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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 532-539
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Surface expression of glycoprotein Ib is dependent on
glycoprotein Ib : evidence from a novel mutation causing
Bernard-Soulier syndrome
Niamh Moran,
Patricia A. Morateck,
Adele Deering,
Michelle Ryan,
Robert R. Montgomery,
Desmond J. Fitzgerald, and
Dermot Kenny
From the Department of Clinical Pharmacology, Royal College of
Surgeons in Ireland, Dublin, Ireland, and the Blood Research Institute,
the Blood Center of Southeastern Wisconsin, Milwaukee, WI.
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Abstract |
Bernard-Soulier syndrome is a rare bleeding disorder caused by a
quantitative or qualitative defect in the platelet glycoprotein (GP)
Ib-IX-V complex. The complex, which serves as a platelet receptor for
von Willebrand factor, is composed of 4 subunits: GPIb , GPIb ,
GPIX, and GPV. We here describe the molecular basis of a novel form of
Bernard-Soulier syndrome in a patient in whom the components of the
GPIb-IX-V complex were undetectable on the platelet surface. Although
confocal imaging confirmed that GPIb was not present on the platelet
surface, GPIb was readily detectable in the patient's platelets.
Moreover, immunoprecipitation of plasma with specific monoclonal
antibodies identified circulating, soluble GPIb. DNA-sequence
analysis revealed normal sequences for GPIb and GPIX. There was a G
to A substitution at position 159 of the gene encoding GPIb,
resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIb,
together with wild-type GPIb and GPIX, demonstrated a failure of
GPIX expression on the surface of HEK 293T cells. Similar results were
obtained with Chinese hamster ovary IX cells, a stable cell line
expressing GPIb that retains the capacity to re-express GPIX. Thus,
we found that GPIb affects the surface expression of the GPIb-IX
complex by failing to support the insertion of GPIb and GPIX into
the platelet membrane.
(Blood. 2000;96:532-539)
© 2000 by The American Society of Hematology.
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Introduction |
Platelet adhesion to damaged blood vessels is a
critical hemostatic mechanism. This adhesion is initiated by the
interaction between von Willebrand factor (vWF) exposed on the
subendothelium and its platelet receptor, the glycoprotein (GP) Ib-IX-V
complex. GPIb-IX-V is a hetero-oligomeric protein complex assembled
from 4 distinct gene products and uniquely expressed on the membranes of platelets and megakaryocytes. GPIb , the largest protein (145 kd),
contains the binding site for vWF1 and
-thrombin2 in its extracellular domain and has binding
sites for 14.3.33 and the actin cytoskeleton4
in its short cytoplasmic region. GPIb is linked by means of an
extracellular disulfide bond to GPIb (25 kd) and exists in a 1:1
covalent complex with this protein. The other components of the
oligomer, GPIX and GPV, are noncovalently linked to the GPIb complex in
a ratio of 2:2:1 (GPIb:GPIX:GPV). The functional roles of GPIb and
GPIX in the complex are not clear, although in vitro evidence suggests
that both these proteins act as complex-specific chaperones that
protect GPIb from lysosomal degradation while it is transported to
the membrane.5 GPV has a role in the high-affinity binding
of thrombin to the platelet6 but does not seem to be
essential for the surface expression of a functional
complex.7
Abnormalities of the GPIb-IX-V protein complex are associated with
abnormal platelet function and appearance, giving rise to a syndrome
first described by Bernard and Soulier in 1948.8 Bernard-Soulier syndrome (BSS) is an autosomal recessive disorder characterized by moderate to severe thrombocytopenia, enlarged (giant)
platelets, and a tendency to have profuse and often spontaneous bleeding.9 Twenty-one causes of BSS have been characterized at a molecular level. Of these, 14 are due to mutations in GPIb, 5 in GPIX, and 2 in GPIb.9-12 There are no reports of BSS
affecting the GPV gene. Furthermore, mice without GPV expressed normal
levels of GPIb-IX protein complex and had no BSS-like
symptoms.7
In this report, we describe a novel, homozygous, single-nucleotide
substitution (G159 A) in the coding region for GPIb at a
tryptophan codon (TGG), which results in the premature termination of
translation (TAG) at amino acid 21. No sequence abnormalities were
observed in either the GPIb or the GPIX subunit of the complex. Although GPIb, GPIX, and GPV were undetectable on the platelet surface, GPIb was readily demonstrated in platelets. Moreover, soluble GPIb was present in plasma. Thus, we found that the
defective GPIb is unable to support the expression or maintenance of
a functional complex at the platelet surface.
