(Received for publication, May 28, 1996, and in revised form, December 23, 1996)
From the Human and murine fibroblasts were found to spread
far more avidly on fibrin monomer monolayers than on immobilized
fibrinogen, indicating that removal of fibrinopeptides by thrombin is a
prerequisite for the fibrin-mediated augmentation of cell spreading. In
fact, cell spreading was not efficiently augmented on monolayers of a
thrombin-treated dysfibrinogen lacking the release of fibrinopeptide A
due to an A Fibrinogen is a 340-kDa glycoprotein consisting of three pairs of
polypeptide subunits, A All chemical reagents were of the highest
analytical grade commercially available and were purchased from the
sources shown below. Bovine serum albumin
(BSA),1 soybean trypsin inhibitor type-1,
trypsin, and sodium dodecyl sulfate (SDS) were from
Sigma, and Microtiter 96- and 24-well flat-bottomed
plates from Costar, Cambridge, MA. Rabbit antisera to the human
vitronectin receptor and the human fibronectin receptor were purchased
from Life Technologies, Inc., rabbit anti-human fibrinogen IgG and
rabbit anti-human fibronectin from Dako Japan, Kyoto, Japan, and
anti-human vitronectin antisera from The Binding Site Ltd., Birmingham,
United Kingdom. The P1F6 antibody directed against the
3T3-fibroblast cells were purchased
from Dainihon Pharma Co., Tokyo, Japan. Human fibroblasts (TIG-3) were
a generous gift from Dr. Tadashi Shimo-Oka of Iwaki Glass Co., Chiba,
Japan. Dulbecco's modified Eagle's essential medium (DMEM) with 10%
fetal calf serum was obtained from Life Technologies, Inc. The cells
were cultured in 10% (w/v) fetal calf serum-containing DMEM at
37 °C in a 6.0% CO2 atmosphere.
Gly-Arg-Gly-Asp-Ser (GRGDS) and
Gly-Arg-Gly-Glu-Ser (GRGES) peptides were purchased from Iwaki Glass
Co., Osaka, Japan, and a
His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val peptide corresponding to the human fibrinogen SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by immunoblotting was carried out according to the
method of Towbin et al. as described previously (10).
Briefly, after separation of fibrinogen on 10% SDS-PAGE slab gels and
electroblotting onto nitrocellulose filters, blots were soaked in 50 mM Tris-HCl, pH 7.4, containing 0.135 M NaCl
(TBS) and 3% BSA for 1 h at 37 °C, rinsed with TBS three
times, and incubated with monoclonal antibodies (3 µg/ml) in TBS
containing 0.1% BSA for 2 h at 22 °C. After washing three
times with TBS, the filters were incubated for 1 h with peroxidase-conjugated rabbit anti-mouse IgG. After rinsing, blots were
immersed in the substrate solution:
4-chloro-1-naphthol/H2O2.
Fibrinogen was purified by repeated precipitation
with 25% ammonium sulfate from human plasma harvested from blood
freshly collected into 0.11 M trisodium citrate, 0.1 M
Vitronectin, a possible contaminant that might affect the study, was
removed by passing the purified fibrinogen through Sepharose CL-4B
conjugated with an anti-vitronectin antibody, as confirmed by ELISA.
Amino acid sequence analyses of fibrinogen and its derivatives were
performed for further characterization, and to establish their
authenticity and purity.
One hundred microliters of 16 mg/ml human fibrinogen was diluted with 3 ml of TBS, and clotted with
0.5 unit/ml thrombin for 2 h at 37 °C, followed by another for
22 h at 4 °C to achieve gel formation as completely as
possible. The fibrin gels formed were washed 5 times with 10 ml of TBS,
dissolved with ~0.5-1.0 ml of 2 M NaBr containing 1 mM GPRP peptide as an antipolymerant (NaBr-GPRP) for 8 h at 20 °C, and further diluted with TBS containing 1 mM
GPRP to appropriate concentrations required for immobilization onto
microtiter wells. When a small portion of a diluted fraction (500 µl,
A280 = 0.98) was subjected to gel filtration on
a Sephacryl CL-6B column (1.0 × 80 cm), proteins were all eluted
in a single peak (fractions 110-125) as fibrin monomer with a relative
molecular mass of 3.4 × 105. Though not shown here,
no measurable fibrin(ogen)-related proteins were identified in
fractions eluted earlier than the peak fractions, indicating that the
NaBr-solubilized fibrin was mostly constituted of monomeric fibrin
molecules.
