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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Medicine and Pathology, Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
Morphologic studies have demonstrated a process by which
Platelets demonstrate a number of characteristics
that render them an important model of membrane trafficking. They are
anucleate, display rare strands of endoplasmic reticulum, do not have
Golgi structures, and synthesize little new protein. They are thought to undergo just one round of granule secretion following activation because they do not have adequate synthetic capacity to generate new
granules. Although receptor-mediated1,2 and
pinocytotic3 endocytosis have been observed in platelets,
constitutive coupled endocytosis-exocytosis cycles that occur in
nucleated cells in general4-8 and in hematopoietic cells
in particular9 have not been documented in platelets.
Thus, the degree to which generalizations regarding the molecular
mechanisms of membrane trafficking can be applied to the platelet is
uncertain. A second distinctive characteristic of the platelet is that
its limiting membrane is characterized by a system of tunneling
invaginations of the plasma membrane, termed the open canalicular
system (OCS).10,11 Evidence that the OCS is open to the
extracellular environment is derived from reports using various
cell-impermeant tracers.12 The fate of the OCS on platelet
activation is the subject of debate. There is evidence that the OCS
undergoes evagination during platelet activation that provides an extra
membrane for the formation of pseudopodia.13,14 Others
have shown that the OCS becomes dilated and acts as a conduit to allow
the exit of Platelets also undergo a dramatic shape change following activation
that is associated with the centralization of granules and the
appearance of masses of cytoplasmic actin filaments. These observations
have led to speculation that cytoskeletal reorganization is responsible
for granule release. However, the inhibition of actin
polymerization18 or microtubule
organization19,20 does not inhibit granule secretion.
Furthermore, granule secretion and shape change can be dissociated
under several experimental conditions.21-23 Morphologic
aspects of activation-dependent membrane fusion have been studied
in detail. Platelet granules appear to undergo homotypic fusion on
activation,24 a phenomenon observed in other cells
of hematopoietic origin.25 Several studies demonstrate that More recently, studies evaluating the molecular mechanisms of platelet
membrane fusion have demonstrated a role for SNARE proteins in these
regulated secretory events.27,28 We29 and others30,31 have demonstrated SNARE proteins in platelets. The tSNAREs syntaxin 2, syntaxin 4, and syntaxin 729-32
and SNAP-2329,31,32 are found in platelets. Platelets also
contain gene products of the VAMP family of vSNAREs29
including a novel VAMP isoform, termed human
cellubrevin.33 The function of SNARE proteins that constitutes the trimeric exocytotic complex in platelet To determine whether the subcellular localization of functional SNARE
proteins could account for the unusual character of platelet granule
secretion, we sought to determine the subcellular localization of 3 functional SNARE proteins of the trimeric exocytotic complex. We first
found that human cellubrevin participates in platelet Materials
Antibodies
Platelet preparation Blood from healthy donors who had not ingested aspirin in the 2 weeks before donation was collected by venipuncture into 0.4% sodium citrate. Citrate-anticoagulated blood was centrifuged at 200g for 20 minutes to prepare platelet-rich plasma. Platelet-rich plasma was then used for ultrastructural studies as described below. For platelet secretion studies, platelets were purified from platelet-rich plasma by gel filtration using a Sepharose 2B column equilibrated in PIPES-EGTA buffer (25 mM PIPES, 2 mM EGTA,137 mM KCl, 4 mM NaCl, 0.1% glucose, pH 6.4). Final gel-filtered platelet concentrations were 1-2 × 108 platelets/mL. Platelet-rich plasma used for subcellular fractionation was obtained from the Beth Israel Deaconess Medical Center Blood Bank.Analysis of P-selectin surface expression For analysis of P-selectin surface expression from permeabilized platelets,36 20 µL gel-filtered platelets (1 × 108/mL-2 × 108/mL) in 5 mM MgATP was incubated with 3 U/mL reduced SL-O in the presence or absence of the indicated concentration of antihuman cellubrevin antibody. Samples were adjusted to pH 6.9 immediately following permeabilization. After a 20-minute incubation with antibody, CaCl2 was added to the reaction mixture. The amount of CaCl2 required to give a free Ca++ concentration of 10 µM in the presence of 2 mM EGTA at pH 6.9 was calculated as described previously.36 Following an additional 10-minute incubation after the addition of Ca++, 10 µL reaction mixture was transferred to 5 µL phycoerythrin-conjugated AC1.2 anti-P-selectin antibody. Phosphate-buffered saline (PBS; 500 µL) was added to the sample after a 20-minute incubation, and the platelets were analyzed immediately by flow cytometry.Immunonanogold labeling and electron microscopy Purified human platelets were fixed in 4% paraformaldehyde in 0.02 M PBS, pH 7.4, for 1 hour at room temperature, washed in 0.02 M PBS, pH 7.4, transferred to microtubes, and centrifuged at 1500g for 1 minute. They were then resuspended in molten 2% agar and quickly recentrifuged. Resultant agar pellets containing the platelets were washed in PBS before immersion in 30% sucrose in 0.02 M PBS, pH 7.4, overnight at 4°C, embedded in OCT compound (Miles, Elkhart, IN), and stored in 176°C liquid nitrogen for subsequent
use. Frozen 10-µm sections were cut with a standard cryostat and were
collected on precleaned glass slides. These sections were air dried for
20 minutes before staining.
