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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4712-4720
Truncation of Glycoprotein (GP) IIIa ( 616-762) Prevents
Complex Formation With GPIIb: Novel Mutation in Exon 11 of
GPIIIa Associated With Thrombasthenia
By
Milagros Ferrer,
Jianming Tao,
Gema Iruín,
Matilde Sánchez-Ayuso,
José González-Rodríguez,
Roberto Parrilla, and
Consuelo González-Manchón
From the Department of Pathophysiology and Human Molecular Genetics,
Centro de Investigaciones Biológicas (CSIC), Madrid, Spain; the
Instituto Rocasolano, Madrid (CSIC), Madrid, Spain; and the Department
of Haematology, Hospital de Cruces, Baracaldo, Bilbao, Spain.
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ABSTRACT |
This work reports the molecular genetic study of a patient who
suffered from Glanzmann thrombasthenia (GT). Structural analysis of the
glycoprotein (GP) IIb and GPIIIa genes showed the presence of a
homozygous G1846 T transversion in exon 11 of
GPIIIa that changes Glu616Stop. Cytometric and
immunochemical analysis indicated that platelet GPIIb-IIIa was absent
in the proband but present at normal levels in the heterozygous
relatives. The following observations indicate that this mutation is
responsible for the thrombasthenic phenotype of the proband. (1) We
failed to detect mutations other than [T1846]GPIIIa in
the coding region of both GPIIb and GPIIIa genes. (2) The
G1846T mutation was observed in either parent and
a brother of the proband, but none of 100 unrelated individuals carried
this defect. (3) Pulse-chase and immunoprecipitation analysis of
GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs
encoding normal GPIIb and [T1846]GPIIIa showed neither
maturation of GPIIb nor complex formation and surface exposure of
GPIIb- GPIIIa. These observations indicate that the sequence from
Glu616 to Thr762 in GPIIIa is essential for
heterodimerization with GPIIb. Polymerase chain reaction-based analysis
demonstrated the presence of normal levels of full-length GPIIIa-mRNA
in the proband and in heterozygous relatives. In addition, a shortened
transcript, with a 324-nucleotide deletion, resulting from in-frame
skipping of exons 10 and 11, was detectable upon reamplification of the
DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa
does not lead to abnormal processing or reduction in the number of
transcripts with the termination codon.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
GLANZMANN described in 1918 a bleeding
disorder manifested immediately after birth, characterized by a
prolonged bleeding time and abnormal clot retraction, a reason why it
was named thrombasthenia.1 Later on, it was recognized that
platelets from these patients would not spread or aggregate and their
fibrinogen content was low or absent,2-5 indicating that
the fibrinogen receptor was absent or functionally
defective.6,7 The Glanzmann thrombasthenia (GT) has been
classified into types I and II.8 In type I GT, platelets
lack fibrinogen and clot retraction and they show absence of
glycoprotein (GP) IIb-IIIa complexes at their surface. In type II GT,
the platelets contain detectable amounts of fibrinogen, the clot
retraction capability may vary from low to moderate, and the expression
of GPIIb-IIIa complexes is 10% to 20% of the control values. In cases
of GT termed variants, the platelets possess normal or near normal
(60% to 100%) expression of dysfunctional receptors.7,9
These differences may justify the clinical heterogeneity of this
bleeding disorder.10 Knowledge of the structural
organization of the GPIIb and GPIIIa genes11-15 has made it
possible to establish the association of thrombasthenic phenotypes with
distinct genetic lesions in one or both genes (for a recent review, see
French and Coller16).
The present investigation is aimed at elucidating the molecular genetic
lesion in a 20-year-old Caucasian woman who suffered from GT.
Structural analysis of the GPIIb and GPIIIa genes showed the existence
of a novel homozygous G1846T transversion in exon
11 of GPIIIa that changes Glu616Stop. Reverse
transcription-polymerase chain reaction (RT-PCR) analysis
demonstrated that full-size [T1846]GPIIIa-mRNA was the
only detectable transcript. Transfection and immunoprecipitation
analysis demonstrated that the translational product of
[T1846]GPIIIa does not complex with GPIIb; therefore, it
is the molecular lesion underlying the lack of platelet expression of
GPIIb-IIIa receptor and the thrombasthenic phenotype of the patient.
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MATERIALS AND METHODS |
Clinical data.
The proband is a 20-year-old Caucasian woman clinically diagnosed of GT
who referred a history of mucocutaneous bleeding episodes and
unprovoked bruising that started soon after birth and copious menstrual
hemorrhages. Her parents neither suffer from any hematological disorder
nor recognize consanguinity. The hematological studies of the
propositus showed a bleeding time of 18 minutes. The platelet count was
305,000/µL, the platelet adhesion capacity was reduced (7%), and no
aggregation was detected either spontaneously or in response to
agonists. Plasma fibrinogen content was 250 mg/dL and there was no clot
retraction.
Preparation of platelets and analysis of GPIIb-IIIa and fibrinogen
content.