Two types
of heterozygous dysfibrinogens were utilized, i.e. a
dysfibrinogen with an A Vitronectin was
purified by the method of Dahlbäck and Podack (17), and
fibronectin by the method of Engvall and Ruoslahti (18). All proteins
were divided into aliquots and stored at Protein concentrations were determined spectrophotometrically by using
absorption coefficients (A1 cm1% at
280 nm) of 15.1 for fibrinogen (11), 9.0 for vitronectin (19), and 13 for fibronectin (20).
Microtiter wells (96-well) were coated with 50 µl of
various concentrations of fibrin monomer and fibrinogen for 4 h at
22 °C, and the wells were washed with TBS followed by blocking with
TBS containing 1% BSA. After washing three times with TBS, an
anti-human fibrinogen polyclonal antibody (1:2000 dilution in TBS-Tween
80) was added to each well and the reaction was allowed to proceed for
3 h at 22 °C. The wells were rinsed three times with TBS, and
then incubated with 200 µl/well of a 1:1000 dilution of anti-rabbit IgG goat antibody conjugated with horseradish peroxidase in TBS for
3 h at 22 °C. The wells were rinsed three times with TBS and finally incubated with a substrate for horseradish peroxidase at
37 °C. The reaction was analyzed by a kinetic ELISA. Data were expressed as the means of quadruplicate determinations. The
concentrations of half-maximal saturation of fibrinogen and fibrin
monomer to the surface are 0.04 µg/ml and 0.2 µg/ml,
respectively (Fig. 2).
The cell adhesion, i.e. the
initial attachment of cells to the substratum, and subsequent spreading
thereon were measured by the method of Grinnell (21) with some
modifications as described previously (22). Briefly, 2-cm2
well (24-well Costar plates) polystyrene plates were coated with various concentrations of fibrinogen, fibrin monomer, fragment X, XDP,
or heat-denatured BSA in TBS containing a synthetic antipolymerant GPRP
peptide at 1 mM. After blocking with 3% heat-denatured BSA in TBS for 1 h at 37 °C, plates were washed with TBS containing 0.2% BSA three times, 0.5-ml aliquots of suspensions of fibroblasts and HUVECs (2 × 104 cells/ml) were pipetted into the
coated wells. Cells were allowed to adhere to the vitronectin- or
BSA-coated surface for 60-90 min at 37 °C. Non-adherent cells were
removed by washing with TBS, and the plates were examined by phase
contrast microscopy and photographed. Spread cells were counted as
described previously (22) and when necessary, they were analyzed using
a computer image processing package to determine cell areas and
diameters as described previously.2 The
spread cells were defined essentially as described elsewhere (22),
namely being polygonal in shape with a dark surface under phase-contrast microscopy and a larger surface area than round cells + 2 S.D. Vitronectin and fibronectin were used as control proteins for
the cell adhesion assay.
After incubation of fibrin
monomer, fibrinogen, fragments D, E, X, and XDP with DMEM and 0.2% BSA
containing 0.5 mM GPRP peptide, cells were mixed with these
solutions, and were added to the wells coated with fibrinogen or fibrin
monomer. We applied the same assay conditions as those for the adhesion
assay. All experiments were performed at least three times with two
independent isolates.
Human fibroblasts were found to spread on wells coated
with fibrin monomer, but not on those coated with fibrinogen below the
concentration of 10 µg/ml in DMEM-solution (Fig. 3).
The cell spreading on the fibrin monomer substratum was apparently
dependent on the concentration of fibrin monomer immobilized to the
wells, as noted in different grades of morphological changes observed under microscopy. Since spontaneous aggregation of fibrin monomer was
efficiently hindered by the addition of an antipolymerant GPRP peptide,
the cell spreading on the fibrin monomer substratum is attributed most
likely to specific changes induced in the molecule upon conversion to
fibrin monomer rather than polymerized fibrin. Indeed, fibrinogen in
solution added to cell suspensions was unable to block the cell
spreading on the fibrin monomer substratum, but fibrin monomer
inhibited it concentration-dependently (Fig. 4). However, both attachment and spreading of cells were
inhibited by an RGD-containing peptide, GRGDSP, but not by a GRGESP
peptide, indicating that the receptor for the RGD-containing peptide,
i.e. an integrin, was involved in the adhesion and spreading
of cells on the fibrin monomer substratum (Fig. 5).