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Patients, materials, and methods |
Case history
A 57-year-old Irish man with a severe bleeding diathesis was given a
tentative diagnosis of BSS because of the presence of thrombocytopenia
(platelet count, 20-120 × 109/L),
large platelets on peripheral blood smear, and a profuse bleeding
tendency requiring transfusion. Bleeding occurred both spontaneously
and after minor surgical procedures. The spontaneous bleeding
apparently stopped when the patient was about 20 years of age, but the
thrombocytopenia persisted. The patient's bleeding time was longer
than 15 minutes. During childhood, the patient was treated with
steroids. The patient had 2 living siblings, neither of whom had any
history of abnormal bleeding. Two other siblings died in infancy, 1 from pneumonia and 1 from unknown causes. The patient's parents were
first cousins and had no history of abnormal bleeding. Investigations
of the patient's thrombocytopenia showed that the bone marrow aspirate
was normal except for the presence of megakaryocytes with an
excessively granular appearance. Coagulation studies showed no
deficiency of factor V, VII, or VIII.
Monoclonal antibodies (mAbs) and reagents
The anti-GPIb antibody AP1 blocks vWF binding to GPIb. MBC
142.2, 142.6, and 142.11 are mAbs raised against purified GPIb that
do not inhibit the binding of vWF to GPIb. GC is a rabbit polyclonal
antibody raised against purified GPIb. Anti-GPIX mAbs FMC 25 and GRP
were purchased from Harlan Bioproducts (Indianapolis, IN). Antibodies
SZ2, SZ1, and SW16 (Beckman Coulter Immunotech, France) are mAbs that
bind GPIb, GPIX, and GPV, respectively, and are components of a kit
for diagnostic estimation of these antigens on platelet surfaces
(BioCytex, Marseilles, France). AP2 is a mAb against the GPIIb-IIIa
complex, and LYP18 is a mAb that recognizes GPIIIa in complex with
GPIIb. AK1, a mAb that recognizes an epitope that requires the intact
GPIb-IX complex,13 was a generous gift of Dr Michael C. Berndt (Baker Medical Research Institute, Victoria, Australia).
Platelet isolation
Venous blood samples from the patient and healthy controls were
collected into 0.15- vol ACD (38 mmol/L citric acid anhydrous, 75 mmol/L sodium citrate, and 124 mmol/L dextrose) and centrifuged to
obtain platelet-rich plasma (PRP). The PRP was acidified to pH 6.5 with
ACD, and prostaglandin E1 (final concentration, 1 µmol/L)
was added. The platelets were then pelleted through the plasma by
centrifugation at 900g for 12 minutes at room temperature. The
supernatant was removed, and platelets were resuspended in a modified
Tyrode buffer containing 130 mmol/L sodium chloride, 10 mmol/L
trisodium citrate, 9 mmol/L sodium bicarbonate, 6 mmol/L dextrose, 0.9 mmol/L magnesium chloride, 0.81 mmol/L potassium phosphate (monobasic),
and 10 mmol/L Tris (pH 7.4). Calcium chloride (final concentration, 1.8 mmol/L) was added to the washed platelets for aggregation and
agglutination studies.
Flow cytometric analysis of whole blood
Platelets were analyzed by flow cytometry with a platelet GP
quantification kit (BioCytex). Briefly, 50 µL of whole blood was
diluted 1:4 in modified Tyrode buffer and incubated for 10 minutes at
room temperature with 20 µL of the following antibodies: negative
control mouse IgG, LYP18 (anti-GPIIIa), SZ2
(anti-GPIb),14,15 SZ1 (anti-GPIX),13,16 and
SW16 (anti-GPV).17,18 Secondary antibody (polyclonal
antimouse IgG [fluorescein isothiocyanate, conjugated], 20 µL) was
incubated with each sample for an additional 10 minutes. Samples were
diluted by adding 2 mL of buffer and were then analyzed on a flow
cytometer (FACS Star; Becton Dickinson, San Jose, CA). A
calibration-bead suspension coated with increasing and known quantities
of mouse IgG was incubated in parallel with secondary antibody and used
to construct a standard curve for fluorescence intensity compared with
known numbers of binding sites. This standard curve was used to convert
results from test samples to number of sites per platelet, and
histogram analysis was performed with CellQuest (version 3.1f; Becton Dickinson).
