Fibroblasts Spread on Immobilized Fibrin Monomer by Mobilizing a beta 1-class Integrin, Together with a Vitronectin Receptor alpha vbeta 3 on Their Surface*

(Received for publication, May 28, 1996, and in revised form, December 23, 1996)

Shinji Asakura Dagger §, Kazuki Niwa Dagger , Takako Tomozawa Dagger , Yong-ming Jin Dagger , Seiji Madoiwa Dagger , Yoichi Sakata Dagger , Takao Sakai , Hiroshi Funayama par , Gilbu Soe **, Fran Forgerty Dagger Dagger , Hajime Hirata §§ and Michio Matsuda Dagger

From the Dagger  Division of Hemostasis and Thrombosis Research,  Division of Hamatopoiesis, Institute of Hematology, and par  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 Dagger Dagger  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


ABSTRACT

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 Aalpha Arg-16 right-arrow 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 Aalpha -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 gamma  Met-89 at its amino terminus, which is located in close association with the RGD segment at Aalpha 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 beta 1 subunit of integrin (mAb13), suggesting that the beta 1-class integrin together with a vitronectin receptor, alpha vbeta 3, is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.


INTRODUCTION

Fibrinogen is a 340-kDa glycoprotein consisting of three pairs of polypeptide subunits, Aalpha , Bbeta , and gamma , 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 alpha -helical coiled-coil (4, 6). The distal part of the D domain is the carboxyl terminus of the gamma -chain, while the proximal part is the carboxyl terminus of the Bbeta -chain. The carboxyl-terminal two thirds of the Aalpha -chains fold back from the D domain and form two independently folded domains (alpha C domains) at their carboxyl-terminal parts. In the native fibrinogen molecule, the alpha C domains interact with each other and form an additional small globular (alpha C-alpha C) domain that is closely associated with the central E domain (4, 6, 7). Upon thrombin cleavage of fibrinopeptides A and B, the globular alpha C-alpha C domain is released from the E domain, and subsequently dissociated into individual alpha 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 Aalpha 95-97 and Aalpha 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.


EXPERIMENTAL PROCEDURES

Materials

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 alpha vbeta 5 integrin receptor was from Telios/Life Technologies, Inc., and the KH33 antibody directed against the alpha 5 chain of integrin receptor was from Seikagaku Corp, Tokyo, Japan. Monoclonal antibody to the beta 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 alpha vbeta 3 complex and was previously shown to block the function of alpha vbeta 3 (9).

Cells and Culture Media

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.

Synthetic Peptides

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 gamma -fragment (400-411), Gly-Pro-Arg-Pro (GPRP), and Gly-His-Arg-Pro (GHRP) peptides were from Sigma.

Immunoblotting

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 Purification and Plasmic Fragmentation

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 epsilon -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 gamma -chain carboxyl-terminal dodecapeptide gamma  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 gamma  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 Aalpha 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 gamma  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 gamma -chain remnants of fragment D species, i.e. gamma /D1, gamma /D2, and gamma /D3 (14). In this study, we were able to localize the epitope to a much smaller 12-kDa peptide segment with gamma  Met-89 at its amino terminus (12-kDa gamma  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 gamma -chain sequence, we found that these seven residues corresponded to the gamma  89-95 residues, the amino-terminal segment of the gamma -chain remnant of fragment D species. Thus, the epitope for JIF-25 was localized to the amino-terminal 12-kDa peptide segment of the gamma -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.
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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.

Preparation of Fibrin Monomer

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.

Structurally Elucidated Hereditary Dysfibrinogens

Two types of heterozygous dysfibrinogens were utilized, i.e. a dysfibrinogen with an Aalpha Arg-16 right-arrow 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 gamma  Arg-275 right-arrow Cys substitution, manifesting impaired D:D self-association (16).

Preparation of Vitronectin and Fibronectin

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 -70 °C.

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).

Coating Efficiency of Fibrin Monomer and Fibrinogen

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).


Fig. 2. Antigenicity of fibrinogen detected by ELISA using polyclonal anti-fibrinogen antiserum. The wells were coated with fibrinogen (bullet ) or fibrin monomer (black-square); 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."
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Cell Adhesion Assay

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.

Adhesion Inhibition 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.


