From the
The glycoprotein (GP) Ib-IX-V complex comprises four
polypeptides: the subunits of the GP Ib-IX complex (GP Ib
Glycoprotein (GP)
The GP
Ib-IX-V polypeptides each span the plasma membrane once and are present
in a stoichiometry of 2:2:2:1 for GP Ib
All of the leucine-rich
polypeptides of the platelet membrane are missing in classic forms of
the Bernard-Soulier syndrome, an autosomal recessive bleeding disorder
in which the hemorrhagic episodes can be life-threatening(6) .
Because each polypeptide is encoded by its own gene, this finding
strongly suggests that surface expression may require coordinate
expression of all of the polypeptides. Studies in transfected cells and
mutations described in Bernard-Soulier syndrome support this
hypothesis. We have shown that a functional GP Ib-IX complex (lacking
GP V) is expressed on the surfaces of transfected mammalian cells by
cotransfection of its three subunits and that transfection of less than
the full complex decreases the levels of GP Ib
Although GP V is
also missing in Bernard-Soulier syndrome, these studies indicate that
it is less likely than the other polypeptides to be required for the
cell-surface expression of GP Ib-IX, but it may nonetheless contribute
to its stability. On the other hand, the findings that platelets from
patients with Bernard-Soulier syndrome caused by mutations of either GP
Ib
We report here the requirements for plasma membrane
expression of GP V and its associations with the other polypeptides of
the GP Ib-IX-V complex. Glycoprotein V did not require an association
with the GP Ib-IX complex to reach the plasma membrane in transiently
transfected cells, but this association was required for its most
efficient expression on the plasma membrane. The association of GP V
with GP Ib-IX appears to be mediated by a direct interaction with GP
Ib
Thus, an understanding is emerging of the
interrelationships of the leucine-rich polypeptides on the platelet
plasma membrane. Recently, we showed that GP Ib
The questions
of whether association with GP Ib-IX increases plasma membrane
expression of GP V and vice versa were both addressed in the current
article. Glycoprotein V was clearly expressed on the plasma membranes
of transiently transfected CHO cells in the absence of any of the
subunits of the GP Ib-IX complex. This may reflect the situation in
platelets and megakaryocytes, or may indicate that GP V surface
expression or stability in CHO cells is affected by proteins not
present in platelets. Two lines of evidence indicate that this
expression on the plasma membrane is not very efficient. The first is
the observation that much higher levels of GP V on the plasma membrane
are achieved in the presence of GP Ib
In contrast to the important role of GP Ib-IX in cell-surface
expression of GP V, GP V did not greatly increase surface levels of GP
Ib-IX in our studies. However, these results may not adequately account
for the potential contribution of GP V to cell-surface expression of GP
Ib
In summary, in the
current report we have defined the requirements for cell-surface
expression of platelet GP V, one of four leucine-rich proteins on the
platelet plasma membrane that make up the receptor for von Willebrand
factor. These data provide further insight into the molecular nature of
Bernard-Soulier syndrome and help to elucidate the structure of this
receptor complex on the cell surface.
We acknowledge the technical help of Martine Morales
in cloning the GP V gene, Drs. Robert W. Mahley and Robert Pitas for
carefully reading the manuscript, Alana Koehler for help in preparing
the manuscript, Liliana Jach and Amy Corder for graphics and
photography, and Dawn Levy for editorial assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, GP
Ib
, GP IX) and GP V. To determine the requirements for
cell-surface expression of GP V, we transiently expressed the
recombinant polypeptide in wild-type Chinese hamster ovary (CHO) cells
by cotransfection with plasmids for the subunits of the GP Ib-IX
complex and in CHO cells that stably express different combinations of
the GP Ib-IX complex subunits. Glycoprotein V expressed alone was
detectable on the cell surface, and the level was not augmented by
cotransfection with any one of the subunits of the GP Ib-IX complex.
However, when GP V was expressed in cells that stably express
combinations of GP Ib-IX complex subunits, its expression on the cell
surface was greater in all the cell lines that contained GP Ib
than in wild-type CHO cells. That GP V associates with GP Ib
was
also suggested by confocal microscopy studies: GP V colocalized with GP
Ib
in CHO
IX (cells that express GP Ib
, GP Ib
,
and GP IX), CHO
, and CHO
IX cells, but did not
colocalize with GP Ib
in CHO
IX cells. Similarly,
immunoprecipitation of GP V from cells expressing GP Ib
led to
coprecipitation of the latter polypeptide; neither GP Ib
nor GP IX
coprecipitated with GP V from CHO
IX cells. Taken together, these
data indicate that GP V associates with the GP Ib-IX complex through a
direct interaction with GP Ib
and establish the topology of the GP
Ib-IX-V subunits on the cell surface.