Polymerase chain reaction (PCR) amplification of genomic DNA
Genomic DNA was isolated from peripheral blood lymphocytes as
described previously.19 DNA was amplified by
PCR20 using primer pairs based on the published genomic
sequence of GPIb, GPIb, and GPIX.21-23 For
DNA-sequence analysis, the full-length coding region for mature GPIb
was amplified with primers 162 to 181 and 2634 to 2653.22
For GPIb, the primers 8 to 30 and 767 to 79121 were used
for amplification; for GPIX, we used primers 792 to 816 and 1547 to
1560.23 The target sequences were amplified in a 50-µL
reaction volume containing 500 to 1000 ng of genomic DNA, 30 pmol of
each primer, and 0.2 mmol/L of each nucleoside triphosphate in a
reaction buffer consisting of 60 mmol/L Tris-hydrochloric acid (pH
9.0), 15 mmol/L ammonium sulfate, 2 mmol/L magnesium chloride, 1 U Taq
polymerase (Perkin Elmer, Foster City, CA), and 4% (vol/vol) dimethyl
sulfoxide. PCR amplification was performed in a programmable thermal
cycler (model 9600; Perkin Elmer) for 35 cycles of 45 seconds of
denaturation at 96°C, annealing for 1 minute at 60°C, and
extension for 1 minute at 72°C. PCR products containing the entire
coding regions of GPIb, GPIb, and GPIX were cloned into the pCRII
cloning vector by using the TA cloning kit (Invitrogen, San Diego, CA).
DNA sequencing
Direct sequence analysis of the entire coding region of
PCR-amplified GPIb, GPIb, and GPIX from the patient was performed with the Prism Ready Reaction DyeDeoxy terminator cycle sequencing kit
and a DNA sequencer (model 373A; Applied Biosystems, Foster City, CA).
Sequencing primers were synthesized on a DNA synthesizer (model 394;
Applied Biosystems).
Fluorescent in situ hybridization (FISH)
FISH analysis was performed on G-banded metaphase chromosomes by
using the D22S75 probe that maps to the region
22q11.2.24,25 This chromosomal region is frequently deleted
in DiGeorge syndrome, velocardiofacial syndrome, and in platelets from
patients with familial or isolated congenital heart defects.
Confocal imaging of platelets
Glass slides were coated with 100 µL fibrinogen (20 µg/mL) for 2 hours at 37°C and blocked with 1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS; pH 7.4) for 1 hour in a humidified staining tray. Washed platelets (50 µL), prepared as described above,
were diluted to 3.0 × 108/mL and allowed to adhere
for 60 minutes at room temperature to the fibrinogen-coated glass
slides. Nonadhered platelets were removed by washing in PBS (pH 7.4),
and adhered platelets were either stained immediately or fixed and
permeabilized in ice-cold methanol (7 minutes) followed by ice-cold
acetone (2 minutes). Primary antibodies (1:50 dilutions of monoclonal
LYP18 for GP IIb-IIIa or polyclonal anti-GC for GPIb) were incubated
for 45 minutes at room temperature for fixed preparations or for 90 minutes at 4°C for nonpermeabilized platelets. Goat antimouse Alexa
488-conjugated IgG or goat antirabbit Alexa 546 IgG was then added
(Molecular Probes, Leiden, Netherlands). The immunostained slides were
washed 3 times in PBS and mounted in fluorescent mounting medium (Dako, Carpinteria, CA) before imaging on a Zeiss Axioplan 2 confocal microscope with a 63 × (1.4 n/a) lens.
Immunoprecipitation of plasma GPIb
GPIb mAb AP1 was coupled to cyanogen bromide-activated Sepharose
4B beads (Sigma, St Louis, MO). Platelet-poor plasma was precleared by
incubating it with uncoupled Sepharose CL-4B beads for 1 hour at room
temperature. The beads were centrifuged at 1000g, and the
plasma was added to the antibody-coupled beads and incubated overnight
at 4°C. The beads were washed, and the immunoprecipitated complexes
from plasma were eluted in a 1 × nonreducing lane marking
sample buffer (Pierce, Rockford, IL). All samples were boiled at
100°C for 3 minutes. The samples were then analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis on a 4% to 20%
exponential gradient in the presence of 5% -mercaptoethanol. The
separated proteins were electroblotted onto a polyvinylidene fluoride
membrane (Novex, San Diego, CA) and detected as described previously.11
Transient expression
For transient-expression studies, HEK 293T cells were used. The
parent 293T cell line is a human renal epithelial cell transformed with
SV40 large T antigen.26 The 293T cells were maintained at
37°C in a 5% carbon dioxide humidified chamber in modified Eagle
medium (Sigma) supplemented with 10% fetal calf serum.
