From the
The interaction between von Willebrand factor (vWF) and the
platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for
platelet adhesion at sites of vascular injury under high shear stress
flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms
of platelet activation through its high affinity binding of
Glycoprotein (GP)
Previous studies with
synthetic peptides
(3) have led to the conclusion that GP
Ib
With this information as a background, we
have employed site-directed mutagenesis to verify the effect of
selected amino acid substitutions on the ligand binding activity of the
recombinant GP Ib
Antibody
LJ-Ib
The results presented here demonstrate that the three
tyrosine residues at position 276, 278, and 279 of mature GP Ib
The
mutation of Tyr residues to Phe is not likely to cause any major
rearrangement in the polypeptide backbone, since the two amino acids,
apart from the presence of the hydroxyl group in the Tyr side chain,
have essentially identical structure. In fact, normal reactivity with
the conformation sensitive antibody, LJ-Ib1, which fails to react with
denatured GP Ib
The potential
functional relevance of Tyr sulfation in GP Ib
In addition to decreased vWF
binding, the recombinant GP Ib
Pronounced loss of vWF binding function
resulted from the concurrent substitution of three negatively charged
Asp residues with noncharged Asn at positions 272, 274, and 277 of
mature GP Ib
The
conclusions discussed here are based on data obtained with a
recombinant fragment comprising residues 1-302 of mature GP
Ib
We thank Dr. John W. Fenton II for the generous gift
of human
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-thrombin. There are two distinct but partially overlapping
regions of GP Ib
thought to be involved in interacting with vWF
(residues 251-279) and
-thrombin (residues 271-284);
they share three tyrosine residues (positions 276, 278, and 279) that
have recently been shown to be sulfated (Dong, J., Li, C. Q., and
Lopez, J. A. (1994) Biochemistry 33, 13946-13953). To
define the functional role of these three residues, we have introduced
selected mutations in a soluble recombinant GP Ib
fragment
(corresponding to the sequence 1-302 of the mature protein) that
binds vWF and
-thrombin with the same attributes as intact GP
Ib-IX-V complex. Fragments containing a single Tyr
Phe
substitution either at position 276 or 278 or 279 exhibited normal
interaction with vWF but markedly reduced or absent binding of
-thrombin. GP Ib
fragment with normal sequence but
synthesized under sulfate-free conditions also failed to bind
-thrombin and, in addition, had markedly reduced interaction with
vWF. The simultaneous substitution of three neighboring Asp residues
with Asn at positions 272, 274, and 277, a multiple mutation that may
impair Tyr sulfation, also resulted in loss of binding of both ligands.
These results define distinct structural features of GP Ib
selectively involved in supporting the interaction with vWF or
-thrombin.
(
)
Ib, composed of the
disulfide-linked
and
chains, associates with GP IX
(1) and GP V
(2) to form a noncovalent hetero-oligomeric
complex expressed on the platelet membrane. The amino-terminal
extracytoplasmic domain of GP Ib
contains binding sites for the
adhesive protein, von Willebrand factor (vWF)
(3) , and the
platelet agonist,
-thrombin
(4) , thus supporting two
interactions potentially relevant for normal hemostasis as well as the
development of pathological thrombosis. The amino-terminal domain of GP
Ib
has been expressed in Chinese hamster ovary (CHO) cells as an
isolated soluble fragment and, with regard to the interaction with vWF,
is known to possess the same binding specificity and affinity of the
intact platelet receptor
(5) .
residues within the sequence 251-279 are involved in vWF
binding, a fact confirmed subsequently by site-directed mutagenesis
experiments highlighting the activity of a cluster of negatively
charged residues located between Asp
and Asp
(5) . Moreover, residues within the sequence 271-284
have been shown to participate in the binding of
-thrombin
(6) . These results suggest that the GP Ib
sites
interacting with vWF and
-thrombin may overlap, but additional
experimental evidence indicates that each has unique features. Indeed,
anti-GP Ib
monoclonal antibodies can selectively inhibit binding
of one or the other ligand
(4, 7) ; and a single GP
Ib
point mutation identified in a patient with Bernard-Soulier
syndrome impairs vWF but not
-thrombin binding
(8, 9) .
amino-terminal domain. In particular, we have
focused on three contiguous Tyr residues (positions 276, 278, 279)
that, as mentioned above, are shared by synthetic peptides that inhibit
either vWF or
-thrombin binding to platelets. These Tyr residues
meet the consensus criteria for the occurrence of post-translational
sulfation
(10, 11) and, indeed, have recently been
shown to be sulfated when expressed in eukaryotic cells
(12) .
The results presented here provide initial information on the
distinctive structural features that characterize the two GP Ib
sites interacting with vWF and
-thrombin.