Immunonanogold staining and processing for electron microscopy was performed at room temperature on cryostat sections mounted on glass slides, as follows37: (1) wash in 0.02 M PBS, pH 7.4, 5 minutes; (2) immersion in 50 mM glycine in 0.02 M PBS, pH 7.4, 10 minutes; (3) wash in 0.02 M PBS, pH 7.4, 5 minutes; (4) immersion in 5% normal goat serum (Vector Laboratories, Burlingame, CA), 20 minutes; (5) incubation in the primary antibody, an affinity-purified rabbit polyclonal antibody against the 12 N-terminal amino acids of human cellubrevin, at a dilution of 1:30-50, in a rabbit polyclonal antiserum against the C-terminal end of SNAP-23 at a dilution of 1:50, or in an affinity-purified goat polyclonal antibody against the C-terminal end of syntaxin 1A, 1B, 2, and 3 at a dilution of 1:10 in 0.02 M PBS, 60 minutes; (6) 3 washes in 0.02 M PBS, pH 7.4, 5 minutes each; (7) incubation in the secondary antibody (affinity-purified Fab' fragment from goat anti-rabbit IgG conjugated with 1.4 nm nanogold for human cellubrevin and SNAP-23 staining or affinity-purified Fab' fragment from rabbit anti-goat immunoglobulin G (IgG) conjugated with 1.4 nm nanogold for syntaxin staining) (Nanoprobes, Stony Brook, NY), 1:50-100 in 0.02 M PBS, pH 7.4, 60 minutes; (8) 3 washes in 0.02 M PBS, pH 7.4, 5 minutes each; (9) after fixation in 1% glutaraldehyde in 0.02 M PBS, pH 7.4, 2 minutes; (10) 3 washes in distilled water, 5 minutes each; (11) development with highest quality (HQ) silver enhancement solution (Nanoprobes) for 6 to 10 minutes in the darkroom; (12) 2 washes in distilled water, 2 minutes each; (13) immersion in 5% sodium thiosulfate, 1 minute; (14) 3 washes in distilled water, 5 minutes each; (15) after fixation in 1% osmium tetroxide in Sym-Collidine buffer, pH 7.4, 10 minutes; (16) one wash in 0.05 M sodium maleate buffer, pH 5.2, 5 minutes; (17) staining with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0, 5 minutes; (18) one wash in distilled water, 5 minutes; (19) dehydration in graded ethanols and infiltration with a propylene oxide-eponate (Ted Pella, Redding, CA) sequence; (20) embedment by the inversion of eponate-filled plastic capsules over the slide-attached tissue sections; (21) polymerization at 60°C for 16 hours; (22) separation of eponate blocks from glass slides by brief immersion in liquid nitrogen; (23) cutting of thin sections with a diamond knife with an ultratome (Reichert, Vienna, Austria) and collection of sections on uncoated 200-mesh copper grids (Ted Pella); and (24) viewing of unstained grids with a transmission electron microscope (CM 10; Philips, Eindhoven, The Netherlands). The following 5 controls were performed to ensure the specificity of immunostaining: absorption of primary antibody by affinity chromatography to solid-phase human cellubrevin peptide; replacement of primary antibody by an irrelevant rabbit IgG or goat IgG; omission of specific primary antibody; omission of secondary antibody; and omission of HQ silver enhancement solution. For quantitation, immunogold particles on randomly imaged platelets
were counted manually and were assigned to various subcellular membrane
compartments that included the
Subcellular fractionation Platelet-rich plasma (approximately 1 × 109 platelets/mL) obtained from the Beth Israel Deaconess Medical Center Blood Bank was transferred to a Fenwal transfer pack (Baxter, Deerfield, IL) and was incubated with 25 µg/mL sulfo-NHS-biotin (Pierce) at 4°C for 1 hour. Platelets were subsequently washed 3 times with ice-cold buffer P (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 25 mM HEPES, 0.5 mM EGTA, 2.5 µM PGE1, 100 µg/mL apyrase, and 100 µM aspirin). Following the final wash, platelets were resuspended in 50 mL ice-cold buffer P and subjected to nitrogen cavitation for 20 minutes at 100 atm on ice, as previously described.38 The cavitate was subsequently processed according to a modification of the procedures described by Gogstad et al.39,40 Briefly, the cavitate was collected and pelleted at 4000g for 20 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 50 mL ice-cold buffer P and