
Handagama p, Scarborough R, Shuman M, Bainton D:
Endocytosis of fibrinogen into megakaryocyte and platelet -granules is mediated by iib3 (glycoprotein II-III).
Blood
82:135, 1993[Abstract/Free Full Text]
43.
Klinger M, Kluter H:
Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: Further evidence for coated vesicle-mediated transport of von Willebrand factor, fibrinogen and fibronectin.
Cell Tissue Res
279:453, 1995[Medline]
[Order article via Infotrieve]
44.
Bretscher M:
Circulating integrins: 51, 64 and MAC-1, but not 31, 4 1 or LFA-1.
EMBO J
11:405, 1992[Medline]
[Order article via Infotrieve]
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Top
Abstract
Introduction