Recombinant Expression of the Amino-terminal Domain of
GP Ib
The characterization of a recombinant plasmid
directing the synthesis of the amino-terminal domain of GP Ib has
been described in detail elsewhere
(5, 9) . Briefly, a
mammalian cell expression plasmid was constructed from a fragment of
the GP Ib
gene (coding for the signal peptide and mature residues
His
-Ala
) synthesized in a polymerase
chain reaction that added BamHI restriction sites on the ends
of the amplified fragment. This was cloned into M13mp19 as a
BamHI fragment and sequenced to verify that no spontaneous
mutations had arisen during the polymerase chain reaction. When
indicated, mutations within the expression plasmid were constructed
using the appropriate oligonucleotides and site-directed mutagenesis on
uracil-containing templates of the original M13 construct
(13) .
The DNA sequence of all mutants was verified to ensure that it
corresponded to the predicted one. The GP Ib
insert was removed
from the M13 construct by digestion with EcoRI and
XbaI and cloned into the corresponding restriction sites of
the polylinker region of pBS/KS
(Stratagene). As a
result of this, restriction sites for XhoI (5` to the GP
Ib
-initiating Met codon) and NotI (3` to the Ala
codon) were acquired from the vector and were used to clone the
fragment into the mammalian cell expression plasmid
pCDM8
. The latter is identical to pCDM8
(14) except that it contains a neomycin gene for conferring
resistance to the aminoglycoside antibiotic, G418, or Geneticin
(Sigma). CHO-K1 cells were grown in 5% CO
in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
0.5 m
M non-essential amino acids, 2 m
M
L-glutamine (Whittaker Bioproducts) and 10% fetal calf serum
(10%-DMEM). DNA (10 µg/dish) was introduced into cells (subcultured
at a density of 1.5
10
per 60-mm dish 24 h prior to
transfection) using a calcium phosphate-mediated transfection procedure
(15) ; transfected cells were then maintained in 10%-DMEM.
Medium to be used as a source of soluble recombinant GP Ib
fragment was collected from confluent cultures of cells grown for 24 h
in the absence of fetal calf serum. A sulfate-free recombinant GP
Ib
fragment was obtained from cells grown for 24 h in minimum
essential medium (MEM) prepared from stock solutions devoid of sulfate
ions (2.5 m
M Hepes, 1.35 m
M CaCl
, 5.4
m
M KCl, 116 m
M NaCl, 1 m
M
NaH
PO
, 0.5 m
M MgCl
, 26
m
M NaHCO
, 5 m
M
D-glucose, 20
m
M sodium pyruvate, pH 7.4); the medium also contained MEM
amino acid solution (Life Technologies, Inc.) supplemented with 4
m
M
L-glutamine, non-essential amino acids
(Whittaker), MEM vitamin solution (Life Technologies, Inc.), and 10
mg/ml phenol red solution as an indicator of pH. Moreover, the
sulfate-free medium contained a final concentration of 5 m
M
NaClO
, an inhibitor of protein sulfation
(16) .
Monoclonal Anti-GP Ib
The
production and characterization of anti-GP Ib Antibodies
monoclonal
antibodies have been described elsewhere
(7, 17, 18) . As demonstrated previously using
proteolytic fragments of GP Ib
generated by trypsin or
Serratia marcescens protease
(7) , antibodies LJ-Ib1
and LJ-P3 interact with distinct epitopes present only in native GP
Ib
and located between residues 1 and 290; in contrast, antibody
LJ-Ib10 interacts with an epitope not affected by denaturation with SDS
nor reduction of disulfide bonds and located between residues 238 and
290. Using the same methods, we have also found
(
)
that antibody LJ-Ib
1 recognizes an epitope not
affected by denaturation with SDS nor reduction of disulfide bonds and
located within residues 1-237, and antibody LJ-P19 reacts with an
epitope present only in native GP Ib
and located within residues
1-290. Monoclonal antibodies (all IgG
k) were
purified by binding to protein A-Sepharose, as described previously
(19) . When indicated, purified IgG were radiolabeled with
I using the IODO-GEN procedure
(20) .