Thrombin-cleavage of fibrinopeptide A seemed to be mandatory for
supporting the spreading of human fibroblasts on the fibrin monomer
substratum, as shown by a study utilizing a hereditary dysfibrinogen
with defective thrombin-catalyzed cleavage of fibrinopeptide A due to
an A
There are two RGD-containing peptide segments assigned to the A
When adhesion and spreading was tested on immobilized fragment X
lacking both RGD-2 segments, spreading of fibroblasts was less
extensive than on fibrin monomer (data not shown). Thus, RGD-2 may also
be involved in cell spreading.
To identify the
cell-surface receptor responsible for cell adhesion to the fibrin
monomer substratum, human fibroblasts were allowed to adhere to
immobilized fibrin monomer in the presence of LM609, P1F6, or mAb13
recognizing
Effects of monoclonal antibodies to integrins on human fibroblast
spreading to immobilized fibrin-monomer and fibronectin
Polystyrene wells were coated with fibrin-monomer (10 µg/ml) and
fibronectin (10 µg/ml) at 4 °C overnight. After blocking with 1%
BSA, wells were washed with TBS and human fibroblasts were added with
monoclonal antibodies against integrins or mouse IgG (50 µg/ml,
respectively). Two hours later, spread cells were counted. This
experiment was repeated four times, and results of one representative
experiment were shown. Data are expressed as the means ± S.D. of
three wells.
Division of Hemostasis and Thrombosis
Research, ¶ Division of Hamatopoiesis,
Department of Cardiology, Jichi Medical
School, Minamikawachi-Machi, Tochigi-Ken 329-04, Japan, ** The
Central Research Laboratories, Iatron Laboratories Inc., 1460-6, Mitodai, Mito, Takomachi, Katori-Gun, Chiba 289-22, Japan, the
Department of Biomolecular Chemistry,
University of Wisconsin-Madison, Madison, Wisconsin 53706, and the
§§ Laboratory of Biosignaling, Department of
Life Science, Faculty of Science, Himeji Institute of Technology,
Harima Science Garden City, Hyogo 0205, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Arg-16
Cys substitution. Since a synthetic
Arg-Gly-Asp (RGD)-containing peptide inhibited the fibrin-mediated cell
spreading, subsequent dissociation of the carboxyl-terminal globular
domain of the A
-chains appears to render the RGD segments accessible to the cell-surface integrins. In support of this, fibrin-augmented cell spreading was inhibited by an antibody recognizing a 12-kDa peptide segment with
Met-89 at its amino terminus, which is located
in close association with the RGD segment at A
95-97 in the helical
coiled-coil interdomainal connector. The fibrin-mediated augmentation
of cell spreading was inhibited not only by an antibody against human
vitronectin receptor (LM 609) but also by an antibody against the
1 subunit of integrin (mAb13), suggesting that the
1-class integrin together with a vitronectin receptor,
v
3, is mobilized onto the surface of
fibroblasts upon contact with the fibrin monomer monolayer.
, B
, and
, linked together by multiple
disulfide bonds (1). By structural studies including electron
microscopic analysis together with biochemical data, there is now
general agreement on the shape of the fibrinogen molecule (2-7). The
fibrinogen molecule is composed of three major globular domains,
i.e. one central E domain and two identical outer D domains
connected by a three chain
-helical coiled-coil (4, 6). The distal
part of the D domain is the carboxyl terminus of the
-chain, while
the proximal part is the carboxyl terminus of the B
-chain. The
carboxyl-terminal two thirds of the A
-chains fold back from the D
domain and form two independently folded domains (
C domains) at
their carboxyl-terminal parts. In the native fibrinogen molecule, the
C domains interact with each other and form an additional small
globular (
C-
C) domain that is closely associated with the central
E domain (4, 6, 7). Upon thrombin cleavage of fibrinopeptides A and B,
the globular
C-
C domain is released from the E domain, and
subsequently dissociated into individual
C domains (4, 6, 7). The fibrinogen molecule thus undergoes distinct conformational changes upon
conversion to the fibrin monomer molecule (4), and thereby exposes
several fibrin-specific regions that may participate in the functions
of fibrin. The Arg-Gly-Asp (RGD) segments residing at A
95-97 and
A
572-574, tentatively designated as RGD-1 and RGD-2, respectively,
may also be categorized into this type of fibrin-specific segments. In
this paper, we describe the binding of cultured human and murine
fibroblasts to immobilized fibrin monomer, but not to immobilized
fibrinogen, by focusing on the conformational changes induced in the
fibrinogen molecule upon its conversion to fibrin.
