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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-07-2144.
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Blood, 1 May 2003, Vol. 101, No. 9, pp. 3485-3491
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
A naturally occurring Tyr143His IIb mutation
abolishes IIb 3 function for soluble
ligands but retains its ability for mediating cell adhesion and clot
retraction: comparison with other mutations causing ligand-binding
defects
Teruo Kiyoi,
Yoshiaki Tomiyama,
Shigenori Honda,
Seiji Tadokoro,
Morio Arai,
Hirokazu Kashiwagi,
Satoru Kosugi,
Hisashi Kato,
Yoshiyuki Kurata, and
Yuji Matsuzawa
From the Department of Internal Medicine and Molecular
Science, Graduate School of Medicine, Osaka University; the Department
of Blood Transfusion, Osaka University Hospital; and the Department of
Laboratory Medicine, Tokyo Medical University, Japan.
 |
Abstract |
The molecular basis for the interaction between a prototypic
non-I-domain integrin, IIb 3, and its
ligands remains to be determined. In this study, we have characterized
a novel missense mutation (Tyr143His) in IIb associated
with a variant of Glanzmann thrombasthenia. Osaka-12 platelets
expressed a substantial amount of IIb 3
(36%-41% of control) but failed to bind soluble ligands, including a
high-affinity IIb 3-specific
peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is
a compound heterozygote for a single 521T>C substitution
leading to a Tyr143His substitution in IIb and for the
null expression of IIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the -propeller domain of IIb, we examined the effects of Tyr143His or
Tyr143Ala substitution on the expression and function of
IIb 3 and compared them with KO (Arg-Thr
insertion between 160 and 161 residues of IIb)
and with the Asp163Ala mutation located in the same loop by using
293 cells. Each of them abolished the binding function of
IIb 3 for soluble ligands without
disturbing IIb 3 expression. Because
immobilized fibrinogen and fibrin are higher affinity/avidity ligands
for IIb 3, we performed cell adhesion and
clot retraction assays. In sharp contrast to KO mutation and
Asp163Ala IIb 3, Tyr143His IIb 3-expressing cells still had
some ability for cell adhesion and clot retraction. Thus, the
functional defect induced by Tyr143His IIb is likely
caused by its allosteric effect rather than by a defect in the
ligand-binding site itself. These detailed structure-function analyses
provide better understanding of the ligand-binding sites in integrins.
(Blood. 2003;101:3485-3491)
© 2003 by The American Society of Hematology.
 |
Introduction |
Integrins are a family of noncovalently associated
 heterodimeric adhesion receptors that mediate cellular
attachment to the extracellular matrix and cell-cell
cohesion.1,2 Integrins are involved in a variety of
physiological processes, including development, immune response, wound
healing, and hemostasis.1 They are also involved in
pathologic processes, such as tumor metastasis, thrombosis, and
athelosclerosis.1
Although its expression is restricted to the megakaryocyte/platelet
lineage, IIb 3 (GPIIb-IIIa) is a
prototypic integrin that functions as a physiologic receptor for
fibrinogen and von Willebrand factor and that plays a crucial role in
platelet aggregation, normal hemostasis, and pathologic
thrombosis.3 Indeed, Glanzmann thrombasthenia (GT) is an
autosomal recessive bleeding disorder caused by a defect in the
expression or the function of integrin IIb 3.4 Recent clinical
studies have shown the beneficial effects of
IIb 3 antagonists in patients undergoing
coronary angioplasty and in those with unstable angina.5,6
Integrin subunits are grouped into 2 classes based on the presence
or absence of an inserted domain of approximately 200 amino acid residues (I- or A-domain); IIb and v
subunits do not have the I-domain.7-9 Recently, the
crystal structures of the extracellular segment of the other
3 integrin, v 3 in the
presence or absence of its small ligand have been
described.10,11 As predicted, the putative
ligand-binding head is primarily formed by a 7-bladed -propeller
domain from v and a I-domain from
3.10-12 The -propeller domain contains 7 4-stranded -sheets (W1-W7) arranged in a torus around a
pseudosymmetry axis. However, the ligand-binding sites in the
-propeller domain of the IIb subunit remain to
be determined.
The characterization of molecular defects in GT from dysfunctional
IIb 3 (variant GT) has succeeded in
pinpointing ligand-binding sites and functionally important
domains.13-15 We have demonstrated that 2-amino acid
insertion (Arg-Thr) between amino acid residues 160 and 161 in
IIb is responsible for the ligand-binding defect in a
Japanese variant GT known as KO.16 The insertion is
located within the small loop (Cys146-Cys167) between W2 and W3 (W3 4-1 loop) located on the upper face of the -propeller. Alanine
substitutions further indicate that Asp163 within the Cys146-Cys167
loop is one of the critical residues for ligand binding.16
In this context, 2 other naturally occurring missense mutations,
Pro145Ala and Leu183Pro, which impair
IIb 3 expression and its ligand-binding function, have been identified in the W3 4-1 loop and the W3 2-3 loop
of the -propeller, respectively.17,18
In this study we demonstrate that a novel naturally occurring missense
mutation (Tyr143His) within the W3 4-1 loop in IIb is
responsible for a binding defect in IIb 3
for soluble ligands. Because Tyr143His, KO, and Asp163Ala mutations are
located within the same loop in IIb, we further compared
functions of these mutant IIb 3 by using
cell adhesion and clot retraction assays. Compared with the KO mutation
and Asp163Ala IIb 3,
Tyr143His IIb 3-expressing cells still had
some ability for cell adhesion and clot retraction. Our results
indicate that the KO mutant and
Asp163Ala IIb 3 impair ligand-binding
function in IIb 3 more severely than
Tyr143His IIb 3.