Evaluation of Immunochemical Reactivity
The
nondenaturing dot-blot technique employed for analysis of antibody
binding to different recombinant GP Ib fragments has been
described in detail in a previous publication
(5) . In brief,
recombinant fragment was immobilized onto nitrocellulose membrane (0.45
µm pore size; Bio-Rad) by filtering culture medium through the
membrane with a peristaltic pump applied to a device (ELIFA, Pierce)
that delimits a circular area of application
(5) ; filtration
time was 5 min for each 200 µl of medium applied. The membrane was
then removed from the apparatus, and its protein binding capacity was
blocked with Blotto solution
(21) , prepared with 50 g/liter
fat-free dry milk and 0.5 ml/liter of Antifoam A emulsion (Sigma); the
membrane was then incubated with the appropriate specific antibody,
washed with Blotto solution, and finally incubated with
I-labeled rabbit anti-mouse IgG. Positive reactivity was
detected by autoradiography; a quantitative estimate of bound antibody
was obtained by counting in a
-scintillation spectrometer the
radioactivity associated with each dot cut out from the nitrocellulose
membrane.
vWF Binding to Immobilized Recombinant GP Ib
vWF was purified and characterized as reported
previously
(22) ; it was radiolabeled with
Fragments
I using
the IODO-GEN procedure
(20) . Serum-free culture medium from
cell lines to be tested was used as such or diluted with the
appropriate amount of serum-free medium from non transfected CHO-K1
cells to obtain comparable concentrations of different recombinant
fragments, as judged by immunochemical dot-blot analysis with antibody
LJ-Ib
1. Proteins in culture medium were immobilized onto
nitrocellulose membrane by filtering 100-800 µl through the
ELIFA apparatus, as described above; this and all subsequent steps of
the assay were performed by filtering reagents through the same device
at room temperature (22-25 °C). Filtration time was 5 min for
each 200 µl of medium applied, with variation between different
samples not greater than 30 s. It should be noted that the
immunoadsorption procedure used to measure the interaction of
-thrombin with recombinant GP Ib
fragments (see below) was
not used for vWF, because it gave lower levels of specific binding,
presumably as a result of steric hindrance caused by the immobilizing
antibody. Following insolubilization of the proteins contained in the
culture media, the membrane was blocked with three washes (200 µl
each; filtration time: 3 min) of Hepes buffer containing 1% bovine
serum albumin and 1% bovine
-globulin (Sigma), the latter added to
reduce nonspecific binding. After blocking, 50 µl of
I-labeled vWF, preincubated for 20 min with botrocetin or
immediately mixed with ristocetin as modulators of binding
(5) ,
was filtered through the nitrocellulose membrane in 5 min. When
indicated, appropriate anti-GP Ib
monoclonal antibodies or
unlabeled vWF were also present in these mixtures. Ristocetin (Sigma)
was used at the final concentration of 1 mg/ml; botrocetin (two-chain
form) was purified as described previously in detail
(23, 24) from the crude venom of Bothrops jararaca (Sigma)
and was used at the final concentration of 4 µg/ml. The membrane
was then washed two additional times with blocking solution (200 µl
each wash; filtration time: 3 min); in the assays employing the
modulator ristocetin, the latter was also present during this step at
the same concentration used with vWF. Finally, the membrane was dried,
the spots corresponding to each application well were cut out, and the
bound radioactivity was measured in a
-scintillation spectrometer.
-Thrombin Binding to Immobilized Recombinant GP
Ib
Fragments
-Thrombin, a generous gift of Dr. John W.
Fenton II (Wadsworth Center for Laboratory and Research and Departments
of Physiology and Biochemistry, Albany Medical College, Union
University, Albany, NY), was purified and characterized as described
previously
(25) ; it was radiolabeled with
I using
the IODO-GEN technique
(20) , as reported in detail in a
previous publication
(4) . The binding assay was performed using
recombinant GP Ib
fragment insolubilized onto Sepharose beads by
binding to the anti-GP Ib
monoclonal antibody, LJ-P3, covalently
coupled to the beads; the antibody was selected for this application,
because it has no inhibitory effect on
-thrombin binding to
platelets. As determined in preliminary experiments not shown here,
this method gave considerably lower nonspecific binding of
-thrombin than the one, described above for vWF, performed with
recombinant fragment immobilized directly onto nitrocellulose. Purified
IgG of LJ-P3 was coupled to cyanogen bromide-activated Sepharose CL-4B
at a density of 4 mg/ml of packed beads. The LJ-P3/Sepharose beads were
washed twice with a buffer composed of 0.1
M Tris-HCl, pH 7.3,
containing 0.5
M LiCl and 1 m
M EDTA, and then
incubated with culture medium containing recombinant GP Ib
fragment (or medium of non transfected cells as control) at a ratio of
800 µl of packed beads/2 ml of medium. After 1 h at 22-25
°C with constant mixing, the beads were again washed twice and
finally resuspended into 25 m
M Tris-HCl, 136 m
M
CH
CO
Na, pH 7.3, containing 0.6% polyethylene
glycol 6000 and 4.1% bovine serum albumin (binding buffer) at a ratio
of 1 volume of packed beads and 6 volumes of buffer. To ensure that
different fragments bound to LJ-P3 beads with equivalent efficiency,
the procedure was monitored by measuring the binding to immobilized
fragments of
I-labeled IgG of another anti-GP Ib
monoclonal antibody, LJ-P19, that does not cross-react with LJ-P3. The
interaction of
-thrombin with recombinant GP Ib
fragments
immobilized onto LJ-P3-Sepharose beads was measured by mixing 20 µl
of bead suspension with 45 µl of binding buffer, 20 µl of a 10
mg/ml solution of bovine IgG (to reduce nonspecific binding), and 40
µl of the desired concentration of
I-labeled
-thrombin. When indicated, appropriate anti-GP Ib
monoclonal
antibodies or unlabeled
-thrombin were also present in the
mixtures. After a 30-min incubation at 22-25 °C, the whole
volume of suspension was layered onto 250 µl of a 20% sucrose
solution (in binding buffer) placed in microcentrifuge tubes;
radiolabeled ligand bound to the beads was separated from free ligand
by centrifugation at 12,000
g for 4 min. The
quantitative parameters of binding were calculated on the basis of the
specific activity of the radiolabeled ligand; binding isotherms were
analyzed using the computer-assisted program LIGAND
(26, 27) .
