Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4207-4211
Identification of Peptides, Selected by Phage Display Technology,
That Inhibit von Willebrand Factor Binding to Collagen
By
H. Depraetere,
A. Viaene,
S. Deroo,
S. Vauterin, and
H. Deckmyn
From the Laboratory for Thrombosis Research, Interdisciplinary
Research Center, KU Leuven Campus Kortrijk, Belgium.
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ABSTRACT |
A repeated selection of phages from a cyclic hexapeptide phage
display library resulted in an enrichment of phages that bound to the
monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor
[vWF] antibody that inhibits binding of vWF to collagen). Two clones
were selected that bound both to MoAb 82D6A3 and to rat tail collagen
type I in a specific and dose-dependent manner. The two phage clones
were further used in a two-direction competition experiment with vWF.
vWF was able to displace phages from collagen in a dose-dependent
manner with an IC50 of 35 µg/mL and phages were able to
inhibit vWF binding to collagen. With the use of specific primers, the
sequence of the cysteine-flanked hexapeptide inserts could be deduced.
The two phage clones carried an almost identical sequence, CVWLWEQC and
CVWLWENC, with a substitution of an N for a Q at position 6 of the
hexapeptide. Sequence comparison with the known vWF sequence showed the
presence of a comparable sequence at position 1129-1136 (VWTLPDQC),
located between the collagen-binding A3-domain and the
D4-domain. The two cyclic peptides, the putative
corresponding vWF peptide, and a peptide with a scrambled cyclic
sequence were synthesized. The two cyclic peptides inhibited vWF
binding to rat tail collagen type I in a dose-dependent manner, whereas
the linear vWF peptide and the scrambled cyclic peptide were inactive.
For half maximal inhibition, 100 ± 12.7 µmol/L and 34.8 ± 8.59 µmol/L (mean ± SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to
inhibit vWF binding to calfskin and human collagen type I, but
effective concentrations were some 5 to 10 times higher.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
PLATELET ADHESION TO subendothelial
structures exposed on damage of the vessel wall is one of the first
steps in a sequence of reactions that can lead to arterial thrombosis.
One of the more abundant thrombogenic compounds to which platelets adhere are the collagens. Platelets bind to collagen both in a direct
manner via their collagen receptors
2
1,1,2 glycoprotein IV3 and VI,4 and indirectly, with von
Willebrand Factor (vWF) forming the bridge between collagen and its
platelet receptor, the glycoprotein (GP) Ib/IX/V-complex.5
Both binding via 21 and vWF is necessary
to sustain platelet adhesion under high shear forces.6,7
Both 21 and vWF bind to collagen with
their I domains, in vWF known as A-domains.8-14 I-domains
form independent globular modules of some 200 amino acid residues. In
vWF three such domains have been identified. The A1-domain
(497-716) contains the binding site for GPIb,15,16
sulfatides,17 heparin,18 and collagen VI,19 which constitutes the main reactive collagen in the
extracellular matrix of endothelial cells. The A2-domain
(717-909) has no clear binding function, but is relatively sensitive to
enzymatic degradation, whereas the A3-domain (910-1111)
contains the main binding site for fibrillar collagens such as type I
and III.14,20 Recombinant A3-domain also binds
to collagen,20 whereas deletion of A3 results in a vWF that binds 40 times less to collagen.14
Both direct and indirect vWF-mediated binding to collagen can be
inhibited by the collagen binding leech products leech
antiplatelet protein21,22 and Calin.23,24
Furthermore, Calin was able to prevent platelet-rich thrombus formation
in a hamster model. Apart from these products, an IgM apparently
interacting with both the A1- and A3-domain and
inhibiting vWF interaction with collagen was identified in a patient
with an acquired bleeding disorder,25 and a number of
murine anti-vWF monoclonal antibodies (MoAbs) have been described that
also block the binding to collagen. One of these, MoAb
82D6A3,19 inhibits vWF binding to fibrillar collagens (type
I and III) but not to collagen VI and also recognizes vWF after sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and Western
blotting. With this antibody, we identified a new collagen-binding
eight amino acid sequence (cyclic hexapeptide). The selection was based
on an anti-idiotypic approach to select phages from a cyclic hexamer
phage display library on both MoAb 82D6A3 and on rat tail collagen type
I.
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MATERIALS AND METHODS |
Products.