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Patients, materials, and methods |
Patient history
Patient Osaka-12, a product of nonconsanguineous parents, was a
21-year-old Japanese woman who had a history of moderate mucocutaneous bleeding in her childhood. Hematologic examinations revealed a prolonged bleeding time (more than 15 minutes) and an absence of
platelet aggregation in response to adenosine diphosphate (ADP), epinephrine, and collagen but a normal response to ristocetin. Clot
retraction using the MacFarlane method was 46% (normal range, 40%-70%).19 Informed consent for analyzing their
molecular genetic abnormalities was obtained from Osaka- 12 and
her parents.
Antibodies and synthetic ligands
AP1 (GPIb-specific monoclonal antibody [mAb]), AP2
( IIb 3-specific mAb), and AP5
( 3-specific mAb) were generously provided by Dr Thomas
J. Kunicki (Scripps Research Institute, La Jolla, CA).20,21 AP3 ( 3-specific mAb) was a
generous gift from Dr Peter J. Newman (Blood Center of Southeastern
Wisconsin, Milwaukee).22 OP-G2 is a ligand-mimetic
IIb 3-specific mAb that binds to
nonactivated and activated
IIb 3.23 PAC-1, a
ligand-mimetic IIb 3-specific mAb that
binds specifically to activated IIb 3, was
kindly provided by Dr Sanford J. Shattil (Scripps Research
Institute).24 PT25-2 ( IIb 3-specific mAb) activates
IIb 3 and was a kind gift from Drs Makoto
Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).25 PMI-1 ( IIb-specific mAb), anti-LIBS1
( 3-specific mAb), and anti-LIBS6 ( 3-specific mAb) were generously provided by Dr Mark H. Ginsberg (Scripps Research Institute).26,27 PMI-1, AP5,
anti-LIBS1, and anti-LIBS6 recognize ligand-induced conformational
changes on IIb 3, termed ligand-induced
binding sites (LIBS).21,26,27 TP80
( IIb-specific mAb) and MOPC21 (mouse myeloma
immunoglobulin G1 [IgG1]) were purchased from
Nichirei (Tokyo, Japan) and Sigma Chemical (St Louis, MO),
respectively. FK633 ( IIb 3-specific peptidomimetic antagonist) was generously provided by Dr Jiro Seki
(Fujisawa Pharmaceutical, Osaka Japan),28 and cyclo(RGDfV) [cyclo(-Arg-D-Gly D-Asp-D-Phe-L-Val-D-)] peptide
( v 3-specific antagonist) was a generous
gift from Merck KGaA (Darmstadt, Germany).29 Fibrinogen
was purchased from Calbiochem-Novabiochem (San Diego, CA). MOPC21, AP1,
AP2, TP80, AP3, PAC-1, and fibrinogen were labeled with fluorescein
isothiocyanate (FITC), as previously described.28
Flow cytometry
Flow cytometric analysis using various mAbs was performed as
previously described.30 FITC-labeled mAbs were used to
quantify the expression levels of IIb 3 on
293 cells and on platelets.
Analysis of platelet messenger RNA
Total cellular RNA of platelets was isolated from 30 mL whole
blood, and IIb or 3 messenger RNA (mRNA)
was specifically amplified by reverse transcription-polymerase chain
reaction (RT-PCR), as previously described.30 Primers for
the amplification of IIb or 3 mRNA and
conditions for RT-PCR were described elsewhere.30,31 Nucleotide sequences of PCR products were determined by using Taq
DyeDeoxy Terminator Cycle Sequencing kit and an ABI373A DNA sequencer
(Applied Biosystems, Foster City, CA).
Allele-specific restriction enzyme analysis
Amplification of the region around exon 4 of the
IIb gene was performed by using primers IIbE3,
5'-GTCGGTCGTCAGCTGGAGC-3' (sense, nucleotide [nt] 3940-3958 in the
IIb gene), and IIbE4, 5'-CAGGTCGTAGCTGGCGCTTAC-3'
(antisense, nt 4192-4172) and by using 250 ng DNA as a template.
First-round PCR products were reamplified using primers IIbg3947S,
5'-GTCAGCTGGAGCGACGTCATTGTG-3' (sense, nt 3947-3970) and IIbE4 and then
were digested with restriction enzyme ScaI. Resultant
fragments were electrophoresed in a 1.5% agarose gel.