Blood samples from the patient and relatives were obtained after
informed consent was obtained by Dr G. Iruín (Department of
Hematology, Hospital Cruces, Bilbao, Spain). Platelets were separated
by differential centrifugation at 120g for 20 minutes at room
temperature, sedimented at 1,000g for 10 minutes, and washed
several times with an isoosmotic buffer (10 mmol/L Tris, 150 mmol/L
NaCl, 1 mmol/L EDTA, pH 7.4). The platelets were sonicated at 4°C
and centrifuged at 100,000g for 1 hour to separate the soluble
and particulate fractions. Competitive solid-phase enzyme immunoassays17 were performed to determine the platelet
content of GPIIb, GPIIIa, and fibrinogen using monoclonal antibodies
(MoAbs) for GPIIb (M3), GPIIIa (P37 and P95.2), and
fibrinogen.18,19 Fibrinogen was determined in the
100,000g supernatant of sonicated platelets, and GPIIb-GPIIIa
in detergent (3% sodium dodecyl sulfate [SDS]) solubilized
particulate fraction. The mean ± SD of control platelet fibrinogen
content was 6% ± 0.6% (wt/wt) of the total 100,000g supernatant protein. The mean ± SD of
the GPIIb-IIIa content in control platelets was as follows: for GPIIb,
3.7 ± 0.7 (n = 17); for GPIIIa, 2.4 ± 0.7 (n = 18), expressed
as the percentage of the total protein of the solubilized particulate fraction. The total protein content was determined using the method of
Markwell et al.20
The detergent-solubilized particulate fraction of sonicated platelets
was also used for Western blot analysis of the GPIIb-IIIa content.
Protein (2.5 to 10 µg) was electrophoresed on 7.5%
SDS-polyacrylamide gels under reducing and nonreducing
conditions and electrotransferred on nitrocellulose membranes. The
blotted membranes were blocked by incubation in phosphate-buffered
saline (PBS)-10% defatted milk powder and then incubated with
anti-GPIIIa (P37 or P95.2) and anti-GPIIb (M3) specific antibodies.
After incubation in a 1:3,000 dilution of antimouse IgG-horseradish
peroxidase (Bio-Rad Laboratories, Hercules, CA), immuno-reactive bands
were shown with H2O2/4-chloro-1-naphtol and
quantitated by computerized image analyzer.
Flow cytometry.
Platelets were harvested using 0.5 mmol/L EDTA in PBS, washed twice
with the same buffer, resuspended at a density of 106
cells/100 µL, and incubated with a MoAb directed against either GPIIb
(M3), GPIIIa (P37 or P95.2), or GPIb at 4°C for 20 minutes. Cells
were then washed and resuspended in 50 µL of PBS containing a 1:20
dilution of fluorescein isothiocyanate (FITC)-conjugated F(ab )2 fragment of rabbit antimouse Ig (Dako A/S,
Glostrup, Denmark), followed by incubation at 4°C for
20 minutes. Finally, after three washes with PBS, the cell suspension
was adjusted to a density of 2.5 × 106 cells/mL and
the surface fluorescence was analyzed in a Coulter flow cytometer,
model EPICS XL (Coulter, Hialeah, FL).
Single-stranded conformational polymorphism (SSCP) analysis,
cloning, and sequencing of PCR-amplified genomic DNA fragments of the
GPIIb and GPIIIa genes.
Genomic DNA was isolated from peripheral blood specimens. PCR
amplification of DNA fragments encompassing one or more exons of GPIIIa
or GPIIb was performed with oligonucleotides complementary to the
intronic flanking regions using Taq polymerase according to the
protocol recommended by Perkin-Elmer Cetus (Norwalk, CT). MgCl2 concentration and annealing temperatures were
optimized for each pair of primers. Screening for mutations in GPIIb
and GPIIIa was performed using cold SSCP analysis.21,22 A
fragment of 198 bp comprising exon-11 of GPIIIa was amplified with the following oligonucleotides: sense,
5-GGGATACGCTTAGGCTTGCT-3; antisense,
5-AACCTGGGTGTGTGCAACTCT-3. To increase the sensitivity of
the method, the amplification products were digested with HinfI to yield two fragments of 140 and 58 bp, and the digests were electrophoresed at 12°C in a nondenaturing 16% acrylamide gel containing 8.7% glycerol. DNAs showing altered electrophoretic mobility patterns of single-stranded bands were cloned directly into a
T vector,23 and at least 10 positive clones from each amplification were pooled and their sequence was
determined24 with the T7 sequencing kit of Pharmacia
Biotech (Uppsala, Sweden). Sequence analyses were performed as
described by Marck.25
The carrier status for the G1846T mutation in the
kindred was determined by allele-specific PCR (ASPCR)26
analysis. Genomic DNA was used as template for the amplification of a
148-bp fragment comprising exon 11, using as sense primer either
5-TCCTTCAGAGAATGTGTGG-3 (normal) or
5-TCCTTCAGAGAATGTGTGT-3 (mutant), whose 3 ends are
complementary to the normal or the mutated base, respectively, and the
antisense primer 5-AACCTGGGTGTGTGCAACTCT-3. Each
amplification cycle consisted of 30 seconds of denaturation at
94°C, 1 minute of annealing at 64°C, and 2 minutes of extension
at 72°C. Portions of the PCR products were analyzed by agarose gel
electrophoresis.
Construction of mammalian expression vectors containing normal or
mutant [T1846]GPIIIa cDNA.