An XhoI/MluI restriction fragment containing the entire
coding region for GPIb was inserted into the mammalian expression vector pCI-Neo (Promega, Madison, WI). EcoRI restriction
fragments containing the entire coding region of both GPIb and GPIX
amplified from genomic DNA were inserted into pCI-Neo. Constructs
containing wild-type GPIb, GPIb, and GPIX, and mutant GPIb
were sequenced to ensure that no additional mutations had been
introduced and that they were inserted in the expression vector in the
correct orientation. Expression plasmids were introduced into
293T cells in the presence of lipofectamine (Gibco BRL,
Gaithersburg, MD).
Transient-expression studies were also performed in Chinese hamster
ovary (CHO) IX cells (kindly provided by Dr José A. López, Baylor College of Medicine, Houston TX). CHOIX cells are CHO cells that stably express human GPIb on their
surface.16 When these cells are additionally transfected
with GPIb, the surface expression of GPIX becomes readily
detectable.27 Cells from this stable cell line were also
transiently transfected with either the plasmid pCI-Neo alone,
wild-type GPIb, or the construct containing the mutation in GPIb.
Expression plasmids were introduced into CHOIX cells in the presence
of lipofectamine and lipofectamine plus (Gibco BRL) by following the
protocol of Felgner et al.28 Briefly, either
1.5 × 106 CHOIX cells or
4 × 106 293T cells were plated in 100-mm dishes and
grown overnight. Then, 8 mL of OPTI-MEM-reduced serum medium (Gibco
BRL) containing 36 µg lipofectamine and 6 µg of the appropriate
plasmid DNA was added to the CHOIX cells and 120 µg lipofectamine
and 8 µg DNA was added to the 293T cells. After transfection and 5 hours of incubation, the transfection medium was removed, 8 mL of
culture medium was added, and incubation was reinitiated at 37°C
and continued for 60 hours.
Flow cytometry studies in transfected cells
Transfected cells were detached from tissue culture plates with 3 mmol/L EDTA, centrifuged at 250g, and resuspended in Hanks balanced salt solution with 1% BSA and 1% normal donkey serum. Then,
3 × 105 cells were transferred to each well of a
96-well V-bottomed plate (Dynatech, Chantilly, VA) and incubated with
either a rabbit antiglycocalicin polyclonal antibody (5 µg/mL), the
anti-IX mAb FMC-25 or GRP (5 µg/mL), or the complex-specific mAb AK1
(ascites 1:1200). The cells were washed twice and
incubated for an additional 30 minutes in a darkened room with a 1:100
dilution of phycoerythrin-conjugated affinity-purified
F(ab')2 donkey antimouse or antirabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA). The cells were washed twice, resuspended in 2% paraformaldehyde, and incubated for at least
1 hour at 4°C before analysis on a flow cytometer (FACScan; Becton Dickinson).
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Results |
Platelet GPs
Platelet function studies showed normal aggregation in the presence
of ADP (10 µmol/L), collagen (10 µg/mL), and thrombin receptor
actuating peptide (TRAP) (10 µmol/L), but platelets
failed to agglutinate in the presence of 1.2 mg/mL ristocetin. The
diagnosis of BSS was confirmed by the presence of clinically
insignificant binding of antibodies to GPIb, GPIX, and GPV on flow
cytometric analysis (Figure 1). Binding of
LYP18 to the unrelated platelet GP, GPIIb-IIIa, was increased. This
finding is consistent with other reports of BSS9 and
probably reflects the larger size of the platelets. When calibration
beads were used to estimate the number of sites per platelet, platelets
from healthy controls were found to express 54 000 ± 7000 (n = 3) GPIIb-IIIa molecules on their surface, whereas platelets from
the patient with BSS expressed 98 000 ± 10 000 sites per platelet
(n = 3; Table 1). All these findings are
consistent with a diagnosis of BSS.
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| Fig 1.
Flow cytometric analysis of platelets from a patient with
Bernard-Soulier syndrome (BSS).
Analysis was performed on whole blood with monoclonal antibodies (mAbs)
against glycoprotein (GP) IIb-IIIa (A), GPIb (B), GPIX (C), and GPV
(D). As expected BSS patients' platelets (BSP), which are larger than
normal, this patient's platelets (clear area) showed an increase in
surface fluorescence compared with platelets from healthy controls
(CTL, shaded area) in reaction to the anti-GPIIb-IIIa mAb LYP18. There
was no detectable GPIb (B) or GPIX (C) on the platelets of the
patient (clear area), whereas both were present on control platelets
(shaded area).