Construction of IIb expression vectors and cell
transfection
Wild-type IIb and 3 complementary
DNAs (cDNAs) cloned into a mammalian expression vector pcDNA3
(Invitrogen, San Diego, CA) were generously provided by Dr Peter J. Newman. To construct the expression vector containing the
521C (His143) form of IIb, PCR-based
cartridge mutagenesis was performed as previously
described.16 Platelet IIb cDNA from
Osaka-12 was amplified by RT-PCR using primers IIb1 and IIb3 AS,
5'-CCCACGATCAGGTCTGGGTATCCG-3' (antisense, nt 1407-1384). Second-round
amplification was then performed using 1 µL first-round PCR products
as a template with nested primers IIb187S, 5'-GAGAGTGGCCATCGTGGTGG-3'
(sense, nt 187-206), and IIb3 AS2C, 5'-CCGTTGTCATCGATGTCTACGGC-3'
(antisense, nt 1364-1386). To construct the expression vector for
Tyr143Ala IIb substitution, we carried out the
overlapping extension PCR as previously described.16
Mismatched sense primer IIb143Ala-S, 5'-GAGCGGCCGCCGCGCCGAGGCCTCCCCCTG-3' (sense, nt 502-531, 2 bp mismatched), and antisense primer IIb143Ala-AS,
5'-CAGGGGGAGGCCTCGGCGCGGCGGCCGCTC-3' (antisense, nt
531-502, 2 bp mismatched) were synthesized. PCR was performed by using
IIb cDNA as a template and primers IIb1 and
IIb143Ala-AS, or primers IIb143Ala-S and IIb3 AS. The 2 individually amplified PCR products were mixed and used as a template of PCR using
primers IIb1 and IIb3 AS. Amplified fragments were digested with
restriction enzyme SacII and ClaI, and the
resultant fragments were extracted using GeneClean II kit (Bio 101, La
Jolla, CA). Fragments were introduced into the pcDNA3 that had been
digested with SacII and ClaI. Inserted fragments
were characterized by sequence analysis to verify the absence of any
other substitutions and the proper insertion of the PCR cartridge into
the vector. Expression vectors containing the KO mutant (2-amino acid
[Arg-Thr] insertion between residues 160 and 161) IIb
cDNA and Asp163Ala IIb cDNA were constructed as previously
described.16
The wild-type or mutant IIb construct was cotransfected
into 293 cells with the wild-type 3 construct by the
calcium phosphate method, as previously described.16 The
293 cells transiently expressing mutant
IIb 3 were obtained and analyzed 2 days
after transfection. In selected experiments, CD36 expression vector was
cotransfected with IIb and 3 constructs
to monitor transfection efficiency.32 In addition, stable
transfectants expressing wild-type, Tyr143His IIb 3, or KO mutant
IIb 3 were selected for G418
resistance (Gibco BRL, Grand Island, NY) and were cultured in Dulbecco
modified Eagle medium (DMEM) with 10% heat-inactivated fetal calf
serum (Life Technologies, Gaithersburg, MD).
Adhesion assays
Adhesion assays were performed as described by Faull et
al.33 Wells from 96-well microtiter plates were coated
with up to 0.5 µg fibrinogen per well in 100 µL phosphate-buffered
saline (PBS) and were incubated at 4°C overnight. After washing with PBS, wells were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma) for 90 minutes at 22°C. To determine background adhesion, control wells were coated with 1% BSA. Then 293 cells transiently expressing wild-type or mutant
IIb 3 were washed twice with PBS and
resuspended in DMEM containing 0.1% BSA at a concentration of
1 × 106 cells/mL, and 100 µL aliquots cell suspension
were added to wells in triplicate. The plate was incubated in a
humidified 37°C incubator for 60 minutes. After washing with PBS 3 times, the adherent cells were quantified by measuring endogenous
cellular acid phosphate activity in an enzyme-linked immunosorbent
assay.34
For morphologic analysis, 293 cells stably expressing wild-type or
mutant IIb 3 were added on
fibrinogen-coated glass coverslips for 60 minutes at 37°C. After they
were washed with PBS, adherent cells were fixed in 3.7% formaldehyde
for 10 minutes, permeabilized in 0.5% Triton X-100 in PBS for 5 minutes at room temperature, and washed twice with PBS. Cells on
coverslips were stained with rhodamine-phalloidin (Sigma) and were
analyzed under a fluorescence microscope (Olympus, Tokyo,
Japan).35
Tyrosine phosphorylation of pp125FAK
Adherent cells on fibrinogen were lysed in Triton X-100 buffer
(1% Triton X-100, 25 mM Tris-HCl, 100 mM NaCl, pH 7.4, 0.1 mg/mL leupeptin, 4 µg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamide) containing sodium vanadate and were
scraped into microcentrifuge tubes. Lysates were incubated on ice for
30 minutes, and clarified supernatants were processed for
immunoprecipitation. Focal adhesion kinase (pp125FAK) was
immunoprecipitated with 1 µg rabbit polyclonal antibody specific for
FAK (Santa Cruz Biotechnology, CA), and protein-G Sepharose (Pharmacia,
Uppsala, Sweden). Precipitates were separated on 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were
transferred to a polyvinylidene difluoride (PVDF) membrane.