Materials
v
5 integrin receptor was from Telios/Life
Technologies, Inc., and the KH33 antibody directed against the
5 chain of integrin receptor was from Seikagaku Corp,
Tokyo, Japan. Monoclonal antibody to the
1 subunit of
the human fibroblast fibronectin receptor, mAb13 (8), was a gift from
Dr. Steven K. Akiyama (Howard University Cancer Center, Howard
University College of Medicine, Washington, DC). Mouse monoclonal
antibody LM609 was a gift from Dr. David A. Cheresh (Research Institute
of Scripps Clinic, La Jolla, CA). This antibody was reported to be
specific for the
v
3 complex and was
previously shown to block the function of
v
3 (9).
-fragment (400-411), Gly-Pro-Arg-Pro (GPRP), and Gly-His-Arg-Pro (GHRP) peptides were from
Sigma.
-aminocaproic acid and 500 kallikrein inhibitor units
(KIU)/ml aprotinin after removal of fibronectin and plasminogen by
passing the plasma through columns of gelatin-Sepharose CL-6B and
lysine-Sepharose CL-6B connected in tandem as described previously
(11). High molecular weight contaminants were removed by gel filtration
chromatography using Sepharose CL-4B. Fibrinogen fractions thus
prepared were found to be >96% pure as determined by SDS-PAGE. They
were found to contain neither von Willebrand factor nor fibronectin as
determined by an enzyme-linked immunosorbent assay (ELISA) using
monoclonal antibodies directed against respective proteins. Fibrinogen
fragments were prepared by limited plasmin digestion of the purified
protein in 20 mM Tris-HCl, pH 7.4, containing 135 mM NaCl. Cleavage conditions were optimized after
monitoring the fragments by reduced SDS-PAGE. Early fragment X was
obtained as described (12). Fragment D1 containing the
-chain
carboxyl-terminal dodecapeptide
400-411 (D1) and fragment E were
prepared using 0.02 units of plasmin/mg of fibrinogen in the presence
of 1 mM CaCl2 at 37 °C for 2 h. Fragment D3 lacking the
303-411 segment was prepared using 0.1 unit of plasmin/mg of fibrinogen in the presence of 10 mM
EGTA at 37 °C for 22 h. The fragment X preparation was
gel-filtered through an HPLC-3000SW system to remove the
carboxyl-terminal segment of the A
chain and dialyzed against TBS.
Fragments D1 and D3 and E were separated by ion-exchange HPLC as
described previously (11). The plasmic phase-3 digests of cross-linked fibrin (XDP) was prepared according to Francis et al. (13). The JIF-23 antibody was described to recognize the amino-terminal conformation of fragment D species, which was exposed when the
63-85/88 residue segment had been removed from fragment D1A on its
conversion to fragment D1 (14). The JIF-25 antibody was shown to react
with all
-chain remnants of fragment D species, i.e.
/D1,
/D2, and
/D3 (14). In this study, we were able to
localize the epitope to a much smaller 12-kDa peptide segment with
Met-89 at its amino terminus (12-kDa
89~peptide). Namely, after
SDS-PAGE run under reducing conditions, the 12-kDa polypeptide was
transferred to polyvinylidene difluoride membranes and subjected to
direct sequencing as described by Matsudaira et al. (15) (Fig. 1). By assigning the first seven cycles, MLEEIMK,
to the known
-chain sequence, we found that these seven residues
corresponded to the
89-95 residues, the amino-terminal segment of
the
-chain remnant of fragment D species. Thus, the epitope for
JIF-25 was localized to the amino-terminal 12-kDa peptide segment of
the
-remnant of fragment D species.
