(Received for publication, October 23, 1996, and in revised form, January 3, 1997)
From the Strangeways Research Laboratory, Worts Causeway, Cambridge
CB1 4RN, United Kingdom and the Department of
Biochemistry, Cambridge University,
Cambridge CB2 1QW, United Kingdom
The platelet-reactive collagen III-derived
fragment 1(III)CB4 has been synthesized as seven overlapping
peptides, each as a homotrimeric triple-helical species covalently
linked at the C terminus. Additional Gly-Pro-Hyp triplets were
introduced at each end of the peptide sequence to ensure a stable
triple-helical conformation at 20 °C, the temperature at which cell
reactivity was measured. A Cys-containing triplet was included at each
end to allow intermolecular cross-linking. All seven peptides in
triple-helical, cross-linked form were able to cause platelet
aggregation. Peptide 6, the most reactive species, was more aggregatory
than collagen fibers. Platelet adhesion occurred to all peptides
immobilized on plastic in monomeric form. Adhesion was integrin
2
1-independent except in the case of
peptide 6, adhesion to which was partially reduced by anti-integrin
2
1 monoclonal antibodies. The presence of
an
2
1 recognition site in peptide 6 was
confirmed using HT 1080 cells, which express
2
1 as their major or sole collagen receptor. HT 1080 adhesion to both peptide 6 and collagen was strongly
inhibited by anti-integrin
2
1 monoclonal
antibodies. These cells did not adhere to any of the other peptides.
Comparison of the structure of peptide 6 with that of adjacent peptides
indicates that the sequence Gly-Gly-Pro-Hyp-Gly-Pro-Arg, residues
522-528 of the collagen
1(III) chain, represents the minimum
structure required for the recognition of
2
1. Our findings support the view that
the collagen triple helix possesses an intrinsic platelet reactivity
that can be expressed independently of integrin
2
1 and the precise level of which is
governed by the exact nature of the primary sequence. Sequences such as
those recognizing
2
1 may potentiate the
activity, whereas others may have the opposite effect.
Cell interaction with collagens is mediated by specific
cell-surface receptors, important among which are the integrins
1
1 and
2
1
(1). Integrins recognize discrete amino acid sequences, the best known
of which, perhaps, is the RGD1 sequence,
which serves as a cell-binding site in several matrix proteins and is
recognized by a number of integrins, including the
1-integrins
3
1,
4
1,
5
1, and
v
1, and the
3 integrins
IIb
3 and
v
3
(1-4). In collagen IV, a triple-helical-dependent
1
1-binding site has been described,
involving Arg461 of the
2(IV) chain and
Asp461 in the
1(IV) chain (5).
2
1 is a platelet receptor that may be
important in the activation of platelets by collagen in hemostasis, an
event that may be expressed pathologically as thrombosis (6-8).
Fragmentation studies have indicated the presence in collagen I of
several platelet
2
1-binding sites whose
recognition is dependent on collagen triple-helical conformation (9).
On the basis of inhibition of platelet adhesion to this collagen by
short, linear (non-triple-helical) peptides, an
2
1-binding site has been assigned to the
sequence DGEA, which corresponds to residues 435-438 of the
1(I)
chain and is found in the CNBr-derived fragment
1(I)CB3
(10).2 However, others have observed no
inhibition of
2
1-mediated cell adhesion
to collagen by DGEA-containing peptides (9, 11-15). Platelet adhesion
to
1(I)CB3 is
2
1-dependent
(9, 16), but the fragment exhibits little platelet aggregatory activity (17). In contrast, the highly structurally homologous collagen III
fragment
1(III)CB4 not only supports
2
1-mediated adhesion, as reported here,
but is also highly aggregatory (17). This suggests that recognition of
2
1 may be insufficient for platelet activation and that recognition of additional reactive sequences in
collagen by other receptors may be required. Pertinent to this, we have
recently described potent platelet activation by simple collagen-like
synthetic peptides comprising a GPP* repeat sequence, whose activity is
totally
2
1-independent (18). The DGEA
sequence reported to serve as an
2
1 site
in
1(I)CB3 is not present in
1(III)CB4. To define more precisely
the primary structural requirements of collagen for platelet reactivity
and to identify the
2
1 site in
1(III)CB4, we have synthesized this fragment as an overlapping series of triple-helical peptides. Here we describe studies on the
ability of these peptides to support platelet adhesion and activation,
and the adhesion of HT 1080 cells, which also express the integrin
2
1. A preliminary account of some of this
work has been given (14).
Collagens I and III were purified from bovine
skin, following limited pepsin digestion, and the collagen type
III-derived fragment 1(III)CB4 isolated from the purified parent
collagen, as described previously (9, 17). Bovine tendon collagen (type I) fibers, dialyzed and diluted using 0.01 M acetic acid
(17), were a gift from Ethicon Inc., Somerville, NJ, and were used as a
standard platelet aggregatory agent.