Rat tail collagen type I was purchased from Collaborative Biomedical
Products (Bedford, MA), human type I and III, and calfskin type I from
Sigma (St Louis, MO). The collagens were solubilized in 50 mmol/L
acetic acid and subsequently dialyzed against phosphate-buffered saline
PBS (48 hours, 4°C) to obtain fibrillar collagen. The phage display
library with the random hexapeptides flanked by cysteine residues was
obtained from Corvas (Gent, Belgium). MoAb 82D6A3 (a kind gift of Dr M. Hoylaerts, Leuven, Belgium), a MoAb against vWF, was purified from
ascites by protein A chromatography. Peptides were custom synthesized
by Ansynth (Roosendaal, The Netherlands). vWF was purchased from the
Red Cross (Belgium).
Isolation of MoAb 82D6A3-binding phages.
Selection of phages was performed essentially as
described.26 Briefly a high-binding polystyrene tube (Nunc,
Roskilde, Denmark) was coated overnight with purified MoAb 82D6A3 (10 µg/mL, Tris-buffered saline [TBS]). After blocking the
tubes with 3% milkpowder (2 hours, TBS), 2.1012 phages
(0.25% Tween 20, 2% milkpowder, TBS) from the amplified cyclic
hexapeptide library were added. After 150 minutes (90 minutes rotating,
60 minutes standing) the input phages were removed and the tube was
washed seven times with TBS (0.4% Tween 20, 0.25% milkpowder) to
remove the nonspecific binders. The bound phages were eluted with 0.1 mol/L glycine, pH 2.2, and the eluate was immediately neutralized with
1 mol/L Tris, pH 8. After amplification of the phages, two additional
rounds of panning were performed. Phages were amplified by infection of
Escherichia coli TG1 cells and partially purified from the
supernatant by polyethylene glycol precipitations. Phage DNA was
prepared using the QIA-prep Spin M13 Kit (Westburg, Leusden, The
Netherlands) and sequencing reactions were performed according to the
autoread sequencing kit (Pharmacia, Roosendaal, The Netherlands) using the primer 5
-TGAATTTTCTGTATGAGG-3 and the Automatic
Laser Fluorescence Detection Unit (A.L.F. Sequencer; Pharmacia). After
the third round of panning, individual phage-bearing E coli
cells were grown in a 96-well plate, and the supernatant was tested for
the presence of phages, binding to both MoAb 82D6A3 and collagen.
Measurement of phage binding to MoAb 82D6A3 and to rat tail collagen
type I.
A 96-well plate was coated overnight with purified MoAb 82D6A3 (10 µg/mL) or with rat tail collagen type I (50 µg/mL). After 2 hours
blocking with 4% milkpowder, a dilution series of the individual phage
clones in TBS with 0.3% milkpowder was added to the wells and phages
were incubated at room temperature for 2 hours. Bound phages were
detected after a 1-hour incubation with a polyclonal
anti-M13-horseradish peroxidase (HRP)
conjugated antibody (Pharmacia)
and visualization was performed with ortho-phenylenediamine (OPD,
Sigma). The reaction was stopped with 4 mol/L
H2SO4 and absorbance was determined at 490-630
nm. In between each incubation step the plates were washed three to
nine times with TBS (0.1% Tween 20).
Inhibition of vWF binding to collagen by phages or peptides.
Collagen was coated at a concentration of 30 to 50 µg/mL (overnight,
4°C). After blocking the plates with 3% milkpowder, a dilution
series of phages in TBS with 0.3% milkpowder or peptides in PBS with
0.3% milkpowder was added. After a preincubation for 30 minutes, a
constant amount of vWF was added to the wells and mixed with the
peptide or phage solution. Unbound vWF was removed after 90 minutes and
bound vWF was detected with a polyclonal anti-vWF-HRPconjugated
antibody (Dako, Glostrup, Denmark). Visualization was performed with
OPD. The reaction was stopped with 4 mol/L H2SO4 and absorbance was determined at 490-630
nm. In between each incubation step the plates were washed three to
nine times with TBS (0.002% Tween 80).
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RESULTS |
Selection of phages that bind to the MoAb 82D6A3 and to collagen.