was subjected to a second round of cavitation. This cavitate was pelleted at 4000g for 20 minutes at 4°C, and the supernatant was collected for analysis. Metrizamide was added to the supernatant of the second cavitation to a final concentration of 11% metrizamide. This material (10 mL) was then loaded onto 2 mL beds of 30% metrizamide. The material was subjected to centrifugation at 40 000g for 2 hours at 4°C. This procedure resulted in a band at the 11% to 30% metrizamide interface that was collected and dialyzed into PBS at 4°C. Metrizamide was added to the dialysate to a final concentration of 11% metrizamide, and 2.5 mL (2 mg/mL protein) was loaded onto 12-mL step gradients consisting of 15%, 20%, 25%, and 30% metrizamide. Step gradients were subjected to centrifugation at 40 000g for 2 hours at 4°C. Following centrifugation, the interfaces of the gradients were collected by fractionation. Protein concentrations of the fractions were determined with a DC Protein Assay kit (Bio-Rad) using bovine serum albumin-metrizamide standards. Samples were analyzed by immunoblot analysis using the indicated antibody.14C-serotonin was used as a marker for platelet-dense granules to evaluate the migration of dense granules in the metrizamide step gradient. In these experiments, 50 µCi (1.85 MBq) 14C-serotonin was added to 150 mL platelet-rich plasma for 30 minutes. Platelets were subsequently washed and processed as described above. Following fractionation, 100-µL samples from the step gradient were mixed with 5 mL Aquasol scintillation cocktail (Packard, Meriden, CT). Radioactivity in each sample was then quantified using a Tri-carb 2100TR Liquid Scintillation Analyzer (Packard Instrument, Downers Grove, IL). Immunoblot analysis Aliquots of the subcellular fractions were diluted in sample buffer (62.5 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), 5% -mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) at 95°C for 5 minutes. Proteins were then separated by SDS-polyacrylamide gel
electrophoresis (PAGE). Biotin-labeled samples were developed using an
avidin-HRP conjugate. Surface labeling is represented by a 65-kd band
previously shown to be a dominant protein on the surface of
biotin-labeled, resting platelets.41
Immunoblotting was performed using anti-P-selectin
cytoplasmic tail, CD53, human cellubrevin, SNAP-23, or syntaxin 2 antibodies, as indicated, and was visualized using enhanced chemiluminescence.
Human cellubrevin mediates  -granule secretion.29 A novel isoform of VAMP, human
cellubrevin (VAMP 3), was initially discovered in
platelets.33 A function for human cellubrevin in platelet
-granule secretion, however, has not been previously demonstrated.
We therefore sought to determine whether this VAMP isoform mediated
platelet -granule secretion. For these experiments, an antipeptide
antibody directed against the unique N-terminal portion of human
cellubrevin was tested in an SL-O-permeabilized model of platelet
-granule secretion. The antihuman cellubrevin antibody recognized a
single band that migrated with an apparent molecular mass of 15 kd in
platelet lysate. In addition, the protein recognized by this antibody
was sensitive to cleavage by tetanus toxin, consistent with human cellubrevin (Figure 1A). Previous studies
have demonstrated that antibodies directed at this peptide fail to
recognize VAMP 1 or VAMP 2.33 Consistent with this
previous work, the antihuman cellubrevin antibody used in these
experiments failed to recognize any VAMP isoforms in brain lysate,
which contains predominantly VAMP 1 and VAMP 2,42 and it
only recognized a band that migrated with an apparent molecular mass of
15 kd in fibroblast lysate, which contains VAMP 2 and VAMP
343 (Figure 1B). In contrast, an antibody directed at an
epitope that is common among VAMP isoforms recognized bands that
migrated with an apparent molecular mass of 18 kd in brain and
fibroblast lysates and bands that migrated with an apparent molecular
mass of 15 kd in fibroblast and platelet lysates (Figure 1B). Thus, the
antihuman cellubrevin used in these studies does not recognize VAMP 1 or VAMP 2.
