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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3260-3267
Adhesive and Signaling Properties of a Naturally Occurring Allele
of Glycoprotein IIIa With an Amino Acid Substitution Within the
Ligand Binding Domain The Pena/Penb
Platelet Alloantigenic Epitopes
By
Ronggang Wang and
Peter J. Newman
From the Blood Research Institute, The Blood Center
of Southeastern Wisconsin, Milwaukee; and the Departments
of Cellular Biology and Pharmacology, Medical College of
Wisconsin, Milwaukee, WI.
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ABSTRACT |
Platelet membrane glycoprotein IIIa (GPIIIa) is the most polymorphic
integrin subunit in man, with at least seven recognized allelic
isoforms present in the human gene pool. Whether these allelic variants
of the GPIIb-IIIa complex differ in the ability to interact with the
adhesive ligand fibrinogen (Fg) is still unknown. Since the
Pena and Penb allelic forms of GPIIIa are
distinguished by a single Arg143Gln amino acid substitution within the
RGD binding domain of GPIIIa and anti-Pena human
alloantibodies have been shown to bind GPIIb-IIIa on the platelet
surface and inhibit ADP-induced platelet aggregation, we expressed both
forms of this integrin in Chinese hamster ovary (CHO)
cells and examined the relative adhesive properties. Both allelic forms
of GPIIb-IIIa were expressed on the cell surface and were recognized by
a well-characterized panel of murine and human monoclonal and
polyclonal antibodies. Like Pena, the Penb form
of GPIIb-IIIa could undergo conformational changes in response to RGD
peptide binding, and could be induced by activating antibodies to bind
Fg and the Fg mimetic antibody P1-55. The binding affinity for Fg of
the Pena form of the GPIIb-IIIa complex was not
significantly different from that of the Penb form, nor was
its ability to signal to focal adhesion kinase, suggesting that
Arg143Gln polymorphism has little or no effect on integrin function.
Examination of the functional consequences of other integrin
polymorphisms may be necessary to determine whether they constitute a
risk factor for thrombosis or hemorrhage.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE INTEGRINS are a family of
heterodimeric cell surface receptors that mediate cell-cell and
cell-matrix interactions.1 A prototype integrin, platelet
glycoprotein (GP)IIb-IIIa ( IIb 3), is
essential for normal hemostasis and plays a central role in platelet
aggregation by binding to adhesive ligands such as fibrinogen (Fg) and
von Willebrand factor. The GPIIb-IIIa complex is restricted to
platelets and is the most abundant member of the integrin family found
on the platelet surface, with 80,000 molecules per
platelet.2 Mature GPIIIa is a single-chain polypeptide
composed of 762 amino acids.3-5 GPIIIa contains 56 cysteine
residues in highly conserved locations within the extracellular domain
of the molecule, all of which are normally disulfide-linked to form 28 nonconsecutive disulfide bridges. Two large loops extending from amino
acids Cys5-Cys435 and
Cys405-Cys655 have been proposed by Calvete et al.6 Mature GPIIb has 1,008 amino acids containing nine
disulfide bonds formed by 18 cysteines,7 and exists as a
two-chain molecule consisting of a heavy and a light chain with a
molecular mass of 125 and 22 kD, respectively.
In addition to their physiologic role, both GPIIb and GPIIIa are known
to bear a number of clinically important alloantigenic determinants
that can induce an alloimmune response in two immunopathologic syndromes, posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenic purpura (NATP).8 These antigenic
determinants are mostly the result of single amino acid substitutions
that result in subtle conformational changes in the platelet membrane GP that bears them.
Although the PlA1/PlA2 (Leu33Pro) polymorphism
of GPIIIa is by far the one most often associated with development of
PTP and NATP, the Pena and Penb antigenic
epitopes, also found on GPIIIa,9 may be a more frequent cause of both alloimmune disorders in individuals of Asian descent. NATP, due to anti-Pen alloantibodies, is particularly severe. Moreover,
human anti-Pena alloantibodies have been shown to bind to
the surface of platelets and inhibit ADP-induced platelet aggregation.
Taking these clues, we used RNA-polymerase chain reaction (PCR) to
amplify the region of GPIIIa encoding the ligand binding domain of
GPIIIa (amino acids 109 to 171) as identified by RGD cross-linking
experiments,10 and found an Arg143Gln polymorphism that was
responsible for formation of the Pena and Penb
alloantigenic epitopes.11 Interestingly, this amino acid
substitution is only 24 residues away from an Asp119Tyr mutation that
abolishes ligand binding and results in a variant form of Glanzmann
thrombasthenia.12 Mutations of oxygenated residues within
this binding domain of GPIIIa have also been shown to affect ligand
binding function.13
Albeit still controversial, recent reports on the association of GPIIIa
polymorphisms with the development of coronary disease suggest
potential functional consequences of these platelet adhesive molecule
polymorphisms.14 However, the relative paucity of
information regarding the adhesive properties of these allelic forms of
GPIIb-IIIa has left open the important issue of whether the GP
polymorphisms are merely associated with or actually contribute to the
increased risk of coronary disease in individuals that inherit them.
Since the Pen polymorphism is located at GPIIIa amino acid residue 143 within the previously defined ligand binding site and anti-Pen antibodies bind to GPIIIa and block platelet aggregation, it is reasonable to hypothesize that changes in this region might have the
potential to change the shape of the ligand binding pocket and thereby
positively or negatively affect the affinity of the adhesive receptor
for its ligand. Because individuals homozygous for the Penb
form of GPIIIa are generally not available for study, we have produced
Chinese hamster ovary (CHO) cell lines stably transfected with either
the Pena (Arg143) or Penb
(Gln143) forms of the GPIIb-IIIa complex, and examined
the effects of this amino acid substitution on cell surface expression,
signal transduction, and ligand binding properties.