(
)V is one of four
homologous polypeptides complexed on the platelet surface to form a
receptor for von Willebrand factor(1, 2, 3) .
This receptor, the GP Ib-IX-V complex, mediates adhesion of platelets
to the subendothelial matrix at sites in the vasculature where the
endothelium has been removed(4) . Adhesion by this mechanism
increases as shear rates increase, which explains its importance in
preventing hemorrhage from capillary or arteriole injury.
, GP Ib
, GP IX, and GP
V, respectively(1) . All of these polypeptides are also
evolutionarily related to one another, belonging to a family of
proteins whose common feature is the presence of a structural motif
with one or several tandem repeats of a sequence containing periodic
spacing of leucines and with disulfide loops that flank the
leucine-rich repeats(5) .
expressed on the
cell surface(7) . This complex retains many of the functions and
structural features of the platelet complex, including its ability to
bind von Willebrand factor and a number of conformation-sensitive
antibodies and to associate with actin-binding protein in the
cytoskeleton. Recent studies have shown that GP Ib
can form
partial complexes with both GP Ib
and GP IX, but the latter two
polypeptides associate only loosely, if at all(8) . Association
with GP Ib
also increases the expression on the cell surface of
both GP Ib
(7) and GP IX(8) .
(9) or GP IX (10) express less GP V than do
normal platelets suggest that GP V requires the GP Ib-IX complex for
its expression on the cell surface. In the studies reported here, we
examined the requirements for cell-surface expression of GP V and
studied the subunits of the GP Ib-IX complex with which it associates.
Plasmids
The expression plasmid ZEM229R was a
kind gift from Dr. Eileen Mulvihill of ZymoGenetics, Inc. and has been
described in detail(11) . Briefly, it contains bacterial
sequences and the -lactamase gene for propagation in bacteria and
growth in ampicillin-containing media. Transcription of cloned DNA is
under the control of the modified mouse MT-1 promoter (which permits
constitutive expression in mammalian cells) and an SV40 enhancer. A BamHI fragment of the gene for GP V, corresponding to
nucleotides 2311 to 7452 of the sequence published by Lanza and
colleagues(3) , was ligated into ZEM229R to generate the
expression vector ZEMGPV.
Cell Lines
CHO DUK cells (ATCC
CRL 9096, American Type Culture Collection, Rockville, MD) and
transfected cells stably expressing various combinations of the GP
Ib-IX subunits were used for transient GP V expression. The stable
lines have been described (7, 8) and include CHO
IX (expressing GP Ib
, GP Ib
, and GP IX), CHO
(expressing GP Ib
and GP Ib
), CHO
IX
(expressing GP Ib
and GP IX), CHO
IX (expressing GP Ib
and GP IX), and L2H cells (an L-cell clone that expresses the GP Ib-IX
complex). In the latter three CHO cell lines, expression of the
transfected polypeptides has been amplified by growth in progressively
increasing concentrations of methotrexate(8, 12) ; CHO
IX cells were not subjected to amplification(7) . CHO
IX were grown in a 1:1 mixture of Dulbecco's modified
Eagle's medium and F-12 medium supplemented with 10% fetal bovine
serum and 400 µg/ml G418 (Sigma). CHO
, CHO
IX, and
CHO
IX were grown in
-minimal essential medium without
nucleosides and containing 400 µg/ml G418 and 80 µM methotrexate (Sigma).
Transfections
The cells were transfected as
described previously (8, 13) using lipofection to
deliver the plasmid DNA. Analysis of GP V expression was carried out
48-72 h after transfection. Four types of transfections were
carried out: 1) transfection of ZEMGPV alone into CHO
DUK, CHO
IX, CHO
, CHO
IX,
and CHO
IX cells; 2) cotransfection into CHO DUK
cells of ZEMGPV with plasmids for each of the other subunits of
the GP Ib-IX-V complex; 3) cotransfection of ZEMGPV with a plasmid for
GP Ib
or with ZEM229R into CHO
IX cells; and 4) transfection
of L2H cells with the GPV plasmid or with ZEM229R.
