Polypeptides with type IV collagen activity

A composition which can bind heparin and promote cellular adhesion is provided which consists essentially of a polypeptide of the formula: PA1 met-phe-lys-lys-pro-thr-pro-ser-thr-leu-lys-ala-gly-glu-leu-arg, PA1 thr-ala-gly-ser-cys-leu-arg-lys-phe-ser-thr-met, PA1 asn-pro-leu-cys-pro-pro-gly-thr-lys-ile-leu, or mixtures thereof. Medical devices such as prosthetic implants, percutaneous devices, bandages and cell culture substrates coated with the polypeptide composition are also provided.

BACKGROUND OF THE INVENTION 
Type IV collagen is a distinctive glycoprotein which occurs almost 
exclusively in basement membranes. It differs from the other types of 
collagen, which are found mainly in interstitial connective tissue, with 
regard to several structural properties. See New Trends in Basement 
Membrane Research, K. Kuehn et al., eds., Raven Press, N.Y. (1982) at 
pages 57-67. It has a molecular weight (MW) of about 500,000 and consists 
of three polypeptide chains: two .alpha.1 (MW 185,000) chains and one 
.alpha.2 (MW 170,000) chain. Type IV collagen has two major domains: a 
large, globular, non-collagenous, NC1 domain and another major 
triple-helical, collagenous domain. The latter domain is only partially 
short, non-collagenous sequences. The amino acid sequence of this 
collageous domain is only partially known; however, the sequences of both 
the .alpha.1- and .alpha.2-chains of the non-collagenous NC1 domain is 
known. See U. Schwartz-Magdolen et al., FEBS Letters, 208, 203 (1986), and 
references cited therein. A diagrammatic representation of the type IV 
collagen molecule is shown in FIG. 1. Apparently, type IV collagen is a 
very complex and multidomain protein with different biological activities 
residing in different domains. 
Type IV collagen is an integral component of basement membranes because it 
self-assembles to higher forms which make up the supportive matrix of 
these structures. Various other macromolecular components of basement 
membranes are thought to assemble on this supportive framework. For 
example, laminin, nidogen and haparan sulfate proteoglycan have been 
reported to bind to type IV collagen. Laminin was observed to bind to two 
distinct sites along the length of the helix-rich, collagenous domain of 
type IV collagen. Nidogen and heparan sulfate proteoglycan were observed 
to bind specifically to the non-collagenous NC1 domain. Another property 
of type IV collagen is the ability to self-assemble by end-to-end and 
lateral associations, as mentioned hereinabove. The end-product of the 
polymerized structure is an irregular polygonal network. The NC1 domain is 
required for network formation because it binds along the length of the 
helix-rich domain and brings adjacent molecules together, thus initiating 
lateral assembly. In the absence of lateral assembly, only end-to-end 
associations occur and the network-structure cannot be formed. 
An additional function of type IV collagen is the binding to various cell 
types via cell surface receptors [M. Kurkinen et al., J. Biol. Chem., 259, 
5915 (1984)]. M. Kurkinen et al. have reported that a major surface 
receptor protein with a molecular weight of 47,000 mediates this binding 
in the case of mouse embryo parietal endodermal cells. 
The variety of functions attributed to type IV collagen indicates that this 
protein is an important reactant in many diverse and clinically important 
processes such as basement membrane assembly, cell migration, wound 
healing, tumor cell metastasis, diabetic microangiopathy, vascular 
hypertrophy due to hypertension and several kidney diseases. For example, 
Goodpastures's syndrome, a disease characterized by hemoptysis and 
hematuria due to alveolitis and nephritis, respectively, is associated 
with the presence of and antibody to the NC1 domain of type IV collagen in 
the serum of all Goodpasture's patients. Another hereditary kidney 
disease, Aport's familial nephritis is apparently due to a genetic defect 
of the NC1 domain of type IV collagen. Finally, in diabetes mellitus, 
intact type IV collagen, as well as the helix-rich domains, are chemically 
modified and functionally impaired by the increased amounts of glucose in 
the plasma and in the immediate vicinity of the basement membranes, i.e., 
in the extracellular matrix. 