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Table 1.
Characteristics of platelets from healthy (control)
donors and from a patient with Bernard-Soulier syndrome (BSS)
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Sequence analysis of GPIb-IX complex
Sequence analysis of the entire coding regions of the genes for
GPIb and GPIX identified no abnormalities. However, a novel G to A
mutation in codon 21 of the gene for GPIb was observed. This
mutation results in premature termination of protein synthesis at W21
by replacing a TGG codon specifying a tryptophan residue with a TAG
codon that specifies termination of translation
(Figure 2). BSS due to a single-allele
mutation in GPIb was reported previously to be accompanied by a
deletion in the DiGeorge-velocardiofacial chromosomal region in
22q11.2. The gene for GPIb is in the center of this
region.29 Our patient had no clinical signs or symptoms of
this syndrome. However, because the syndrome may be subtle, FISH
studies were performed. Using G-banded metaphase chromosomes, we
detected no deletion on either of the chromosome 22 homologues by FISH
analysis with a probe (D22S75) that mapped to the region 22q11.2 (data not shown).
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| Fig 2.
Mutation in GPIb.
DNA-sequence analysis of GPIb from a healthy volunteer (A) and the
patient with BSS (B). Sequence analysis of DNA amplified by polymerase
chain reaction showed that nucleotide 159, part of a TGG codon, was
replaced by an A, yielding a TAG stop signal.
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Confocal microscopical analysis of BSS platelets
Initial studies to investigate the diagnosis of BSS included
analysis of platelet characteristics. Platelet size was determined by
assessing the maximal diameter of a random selection of 40 normal
platelets and 40 platelets from the patient, imaged by confocal
microscopy. All the platelets in a field were measured to avoid the
possibility of selection bias. The mean (± SD) diameter of the BSS
platelets was 5.3 ± 1.5 µm (range, 3.1-9.1 µm), whereas that of
control platelets was 3.3 ± 0.8 µm (range, 1.3-5.7 µm) (Table
1). Interestingly, the BSS platelets and the platelets from healthy
volunteers adhered to and spread on the immobilized fibrinogen in an
identical manner. As a measure of cell spreading, we estimated the
ratio of height to diameter for a random selection of platelets (Table
1), with selection bias ruled out by including all platelets in a
field. The ratio in the 2 platelet populations was almost identical,
indicating that spreading on fibrinogen, a function of the GPIIb-IIIa
integrin adhesion protein, was normal in our patient.
Normal platelets plated on fibrinogen-coated glass slides were stained
for GPIb (anti-GC) and for the integrin GPIIb-IIIa (CD41). These
platelets showed normal surface expression of both antigens on
fluorescent microscopical imaging (Figure
3A-B). BSS platelets also stained normally
for GPIIb-IIIa on their surface and in their cytoplasm (Figure 3H,K).
This staining showed a focal distribution, consistent with activation
of the platelet by the immobilized fibrinogen substrate. However,
staining for GPIb was absent in these platelets (Figure 3G). In
contrast, when platelets were permeabilized before staining, GPIb
staining was readily detectable in the BSS platelets, though not with a
distribution equivalent to that of GPIIb-IIIa, suggesting a cytosolic
distribution (Figure 3J). To investigate a role for GPIb in
intracellular complex formation, we used fluorescent microscopy to
determine whether GPIb and GPIX formed a complex in the BSS
platelets in the absence of functional GPIb. The antibody AKI
recognizes the GPIb-GPIX complex but not its individual components
alone.13 AK1 failed to bind to either permeabilized or
nonpermeabilized BSS platelets but did bind substantially to normal
platelets (Figure 4). These data show that
the expression of GPIb-GPIX complex is dependent on functional
GPIb. The fluorescent images of GPIb staining in the BSS
platelets demonstrated a circumferential pattern, suggesting that
GPIb is transported toward the platelet membrane even in the absence
of an association with GPIb or GPIX. Flow cytometric analysis of the
BSS platelets (Figure 1 and Table 1) found minimal expression of
GPIb on the surface of BSS platelets, indicating that there is a
residual capacity for this protein to be expressed alone on the
cell surface.
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| Fig 3.
Fluorescent imaging of platelet GPs.
Washed platelets from healthy volunteers (A-F) and the patient with BSS
(G-L) were plated onto glass slides precoated with fibrinogen (20 µg/mL) and allowed to adhere for 1 hour at room temperature.
