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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratory for Thrombosis Research, IRC, K U
Leuven Campus Kortrijk, Belgium; the Department of Haematology,
University Hospital Utrecht, The Netherlands; the Thrombosis Research
Section, Baylor College of Medicine, Houston, TX; the Baker Medical
Research Institute, Melbourne, Australia; and the Department of
Clinical Biochemistry and Molecular Pathology, University Medical
School, University of Debrecen, Hungary.
The interaction of von Willebrand factor (vWF) with the platelet
receptor glycoprotein Ib Adhesion of platelets to sites of vascular injury
is critical for hemostasis and thrombosis and is dependent on the
binding of von Willebrand factor (vWF) to the glycoprotein Ib
(GPIb)/IX/V complex on the platelet surface.1 The GPIb-vWF
interaction mediates the initial tethering and subsequent rolling of
platelets over collagen in the subendothelium.2 This
interaction is of major importance for platelet adhesion at high shear
stress and leads to activation of the integrin
There are approximately 25 000 copies of the GPIb/IX/V complex
on the membrane of resting platelets. GPIb is a heterodimer consisting
of 2 subunits, GPIb Several studies in the past 10 years have attempted to identify the
crucial interaction sites on both GPIb By using synthetic peptides, the vWF-binding site was localized within
different areas on the N-terminal domain of GPIb Mutations within the LRR, identified by studies of patients with
Bernard-Soulier syndrome, result in either defective or decreased binding of vWF12-14 highlighting the role of the LRR (aa
36-200) in the correct exposure and function of the vWF binding site. This was further demonstrated by Shen et al,15 who used
Chinese hamster ovary (CHO) cells expressing canine-human chimeras of GPIb Finally, within the disulfide loop C209-C248 of GPIb To further investigate the GPIb-vWF interaction, we produced a new
panel of murine anti-GPIb mAbs that block the binding of GPIb to vWF.
The inhibitory characteristics of these mAbs were studied under shear
conditions and under static conditions using the modulators ristocetin
and botrocetin. Several approaches were also undertaken to localize the
binding site of these mAbs on GPIb Production and purification of novel anti-GPIb mAbs and source
of others
Production and purification of rGPIb Shear-independent platelet aggregation Blood was drawn from healthy volunteers on 3.8% sodium citrate (9:1 vol/vol), and platelet rich plasma (PRP) was prepared by centrifugation at 200g for 10 minutes. Aggregation studies were performed in an Elvi-840 dual-channel aggregometer from Pabish (Brussels, Belgium). Briefly, 200 µL PRP (final concentration, 2 × 108 platelets/mL) was preincubated with serial dilutions of various anti-GPIb mAbs for 3 minutes at room temperature (RT), followed by the addition of ristocetin (1.25 mg/mL) or botrocetin (0.5 µg/mL), and the aggregation response was followed for 5 minutes. Botrocetin was purified from crude Bothrops jararaca venom (Sigma).27 Platelet aggregations in response to -thrombin (0.2 nmol/L) were performed by Dr M. Jandrot-Perrus
(Paris, France).28
Effect of mAbs on shear-induced platelet adhesion The inhibitory effect of the anti-GPIb mAbs was studied at a shear rate of 2600 seconds 1 in a parallel-plate flow
chamber with a slit height of 0.4 mm.29 Human collagen
type I (Sigma) was dissolved in 50 mM acetic acid (1 mg/mL), dialyzed
extensively for 48 hours against phosphate-buffered saline, and
subsequently sprayed onto plastic Thermanox coverslips (Nunc,
Rochester, NY). Perfusions were carried out at 37°C with anticoagulated whole blood (low-molecular-weight heparin, 25 U/mL; Clexane, Rhône-Poulenc Rorer, France) obtained from healthy
volunteers. Twelve milliliters whole blood was preincubated with
various concentrations of the anti-GPIb mAbs for 5 minutes at 37°C,
after which it was circulated over the collagen-coated coverslips.
After 5-minute perfusion, the coverslips were immersed in methanol and
stained with May-Grünwald-Giemsa. Platelet adhesion was evaluated
with a light microscope connected to an image analyzer and was
expressed as percentage surface coverage with platelets. Before and
after every perfusion experiment the platelet count was measured.