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MATERIALS AND METHODS |
Monoclonal antibodies and peptides.
The monoclonal antibody Tab specific for GPIIb15 was kindly
provided by Dr Rodger McEver (University of Oklahoma, Oklahoma City,
OK). The GPIIb-IIIa complex-specific antibody
AP2,16 which inhibits Fg binding to GPIIb-IIIa, and
AP3,17 specific for GPIIIa, were produced at the Blood
Research Institute (BRI) Hybrodoma Core Laboratory and purified by
affinity chromatography. LIBS antibody D3,18 kindly
provided by Dr Lisa Jennings (University of Memphis, Memphis, TN), has
been mapped to a GPIIIa epitope at residue 422-692.19
Pl-55, an activation-dependent antibody,20 was provided by
Dr Beat Steiner (Hoffmann-La Roche Ltd, Basel, Switzerland).
FITC-conjugated Pl-55 was labeled with FITC as follows: 1 mg Pl-55 in
0.1 mol/L sodium bicarbonate buffer, pH 9.0, was mixed with 2 mg
FITC-Celite (Molecular Probes, Eugene, OR) in the dark at room
temperature. After a 30-minute incubation, the reaction was stopped by
adding 1 mol/L NH4Cl, and the mixture was dialyzed against
phosphate-buffered saline (PBS). RGDW and RGEW peptides were
synthesized and purified by high-performance liquid chromatography in
the BRI Protein Core Laboratory. The mass of all peptides was verified
by mass spectrometry before use.
Generation of stable cell lines expressing allelic forms of
GPIIb-IIIa in CHO cells.
Allele-specific recombinant forms of GPIIIa cDNA were produced as
described previously,11 and subcloned into the
EcoRI cloning site of the mammalian expression vector EMC-3
containing a methotrexate resistance gene (generously provided by Dr
Glenn Larsen, Genetics Institute, Cambridge, MA) or pcDNA3, which
contains a neomycin resistance gene (Invitrogen, San Diego, CA).
Wild-type full-length GPIIb cDNA was cloned into the EcoRI site
of the same two vectors. Cotransfection of CHO cells with the GPIIb
cDNA construct and either the Pena (Arg143)
or Penb (Gln143) form of GPIIIa cDNA was
performed using the calcium phosphate method.21 Forty-eight
hours after transfection, cells were subjected to drug selection in
media containing 500 to 600 µg/mL G418 (geneticin; GIBCO,
Gaithersburg, MD) or minimum essential media without
ribonucleotides and deoxyribonucleotides, depending on the vector used,
for 2 weeks. Resistant colonies were isolated with a cloning cylinder
and assayed for expression of GPIIb-IIIa by flow cytometry using a
variety of monoclonal antibodies against single subunits or the intact
complex. To obtain a homogeneous population, cells were sorted in a
FACStar (Becton Dickinson, San Jose, CA) using the complex-specific
antibody AP2 and maintained in the same selection media.
Flow cytometric analysis.
Cultured cells were harvested by adding 3.5 mmol/L EDTA and 0.01%
trypsin for 2 to 3 minutes. Trypsin was neutralized by adding serum-containing media, and the cells were washed twice in D-PBS (GIBCO). Cells (3 to 5 × 105) in PBS containing 0.5%
bovine serum albumin (BSA) and 0.01% NaN3 were incubated
with 20 µg/mL AP3, Tab, AP2, or an isotype control antibody for 1 hour at room temperature. The cells were then washed with PBS twice and
incubated with a FITC-conjugated F(ab )2 fragment of goat
anti-mouse IgG for 30 minutes. The cells were washed, resuspended in
PBS, and assayed with a FACS-scan (Becton Dickinson). Binding of the
LIBS antibody D3 in the absence or presence of RGDW or RGEW peptides
was assessed as already described. To determine the conformational
state of the recombinant GPIIb-IIIa in CHO cells, binding of
FITC-conjugated Pl-55 was measured in the absence or presence of the
LIBS antibody D3. A non-LIBS antibody, AP3, was used as a negative
control. Nonspecific binding of FITC-Pl-55 to the recombinant
GPIIb-IIIa was defined by measuring the mean fluorescence intensity in
the presence of RGDW peptide.
Immunoprecipitation analysis of cell lysates from transfected CHO
cells.
Stably transfected or nontransfected CHO cells were washed twice with
PBS. After washing, the cells were surface-labeled with 5 mmol/L
NHS-LC-biotin in PBS for 30 minutes at room temperature and solubilized
for 30 minutes on ice in lysis buffer containing 20 mmol/L Tris, 100 mmol/L NaCl, 1% Triton X-100, 2mmol/L PMSF, and 100 µg/mL leupeptin.
The supernatant was obtained by centrifugation at 15,000g for
30 minutes at 4°C. Aliquots of labeled cell extracts were incubated
overnight at 4°C with a variety of monoclonal antibodies, as well as
human alloantibodies. Rabbit anti-mouse IgG was added to tubes
containing mouse IgG, and the immune complexes were recovered with
protein A Sepharose beads (Pharmacia, Uppsala, Sweden). The beads were
washed five times with lysis buffer and resuspended in reducing sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer. The samples were separated on 7% SDS-PAGE and transferred to
polyvinylidene difluoride (PVDF) membranes. The membranes were blocked
with 3% BSA in Tris-buffered saline (TBS) for 1 hour at room
temperature. After washing, the membrane was incubated for 1 hour with
a 1:4,000 dilution of streptavidin conjugated to horseradish peroxidase
(HRP), washed, and detected by ECL as described
previously.22
Cell adhesion assays.