The anti-(human integrin 2-subunit) mAb 6F1 (19) was a
generous gift from Dr. B. S. Coller, Mount Sinai Hospital, New York, NY. Anti-(human integrin
2-subunit) mAb, clone A2-IIE10,
was purchased from TCS Biologicals Ltd., Botolph Claydon, Bucks., United Kingdom (UK), and the anti-(human integrin
1-subunit) mAb 13 from Becton Dickinson UK Ltd., Oxford,
UK.
Platelet adhesion was measured in Falcon 1008 35-mm Petri dishes using 51Cr-labeled gel-filtered human platelets as described (18). When testing mAbs for inhibitory activity, platelets were preincubated with antibody for 15 min. The effect of mAbs on platelet adhesion was tested by one-way ANOVA (analysis of variance), either within experiments or using the mean values from at least three separate experiments.
Platelet aggregation was measured turbidimetrically using human citrated platelet-rich plasma as previously (18).
Adhesion of Human Fibrosarcoma (HT 1080) CellsHT 1080 cells, from the European Collection of Animal Cell Cultures, Porton Down, Wilts., UK, were maintained in Eagle's minimal essential medium containing 15% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin. Cells were harvested with trypsin/EDTA, suspended in Eagle's minimal essential medium containing 20% fetal bovine serum, washed four times with Dulbecco's phosphate-buffered saline solution (Ca2+- and Mg2+-free) and finally suspended in adhesion buffer (Tris-buffered saline solution) containing 1 mM Mg2+. Immulon 2 multiwell plates were coated with collagen or peptide, normally at 10 µg/ml, for 1 h at 20 °C. Cell suspension (0.1 ml, 3 × 104 cells) was added to each well and adhesion measured after 90 min at 20 or 37 °C, as required. Adhesion was determined using a Coulter Counter (model ZF) to count unattached cells. Cells were preincubated with antibody, when testing for inhibition, for 15 min. The significance of any inhibition of adhesion by mAbs was tested using the same statistical analysis as for platelets.
Peptide SynthesisPeptides were synthesized as homotrimers
covalently bonded at the C terminus, as shown in Fig. 1A,
essentially as described by Fields and colleagues (20, 21), and
employing standard Fmoc chemistry. The initial synthesis of a branched
peptide structure on the resin was undertaken manually. For each
synthesis, 0.25 g of Fmoc-peptide amide linker-polyethylene
glycol-polystyrene resin (PerSeptive Biosystems, Hertford, UK; 0.2 mmol/g), preswollen in DMF, were loaded into a 10-mm diameter column
and washed with DMF. Subsequent DMF washes, at 3.3 ml/min, were for 10 min prior to deprotection and for 20 min after deprotection. Fmoc amino acids were double-coupled using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N,N-diisopropylethylamine chemistry (22) for 60 min employing a 4-fold excess of reagents. Fmoc groups were
removed with a mixture of 2% 1,8-diazabicyclo(5.4.0)undec-7-ene and
2% piperidine in DMF for 30 min. The Dde protecting group was removed
with 2% hydrazine hydrate in DMF for 30 min. Coupling and deprotection
at each step was monitored using the appropriate color tests.
The resin was initially coupled to Fmoc-6-aminohexanoic acid-OH to introduce a spacer arm. The trimeric frame was then generated by first coupling Fmoc-Lys(Dde)-OH, followed by Fmoc-Lys(Fmoc)-OH. Removal of both Fmoc groups and the Dde group produced three amino groups to each of which was attached an 6-aminohexanoic acid spacer arm, providing three foci for subsequent peptide synthesis. Amino acid analysis at this stage verified the structure of the branched peptide resin.
The remainder of the synthesis was carried out automatically on a PerSeptive Biosystems 9050 Plus Pepsynthesizer using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate chemistry and deprotection with 20% piperidine in DMF. The completed peptide was cleaved from the resin with 88% trifluoroacetic acid, 5% phenol, 5% water and 2% triisopropylsilane. Peptides were purified using reverse-phase chromatography (23) and the composition verified by amino acid analysis.
In all, seven overlapping peptides, designated 1-7, based on the
sequence of the fragment 1(III)CB4, were synthesized. The triple-helical stability of each peptide was assessed by polarimetry as
described previously (18).
Peptides were cross-linked with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester as before (18).
The sequence of each of the seven peptides based on the known
sequence of bovine 1(III)CB4 (24) is shown in Fig.
1B. Each sequence was constructed as a
homotrimer covalently bound at the C terminus, as indicated in Fig.