Fig. 1.
Immunoblot analysis of fibrinogen and its
plasmic digests. A, protein staining. B,
Immunostaining by JIF-25. Lanes 1 and 3,
fibrinogen; lanes 2 and 4, plasmic digests of
fibrinogen. Positions of marker proteins are indicated at the
left.
[View Larger Version of this Image (37K GIF file)]
Arg-16
Cys substitution resulting in
defective thrombin-catalyzed release of fibrinopeptide A due to the
mutation at the P1 site for thrombin, and a dysfibrinogen with a
Arg-275
Cys substitution, manifesting impaired D:D self-association
(16).
70 °C.
Fig. 2.
Antigenicity of fibrinogen detected by ELISA
using polyclonal anti-fibrinogen antiserum. The wells were coated
with fibrinogen () or fibrin monomer (
); after blocking with 1%
BSA-TBS, plates were incubated with polyclonal antibody to fibrinogen
(1:1000) at room temperature for 3 h. After extensive washing with
TBS, 0.02% Tween 20, horseradish peroxidase-conjugated second antibody was added and incubated at room temperature for 3 h. Plates were then washed three times with TBS, developed, and examined by ELISA as
described under "Experimental Procedures."
[View Larger Version of this Image (17K GIF file)]
Characterization of Fibrin Monomer-dependent Cell
Adhesion
R-16 to C substitution as compared with fibrinogen molecules
manifesting normal thrombin-cleavage of fibrinopeptide A,
i.e. normal fibrinogen and a dysfibrinogen with a
R-275
to C substitution (Fig. 6). The newly exposed amino
terminus of the fibrin
-chain appeared to be indifferent to the
reactions because the synthetic GPRP peptide failed to inhibit both
attachment and spreading of cultured fibroblasts (Fig. 5). The result
thus suggested that a specific conformation newly induced in the fibrin
monomer was responsible for the interaction with the cell-surface
integrins.
Fig. 3.
Cell adhesion of human fibroblasts to
immobilized fibrin substratum. A, normal fibrinogen ()
and fibrin derived from normal fibrinogen (
) were used to coat the
wells of multi-well dishes and incubated at 4 °C overnight. After
blocking with 1% bovine serum albumin-TBS, fibroblasts were added to
the wells as described under "Experimental Procedures." Two hours
later, cells were fixed, stained, and counted. B, cells
spread on fibrin monomer-coated wells (a), but did not
spread on fibrinogen-coated wells (b). Bar, 20 µm.
[View Larger Version of this Image (57K GIF file)]
Fig. 4.
Effect of solubilized fibrinogen-derivatives
on fibrin-dependent cell adhesion. A, fibrin
monomer was used to coat on the wells of multi-well dishes at 4 °C
overnight. After blocking with 1% BSA, wells were washed with TBS, and
human fibroblasts were added with various fibrinogen-derivatives as
outlined in the figure. After incubation for 1 h, wells were
washed with PBS, and fixed with 4% of paraformaldehyde solution and
spread cells were counted as described under "Experimental
Procedures." B, a, cell spreading on fibrin
monomer substratum (30 µg/ml); b, cells with 100 µg/ml
soluble fibrinogen; c, cells with 100 µg/ml soluble fibrin
monomer. Bar, 100 µm.
[View Larger Version of this Image (67K GIF file)]
Fig. 5.
Effect of RGD peptide and fibrin fragments
(GPRP, GHRP) on fibrin-dependent cell adhesion of human
fibroblasts. Wells were coated with fibrin monomer at 4 °C
overnight. After blocking with 1% BSA, wells were washed with TBS, and
fibroblasts were added with various concentrations of synthetic
peptides. After incubation for 1 h, wells were washed with PBS and
fixed with 4% of paraformaldehyde solution, and spread cells were
counted as described under "Experimental Procedures." Figure shows
GRGDSP (), GRGESP (
), GPRP (
), and GHRP (
).
[View Larger Version of this Image (19K GIF file)]
Fig. 6.