Flow Cytometry
The cells were analyzed for surface
expression of GP V or GP Ib by flow cytometry after being labeled
with monoclonal antibodies against either of the two polypeptides.
Cells transfected with the expression plasmid alone served as negative
controls for transfection. The antibodies used were SW16 (1) for GP V
(Accurate Chemical and Scientific Corp., Westbury, NY) and AN51 (14) (DAKO, Carpinteria, CA) for GP Ib
. Cells were labeled
with the antibodies as described previously(8) , and their
fluorescence was analyzed on a Becton-Dickinson FACStar flow cytometer
after stimulation of the fluorescein with an argon ion laser at a
wavelength of 488 nm. Emission was detected at 520 nm.
Confocal Microscopy
In CHO IX, CHO
, CHO
IX, and CHO
IX cell lines transfected with GP
V, subcellular localization of GP V was compared to the localization of
one of the polypeptides that is expressed constitutively. In all of the
cells that contain GP Ib
, this polypeptide was detected with a
polyclonal antibody(7) , and in CHO
IX cells, polyclonal
anti-GP Ib
(7) was used. SW16 was used to detect GP V. The
monoclonal antibody was detected with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG, and the polyclonal
antibodies were detected with biotin-labeled goat anti-rabbit IgG
followed by streptavidin-conjugated Texas Red (Amersham, Arlington
Heights, IL). Forty-eight to 72 h after transient transfection with
ZEMGPV, the cells, which were grown directly on glass chamber slides
(Titer-tek, Nunc, Naperville, IL), were washed in phosphate-buffered
saline, fixed with 4% paraformaldehyde (Sigma), and permeabilized in
0.1% Triton X-100 (Sigma). The cells were then incubated with the two
primary antibodies for 1 h, washed several times with
phosphate-buffered saline, and incubated with the secondary antibodies
for an additional 30 min. The secondary antibodies were then removed,
the cells washed again several times with phosphate-buffered saline,
and the streptavidin-conjugated Texas Red was added and incubated for
30 min. After several more washes, the cells were either examined
immediately by microscopy or stored in the dark at 4 °C until they
were examined. Confocal microscopy was performed with a Bio-Rad MRC-600
Laser Scanning Confocal Imaging System.
Metabolic Labeling, Immunoprecipitation, and
SDS-Polyacrylamide Gel Electrophoresis
These experiments were
performed on cells transiently transfected with ZEMGPV 48-72 h
after the transfection. The cells, which were grown in 35-mm cell
culture dishes, were washed twice in cysteine-free -minimal
essential medium, then incubated in this medium for 30 min before
adding to the medium 100 µCi/dish of
[
S]cysteine (ICN, Irvine, CA). After incubating
the cells in radioactive medium for 4 h, the medium was removed and the
cells washed twice in phosphate-buffered saline. Cells were lysed in
digitonin lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mg/ml leupeptin, 1.6 mg/ml benzamidine, 0.1 mg/ml soybean
trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 1%
digitonin) and centrifuged at 14,000
g for 5 min to
remove debris. The lysate was then incubated overnight at 4 °C with
fixed Staphylococcus aureas cells (Pansorbin, Calbiochem
Corp., La Jolla, CA) to preclear the lysate. After the cells were
removed by centrifugation, the lysate was incubated in 10 µg/ml
SW16 before adding Pansorbin beads that had been pre-equilibrated with
rabbit anti-mouse IgG (5 µg/ml, 4 h, 37 °C). The beads were
incubated with the lysate for 4 h at 4 °C, then pelleted by
centrifugation at 14,000
g for 5 min and resuspended
and washed twice in digitonin lysis buffer. Immunoprecipitated,
radiolabeled proteins were removed from the beads by boiling in SDS
sample buffer (15) for 5 min and were then subjected to SDS-PAGE
on a 7.5% polyacrylamide gel. After electrophoresis, the dried gel was
evaluated by autoradiography on a Fuji phosphorimager (Model BAS 1000).