1A, to promote correct alignment of the three chains as they
adopt a triple-helical conformation, necessary for the expression of
platelet reactivity (25). Initial studies indicated that each sequence
as a covalently bonded trimer was only able to form a triple-helical
structure at relatively low temperatures. Additional GPP* triplets were
therefore added at each end, as indicated in Fig. 1B, to
allow the adoption of a triple helix stable at 20 °C, the minimum
temperature at which cell reactivity of the peptides could be measured.
A GCP* triplet was also included at each end to allow cross-linking to
produce a polymer, since collagen quaternary structure is also
essential for the expression of its aggregatory activity (25). Melting temperatures (Tm1/2) were as follows:
peptide 1, 33 °C; 2, 25 °C; 3, 25 °C; 4, 34 °C; 5, 26 °C; 6, 27 °C; 7, 26 °C. A representative melting
curve is shown in Fig. 2.
Platelet Reactivity: Aggregation
Following cross-linking, all
seven peptides exhibited platelet aggregatory activity at 20 °C. The
minimum concentration (µg/ml) required for activity was as follows:
peptide 1, 20; peptide 2, 30; peptide 3, 30; peptide 4, 1; peptide 5, 500; peptide 6, 0.5; and peptide 7, 5. This represents a 1000-fold
difference in activity between the most and the least reactive. In
accord with the melting temperatures and the need for a collagen
triple-helical conformation for platelet reactivity (25), none of the
peptides exhibited aggregatory activity at 37 °C. In accord with the
requirement for a quaternary structure for collagen to express
aggregatory activity (25), none of the uncross-linked peptides showed
activity. For any given peptide, aggregatory activity recorded from one cross-linked sample to another was relatively consistent. Thus peptides
1-3 were always of moderate activity, whereas peptide 5 showed low
activity. Peptide 6 was highly active (more so than collagen fibers,
which were active at 2 µg/ml, and the parent collagen type III
polymerized by random cross-linking, active at 10-20 µg/ml; Ref. 17)
and was of comparable activity to the collagen type III fragment
1(III)CB4 (randomly cross-linked) from which it is derived (17).
Peptide 7 showed activity intermediate between peptides 1-3 and
peptide 6. For reasons unknown, the activity of peptide 4 was variable.
Usually, the peptide showed activity comparable to or greater than
collagen fibers, but occasionally the peptide showed no activity even
when tested at concentrations up to 250 µg/ml. Platelet aggregation
by peptide 6 is shown by way of example in Fig. 3.
Platelet Reactivity: Adhesion
Mg2+-dependent platelet adhesion
to monomeric collagen type I immobilized on plastic and to the type
I-derived fragment 1(I)CB3 at 20 °C is strongly inhibited by
anti-integrin
2
1 mAbs (9, 16, 19).
Adhesion to both monomeric collagen III (26) and (as observed in this
study) the type III-derived fragment
1(III)CB4, which is
structurally highly homologous with
1(I)CB3, is also Mg2+-dependent and equally well inhibited by
anti-integrin
2
1 mAbs (data not shown).
To identify the
2
1 recognition site(s) in
1(III)CB4, we examined adhesion to seven
1(III)CB4-based
peptides. Good adhesion occurred at 20 °C to all the peptides in the
presence of Mg2+ and was strongly decreased in the presence
of EDTA (Table I). In accord with the need for a
triple-helical conformation for adhesion (9), no adhesion to peptides
occurred at 37 °C (data not shown). mAbs against the integrin
2
1 failed to inhibit adhesion except in
the case of peptide 6, adhesion to which was reduced by a maximum of
around 30%. Typical inhibition data are shown in Table
II. Analysis of pooled data from Table II and other
experiments showed that anti-
2 mAbs reduced the adhesion
of platelets to monomeric collagen (type I) from 44 ± 1.5% to
2.3 ± 0.5% (p < 0.001) and to peptide 6 from
44 ± 1.4% to 34 ± 0.9% (p < 0.01). The
anti-
2 mAbs exerted no significant effect on platelet
adhesion to any other peptide. Higher concentrations of mAb beyond
those utilized here with peptide 6 (as shown in Table II) gave the same results (data not shown).
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To verify the presence of an
2
1 recognition sequence in peptide 6, we
also examined the adhesion of HT 1080 cells, which are known to adhere
to collagen (type I) via
2
1 (11, 15, 27-29). We found good adhesion (up to 80% at 37 °C at 90 min) to both collagens I and III, that was
Mg2+-dependent and was inhibited by
anti-integrin
2
1 mAbs. Inhibition by
antibody was generally
90%, but on occasion a residual adhesion, up
to around 30% of the total, remained (results not shown), as reported
by others (11). Adhesion to the type III CNBr-derived fragment
1(III)CB4 at 20 °C occurred at about 75% of that to the parent
collagen, was similarly Mg2+-dependent, and was
as effectively blocked by anti-
2
1 mAbs
(data not shown). No significant adhesion occurred to any of the seven synthetic triple-helical peptides except peptide 6. As can be seen from
the data presented in Table III, adhesion occurred to this peptide although some variation in level was noted between experiments. Irrespective of the actual level, adhesion was strongly inhibited by anti-
2 mAbs, confirming the presence of an
2
1 recognition site in peptide 6.