We next tested the activity of this antibody in a
Ca++-triggered, SL-O-permeabilized platelet model of
Subcellular localization of human cellubrevin in platelets Given that human cellubrevin participates in-granule
secretion, we sought to determine the subcellular localization of this VAMP isoform. A pre-embedding immunonanogold staining technique with
analysis by transmission electron microscopy was used in these studies
because this technique can reliably distinguish among the various
membrane compartments of platelets. Immunolocalization demonstrated
that most human cellubrevin is associated with granule membranes
(Figure 3A-B). A small amount of human
cellubrevin was found associated with the OCS and the plasma membrane
compartments. Although the amount of human cellubrevin associated with
the OCS and the plasma membrane compartments amounted to less than 15% of the total, human cellubrevin staining of these membrane compartments was consistently observed in randomly selected, resting platelets. The
amount of human cellubrevin associated with the platelet plasma membrane was less than that associated with the OCS (Table 1). Control
samples processed with either substitution of irrelevant rabbit IgG for
immune primary antibody (Figure 3D) or omission of immune primary
antibody demonstrated no significant staining. Samples processed with
immune antibody subjected to affinity chromatography to
solid-phase human cellubrevin peptide also showed no significant staining (Figure 3C). Similarly, no significant staining was observed when the secondary antibody or the HQ silver enhancement solution was omitted from the processing. Additionally, staining was virtually absent in the -granule matrix and in the mitochondria.
To determine the relative distribution of SNARE proteins in platelets
by an independent method, subcellular fractionation was
performed. Although this technique cannot separate OCS from plasma
membranes, subcellular fractionation is capable of separating OCS and
plasma membranes from
Analysis of the nitrogen cavitate of biotin-labeled platelets
demonstrates complete separation of biotin-labeled membranes from
P-selectin-containing fractions (Figure 4B). OCS and plasma membranes
were recovered primarily in the 11% fraction and the 11% to 15%
interface, whereas Subcellular localization of SNAP-23 in platelets SNAP-23 is the SNAP isoform demonstrated to function in platelet-granule release.34 Pre-embedding immunonanogold
ultrastructural studies were performed to define the subcellular
distribution of SNAP-23 in platelets. These studies demonstrated that
this SNARE protein is predominantly associated with OCS and plasma membranes (Figure 5A-C). Furthermore,
4-fold more SNAP-23 was associated with plasma membrane than with OCS
(Table 1). The amount of SNAP-23 associated with the granule membrane
was similar to the amount associated with the OCS. Control
samples processed with the substitution of irrelevant rabbit IgG for
immune primary antibody (Figure 5D) or the omission of immune primary
antibody demonstrated no significant staining. Similarly, no
significant staining was observed when either the secondary antibody or
the HQ silver enhancement solution was omitted from the processing. Additionally, staining was virtually absent over the -granule matrix and over the mitochondria. Localization of SNAP-23 by
subcellular fractionation demonstrated that most of this SNARE protein
comigrated with biotin-labeled membranes (Figure 4). This observation
is consistent with the immunonanogold staining that shows most SNAP-23 is associated with OCS and plasma membranes. A fraction of SNAP-23, however, was consistently found in the 25% to 30% interface, as was
observed with the antihuman cellubrevin antibody. These observations demonstrate that SNAP-23 is present in the -granule-enriched fractions of the step gradient, which concurs with the ultrastructure study findings and suggests that some SNAP-23 is present on platelet -granules.
