The Platelet Reactivity of Synthetic Peptides Based on the Collagen III Fragment alpha 1(III)CB4
EVIDENCE FOR AN INTEGRIN alpha 2beta 1 RECOGNITION SITE INVOLVING RESIDUES 522-528 OF THE alpha 1(III) COLLAGEN CHAIN*

(Received for publication, October 23, 1996, and in revised form, January 3, 1997)

Laurence F. Morton , Anthony R. Peachey , C. Graham Knight , Richard W. Farndale Dagger and Michael J. Barnes §

From the Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, United Kingdom and the Dagger  Department of Biochemistry, Cambridge University, Cambridge CB2 1QW, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Addendum
REFERENCES


ABSTRACT

The platelet-reactive collagen III-derived fragment alpha 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 alpha 2beta 1-independent except in the case of peptide 6, adhesion to which was partially reduced by anti-integrin alpha 2beta 1 monoclonal antibodies. The presence of an alpha 2beta 1 recognition site in peptide 6 was confirmed using HT 1080 cells, which express alpha 2beta 1 as their major or sole collagen receptor. HT 1080 adhesion to both peptide 6 and collagen was strongly inhibited by anti-integrin alpha 2beta 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 alpha 1(III) chain, represents the minimum structure required for the recognition of alpha 2beta 1. Our findings support the view that the collagen triple helix possesses an intrinsic platelet reactivity that can be expressed independently of integrin alpha 2beta 1 and the precise level of which is governed by the exact nature of the primary sequence. Sequences such as those recognizing alpha 2beta 1 may potentiate the activity, whereas others may have the opposite effect.


INTRODUCTION

Cell interaction with collagens is mediated by specific cell-surface receptors, important among which are the integrins alpha 1beta 1 and alpha 2beta 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 beta 1-integrins alpha 3beta 1, alpha 4beta 1, alpha 5beta 1, and alpha vbeta 1, and the beta 3 integrins alpha IIbbeta 3 and alpha vbeta 3 (1-4). In collagen IV, a triple-helical-dependent alpha 1beta 1-binding site has been described, involving Arg461 of the alpha 2(IV) chain and Asp461 in the alpha 1(IV) chain (5).

alpha 2beta 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 alpha 2beta 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 alpha 2beta 1-binding site has been assigned to the sequence DGEA, which corresponds to residues 435-438 of the alpha 1(I) chain and is found in the CNBr-derived fragment alpha 1(I)CB3 (10).2 However, others have observed no inhibition of alpha 2beta 1-mediated cell adhesion to collagen by DGEA-containing peptides (9, 11-15). Platelet adhesion to alpha 1(I)CB3 is alpha 2beta 1-dependent (9, 16), but the fragment exhibits little platelet aggregatory activity (17). In contrast, the highly structurally homologous collagen III fragment alpha 1(III)CB4 not only supports alpha 2beta 1-mediated adhesion, as reported here, but is also highly aggregatory (17). This suggests that recognition of alpha 2beta 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 alpha 2beta 1-independent (18). The DGEA sequence reported to serve as an alpha 2beta 1 site in alpha 1(I)CB3 is not present in alpha 1(III)CB4. To define more precisely the primary structural requirements of collagen for platelet reactivity and to identify the alpha 2beta 1 site in alpha 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 alpha 2beta 1. A preliminary account of some of this work has been given (14).


EXPERIMENTAL PROCEDURES

Materials

Collagens I and III were purified from bovine skin, following limited pepsin digestion, and the collagen type III-derived fragment alpha 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 alpha 2-subunit) mAb 6F1 (19) was a generous gift from Dr. B. S. Coller, Mount Sinai Hospital, New York, NY. Anti-(human integrin alpha 2-subunit) mAb, clone A2-IIE10, was purchased from TCS Biologicals Ltd., Botolph Claydon, Bucks., United Kingdom (UK), and the anti-(human integrin beta 1-subunit) mAb 13 from Becton Dickinson UK Ltd., Oxford, UK.

Platelet Adhesion and Aggregation

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) Cells

HT 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 Synthesis

Peptides 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.