Platelets were then either stained directly (A-C and G-I) or
permeabilized with ice-cold methanol and acetone (D-F and J-L) before
staining. Slides were dual stained for GPIb with a rabbit anti-GC
polyclonal antibody and for GPIIb-IIIa with a mouse mAb, LYP18.
Platelets were imaged on a Zeiss Axioplan II confocal microscope with a
63 × oil immersion lens (1.4 n/a). The platelets were
permeabilized in the images shown in A to C and G to I to allow
antibody probes to react with cytoplasmic epitopes. Data are presented
as triplets of images separated to show GPIb staining with an Alexa
546-conjugated anti-rabbit antibody (red: A, D, G, and J), GPIIb-IIIa
staining with Alexa 488 antimouse antibody (green: B, E, H, and K), or
the confocal image showing both colors (C, F, I, and L). One image is
shown for each treatment; this is representative of 3 independent experiments in which up to 50 platelets were
analyzed. GPIb was present in permeabilized and nonpermeabilized
normal platelets (A, D) and permeabilized BSS platelets (J) but absent
from the surface of nonpermeabilized preparations of the BSS
platelets (G). In contrast, the platelet integrin GPIIb-IIIa was
present on the surface of both normal and BSS platelets,
regardless of permeabilizing treatment of the cell membrane (B,
E, H, and K).
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| Fig 4.
GPIb-IX complex formation in BSS platelets.
Washed platelets from healthy volunteers (A-D) and the patient with BSS
(E-H) were plated onto glass slides precoated with fibrinogen (20 µg/mL) and allowed to adhere for 1 hour at room temperature.
Platelets were then either permeabilized with ice-cold methanol and
acetone before staining (A, B and E, F) or stained directly without
permeabilizing (C, D and G, H). Slides were stained for GPIb-IX with
the mAb AK1. Platelets were imaged in parallel in fluorescent (A, C, E,
and G) and differential-interference contrast (DIC) mode (B, D, F, and
H). The platelets were permeabilized to allow antibody probes to react
with cytoplasmic epitopes. Data are presented as paired images
separated to show AK1 staining with an Alexa 546-conjugated antimouse
antibody or DIC images to indicate the platelet position in the
nonstaining cells. One image is shown for each treatment; this is
representative of 3 independent experiments in which more than 20 platelets were analyzed. GPIb-IX complex was present in the
cytoplasm and on the surface of normal platelets (A, C) but absent from
BSS platelets (E, G). The DIC images show the larger size of the BSS
platelets.
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Soluble GPIb in the plasma
Because GPIb was readily detectable in the patient's platelets
but not on their surface, we hypothesized that circulating, soluble
GPIb might be detectable in the plasma. There was less soluble
GPIb in plasma from the patient than in that from healthy volunteers (Figure 5). This finding is
consistent with the reduced number of platelets in the patient's blood
and the reduced surface expression of the GP complex.
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| Fig 5.
Western blot analysis of plasma GPIb.
Platelet-poor plasma from 2 healthy volunteers and the patient with BSS
was immunoprecipitated with antibody AP1, analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20%
gradient gel in the presence of -mercaptoethanol, and immunoblotted
with anti-GPIb monoclonal antibody MCB142.11. The
glycocalicin-positive control (3.8 ng) is shown in lane 1; BSS plasma
is shown in lane 2. The control plasma samples were diluted (1:4)
before SDS-PAGE to avoid overloading of the gel (lanes 3 and
4). There was less soluble GPIb in the plasma from the patient
with BSS.
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Expression of GPIb-IX complex
To test the hypothesis that the observed mutation in the GPIb
gene was responsible for the defects in the expression of the GPIb-IX
complex on the platelet surface, the effect of this mutation was
evaluated in HEK 293T cells transiently transfected with either wild-type GPIb or W21stop-GPIb. GPIb, GPIb, and GPIX are
all required for efficient expression of the ligand-binding entity on
the surface of transfected cells.30 Therefore, to
investigate the effects of W21stop-GPIb on complex expression by the
cells, plasmids encoding GPIb, GPIb, and GPIX were transiently
transfected into HEK 293T cells. Transient coexpression of normal
GPIb in HEK 293T cells with wild-type GPIb and GPIX resulted in
enhanced surface expression of GPIb and expression of GPIX (Figure
6). In contrast, the mutant W21stop-GPIb
gave rise to a lesser expression of GPIb and failed to support GPIX
expression. In these cells, in the presence of wild-type GPIb but
not W21stop-GPIbGPIb and GPIX were expressed as a complex on
the platelet surface and recognized by a complex-specific antibody,
AKI. Thus, it appears that wild-type GPIb facilitates the transport
and expression of GPIb and GPIX on the cell surface but
W21stop-GPIb does not achieve this function.