[T1846]GPIIIa cDNA was prepared by the splicing by
overlap extension procedure (SOE).27 Human GPIIIa cDNA
contained in the plasmid pBluescript KS was used as
template to generate two PCR overlapping fragments, herewith referred
to as 5 and 3 segments: the 5 segment was
amplified using the oligonucleotide sense IIIa (1670-1689), 5-ACTGCAACTGTACCACGCGT-3, and the mutated antisense
primer (1861-1839), 5-AACTTCTTACACTACACACAT-3;
the 3 segment was obtained using as sense mutant primer
(1837-1857), 5-GAATGTGTGTAGTGTAAGAAG-3, and the
primer Xho I antisense IIIa (2319-2296),
5-TGATAATGACTCGAGGATGACTGC-3. Bases substituted to
generate mutation in overlapping primers are underlined. The 5
and 3 PCR products were used as template in a new round of PCR
amplification with the oligonucleotides sense (1670-1689) and
Xho I antisense (2319-2296) described above; the amplified DNA
was digested with Xho I and partially with Not I,
because another internal Not I site is found in the cDNA
sequence. The 581-bp digestion product was then exchanged for the wild
sequence in the pBluescript KS+-GPIIIa. Both wild-type and
mutated GPIIIa-containing plasmids were subjected to total digestion
with Xho I and partial digestion with BamHI, and the
released complete cDNA sequences were subcloned into the expression
vector pcDNA3 digested with the same enzymes. Nucleotide sequence
analysis was performed to confirm the proper insertion of the amplified
mutant product and the absence of errors potentially caused by the Taq
polymerase.
Wild-type GPIIb-cDNA in pBluescript, obtained from Dr. D. Phillips (COR
Therapeutics, Inc, San Francisco, CA), was subcloned into the
HindIII site of pcDNA3.
Cell culture and transfection.
CHO cells were grown in Dulbecco's modified eagle medium (DMEM)
supplemented with 10% fetal calf serum in 5% CO2-95% air
at 37°C. Cells were incubated with 3.5 µg of the plasmid
pcDNA3-GPIIb and/or 3.5 µg of either pcDNA3-GPIIIa or
pcDNA3-[T1846]GPIIIa in the presence of 100 µg/mL
diethyl aminoethyl (DEAE)-dextran and 100 µmol/L
chloroquine diphosphate.28 After 4 hours, cells were
exposed to 10% dimethyl sulfoxide (DMSO) in PBS for 6 minutes and
rinsed with PBS; completed medium was then added and the incubation was
continued for 48 or 72 hours.
Biotin labeling and immunoprecipitation analysis of GPIIb-IIIa
complexes from CHO cells cotransfected with cDNAs encoding GPIIb and
either normal or mutant [T1846]GPIIIa.
Biotin labeling and immunoprecipitation of cell surface GPIIb-IIIa
complexes was performed as follows. CHO cells transfected with cDNA
encoding normal GPIIb and either normal or mutated
[T1846]GPIIIa were washed twice with PBS and incubated in
2 mL of PBS containing 5 mmol/L biotin-NHS (D-biotin-N-hydroxy
succinimidester; Boehringer Mannheim, Mannheim, Germany).
At the end of the incubation, the cells were washed with PBS and
treated 30 minutes at 4°C with lysis buffer (50 mmol/L Tris, pH
7.4, 150 mmol/L NaCl, 2 mmol/L phenylmethyl sulfonyl fluoride [PMSF],
1% Triton X-100, 0.05% Tween 20, and 0.03% sodium azide) and GPIIb,
GPIIIa, or GPIIb-IIIa complexes were immunoprecipitated as described
before.29 The immunoprecipitates were separated by
electrophoresis at 40 V in 0.1% SDS-7.5% polyacrylamide slab gels.
Proteins were transferred to a nitrocellulose membrane in the presence
of Towbin buffer (25 mmol/L Tris, 192 mmol/L glycine) containing 15%
methanol and 1.3 mmol/L SDS. The membranes were blocked by incubation
in PBS-10% defatted milk powder, washed with PBS-0.1% Tween 20, and
incubated in a 1:3,000 dilution of avidin-horseradish peroxidase
(Bio-Rad Laboratories, Hercules, CA) for 1 hour at room temperature.
The color of biotin-containing materials was developed by incubation in
PBS containing 0.015% H202 and 0.5 mg/mL of
4CN (4-chloro-1-naphtol).
For biotin labeling and detection of total (surface and intracellular)
GPIIb-IIIa complexes, detergent lysates of transfected CHO cells were
incubated with 5 mmol/L biotin-NHS for 2 hours at room temperature,
followed by immunoprecipitation with specific MoAbs against GPIIIa
(P37), GPIIb (M3), or GPIIb-IIIa (CII), as described above.
Metabolic labeling of transfected CHO cells.
CHO cells were transiently transfected as described above. Forty-eight
hours after transfection, the cells were incubated in DMEM without
methionine for 30 minutes and then pulsed for 30 minutes with
[35S]methionine (200 µCi/mL). The plates were washed 3 times with PBS containing 1 mg/mL cold methionine and incubated with
DMEM containing unlabeled methionine for 0, 0.5, 2, and 8 hours. Cells were washed 5 times with PBS containing cold methionine and extracted after 30 minutes in 1 mL of lysis buffer. Immunoprecipitation analysis
was performed as described for biotin-labeled cells. The
immunoprecipitates were electrophoresed at 50 V in 0.1% SDS-7.5% polyacrylamide slab gels. The gels were vacuum dried and exposed to
hypersensitive x-ray film.