In order to better understand the pathophysiology of these processes at a 
molecular level, there is a need to try to assign each of the biological 
activities that type IV collagen exhibits to a specific subdomain (i.e., 
NC1, helix-rich) or aligopeptide of type IV collagen. If this can be 
achieved, it may be possible to synthesize small peptides which can 
provide the basis for important pharmaceutical compositions. 
BRIEF DESCRIPTION OF THE INVENTION 
The present invention provides three polypeptides which formally represent 
fragments of the .alpha.1-NC1 chain of type IV collagen. The polypeptides, 
which can be prepared by conventional solid phase peptide synthesis, can 
be represented by the formulas: 
EQU met-phe-lys-lys-pro-thr-pro-ser-thr-leu-lys-alagly-glu-leu-arg (I), 
EQU thr-ala-gly-ser-cys-leu-arg-lys-phe-ser-thr-met (II), 
EQU and 
EQU asn-pro-leu-cys-pro-pro-gly-thr-lys-ile-leu (III). 
Polypeptide I, (hereinafter "TS-1") formally represents amino acids 201-216 
of the .alpha.1-NC1 domain, and can be represented by the single letter 
code: MFKKPTPSTLKAGELR. Polypeptide II formally represents isolated 
.alpha.1-NC1 amino acid residues 49-50, while polypeptide III formally 
represents isolated .alpha.1-NC1 amino acid residues 17-27. The single 
letter amino acid codes for these polypeptides are TAGSCLRKFSTM and 
NPLCPPGTKIL, respectively. For brevity, polypeptide II will be hereinafter 
designated as "TS-2". and polypeptide III will be designated as "TS-3". 
Some of their properties, such as hydropathy index, net charge and number 
of lysines, are shown in Table I, below. 
TABLE I 
______________________________________ 
Position in 
the .alpha.1--NCl Chain 
Hydro- Number of 
(a.a. No. from 
pathy* Net Lysine 
Peptide 
the NM.sub.2 --terminus) 
lndex Charge Residues 
______________________________________ 
TS--1 201-216 -149 +3 3 
TS--2 49- 60 +11 +2 1 
TS--3 17- 27 -26 0 1 
______________________________________ 
*Kyte and Doolittle, J. Mol. Biol., 157, 105 (1982). 
These synthetic polypeptides were assayed for bioactivity and TS-2 and TS-3 
were found to (a) bind to type IV collagen and to the isolated NC1 domain 
thereof (TS-2 only), (b) bind to heparin, and (c) to inhibit the binding 
of heparin to type IV collagen. TS-1, TS-2 and TS-3 promote the adhesion 
of aortic endothelial cells and bind to melanoma cells (TS-3 only). 
Therefore it is believed that TS-1, TS-2 and TS-3 may be useful to (a) 
promote wound healing and implant acceptance, (b) promote cellular 
attachment to culture substrata and/or (c) inhibit the metastasis of 
malignant cells. Due to the difference in the spectra of biological 
activities exhibited by polypeptides TS-1, TS-2 and TS-3, mixtures of 
TS-1, TS-2 and TS-3 are also within the scope of the invention. 
Furthermore, since it is expected that further digestion/hydrolysis of 
polypeptides TS-2 and TS-3 in vitro and in vivo will yield fragments of 
substantially equivalent bioactivity, such lower molecular weight 
polypeptides are considered to be within the scope of the present 
invention.

DETAILED DESCRIPTION OF THE INVENTION 
The amino acid sequence of the helix-rich collagenous part of the .alpha.1 
chain has been partially described by W. Babel et al., Eur. J. Biochem., 
143, 545 (1984). The full sequence of the noncollagenous .alpha.1-NC1 is 
shown in FIG. 2. The sequence information available on the globular NC1 
domain of the .alpha.1 chain was examined and the three polypeptides of 
the invention, designated TS-1, TS-2 and TS-3, were synthesized for 
further evaluation. 