Cross-blocking analysis for monoclonal antibody binding to platelets A binding curve of all mAbs to human platelets was determined by ELISA. Two hundred-microliter aliquots of 2 × 108/mL formaldehyde-fixed platelets were added to microtiter plates precoated with 10 µg/mL poly-L-lysine (100 µL/well; Sigma). The plates were centrifuged at 4°C for 15 minutes at 150g, washed with phosphate-buffered saline, blocked with 3% skimmed milk (2 hours), and incubated with serial dilutions of biotinylated anti-GPIb mAbs (bmAbs) for 1 hour at RT. After another washing step, streptavidin-horseradish peroxidase (HRP; 1/10 000 dilution; Boehringer, Mannheim, Germany) was added, and binding was detected after the addition of ortho-phenylenediamine (OPD; Sigma). The reaction was stopped with 4 M H2SO4, and absorbance was determined at 492 nm.Competitive inhibition assays between the different anti-GPIb mAbs were performed by preincubating a serial dilution of one unlabeled mAb (50 µL/well) with the platelets for 30 minutes at RT. Then, 50 µL constant concentration of the same or another anti-GPIb bmAb was added to the wells for 1 hour at RT. bmAb was detected after incubation with streptavidin-HRP as described above and was used at a constant concentration experimentally established to saturate 50% of the binding sites. Inhibition of binding of vWF to a rGPIb fragment (H1-V289) (2 µg/mL) was captured for 2 hours (RT) onto a microtiter plate, precoated with mAb 2D4 (5 µg/mL), and blocked with 3% skimmed milk. mAbs 2D4 and 26D1 (negative control
mAb) were nonblocking anti-GPIb mAbs produced in-house. A serial
dilution of the inhibitory anti-GPIb mAb was added to the wells,
together with purified human vWF, at a concentration of 0.5 µg/mL or
0.15 µg/mL, when ristocetin (300 µg/mL) or botrocetin (0.25 µg/mL) was used as a modulator, respectively. Wells were incubated
for 2 hours at RT. Bound vWF was detected after 1-hour incubation with
HRP-labeled anti-vWF antibody (DAKO, Glostrup, Denmark) using OPD as
described above.
Screening of the antibodies with the canine-human chimeras
of rGPIb  IX cells has been described in detail by Shen et al.15 In Figure 3A, an overview of the
different chimeras is given. Binding of the anti-GPIb mAbs to the
transfected CHO cells was analyzed using fluorescein
isothiocyanate-labeled secondary antibody and flow
cytometry.15
Screening of antibodies with a phage-display peptide library A linear 15-mer library (L15; L. Jespers, Leuven, Belgium) and a C-C-linked circular 7-mer library (C7; E. Ruoslahti, La Jolla, CA) were used. Selection for antibody-binding peptides was made using a modified bio-panning technique, with the exception that beads rather than tubes were used for coating mAbs.30 In the first panning round, 2 × 1012 phages from each library (L15 and C7) were added, and bound phages were eluted with 0.1 M glycine-HCl. For the subsequent panning rounds, 1 × 1011 eluted phages of the previous round were used as input phages, and a competitive elution was performed by adding 10 µg rGPIb fragment
for 30 minutes at RT.
Phage ELISA Three types of ELISA were performed to evaluate whether the selected phage bound specifically to the target mAb. In the first ELISA, each pool of phage eluted in the different rounds was screened for binding to the mAb used for selection. In the second ELISA, positive individual colonies from these pools were identified. To this end, a dilution of the eluted phage pool of the last positive panning round was plated on Luria broth (LB) agar (+tetracycline [Tc]) plates. Ninety-six single colonies were picked from these plates and grown overnight in 2 × TY medium (+Tc) in a sterile culture microtiter plate. After centrifugation of the plates, the phage containing supernatant of each individual clone was evaluated in the ELISA. The third type was a competition ELISA both the
monoclonal phage and the rGPIb fragment competed with each other for
binding to the coated mAb. In these competition ELISAs, a serial
dilution of the rGPIb fragment (H1-V 289) was added with a constant
concentration of the phages. Phages were used at a concentration
sufficient to obtain 50% binding to the respective mAbs.
