From the
The goal of the present study was to determine whether platelet
glycoprotein (GP) V interacts directly with the von Willebrand factor
receptor GP Ib-IX and, if so, whether it affects the expression and
function of this receptor. A melanoma cell line that does not contain
actin-binding protein was transfected with the cDNAs coding for GP V
and for each of the three subunits of GP Ib-IX. GP V
co-immunoprecipitated and co-localized with GP Ib-IX. Although GP V
could be expressed in the absence of GP Ib-IX, the amount incorporated
in the membrane was markedly increased when GP Ib-IX was present.
Similarly, there was an enhanced expression of GP Ib-IX on the cell
surface in the presence of GP V. The binding affinity of
botrocetin-induced von Willebrand factor to GP Ib-IX was unaffected by
the presence or absence of GP V. However, the binding capacity was
increased by the presence of GP V. We conclude that GP V interacts
directly with GP Ib-IX, that GP V must associate with GP Ib-IX to be
efficiently expressed in the membrane, and that GP V increases the
binding capacity of the cells for von Willebrand factor by enhancing
the surface expression of the GP Ib-IX complex.
Platelets play a fundamental role in the formation of the
hemostatic plug at sites of vascular injury(1, 2) . One
of the critical interactions mediating the initial contact of platelets
with the damaged vessel wall is the interaction of the platelet
glycoprotein (GP)
The GP Ib-IX complex
consists of two disulfide-linked subunits, GP Ib
The function
of GP V is not known. Because it is cleaved by thrombin, it was
originally thought to be the functional thrombin
receptor(26, 27, 28) . However, it is now known
that it is cleaved from the surface of activated platelets by a
protease other than thrombin; thrombin appears to cleave it after it
has been removed from the platelet membrane by other mechanisms (for
review, see Ref. 29). Based on the finding that decreases in GP Ib-IX
in patients with Bernard-Soulier syndrome are also accompanied by
decreased surface expression of GP
V(15, 16, 17, 18) , it appears possible
that GP Ib-IX is required for the expression of GP V on the surface.
Conversely, it appears possible that if GP V and GP Ib-IX form a
complex, GP V might regulate the expression of the GP Ib-IX subunits on
the surface. If so, this would not only provide the first known
function of GP V, but it would also raise the possibility that some
cases of Bernard-Soulier syndrome could potentially arise from
mutations in GP V rather than from mutations in one of the subunits in
GP Ib-IX.
In the present studies, GP V and GP Ib-IX were expressed
in a melanoma cell line that lacks actin-binding protein(30) .
This approach eliminated the potential problem of GP Ib-IX and GP V
co-immunoprecipitating because they are both associated with the
cytoskeleton and allowed us to determine whether GP V interacts with GP
Ib-IX. Further, confocal microscopy was used to determine whether the
glycoproteins co-localize in the intact cell, an approach that is not
feasible in platelets because of the small size of the cells. By
transfecting cells with combinations of cDNAs coding for the
glycoproteins, we have determined whether the expression of one
glycoprotein influences the expression of others. We show that GP V
interacts directly with GP Ib-IX in the membrane of the cells and that
this interaction is needed for the efficient expression of both GP V
and GP Ib-IX in the membrane. Further, our studies suggest that GP V
increases the binding capacity of the cells for von Willebrand factor
by increasing the surface expression of the GP Ib-IX complex.
Stable
transfections were performed by the calcium phosphate method described
by Graham and van der Eb(32) . The cells were co-transfected
with 10 µg of each linearized construct containing the cDNAs
encoding GP Ib
In one experiment, as indicated in the text, GP V-expressing cells
were transiently transfected with 3 µg of each expression plasmid
containing the cDNAs coding for GP Ib
To determine whether GP V associates with GP Ib-IX, cells
were stably transfected with the cDNA for GP V and the three cDNAs
encoding the subunits of GP Ib-IX (GP Ib
Previously, the observation that the platelets from patients
with Bernard-Soulier syndrome lacked GP V in addition to GP Ib-IX
suggested that GP V and GP Ib-IX may exist in a complex. The finding
that the four glycoproteins co-immunoprecipitated from detergent-lysed
platelets provided some support for this possibility. However, GP Ib-IX
interacts with high affinity with actin-binding protein, so a variety
of cytoskeletal proteins are always present in a GP Ib-IX
immunoprecipitate. In the present study, we provide several lines of
evidence that GP V interacts directly with GP Ib-IX. First, GP V
co-immunoprecipitated with GP Ib-IX from lysates of cells that did not
contain actin-binding protein. Second, platelets are too small to
provide convincing evidences for co-localization of proteins by
immunofluorescence microscopy. However, by expressing the glycoproteins
in melanoma cells we were able to visualize their distribution by
confocal microscopy; this approach revealed a precise co-localization
of GP V and GP Ib-IX in the membrane, showing that the interaction
between the glycoproteins occurs in the intact cell and is not an
artifact induced in cell lysates. Finally, in cells expressing both GP
Ib-IX and GP V, the cells expressing most GP Ib-IX on the surface
expressed most GP V and vice versa.