Effect of Different Mutations and Sulfate Depletion on
the Immunochemical Reactivity of the Recombinant GP Ib
The molecules tested in these
experiments included normal GP Ib
Amino-terminal Fragment
fragment (comprising residues
1-302 of the mature protein and secreted by transfected CHO cells
grown under standard conditions) and four different mutants, three
containing single Tyr
Phe substitutions either at residue 276
(Y276F) or 278 (Y278F) or 279 (Y279F) and one containing three Asp
Asn substitutions at positions 272, 274, and 277
(D272N,D274N,D277N); the latter mutations may affect tyrosine sulfation
(see ``Discussion''). Moreover, we tested the GP Ib
fragment with native sequence but synthesized by transfected CHO cells
grown under sulfate-free conditions. Three different monoclonal
antibodies were used to evaluate the immunochemical reactivity of the
GP Ib
fragments; two of the antibodies, LJ-Ib1 and LJ-Ib10, were
chosen because of their selective inhibitory effect on vWF or
-thrombin binding to GP Ib, respectively
(4, 9) ;
the third, LJ-Ib
1, a non-inhibitory antibody, because of its
ability to interact with fully denatured GP Ib
and because the
corresponding epitope, is located between residues 1-237, thus at
a distance from the area targeted for mutagenesis.
1 reacted in a similar manner with the native GP Ib
fragment and all the mutant molecules tested, as well as with the
sulfate-free fragment; thus, it was assumed to report on the amount of
recombinant protein present in each culture medium (Fig. 1,
top panel). In contrast, different mutant GP Ib
fragments
exhibited distinct reactivity with antibodies LJ-Ib1 and LJ-Ib10. In
particular, LJ-Ib1, the selective inhibitor of vWF binding to GP Ib,
showed markedly reduced interaction with the mutant D272N,D274N,D277N,
but only a modestly reduced reactivity with the mutant Y279F and normal
reactivity with all the other mutant molecules as well as with the
sulfate-free fragment (Fig. 1, middle panel). Antibody
LJ-Ib10, the selective inhibitor of
-thrombin binding to GP Ib,
reacted well with the mutant Y276F but exhibited markedly reduced
interaction with all the other mutant molecules and with the
sulfate-free fragment (Fig. 1, bottom panel).
Figure 1:
Immunochemical analysis of native and
modified GP Ib fragments using three different monoclonal
antibodies. Volumes of 100-800 µl of culture medium
containing different types of recombinant GP Ib
fragment,
precleared with a 0.45-µm filter, were aspirated through
nitrocellulose membrane in a circular application area, in duplicate as
indicated. For testing with antibodies LJ-Ib
1 and LJ-Ib10 (see
below), disulfide bond reduction in the sample was achieved by
treatment with 60 m
M dithiothreitol at 37 °C for 1 h
before filtration. All steps of the assay procedure were performed at
22-25 °C. Native indicates the fragment with normal
GP Ib
sequence (residues 1-302) derived from cells grown
under standard conditions; D272,274,277N indicates the
fragment with three Asp
Asn mutations at positions 272, 274, and
277; Y276F indicates the fragment with a Tyr
Phe
mutation at residue 276; Y278F indicates the fragment with a
Tyr
Phe mutation at residue 278; Y279F indicates the
fragment with a Tyr
Phe mutation at residue 279; sulfate-free
indicates the fragment with normal sequence but derived from cells
grown under sulfate-free conditions; CHO indicates the medium of
nontransfected cells used as control. The membrane with bound
recombinant fragments was soaked for 1 h in Blotto solution, followed
by a 2-3-h incubation with a 1:1000 dilution (in Blotto) of
ascitic fluid containing the monoclonal antibody to be tested, as
indicated (all mouse IgG). At the end of the incubation, the membrane
was washed three times (10 min each) with fresh Blotto solution and
then incubated for 1 h in Blotto solution containing
I-labeled rabbit anti-mouse IgG (0.05 mCi of total
radioactivity). The membrane was again washed three times with fresh
Blotto solution and one last time with 20 m
M Hepes buffer, pH
7.4, containing 150 m
M NaCl and 500 µl/liter of Tween 20
(Sigma). At this point the membrane was dried and an autoradiograph was
obtained by exposure for 12-18 h to a Kodak X-Omat RP XRP-1 film
with a Dupont Cronex Quanta III intensifying screen. Although not all
fragments were tested at the same time, the results from different
experiments are readily comparable, since the native GP Ib
fragment used as control on each membrane always gave reactivity
similar to that shown in this figure.