GPV Is Expressed Alone on the Plasma Membrane But Its
Expression Is Greatest in the Presence of GP Ib
To examine
the requirements for cell-surface expression of GP V, a plasmid
containing a fragment of its gene that includes the entire coding
region (designated ZEMGPV) was transfected into CHO DUK cells or CHO cells expressing various combinations of the
subunits of the GP Ib-IX complex (Fig. 1A). Plasma-membrane expression of GP V was examined by flow
cytometry 48-72 h after transfection using the anti-GP V
monoclonal antibody SW16. As a control for the nonspecific effects of
transfection on fluorescence, CHO DUK
cells
sham-transfected with the expression plasmid ZEM229R were also
examined. Transient expression of GP V by itself resulted in
significant expression of the polypeptide on the cell surface compared
to the level of background fluorescence in sham-transfected cells (Fig. 1B). When the level of expression in cells
expressing different GP Ib-IX complex subunits was examined, only cells
that expressed GP Ib
displayed GP V on their surfaces at levels
greater than the cells transfected with GP V alone (Fig. 1C). The level of expression in CHO
IX cells
was not significantly different from that in CHO DUK
cells.
Figure 1:
A, scheme for GP V transfection into
cell lines with stable expression of combinations of the polypeptides
of the GP Ib-IX complex. The plasmid containing a fragment of the GP V
gene, designated ZEMGPV, was transiently transfected into CHO
DUK (CHO) cells and into CHO cell lines stably
expressing every combination of the subunits of the GP Ib-IX complex.
Plasma membrane expression of GP V was evaluated 48-72 h after
the transfection with the anti-GP V monoclonal antibody, SW16. The
control for the nonspecific effects of transfection on cell
fluorescence was obtained by transfecting the parent plasmid, ZEM229R,
into wild-type CHO DUK
cells (sham-transfected). B, flow cytometry analysis of GP V expression on the surfaces
of transfected cells. Cytometry histogram of CHO cells transiently
expressing GP V. Glycoprotein V expressed alone appears on the cell
surface. C, comparison of mean fluorescence intensities of the
total cell populations of GP V-transfected cells. Glycoprotein V was
expressed in CHO DUK
cells or in CHO cells expressing
combinations of GP Ib-IX subunits. To obtain these values, the
background fluorescence value of sham-transfected cells was subtracted.
These results are representative of four separate
experiments.
Cotransfection of GP V with Any Single Subunit of the GP
Ib-IX Complex Does Not Increase Its Level on the Plasma
Membrane
To determine if any one of the GP Ib-IX complex
polypeptides enhances GP V expression on the plasma membrane, ZEMGPV
was cotransfected individually with plasmids for each of the other
three polypeptides. None of the three GP Ib-IX polypeptides enhanced GP
V surface levels by transient coexpression (Fig. 2), which may
indicate that higher levels of the auxiliary polypeptide are needed
than can be achieved with transient expression, or that GP Ib must
be coexpressed with either GP Ib
or GP IX to associate with GP V.
Figure 2:
Cotransfection of GP V with plasmids for
each of the GP Ib-IX complex polypeptides. To evaluate whether any one
of the GP Ib-IX polypeptides is able to enhance GP V surface
expression, each was cotransfected into CHO DUK cells
with GP V, and the level of GP V on the plasma membrane was assessed
using the monoclonal antibody SW16 followed by fluorescein
isothiocyanate-conjugated rabbit anti-mouse IgG. Surface levels of GP V
in these cells were compared to levels in sham-transfected
cells.
Metabolic Labeling and Immunoprecipitation of GP
V
GP V was immunoprecipitated with SW16 from metabolically
labeled CHO cells transiently expressing only GP V or coexpressing GP V
with one of the other GP Ib-IX polypeptides. Cell lysis and
immunoprecipitation studies were performed using a digitonin-containing
buffer, a condition that has been shown in platelets to maintain the
association between GP V and the GP Ib-IX complex(1) . One major
radiolabeled polypeptide, with an approximate molecular mass of 70 kDa,
was immunoprecipitated, whether the cells had been transfected with GP
V alone or cotransfected with GP V and one of the GP Ib-IX polypeptides (Fig. 3A).
Figure 3:
GP V
immunoprecipitation. SW16 was used to immunoprecipitate GP V from S metabolically labeled transiently transfected CHO cells. A, GP V transfected alone or with one of the three GP Ib-IX
polypeptides indicated at the top of the gel. B, GP V
transiently expressed in either wild-type CHO cells or CHO cells that
stably express the GP Ib-IX subunits indicated at the top of the
gel.
A different pattern of immunoprecipitated
polypeptides emerged when GP V was transiently expressed in cells that
stably express either the full GP Ib-IX complex or combinations of two
subunits. The immunoprecipitates from cells that express GP Ib
contained two other specific radioactive bands (Fig. 3B). The smaller of these bands, at about 83 kDa,
corresponds to the molecular mass of mature GP V found in platelets.