These results suggest
that GP V is an additional component of the GP Ib-IX complex in intact
cells. In the present study, GP V and GP Ib-IX remained associated when
the cells were lysed with the mild detergent, digitonin, but
dissociated in a stronger detergent, Triton X-100. Modderman et al.(19) also found that GP V and GP Ib-IX
co-immunoprecipitated from lysates prepared with digitonin but did not
co-immunoprecipitate when lysates were prepared with Nonidet P-40 or
octyl glucoside. In contrast, the three subunits of the GP Ib-IX
complex remain tightly associated when platelets are lysed in strong
detergents such as Triton X-100(6, 23) . Thus, GP
Ib
Binding studies using monoclonal antibodies
have indicated that each platelet contains approximately 24,000
molecules of GP Ib-IX but only 11,000 molecules of GP
V(19, 46, 47) . It has been suggested that two
GP Ib-IX molecules may associate with one GP V molecule(19) . An
alternative possibility might be that some of the GP Ib-IX complexes
are not associated with GP V and that GP V confers a special function
on a subpopulation of GP Ib-IX. It was originally thought that GP V was
the functional thrombin receptor on
platelets(26, 27, 28) , but it is now clear that
this is not the case (for review, see Ref. 29). Elucidation of other
potential functions of GP V awaits further study.
Interestingly, GP
V, like GP Ib
Previously, it has been shown that although GP
Ib
The finding that GP Ib-IX is needed for the
efficient surface expression of GP V supports the idea that all four
subunits of the GP Ib-IX-V complex are needed for the efficient
expression of the others in the membrane. Further support for this came
from experiments in which we transfected melanoma cells with the cDNAs
encoding GP Ib
The finding that GP V increases the surface
expression of GP Ib-IX represents the first known function of GP V.
This function is important in that it raises the possibility that some
cases of Bernard-Soulier syndrome could arise from mutations in GP V
rather than in one of the subunits of the GP Ib-IX complex. Further, it
raises the possibility that GP V could be important in regulating the
ability of cells to bind von Willebrand factor. To address this
question, we measured botrocetin-induced von Willebrand factor binding
to the GP Ib-IX- and GP Ib-IX plus GP V-expressing cells. The binding
affinity of von Willebrand factor to GP Ib-IX was not affected by the
presence of GP V. However, the binding capacity of the cells was
markedly increased by the presence of GP V. Thus, we suggest that GP V
serves to increase the binding of von Willebrand factor by regulating
the expression of the GP Ib-IX complex on the surface of the cells. In
the present study, the binding of von Willebrand factor was induced by
botrocetin; in vivo, the binding of von Willebrand factor to
platelets is induced by immobilization of von Willebrand factor and is
very dependent on shear forces (for review, see Refs. 54 and 55). While
we could not detect an effect of GP V on the binding affinity of von
Willebrand factor in the present study, the interaction of GP Ib-IX
with von Willebrand factor is especially important at high wall shear
conditions (56). It appears possible that GP V might affect the
accessibility of von Willebrand factor for the binding site on GP
Ib
Stably transfected cells expressing GP V or GP V
and GP Ib-IX were plated in six-well tissue culture dishes (10
We are grateful to Drs. C. Cunningham and J. Hartwig
for providing the melanoma cells; Dr. J. López for the
constructs coding for GP Ib-IX; Dr. F. Lanza for the GP V cDNA; Drs. B.
Steiner, D. Phillips, P. Modderman, and K. Fujimura for antibodies; and
Dr. J. Moake for purified vWf. We thank Dr. S. Ruzin for assistance
with confocal microscopy and W. Hyun for assistance with cell sorting.