A repeated selection of phages from the cyclic hexapeptide phage
display library resulted in an enrichment of phages that bound to the
MoAb 82D6A3. Because this anti-vWF MoAb inhibits the binding of vWF to
collagen, then, in line with the anti-idiotypic theory, a phage that
binds to MoAb 82D6A3 could be a mimic of vWF and in this case could
bind to collagen. Therefore, after the third round of panning
individual colonies were tested for binding both to MoAb 82D6A3 and to
rat tail collagen.
Two double-positive clones were identified and amplified for further
characterization. The two selected clones bound both to MoAb 82D6A3
(Fig 1A) and to rat tail collagen type I (Fig
1B) in a specific and dose-dependent
manner, whereas they did not bind to uncoated wells. The two clones
were further used in a two-direction competition experiment with vWF.
We found that vWF displaced phages from collagen in a dose-dependent
manner (Fig 2A) with an IC50 of
35 µg/mL at 5 × 1011 phages/mL. Moreover, these phages were also able to inhibit vWF binding to collagen (Fig
2B). However, because of limitations in the number of phages that can
be used, no full inhibition curve could be constructed.
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| Fig 1.
Binding of two different phage clones ( , Q-phage;  ,
N-phage) to microtiterplates coated with 10 µg/mL MoAb 82D6A3 (A)
or with 50 µg/mL rat tail collagen type I (B). The figures are
representative of three experiments each.
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| Fig 2.
(A) Inhibition of the binding of 5 × 1011
N-phages/mL to rat tail collagen type I by increasing concentrations of
vWF. (B) Inhibition of the binding of 25 µg/mL vWF to rat tail
collagen type I by increasing concentrations of the N-phage.
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Sequence determination and comparison.
With the use of specific primers, the sequence of the Cys-flanked
hexapeptide inserts could be deduced: the two collagen binding phage
clones carried an almost identical sequence, with a substitution of an
N (N-peptide) for a Q (Q-peptide) at position 6 of the hexapeptide (Fig
3). Sequence comparison with the known vWF
sequence showed the presence of a comparable sequence at position
1129-1136 (Fig 3). This vWF sequence also contained a cysteine, which
in the phage peptide is expected to be part of a disulphide bond.
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| Fig 3.
Sequence comparison of the Q- and N-peptide and the
scrambled peptide with vWF. Identical and similar amino acids are given
in bold and in italics, respectively. A gap is introduced in the Q- and
N-peptide to optimize the alignment.
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Peptide analysis.
Four peptides were synthesized based on the two cyclic sequences, the
putative corresponding vWF sequence, and a scrambled cyclic sequence,
containing the same amino acids as the Q-peptide, but in a random
sequence. Whereas the peptides were not able to compete with vWF for
the binding to the MoAb, the two cyclic peptides inhibited vWF binding
to rat tail collagen type I in a dose-dependent manner. The linear vWF
peptide and the scrambled cyclic peptide on the other hand were
inactive (Fig 4). Concentrations needed for
halfmaximal inhibition of the binding of 25 µg/mL vWF to rat tail
collagen type were 100 ± 12.7 µmol/L and 34.8 ± 8.59 µmol/L (mean ± SEM, n = 3) for the N- and the Q-peptide, respectively. Next, the inhibitory effect of the two cyclic peptides and the scrambled peptide was tested on different types of collagen, such as
calfskin type I and human type I and type III. Both the N- and the
Q-peptide were able to inhibit vWF binding to calfskin type I collagen
(Fig 5A) and to human type I collagen (Fig
5B) but inhibitory concentrations were 5 to 10 times higher than the ones needed for inhibition of vWF binding to rat tail collagen type I. We were not able to detect any inhibition on human type III collagen.
Finally the inhibitory effect of those peptides was tested in an
experiment where vWF in diluted plasma was used. Also, in this test
both peptides showed an inhibitory effect with halfmaximal
concentrations in the same range as in the inhibition assays described
above.
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| Fig 4.
Inhibition of the binding of 25 µg/mL vWF to rat tail
collagen type I by increasing concentrations of peptide. (,
Q-peptide; , N-peptide;  , linear vWF peptide; and  , scrambled
peptide) (mean ± SEM, n = 3).
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| Fig 5.
Inhibition of the binding of 150 ng/mL vWF to calfskin
collagen type I (A) and of 50 ng/mL vWF to human collagen type I (B) by
increasing concentrations of Q- () and N-peptide ()
(mean ± SEM, n = 3).