Synthesis of Polypeptides 
The polypeptides of the invention were synthesized using the Merrifield 
solid phase method. This is the method most commonly used for peptide 
synthesis, and it is extensively described by J. M. Stewart and J. D. 
Young in solid Phase Peptide Synthesis, Pierce Chemical Company, pub., 
Rockford, Il. (2d ed., 1984), the disclosure of which is incorporated by 
reference herein. 
The Merrifield system of peptide synthesis uses a 1% crosslinked 
polystyrene resin functionalized with benzyl chloride groups. The 
halogens, when reacted with the salt of a protected amino acid will form 
an ester, linking it covalently to the resin. The benzyloxy-carbonyl (BOC) 
group is used to protect the free amino group of the amino acid. This 
protecting group is removed with 25% trifluoroacetic acid (TCA) in 
dichloromethane (DCM). The newly exposed amino group is converted to the 
free base by 10% triethylamine (TEA) in DCM. The next BOC-protected amino 
acid is then coupled to the free amine of the previous amino acid by the 
use of dicyclohexylcarbodiimide (DCC). Side chain functional groups of the 
amino acids are protected during synthesis by TFA stable benzyl 
derivatives. All of these repetitive reactions can be automated, and the 
peptides of the present invention were synthesized at the University of 
Minnesota Microchemical facility by the use of a Beckman System 990 
Peptide synthesizer. 
Following synthesis of a blocked polypeptide on the resin, the resin-bound 
polypeptide is treated with anhydrous hydrofluoric acid (HF) to cleave the 
benzyl ester linkage to the resin and thus to release the free 
polypeptide. The benzyl-derived side chain protecting groups are also 
removed by the HF treatment. The polypeptide is then extracted from the 
resin, using 1.0 M acetic acid, followed by lyophilization of the extract. 
Lyophilized crude polypeptides are purified by reparative high performance 
liquid chromatography (HPLC) by reverse phase technique on a C-18 column. 
A typical elution gradient is 0% to 60% acetonitrile with 0.1% TFA in 
H.sub.2 O. Absorbance of the eluant is monitored at 220 nm, and fractions 
are collected and lyophilized. 
Characterization of the purified polypeptide is by amino acid analysis. The 
polypeptides are first hydrolyzed anaerobically for 24 hours at 
110.degree. C. at 6M HCl (constant boiling) or in 4N methanesulfonic acid, 
when cysteine or tryptophane are present. The hydrolyzed amino acids are 
separated by ion exchange chromatography using a Beckman System 6300 amino 
acid analyzer, using citrate buffers supplied by Beckman. Quantitation is 
by absorbance at 440 and 570 nm, and comparison with standard curves. The 
polypeptides may be further characterized by sequence determination. This 
approach is especially useful for longer polypeptides, where amino acid 
composition data are inherently less informative. Sequence determination 
is carried out by sequential Edman degradation from the amino terminus, 
automated on a Model 470A gas-phase sequenator (Applied Biosystems, Inc.), 
by the methodology of R. M. Hewick et al., J. Biol. Chem., 256, 7990 
(1981). 
The invention will be further described by reference to the following 
detailed examples. 
EXAMPLE 1 
Liquid Phase Collagen Binding Assay 
The ability of these peptides to bind to type IV collagen and to the NC1 
domain was evaluated by a variety of approaches. In the first place, 
network formation by prewarmed intact type IV collagen was examined via 
the technique of rotary shadowing at the electron microscopic level. Table 
II lists the permutations which were tested and statistically evaluated 
(by counting the number of fields which contained irregular polygonal 
networks). 
TABLE II 
______________________________________ 
Run Material 
______________________________________ 
(1) type IV collagen alone (control). 
(2) type IV collagen and albumin (BSA; 
another control). 
(3) type IV collagen and isolated NCl 
domain (control NCl).* 
(4) type IV collagen and diabetically 
modified or glucosylated NCl domain. 
(5) type IV collagen and peptide TS--1. 
(6) type IV collagen and peptide TS--2. 
(7) type IV collagen and peptide TS--3. 
______________________________________ 
*The NCl domain was isolated by collagenase treatment of murine, 
EHSderived, purified type IV collagen, as described by Tsilibary et al. 