RT-PCR analysis of GPIIb and GPIIIa mRNAs.
Lymphocytes from the proband, her father, and normal individuals were
immortalized with Epstein-Barr virus as previously
described.30 Total RNA was extracted using the guanidinium
thiocyanate method,31 reverse-transcribed with the primer
IIIa-AS (2329-2309; 5-TGGCACAGGCTGATAATGATC-3), and used
as template for the PCR amplification of two overlapping GPIIIa cDNA
fragments. The 5 segment was amplified using the oligonucleotides IIIa-S (63-84),
5-ATGTGTGCCTGGTGCTCTGAT-3, and IIIa-AS (1439-1419),
5-GGGCGATAGTCCTCCTCTGA-3; the 3 cDNA fragment was
amplified with oligo IIIa-S (1366-1385),
5-GAGTGTGGGGTATGCCGTTG-3, and IIIa-AS (2329-2309). To
search for alternative spliced forms of GPIIIa-mRNA, portions of the
PCR products were reamplified with the same primers. The amplified DNA
was analyzed by electrophoreses in agarose gels and cloned in a T
vector23 for further sequence analysis.
PCR-based quantitation of platelet GPIIIa-mRNA with the TaqMan
system.
The instrumentation and the fluorogenic probes of the Perkin-Elmer
Cetus (Norwalk, CT) LS-50B TaqMan System were used for PCR-based
quantitation of GPIIIa mRNA in platelets from the proband, her
heterozygous relatives, and normal individuals, as previously described.32 Specific oligonucleotide probes,
R-CTCTGGCGCGTTCTTCCTCAAATTTAGC-Q and R-ATGCCCT-Q-CCCCCATGCCA TCCTGCGT,
were designed to anneal to targets located within PCR-amplified
fragments of 132 bp (2157-2288) of GPIIIa or 295 bp (2141-2435) of
 -actin cDNA, respectively. The location of the reporter and quencher
dyes are indicated by R and Q, respectively. Because of the scant
amount of material made available to us, only two different amounts of
RNA were used for amplification of GPIIIa and -actin DNA fragments
using the rTth polymerase XL from Perkin-Elmer Cetus. Briefly, in a
first step, mRNA was reverse-transcribed with the antisense primers in
the presence of 1.1 mmol/L Mn(OAc)2 for 30 minutes at
60°C. The PCR amplification was then performed by chelating the
Mn2+ and adding 0.8 mmol/L Mg(OAc)2, the sense
primer, and the specific TaqMan probe. Thirty amplification cycles were
performed, consisting of 15 seconds at 95°C and 15 seconds at
65°C. After PCR cycling, 25-µL portions were taken from each
sample and the fluorescence was measured using a 488-nm excitation
wavelength and 518- and 580-nm emission wavelengths for the reporter
and quencher dyes, respectively. Values were corrected for internal
quenching and expressed as GPIIIa/-actin fluorescence ratios.
Materials.
Restriction enzymes were obtained from Boehringer (Mannheim, Germany)
and DNA sequencing reagents were from Pharmacia Biotech (Uppsala,
Sweden). The pcDNA3 expression vector was from Invitrogen (San Diego,
CA). Most other reagents were purchased from Sigma Chemical Co (St
Louis, MO) or from Merck (Darmstadt, Germany). [35S]-methionine (specific activity [SA],
1,000 Ci/mmol) and [35S]-dATP (SA, 1,000 Ci/mmol) were
obtained from Amersham Ibérica (Madrid, Spain). MoAbs specific
for GPIIIa, GPIIb, GPIIb-IIIa heterodimer, and fibrinogen were provided
by Dr J. González (Instituto Roscasolano (CSIC), Madrid, Spain).
Anti-GPIb/IX MoAb was purchased in Sigma Hispania (Madrid, Spain).
| |
RESULTS |
The proband refers a history of frequent bleeding episodes,
particularly mucocutaneous, that started immediately after birth. However, no family history of hemorrhage disorders or consanguinity was
recognized by her parents. Analytical features were compatible with a
diagnostic of GT. The absence of platelet GPIIb-IIIa complexes by flow
cytometric analysis using specific MoAbs
(Fig 1) confirmed that the proband was a
type I case of GT. Her parents and her brother showed platelet
expression of GPIIb-IIIa within the range of normal individuals.
Moreover, enzyme immunoassay and immunoblotting analysis using a
different anti-GPIIIa (P95.2) MoAbs confirmed the absence of platelet
GPIIb-IIIa in the proband and levels within the normal range in her
brother and her parents (Table 1). In agreement with the latter observation, intraplatelet fibrinogen content
was 15% of the platelet control in the proband and within normal
levels in the other family members. The apparently normal, or near
normal, levels of platelet GPIIb-IIIa in the brother and parents of the
proband contrast with previous reports showing a marked reduction in
the surface exposure of this heterodimer in heterozygous individuals
for other mutations.
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| Fig 1.