The larger one, migrating at about 125 kDa, is identical to the
molecular mass of GP Ib
expressed in CHO cells(7) . In
contrast, only the 70-kDa band was observed in the immunoprecipitate
from CHO
IX cells, which express high levels of both GP Ib
and GP IX(8) . Several faster-migrating bands were also
precipitated from the GP Ib
-containing cells; their identities are
unclear, but they may represent degradation products. Thus, it appears
that association of GP V with the GP Ib-IX complex is mediated through
GP Ib
, and that the association probably allows GP V to traverse
intracellular compartments, allowing for the complete maturation of its N-glycan chains.
GP V Colocalizes with GP Ib
Our data to this
point indicated that GP V associates with the complex solely through GP
Ib and that neither GP Ib
nor GP IX associate with GP V in
the absence of GP Ib
. Another explanation for our findings,
however, could be that GP V associates weakly with GP Ib
-GP IX but
that this association neither increases plasma membrane expression of
GP V nor can be detected by immunoprecipitation under the conditions we
used. We therefore used confocal microscopy to examine the subcellular
locations of GP V and one of the other polypeptides in cells expressing
the different GP Ib-IX subunits. In cells expressing GP Ib
(CHO
IX, CHO
, and CHO
IX), expression of GP
Ib
was examined concomitantly with expression of GP V (Fig. 4), whereas in CHO
IX cells expression of GP Ib
and GP V was examined (Fig. 5). In all cases the cells were
rendered permeable to the antibodies with 0.1% Triton X-100.
Glycoprotein V and GP Ib
colocalized perfectly in the three cell
lines that express GP Ib
; in CHO
IX cells a major
portion of both polypeptides was found on the plasma membrane (Fig. 4A). In contrast, although GP V and GP Ib
were both expressed in CHO
IX cells transfected with GP V and were
both found in what appears to be the Golgi complex and endoplasmic
reticulum, there were extensive regions where the two polypeptides did
not colocalize (Fig. 5).
Figure 4:
Confocal
microscope immunofluorescence images of CHO cells transiently
expressing GP V. Glycoprotein V was transiently expressed in CHO
IX, CHO
, and CHO
IX cells, and its
subcellular distribution in these cells was compared to the
distribution of GP Ib
. SW16 was used to detect GP V, and a
polyclonal antiserum was used for GP Ib
. Each micrograph
represents a 1-µm thick optical section through the
cell.
Figure 5:
GP V and
GP Ib do not colocalize in CHO
IX cells transiently
expressing GP V. Confocal microscope images of immunostained cells
prepared as described in the legend to Fig. 4. Glycoprotein V was
detected with SW16, and GP Ib
with a polyclonal antiserum. The
distributions of GP V and GP Ib
are represented in pseudocolor,
red for GP V and green for GP Ib
. The center panel shows
the two images merged. In this image red represents regions containing
GP V and devoid of GP Ib
; vice versa for green. Yellow represents
regions of overlap. There are numerous regions where the polypeptides
do not colocalize, in both the juxtanuclear and peripheral
regions.
Effect of GP V Expression on GP Ib
By examining the expression of GP
Ib Levels on the
Plasma Membrane in L2H Cells
in L2H cells 48 h after they were transfected with a plasmid
for GP V, we tested the possibility that, like GP Ib
and GP IX, GP
V might further increase expression of GP Ib
on the cell surface.
No significant increase in the plasma membrane level of GP Ib
was
detected in these cells (Fig. 6A) despite the fact that
GP V was expressed quite efficiently (Fig. 6B).
Figure 6:
Effect of GP V expression on GP Ib
levels on the plasma membrane in L2H cells expressing the GP Ib-IX
complex. A, L2H cells were transiently transfected with GP V
(
IX/V), and their plasma membrane levels of GP Ib
,
detected with AN51, compared to the levels in sham-transfected cells
(
IX/ZEM). B, the levels of GP V, detected with SW16,
are shown in the same cells.
This result may have been due to a pre-existing high plasma
membrane level of GP Ib on the cell surface, which would have made
an additional contribution from GP V on the surface level of GP Ib
difficult to detect. We therefore also examined the surface expression
of GP Ib
after transient expression in CHO
IX cells, with
cotransfection either of a control plasmid or of ZEMGPV. In this case,
although the efficiency of transfection was poor, the surface level of
GP Ib
did appear to be augmented slightly by GP V (Fig. 7).