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The ability of all seven peptides to cause platelet aggregation is
consistent with our proposal that the collagen triple helix possesses
an intrinsic platelet reactivity (18). The large variation in
aggregatory activity between peptides further emphasizes the important
influence of the primary sequence on this activity. We have previously
argued that the intrinsic activity of the helix will be modified by the
presence of stimulatory or inhibitory sequences within the collagen
primary structure (18). The high activity of peptide 4 is consistent
with the presence of the sequence GKP*GEP*GPKGEA, which we and others
have previously proposed may serve as a platelet activation signal in
collagen III (17, 30-32). Likewise, the potency of peptide 6 is
consistent with the evidence presented here that this peptide contains
an 2
1 recognition sequence. Our data lend
support to the view (33, 34) that collagen-platelet interaction is a
two-step process involving an initial recognition of adhesive
sequences, such as those recognized by
2
1, as a primary interaction that may be
especially important to retain platelets under flow conditions (7, 35).
Subsequent engagement with a second receptor induces signaling leading
to platelet activation (aggregation). We consider that this signaling receptor recognizes the collagen triple helix. In support of this, it
has been found that the platelet-reactive (GPP*)10-based
peptides (18) induce in platelets signaling events identical to those evoked by collagen (36-39). Platelet activation in response to recognition of the triple helix may be enhanced by the presence within
the helix of specific "activation" sequences such as the putative
activation sequence in peptide 4.
All seven peptides were able to support
conformation-dependent platelet adhesion under static
conditions at a level comparable to that occurring to collagen.
Adhesion to triple-helical monomeric collagen is
Mg2+-dependent and mediated by the integrin
2
1 (7-10, 16, 19). Adhesion to the
peptides was also Mg2+-dependent, but only
peptide 6 revealed any dependence on integrin
2
1 for adhesion, indicating the existence
of an additional divalent-cation dependent mechanism of adhesion that
is not mediated by
2
1. We have previously
noted that polymeric collagen, i.e. collagen fibers, and the
highly reactive synthetic (GPP*)10-based peptides, which
spontaneously form micropolymers, support substantial adhesion by a
divalent cation-independent mechanism not involving
2
1 (9, 18). At least three mechanisms of
platelet adhesion under static conditions can therefore be discerned:
two cation-dependent, of which only one is
2
1-dependent; and a third,
which is both cation- and
2
1-independent.
Partial inhibition of platelet adhesion to peptide 6 by
anti-2
1 mAbs indicated the presence of an
2
1 recognition sequence in this peptide,
which was confirmed with HT 1080 cells. In contrast to platelets, these
cells appear only to utilize
2
1-dependent adhesion since,
of the seven peptides, only peptide 6 was able to support significant
adhesion and this was inhibitable with anti-integrin
2
1 mAbs, confirming the presence of an
2
1 recognition site in this peptide. The
reason for the variation between experiments in the level of adhesion
to this peptide is not known, but may suggest that presentation of the
2
1 binding site in the correct conformation is crucial for these cells, and that the conformation may
be affected or obscured in some way during the coating procedure.
From a comparison of the primary structure of peptide 6 with those of
the two adjacent inactive peptides 5 and 7 (see Fig. 1), it is possible
to deduce that GGPP*GPR, equivalent to residues 522-528 of the
1(III) collagen triple-helical chain (24), is the only sequence
unique to peptide 6 and must represent the minimum structure involved
in the recognition of integrin
2
1. In
support of the importance of this sequence in collagen-platelet
interaction, it has been found that a mAb directed against human
collagen III (40) and able to inhibit collagen III-induced platelet
aggregation recognizes an epitope corresponding to residues 520-528 of
the type III molecule.3 Integrin
recognition sites generally contain a negatively charged residue,
normally Asp (3). Conceivably the Glu residue in peptide 6 at residue
position 515 or, perhaps less likely, that at position 537 may fulfill
this requirement.
After completion of this work, Fields and colleagues
(41) reported that the sequence GPQGIAGQRGVVGLP*, residues 772-786 of
the collagen type I 1(I) chain, could support
conformation-dependent adhesion of skin fibroblasts.
Adhesion was partially blocked by anti-
1 integrin
subunit mAb, and it was tentatively concluded that the sequence may
represent an integrin recognition site.