Phosphotyrosine was detected with mAb 4G10. To monitor the loading of
gel lanes, the blots were stripped (2% SDS, 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol for 30 minutes at 70°C) and reprobed with
anti-FAK.36
Clot retraction
Clot retraction of stable cell lines was performed based on the
method described by Katagiri et al.37 In brief, 293 cells stably expressing IIb 3 in Tyrode/HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer
(2 × 106 cells/mL) were incubated with 10 mM
tranexamic acid, 250 µg/mL fibrinogen, and 2 mM CaCl2 at
37°C in a siliconized glass tube. To block the effect of
v 3 composed of endogenous
v and transfected 3, these stable
transfectants were preincubated with 50 µM cyclo(RGDfV) ( v 3-specific antagonist) at room
temperature for 30 minutes. Then 1 U thrombin was added to 1 mL cell
suspension. Fibrin gels began to form immediately after the addition of
thrombin, and the tubes were kept at 37°C. Clot retraction was
monitored by taking photographs every 30 minutes using a digital
camera. Quantification of retraction was performed by an assessment of
the clot area using the NIH Image 1.67e software (Bethesda, MD). Data
were expressed as follows: % clot retraction = [(area
t0 area t)/area
t0] × 100.
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Results |
Platelets from thrombasthenic patient Osaka-12 show impaired
ligand-binding function
We first examined the surface expression of
IIb 3 on platelets from patient Osaka-12
and 3 control subjects using flow cytometry. Although FITC-AP1
(GPIb-specific mAb) bound to Osaka-12 platelets slightly higher than to
control platelets (113% of control value [mean value obtained from 3 control subjects]), FITC-AP3 ( 3-specific mAb) and
FITC-TP80 ( IIb-specific mAb) revealed a significant reduction in the expression of IIb 3 on
Osaka-12 platelets (AP3 binding, 41% of control value; TP80 binding,
36% of control value; n = 2) (Figure
1). Compared with AP3 and TP80 binding to
Osaka-12 IIb 3, AP2 binding was markedly
impaired (13% of control value; n = 2) probably because of a
disturbance of AP2 epitope formation. Because Osaka-12 platelets
expressed 36% to 41% of normal amounts of
IIb 3 on their surfaces, we examined their
ligand-binding function. OP-G2 and PAC-1 mAbs are
activation-independent and activation-dependent, ligand-mimetic mAbs
specific for IIb 3, respectively. Neither
OP-G2 nor PAC-1 in the presence of an activating PT25-2 mAb bound to
Osaka-12 platelets (Figure 1). Thus, Osaka-12 IIb 3 seems to have a ligand-binding
defect and a quantitative defect.

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| Figure 1.
Flow cytometric analysis of the surface expression and
ligand-binding function of IIb 3 Osaka-12
platelets.
Washed platelets obtained from Osaka-12 or 3 control subjects were
incubated with FITC-AP1 (GPIb-specific mAb), FITC-AP2
( IIb 3-specific mAb), FITC-AP3
( 3-specific mAb), and FITC-TP80 ( IIb-specific mAb) at
a concentration of 10 µg/mL for 30 minutes at room temperature.
FITC-MOPC21 (mouse myeloma IgG1) was used as a negative
control. For OP-G2 binding (activation-independent ligand-mimetic
IIb 3-specific mAb), bound antibodies were
detected by FITC-conjugated goat F(ab')2 antimouse IgG.
Filled and open histograms represent the binding of specific and
control antibodies, respectively. For PAC-1 binding, washed platelets
were preincubated with 10 µg/mL PT25-2 (activating
IIb 3-specific mAb) for 30 minutes, and
then 10 µg/mL FITC-labeled PAC-1 was added. Closed and open
histograms represent the PAC-1 binding in the absence and presence of
10 µM FK633 ( IIb 3-specific antagonist),
respectively. Results are representative of 2 separate
experiments.
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IIb 3 LIBS expression on Osaka-12
platelets by IIb 3-specific peptidomimetic
antagonist FK633
PMI-1, AP5, anti-LIBS1, and anti-LIBS6 mAbs preferentially
recognize LIBS on IIb 3, which are exposed
following occupancy of the receptor by ligands. To further investigate
the ligand-binding function of Osaka-12
IIb 3, we examined the effect of FK633, IIb 3-specific peptidomimetic antagonist
on the expression of these LIBS. Compared with Arg-Gly-Asp-Trp peptide,
FK633 has approximately 100-fold higher potency for LIBS
induction.28 Although we could not simply compare the
binding of mAbs recognizing LIBS between Osaka-12 and control platelets
because of the different expression levels of
IIb 3, even 10 µM FK633 showed a
negligible effect on the expression of LIBS1 of Osaka-12
IIb 3 (Table
1). On the other hand, Table 1 shows that
Osaka-12 IIb 3 aberrantly expressed LIBS
recognized by PMI-1, AP5, and anti-LIBS6. These data further suggested
that the ligand-binding sites of Osaka-12
IIb 3 were markedly impaired.
Nucleotide sequence and allele-specific restriction enzyme analyses
for Osaka-12 IIb 3
To identify the molecular genetic defect responsible for Osaka-12,
all coding regions of IIb and 3 cDNA
amplified by RT-PCR from Osaka-12 and control platelet mRNA were
analyzed. Direct sequence of the PCR fragments showed a single T>C
substitution at nt 521 in IIb cDNA that would lead to a
Tyr143His substitution in exon 4 of IIb (Figure
2A). No other nucleotide substitutions were detected in the coding regions of either IIb or
3 cDNAs. The T>C substitution abolished a restriction
site for ScaI, and allele-specific restriction enzyme
analysis for the amplified IIb cDNA around exon 4 suggested that Osaka-12 might be homozygous for the substitution.