Effect of Different Mutations and Sulfate Depletion on
vWF Binding to Recombinant GP Ib
The interaction
of vWF with immobilized recombinant GP Ib Fragment
fragment, like with
intact GP Ib-IX-V receptor complex on platelets, requires exogenous
modulators such as ristocetin or botrocetin
(5) ; both were used
to evaluate the vWF-binding function of the mutant molecules produced.
Binding of antibody LJ-Ib
1 was measured in parallel to monitor the
content of expressed recombinant GP Ib
amino-terminal fragment in
the culture media used; expression levels were found to be within a
2-fold limit of variation for all the molecules tested (Fig. 2).
The three single Tyr
Phe mutants bound vWF in the presence of
ristocetin at levels equal to or greater than those observed with the
native fragment (Fig. 3); their binding capacity was in
accordance with the levels of recombinant protein expressed, as
measured by interaction with antibody LJ-Ib
1 (compare
Fig. 3
with Fig. 2). In contrast, both the mutant
D272N,D274N,D277N and the sulfate-free fragment exhibited a marked
reduction of ristocetin-mediated vWF binding (Fig. 3), in spite
of expression levels of recombinant protein equal to or greater than
those of the normal control fragment (Fig. 2). Seven different
preparations of sulfate-free fragment were tested for
ristocetin-mediated vWF binding using a single ligand concentration of
either 2 or 4 µg/ml: the average binding, relative to normal GP
Ib
fragment tested in parallel, was 64% (range from 23 to 86%).
Measurement of botrocetin-mediated vWF binding confirmed the normal
activity of the three single Tyr
Phe mutants as well as the
markedly reduced function of the mutant D272N,D274N,D277N and of the
sulfate-free fragment (Fig. 4). Six different preparations of
sulfate-free fragment were tested for botrocetin-mediated vWF binding
using a single ligand concentration of either 2 or 4 µg/ml: the
average binding, relative to normal GP Ib
fragment tested in
parallel, was 13% (range from 7 to 19%). As verified with normal GP
Ib
fragment, vWF binding was inhibited at least 80% by the
antibody LJ-Ib1, regardless of the modulator used. Moreover, the
binding was saturable, as demonstrated by the fact that addition of
100-fold excess unlabeled vWF together with the labeled ligand resulted
in 85% inhibition of ristocetin-mediated binding and 98% inhibition of
botrocetin-mediated binding.
Figure 2:
Binding of the monoclonal antibody
LJ-Ib1 to different GP Ib
fragments. This assay was performed
as described in the legend to Fig. 1 (see also for the nomenclature
used for the different fragments) except that, at the end of the
procedure, each circular area corresponding to the sample application
point was cut out and the associated radioactivity was measured in a
-scintillation spectrometer to obtain a quantitative estimate of
antibody bound. The results shown are the mean (±S.E.) of four
distinct experimental observations. In interpreting these values it
should be noted that the level of maximum antibody binding may be
influenced by the concentration of other proteins in each culture
medium relative to that of GP Ib
fragment, since all proteins
contribute to saturation of the membrane; regardless of this problem,
however, the data allow us to compare the amount of immunoreactive
material ( i.e. GP Ib
fragment) bound to the
nitrocellulose with each medium.
Figure 3:
Ristocetin-mediated binding of
I-labeled vWF to different GP Ib
fragments. Culture
medium containing native or modified GP Ib
fragments was
immobilized onto nitrocellulose membrane, in duplicate application
points and in variable volume, as described in the legend to Fig. 1
(see also for the nomenclature used for the different fragments). The
membrane was then saturated and washed by filtering through it 200
µl of Hepes buffer containing 1% bovine serum albumin and 1% bovine
-globulin; the procedure was repeated three times. At this point,
50 µl of
I-labeled vWF (4 µg/ml final
concentration, corresponding to 0.7 pmol/well) mixed with ristocetin (1
mg/ml final concentration) was filtered through each well. The membrane
was then washed twice by filtering through each well 200 µl of
Hepes/bovine serum albumin/
-globulin solution containing 1 mg/ml
of ristocetin. All steps of this assay were performed at 22-25
°C. Each circular area corresponding to the application wells was
then cut out from the membrane, and the associated radioactivity was
measured in a
-scintillation spectrometer. Bound vWF was
calculated based on its specific radioactivity. The results shown are
the mean (±S.E.) of four distinct experimental
observations.