(J. Cell. Biol., 103, 2467-2473 (1986)). 
The comparative assembly data obtained from permutations 1-4 are shown in 
FIG. 3A. The comparative assembly data obtained from permutations 5-7 are 
shown in FIG. 3B, which also shows the results obtained using type IV 
collagen with control NC1 domain and with glucosylated NC1 domain. 
The data summarized on Tables 3A and 3B confirm that: (a) control NC1 
domain binds to type IV collagen and competes for network formation; and 
(b) diabetically modified or glycosylated NC1 domain cannot bind to type 
IV collagen because it cannot compete for network formation. Specifically, 
it was observed that polypeptide TS-2 can mimic the effect of intact, 
control NC1 domain, since it can effectively prevent network formation. 
The other two peptides, TS-1 and TS-3, do not have any effect on the 
assembly of type IV collagen (FIG. 3B). 
EXAMPLE 2 
Solid Phase Collagen Binding Assay 
The binding of the intact NC1 domain and polypeptides TS-2 and TS-3 to type 
IV collagen was also examined by solid phase binding assays. Type IV 
collagen was coated onto a plastic surface and the binding of isolated NC1 
domain was examined by adding increasing concentrations of .sup.125 
I-labelled NC1 domain (isolated from purified type IV collagen by 
treatment with collagenase). These data are shown in FIG. 4. It was 
observed that increasing amounts of .sup.125 I-NC1 bound to type IV 
collagen as the concentration of NC1 added was increased, until a plateau 
was reached, indicating that all available sites for binding to the NC1 
domain were occupied. This saturable binding of .sup.125 I-NC1 to type IV 
collagen was specific since it could be competed for by an excess of 
unlabelled NC1 domain (FIG. 4). 
Analysis of the affinity of the binding of NC1 domain to type IV collagen 
by a Scatchard plot indicated two classes of binding sites with two 
different binding constants were present (FIG. 5). The two binding 
constants were 40 nM and 330 nM, respectively. 
Type IV collagen in PBS (10 .mu.g/ml, 50 .lambda.) was added to plastic 
wells and allowed to bind to the plastic surface by incubation overnight 
at 4.degree. C. Subsequently, .sup.125 I-labelled NC1 domain was allowed 
to interact with the immobilized type IV collagen in the presence of BSA, 
peptide TS-2 or peptide TS-3. When peptide TS-3 was added in solutions of 
.sup.125 I-NC1 , it did not have any effect on the subsequent binding of 
.sup.125 I-NC1 to type IV collagen (FIG. 6). The presence of peptide TS-2 
in solutions of .sup.125 I-NC1 caused a dramatic increase of the binding 
of .sup.125 I-NC1 to type IV collagen until a plateau was reached (FIG. 
6). These data indicate that peptide TS-2 can bind to type IV collagen and 
also to the isolated NC1 domain. This double binding ability of the 
peptide would account for the observed increase in binding of the NC1 
domain to type IV collagen when higher concentrations of peptide TS-2 are 
used. 
EXAMPLE 3 
Heparin Binding Assays 
In order to test the ability of the present polypeptides to bind to 
heparin, a solution of 2 mg/ml of each polypeptide in 50 mM ammonium 
bicarbonate (pH 7.8) was prepared and then serially diluted 1:1 in the 
same buffer to produce concentrations from 2 mg/ml to 1 .mu.g/ml. One 
hundred .alpha.1 from each dilution were incubated with .sup.3 H-heparin 
(50,000 dpm/ml) for two hours at 37.degree. C. and the mixtures were then 
added to nitrocellulose-coated wells. The wells were then washed in 10 mM 
Tris-HCl pH 8.0 (4.times., 2 min each time). The amount of .sup.3 
H-heparin bound to each peptide at each dilution is shown in FIG. 7. The 
results of this experiment indicate that peptides TS-2 and TS-3 bind to 
heparin (TS-2 to a greater extent than TS-3), whereas TS-1 does not 
interact with heparin. 