However, allele-specific restriction enzyme analysis for the PCR
fragments from genomic DNA revealed that Osaka-12 was heterozygous for
the T>C substitution. The substitution was inherited from her father.
The molecular genetic defect inherited from her mother remains
determined. However, only the allele having 521C from her
father was amplified by RT-PCR from Osaka-12 IIb mRNA, suggesting that the expression levels of maternal IIb
mRNA seem to be extremely low in Osaka-12 platelets (Figure
2B-C).

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| Figure 2.
Identification of the genetic defect responsible for
Osaka-12.
(A) Nucleotide sequence analysis of IIb cDNA from
Osaka-12. Platelet IIb mRNA was amplified by RT-PCR.
Nucleotide sequence of the amplified fragments was determined by using
Taq DyeDeoxy Terminator Cycle Sequencing kit and an ABI 373A DNA
sequencer. (B) Allele-specific restriction enzyme analysis of
IIb cDNA. The region around exon 4 of the
IIb mRNA was amplified by PCR, followed by digestion
with ScaI. ScaI digestion of the PCR products
yielded 517-bp, 264-bp, and 316-bp fragments in the healthy allele. The
T>C substitution abolished one of the restriction sites for
ScaI. Resultant fragments were electrophoresed in a 1.5%
agarose gel. Marker indicates 100-bp DNA ladder. (C) Allele-specific
restriction enzyme analysis of IIb genomic DNA. The
region around exon 4 of the IIb gene was amplified by
PCR using primers IIb3945S and IIbE4, followed by digestion with
ScaI. ScaI digestion of the PCR products yielded
241-bp and 75-bp fragments in the healthy allele. The T>C substitution
abolished the restriction site for ScaI. Resultant fragments
were electrophoresed in a 6% polyacrylamide gel. Marker indicates
X174 digested with HaeIII.
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Effect of Tyr143His substitution in IIb on
IIb 3 expression and function
To confirm that the 521T>C substitution leading to
Tyr143His substitution in IIb is responsible for the
functional defect, we constructed an expression vector that contained
the wild-type or mutant His143 form of IIb and
cotransfected it with wild-type 3 vector into 293 cells.
Effects of a Tyr143Ala substitution in IIb on
IIb 3 expression and function were also
examined. Because Tyr143 is located within the W3 4-1 loop of the
-propeller domain, close to the KO mutation (the Arg-Thr insertion
between 160 and 161 residues of IIb) and the Asp163Ala
IIb mutation, we compared the expression and function of
these mutant IIb 3 in parallel.
Transfection efficiency monitored by the cotransfected CD36 expression
vector was essentially the same between wild-type and mutant
IIb 3 (Figure
3). Flow cytometric analysis using
FITC-AP3 or FITC-TP80 mAb showed that the expression levels of
Tyr143His IIb 3 were essentially the same
as wild-type IIb 3 (AP3 binding,
91% ± 15% of wild-type; TP80 binding, 102% ± 2% of wild-type;
mean ± SD; n = 3). In sharp contrast,
Tyr143His IIb 3 failed to bind OP-G2 mAb,
FITC-PAC-1 mAb, or FITC-fibrinogen in the presence of PT25-2 mAb
(Figure 3). We also confirmed that the binding failure of these
activation-dependent ligands was not caused by the binding failure of
the activating mAb, PT25-2. In addition, FITC-AP2 binding was
significantly impaired compared with TP80 binding (AP2 binding; 15% ± 3% of wild-type; mean ± SD; n = 3), which is
consistent with the data obtained from Osaka-12 platelets (Figures 1
and 3). Given that the expression levels of abnormal IIb
mRNA derived from her mother were so low that they were not detected in
Osaka-12 platelets by RT-PCR, this maternal abnormality would reduce
the expression of IIb 3 by 50%. As
expected, the expression level of
Tyr143His IIb 3 on 293 cells was roughly
twice as much as that on Osaka-12 platelets. These data indicate that
the Tyr143His mutation is responsible for the variant GT phenotype in
Osaka-12. The Tyr143Ala substitution induced essentially the same
effects on IIb 3 as the Tyr143His mutation
(AP3 binding, 117% ± 14% of wild-type; TP80 binding,
113% ± 8% of wild-type; AP2 binding, 14% ± 1% of wild-type;
mean ± SD; n = 3). Because Tyr143Ala abolished the ligand
binding, it is likely that the presence of Tyr143 is critical for
IIb 3 function. Interestingly, the
phenotype of Asp163Ala IIb 3 appears the
same as that of Tyr143His IIb 3. The phenotype of KO IIb 3 was essentially the
same as wild-type IIb 3 except for the
failure in OP-G2 and PAC-1 binding, as previously described.15 Thus, none of these mutant
IIb 3 showed a binding ability for soluble
ligands (Figure 3).