Ninety-six-well plates (Immunlon 2; Dynatech, Chantilly, VA) were
coated with 0.1 mL PBS containing different concentrations of Fg, 10 µg/mL AP2, or 1% BSA at 4°C overnight and blocked with 1% BSA in
PBS at room temperature for 1 hour before use. Transfected CHO cells
were harvested as already described. Cells were washed, resuspended in
media, and labeled with 2 µmol/L calcein-AM (Molecular Probes) at
37°C for 30 minutes. The cells were then washed and resuspended in
serum-free media. One hundred microliters of cell suspension
(2 × 105 cells) was added to each well. Cells were then
allowed to adhere for 1 hour at 37°C. Following incubation, the
plates were inverted and flicked to remove nonadherent cells. Excess
liquid was blotted on paper towels. The wells were washed three times
with media containing 1% BSA. Finally, 200 µL wash media was added
to the wells, and the plates were read in a microplate fluorescence
reader (CytoFluor II; PerSeptive Biosystem, Bedford, MA) at an
excitation wavelength of 485 nm and an emission wavelength of 530 nm.
To assess the effects of antibody on cell adhesion, cells were
pretreated with AP2 or an anti-PECAM-1 antibody at 20 µg/mL at room
temperature for 30 minutes. The cells were then washed before plating
onto coated wells.
Tyrosine phosphorylation of pp125FAK.
Tissue culture plates (100-mm) were coated overnight at 4°C with
either Fg (100 µg/mL) or BSA (10 mg/mL). All plates were blocked with
1% BSA for 2 hours at room temperature. Transfected CHO cells in
serum-free media were added to these plates and incubated at 37°C for
60 minutes. After washing, adherent cells were lysed in RIPA buffer
containing 1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, 1 mmol/L
EGTA, 1 mmol/L Na3VO4, 0.5% Nonidet P40, 1%
sodium deoxycholate, 2 mmol/L PMSF, and 10 µg/mL each of aprotinin and leupeptin. Lysates were clarified by centrifugation in a microfuge at 15,000g at 4°C for 30 minutes, and protein content was
quantified using the BCA protein assay (Pierce Chemical Co, Rockford,
IL). Equal amounts of protein from each lysate (500 µg) were
immunoprecipitated with 4 µg polyclonal anti-pp125FAK
antibody (Upstate Biotechnology) overnight at 4°C. The tyrosine phosphorylation state of pp125FAK was determined using
HRP-conjugated PY20 as described previously.23
Fg labeling and binding to transfected CHO cells.
Fg was labeled with FITC as described for antibody labeling. After
dialysis against PBS to remove free FITC, the concentration of labeled
Fg was determined using a BCA reagent kit (Pierce). The clottability of
labeled Fg was greater than 93% as determined by measuring the OD at
280 and 320 nm after Fg was incubated with human thrombin (1 U/mL) for
2 hours at 37°C. FITC-Fg (100 µg/mL) was incubated with transfected
CHO cells in the presence of D3 for the times indicated. Then, aliquots
of cells were analyzed in a FACScan.
Fg was also labeled with radioactive iodine using Iodo beads (Pierce)
according to the manufacturer's directions. Briefly, 5 mg Fg (peak I;
kindly provided by Dr M. Mosesson, Mount Sinai Medical Center,
Milwaukee, WI) was labeled with 0.3 mCi carrier-free sodium
125I (Amersham Corp) using two Iodo beads per tube at room
temperature for 10 minutes. Following iodination, labeled Fg was
separated from free iodine by chromatography using Sephadex G-50 (PD10
column; Pharmacia Biotech, Uppsala, Sweden) equilibrated in Hanks'
buffered salt solution (HBSS). The specific activity of Fg used in the binding assays was approximately 50 to 80 dpm/ng. The clottability of
labeled Fg was greater than 95% at 37°C in the presence of human
thrombin (1 U/mL). The concentration of labeled Fg was determined spectrophotometrically at 280 nm assuming an extinction coefficient of
1.51 and a molecular mass of 340 kD. The labeled proteins were stored
at  20°C until use. Transfected CHO cells were prepared as
described earlier and suspended in HBSS buffer containing 2 mmol/L
Ca2+, 1 mmol/L Mg2+, and 4 mg/mL BSA. Fg
binding experiments were performed in triplicate in presiliconized
1.5-mL polypropylene tubes (Denville Scientific, Denville, NJ). The
amount of antibody D3 used for activating GPIIb-IIIa was determined by
preliminary studies, and the optimal concentration was 150 to 200 µg/mL. Cells (1 × 106) were preincubated with D3
(200 µg/mL) at room temperature for 30 minutes with occasional
shaking. Increasing concentrations of 125I-labeled Fg were
then added and incubated for 45 minutes in a total volume of 150 µL.
After incubation, 40 µL of the reaction mixture was removed and
layered onto 400 µL 20% sucrose in siliconized 1.5-mL polypropylene
tubes, and cell-bound Fg was separated from free Fg by centrifugation
at 15,000g for 3 minutes. After careful aspiration of the
supernatant, the tip of each tube was cut off, and the bound
radioactivity was determined using a gamma counter. For nonspecific
binding, 3 mmol/L RGDW peptide and/or 10 mmol/L EDTA were added
to the tubes with labeled fibrinogen Fg. In some cases, cells without
D3 treatment were used to determine nonspecific binding. Specific
binding was determined by subtracting nonspecific binding from total
ligand binding. Specific binding isotherms were generated by plotting
the specific binding of 125I-Fg to transfected CHO cells
versus the 125I-Fg concentration using the GraphPad PRIZM
program (Graph Pad Software Inc, San Diego, CA).
Dissociation constants (kd) were determined from Scatchard plots using
standard linear regression.
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RESULTS |
Expression of the Pena and Penb allelic forms
of GPIIIa.