We are grateful to S. Zuerbig for technical assistance and graphic
services, G. Santos for his help with the confocal microscopy, and Drs.
P. Gascard and J. Cunningham for critical review of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)Ib-IX complex with von
Willebrand factor (vWf)(3, 4) .
(M
= 140,000), and GP Ib
(M
= 25,000) that are noncovalently
associated in a 1:1 ratio with GP IX (M
=
22,000)(5, 6) . Each of these glycoproteins is a
transmembrane protein(7, 8, 9) . An interesting
feature of the three components of the GP Ib-IX complex is that they
each contain leucine-rich motifs in their extracellular
domains(7, 8, 9) . A fourth platelet membrane
glycoprotein, GP V (M
= 82,000), is an
additional member of this family of leucine-rich
glycoproteins(10, 11, 12, 13) . All
three subunits of the GP Ib-IX complex are missing or are present in
significantly decreased amounts in platelets from Bernard-Soulier
patients(14, 15, 16) ; the finding that the
amount of GP V is also decreased in the membranes of these
platelets(15, 16, 17, 18) raised the
possibility that GP V may be part of a complex with the other three
leucine-rich glycoproteins. Evidence that such a complex may exist
comes from a report that GP V can be co-immunoprecipitated with GP
Ib-IX from platelets lysed with the mild detergent digitonin (19).
However, GP Ib-IX associates with actin-binding protein, thus mediating
attachment of this adhesion receptor to the cytoskeleton. Actin-binding
protein and GP Ib-IX interact with such high affinity that they remain
associated in all of the detergents used to lyse
platelets(20, 21, 22, 23, 24, 25) .
Thus, actin-binding protein and associated cytoskeletal proteins always
co-immunoprecipitate with GP Ib-IX from detergent-lysed platelets,
making it difficult to determine whether GP V and GP Ib-IX
co-immunoprecipitate because they associate directly with each other or
because they are both associated with the cytoskeleton.
Cell Culture and Transfections
The melanoma
cells used in these studies were derived from a human malignant
melanoma lacking actin-binding protein(30) . Actin-binding
protein-deficient cells were used in the immunoprecipitation
experiments described below. For all other experiments, cells that had
been stably transfected with the cDNA coding for actin-binding protein
were used(30) . Both the actin-binding protein-deficient cells
and the actin-binding protein-containing cells were kindly provided by
Dr. C. Cunningham (Brigham Women's Hospital, Boston, MA). The
actin-binding protein-deficient cells were grown in Dulbecco's
Modified Eagle Medium (Life Technologies, Inc.) with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life
Technologies, Inc.) at 37 °C in a 5% CO atmosphere. The
actin-binding protein-containing cells were grown in the same medium
supplemented with 0.5 mg/ml G418. The constructs containing the cDNAs
coding for GP Ib
, GP Ib
, and GP IX
subcloned into the eukaryotic expression vector pDX were a gift from
Dr. J. López (Gladstone Institutes, San Francisco,
CA)(31) . The cDNA encoding GP IX (9) was previously
obtained from Dr. G. Roth (Seattle Veterans Administration Hospital).
The cDNA encoding GP V was provided by Dr. F. Lanza (Centre
Régional de Transfusion Sanguine, Strasbourg, France) (12) and was subcloned into the pDX vector.
, GP Ib
, and GP IX or GP
V and 0.5 µg of an appropriate selection marker. Individual
colonies were isolated using a cloning cylinder. About 20 clones were
grown and evaluated by flow cytometry for surface expression of the GP
Ib-IX complex or GP V. The clones with the higher expression levels of
the proteins of interest were used in the experiments described below.
, GP
Ib
, and GP IX and 40 µl of liposome suspension
(LipofectAMINE, Life Technologies, Inc.)(33) .