Binding isotherms obtained in the
presence of increasing concentrations of added vWF confirmed the
reduced binding activity of the sulfate-free GP Ib fragment, both
in the presence of ristocetin and botrocetin, as well as the more
marked abnormality with the latter modulator (Fig. 5). The interaction
of vWF with GP Ib
, whether measured with intact platelets or under
the experimental conditions described here, is not reversible in the
presence of exogenous modulators; thus, these data were not subjected
to Scatchard-type analysis. At the highest concentration of ligand
added, maximal binding in the presence of ristocetin was (mean ±
S.E. of two separate experiments): 4.71 ± 0.19 pmol of vWF
subunit/well coated with normal fragment, 2.12 ± 0.03 pmol of
vWF subunit/well coated with sulfate-free fragment, and 1.37 ±
0.14 pmol of vWF subunit/well coated with nontransfected CHO cell
control medium. The corresponding values in the presence of botrocetin
were: 5.37 ± 0.30 pmol of vWF subunit/well coated with normal
fragment, 1.05 ± 0.02 pmol of vWF subunit/well coated with
sulfate-free fragment, and 0.58 ± 0.02 pmol of vWF subunit/well
coated with nontransfected CHO cell control medium. The binding to
nontransfected CHO cell control medium was assumed to be nonspecific
and was subtracted from each point shown in Fig. 5; such value
was approximately twice as high in the presence of ristocetin than
botrocetin.
Figure 5:
Dose-dependent binding of
I-labeled vWF to normal or sulfate-free GP Ib
fragment. These experiments were performed as described in the legend
to Figs. 3 and 4, with the exception that labeled vWF was used in
increasing concentrations, as indicated on the abscissa, and
each culture medium was used at the fixed amount of 800 µl/well.
Different symbols indicate vWF binding to GP Ib
fragment
synthesized under normal culture conditions (
) or under
sulfate-free conditions (
), either in the presence of ristocetin
( upper panel) or botrocetin ( lower
panel).
Effect of Different Mutations and Sulfate Depletion on
the Binding of
Recombinant fragments were immobilized by binding to
the anti-GP Ib-Thrombin to Recombinant GP Ib
Fragment
monoclonal antibody, LJ-P3, covalently coupled to
Sepharose beads. Coupling efficiency was comparable for the native and
all but one of the modified recombinant GP Ib
fragments tested, as
shown by binding of another anti-GP Ib
monoclonal antibody,
LJ-P19, labeled with
I (results not shown); only the
mutant D272N,D274N,D277N failed to interact with the antibody coupled
to Sepharose beads and could not be tested in this assay. The normal GP
Ib
fragment bound
-thrombin in a saturable manner, as
demonstrated by the fact that addition of 100-fold excess unlabeled
-thrombin to a reaction mixture containing labeled ligand resulted
in >85% inhibition of binding. Moreover, greater than 80% of the
I-labeled
-thrombin bound to normal GP Ib
fragment was displaced within 5 min after addition of a 100-fold excess
of unlabeled ligand, a result indicative of equilibrium binding.
Scatchard-type analysis of binding isotherms revealed one class of
binding sites and demonstrated that nonspecific binding calculated as a
fitted parameter corresponded closely to that measured with control
beads prepared with the culture medium of nontransfected CHO cells
(Fig. 6). Analysis of the binding data obtained with normal GP
Ib
fragment in four separate experiments (23 points, in duplicate,
corrected by subtracting the nonspecific binding measured with culture
medium of nontransfected CHO cells; Fig. 7) showed a mean binding
at saturation of 50 ± 7 (S.E.) fmol of
-thrombin per 3
µl of packed beads carrying GP Ib
fragment ( i.e. the
volume of beads present in each experimental mixture); the
corresponding k
value of 5.64 ± 1.15 (S.E.)
10
M
is similar to that
(2.17
10
M
) obtained
for
-thrombin binding to glycocalicin ( i.e. the purified
extracytoplasmic domain of GP Ib
derived from platelets; not
shown). In contrast to their normal ability to interact with vWF, the
three mutant molecules containing a single Tyr
Phe substitution
exhibited either markedly reduced (Y278F) or undetectable (Y276F and
Y279F)
-thrombin binding, as did the sulfate-free fragment
(Fig. 7). Only the Y278F mutant allowed calculation of binding
parameters at saturation: B
was 12.7 ± 2.4 (S.E.) fmol/3 µl of packed beads, i.e. approximately
25% of the value obtained with normal control fragment, and the
corresponding K
was 3 ± 0.88
(S.E.)