Stable cell lines expressing the Pena and Penb
allelic forms of GPIIb-IIIa were produced by cotransfection of GPIIb
cDNA with either Pena (Arg143) or
Penb (Gln143) GPIIIa cDNA into CHO cells.
Surface-expressed GPIIb-IIIa was examined by immunoprecipitation and by
immunofluorescence flow cytometric analysis using a variety of
anti-GPIIb and anti-GPIIIa antibodies and human alloantibodies specific
for the Pen alloantigenic determinants. Both allelic forms of
GPIIb-IIIa were immunoprecipitated by monoclonal antibodies AP3
(specific for GPIIIa), Tab (specific for GPIIb), and AP2 (specific for
the intact complex) but not by normal mouse IgG, as expected (Fig
1A). Anti-PlA1 human
alloantibodies reacted with GPIIIa independently of Pen phenotype (Fig
1B). In contrast, human anti-Pena alloantisera reacted with
the Arg143 form of GPIIIa but not with the
Gln143 form, and human anti-Penb alloantibody
bound to the Gln143 but not to the Arg143
GPIIIa allele. Indirect immunofluorescence staining analysis using the complex-specific antibody AP2 showed that the GPIIb-IIIa complex was
surface-expressed on both Pena and Penb stable
cell lines (not shown). These data demonstrate that both the
Pena and Penb forms of the GPIIb-IIIa complex
are expressed normally on the cell surface and recognized by
allele-specific anti-Pen alloantibodies.
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| Fig 1.
Immunoprecipitation analysis of biotin-labeled
surface-expressed proteins from transfected CHO cells expressing
Pena (Arg143) or Penb
(Gln143) alloantigens. Stably transfected CHO cells were
surface-labeled with biotin. Cell extracts were immunoprecipitated with
the indicated antibodies. (A) Surface-expressed GPIIb-IIIa was
recognized by different monoclonal antibodies. MoAb AP3 (specific for
GPIIIa, lane 1) and Tab (specific for GPIIb, lane 2) immunoprecipitated
the respective subunit and its associated subunit. AP2 recognized the
intact mature complex (lane 3). Normal mouse IgG was used as a negative
control (lane 4). (B) Surface-expressed GPIIb-IIIa was recognized by
allele-specific alloantibodies. Whereas PlA1 (lane 3)
reacted with GPIIb-IIIa derived from both stable cell lines,
anti-Pena alloantibodies (lane 1) recognized the
Arg143 form of GPIIb-IIIa but not the Gln143
form. Anti-Penb alloantibodies (lane 2) only reacted with
the Gln143 form, as expected. Normal human IgG (lane 4) was
used as a control.
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Adhesive properties of the Pena and Penb
allelic forms of GPIIb-IIIa.
To examine whether the Pena and Penb allelic
forms of GPIIb-IIIa could undergo conformational changes and bind to
ligand in a similar manner, we measured the binding of the LIBS
antibody D3 in the presence and absence of RGD peptides. D3 recognizes a conformation of the integrin complex induced by ligand
binding.18 RGD, but not RGE, peptides induced exposure of
the D3 epitope in both the Pena and Penb
allelic forms of GPIIIa (Fig 2). In
contrast, binding of the non-LIBS antibody AP3 was not affected by
either RGDW or RGEW peptides, as expected. We also measured the binding
of the activation-dependent antibody Pl-55 to GPIIb-IIIa upon induction
with LIBS antibody. Binding of the Pl-55 antibody to both
Pena/CHO and Penb/CHO cells was increased by
the LIBS antibody D3 but not by the non-LIBS antibody AP3 (Fig 2C). The
binding of Pl-55 was specific, as it could be inhibited by RGD
peptides. Thus, the Gln143 form of GPIIb-IIIa expressing
the Penb antigenic determinant is capable of undergoing
conformational changes and binding ligand.
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| Fig 2.
Flow cytometric analysis of surface-expressed allelic
forms of GPIIb-IIIa. (A) Binding of LIBS antibody D3 to
Pena (Arg143)-transfected CHO cells. D3
binding was measured in the presence or absence of RGDW or RGEW
peptides. Whereas RGDW peptides increased the binding of D3 to
transfected CHO cells, RGEW did not. As a control, binding of AP3 to
the cells was also measured. Neither RGDW nor RGEW affected the binding
of AP3. (B) Binding of LIBS antibody D3 to Penb
(Gln143)-transfected CHO cells. D3 binding to
Penb-transfected CHO cells was increased in the presence of
RGDW peptides. AP3 binding was not affected by RGDW peptides. The
number above each histogram represents the mean fluorescence intensity.
(C) Binding of activation-dependent antibody Pl-55. Transfected CHO
cells were incubated with FITC-labeled Pl-55 in the presence of AP3 or
D3 (200 µg/mL) for 30 minutes. The samples were diluted with PBS and
analyzed by a FACScan. Nonspecific binding of the antibody was defined
in the presence of 1 mmol/L RGD peptides. D3 increased the binding of
Pl-55 to both Pena- and Penb-transfected CHO
cells. Pl-55 binding was inhibited by 1 mmol/L RGDW peptides.
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In addition, we examined the ability of the stable CHO cell
transfectants to bind to soluble and immobilized Fg. Soluble Fg bound
to both Pena and Penb CHO cells upon induction
of the ligand binding site with the LIBS antibody D3 (Fig
3A). Cell adhesion to immobilized Fg
mediated by allele-specific forms of this integrin complex was examined using calcein AM-labeled cells. To directly compare the ability of
these cells to adhere to substrates, cell adhesion to immobilized Fg
was normalized to cell adhesion on AP2. Pena- and
Penb-transfected CHO cells showed a similar pattern of
adhesion to immobilized Fg following a 60-minute incubation (Fig 3B).