Flow cytometric analysis of GPIIb-IIIa content in
platelets from the proband, her parents, and her brother. The
fluorescence analysis was performed in a Coulter cytometer, model Epics
XL. Results are expressed as semilog plots of cell number versus
fluorescence intensity. The upper panel shows the fluorescence signals
of GPIIIa and GPIIb from control platelets. The negative control
represents the fluorescent signal of platelets without antibodies. The
middle panel shows the labeling of GPIIIa with the MoAb P95.2 in
platelets from the proband, her parents, and her brother. The lower
panel shows the results of labeling GPIIb with the MoAb M3 in platelets
from the proband, her parents, and her brother. For the sake of
clarity, the original plots have been redrawn.
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Structural analysis of the GPIIb and GPIIIa genes.
GPIIb and GPIIIa genes were screened for mutations by the cold
SSCP21,22 of PCR-amplified exons from genomic DNA. Only the
amplification product of exon 11 of GPIIIa showed a distinct pattern of
bands in the proband. Figure 2 depicts the
electrophoretic analysis of HinfI digests of exon 11. All of
the analyzed members of the kindred showed a distinct band, indicated
by an arrow, that is not observed in the control. The relatives, but
not the proband, show an additional band that is also present in the
control. This observation indicated that the three members of the
kindred appeared to be heterozygous for the mutation carried by the
proband. Sequence analysis showed the presence of a homozygous
G1846T transversion that changes
Glu616Stop (Fig 3).
The predicted translational product of this messenger is a truncated
protein ( GPIIIa) in which a segment encompassing 76 amino acid
residues of the extracellular domain, the transmembrane domain,
and the intracellular carboxyterminal end of the protein would be
absent. The carrier status of the kindred members was further verified
by allele-specific PCR amplification (Fig
4). As expected, amplification from the proband's DNA was only
observed with the mutant primer.
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| Fig 2.
SSCP analysis of exon 11 of GPIIIa amplified from genomic
DNA. Genomic DNA fragment of 198 bp comprising exon 11 of GPIIIa and
intronic flanking regions was amplified as described in Materials and
Methods. The PCR products were digested with HinfI to yield
fragments of 140 and 58 bp and electrophoresed in nondenaturing 16%
acrylamide slab gels containing 8.7% glycerol. The arrow points to a
distinct band shown by the patient, her parents, and her brother that
is absent in the control (wt) DNA.
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| Fig 3.
Identification of a G1846T mutation
in exon 11 of GPIIIa. Exon 11 of GPIIIa was amplified from genomic DNA
as described in Materials and Methods. The amplification products were
cloned in a T-vector and the primary nucleotide sequence of pooled DNA
was determined in both directions. The figure shows a fragment of the
sequencing ladder of the sense strand. The arrows point to a homozygous
G1846T transversion that changes
Glu616Stop.
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| Fig 4.
Specific amplification of normal and
[T1846]-GPIIIa alleles. A DNA fragment of 148 bp
encompassing exon 11 and intronic flanking regions of GPIIIa was
amplified from genomic DNA of a control, the proband, her parents, and
her brother. Each DNA was amplified using a sense primer whose 3
end was complementary to either the normal sequence (Wt) or to the
mutant base (Mut). The amplification products were electrophoresed in a
3% agarose gel and the DNA bands were stained with ethidium bromide.
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PCR-based analysis of mRNA-GPIIIa.
Because of difficulties in obtaining platelets from this kindred when
this study was started and because GPIIIa is expressed in
lymphocytes,33 to have an endless source of genetic
material, we immortalized cells from the proband and her father with
the Epstein-Barr virus. In agreement with the cytometric analysis performed in platelets (Fig 1), the lymphoblasts from the proband showed absence of GPIIIa when compared with her father who is heterozygous for the same mutation (results not shown).
PCR amplification of reverse-transcribed RNA from the proband and one
heterozygous relative was performed with oligonucleotides encompassing
the entire coding sequence of GPIIIa. Sequence analysis showed that
[T1846]GPIIIa was the only form of mRNA found in the
proband, whereas apparently similar proportions of mutant
[T1846] and normal [G1846]GPIIIa messengers
were found in heterozygous individuals. To search for less represented
alternative spliced transcripts, we reamplified portions of the first
PCR with the same primers. In these conditions, we detected an
additional shortened product in the proband and her father that was not
observed in the control (results not shown). Sequencing of this
shortened transcript showed an in-frame internal deletion of 324 nucleotides as a result of skipping exons 10 and 11; in addition, the
first codon of exon 12 changed from AAG(Lys) to GAG(Glu). The
translation of this transcript should yield a protein with a deletion
of residues 538 to 645.
To determine the level of platelet GPIIIa-mRNA, we performed a
quantitative TaqMan PCR-based determination of messenger.32 The assays were performed under predetermined conditions of cycle number and amount of RNA in which the TaqMan fluorescence emission was
a function of the DNA target concentration.
Figure 5 depicts the results obtained at
one of the RNA concentrations used. RQ represents the ratio of
fluorescence emission of the reporter dye to that of a quencher that is
a passive internal standard. The fluorescence dye is located in a probe
that hybridizes within the target sequence and its signal intensity is
proportional to the number of amplification cycles and amount of PCR
product. The RQ is calculated by substracting the RQ value obtained
by a PCR amplification without template. The RQ-GPIIIa to
RQ--actin ratio in the proband was virtually identical to the
values of the other kindred members and to the normal controls (Fig 5,
bottom). This observation indicates that mutant
[T1846]GPIIIa allele was expressed at similar rates as
the normal one.