The ability of the present polypeptides to interact with heparin when 
coated on 96-well plastic plates was evaluated. Stock solutions of the 
polypeptides at a maximum concentration of 2 mg/ml were prepared and 
serially diluted in phosphate-buffered saline (PBS)+NaN.sub.3, producing 
final concentrations from 2 mg/ml to 1 .mu.g/ml. Fifty .mu.l from each 
dilution were coated on the 96-well plates and left to dry overnight at 
28.degree. C. Then, the wells were treated for two hours with 2 mg/ml BSA 
in order to minimize nonspecific ligand binding. .sup.3 H-heparin was 
added (50,000 dpm/well) and the mixture incubated for two hours. After 
extensive washing, the .sup.3 H-heparin bound at each peptide 
concentration was removed with sodium dodecyl sulfate (SDS) and counted in 
a scintillation counter. The results shown in FIG. 8 indicate that peptide 
TS-2 binds heparin strongly. Peptide TS-3 also binds heparin, but less 
extensively (FIG. 9). Peptide TS-1 does not bind heparin above background 
(BSA) values. 
It was then established that .sup.3 H-heparin binds both to intact, native 
type IV collagen and to the isolated NC1 domain. FIGS. 10 and 11 show the 
binding of a constant amount of .sup.3 H-heparin (85,000 dpm) to 
increasing concentrations of type IV collagen and the NC1 domain, 
respectively. A saturable binding is observed in both cases. Type IV 
collage binds 5-6 times more .sup.3 H-heparin than does the NC1 domain. 
Next, the binding of increasing concentrations of .sup.3 H-heparin to a 
constant amount of type IV collagen (3 .mu.g) and an equimolar amount of 
the NC1 domain (1 .mu.g) was tested. Again, a saturable binding of .sup.3 
H-heparin to type IV collagen (FIG. 12) and to the NC1 domain (FIG. 13) 
was observed. In this type of experiment, approximately 4 times more 
.sup.3 H-heparin bound to type IV collagen than to the NC1 domain. These 
data indicate that at least two binding sites for heparin exist in type IV 
collagen: one in the NC1 domain and the other(s) in the helix-rich part of 
the molecule. 
Next, solutions of the three peptides of the invention (not absorbed to 
plastic) were screened for ability to inhibit the binding of heparin to 
intact, native type IV collagen coated on plastic. This experimental 
approach is intended to obviate problems due to any differential coating 
of peptides in heparin binding assays. 
Type IV collagen at 60 .mu.g/ml in PBS +0.1% Triton-X was coated on 96-well 
plates (use of 50 .mu.l or 3 .mu.g of laminin per well), dried overnight 
at 28.degree. C. and then the wells were coated with 2 gm/ml bovine serum 
albumin (BSA) in PBS. Peptides at various dilutions ranging from 2 mg/ml 
to 1 .mu.g/ml were co-incubated with a standard amount of .sup.3 H-heparin 
(30,000 dpm/well) for two hours and the mixture was then transferred to 
the type IV collagen-coated plates and allowed to incubate for another two 
hours. After extensive washing, the radioactivity retained in each well 
was counted. 
The results shown in FIG. 14 indicate that peptides TS-2 (FIG. 14B) and 
TS-3 (FIG. 14C) interact with heparin in this assay, since they both 
exhibited an about 43% inhibition of the binding of .sup.3 H-heparin to 
collagen at the highest concentration tested. However, peptide TS-2 was 
able to significantly inhibit the binding of .sup.3 H-heparin to type IV 
collagen at lower concentrations than TS-3, and therefore, bound to 
heparin with higher affinity. TS-1 does not interact with heparin by this 
assay (FIG. 14A). 
Peptides TS-2 and TS-3 were able to bind to heparin in all the assays which 
were performed. It is interesting that peptide TS-2 bind both to heparin 
and to type IV collagen. These data indicate the presence of more than one 
binding site in this peptide. Therefore, it is possible that binding of 
heparin to one binding site of this peptide competes for the binding of 
the peptide to type IV collagen. Also, this peptide could be used to link 
heparin and type IV collagen together, as well as to bind them to various 
biomaterials. 