Similar results were obtained when cells were allowed to adhere for
only 10 or 30 minutes (not shown). Adhesion of these cells to
immobilized Fg was mediated by GPIIb-IIIa, as the complex-specific
antibody AP2 blocked attachment of cells to the matrix (Fig 3C).
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| Fig 3.
Binding of transfected CHO cells to soluble and
immobilized Fg. (A) Binding of transfected CHO cells to FITC-labeled
Fg. Soluble Fg was labeled with FITC. Transfected CHO cells were
incubated for 45 minutes at room temperature with FITC-labeled Fg at a
concentration of 100 µg/mL in the presence or absence of D3 (200 µg/mL) followed by flow cytometry. The left panel shows the binding
of FITC-Fg in the presence or absence of antibody D3. The right panel
shows the surface expression of GPIIb-IIIa complexes as detected by
AP2. (B) Cell adhesion to immobilized Fg ligand. Transfected CHO cells
expressing different allelic forms of GPIIb-IIIa complex were labeled
with calcein-AM at 37°C for 30 minutes. After washing, the cells were
allowed to attach for 60 minutes to wells coated with various
concentrations of Fg, BSA, or anti-GPIIb-IIIa complex antibody at
37°C. After adhesion, nonadherent cells were removed by washing, and
adherent cells were measured in a fluorescence plate reader.
Nonspecific cell adhesion on BSA-coated wells was subtracted. Cell
adhesion was normalized to cell adhesion on AP2 (Fg/AP2 ratio). CHO
cells expressing both allelic forms of GPIIb-IIIa bound to immobilized
Fg in a dose-dependent manner. (C) Adhesion of transfected CHO cells to
immobilized Fg is mediated by GPIIb-IIIa. Cells were pretreated with
AP2 or anti-PECAM-1 antibodies (20 µg/mL) for 30 minutes at room
temperature. After washing, cell adhesion to 2 µg/mL Fg was measured
and plotted by fluorescence intensity. AP2 but not anti-PECAM-1
antibody blocked cell adhesion mediated by both allelic forms of
GPIIb-IIIa to immobilized Fg.
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Determination of affinity constants of the Pena and
Penb allelic forms of the GPIIb-IIIa complex for soluble
Fg.
To quantitatively examine the affinity of these two forms of GPIIb-IIIa
for soluble ligand, we performed binding isotherm analysis using
125I-labeled Fg in the presence of the activating LIBS
antibody D3. Recombinant GPIIb-IIIa expressed on CHO cells or COS-7 is
in the resting state, as these cells cannot bind to soluble Fg, but can be induced into an activated conformation using LIBS
antibodies.24 LIBS antibody-activated GPIIb-IIIa complex
binds to Fg in a manner similar to physiologically activated
GPIIb-IIIa.25 To directly measure the affinity of the
receptor for Fg, we used the LIBS antibody D3 to activate and induce Fg
binding to the GPIIb-IIIa complex. Specific and saturable binding of Fg
to GPIIb-IIIa-transfected CHO cells was reached at a concentration of
2,000 nmol/L (Fig 4). Scatchard plot
analyses of Fg binding to both Pena and Penb
forms of GPIIb-IIIa showed a kd of 400 nmol/L for Pena/CHO
cells and 455 nmol/L for Penb/CHO cells, respectively (Fig
4, inset). Data from five experiments are summarized in Fig 4C. The
Pena and Penb forms of the GPIIb-IIIa complex
have a similar affinity for Fg (P = .78).
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| Fig 4.
Saturation binding isotherm and Scatchard plot analysis
of 125I-Fg binding to D3-activated transfected CHO cells.
Transfected CHO cells expressing (A) Pena or (B)
Penb forms of GPIIb-IIIa were treated with D3 at a
concentration of 200 µg/mL and then incubated with increasing
concentrations of 125I-Fg at room temperature for 45 minutes. Data are plotted as molecules of 125I-Fg
specifically bound versus fibrinogen concentration. Scatchard plot
analyses of the specific binding data are shown in the insets. Data are
replotted according to the Scatchard method using the GraphPad linear
regression program. kd Values for both Pena and
Penb forms of GPIIb-IIIa are 400 and 455 nmol/L,
respectively. (C) Summary of kd values from five different experiments.
kd Values derived from the Scatchard plots are summarized. Both forms
of GPIIb-IIIa bound to Fg with similar affinity, 428 nmol/L for
Pena- and 457 nmol/L for Penb-transfected CHO
cells, respectively (P = .78). Note that there is no
significant difference in affinity for Fg between the
Arg143 (Pena) and Gln143
(Penb) forms of the GPIIb-IIIa complex.
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Signaling properties of the Pena and Penb
allelic forms of GPIIb-IIIa.
Previous studies have shown that adhesion to immobilized ligands of CHO
cells expressing GPIIb-IIIa induces tyrosine phosphorylation of
pp125FAK.26 To examine whether the Pen
polymorphism affects the signaling properties of the integrin, we
measured tyrosine phosphorylation of pp125FAK after cell
adhesion to immobilized Fg. pp125FAK became
tyrosine-phosphorylated in both adherent Pena- and
Penb-transfected CHO cells, but not in nonadherent cells
over a BSA-coated surface (Fig 5). Similar
results were observed when GPIIb-IIIa-transfected CHO cells adhered to
an immobilized monoclonal antibody to GPIIb-IIIa (not shown). Thus, not
only are the adhesive properties of the Pena and
Penb allelic forms of GPIIIa similar, but outside-in
signaling appears to be unaffected by the polymorphism as well.
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| Fig 5.