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| Fig 5.
PCR-based determination of platelet mRNA-GPIIIa.
PCR-based quantitation of -actin and GPIIIa mRNAs in platelets from
the proband, heterozygous relatives, and normal individuals was
performed using the instrumentation and the fluorogenic probes of the
Perkin-Elmer Cetus LS-50B TaqMan System as described in Materials and
Methods. R and Q denote the fluorescence of the reporter and the
quencher dyes, respectively. Values were corrected for internal
quenching and expressed as GPIIIa/-actin fluorescence ratios.
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Heterologous expression of normal or [T1846]GPIIIa.
CHO cells were transiently cotransfected with the plasmid pcDNA3-GPIIb
and either pcDNA3-GPIIIa or pcDNA3-[T1846]GPIIIa. To
investigate the surface exposure of GPIIb-IIIa, we labeled intact cells
with biotin, and the GPIIb-IIIa complexes were immunoprecipitated with
anti-GPIIb (M3) or anti-GPIIIa (P37) MoAbs. Bands migrating as native
GPIIbH (the disulfide-linked heavy chain of GPIIb) and GPIIIa were
found in the immunoprecipitates from cells transfected with normal
GPIIb and GPIIIa cDNAs (Fig 6A); however,
no biotin-labeled products were found in cells transfected with the
void pcDNA3 plasmid or cotransfected with normal GPIIb and mutant
[T1846]GPIIIa.
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| Fig 6.
Immunoprecipitation of biotin-labeled surface or total
(surface and intracellular) proteins from CHO cells cotransfected with
cDNAs encoding GPIIb and either normal or [T1846]GPIIIa.
CHO cells were transiently cotransfected with cDNAs encoding GPIIb and
either normal or [T1846]GPIIIa in the expression plasmid
pcDNA3. (A) Protein labeling was performed by exposure of intact cells
to biotin-NHS, and the GPIIb-IIIa complexes were immunoprecipitated
with either anti-GPIIIa (P37) or anti-GPIIb (M3) MoAbs as described in
Materials and Methods. (B) The experimental design was similar to that
described in (A), except that total cell lysates rather intact cells
were exposed to biotin-NHS and the immunoprecipitation was also
performed with a GPIIb-IIIa complex-specific antibody (CII). Mock cells
were transfected with void pcDNA3 plasmid.
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To examine the intracellular presence of GPIIb and/or GPIIIa as
either monomers or heterodimers, we labeled total cell lysates with
biotin, and GPIIb, GPIIIa, or GPIIb-IIIa complexes were
immunoprecipitated as described for the biotin surface-labeled cells
using anti-GPIIb (M3), anti-GPIIIa (P37), or GPIIb-IIIa
complex-specific (CII) MoAbs. Immunoprecipitates with apparent
molecular weights corresponding to proGPIIb, GPIIIa, or GPIIIa were
observed in extracts of cells transfected with either normal GPIIb,
normal GPIIIa, or mutant GPIIIa, using antibodies directed against
each subunit, but not with a complex-specific antibody (Fig 6B). As
expected, whether subunit- or complex-specific antibodies were used,
GPIIbH and GPIIIa were detected in extracts of cells coexpressing
normal GPIIb and GPIIIa (Fig 6B); proGPIIb was detectable only using the anti-GPIIb MoAb, suggesting that it was present mainly as a
monomer. Immunoprecipitation using a MoAb against either GPIIb or
GPIIIa yielded proGPIIb or GPIIIa, respectively, in cells coexpressing normal GPIIb and mutant GPIIIa. Unlike in extracts from
cells coexpressing normal subunits, we failed to immunoprecipitate heterodimers using a complex-specific MoAb (Fig 6B), suggesting that
the mutant GPIIIa does not complex with GPIIb. The absence of GPIIb
cleavage into heavy and light chains suggested either that GPIIIa
did not complex with GPIIb or that heterodimers had not been
transported into the Golgi complex, where this process takes
place.34,35 To test whether the lack of transport of GPIIb-GPIIIa out of the endoplasmic reticulum was due to formation of unstable heterodimers, we performed a pulse-chase analysis of cells
transiently cotransfected with both subunits. Cells were pulse-labeled
for 30 minutes with [35S]methionine and then chased with
medium containing unlabeled methionine for 0.5, 2, or 8 hours before
GPIIb-IIIa complexes were immunoprecipitated with an anti-GPIIb MoAb.
Figure 7 shows the reciprocal changes in
the labeling of proGPIIb and GPIIbH as a function of the chasing time
in cells coexpressing normal GPIIb and GPIIIa proteins, indicating
stability and normal transport of GPIIb-IIIa complexes into the Golgi
apparatus. In contrast, proGPIIb was immunoprecipitated with anti-GPIIb
in cells cotransfected with normal GPIIb and mutant GPIIIa, but
GPIIbH or GPIIIa did not. However, labeled material migrating like
GPIIIa was detected upon precipitation with an anti-GPIIIa antibody.
These observations further verify that the mutant GPIIIa does not
complex to GPIIb.
View larger version (31K):
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| Fig 7.