For all 3 ELISAs, mAbs were coated onto microtiter plates (5 µg/mL in
Tris-buffered saline [TBS] overnight at 4°C). Plates were blocked
with TBS + 2% skim milk (2 hours, RT). After washing with
TBS + 0.1% Tween 20, phage, rGPIb Immunoblot analyses Purified phage clones were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), blocked with 3% skim milk in TBS, washed with TBS + 0.05% Tween 80, and incubated with bmAbs (5 µg/mL in TBS + 0.05% Tween 80, 2 hours). After another washing step, the nitrocellulose membranes were incubated with streptavidin-HRP and were developed using the enhanced chemiluminescence detection system from Amersham (Buckinghamshire, United Kingdom).Sequencing of phage clones Phages selected for sequencing were amplified overnight in LB (+Tc) medium and were purified twice by precipitation with polyethylene glycol/NaCl. DNA was purified by phenol/chloroform extraction and was sequenced with the Sequenase version 2.0 T7 DNA Polymerase Sequencing Kit (Pharmacia) according to the manufacturer's instructions using 32S-dATP (Amersham), 5'-CTCATAGTTAGCGTAACG-3' for the 15-mer library, and 5'-CCCTCATAGTTAGCGTAACG-3' for the 7-mer library.Production of wild-type rGPIb fragments used in the characterization of
antibody binding were produced in insect cells. Wild-type and mutant
GpIb were expressed with a C-terminal histidine-tag and were
purified from the harvested secretion medium by metal-affinity chromatography and anion-exchange chromatography (D.F.W. et al, unpublished results, 2001). In ELISA the wild-type fragment
does not bind to plasma vWF in the absence of botrocetin and
ristocetin, whereas the platelet-type vWD GpIb fragments bind vWF
spontaneously, reflecting the in vivo characteristics of wild-type and
platelet-type vWD GpIb.
Binding of anti-GPIb mAbs to the fragments was determined using plasmon
resonance technology (Biacore, Uppsala, Sweden). A standard flow buffer
(50 mM Tris pH 7.4, 150 mM NaCl, 0.005% surfactant P-20) was used, and
the experiments were performed at RT. Capturing anti-GPIb mAb 2D4 was
coupled to a sensor chip-type CM5 using conventional amine-coupling
chemistry,31 resulting in a signal of ±1500 resonance
units. The wild-type (WT)-rGPIb
Identification of inhibitory anti-GPIb monoclonal antibodies Out of 2 fusions, a panel of 50 anti-GPIb mAbs was obtained, from which 5 mAbs were identified as inhibitory because they blocked ristocetin-induced human platelet agglutination. To obtain 50% saturation of binding to washed fixed platelets, 0.1, 0.04, 0.08, 0.1, and 0.02 µg/mL 27A10 (IgG1), 12G1 (IgG1), 12E4 (IgG2b), 24G10 (IgG2a), and 6B4 (IgG1), respectively, were required. All mAbs recognized the rGPIb fragment (H1-V289) by ELISA, but none recognized GPIb from platelet lysate or the rGPIb fragment
(H1-V289) after SDS denaturation and Western blot analysis.
Binding of vWF to the rGPIb
These ELISA results are in agreement with aggregation studies in which
mAbs 12E4, 12G1, and 27A10 could not block botrocetin (0.5 µg/mL)-induced human platelet aggregation in PRP, in contrast to mAbs
6B4 and 24G10, which have an IC50 value of 0.8 µg/mL and 2.5 µg/mL, respectively. The IC50 values for inhibition
of ristocetin-induced (1.2 mg/mL) platelet aggregation are 2.5 µg/mL,
1.25 µg/mL, 1.3 µg/mL, 0.3 µg/mL, and 0.8 µg/mL for mAbs 27A10,
12G1, 12E4, 6B4, and 24G10, respectively. Only mAb 24G10 showed some
inhibitory activity versus platelet aggregation induced by low doses of
thrombin: 50 µg/mL 24G10 inhibited 0.2 nmol/L thrombin-induced
aggregation by 62% (data not shown). Finally, at a concentration of 5 µg/mL, all the mAbs
Cross-blocking analysis for mAb binding to platelets We evaluated whether the 5 anti-GPIb mAbs competed with each other for binding to human platelets (Figure 2A). Binding of each biotinylated bmAb could be displaced by its unlabeled counterpart in a dose-dependent manner. Monoclonal antibodies 12G1, 12E4, and 27A10 did compete with each other, nor did they compete with mAbs 6B4 and 24G10, yet mAbs 6B4 and 24G10 cross-blocked the binding of each other. Based on these competition experiments, the mAbs can be divided into 2 groups: 12G1, 12E4, and 27A10 in the first group and 24G10 and 6B4 in the second group. This grouping correlates with their different inhibitory effects on the GPIb-vWF binding. The second group of mAbs (24G10, 6B4) were defined as overall inhibitors, blocking ristocetin-, botrocetin-, and shear-induced GPIb-vWF interaction. The first group of mAbs (12G1, 12E4, 27A10) was clearly less inhibitory with respect to ristocetin-dependent binding of vWF and could not block botrocetin-induced binding but did inhibit the shear-induced binding of vWF.