Adhesion of human fibroblasts to immobilized
fibrin substratum derived from dysfibrinogens. Fibrins derived
from normal fibrinogen and dysfibrinogens ( R275C and A
R16C) were
used to coat the wells of multi-well plates by incubation at 4 °C
overnight. After blocking with 1% bovine serum albumin-TBS,
fibroblasts were added to the wells as described under "Experimental
Procedures." Two hours later, cells were fixed and stained, and
spread cells were counted.
[View Larger Version of this Image (21K GIF file)]
95-97, RGD-1, and the A
575-577, RGD-2, residues in each fibrinogen A
-chain. To see whether or not either one or both of the
two RGD segments, RGD-1 and RGD-2, are masked in native fibrinogen, and
exposed on its conversion to fibrin monomer to support adhesion and
spreading of cells, we utilized the JIF-25 antibody recognizing the
89-173 residue segment. Interestingly, by assignment of the amino acid
residues of the A
-and
-chains in the helical coiled-coil regions
based on the two ring-like disulfide bridges connecting the three
polypeptides called disulfide swivels (1), RGD-1 is located close to
the
(73-75) residue segment, which is only 16 residues apart from
the amino terminus of the 12-kDa
89~segment recognized by JIF-25.
Thus, binding of JIF-25 to the 12-kDa
89~segment may affect the
local conformation surrounding the RGD-1 segment. Indeed, when
fibroblasts were seeded together with JIF-25, adhesion of fibroblasts
to the fibrin substratum was substantially inhibited (>85%) (Fig.
7, A and B), indicating that RGD-1
was hidden in fibrinogen but exposed on immobilized fibrin monomer to
modulate cell adhesion and spreading.
Fig. 7.
Effects of monoclonal antibodies (JIF-23,-25)
to fibrin or fibrinogen on cell adhesion to immobilized fibrin.
A, 100 nM monoclonal antibodies (JIF-23, -25) or
mouse IgG were incubated with fibrin monomer-coated wells for 2 h
at room temperature. After washing with TBS, cells were added to the
wells and spread cells were counted. Number of spread cells on 100 µg/ml fibrin derived from normal fibrinogen was used as a control.
B, various concentrations of JIF-25 were incubated with
fibrin monomer-coated wells for 2 h at room temperature. After
washing with TBS, cells were added to the wells. Attached cells ()
and spread cells (
) were counted.
[View Larger Version of this Image (23K GIF file)]
v
3,
v
5, and the
1-subclass integrin, respectively. Cell adhesion was inhibited by mAb13 (85.0%) and LM609 (42%) (Table I). The data indicate that both
VNR and a
1-subclass integrin are involved in the fibrin
monomer-dependent cell adhesion.
Spread cells
Fibrin-monomer
Fibronectin
% of
control
Mouse IgG
100.0
100.0
LM609(
v
3)
48.0
± 4.5
110.0 ± 13.5
P1F6(
v
5)
109.5 ± 5.5
111.0
± 14.0
mAb13(
1)
15.0 ± 1.0
18.3
± 3.0
Adhesion and spreading of cells on immobilized thrombin-treated and non-treated fibrinogen have been studied extensively (24-34), but there still remain controversies on the mechanism of cell adhesion to the fibrinogen and fibrin substrata. Although experimental conditions are not necessarily comparable from one experiment to another, the controversies may largely arise from inconsistency regarding the configuration of individual fibrinogen or fibrin molecules immobilized onto tissue culture wells.
In this study, we established an assay system using NaBr-solubilized preformed fibrin, fibrin monomer, which had been immobilized onto tissue culture wells in the presence of a synthetic antipolymerant GPRP peptide. This system allowed us to achieve uniform immobilization of the fibrin monomer molecules on the wells as verified by homogeneously distributed adherent cells. We were thus able to count the adherent cells more accurately than by conventional assay systems.
Utilizing this assay system, we found that both human and murine
fibroblasts were avidly adherent to immobilized fibrin monomer but not
to immobilized fibrinogen (Figs. 3 and 4), and that the adhesion was
dose-dependently inhibited by soluble fibrin monomer (Fig.
4) and a synthetic RGD peptide (Fig. 5). The results together indicate
that the fibrin-specific regions are preserved satisfactorily after
immobilization and that they are most likely two RGD segments, RGD-1
and RGD-2. Failure to inhibit the adhesion of fibroblasts to fibrin
monomer by fragments X, D, and E may be explained at least partly by
lack of one or both of the RGD segments in these plasmic fragments.