Fluorescence and Confocal Microscopy
The cells
were plated on Lab-Tek tissue culture chamber slides (Nunc, Inc.,
Naperville, IL), fixed in the absence of detergent, and stained as
described previously (31). The cells were labeled with a monoclonal
antibody against GP Ib (monoclonal antibody Ib-23 (34) courtesy of Dr. B. Steiner, Hoffmann-La Roche, Basel,
Switzerland) and/or serum against GP V (courtesy of Dr. D. Phillips,
COR Therapeutics Inc., San Francisco, CA). The polyclonal GP V antibody
was detected with Texas red-labeled goat anti-rabbit IgG (Vector
Laboratories, Burlingame, CA), and the monoclonal GP Ib
antibody was detected with biotinylated goat anti-mouse IgG and
fluorescein isothiocyanate (FITC)-conjugated streptavidin (Amersham
International plc, Buckinghamshire, United Kingdom). When the labeling
was performed only with the serum against GP V, biotinylated goat
anti-rabbit IgG (Amersham), and FITC-conjugated streptavidin were used.
Fluorescence microscopy was performed on an inverted microscope
(DIAPHOT-TMD, Nikon, Japan). Confocal microscopy was performed using a
Sarastro 1000 confocal microscope (Molecular Dynamics, Sunnyvale, CA).
Flow Cytometry and Cell Sorting
Cells were
harvested by treatment with 1 mM EDTA (Sigma), washed in PBS,
and incubated with the relevant polyclonal or monoclonal antibody for
30 min at 4 °C. The cells were washed and then incubated for 30 min
at 4 °C with FITC-conjugated goat anti-mouse or anti-rabbit IgG (5
µg/ml in PBS containing 1% bovine serum albumin) (Vector
Laboratories). Following washes, the samples were resuspended in PBS
and analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose,
CA). In some cases, cell sorting was performed using a modified FACS
440 cell sorter (Becton Dickinson). The 5% of cells with the highest
levels of fluorescence intensity were collected.
Western Blot Analysis
Cells (2
10
) were solubilized in 40 µl of Laemmli sample
buffer(35) , and solubilized proteins were electrophoresed
through a 7.5% SDS-polyacrylamide gel. The proteins were transferred
onto nitrocellulose paper(36) , which was incubated with a serum
against GP V or with a monoclonal antibody against GP
Ib
, monoclonal antibody Ib-4 (34) (courtesy of
Dr. B. Steiner). The nitrocellulose paper was sequentially incubated
with either a biotinylated goat anti-rabbit IgG or an anti-mouse IgG
and streptavidin-horseradish peroxidase conjugate (Amersham). The
signal was detected by enhanced chemiluminescence using
Amersham's ECL system.
Co-immunoprecipitation of GP Ib-IX with GP
V
Polyclonal antibodies against GP V or rabbit IgG (Sigma) were
covalently coupled to polyacrylamide immunobeads (Bio-Rad Laboratories,
Richmond, CA) (average diameter, 10 µm) according to the
manufacturer's instructions. The cells were harvested with EDTA,
washed twice with PBS and lysed in an ice-cold lysis buffer containing
50 mM Tris-HCl, pH 7.4, 1% digitonin (Sigma), 5 mM CaCl (Sigma), 1 mg/ml DNase I (Boehringer Mannheim), 1
mg/ml leupeptin (Vega Biotechnologies, Tucson, AZ), 1 mM phenylmethylsulfonyl fluoride (Sigma), 50 mM benzamidine
(Sigma), 1 mM sodium orthovanadate (Fisher Scientific, Fair
Lawn, NJ), 20 µg/ml soybean trypsin inhibitor (Sigma) for 1 h at 4
°C under agitation (2.5
10
cells/ml of lysis
buffer). The samples were centrifuged at 14,000
g for
20 min at 4 °C. The supernatant (1 ml) was agitated for 1 h at 4
°C with 50 µl of rabbit IgG-coupled immunobeads. The beads were
sedimented by centrifugation at 14,000
g for 4 min at
4 °C. The precleared supernatant was agitated with 50 µl of
anti-GP V-coated beads or rabbit IgG-coated beads for 16 h at 4 °C.
The immunobeads were washed four times in a buffer similar to the lysis
buffer but containing only 0.1% digitonin and lacking DNase I.
Immunoprecipitated proteins were removed from the beads by boiling the
samples in an SDS-containing buffer (35) and were analyzed on
Western blots.
Binding of Botrocetin-induced von Willebrand Factor to
the Transfected Cells
Purified vWf (generously provided by Dr.