Tyrosine phosphorylation of pp125FAK
following cell adhesion to immobilized Fg. Adherent CHO cells bound to
Fg-coated wells or nonadherent cells on BSA-coated wells were lysed in
RIPA buffer and immunoprecipitated using an anti-FAK antibody (FAK) or
normal rabbit IgG (NR). Immunoprecipitates were separated on SDS-PAGE,
transferred to a PVDF membrane, and probed with HRP-conjugated PY20 to
assess the tyrosine phosphorylation state of FAK. Note that adhesion of
Pena and Penb transfectants to immobilized Fg
induces similar levels of tyrosine phosphorylation of FAK. The gel is
representative of 3 experiments.
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DISCUSSION |
The GPIIb-IIIa complex is the most abundant protein present on the
platelet surface. As a receptor for Fg and von Willebrand factor, the
GPIIb-IIIa complex plays an important role in platelet aggregation and
adhesion during the process of normal hemostasis and the pathogenesis
of thrombotic diseases. Certain amino acid changes in the receptor
resulting in a loss of either the surface expression or function of the
integrin complex have been implicated in an inherited bleeding
disorder, Glanzmann's thrombasthenia (GT). In contrast, other amino
acid changes create polymorphisms in the receptor, which can be
recognized as alloantigens or autoantigens in an immunopathologic
response. To date, there are currently seven known molecular variants
of GPIIIa in the human gene pool, including Leu33
(PlA1), Pro33
(PlA2),27
Pro33Arg40,28 Gln143
(Penb),11,29 Ala407
(Mo),30 Gln489 (Ca),22 and
Cys636 (Sra).31 Unlike the
dysfunctional GPIIb-IIIa found in GT platelets, these variant forms of
GPIIb-IIIa seem to function normally, as the individuals bearing these
alleles show no known difference in normal hemostasis. However, the
fact that certain antibodies directed against these polymorphisms, for
example, anti-Pena antibodies, are potent inhibitors of
platelet aggregation implies the importance of these regions containing
the polymorphisms in receptor function. Indeed, an Arg143
 Gln143 dimorphism within the RGD binding domain of
GPIIIa has been shown to be responsible for the formation of the
Pena and Penb alloantigenic
epitopes.11
Weiss et al14 reported that individuals bearing the
Pro33 variant form of GPIIIa (PlA2) might be
at increased risk to develop coronary thrombosis, especially individuals under the age of 60. This finding poses an interesting question about the role of receptor polymorphism in integrin function. However, a recent study in a large cohort of apparently healthy men has
indicated that inheritance of the GPIIIa PlA2 allele may
not be associated with an increase in the subsequent risk of myocardial
infarction, stroke, or venous thrombosis.32 Although the
association between the PlA2 allele and coronary heart
disease is still controversial, the functional consequence of the
PlA2 allelic form of GPIIIa in contributing to the
formation of a more adherent Fg receptor remains to be elucidated.
Expression of PlA1 and PlA2 antigens is
controlled by a single point mutation at GPIIIa nucleotide 196, resulting in a Leu33Pro dimorphism at the amino-terminal region of
GPIIIa.27 However, the actual structural and functional differences between the GPs bearing these two antigens have not been
identified.
The purpose of the present investigation was to identify potential
functional differences between the Pena and
Penb forms of GPIIb-IIIa in a controlled mammalian
expression system. Previously, we demonstrated that residue 143 of
GPIIIa is both necessary and sufficient to control expression of the
Pena and Penb epitopes in a single GPIIIa
subunit expression system.11 To determine whether these
epitopes could be expressed in a GPIIb-IIIa complex on the surface of
CHO cells, we established two stable cell lines expressing these two
allelic forms of GPIIb-IIIa by cotransfection of GPIIb with either the
Pena or Penb form of GPIIIa cDNA.
Immunoprecipitation and flow cytometric analysis demonstrated that
Pena and Penb forms of GPIIb-IIIa were
expressed normally on the CHO cell surface and recognized by
alloantibodies specific for each allelic form of GPIIIa (Fig 1).
The Pen polymorphism is located proximal to one of the previously
defined ligand binding domains of the GPIIb-IIIa complex (residues 109 to 171 of GPIIIa), where both an Asp119Tyr substitution and a flanking
Ser162Leu amino acid change have been shown to abolish ligand binding
or affect complex maturation in two GT patients.12,33
Whether the Gln143 form of GPIIIa (Penb) has
an advantage or disadvantage over the Arg143
(Pena) form with respect to affinity for Fg has not been
established. In this study, we examined the function of the
Penb form of GPIIIa in ligand binding and post-ligand
binding events of GPIIb-IIIa. Using a LIBS antibody as a reporter for
conformational changes in the receptor upon ligand occupancy, we found
that the Penb form of GPIIb-IIIa was capable of assuming an
activated conformational state upon ligand binding. We also
demonstrated that the Penb form of GPIIb-IIIa could be
induced into an activated state capable of binding soluble ligand in an
RGD-inhibitable manner. Furthermore, the Penb form of
GPIIb-IIIa could mediate downstream signaling events such as cell
spreading and tyrosine phosphorylation of pp125FAK.
Together, these data suggest that the Penb form of
GPIIb-IIIa is functionally normal compared with the Pena
form.
To examine more precisely the adhesive properties of these two stable
cell lines for soluble Fg, we performed ligand binding isotherm
analysis using radioactively labeled Fg. We found that the binding
affinity for Fg of the Pena form of the GPIIb-IIIa complex
was not significantly different from that of the Penb form,
suggesting that the Arg143Gln polymorphism has little or no effect on
integrin function. This observation is consistent with our previous
finding that platelets derived from a Penb/b homozygous
individual aggregate normally in response to ADP.11
We would have liked to study the adhesive properties of these two
integrin variants using human platelets. Unfortunately, Penb/b homozygous individuals are rare in the Western
hemisphere and difficult to obtain in Asia. Therefore, we expressed the
Pena (Arg143) or Penb
(Gln143) forms of the GPIIb-IIIa complex on CHO cells.