Subcellular localization of syntaxin 2 Syntaxin 2 has been shown to mediate-granule
secretion34 along with syntaxin 4.29,34
Therefore, we sought to determine the subcellular localization of this
member of the platelet trimeric exocytotic complex. Immunonanogold
ultrastructural studies demonstrated that syntaxin 2 was found on OCS,
plasma, and granule membranes (Figure
6A-D; Table 1). Syntaxin 2 was localized
more evenly than human cellubrevin or SNAP-23 among these 3 membrane
compartments. Control samples processed with the substitution of
irrelevant goat IgG (Figure 6E) for immune primary antibody or the
omission of immune primary antibody demonstrated no significant
staining. In addition, no significant staining was observed when the
secondary antibody or the HQ silver enhancement solution was omitted
from the processing. As with the other antibodies, staining was
virtually absent in the -granule matrix and in the mitochondria.
Immunoblot analysis of subcellular fractions from the step gradient
demonstrated that syntaxin 2 comigrates with biotin-labeled fractions
and with fractions containing P-selectin (Figure 4). This pattern of
staining suggests that syntaxin 2 is present on OCS and plasma
membranes and on -granules. Thus, antihuman cellubrevin,
anti-SNAP-23, and anti-syntaxin 2 antibodies all react with an
antigen in the -granule-enriched fractions. Consistent with the
immunonanogold staining, this observation suggests that all 3 SNARE
proteins of the trimeric exocytotic complex are present on platelet
-granules.
With respect to regulated vesicle exocytosis, the original SNARE hypothesis predicted that vSNAREs located on vesicles interact with tSNAREs located on plasma membranes. Detailed subcellular localization of SNARE proteins in synaptosomal preparations and yeast, however, have demonstrated that the assignment of tSNAREs to plasma membrane and vSNAREs to vesicle membranes cannot be generalized. Immunogold labeling of synaptosomal preparations demonstrated that syntaxin and SNAP-25 colocalize on vesicles.49 In fact, syntaxin 1 and SNAP-25 comprise 3% of the total protein of purified synaptic vesicles.50 A ternary cis complex of syntaxin, SNAP-25, and VAMP has been demonstrated to assemble and disassemble on purified synaptic vesicles in the absence of plasma membrane.51 In yeast, isolated vacuoles were also found to contain tSNAREs and vSNAREs, which were demonstrated to form a pentameric cis complex required for membrane fusion.52 Subcellular localization of SNARE proteins has also been performed in some hematopoietic cells. In neutrophils, VAMP-2 was concentrated in tertiary granules and secretory vesicles, whereas syntaxin 4 was found almost exclusively in plasma membrane.53 In resting mast cells, subcellular fractionation showed that VAMP 2 and syntaxin 3 were associated with granules, whereas syntaxin 4 and SNAP-23 were absent from the granules.54 Ultrastructural studies showed that on activation, SNAP-23 relocated from plasma membrane lamellipodialike projections to mast cell granule membranes. To better understand the molecular basis for the unusual morphologic
features of the platelet-release reaction, we determined the
subcellular localization of 3 functional SNARE proteins of the trimeric
exocytotic complex. SNAP-23 and syntaxin 2 have been demonstrated to function in platelet granule
secretion.29,32,34 Given that a function for human
cellubrevin in platelet granule secretion had not previously been
demonstrated, we sought to determine whether this vSNARE contributed to
granule secretion before we performed subcellular localization of this
VAMP isoform. Antibody directed at human cellubrevin inhibited
Ca++-induced Immunonanogold staining and subcellular fractionation demonstrate human cellubrevin, SNAP-23, and syntaxin 2 on platelet granule membranes. It is possible that trans complexes of vSNAREs and tSNAREs exist between granules facilitating homotypic granule fusion. An alternative possibility is that these 3 SNARE proteins exist in a cis complex on isolated granules in the resting platelet, as has been found in neurons and yeast.51,52 In this scenario, activation of the platelet may lead to the disassembly of cis complexes and the formation of trans complexes that drive homotypic granule fusion. In either case, the observation that platelet granules contain vSNAREs and tSNAREs forms a molecular basis for homotypic granule fusion. Similarly, the distribution of functional SNARE proteins of the trimeric exocytotic complex provides a molecular mechanism whereby platelet granules can fuse with the plasma membrane and membranes of the OCS. Although the composition of SNARE proteins, as detected by immunonanogold electron microscopic labeling, differs between OCS and plasma membranes (Table 1), both membranes are relatively enriched for tSNAREs that could interact with vSNARE isoforms, such as human cellubrevin, on granule membranes. One important observation regarding the subcellular localization of human cellubrevin, SNAP-23, and syntaxin 2 is that SNARE protein distribution in an