Pulse-chase analysis of the stability of normal or
GPIIIa-IIb complexes. CHO cells were transiently cotransfected with
cDNAs encoding GPIIb and either normal or [T1846]GPIIIa
in the expression plasmid pcDNA3. Cells were pulse-labeled with
[35S]-methionine for 30 minutes and then chased with
medium containing unlabeled methionine. At the indicated times, the
cells were lysed and labeled proteins were immunoprecipitated with the
indicated antibodies. The immunoprecipitates were analyzed by
electrophoresis as described in Materials and Methods.
|
|
| |
DISCUSSION |
Pathophysiological significance of the G1846T
mutation of GPIIIa.
Clinical and analytical studies supported the conclusion that the
proband suffered from a type I GT. We screened the GPIIb and GPIIIa
genes for mutations and the only structural change found was a novel
mutation, a G1846T transversion, lying within
exon 11 of GPIIIa, that changes Glu616 to Stop.
Heterozygosity of both parents and the brother of the proband for this
mutation was verified by different analytical procedures. Because both
ancestors denied consanguinity, their heterozygosity for the
[T1846]GPIIIa mutation could indicate a high incidence of
this mutation. However, inasmuch as the ancestors of either parent were
from two little villages in the northeastern part of Spain, separated only by 10 Km, it is probable they could be consanguineous without knowing. Moreover, our inability to find [T1846]GPIIIa in
more than 100 DNAs from unrelated individuals indicated that this
substitution was not a polymorphism and, together with the transfection
data, strongly suggested that it was associated with the thrombasthenic
phenotype of the patient.
RT-PCR analysis of mRNA GPIIIa.
Because lymphocytes express GPIIIa associated with  subunits other
than GPIIb,33 we decided to use lymphoblasts as an endless source of genetic material as well as a convenient mean of
investigating the functional repercussion of structural changes in
GPIIIa.36,37 Lymphoblasts from the proband did not show
surface GPIIIa. In contrast, her father, heterozygous for the
G1846T mutation, showed exposure of GPIIIa
similar to either a normal control or a GT patient carrying a mutation
in GPIIb (results not shown).
Nonsense mutations are known to force alternate forms of splicing with
the frequent result of skipping of one or more exons.38 PCR-based analysis of mRNAs indicate that the
G1846T mutation does not alter the processing of
GPIIIa-mRNA, because both normal and mutant alleles seem to be
expressed at similar rates. The scarce representation of a shortened
messenger, with an in-frame deletion of 324 nucleotides, found upon
reamplification, suggests that its translational product may not reach
a sufficient level as to play a significant functional role.
Intraexonic sequences are known to influence splice site selection. The
importance of the first two and the last three bases of the exon in the
splicing process has been recognized,39 but the role of
more internal sequences, as it is the case in the present
investigation, has not been yet elucidated. Moreover, whether a
nonsense mutation would alter the amount of transcript carrying the
stop codon is unclear. Nonsense mutations in GPIIb have been reported
to cause either no change40,41 or marked
reduction42 in the amount of messenger. These differences
appear to be consistent with the proposal that the effect of nonsense
mutations is position related, in that the closer to the 5 end
of the coding sequence, the lower the amount of
transcript.43
Heterologous expression of normal or [T1846]GPIIIa
cDNA.
In principle, the lack of surface exposure of GPIIb-GPIIIa could be
the result of either a lack of heterodimerization or a perturbation in
the processing and/or intracellular trafficking of
heterodimers. Data in this work support the conclusion that the
Glu616Stop mutation in GPIIIa prevents the
formation of heterodimers with GPIIb. The lack of immunoprecipitable
GPIIbH indicates a deficient association of proGPIIb with GPIIIa and
agrees with the postulate that endoproteolytic cleavage of
intracellular GPIIb requires its association with GPIIIa and transport
from the endoplasmic reticulum into the Golgi complex.44
Pulse-chase analysis to determine the stability of GPIIb-GPIIIa
heterodimers further verified that the mutated GPIIIa does not
complex to GPIIb. The predicted translational product of
[T1846]GPIIIa-cDNA is a protein truncated from
Glu616 to Thr762, 85% of the normal size, in
which the cysteine-rich repeat domain45 is conserved but
lacks the transmembrane and cytoplasmic regions. Truncated forms of
GPIIb and GPIIIa seem to form soluble GPIIb-GPIIIa heterodimers
capable of binding ligand.46,47 The transmembrane region of
GPIIIa was reported to be required for cellular retention of the
monomeric subunit.44 However, in agreement with a previous observation on the 1 integrin,48 a
truncated GPIIIa from Ile693 to Thr762, lacking
the transmembrane and cytoplasmic domains, was capable of assembly and
surface exposure of GPIIb-GPIIIa complexes.46 Based on
this observation, it was concluded that regions located in the
extracellular domain of GPIIIa were sufficient to achieve a correct
processing and surface expression of GPIIb-GPIIIa complexes, indicating that transmembrane interactions were not essential. These
observations seem to be in conflict with the data reported in this work
demonstrating that neither platelet from the proband nor CHO cells
coexpressing normal GPIIb and mutant GPIIIa showed surface exposure
of GPIIb-GPIIIa. Because the proband showed normal levels of
platelet [T1846]GPIIIa-mRNA, it is unlikely that the lack
of platelet expression of GPIIb-GPIIIa was the result of a limited
availability of GPIIIa subunits. The discrepancy between our case
and previous work showing surface exposure of the
GPIIIa46 could find a justification in the extension of
the truncation in each condition, 616-762 in our case versus
693-762 in the recombinant protein. The absence of the
Glu616 to Ile693 region implies the loss of
several cysteines and disruption of the long-range S406-S655 disulfide
bridge45 that could result in an abnormal protein folding.