This approach has been widely used for studying integrin function,
including ligand binding and signal transduction. Effects of many
naturally occurring mutations of GPIIb or GPIIIa on integrin function
have been recapitulated in CHO cell lines expressing mutant forms of integrin constructs,23,34-35 despite the fact that a few
differences exist between GPIIb-IIIa expressed on CHO cells versus
naturally presenting cells such as platelets. For example, CHO cell
GPIIb-IIIa is unable to become activated from within the cell and bind
to soluble ligand.24 However, activation of GPIIb-IIIa on
CHO cells can be achieved from outside the cell using activating LIBS
antibody, and the GPIIb-IIIa complex activated in this fashion has been shown to bind Fg in a manner similar to physiologically activated GPIIb-IIIa.25 By using LIBS antibodies as activators, we
examined the adhesive properties of the Pena and
Penb allelic forms of GPIIIa with respect to the ability to
bind to soluble or surface-bound ligands (Figs 3 and 4). Despite the
location of this GPIIIa polymorphism within the ligand binding site, no significant difference in ligand binding was observed between these two
allelic forms of GPIIb-IIIa.
In summary, we have produced and functionally characterized stable cell
lines expressing the Pena and Penb allelic
forms of GPIIb-IIIa in CHO cells. These stable cell lines should
provide useful diagnostic reagents for serologic determination of
platelet alloantigenic epitopes. By direct determination of the ligand
binding affinity of different allelic forms of GPIIb-IIIa for Fg, we
have found that the Arg143Gln polymorphism located proximal to the
important ligand binding site of GPIIIa has little or no effect on
integrin function. The use of this approach should permit other
investigators to directly measure the ligand binding affinity of the
other allelic forms of GPIIb-IIIa, and address the important
mechanistic issue of whether molecular polymorphisms affect integrin
function and subsequently contribute to the development of coronary
artery disease.
| |
FOOTNOTES |
Submitted November 6, 1997;
accepted June 25, 1998.
Supported in part by National Institutes of Health Grant No. HL-44612
(P.J.N.) and American Heart Association (Wisconsin Affiliate) Predoctoral Fellowship Award No. 95F-Pre-16 (R.W.).
Presented in abstract form at the 38th Annual Meeting of the American
Society of Hematology, Orlando, FL, December 6-10, 1996.
Address reprint requests to Ronggang Wang, MD, PhD, Blood Research
Institute, The Blood Center of Southeastern Wisconsin, 638 N 18th St,
PO Box 2178, Milwaukee, WI 53201-2178; e-mail: rwang{at}smtpgate.bcsew.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Lisa Jennings and Dr Beat Steiner for the generous gift of
monoclonal antibodies D3 and P1-55, and Dr Mike Mosesson for the
generous supply of peak I Fg.
| |
REFERENCES |
1.
Hynes RO:
Integrins: Versatility, modulation, and signaling in cell adhesion.
Cell
69:11,
1992[Medline]
[Order article via Infotrieve]
2.
Wagner CL,
Mascelli MA,
Neblock DS,
Weisman HF,
Coller BS,
Jordan RE:
Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets.
Blood
88:907,
1996[Abstract/Free Full Text]
3.
Zimrin AB,
Eisman R,
Vilaire G,
Schwartz E,
Bennett JS,
Poncz M:
Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.
J Clin Invest
81:1470,
1988
4.
Fitzgerald LA,
Steiner B,
Rall SC Jr,
Lo SS,
Phillips DR:
Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin."
J Biol Chem
262:3936,
1987[Abstract/Free Full Text]
5.
Rosa JP,
Bray PF,
Gayet O,
Johnston GI,
Cook RG,
Jackson KW,
Shuman MA,
McEver RP:
Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17.
Blood
72:593,
1988[Abstract/Free Full Text]
6.
Calvete JJ,
Henschen A,
Gonzalez-Rodriguez J:
Assignment of disulfide bonds in human platelet GPIIIa. A disulfide pattern for the beta-subunits of the integrin family.
Biochem J
274:63,
1991
7.
Poncz M,
Eisman R,
Heidenreich R,
Silver SM,
Vilaire G,
Surrey S,
Schwartz E,
Bennett JS:
Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors.
J Biol Chem
262:8476,
1987[Abstract/Free Full Text]
8.
Aster RH:
Alloimmune thrombocytopenias
, in Loscalzo J,
Schafer AI
(eds):
Thrombosis and Hemorrhage.
Boston, MA, Blackwell Scientific
, 1994
, p 529
9.
Furihata K,
Nugent DJ,
Bissonette A,
Aster RH,
Kunicki TJ:
On the association of the platelet-specific alloantigen, Pena, with glycoprotein IIIa. Evidence for heterogeneity of glycoprotein IIIa.
J Clin Invest
80:1624,
1987
10.
D'Souza SE,
Ginsberg MH,
Lam SC,
Plow EF:
Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets.
J Biol Chem
263:3943,
1988[Abstract/Free Full Text]
11.
Wang R,
Furihata K,
McFarland JG,
Friedman K,
Aster RH,
Newman PJ:
An amino acid polymorphism within the RGD binding domain of platelet membrane glycoprotein IIIa is responsible for the formation of the Pena/Penb alloantigen system.
J Clin Invest
90:2038,
1992
12.
Loftus JC,
O'Toole TE,
Plow EF,
Glass A,
Frelinger AL,
Ginsberg MH:
A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation.
Science
249:915,
1990[Abstract/Free Full Text]
13.
Bajt ML,
Loftus J:
Mutation of a ligand binding domain of beta 3 integrin. Integral role of oxygenated residues in alpha IIb beta 3 (GPIIb-IIIa) receptor function.
J Biol Chem
269:20913,
1994[Abstract/Free Full Text]
14.