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| Figure 3.
Effects of Tyr143His IIb,
Tyr143Ala IIb, Asp163Ala IIb, or KO mutant
on the expression and ligand-binding function of
IIb 3 on 293 cells.
Wild-type IIb or each mutant IIb cDNA was
transiently cotransfected with wild-type 3 cDNA into 293 cells. CD36 expression vector was cotransfected with IIb
and 3 constructs, and CD36 expression was measured by
FITC-anti-CD36 mAb to monitor transfection efficiency. The binding of
FITC-AP2, FITC-AP3, FITC-TP80, OP-G2, and PT25-2 was analyzed by flow
cytometry 2 days after transfection (filled histograms). FITC-MOPC21
was used as a negative control (open histograms). For OP-G2 or PT25-2
binding, bound antibodies were detected by FITC-conjugated goat
F(ab')2 antimouse IgG. For PAC-1 or fibrinogen binding,
washed platelets were preincubated with 10 µg/mL PT25-2 (activating
IIb 3-specific mAb) for 30 minutes, and
then 10 µg/mL FITC-labeled PAC-1 or 150 µg/mL FITC-labeled
fibrinogen was added. Closed and open histograms represent PAC-1 or
fibrinogen binding in the absence and presence of 10 µM FK633
( IIb 3-specific antagonist), respectively.
Results are representative of 3 separate experiments.
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Adhesion of the cells to immobilized fibrinogen
To further examine the functions of mutant
IIb 3, we performed cell adhesion assay to
immobilized fibrinogen. In contrast to parent cells, 293 cells
transiently transfected with wild-type IIb 3 or mutant
IIb 3 became adhesive to the immobilized
fibrinogen. Given that endogenous v in 293 cells can
form v 3 with the exogenous 3 and may contribute to the adhesion, we preincubated
the transfected cells with 50 µM cyclo(RGDfV) to block the
v 3 function. Under these conditions, 293 cells transfected with wild-type 3 alone (data not
shown), KO variant IIb 3, or
Asp163Ala IIb 3 failed to adhere to
immobilized fibrinogen (Figure 4).
However, 293 cells expressing
Tyr143His IIb 3 showed a significant
adhesion to immobilized fibrinogen, and the amounts of adherent cells
were approximately 50% compared with 293 cells expressing wild-type
IIb 3. The adhesion of 293 cells
expressing Tyr143His IIb 3 in the presence
of 50 µM cyclo(RGDfV) was mediated solely by
IIb 3 because 10 µM FK633 completely
blocked the cell adhesion (data not shown).

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| Figure 4.
Adhesion of IIb 3 mutants
to immobilized fibrinogen.
Wild-type IIb or each mutant IIb cDNA was
transiently cotransfected with wild-type 3 cDNA into 293 cells. Two days after transfection, wild-type or mutant
IIb 3-transfected cells
(1 × 106 cells/well) were incubated with
immobilized fibrinogen (FBG) in the presence of 50 µM cyclo(RGDfV)
( v 3-specific antagonist) at 37°C. After
washing with PBS, adherent cells were quantified with a colorimetric
reaction using endogenous cellular acid phosphatase activity. Data
represent the mean ± SD of triplicate measures of optical density
at 415 nm. Statistical analysis (2-tailed P values for
paired sample) was performed between 293 cells expressing wild-type
IIb 3 and those expressing each mutant
IIb 3 (**P < .01;
*P < .05).
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We then obtained the cells stably expressing wild-type, Tyr143His, and
KO mutant IIb 3 with G418 selection for
morphologic analysis of the adherent cells. Adhesive functions of 293 cells stably expressing Tyr143His IIb 3 or
KO IIb 3 were the same as those
transiently expressing these mutant IIb 3
(data not shown). Although 293 cells stably expressing wild-type
IIb 3 adhered firmly and showed complete
spreading, 293 cells stably expressing KO mutant
IIb 3 failed to adhere. In contrast to KO IIb 3, 293 cells stably expressing
Tyr143His IIb 3 moderately impaired cell
spreading and cell adhesion (Figure 5).
Because FAK, a 125-kDa cytoplasmic tyrosine kinase, is a component of focal adhesions and is a well-established component of integrin signaling pathways,38 the tyrosine phosphorylation of
pp125FAK in these adherent 293 cells stably expressing
wild-type IIb 3 or
Tyr143His IIb 3 was examined. As shown in
Figure 6, pp125FAK
phosphorylation was observed but impaired in cells expressing Tyr143His IIb 3 compared with wild-type
IIb 3 (Figure 6).

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| Figure 5.
Morphologic analysis of the adherent cells stably
expressing mutant IIb 3 to immobilized
fibrinogen.
293 cells stably expressing (A) wild-type
IIb 3, (B)
Tyr143His IIb 3, or (C) KO mutant
IIb 3 were incubated with immobilized
fibrinogen (5 µg/mL) at 37°C for 60 minutes in the presence of 50 µM cyclo(RGDfV) ( v 3-specific
antagonist). After gentle washing with PBS, adherent cells were fixed
in 3.7% formaldehyde and permeabilized in 0.5% Triton X-100. Cells
were stained with rhodamine-phalloidin and analyzed under a
fluorescence microscope. Original magnification, × 100.