RESULTS

Characterization of Fibrin Monomer-dependent Cell Adhesion

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 Aalpha 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 gamma  R-275 to C substitution (Fig. 6). The newly exposed amino terminus of the fibrin alpha -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 (bullet ) and fibrin derived from normal fibrinogen (black-square) 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.
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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.
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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 (black-square), GRGESP (square ), GPRP (bullet ), and GHRP (black-triangle).
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Fig. 6. Adhesion of human fibroblasts to immobilized fibrin substratum derived from dysfibrinogens. Fibrins derived from normal fibrinogen and dysfibrinogens (gamma  R275C and Aalpha 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.
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There are two RGD-containing peptide segments assigned to the Aalpha 95-97, RGD-1, and the Aalpha 575-577, RGD-2, residues in each fibrinogen Aalpha -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 gamma  89-173 residue segment. Interestingly, by assignment of the amino acid residues of the Aalpha -and gamma -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 gamma  (73-75) residue segment, which is only 16 residues apart from the amino terminus of the 12-kDa gamma  89~segment recognized by JIF-25. Thus, binding of JIF-25 to the 12-kDa gamma  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 (bullet ) and spread cells (black-square) were counted.
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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.

Identification of the Cell-surface Receptor of Fibrin Monomer-dependent Cell Adhesion

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 alpha vbeta 3, alpha vbeta 5, and the beta 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 beta 1-subclass integrin are involved in the fibrin monomer-dependent cell adhesion.

Table I.

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.


Spread cells
Fibrin-monomer Fibronectin

% of control
Mouse IgG 100.0 100.0
LM609(alpha vbeta 3) 48.0  ± 4.5 110.0  ± 13.5
P1F6(alpha vbeta 5) 109.5  ± 5.5 111.0  ± 14.0
mAb13(beta 1) 15.0  ± 1.0 18.3  ± 3.0


DISCUSSION

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 (Aalpha 586-595), which may interact with a cluster of positively charged residues (Aalpha 601-608) in the carboxyl-terminal segment of the Aalpha -chain, RGD-2 may be inaccessible or hidden in the native fibrinogen molecule. When the (alpha C-alpha 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 gamma  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 gamma -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 alpha IIb/beta 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 beta 1-subclass integrin is preferentially involved in the adhesion of fibroblasts to fibrin monomer together with alpha vbeta 3, a vitronectin receptor, expressed on the fibroblast. The receptors for fibrin(ogen) are shown to be alpha IIbbeta 3, which is specific for binding with the gamma  (400-411) residue segment of fibrinogen (26, 39-42) and alpha vbeta 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 alpha 5 chain of integrins failed to block the fibrin monomer-dependent cell adhesion (data not shown).

The mechanism of the involvement of the beta 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 beta 1-subclass integrin due to a newly induced conformational change upon contact with its authentic receptor alpha vbeta 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 beta 1 family expressed on the HD11 chick myoblast cell line functions as a receptor for fibrinogen. They also showed that a mAb to the beta 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 beta 1-subclass integrin.


FOOTNOTES

*   This study was supported by the following Japanese sources: The Cell Science Research Foundation, The Sagawa Foundation for Promotion of Cancer Research, Yamanouchi Foundation for Research on Metabolic Disorders, Mochida Memorial Research Foundation, The Ichiro Kanehara Memorial Research Foundation, The Ryoichi Naito Foundation for Medical Research, Heparin Research Foundation, Nakatomi Health Science Resarch Foundation, The Foundation for the Development of the Communitym and Scientific Research Grants-in-aid 05670925, 06671108, and 07273263 from the Ministry of Education, Science and Culture of the Government of Japan.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 and reprint requests should be addressed: Division of Hemostasis and Thrombosis Research, Institute of Hematology, Jichi Medical School, Minamikawachi-Machi, Kawachi-Gun, Tochigi-Ken, 329-04, Japan Tel.: 81-285-44-2111 (ext. 3378); Fax: 81-285-44-7817.
1   The abbreviations used are: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.
2   S. Asakura, Q. Zhang, I. Ohkubo, J. Sottile, W. Müller-Esterl, M. Sasaki, M. Matsuda, T. Sakurama, H. Uno, and D. F. Mosher, submitted for publication.

ACKNOWLEDGEMENTS

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).


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