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DISCUSSION |
In an attempt to identify peptides that may interfere with vWF binding
to collagen, phages from a phage display library were selected on MoAb
82D6A3, an anti-vWF MoAb that prevents vWF binding to fibrillar
collagen.19 A hexapeptide phage display library was used,
in which the hexapeptides were flanked by two cysteines, thus
potentially forming a disulphide-linked octapeptide loop, with a
restricted conformational freedom.
After three rounds of panning, nearly all selected phages bound to MoAb
82D6A3 in a phage enzyme-linked immunosorbent assay. A more stringent
selection criterion was next applied: if MoAb 82D6A3 recognizes a
collagen-binding sequence in vWF, then some of the selected phages may
mimic vWF and should bind to collagen. This
"anti-idiotypic-like" approach resulted in the identification of
two phage clones that bound to both the antibody and to collagen. That
they indeed were binding to similar sites on collagen was further shown
by competition experiments: vWF could displace the phages and vice
versa.
Sequence analysis of the phage hexapeptide inserts showed two nearly
identical sequences, and a similar sequence could be identified in the
vWF sequence at amino acids 1129-1136. The sequence found is not part
of the known collagen binding areas of vWF, but is located 18 amino
acid residues C-terminal of the A3-domain (910-1111).
Although it is quite possible that this linear sequence in vWF
represents the actual epitope of the antibody, especially because the
antibody recognizes denatured vWF in Western blots, this still is
unclear because neither the synthetic cyclic peptide nor the linear
corresponding vWF peptide were able to prevent vWF binding to the
antibody. However, part of the reason for this could be that the
affinity of the antibody for vWF is quite high: antibody concentrations
at or below 1 µg/mL only are needed to observe binding as well as
inhibitory effects (19 and own observations).
Furthermore, the synthetic linear vWF peptide also was not able to
inhibit vWF binding to collagen. This is probably because of the fact
that the peptide may not represent the active conformation found in
vWF. On the other hand, synthetic cyclic peptides with sequences
corresponding to either of the phage peptides inhibited vWF binding to
rat tail, calfskin, and human collagens type I, but were inactive when
vWF binding to human collagen type III was studied. Whereas so far it
is known that different domains in vWF are involved in its binding to
fibrillar collagens type I or III (A3-domain) and to the
microfibrillar collagen type VI (A1-domain), the present
findings also imply that differences in the binding mechanisms of vWF
to collagens type I and III may exist.
That the vWF A3-domain is involved in the binding of vWF to
fibrillar collagens comes from the observation that an
A3-deletion mutant only minimally interacts with collagen,
whereas a recombinant A3-fragment binds to
collagen.14,20 However, the binding of the fragment was
rather weak, which was believed to be because of a higher avidity of
the multimeric vWF for collagen as compared with the monovalent
A3-domain.20 With the identification of a
possible additional collagen-binding sequence within vWF, we hypothesize that the A3-domain and this sequence, which is
situated some 18 amino acids downstream of the A3-domain,
may be both necessary for and cooperate in the binding of vWF to
collagen type I.
To analyze whether the vWF sequence 1129-1136 is really important for
the binding to collagens, we are constructing a mutant vWF and an
extended recombinant A3-domain. At this moment preliminary evidence seems to indicate that residues 1129-1136 of vWF may indeed be
involved in the binding of vWF to rat tail collagen type
I.27
Whatever the further results will be with these constructs, the two
cyclic peptides that we have identified nevertheless inhibit vWF
binding to collagen and therefore may be useful in the development of
new antiadhesive compounds.
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ACKNOWLEDGMENT |
We thank Dr G. Vlasuk (Corvas San Diego) and Dr M. Hoylaerts (Center
for Molecular and Vascular Biology, KU Leuven) for the generous gift of
the cyclic hexapeptide library and MoAb 82D6A3, respectively.
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FOOTNOTES |
Submitted December 8, 1997;
accepted August 4, 1998.
Supported by the following grants: National Fonds voor Wetenschappelijk
Onderzoek 3.0270.95, OT/95/11, VIS/96/15, and
FWO-PDM/97/59 (to A.V.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H. Depraetere, PhD, Laboratory for
Thrombosis Research, IRC, KU Leuven Campus Kortrijk, E Sabbelaan 53, B-8500 Kortrijk, Belgium; e-mail: Hilde.Depraetere{at}kulak.ac.be.
| |
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Top
Abstract
Introduction