Figure 7:
Effect of GP V expression on GP Ib
levels in CHO
IX cells cotransfected with GP Ib
and GP V. CHO
IX cells were cotransfected with plasmids for GP Ib
and GP V
or with either one of these two plasmids and ZEM229R lacking a cDNA
insert. Seventy-two hours after transfection the surface levels of GP
Ib
and GP V were examined with monoclonal antibodies AN51 and
SW16, respectively. Two gates were set based on the distribution of
cell populations in control cells. In the control cells, gate 2 was
virtually devoid of cells. The control cells for GP Ib
expression
were CHO
IX cells transfected with GP V and ZEM229R
(
IX/VZEM). The control for GP V expression was CHO
IX cells
transfected with GP Ib
and ZEM229R (
IX/
ZEM). The number
of cells in each gate is indicated. Gate 2 is expanded and depicted
below the full histogram.
, a conclusion supported by three lines of evidence: 1) the
cell-surface expression of GP V increases when the polypeptide is
expressed in cell lines that contain GP Ib
; 2) GP V antibodies
coprecipitate GP Ib
from cell lysates; and 3) GP V is colocalized
with GP Ib
in CHO
IX, CHO
, and CHO
IX
cells but in CHO
IX cells significant lack of colocalization with
GP Ib
is observed.
associates
independently with GP IX and GP Ib
but were unable to demonstrate
association of GP IX with GP Ib
(8). When combined with the
finding in the current article that GP V associates with GP Ib
,
these data suggest that the actual topology of the GP Ib-IX-V complex
is as shown in Fig. 8. This scheme shows GP V associating with
two GP Ib
molecules, an arrangement that is not proven but which
is suggested by the relative numbers of the two polypeptides on the
platelet surface(1) .
Figure 8:
Schematic depiction of the probable
spatial relationships between the polypeptides of the GP Ib-IX-V
complex (modified from Ref. 5). The arrangement is based on data
presented in this article, as well as data from Ref. 8 and the
stoichiometry data of Ref. 1.
The precise regions through which the
GP Ib-IX-V complex polypeptides associate have not been defined.
Although each of the four polypeptides contains a leucine-rich motif,
it is not clear how these motifs function in linking the polypeptides.
Mutations in this motif in GP IX associated with Bernard-Soulier
syndrome suggest that this region may have a role in the association
between GP Ib and GP IX(10, 16) .
(
)The leucine-rich motifs are not likely to be involved
in the association between GP V and GP Ib
, however, as GP V
remains associated with the part of the GP Ib-IX complex that remains
on the plasma membrane after the amino-terminal 45-kDa region of GP
Ib
is removed by protease treatment(1) .
. Second, although the GP V
that appears on the cell surface in the single-subunit transfection has
presumably undergone full post-translational modification, the
immunoprecipitation and microscopy studies indicate that fully
processed cell-surface GP V represents only a minor fraction of total
GP V in cells that lack GP Ib
( Fig. 3and Fig. 5).
. If GP Ib
is in excess of GP V and already efficiently
expressed on the plasma membrane (which is the case in the studies
involving transient expression of GP V in L2H cells, a stable cell line
containing GP Ib-IX), the presence of GP V may have only a minor effect
on the levels of GP Ib
that reach the plasma membrane. This
interpretation is supported by the observation that confocal
micrographs of cells expressing the full GP Ib-IX complex show
virtually all of the complex on the cell surface (not shown), which
implies that only a very small intracellular pool remains to be
assisted to the cell surface if the cells are super-transfected with GP
V. Glycoprotein V might significantly increase the surface level of GP
Ib
when the latter polypeptide is not as efficiently expressed on
the plasma membrane, i.e. when expressed in the absence of GP
Ib
or GP IX. We did not examine the effect of GP V on the surface
levels of GP Ib
in this situation. Alternatively, GP V may
increase GP Ib-IX levels when GP V is in excess, for example, if the GP
Ib-IX plasmids were to be expressed transiently in cells that stably
express GP V. In any event, the failure of GP V to significantly
enhance surface expression of GP Ib
in the presence of GP Ib
and GP IX decreases the likelihood that GP V mutations will lead to
Bernard-Soulier syndrome. Nevertheless, the absence of GP V may cause
other functional disorders of the platelets.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.