EXAMPLE 4 
Cell Adhesion Assays 
These assays were performed with the following cell lines: aortic 
endothelial cells, metastatic melanoma murine (M.sub.4) cells, normal rat 
fibroblasts, MM fibrosarcoma cells, C6 glioma cells and breast carcinoma 
(A431) cells. Cell binding assays were performed in the same way for each 
cell line and the three peptides of the invention, TS-1, TS-2 and TS-3 
were individually assayed in each case for cell adhesion. 
Adhesion was tested in each case, using a 96-well microtiter plates 
absorbed with four different amounts (0.5; 5; 50; and 500 .mu.g/ml, 100 
.lambda./well) of peptides TS-1, TS-2 , TS-3 and BSA. Cultures of cells 
which were 60-80% confluent were metabolically labelled for 24 hours by 
the addition of 3 mCi of .sup.3 H-thymidine. On the day of the assay, the 
cells were harvested by trypsinization, the trypsin was inhibited by the 
addition of serum and the cells were washed free of this mixture and 
resuspended in DMEM. The cells were adjusted to a concentration of 
6'10.sup.5 /ml and 100 .mu.l of this cell suspension was added to the 
wells. The assay mixture was then incubated at 37.degree. C. for 90 min. 
At the end of the incubation, the wells were washed with warm PBS 
containing 10 MM Ca.sup.++ and the adherent population was solubilized 
with 0.5M NaOH containing 1% sodium dodecyl sulfate. The solubilized cells 
were then quantitated using a liquid scintillation counter. Each 
determination was done in triplicate. 
A. Aortic Endothelial Cells 
Aortic endothelial cells were obtained from bovine aortas by treatment with 
collagenase and were frozen at -196.degree. C. until use. These cells were 
cultured in primary cultures in the presence of DMEM and 10% fetal calf 
serum at 37.degree. C. in a humid atmosphere. When aortic endothelial 
cells were about 70% confluent, they were released from the tissue culture 
plastic by trypsin and then were added in suspension to the wells of 
96-well plates coated with the following peptides: (a) TS-1, (b) TS-2 and 
(c) TS-3, in the concentrations described above. 
The cells were metabolically labelled with 3.0 mCi of .sup.3 H-thymidine 
for 24 hours prior to the assay. After trypsinization, the cells were 
allowed to attach for a 90-minute incubation period in the peptide-coated 
wells. After extensive washing, the radioactivity associated with each 
well was measured and used as an index of cell attachment (expressed as 
percent adherent). The data summarized on FIG. 15 shows that peptide TS-1 
was the most potent in causing the adhesion of aortic endothelial cells, 
followed by peptides TS-2 and TS-3, respectively. In FIG. 15, the 
background values due to BSA binding were subtracted. 
B. Metastatic Melanoma Cells 
Highly metastatic melanoma cells, K1735M4, were originally provided by Dr. 
I. J. Fidler of Houston, Tex. When the cells were received, a large number 
of early passage cells were propagated and frozen in liquid nitrogen. the 
tumor cells are usually cultured in vitro for no longer than six weeks. 
Following this period, the cells are discarded and new cells withdrawn 
from storage for use in further in vitro or in vivo experiments. This 
precaution is taken to minimize phenotypic drift that can occur as a 
result of continuous in vitro passage. The cells were cultured in 
Dulbecco's Modified Eagle's Medium (DMEM) containing 5% heat inactivated 
fetal calf serum. The cultures were grown in 37.degree. C. incubators 
under a humidified atmosphere containing 5% CO.sub.2. Cells were 
subcultured twice weekly by releasing cells gently from the flask, using 
0.05% trypsin and 1 mM EDTA. 
The melanoma cells were metabolically labelled in the same fashion as the 
endothelial cells described hereinabove, using 2 .mu.Ci/ml .sup.3 HTdR 
(tritiated thymidine). The labelled cells were harvested as described for 
the endothelial cells. The cell adhesion assay was identical to that 
described hereinabove for the bovine aortic endothelial cell assay. 