Fig. 1. Basic structure of the synthetic homotrimers covalently linked at the C termini (A) and sequences of peptides 1-7 (B). The bold sequence is that found in alpha 1(III)CB4, and the numbers refer to residue positions in the alpha 1(III) chain. The overlap between adjacent peptides is underlined. Peptide 7 terminates at residue position 558; the fragment alpha 1(III)CB4 commences at residue 412 and terminates at the methionyl residue at position 560. Ahx in (A) = 6-aminohexanoic acid.
[View Larger Version of this Image (28K GIF file)]


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 alpha 1(III)CB4, were synthesized. The triple-helical stability of each peptide was assessed by polarimetry as described previously (18).

Cross-linking

Peptides were cross-linked with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester as before (18).


RESULTS

The sequence of each of the seven peptides based on the known sequence of bovine alpha 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.


Fig. 2. Melting curve of peptide 6 as a representative example.
[View Larger Version of this Image (14K GIF file)]


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 alpha 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.


Fig. 3. Platelet aggregation by collagen fibers and by cross-linked peptide 6, as a representative example. Aggregation at 20 °C is shown as an increase in light transmittance. The arrows indicate addition of sample at a final concentration (µg/ml) as indicated below. Fibers: a, 0.5; b, 1; c, 2 (6.7 × 10-9 M). Peptide 6: d, 0.2; e, 0.3; f, 0.5; g, 3 (2.4 × 10-7 M).
[View Larger Version of this Image (15K GIF file)]


Platelet Reactivity: Adhesion

Mg2+-dependent platelet adhesion to monomeric collagen type I immobilized on plastic and to the type I-derived fragment alpha 1(I)CB3 at 20 °C is strongly inhibited by anti-integrin alpha 2beta 1 mAbs (9, 16, 19). Adhesion to both monomeric collagen III (26) and (as observed in this study) the type III-derived fragment alpha 1(III)CB4, which is structurally highly homologous with alpha 1(I)CB3, is also Mg2+-dependent and equally well inhibited by anti-integrin alpha 2beta 1 mAbs (data not shown). To identify the alpha 2beta 1 recognition site(s) in alpha 1(III)CB4, we examined adhesion to seven alpha 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 alpha 2beta 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-alpha 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-alpha 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).

Table I.

Divalent cation-dependent platelet adhesion to peptides 1-7

Adhesion was measured at 60 min to collagen I (as the monomer isolated from bovine skin) or peptide adsorbed to Falcon 1008 Petri dishes (at 10 µg/ml for 1 h at 20 °C). Adhesion at 20 °C was in the presence of 2 mM Mg2+ or 2 mM EDTA as indicated. Results are expressed as the mean ± S.E. of three determinations. Exp. a-c, three representative experiments.


Adhesion
Mg2+ EDTA

%
Exp. a
  Collagen 50  ± 1.3 0
  Peptide 1 42  ± 3.8 8  ± 2.0
  Peptide 2 46  ± 4.0 8  ± 0.9
  Peptide 3 43  ± 2.8 6  ± 0.5
  Peptide 4 51  ± 0.7 4  ± 0.1
Exp. b
  Collagen 44  ± 1.5 1  ± 0.2
  Peptide 5 28  ± 1.0 3  ± 0.3
  Peptide 6 31  ± 1.8 4  ± 0.1
Exp. c
  Collagen 49  ± 3.6 0
  Peptide 5 42  ± 1.0 13  ± 0.1
  Peptide 6 42  ± 0.6 3  ± 1.0
  Peptide 7 53  ± 0.4 11  ± 0.4

Table II.

alpha 2beta 1-dependent platelet adhesion to peptide 6 

Adhesion in the presence of 2 mM Mg2+ was measured as described in the legend to Table I. Anti-alpha 2 mAb 6F1 was used at 2 µg/ml and the anti-alpha 2 mAb A2-IIE10 at 5 µg/ml. Exp. a-f, six representative experiments.


Adhesion
Control + mAb 6F1

%
Exp. a
  Collagen 41  ± 0.9 1  ± 0.2
  Peptide 1 40  ± 1.5 42  ± 0.6
  Peptide 2 39  ± 2.0 38  ± 2.2
Exp. b
  Collagen 44  ± 0.2 6  ± 0.1
  Peptide 3 36  ± 0.1 36  ± 0.7
  Peptide 4 40  ± 1.7 38  ± 1.9
Exp. c
  Collagen 45  ± 0.5 1  ± 0.1
  Peptide 5 43  ± 0.9 44  ± 1.4
  Peptide 6 46  ± 2.5 34  ± 3.4
Exp. d
  Collagen 41  ± 2.1 1  ± 0.1
  Peptide 6 44  ± 0.5 32  ± 2.5
Exp. e
  Collagen 47  ± 0.5 7  ± 0.6
  Peptide 4 51  ± 1.9 50  ± 1.6
  Peptide 7 50  ± 0.6 49  ± 1.0
Control + mAb A2-IIE10