We also performed competition studies between our 5 mAbs and the mAbs LJ-Ib1, LJ-Ib10, HIP1, 6D1, TM60, and AK2 for binding to platelets (Figure 2B). Binding of mAbs of the first group (12G1, 12E4, 27A10) was not blocked by any of the investigated competing mAbs. However, mAbs 6B4 and 24G10 did compete with mAbs LJ-Ib1, AK2, HIP1, and TM60. The binding of TM60 itself could be equally blocked by LJIb1, AK2, HIP1, and TM60. Screening the monoclonal antibodies by using canine-human chimeras Recently, the binding sites for several other inhibitory anti-GPIb mAbs, characterized by other laboratories, were identified using the same canine-human chimeric system.15 In one set of chimeras, the human part (aa 1-282) was progressively replaced with the canine sequence starting from the N-terminus (Figure 3A). Another set of chimeras was constructed such that the canine sequence (aa 1-282) was used as a template and was incrementally replaced with the human sequence. Shen et al15 demonstrated that all chimeras were functionally expressed because they bound to human vWF in the presence of botrocetin, a modulator that does not discriminate between human and canine GPIb.
Our 5 anti-human GPIb mAbs did not bind to canine platelets,
indicating that this system can be used for epitope mapping. Binding of
the mAbs to the canine-human chimeras was checked by flow cytometry,
and the results are summarized in Table 2
and Figure 3B. As described by Shen et al,15 the binding
domains for each mAb were assigned from the first domain at which
the replacement of the human sequence with the canine sequence
abolished binding, to the last domain where replacement of the canine
sequence by the human sequence resulted in recovered binding.
Binding of mAb 27A10 was lost when the first 59 human aa were changed
into canine and binding was re-acquired when the canine sequence was
rehumanized to aa 59. Therefore, the epitope is likely to be contained
between aa 35 and 59 (first LRR) of GPIb Selection of phage peptides, phage ELISA, sequencing, and sequence alignment Phages were selected from a linear pentadecamer library (L15) on mAbs 6B4, 4G10, 27A10, 12G1, and 12E4 and, in addition, from the cyclic heptamer library (C7) on mAb 6B4. After 3 rounds of panning, a significant enrichment for phages binding to mAbs 27A10, 12G1, 12E4, and 6B4 was obtained. No phages could be selected for binding to mAb 24G10, despite several selection protocols with varying elution and washing procedures. We do not have a clear explanation for this nonselection. It may be that the epitope of mAb 24G10 is unavailable, though the diversity of the library is high. The same phenomenon has been observed by others.19,20 It might be desirable to screen this particular mAb using a panel of peptide phage libraries or using libraries containing larger peptide inserts.Individual phage clones from the third selection round (using the L15
library) on mAbs 27A10, 12G1, 12E4, and 6B4 were analyzed for binding
to the respective mAb on which they where selected (Figure
4) and for cross-reactivity with one of
the other mAbs. None of the individual phage clones cross-reacted with
other mAbs except for the phage (phage 1) selected on mAb 12G1 that
also bound to mAb 12E4 (Figure 4). Both mAbs belong to the same group; however, it is unlikely that they are identical because a phage selected for binding to mAb 12E4 did not bind to mAb 12G1 (Figure 4).
By means of competition ELISA, we could further show that all phages
selected from L15 and C7 libraries bound specifically to the
antigen-binding site of their respective mAbs because the rGPIb
Sequence analysis of the phage inserts (from the L15 library) revealed
that mAbs 27A10 and 12E4 each recognized a single sequence and that mAb
12G1 recognized 2 sequences (Table 3).
Sequence alignment of the different
sequences with the GPIb
In addition, we tested whether the 2 cysteines present in some L15
phage peptides form a disulfide bridge: purified phages carrying the
corresponding peptide sequence were separated on SDS-PAGE under
nonreducing and reducing conditions, followed by Western blot analysis,
during which phages were detected with the mAb for which they were
selected (Figure 6). Although mAbs 27A10,
12E4, and 12G1 recognized their respective phages, mAbs 12G1 and 27A10
no longer did so when reducing conditions were used. For MoAb 12E4,
recognition was diminished after reduction, and mAb 12E4 could
recognize the cross-reacting 12G1 phage 1 under nonreducing, but not
under reducing, conditions (not shown). Results of these Western blot
experiments indicate that the cysteines present in the peptides
selected on these mAbs (12G1, 27A10, 12E4) indeed form a disulfide
bridge, necessary for recognition by their respective mAbs. All the C7
and L15 phages selected on mAb 6B4 could be detected under both
nonreducing and reducing conditions, indicating that in this case the
disulfide-linked structure is not necessary for recognition by 6B4. As
an example, the results with the C7-6B4 phage clone D3 are shown in
Figure 6.