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| Fig 6.
Analysis of GPIb and GPIX in HEK 293T cells
transfected with GPIb, GPIb, and GPIX.
HEK 293T cells were transiently transfected with wild-type GPIb,
GPIb, and GPIX or with wild-type GPIb and GPIX and mutant
W21stop-GPIb. The cells were analyzed with an anti-GPIb
polyclonal antibody, the anti-GPIX antibody FMC25, or the
complex-specific antibody AK1 (each graph is representative of 4 different experiments). (A) In cells transfected with wild-type
GPIb, GPIb, and GPIX, there was a significant increase in
fluorescence in cells in reaction to the anti-GPIb polyclonal
antibody (boldface lines) compared with mock-transfected cells (shaded
area). In cells transfected with W21stop-GPIb and wild-type GPIb
and GPIX, GPIb was detectable on the cell surface (thin line) but in
significantly smaller amounts than in triple wild-type transfections.
(B) In cells transfected with wild-type GPIb, GPIb, and GPIX,
GPIX was readily detectable on the cell surface (boldface line).
However, GPIX was not detectable when W21stop-GPIb was transfected
with wild-type GPIb and GPIX (thin line). Mock-transfected cells are
shown for comparison (shaded area). (C) There was a marked increase in
surface fluorescence in cells transfected with wild-type GPIb,
GPIb, and GPIX in reaction to the complex-specific antibody, AK1
(boldface lines), compared with mock-transfected cells (shaded area)
and cells transfected with wild-type GPIb, GPIX, and mutant GPIb
(thin line). Thus, GPIb that was expressed on the cell surface (A)
in the transfections involving the mutant GPIb was not recognized by
AK1, thereby confirming the lack of surface expression of GPIX or
complex formation.
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Similar results were obtained when plasmids encoding wild-type GPIb
were transiently transfected into CHOIX cells stably expressing
GPIb (ie, CHO cells that retain the capacity to re-express GPIX).
There was a significant increase in the surface expression of GPIX and
in the binding of the complex-specific antibody. In contrast, when
W21stop-GPIb was transiently transfected into these cells, GPIX was
not re-expressed on the plasma surface (Figure 7), thereby confirming a role for wild-type
GPIb in maintaining functional GPIb-IX complex on the cell surface.
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| Fig 7.
Analysis of GPIb and GPIX in Chinese hamster ovary
(CHO) IX cells transiently transfected with GPIb.
(A) CHOIX cells expressing GPIb and GPIX were additionally
transfected with wild-type GPIb or the mutant W21stop-GPIb or
were mock transfected with the expression plasmid alone. GPIb was
readily detectable in cells transfected with plasmid alone (shaded
area), the mutant W21stop-GPIb (thin lines), or wild-type GPIb
(boldface lines). (B) There was a significant increase in the surface
expression of GPIX when wild-type GPIb (boldface lines) was
transfected into CHOIX cells compared with mock-transfected controls
(shaded area) or cells transfected with the mutant W21stop-GPIb
(thin line). (C) CHOIX cells transfected with wild-type GPIb
(boldface lines) were allowed to react with the complex-specific
antibody AK1. Again, there was a marked increase in surface
fluorescence compared with either the mock-transfected cells (shaded
area) or the cells transfected with W21stop-GPIb (thin lines). Thus,
the mutant GPIb failed to support the efficient expression of the
GPIb-IX complex on the surface of these cells.
|
|
These results suggest that in the absence of normal GPIb,
the larger subunit is not maintained on the platelet surface but is
released into the plasma. Furthermore, GPIb-IX complex formation and surface expression are impaired in the absence of functional GPIb.
| |
Discussion |
We observed a novel, homozygous mutation in the gene for GPIb
that results in BSS. The single base substitution converts the TGG
sequence at codon 21 of the GPIb gene to TAG, which causes a
premature termination of translation. Because the patient's parents
had a consanguineous marriage, the patient might be predicted to be
homozygous. However, 2 other reports of BSS due to mutations in GPIb
identified a single-allele mutation accompanied by a partial
chromosomal deletion at 22q11.2.11,31 This deletion gives
rise to a clinical condition known as DiGeorge-velocardiofacial syndrome and is often referred to as the DiGeorge chromosomal region.
The GPIb gene has been localized to position q11.2 on chromosome
2232,33 and is in the middle of the DiGeorge
region.34 We therefore assessed our patient for deletion of
the DiGeorge region of chromosome 22 by FISH analysis using a D22S75
probe specific for this region. We found a normal male karyotype with dual labeling of both chromosomes 22.