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| Figure 6.
Tyrosine phosphorylation of pp125FAK.
Nonadherent or adherent 293 cells stably expressing wild-type
IIb 3 (lanes 1 and 3) or
Tyr143His IIb 3 (lanes 2 and 4) on
fibrinogen were lysed in Triton X-100 buffer containing sodium
vanadate. pp125FAK was immunoprecipitated with 1 µg
rabbit polyclonal antibody specific for FAK. Precipitates were
separated on 7.5% SDS-PAGE and transferred to a PVDF membrane.
Phosphotyrosine was detected with monoclonal antibody 4G10. To
monitor the loading of gel lanes, the blots were reprobed with
anti-FAK.
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Clot retraction
We examined the capacity of
Tyr143His IIb 3 and KO mutant
IIb 3 to mediate fibrin clot retraction by
using stable transfectants. This process reflects the capacity of
IIb 3 to transduce mechanical force
between the intracellular cytoskeleton and the extracellular fibrin
strands. Parent 293 cells failed to retract fibrin clots. Given that
v 3 is involved in mediating fibrin clot
retraction,37 we preincubated all stable transfectants
with 50 µM cyclo(RGDfV) to block the v 3
function. Under these conditions, 293 cells stably transfected with
wild-type 3 alone failed to mediate fibrin clot
retraction, whereas those with wild-type IIb and
3 showed a marked retraction. Although 293 cells stably
expressing Tyr143His IIb 3 showed some
ability for fibrin clot retraction (Figure
7), 293 cells stably expressing KO mutant
IIb 3 failed to mediate fibrin clot
retraction.

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| Figure 7.
Fibrin clot retraction mediated by the cells stably
expressing mutant IIb 3.
293 cells (control), 293 cells stably expressing wild-type
3 alone, wild-type IIb 3,
Tyr143His IIb 3, or KO mutant
IIb 3 suspended in Tyrode/HEPES buffer
(2 × 106 cells/mL) were incubated with 10 mM tranexamic
acid, 250 µg/mL fibrinogen, and 2 mM CaCl2 at 37°C in a
siliconized glass tube in the presence of 50 µM cyclo(RGDfV)
( v 3-specific antagonist). Then 1 U
thrombin was added to 1 mL cell suspension. Clot retraction was
monitored by digital photography every 30 minutes. Data were expressed
as % clot retraction = [(area t0 area
t)/area t0] × 100. Results
are representative of 3 separate experiments.
|
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 |
Discussion |
The molecular basis for the interaction between
IIb 3 and its ligands remains to be
determined. In this study, we have described a novel missense mutation
(Tyr143His) in IIb associated with a ligand-binding
defect in this receptor. Because the Tyr143His mutation is located in
the W3 4-1 loop of the -propeller domain of IIb, we
compared the effects of Tyr143His IIb on the expression and function of IIb 3 with those of
previously described KO and Asp163Ala mutations located in the same
loop.16 Although GT patient analysis demonstrated that the
surface expression level of Tyr143His IIb 3
on platelets from heterozygous Osaka-12 was 36% to 41% of control,
this reduction in the expression resulted mainly from the null
expression of IIb mRNA from the maternal allele. Indeed,
Tyr143His IIb mutation resulted in almost normal IIb 3 expression on 293 cells (91%-102%
of wild-type IIb 3). In sharp contrast,
this mutation abolished the interaction between IIb 3 and its macromolecular ligands OP-G2
mAb, PAC-1 mAb, and fibrinogen. The failure of LIBS expression by FK633
further suggested that Tyr143His IIb 3 has
a binding defect for high-affinity
IIb 3-specific small ligand. The effects
of Tyr143Ala IIb on the expression and function of
IIb 3 were essentially the same as
Tyr143His IIb, suggesting that the presence of Tyr143,
rather than the presence of a mutated residue such as His143, is
critical for IIb 3 function. Interestingly, the defects for soluble ligands are essentially the same
as those induced by KO or Asp163Ala mutation. However, cell adhesion
and fibrin clot retraction assays revealed distinctive features among
Tyr143His IIb 3, KO
IIb 3, and
Asp163Ala IIb 3. Thus, KO
IIb 3 and
Asp163Ala IIb 3 show a more profound
binding defect for immobilized fibrinogen and fibrin strands than
Tyr143His IIb 3, suggesting that Asp163 is
located at or close to the ligand-binding sites in the -propeller
domain of IIb.