J. Moake, Baylor College of Medicine, Houston, TX) (37) was
labeled by the lactoperoxidase method(38) . The cells (10 cells in 400 µl of PBS containing 1% bovine serum albumin)
were incubated in the presence of 5 µg/ml botrocetin (Pentapharm,
Basel, Switzerland), and various concentrations of
I-labeled vWf (specific activity approximately 6
10
cpm/µg). Samples were rocked gently at room
temperature for 1 h. 225-µl aliquots were removed and layered onto
400 µl of ice-cold 30% sucrose in PBS containing 0.1% bovine serum
albumin. The samples were centrifuged at 15,600
g for
4 min at 4 °C. The sucrose solution was aspirated carefully, and
the amount of
I-labeled vWf associated with the
sedimented cells was determined in a
counter (Iso-Data, Rolling
Meadows, IL). Nonspecific binding at each vWf concentration was
determined by measuring
I-labeled vWf binding to melanoma
cells that had not been transfected with GP Ib-IX. These counts were
subtracted from those obtained with the corresponding transfected
cells. A molecular weight of 1.1
10
was assumed for
vWf. The data were analyzed by Scatchard analysis.
, GP
Ib
, and GP IX). Cells were lysed, and GP V
immunoprecipitated from the lysates. Because GP Ib-IX associates with
actin-binding protein, thus mediating attachment of this adhesion
receptor to the cytoskeleton, actin-binding protein and associated
cytoskeletal proteins always co-immunoprecipitate with GP Ib-IX from
detergent-lysed
cells(20, 21, 22, 23, 24, 25) .
To circumvent this problem, the present experiments were performed
using a lysis buffer that included DNase I to depolymerize actin
filaments(20, 21) . However, when actin filaments are
depolymerized and GP Ib-IX is released into the detergent-soluble
fraction, the glycoprotein complex remains associated in a high
affinity interaction with actin-binding protein(21) . To
eliminate the possibility that the glycoproteins co-immunoprecipitate
simply because they are both associated with actin-binding protein, the
present experiments were performed with a previously described cell
line that lacks actin-binding protein(30) . Western blot
analysis showed that GP Ib
co-immunoprecipitated with
GP V from these cells (Fig. 1B, lane2). The finding that GP Ib-IX co-immunoprecipitated with
GP V even when actin-binding protein was not present indicates that
there is a direct interaction of GP V with one or more of the
glycoproteins in the GP Ib-IX complex. Some proteolysis of the GP
Ib
chain apparently occurred during lysis of the cells
yielding a fragment of M
=
90,000-100,000 (Fig. 1B). This fragment was not
immunoprecipitated by a monoclonal antibody that blocked von Willebrand
factor binding to GP Ib
(monoclonal antibody Ib-23)
(data not shown), suggesting that it represented GP Ib
that has been cleaved at a protease-sensitive site in the
extracellular amino-terminal end of the molecule(39) . This
fragment lacks the 45-kDa domain that contains the leucine-rich repeats
and the ligand-binding
site(7, 40, 41, 42, 43, 44) ;
like intact GP Ib
it co-immunoprecipitated with GP V (Fig. 1B, lane2).
Figure 1:
Co-immunoprecipitation of GP Ib-IX with
GP V in actin-binding protein-deficient melanoma cells. Melanoma cells
that were expressing GP Ib-IX and GP V but were not expressing
actin-binding protein were lysed in a digitonin-containing lysis buffer
in the presence of Ca and DNase I to induce actin
filament depolymerization (see ``Materials and Methods'').
After centrifugation the digitonin-soluble fractions (lanes1) were incubated with either anti-GP V-coupled beads (lanes2) or rabbit IgG-coupled beads (lanes3). Immunoprecipitated proteins were electrophoresed
through 7.5% SDS-polyacrylamide gels under reducing conditions and then
transferred to nitrocellulose. The blots were probed with a polyclonal
serum against GP V (A) or a monoclonal antibody against GP
Ib
(B). The mobility of prestained marker
proteins (kDa) is indicated on the rightside of each panel.
To determine
whether GP V associates with the GP Ib-IX complex in intact cells, we
analyzed the location of the proteins by confocal microscopy (Fig. 2). Each micrograph represents a thin optical
section through the cells. There was a precise co-localization of GP V (Fig. 2A) with GP Ib (Fig. 2B).