However, recent work has demonstrated that disruption of the long-range
GPIIIa S406-S655 disulfide bridge does not prevent the surface exposure
of functional GPIIb-IIIa heterodimers.49 The Iraqi-Jewish
GT is associated with an 11-bp deletion in the exon 12 of GPIIIa that
leads to premature protein termination before the transmembrane domain
(GPIIIa650-762).50 In agreement with our findings, these
patients show detectable proGPIIb51 and absence of platelet
GPIIb-IIIa and vitronectin receptors.51,52 The possibility
should also be considered that the Glu616 to
Ile693 region could encompass a domain(s) not previously
recognized that is essential for the subunit
dimerization. In any case, the agreement of the data on
transfection experiments with the observations made in the patient's
platelets suggests that, in megakaryocytes, residues from
Glu616 to Ile693 may be involved in assembly
and surface exposure of GPIIb-GPIIIa complexes.
Platelet expression of GPIIb-IIIa in heterozygous individuals.
The present study indicates that platelets from heterozygotes for the
[T1846]GPIIIa mutation showed levels of GPIIb-IIIa within
the range of the normal population. The analysis of platelet expression of GPIb receptor (Table 1) seemed to rule out that platelet expression of GPIIb-IIIa was inherently high in this kindred. Our finding contrasts with studies on GPIIb mutations associated with
thrombasthenic phenotypes in which the heterozygous states consistently
showed a marked reduction ( 50%) in the platelet GPIIb-IIIa content. As far as we know, the G1846T is the first
nonsense GPIIIa mutation in which the platelet content of GPIIb-IIIa
has been quantitatively determined in the heterozygous states. However,
heterozygotes for other GPIIIa mutations have also been reported to
show only moderate decreases in the platelet GPIIb-IIIa
content.53,54 The existence of only one functional allele
could imply a reduction in the availability of mRNA so that it could
become limiting for the synthesis of GPIIb and/or GPIIIa.
However, the apparent discrepancy between the platelet expression of
GPIIb-IIIa in heterozygous states for the [T1846]GPIIIa
and other mutations in GPIIb suggests that, at least for this
particular mutation, transcripts from one allele may provide sufficient
GPIIIa to maintain a normal rate of heterodimerization and surface
exposure of GPIIb-IIIa complexes. Alternative explanations could be the
influence of the genetic context of this kindred in the behavior of
this mutation or, perhaps, that the truncated protein inhibited the
degradative pathway of the normal GPIIIa. The possibility should also
be considered that, unlike other mutations that can form stable
complexes with GPIIb, in our case the lack of complex formation would
leave more GPIIb available to complex normal GPIIIa. Overexpression of
the normal allele is improbable, because the overall platelet
GPIIIa-mRNA levels were similar in the proband and the heterozygous
relatives. Thus, our observations seem to suggest that availability of
mRNA-GPIIIa may not be the main rate-limiting step for the synthesis
and/or processing of this subunit. This interpretation seems to
be in conflict with the reported synthesis of a fivefold excess of
pre-GPIIb relative to GPIIIa (cited in Calvete55),
indicating that availability of GPIIIa rather than GPIIb mRNA could be
a limiting step for the surface exposure of GPIIb-IIIa. Precise
quantitative analysis of mRNAs GPIIb and GPIIIa under different
functional conditions will be required to elucidate this point.
To conclude, we report the finding of an homozygous
[T1846]GPIIIa mutation associated with type I GT. This
mutation changes Glu616 to Stop, producing a GPIIIa protein
truncated from Glu616 to Ile762. No other
mutations were found in either GPIIb or GPIIIa genes and
[T1846]GPIIIa accounted for virtually all the platelet
GPIIIa-mRNA found in the proband. Coexpression of normal GPIIb and
GPIIIa in CHO cells showed a lack of GPIIb maturation and surface
exposure of GPIIb-GPIIIa complexes. Moreover, immunoprecipitation
and pulse-chase analysis demonstrated that the translational product of
the [T1846]GPIIIa mutation does not complex to GPIIb.
Thus, the homozygous [T1846]GPIIIa mutation is
responsible for the thrombasthenic phenotype of the proband.
| |
ACKNOWLEDGMENT |
Normal GPIIb/IIIa cDNAs were a gift of Dr D.R. Philips.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted August 12, 1998.
Supported in part by grants from Dirección General de
Investigación Científica y Técnica (PB94-1544),
Fondo de Investigaciones Sanitarias (96/2014), CAM: CO7191, and
European Community concerted action contract BMH1-CT93-1685. M.F. was
the recipient of a predoctoral fellowship from the Comunidad
Autónoma de Madrid. C.G.M. was recipient of a grant from
Fundacion Rodriguez Pascual.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Consuelo González-Manchón, MD,
PhD, Centro de Investigaciones Biológicas, Velázquez 144, 28006-Madrid, Spain; e-mail: CIBP255{at}FRESNO.CSIC.ES.
| |
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Top
Abstract
Introduction