Recently described crystal structure of the extracellular
segment of v 3 shows that the
ligand-binding head is formed by a 7-bladed -propeller domain from
v and a I-domain from
3.10 Moreover, there is mounting evidence
that the W3 4-1 loop, W3 2-3 loop, and W4 4-1 loop of the -propeller
domain are important for ligand binding in non-I-domain subunits.39-41 In terms of the W3 4-1 loop of
IIb, several residues critical for ligand binding have
been suggested by alanine-scanning mutagenesis.16,41 In
addition, KO, Pro145Ala, and Tyr143His (this study) are naturally occurring mutations responsible for the ligand-binding defect in
patients with variant GT. Compared with soluble ligands, immobilized fibrinogen and fibrin are activation-independent ligands for
IIb 3. Immobilized fibrinogen binding to
IIb 3 initiates outside-in signaling that
leads to cellular responses such as cell spreading. In addition to
outside-in signaling, fibrin clot retraction requires inside-out
transmission of the contractile forces from intracellular cytoskeleton
to fibrin strand. Much higher concentrations of
IIb 3-specific antagonists are needed to
block cell adhesion to fibrinogen and fibrin clot
retraction.42-44 Thus, these ligands show higher
affinity/avidity for IIb 3 than soluble
fibrinogen. Because some mutations in the W3 4-1 loop, such as
Pro145Ala, impaired IIb 3 biosynthesis and
its function,17 such mutations are likely to affect the conformation of this receptor, including the W3 4-1 loop. Therefore, we
used these higher affinity/avidity ligands to further examine the
residual function of IIb 3 mutants.
Neither the KO mutant nor
Asp163Ala IIb 3-expressing 293 cells
adhered to immobilized fibrinogen or showed fibrin clot retraction. In
contrast, Tyr143His IIb 3-expressing 293 cells showed some ability for fibrin clot retraction and for cell
adhesion to fibrinogen with a reduction in spreading. Consistent with
these findings, the impaired tyrosine phosphorylation of pp125FAK mediated through
IIb 3 was observed in
Tyr143His IIb 3-expressing 293 cells
compared with wild-type IIb 3. These
findings indicate that Tyr143His IIb 3
still has some ligand-binding function. Although a differential
engagement of IIb 3 in fibrin clot
retraction versus aggregation has been suggested,45,46 KO
mutant and Asp163Ala IIb abolished the interaction with
soluble fibrinogen and immobilized fibrin(ogen), as shown in this
study. These findings suggest that the integrin residues involved in
these phenomena, at least in part, overlap. The close location of
Tyr143 with Asp163 suggests that the defect in soluble ligand binding
induced by the Tyr143His IIb mutation is likely the
result of its allosteric effect rather than of a defect in the
ligand-binding site itself.
More recently, the crystal structure of
v 3 with cyclo(RGDfV) has been
described.11 The Arg and Asp side chains exclusively contact the propeller and I-domain, respectively, confirming the
critical roles of these domains in ligand binding. The Arg side chain
inserts into a narrow groove at the top of the propeller domain, formed
primarily by the W3 4-1 loop and the W4 4-1 loop, and the Arg
guanidinium group is held in place by a salt bridge to Asp218 in the W4
4-1 loop and to Asp150 in the W3 4-1 loop.11 However, our
previous data showed that alanine-scanning mutagenesis in the W3 4-1 loop, including Asp150Ala, did not inhibit the ligand-binding function
of v 3; this is not consistent with the
crystal structure data.34 The role of the W3 4-1 loop
appears to be different between non-I-domain subunits. The W3 4-1 loops in IIb and in 3 (Thr162 and Gly163)
appear critical for ligand binding, whereas those in 4
and in v do not.16,34,39,40 Although alanine-scanning mutagenesis suggested several residues within the W3
4-1 and W3 2-3 loops of IIb may be critical for ligand binding, the same procedure showed only Tyr178 in the W3 2-3 loop of
v as a critical residue.34,41 In addition,
our recent study suggests that the expression and function of
IIb 3 are more strictly regulated than
v 3.47 Among non-I-domain
subunits, IIb has the longest W3 4-1 loop, and it is
possible that differences in the structure between IIb
and v may account for the distinctive role of the W3 4-1 loop in ligand binding between these 3 integrins. Unique
features of the W3 4-1 loop of IIb 3 are
also likely to account for the discrepancy between the ability for
soluble ligand binding and cell adhesion. Detailed structure-function
analyses of 3 integrins and of their crystal structure
would provide a better understanding of integrin function and a new
antagonist design for these integrins to prevent thrombosis.
 |
Acknowledgments |
We thank Dr Thomas Kunicki for mAbs AP1, AP2, and AP5; Dr Peter
Newman for mAb AP3 and the IIb and 3 cDNA
cloned into a mammalian expression vector pcDNA3; Dr Sanford Shattil
for mAb PAC-1; Drs. Makoto Handa and Yasuo Ikeda for mAb PT25-2; Dr
Mark Ginsberg for mAbs PMI-1, anti-LIBS1, and anti-LIBS 6; Dr Jiro Seki
for FK633; and Dr P. Raddatz for cyclo(RGDfV).
 |
Footnotes |
Submitted July 18, 2002; accepted December 18, 2002.
Prepublished
online as Blood First Edition Paper, December 27, 2002; DOI
10.1182/blood-2002-07-2144.
Supported in part by a Grant-in Aid for Scientific Research from
the Ministry of Education, Science and Culture in Japan, the Yamanouchi
Foundation for Research on Metabolic Disorders, Tukuba, Japan, and
Mitsubishi Pharma Research Foundation, Osaka, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Yoshiaki Tomiyama, Department of Internal
Medicine and Molecular Science, Graduate School of Medicine B5, Osaka
University, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan; e-mail:
yoshi{at}hp-blood.med.osaka-u.ac.jp.
 |
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