Figure 2:
Confocal microscopy images showing
co-localization of GP Ib-IX and GP V in the membrane of transfected
cells. Melanoma cells stably transfected with the cDNAs encoding GP
Ib, GP Ib
, GP IX, and GP V were
incubated with serum against GP V and a monoclonal antibody against GP
Ib
. The polyclonal GP V antibody was detected with
Texas red-labeled goat anti-rabbit IgG (A), and the monoclonal
GP Ib
antibody was detected with biotinylated goat
anti-mouse IgG and FITC-conjugated streptavidin (B).
Since in patients
with Bernard-Soulier syndrome, decreases in GP Ib-IX are also
accompanied by decreased surface expression of GP
V(15, 16, 17, 18) , we wanted to know
whether GP V expression requires the presence of the GP Ib-IX complex.
Melanoma cells were stably transfected with the cDNA encoding GP V
alone. In these and all subsequent experiments, melanoma cells that
were expressing actin-binding protein were used. Analysis of
solubilized cells on Western blots showed that even in the absence of
GP Ib-IX, GP V was synthesized and had a molecular weight(82, 0) similar to that of the protein in platelets (Fig. 3, left panel,lane2). As shown by FACS
analysis (Fig. 3, right panel,dashedline) and immunofluorescence of nonpermeabilized cells (Fig. 4B), some GP V was incorporated into the membrane.
To determine whether the surface expression of GP V was more efficient
if the three subunits of the GP Ib-IX complex were present, the
synthesis and surface expression of GP V was also examined in a clone
of cells that had been stably transfected with the cDNAs for GP
Ib, GP Ib
, and GP IX in addition to GP
V. Even though this clone synthesized considerably less GP V than the
clone that had been transfected with the cDNA for GP V alone (Fig. 3, left panel, compare lane3 with lane2, and ), more GP V was
expressed on the surface of these cells (Fig. 3, right
panel, compare solidline with dashedline). These results suggest that in the absence of GP
Ib-IX intact GP V exists within the cell but that it is more
efficiently incorporated into the cell membrane when the GP Ib-IX
complex is present. To directly test this idea, a clone of cells
expressing GP V alone was transiently transfected with the three cDNAs
encoding GP Ib
, GP Ib
, and GP IX. The
cells were harvested 48 h after transfection. The expression of GP
Ib-IX was verified by flow cytometry (data not shown). Fig. 5shows that after addition of GP Ib-IX, GP V reached the
surface at levels significantly higher (solidline)
than those that existed prior to GP Ib-IX addition (dashedline). When GP V was expressed alone, a large amount of
protein with the same molecular weight as intact GP V was also found in
the cell culture medium (). The amount of GP V in the
medium, relative to the amount in the cells, was markedly decreased
when GP Ib-IX was also present ().
Figure 3:
Western blots and FACS analysis showing
that full-length GP V is expressed in transfected cells but that little
is expressed in the membrane unless GP Ib-IX is also present. Left
panel, melanoma cells (2 10
cells/sample) were
solubilized in an SDS-containing buffer. Solubilized proteins were
electrophoresed through a 7.5% SDS-polyacrylamide gel and transferred
to nitrocellulose. The blot was probed with a serum against GP V. Lane1, nontransfected cells; lane2, a clone of cells stably transfected with the cDNA
encoding GP V; lane3, a different clone of cells
that had been stably transfected with the cDNAs for GP V, GP
Ib
, GP Ib
, and GP IX. The mobility of
prestained marker proteins (kDa) is indicated on the right. Right panel, the same cells as those analyzed in the left
panel (5
10
cells/sample) were labeled with a
serum against GP V followed by FITC-conjugated anti-rabbit IgG and
analyzed by flow cytometry. The dottedline represents nontransfected cells, the dashedline represents cells stably transfected with the cDNA encoding GP V
only, and the solidline represents cells stably
transfected with the four cDNAs encoding GP V, GP Ib
,
GP Ib
, and GP IX. The cells that contained the cDNAs
for all four glycoproteins expressed more GP V on the surface than did
the cells containing only the cDNA for GP V even though the latter
cells contained considerably higher amounts of GP V than did the cells
that also expressed GP Ib
, GP Ib
and
GP IX.
Figure 4:
Immunofluorescence microscopy of cells
expressing GP V. Melanoma cells that were not transfected (A)
or that were stably transfected with the cDNA encoding GP V (B) were fixed (in the absence of detergent) and then
incubated with serum against GP V, followed by biotinylated goat
anti-rabbit IgG, and FITC-conjugated streptavidin. Bar, 15
µm.