10
M
(eight
points, in duplicate, in two separate experiments; Fig. 7). In
the case of the other two mutants tested (Y276F and Y279F) and of the
sulfate-free fragment, binding at saturation could not be calculated;
the experimental data indicated that specific binding was less <5% of that measured with the normal control fragment (Fig. 7).
Figure 6:
Binding isotherm and Scatchard-type
analysis of I-labeled
-thrombin binding to normal GP
Ib
fragment immobilized onto Sepharose beads. Purified IgG of the
anti-GP Ib
antibody LJ-P3 was coupled to cyanogen
bromide-activated Sepharose CL-4B at a density of 4 mg/ml of packed
beads. The LJ-P3/Sepharose beads were washed twice with a buffer
composed of 0.1
M Tris-HCl, pH 7.3, containing 0.5
M
LiCl and 1 m
M EDTA, and then incubated with culture medium
containing native recombinant GP Ib
fragment (or medium of
nontransfected cells as control) at a ratio of 800 µl of packed
beads/2 ml of medium. After 1 h at 22-25 °C with constant
mixing, the beads were washed twice with the above mentioned buffer and
then resuspended into a binding buffer composed of 25 m
M
Tris-HCl, 136 m
M CH
CO
Na, pH 7.3,
containing 0.6% polyethylene glycol 6000 and 4.1% bovine serum albumin
(1 volume of packed beads and 6 volumes of buffer). Each experimental
mixture contained 20 µl of bead suspension (corresponding to 3
µl of packed beads), 45 µl of binding buffer, 20 µl of a 10
mg/ml solution of bovine IgG (to reduce nonspecific binding), and 40
µl of
I-labeled
-thrombin (in increasing
concentrations, as indicated). After a 30-min incubation at 22-25
°C, the whole mixture was layered onto 250 µl of a 20% sucrose
solution in binding buffer placed in microcentrifuge tubes; bound and
free ligand were separated by centrifugation of the beads at 12,000
g for 4 min. Bound thrombin was calculated based on
its specific activity. The values shown represent binding to native GP
Ib
fragment (
), binding to control medium of
nontransfected CHO cells (
), nonspecific (nonsaturable) binding
calculated as fitted parameter from the total binding to native GP
Ib
fragment (
). The inset shows the Scatchard plot
(bound/free versus bound) of binding to native GP Ib
fragment after subtraction of calculated nonspecific
binding.
Figure 7:
Dose-dependent binding of
I-labeled
-thrombin to recombinant GP Ib
fragments immobilized onto Sepharose beads. The assays were performed
as described in the legend to Fig. 6. The results shown are the mean
(with S.E.) of four separate experiments for the native fragment
(
) and two separate experiments each for the mutant molecules
Y276F (
), Y278F (
), Y279F (+), and for the
sulfate-free fragment (
); error bars are not shown when
the corresponding values are too small for effective graphic
representation.
,
known to be the only sites of sulfation in the GP Ib-IX complex
(12) , are concurrently necessary for expression of
-thrombin binding activity; however, each one of them can be
mutated individually to phenylalanine, abolishing the possibility of
sulfation, without loss of vWF binding activity. In contrast,
nonselective blockage of sulfation on all three Tyr residues causes
severe impairment of both vWF and
-thrombin binding. Thus, two
sulfotyrosine residues are sufficient for normal GP Ib interaction with
vWF, whereas all three are needed for normal interaction with
-thrombin. As a corollary observation, it is of note that the
mutation of either Tyr
or Tyr
, but not of
Tyr
, resulted in markedly reduced binding of the
monoclonal antibody LJ-Ib10, previously shown to be a specific
inhibitor of
-thrombin binding to GP Ib
(4) . This finding
supports the hypothesis that the antibody epitope overlaps with, but
does not exactly correspond to, the
-thrombin binding site.
(7) , is a good indication that each of the
mutant molecules containing Tyr
Phe substitutions retained an
overall conformation similar to that of the native GP Ib
fragment.
The Tyr hydroxyl group, however, may become involved in hydrogen
bonding and, thus, influence the three-dimensional molecular structure.
Moreover, the hydroxyl group may undergo sulfation, a
post-translational modification occurring in at least 1% of tyrosine
residues in eukaryotic organisms
(28) ; in this case, the added
negative charge may become engaged in electrostatic interactions. Both
these important structural properties of tyrosine may be necessary for
expression of specific molecular activities.
has been recently
highlighted by the demonstration that ristocetin-dependent vWF binding
to heterologous cells expressing the GP Ib-IX complex is partially
decreased when tyrosine sulfation is inhibited
(12) . We have
confirmed and extended that finding by showing that the sulfate-free
recombinant GP Ib
fragment exhibits a more pronounced reduction of
vWF binding capacity supported by botrocetin as compared with
ristocetin. Although unexplained at present, this result reflects the
fact that the mechanisms of ristocetin- and botrocetin-dependent vWF
binding to GP Ib are not the same
(3, 18, 29) .