anucleate system is not random. The quantitation of immunogold labeling of human cellubrevin and SNAP-23 demonstrates that each functional SNARE protein of the trimeric exocytotic complex has a distinct localization (Table 1). A study by Chen et al47 performed in platelets using a postembedding labeling technique demonstrated that syntaxins 2 and 4 associate with granule membranes and with OCS and plasma membranes. Consistent with their results, our study found that the largest portion of syntaxin 2 associates with granule membranes and that the remainder is divided evenly between OCS and plasma membranes. Human cellubrevin is found predominantly in granule membranes. Of the human cellubrevin not associated with granule membranes, approximately twice as much is present in OCS as in plasma membranes. This pattern of distribution differs in several respects from that of SNAP-23. Not only is SNAP-23 found predominantly (approximately 80%) on OCS and plasma membranes, but labeling reveals 4 times more SNAP-23 staining of plasma membranes than of the membranes of the OCS. Thus, SNARE protein staining of platelet granular membranes, OCS, and plasma membranes demonstrates a distinct pattern of distribution for each membrane compartment. Subcellular fractionation confirms a nonrandom distribution of SNARE
proteins of the exocytotic complex in platelets. The abundance of
SNAP-23 and syntaxin 2 detected in fractions that migrate with OCS and
plasma membranes may be secondary to the fact that the subcellular
fractionation strategy described in these studies demonstrates a 2- to
3-fold better yield for biotin-labeled membranes (ie, OCS and plasma
membranes) than for P-selectin- and One explanation for the nonrandom nature of SNARE protein distribution in platelets is that SNARE proteins are targeted differentially to platelet membranes during megakaryocytopoiesis and that this distribution is maintained throughout the lifespan of the platelet. This mechanism alone would require that little membrane trafficking occur in the platelet because rapid membrane turnover would lead to membrane mixing and uniformity of SNARE protein distribution. However, constitutive endocytosis1,3,55 and true phagocytosis56,57 have been reported in platelets. In most cells, endocytosis is accompanied by exocytosis to maintain a relatively constant plasma membrane surface area. For example, the surface area of the monocyte remains relatively stable despite the fact that it ingests 3% of its plasma membrane every minute.58 Even if membrane turnover is an order of magnitude less robust in platelets, substantial membrane mixing would occur over the lifespan of the platelet. An alternative explanation for the nonrandom distribution of SNARE proteins in the platelet is the possibility that platelets have an active sorting mechanism that, for example, preferentially shuffles human cellubrevin to granule membranes and SNAP-23 to plasma membranes. Delivery of soluble proteins to specific platelet granules and sorting of membrane receptors to specific membrane compartments is well documented in platelets.55,59 The molecular mechanisms directing these constitutive sorting events, however, are ill defined. Studies in nucleated cells have demonstrated that SNARE proteins are involved in constitutive membrane targeting.60,61 In addition, SNARE proteins in nucleated cells recycle between membrane compartments.62,63 Constitutive recycling of SNARE proteins may also occur in platelets. This SNARE protein trafficking may contribute to the constitutive membrane and protein sorting observed in platelets.
We thank Kathyrn Pyne for photographic assistance. We thank members of the Beth Israel Deaconess Medical Center Blood Bank for their assistance.
Submitted July 17, 2001; accepted January 29, 2002.
Supported by National Institutes of Health grants AI33372 (A.M.D.),
AI44066 (A.M.D.), and HL63250 (R.F.). R.F. is a Burroughs Wellcome Fund
Career Awardee and is a participant in the Clinical Investigator
Training Program: Beth Israel Deaconess Medical Center
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.
Reprints: Robert Flaumenhaft, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 319, Research East, PO Box 15732, Boston, MA 02215; e-mail: rflaumen{at}caregroup.harvard.edu.
1. Morgenstern E. Coated membranes in blood platelets. Eur J Cell Biol. 1982;26:315-318[Medline] [Order article via Infotrieve].
2.
Handagama P, Scarborough RM, Shuman MA, Bainton DF.
Endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa).
Blood.
1993;82:135-138 3. Behnke O. Degrading and non-degrading pathways in fluid-phase (non-adsorptive) endocytosis in human blood platelets. J Submicrosc Cytol Pathol. 1992;24:169-178[Medline] [Order article via Infotrieve]. 4. Sudhof TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995;375:645-653[CrossRef][Medline] [Order article via Infotrieve]. |
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Abstract
Introduction
human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman
cellubrevin antibody inhibited Ca++-induced 
.001) (Figure 