The data summarized in FIG. 16 demonstrates that peptides TS-1, TS-2 and 
TS-3 promote adhesion of M.sub.4 cells. Peptide TS-1was the most potent in 
this respect. In this figure, adhesion of M.sub.4 cells to intact type IV 
collagen and isolated NC1 domain is shown for comparison. Background 
adhesion to BSA has been subtracted. 
C. Normal Rat Fibroblasts 
These cells were obtained from cultures of rat dermis explants in DMEM 
containing 10% fetal calf serum, in plastic wells. Under these conditions, 
fibroblasts migrated to the bottom of the plastic dish. When the cells 
were confluent, they were harvested by trypsinization and were then 
metabolically labelled, as described above, for the cell adhesion assay. 
FIG. 17 shows that peptide TS-1 was the most potent in promoting adhesion 
of these cells followed by peptides TS-2 and TS-3, which promoted adhesion 
of rat fibroblasts to a minimal extent. Background adhesion to BSA has 
been subtracted. 
D. Isolation of and Cell Adhesion Assay for MM Fibrosarcoma Cell Line 
Murine fibrosarcoma cells (uv-2237-MM) were originally provided by Dr. I. 
J. Fidler of Anderson Hospital, University of Texas health Sciences 
Center, Houston, Tex. Culturing, labelling and harvesting techniques were 
as described in Part A. The results of this assay are summarized in FIG. 
18. Background adhesion to BSA has been subtracted. 
E. Isolation of and Cell Adhesion Assay for C6 Cell Line 
Rat C6 glioma cell line was purchased from the American Type Culture 
Collection (identification number CCL 107). Culturing techniques were as 
described in Part A. Labelling and harvesting techniques were as described 
above, under Example 4. The results of this assay are summarized in FIG. 
19. Peptides TS-1, TS-2 and TS-3 promoted adhesion of C6 glioma cells. At 
the highest concentration, peptide TS-1was the most potent in promoting 
adhesion. Background adhesion to BSA has been subtracted. 
F. A431 Breast Carcinoma Cells 
A431 cells were purchased from the American Type Culture Collection. 
Culturing, labelling and harvesting techniques have been described above 
(see methodology under Example 4, and Part A). The results of this assay 
are summarized in FIG. 20. Background adhesion to BSA has been subtracted. 
FIG. 21 summarizes the data with respect to the adhesion of the 
above-mentioned peptides TS-1, TS-2 and TS-3 to the previously described 
cell lines. Two other irrelevant peptides were also used: JM-8, as a 
positive control (a peptide derived from the sequence of fibronectin, 
which is known to promote cell adhesion) and F-11 (a peptide derived from 
the sequence of the .beta..sub.1 chain of laminin, which does not promote 
cell adhesion and which was used as a negative control). Adhesion to 
isolated domain NC1 is also shown for comparison. Background adhesion to 
BSA has been subtracted. 
In summary, peptide TS-1 promotes adhesion of aortic endothelial cells, 
metastatic carcinoma M.sub.4 cells, normal rat fibroblasts, MM 
fibrosarcoma cells, C6 glioma cells and A431 breast carcinoma cells. 
Peptide TS-2 binds (a) to type IV collagen, (b) to heparin and (c) 
promotes adhesion of the abovementioned cell lines. Peptide TS-3 (a) binds 
to heparin and (b) promotes adhesion of the abovementioned cell lines. 
A number of practical applications for polypeptides TS-1, TS-2 and TS-3 can 
be envisioned. Such applications include the promotion of the healing of 
wounds caused by the placement of natural or synthetic substrata within 
the body. Such synthetic substrata can include artificial vessels, 
intraocular contact lenses, hip replacement implants and the like, where 
cell adhesion is an important factor in the acceptance of the synthetic 
implant by normal host tissue. 