Regardless of the modulator used, however, it is clear that the vWF
binding abnormality exhibited by the sulfate-free GP Ib
fragment
is not shared by any of the single Tyr
Phe mutants, suggesting
that the presence of any two of the three sulfate groups located at
positions 276, 278, and 279 is sufficient to mediate a normal
interaction with vWF. The alternative possibility that lack of
sulfation may cause conformational aberrations in the GP Ib
fragment, and thus explain the loss of function, seems to be excluded
by the observation that the sulfate-free fragment, like the normal
functioning molecules containing single Tyr
Phe mutations,
exhibited normal reactivity with the conformation-sensitive antibody
LJ-Ib1. Also consistent with this conclusion is the fact that sulfation
takes place in the trans-Golgi network at a relatively late stage after
protein synthesis and folding
(30) and that sulfate-free GP
Ib
is normally expressed on the cell surface in complex with GP
Ib
and GP IX
(12) .
fragment secreted by cells grown
under sulfate-free conditions also failed to express
-thrombin
binding activity. This finding is consistent with the hypothesis that
one reason, if not the only one, why single Tyr
Phe mutations
resulted in a similar functional abnormality is the lack of necessary
sulfate groups (Phe residues cannot be modified by sulfation). In fact,
the presence of sulfotyrosine appears to be a recurrent structural
motif in
-thrombin-binding proteins, like the specific inhibitor
hirudin
(31, 32) . Thus, our results with the
recombinant GP Ib
fragment are consistent with the notion that
sulfated tyrosine residues may influence in a significant manner the
affinity of interaction with
-thrombin
(32) . By analogy
with the well established three-dimensional model of the
hirudin-thrombin complex
(33, 34) , it seems reasonable
to hypothesize that the GP Ib
domain containing the sulfated
tyrosine residues interacts with the anion-binding exosite of
-thrombin and that electrostatic forces play a key role in
supporting the binding.
. The functional abnormality associated with this
mutation may simply reflect the abolition of sulfation on the
neighboring Tyr residues at positions 276, 278, and 279; in fact,
clusters of acidic amino acids appear to be essential for the action of
tyrosylprotein sulfotransferase, the enzyme that catalyzes this
post-translational modification
(28) . The D272N,D274N,D277N
mutant, however, unlike the sulfate-free fragment or any of the Tyr
Phe mutants, exhibited markedly reduced reactivity with the
conformation-sensitive antibody LJ-Ib1. This finding may indicate that
the Asp
Asn mutations cause a substantial conformational change
in the GP Ib
fragment or, alternatively, that the mutated Asp
residues are directly part of the antibody combining site and, perhaps,
or the vWF binding site. The latter hypothesis is supported by the
knowledge that antibody LJ-Ib1 is a competitive inhibitor of vWF
binding to GP Ib
(35, 36) , and the corresponding
epitope is thought to overlap, at least in part, with the vWF-binding
site in GP Ib
; and by the known precedent of a single amino acid
substitution (Ala
Val), identified in a variant
form of Bernard-Soulier syndrome
(9) , which results in loss of
vWF binding function as well as of the LJ-Ib1 epitope. It remains to be
established whether the lack of reactivity with antibody LJ-Ib1 is the
reflection of a unique structural requirement that, in alternative or
in addition to sulfation of Tyr residues, is necessary to support vWF
binding. Answering this question may require a more detailed structural
knowledge of the amino-terminal domain of GP Ib
.
; however, they are likely to apply to the intact GP Ib-IX-V
complex expressed on platelets. Indeed, we have shown in previous
studies that the isolated fragment interacts with vWF in a manner
similar to intact GP Ib
(5) and that mutations in the fragment
reproduce functional aberrations of the receptor observed in congenital
diseases, resulting both in decreased
(8, 9) or
paradoxically increased affinity for soluble vWF
(37) . Of note,
the demonstration that each of the sulfated residues Tyr
,
Tyr
, and Tyr
have a concurrent functional
role in mediating
-thrombin binding to GP Ib
, whereas none of
them is individually necessary to support vWF binding, provides a
potential target for either selective or combined inhibition of
-thrombin and vWF binding to platelets.
-thrombin, James R. Roberts and BenjaminGutierrez for
help in the preparation of monoclonal antibodies, and Faye Miller and
Ellye Lukaschewsky for secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.