Although fragment X still retains a pair of RGD-1 segments, both RGD-1
and RGD-2 may be required to cooperate with each other for full
expression of cell-spreading supporting activity. Indeed, the
involvement of separate sites in differentially mediating cell
attachment and spreading has been observed in other adhesion molecules
such as laminin (35) and thrombospondin (36). Thus, concerted action of
the two RGD-segments may be required in order for fibroblasts to attach
to the fibrin monomer substratum and avidly spread thereon. Since RGD-2
resides very close to a cluster of negatively charged residues (A
586-595), which may interact with a cluster of positively charged
residues (A
601-608) in the carboxyl-terminal segment of the
A
-chain, RGD-2 may be inaccessible or hidden in the native
fibrinogen molecule. When the (
C-
C) domain is fully dissociated
upon conversion of fibrinogen to fibrin, RGD-2 may become available for
supporting the cell adhesion. RGD-1 residing in the helical coiled-coil
of the interdomainal connector may also be masked in the native
fibrinogen molecule, and exposed upon fibrinogen to fibrin conversion.
The adhesion of fibroblasts to fibrin monomer was inhibited by JIF-25,
which recognized the 12-kDa
89~segment (Fig. 7, A and
B). The epitope for JIF-25 seems to be buried in native
fibrinogen, because the JIF-25-conjugated Sepharose failed to adsorb
fibrinogen in solution, although the
-chain of SDS-denatured
fibrinogen immobilized to the membrane was clearly visualized by JIF-25
on immunoblotting (14). Therefore, RGD-1 may also be buried in
fibrinogen and exposed upon fibrinogen to fibrin conversion, and
thereby serves as a functional site with the fibroblast in concert with
RGD-2. Interestingly, RGD-1 has been found to be a receptor-induced
binding site of transmembrane
IIb/
3 (37),
and thus it should be cryptic in a native fibrinogen molecule, in good
agreement with our presumption. This conclusion is also supported by an
experiment, in which fibrin monomer derived from a hereditary
dysfibrinogen with defective thrombin-cleavage of fibrinopeptide A
failed to support the adhesion of fibroblasts (Fig. 6).
Of interest is the finding that the 1-subclass integrin
is preferentially involved in the adhesion of fibroblasts to fibrin monomer together with
v
3, a vitronectin
receptor, expressed on the fibroblast. The receptors for fibrin(ogen)
are shown to be
IIb
3, which is specific
for binding with the
(400-411) residue segment of fibrinogen (26,
39-42) and
v
3, which interacts with
vitronectin as well (9).
Dejana et al. (43) reported that cell spreading on the
fibrinogen substratum was mediated by cellular fibronectin synthesized by the cell itself. This possibility still remains to be resolved, but
so far it seems to be unlikely, because a well characterized antibody,
KH33, that inhibits the function of the 5 chain of integrins failed to block the fibrin monomer-dependent cell
adhesion (data not shown).
The mechanism of the involvement of the 1-subclass of
integrin in the interaction with the RGD-segments of fibrin monomer is
not clear at this stage of the investigation, but we speculate that the
fibrin monomer molecule acquired affinity for the
1-subclass integrin due to a newly induced
conformational change upon contact with its authentic receptor
v
3. Acquisition of such extraordinary affinity for non-authentic integrins has been shown in other adhesion molecules. For example, Neugenbauer et al. (23) showed that an integrin heterodimer in the
1 family expressed on the
HD11 chick myoblast cell line functions as a receptor for fibrinogen. They also showed that a mAb to the
1-integrin enhanced
attachment of HD11 cells to fibrinogen and inhibited attachment to
vitronectin. Although the molecular mechanism still remains
speculative, this report seems to be the first to describe acquisition
by fibrin monomer of affinity for the
1-subclass
integrin.
We thank Dr. David Cheresh for providing the antibody to human vitronectin receptor (LM609), Dr. Steven K. Akiyama for providing the antibody to human fibronectin receptor (mAb13), and Dr. Tadashi Shimo-oka for providing human fibroblast cell line (TIG-3).