As described in U.S. Pat. No. 4,578,079, medical devices can be designed 
making use of these polypeptides to attract cells to the surface in vivo 
or even to promote the growing of a desired cell type on a particular 
surface prior to grafting. An example of such an approach is the induction 
of endothelial cell growth on a prosthetic device such as a blood vessel 
or vascular graft, which is generally woven or knitted from a synthetic 
resin such as nitrocellulose, expanded polytetrafluoroethylene or 
polyester fiber, particularly Dacron.TM. (polyethylene tetephthalate) 
fiber. Devices intended for cardiac insertion include temporary left 
ventricular assist devices, heart valves, intraortic balloon pumps and 
artificial hearts. Such devices are preferably formed from synthetic 
resins such as polyether-type polyurethane elastomers (Cardiothane.TM., 
Kontron) or from vulcanized polyolefin rubbers (Hexsyn.TM., Goodyear). 
Most types of cells are attracted to collagen and to the present 
polypeptides, but endothelial cells, epithelial cells and fibroblastic 
cells in particular may be strongly attracted to the present polypeptides. 
The latter point indicates the potential usefulness of these defined 
polypeptides in coating a patch graft or the like for aiding wound closure 
and healing following an accident or surgery. 
In such cases, it may be advantageous to couple the peptide to a biological 
molecule, such as collagen, a glycosaminoglycan or a proteoglycan. 
Collagens, proteoglycans and glycosaminoglycans are major components of 
connective tissues and basement membranes. In some cases, prosthetic 
devices formed entirely or in part from naturally-occurring tissues 
instead of synthetic polymers are used. One example is the use of porcine 
heart valves to replace defective human heart valves. Such artificial 
valves can also comprise human dura matter or bovine pericardium. Another 
example is the use of bovine arteries as vascular grafts. 
It may be useful to coat surfaces of these biological substrata with the 
present polypeptides, in order to modify the cellular response, in vivo, 
thus improving the therapeutic outcome. This can be achieved by a variety 
of methods known to the art, e.g., by direct binding of the polypeptides 
to the target surfaces based on the affinities described hereinabove, or 
by the covalently bonding the polypeptides to the substrate using various 
crosslinking reactions or reagents. For a review of the use of synthetic 
resins and biomaterials in prosthetic devices, see Chem. & Eng. News (Apr. 
14, 1986) at pages 30-48, the disclosure of which is incorporated by 
reference herein. 
It is also indicative of their value in coating surfaces of a prosthetic 
device which is intended to serve as a temporary or semipermanent entry 
into the body, e.g. into a blood vessel or into the peritoneal cavity, 
sometimes referred to as a percutaneous device. Such devices include 
controlled drug delivery reservoirs or infusion pumps. 
Also, polypeptides TS-1, TS-2 and TS-3 can be used to promote endothelial, 
fibroblast or epithelial cell adhesion to naturally occurring or 
artificial substrata intended for use in vitro. For example, a culture 
substrate such as the wells of a microtiter plate or the medium-contacting 
surface of microporous fibers or beads, can be coated with the 
cell-attachment polypeptides. This can obviate the use of fibronectin in 
the medium, thus providing better defined conditions for the culture as 
well as better reproducibility. 
As one example of commercial use of cell-attachment surfaces, Cytodex.TM. 
particles, manufactured by Pharmacia, are coated with gelatin, making it 
possible to grow the same number of adherent cells in a much smaller 
volume of medium than would be possible in dishes. The activity of these 
beads is generally dependent upon the use of fibronectin in the growth 
medium and the present polypeptides are expected to provide an improved, 
chemically-defined coating for such purposes. Other surfaces or materials 
may be coated to enhance attachment, such as glass, agarose, synthetic 
resins or long-chain polysaccharides. 
Finally, TS-1, TS-2 and TS-3 can be used to coat the surface of medical 
devices intended for external application or attachment to the body. Such 
devices include "bandages". which term is also intended to refer to wound 
packs and dressings, which can comprise surfaces formed from absorbent 
cellulosic fibers, from synthetic fibers or form mixtures thereof. These 
surfaces can be coated with amounts of TS-1, TS-2 and/or TS-3 effective to 
promote cellular growth, wound healing, graft attachment and the like. 
The invention has been described with reference to various specific and 
preferred embodiments and techniques. However, it should be understood 
that many variations and modifications may be made while remaining within 
the spirit and scope of the invention.