Figure 5:
Transient expression of GP Ib-IX increases
the surface expression of GP V in melanoma cells. Melanoma cells were
stably transfected with the cDNA encoding GP V (dashedline). In addition, some cells expressing GP V were
transiently transfected with the cDNAs for GP Ib, GP
Ib
, and GP IX (solidline). The cells
were incubated with serum against GP V and FITC-conjugated anti-rabbit
IgG, and they were analyzed by flow cytometry. Dottedline, nontransfected cells.
To determine
whether the presence of GP V regulates the expression of the GP Ib-IX
subunits, the amount of GP Ib-IX present on the surface of the GP
Ib-IX-expressing cells was measured by flow cytometry (Fig. 6)
and compared with the amount present on the surface of the GP Ib-IX
plus GP V-expressing cells. In this experiment, the cells were stained
with an antibody against GP Ib, but similar results
were obtained by using an antibody against GP IX (clone BL-H6) (45) or a complex-specific antibody, SZ1 (46) (data not
shown). As shown in Fig. 6, GP Ib-IX was expressed on the surface
in the absence of GP V (dashedline), but GP Ib-IX
expression was enhanced in cells that also expressed GP V (solidline). Different clones were analyzed, and the shift in
fluorescence was always proportional to the amount of GP V expressed.
Figure 6:
Increased surface expression of GP Ib-IX
after addition of GP V in the melanoma cells. Melanoma cells (5
10
cells/sample) that were not transfected (dottedline) or were stably transfected with the cDNAs encoding
either GP Ib
, GP Ib
, and GP IX (dashedline) or GP Ib-IX plus GP V (solidline) were incubated with a monoclonal antibody against
GP Ib
. Following addition of FITC-conjugated goat
anti-mouse IgG, the cells were analyzed by flow
cytometry.
To determine whether the presence of GP V affects the ability of GP
Ib-IX to bind von Willebrand factor, botrocetin-induced binding of I-labeled von Willebrand factor to either the GP
Ib-IX-expressing cells (Fig. 7A) or to the GP Ib-IX plus
GP V-expressing cells (Fig. 7B) was measured.
Nonspecific binding was determined by measuring binding of
I-labeled von Willebrand factor to non-GP
Ib-IX-expressing cells. The binding was botrocetin-dependent and could
be inhibited by a monoclonal antibody directed against the von
Willebrand factor binding site on GP Ib
(data not
shown). Binding of von Willebrand factor to the cells was saturable.
The data, presented as a Scatchard plot, fit a model of a single class
of binding sites and was representative of four independent
experiments. The dissociation constants were similar for the GP
Ib-IX-expressing cells and for the cells that also expressed GP V (0.16
± 0.01 and 0.18 ± 0.02 nM, respectively). Thus,
GP V did not affect the binding affinity of von Willebrand factor to
the receptor. However, as shown above, GP V enhanced the expression of
GP Ib-IX and by doing so increased the binding capacity of the cells
for von Willebrand factor. Approximately three times more GP Ib-IX
complex was present on the cells that also expressed GP V. The binding
capacity of the cells expressing GP V was higher than that of the cells
expressing only GP Ib-IX (63 ± 20 pM and 26 ± 5
pM, respectively).
Figure 7:
Botrocetin-induced binding of I-von Willebrand factor to GP Ib-IX-expressing melanoma
cells in the presence or absence of GP V. 10
cells were
incubated in the presence of botrocetin and various concentrations of
I-vWf. The specific binding of
I-labeled
vWf to the cells was measured as described under ``Materials and
Methods.'' Each data point represents the mean of a duplicate
determination. The experiment shown is representative of four
independent experiments. A, binding to cells expressing only
GP Ib-IX; B, binding to cells expressing GP Ib-IX plus GP
V.
, GP Ib
, and GP IX associate with
high affinity, but GP V appears to associate with the resulting complex
with a lower affinity.
, GP Ib
, and GP IX is a
member of the leucine-rich family of
proteins(7, 8, 9, 10, 11, 12, 13) .
Thus, all four members of this family that are known to be present in
platelets exist in a complex with each other. In GP Ib
,
the leucine-rich repeats are all in the 45-kDa amino-terminal end that
contains the binding site for von Willebrand
factor(7, 40, 41, 42, 43, 44) .