There are 3 other reports of BSS resulting from defects in GPIb
synthesis.11,29,35 In 2 of these, the mutations were associated with macrodeletions of chromosome 22q11.2, the locus for the
GPIb gene.11,29 The third patient was a compound
heterozygote with 2 independent mutations at amino acid positions 88 and 108 of the GPIb gene.35 This patient was described
as having a variant form of BSS in which giant platelets were present
but neither thrombocytopenia nor a definite tendency to have abnormal bleeding was observed. Notably, however, there was a reduced density of
GPIb-IX complexes on the platelet surface and absence of disulfide linkage between the GPIb and GPIb subunits.
All these reports confirm a critical role for GPIb in the
functioning of the vWF receptor on the platelet surface. However, the
precise role of GPIb in the GPIb-IX complex remains elusive. The
ligand-binding features of the complex reside in the sequence of
GPIb. This large GP protein contains the binding sites for vWF and
-thrombin in its large extracellular domain, and a serine phosphorylation site36 and a binding site for
14-3-33,37 have been identified in its cytoplasmic domain.
Thus, it has been suggested that the role of the other GPs is in the
successful assembly and transport of the intact GPIb-IX-V complex to
the platelet surface. Indeed, mutations in GPIX, which give rise to BSS, were shown to adversely affect the stability of the GPIb-IX-V complex on the platelet surface by inhibiting the association of GPIX
with GPIb. Furthermore, in vitro studies examining the biosynthesis
and assembly of the individual components of the GPIb-IX-V complex in
stably transfected CHO cells confirmed an essential role for GPIX and
GPIb in the assembly of the complex.27,38 In this study,
we found that GPIb was virtually absent from the surface of
platelets from our patient with BSS but present in the
platelet cytoplasm and detectable in plasma. These findings confirm a
role for GPIb in the stability of the GPIb-IX complex on the
platelet surface.
GPIb and GPIb are normally held together by an intermolecular
disulfide bond.27 The W21stop mutation in GPIb reported here cannot sustain this disulfide bridge and is therefore not disulfide linked to GPIb in the membrane. Similar results occur when
GPIb is mutated at Ala80.11 Furthermore, our studies in which W21stop-GPIb was cotransfected into HEK 293T cells with wild-type GPIb and GPIX demonstrated a similar lack of surface expression of the intact complex. Identical results were obtained when
the stable cell line CHOIX was transiently transfected with the
mutant GPIb. The fluorescent images of GPIb staining in the BSS
platelets demonstrated a circumferential pattern, suggesting that
GPIb is transported to the platelet membrane even in the absence of
an association with GPIb or GPIX. Flow cytometric analysis of the
BSS platelets (Figure 1 and Table 1) showed some residual expression of
GPIb on the surface of those platelets, indicating a capacity for
this protein to be expressed alone on the cell surface. This may
explain why the CHOIX cells and the HEK cells transfected with
GPIb had some surface expression of GPIb, even in the absence of
a functional GPIb.
Our study demonstrates that in the absence of a functional GPIb,
GPIb can be synthesized by BSS platelets. The fluorescent images of
GPIb staining in the BSS platelets showed that GPIb is protected
from proteolytic degradation and is transported to the platelet
membrane. There, it is either not inserted into the membrane (which
would result in a complete absence of membrane expression) or it is
inserted and then shed (resulting in transient surface expression). Our
detection of soluble GPIb in the patient's plasma lends support to
the latter alternative. The data also suggest that GPIb acts in
normal platelets to stabilize the GPIb-IX complex in the platelet
cytoplasm and to enhance its tenure in the platelet membrane.
To our knowledge, this is the first report of a novel, homozygous
mutation in the gene for GPIb. This mutation affects the synthesis
of GPIb and the expression and functions of the vWF receptor, the
GPIb-IX complex. The mutation also results in the absence of the
surface expression of the GPIb-IX complex on platelets and the presence
of circulating soluble GPIb, and it accounts for the
BSS phenotype.
| |
Footnotes |
Submitted October 12, 1999; accepted March 2, 2000.
Supported in part by grants from the Higher Education Authority
(Ireland), the Irish Heart Foundation, the RCSI Research Trust, and the
Wellcome Trust, and by US Public Health Service grant HL56027.
Reprints: Dermot Kenny, Department of Clinical Pharmacology,
Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin
2, Ireland; e-mail: dkenny{at}rcsi.ie.
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.
| |
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Abstract
Introduction