Binding of mAbs to wild-type and mutated rGPIb fragments (H1-R280) that contain
the PT-vWD mutations (G233V and M239V) by Biacore (Figure 7). Monoclonal antibodies 6B4, 24G10, and
TM60 associated strongly and dissociated slowly, resulting in low
apparent dissociation constant values. Monoclonal antibodies 27A10 and
TM60 bound equally well to both mutants, G233V and M239V. In contrast,
6B4 had a 5- to 6-fold reduced affinity, whereas 24G10 had a 5-fold
enhanced affinity mainly because of an enhanced on-rate.
The platelet GPIb is an important receptor in the process of
platelet adhesion under arterial levels of blood flow. The vWF binding
site is located within the N-terminal 300 aa of GPIb Studies using the canine-human GPIb The 2 other mAbs (6B4, 24G10) not only competed with each other,
but also with a series of inhibitory mAbs from other laboratories (LJ-Ib1, HIP1, AK2, and TM60). The mAbs 6B4 and 24G10 inhibited vWF-GPIb interaction, regardless of whether this was induced by ristocetin, botrocetin, or shear and whether it was in contrast to the
mAbs 12G1, 27A10, and 12E4. The mAb 24G10, like TM60, also inhibited
thrombin binding to GPIb To further define the amino acid residues important for binding of mAb
6B4, peptides were selected by phage display using a linear 15-mer and
a cyclic 7-mer peptide library. The sequences obtained could be divided
within 2 groups and were aligned to the region previously identified by
the chimeras (aa 201-282). One group could be aligned to position
230-242, where the gain-of-function platelet-type vWD mutations have
been identified. Given that we demonstrated that 6B4 has a
discontinuous epitope, it is not surprising that 2 potential binding
sites were found. Our alignment results were partially confirmed by
demonstrating that mAb 6B4 has a reduced affinity for recombinant
GPIb Surprisingly, mAb 24G10, also belonging to the same group as mAb
6B4 but interacting with aa 1-81 of GPIb Miller and Lyle37 reported an analogous finding when
mapping the epitope of the anti-GPIb mAb C34. This particular mAb binds better to the PT-vWD mutant G233V than the nonmutated GPIb Further evidence that the areas aa 201-268 and aa 1-81 are
topographically associated comes from antibody cross-blocking studies, because mAbs interacting with the first area prevent binding of mAbs to
the second, and vice versa. The anticipated horseshoelike structure of
the leucine-rich repeats of GPIb Other investigators have shown that the region 201-268 of GPIb Our results are also consistent with the hypothesis that the
region aa 230-242 of GPIb On the other hand, it is clear that with the present approaches,
we have not fully identified the epitopes of any of our antibodies. Indeed, because none of the antibodies recognized SDS-denatured rGPIb In conclusion, characterization of a novel panel of anti-GPIb
mAbs has provided further insight into the GPIb-vWF interaction. In
particular, this study has made the novel finding that 2 binding sites,
N-terminal aa 1-59 and aa 1-81 in close contact with aa 201-268, may
play a role in vWF binding to GPIb |
reveals interaction between the leucine-rich
repeat N-terminal and C-terminal flanking domains of
glycoprotein Ib
Top
Abstract
Introduction
) and 6B4 (
) blocked ristocetin-induced binding dose
dependently, whereas mAbs 27A10 (
), 12G1 (
), and 12E4 (
)
inhibited the binding only moderately. mAbs 27A10, 12G1, and 12E4 did
not block the botrocetin-induced binding, in contrast to mAbs 6B4 and
24G10. mAb 26D1 (control, 
) had no effect when both modulators were
used. Binding of vWF was detected with HRP-labeled anti-vWF antibody.
Data are the mean of 2 experiments.
, 12G1 phage 1 and 12G1 mAb; 
, 27A10 phage and 12G1 mAb; 
, 27A10 phage and 27A10 mAb;