In the present study, we observed that GP V still co-immunoprecipitated
with GP Ib-IX in which this 45-kDa domain had been removed
proteolytically. Similarly, GP V still co-immunoprecipitated with GP
Ib-IX from platelet lysates even after the 45-kDa amino terminus of GP
Ib
had been removed by treatment of the platelets with
human leukocyte elastase(40, 19) . Thus, the
leucine-rich repeats of GP Ib
do not appear to be
needed for the interaction of GP Ib-IX with GP V. Interestingly, many
of the known members of the leucine-rich family of glycoproteins are
either adhesive receptors or signaling molecules (for review, see Ref.
49). The GP Ib-IX-V complex serves both of these functions; it binds
the adhesive ligand von Willebrand factor(3, 4) , and
following von Willebrand factor binding it transmits signals across the
membrane such that intracellular signaling molecules are
activated(50, 51, 52, 53) . The
availability of the expression system developed in the present work may
allow the importance of these leucine-rich repeats and the significance
of the association of the four leucine-rich subunits to be
investigated.
could be expressed on the surface of transfected
cells in the absence of the other subunits(34) , the expression
was markedly increased when all three subunits were
present(31, 48) . Together with the finding that in
patients with Bernard-Soulier syndrome, mutations in GP
Ib
, GP Ib
, or GP IX apparently result
in decreased expression of all three subunits of the GP Ib-IX complex,
these observations led to the idea that all three subunits of this
complex must associate in order to obtain efficient surface expression
of any of the subunits. The fact that Bernard-Soulier patients have
little GP V on their surface (15, 16, 17, 18) suggests that GP Ib-IX
may also play a role in the expression of GP V. The finding that GP V
associates with GP Ib-IX suggests that all four subunits of this
complex may be needed for efficient expression of the entire complex.
To directly test this possibility, we transfected melanoma cells with
GP V in the presence or absence of GP Ib-IX. As with GP
Ib
, expression of GP V as a single polypeptide resulted
in the synthesis of a glycoprotein that could be incorporated into the
membrane. However, the presence of the three subunits of the GP Ib-IX
complex markedly increased the amount of GP V that was incorporated
into the membrane. Previous studies have shown that when GP
Ib
is expressed alone only about 5% of the recombinant
protein exists as intact protein(34) . Most of the protein is
degraded and exists in the cells or is secreted into the medium as
fragments. Like GP Ib
, when GP V was expressed alone,
only small amounts of the protein were incorporated into the membrane.
However, in contrast to GP Ib
, the remaining GP V was
present in the cell lysates and in the cell medium with the same
molecular weight as intact GP V. Thus, although it is not possible to
determine whether GP V was present as the intact protein or whether it
had been cleaved at the protease-sensitive site adjacent to the
membrane insertion site(29) , GP V appears to differ from GP
Ib
in that it is not degraded to detectable smaller
fragments when expressed alone. These findings on the importance of GP
Ib-IX in allowing GP V to be expressed in the membrane might explain
the fact that surface labeling of platelets showed that GP V was
missing or present in a decreased amount in Bernard-Soulier patients
with mutations in components of the GP Ib-IX
complex(15, 16, 17, 18) . Western blot
analysis of these platelets would determine if GP V is synthesized but
not incorporated in the platelet membrane, as is the case for
transfected cells.
, GP Ib
, GP IX, and then
GP V. The addition of GP V increased the cell surface expression of GP
Ib-IX. Thus, while it has previously been thought that the three
subunits of GP Ib-IX are sufficient for the efficient expression of
this complex in the membrane(31) , the present results show that
the presence of the fourth component, GP V, increases expression of the
complex further.
differently in a shear dependent system. Future
studies will be needed to address this possibility and to elucidate
additional functions of GP V.
Table: Amount of
GP V secreted in the medium and bound to the cells in the presence or
absence of GP Ib-IX
cells/2 ml culture medium/well). The cell supernatant was
collected after 72 h, centrifuged at 1,000
g for 10
min to eliminate dead cells, and denaturated in Laemmli sample buffer.
The cells were harvested with EDTA, counted, and solubilized in Laemmli
sample buffer. The amount of GP V associated with the cells or present
in the medium was determined on Western blots using a serum against GP
V and quantitated by densitometry. Values shown are the mean (±
S.D.) from the number of determinations shown.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.