Exp. f
  Collagen 58  ± 0.5 5  ± 1.0
  Peptide 1 56  ± 1.5 57  ± 0.7
  Peptide 3 49  ± 1.2 47  ± 2.0
  Peptide 5 45  ± 3.0 46  ± 0.4
  Peptide 6 46  ± 1.0 35  ± 1.0
  Peptide 7 57  ± 1.0 59  ± 1.2

Adhesion of HT 1080 Cells

To verify the presence of an alpha 2beta 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 alpha 2beta 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 alpha 2beta 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 alpha 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-alpha 2beta 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-alpha 2 mAbs, confirming the presence of an alpha 2beta 1 recognition site in peptide 6. 

Table III.

The adhesion of HT 1080 cells to collagen and peptide 6 

The adhesion of HT 1080 cells to either monomeric collagen type I or peptide 6 was measured at 20 °C, as described under "Experimental Procedures," in the presence or absence of anti-alpha 2 mAbs (6F1, 2 µg/ml, or A2-IIE10, 5 µg/ml). Data shown are the means of at least four determinations, each performed in triplicate. Significance was determined using ANOVA; *, p < 0.001; **, p < 0.01. 


Adhesion (mean ± S.E.)
Control + mAb

%
Collagen 74  ± 2.8 9.1  ± 2.4*
Peptide 6 32  ± 14.4 1.5  ± 0.3**


DISCUSSION

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 alpha 2beta 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 alpha 2beta 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 alpha 2beta 1 (7-10, 16, 19). Adhesion to the peptides was also Mg2+-dependent, but only peptide 6 revealed any dependence on integrin alpha 2beta 1 for adhesion, indicating the existence of an additional divalent-cation dependent mechanism of adhesion that is not mediated by alpha 2beta 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 alpha 2beta 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 alpha 2beta 1-dependent; and a third, which is both cation- and alpha 2beta 1-independent.

Partial inhibition of platelet adhesion to peptide 6 by anti-alpha 2beta 1 mAbs indicated the presence of an alpha 2beta 1 recognition sequence in this peptide, which was confirmed with HT 1080 cells. In contrast to platelets, these cells appear only to utilize alpha 2beta 1-dependent adhesion since, of the seven peptides, only peptide 6 was able to support significant adhesion and this was inhibitable with anti-integrin alpha 2beta 1 mAbs, confirming the presence of an alpha 2beta 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 alpha 2beta 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 alpha 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 alpha 2beta 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.


FOOTNOTES

*   This work was supported by the Medical Research Council of the United Kingdom (of which M. J. B., L. F. M., and A. R. P. are members of the External Staff).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 44-1223-243231; Fax: 44-1223-411609.
1   Standard single-letter nomenclature is used to describe peptide sequences, with P* representing hydroxyproline.
2   The abbreviations used are: CB, cyanogen bromide (in collagen fragment nomenclature); Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DMF, N,N-dimethylformamide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; mAb, monoclonal antibody.
3   V. Glattauer, J. A. Werkmeister, A. Kirkpatrick, and J. A. M. Ramshaw, personal communication (submitted for publication).

Addendum

After completion of this work, Fields and colleagues (41) reported that the sequence GPQGIAGQRGVVGLP*, residues 772-786 of the collagen type I alpha 1(I) chain, could support conformation-dependent adhesion of skin fibroblasts. Adhesion was partially blocked by anti-beta 1 integrin subunit mAb, and it was tentatively concluded that the sequence may represent an integrin recognition site.


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  33. Morton, L. F., Peachey, A. R., and Barnes, M. J. (1989) Biochem. J. 258, 157-163 [Medline] [Order article via Infotrieve]
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  36. Achison, M., Joel, C., Hargreaves, P. G., Sage, S. O., Barnes, M. J., and Farndale, R. W. (1996) Blood Coagul. Fibrinolysis 7, 149-152 [Medline] [Order article via Infotrieve]
  37. Asselin, J., Gibbins, J. M., Achison, M., Lee, Y. H., Morton, L. F., Farndale, R. W., Barnes, M. J., and Watson, S. P. (1997) Blood 89, 1235-1242 [Abstract/Free Full Text]
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