Weiss EJ,
Bray PF,
Tayback M,
Schulman SP,
Kickler TS,
Becker LC,
Weiss JL,
Gerstenblith G,
Goldschmidt-Clermont PJ:
A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis.
N Engl J Med
334:1090,
1996[Abstract/Free Full Text]
15.
McEver RP,
Bennett EM,
Martin MN:
Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies.
J Biol Chem
258:5269,
1983[Abstract/Free Full Text]
16.
Pidard D,
Montgomery RR,
Bennett JS,
Kunicki TJ:
Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets.
J Biol Chem
258:12582,
1983[Abstract/Free Full Text]
17.
Newman PJ,
Allen RW,
Kahn RA,
Kunicki TJ:
Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3.
Blood
65:227,
1985[Abstract/Free Full Text]
18.
Kouns WC,
Wall CD,
White MM,
Fox CF,
Jennings LK:
A conformation-dependent epitope of human platelet glycoprotein IIIa.
J Biol Chem
265:20594,
1990[Abstract/Free Full Text]
19.
Kouns WC,
Newman PJ,
Puckett KJ,
Miller AA,
Wall CD,
Fox CF,
Seyer JM,
Jennings LK:
Further characterization of the loop structure of platelet glycoprotein-IIIa Partial mapping of functionally significant glycoprotein-IIIa epitopes.
Blood
78:3215,
1991[Abstract/Free Full Text]
20.
Steiner B,
Trzeciak A,
Pfenninger G,
Kouns WC:
Peptides derived from a sequence within 3 integrin bind to platelet IIb3 (GPIIb-IIIa) and inhibit ligand binding.
J Biol Chem
268:6870,
1993[Abstract/Free Full Text]
21.
Graham FL,
VanderEb AJ:
A new technique for the assay of infectivity of human adenovirus 5 DNA.
Virology
52:456,
1973[Medline]
[Order article via Infotrieve]
22.
Wang R,
McFarland JG,
Kekomaki R,
Newman PJ:
Amino acid 489 is encoded by a mutational "hot spot" on the 3 integrin chain: The CA/TU human platelet alloantigen system.
Blood
82:3386,
1993[Abstract/Free Full Text]
23.
Wang R,
Shattil SJ,
Ambruso DR,
Newman PJ:
Truncation of the cytoplasmic domain of 3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin IIb3 complex.
J Clin Invest
100:2393,
1997[Medline]
[Order article via Infotrieve]
24.
O'Toole TE,
Loftus JC,
Du XP,
Glass AA,
Ruggeri ZM,
Shattil SJ,
Plow EF,
Ginsberg MH:
Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.
Cell Regul
1:883,
1990[Medline]
[Order article via Infotrieve]
25.
Frelinger AL,
Du XP,
Plow EF,
Ginsberg MH:
Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function.
J Biol Chem
266:17106,
1991[Abstract/Free Full Text]
26.
Leong R,
Hughes PE,
Schwartz MA,
Ginsberg MH,
Shattil SJ:
Integrin signaling: Roles for the cytoplasmic tails of IIb3 in the tyrosine phosphorylation of pp125FAK.
J Cell Sci
108:3817,
1995[Abstract]
27.
Newman PJ,
Derbes RS,
Aster RH:
The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing.
J Clin Invest
83:1778,
1989
28.
Walchshofer S,
Ghali D,
Fink M,
Panzer-Grumayer ER,
Panzer S:
A rare leucine40/arginine40 polymorphism on platelet glycoprotein IIIa is linked to the human platelet antigen 1b.
Vox Sang
67:231,
1994[Medline]
[Order article via Infotrieve]
29.
Wang L,
Juji T,
Shibata Y,
Kuwata S,
Tokunaga K:
Sequence variation of human platelet membrane glycoprotein IIIa associated with the Yuka/Yukb alloantigen system.
Proc Jpn Acad
67:102,
1991
30.
Kuijpers RWAM,
Simsek S,
Faber NM,
Goldschmeding R,
van Wermerkerken RKV,
Von dem Borne AEGK:
Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia.
Blood
81:70,
1993[Abstract/Free Full Text]
31.
Santoso S,
Kalb R,
Kroll H,
Walka M,
Kiefel V,
Mueller-Eckhardt C,
Newman PJ:
A point mutation leads to an unpaired cysteine residue and a molecular weight polymorphism of a functional platelet 3 integrin subunit: The Sra alloantigen system of GPIIIa.
J Biol Chem
269:8439,
1994[Abstract/Free Full Text]
32.
Ridker PM,
Hennekens CH,
Schmitz C,
Stampfer MJ,
Lindpaintner K:
PlA1/A2 polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke, and venous thrombosis.
Lancet
349:385,
1997[Medline]
[Order article via Infotrieve]
33.
Newman PJ,
Weyerbusch-Bottum S,
Visentin GP,
Gidwitz S,
White GC:
Type II Glanzmann thrombasthenia due to a destabilizing amino acid substitution in platelet membrane glycoprotein IIIa.
Thromb Haemost
69:1017a,
1993(abstr)
34.
O'Toole TE,
Katagiri Y,
Faull RJ,
Peter K,
Tamura R,
Quaranta V,
Loftus JC,
Shattil SJ,
Ginsberg MH:
Integrin cytoplasmic domains mediate inside-out signal transduction.
J Cell Biol
124:1047,
1994[Abstract/Free Full Text]
35.
Chen Y-P,
O'Toole TE,
Ylänne J,
Rosa J-P,
Ginsberg MH:
A point mutation in the integrin 3 cytoplasmic domain (S752 P) impairs bidirectional signaling through IIb3 (platelet glycoprotein IIb-IIIa).
Blood
84:1857,
1994[Abstract/Free Full Text]
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Abstract
Introduction
Abstract