J Biol Chem, Vol. 274, Issue 30, 20943-20948, July 23, 1999

Requirements for ₅₁ Integrin-mediated Retraction of Fibronectin-Fibrin Matrices^*

Siobhan A. Corbett^§ and Jean E. Schwarzbauer^¶

From the  Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903 and the ^¶ Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Retraction of the blood clot by nucleated cells contributes both to hemostasis and to tissue remodeling. Although plasma fibronectin (FN) is a key component of the clot, its role in clot retraction is unclear. In this report, we demonstrate that the incorporation of FN into fibrin matrices significantly improves clot retraction by nucleated cells expressing the integrin ₅₁. Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. To determine the structural requirements for FN in this process, recombinant FN monomers deficient in ligand binding or fibrin cross-linking were incorporated into fibrin clots. We show that recombinant FN monomers support clot retraction by Chinese hamster ovary cells expressing the integrin ₅₁. This process depends on both the Arg-Gly-Asp (RGD) and the synergy cell-binding sites and on covalent FN-fibrin binding, demonstrating that cross-linking within the clot is important for cell-FN interactions. These data show that ₅₁ can bind to FN within a clot to promote clot retraction and support cell shape change. This provides strong evidence that ₅₁-FN interactions may contribute to the cellular events required for wound contraction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Following tissue injury or inflammation, the formation of a provisional extracellular matrix (ECM)¹ from extravasated plasma proteins plays a critical role both in hemostasis and wound repair (1, 2). The principle protein components of this "injury-associated" matrix are fibrin and plasma fibronectin (FN), and in a variety of wound healing events that have been studied, the proteins are deposited together to provide a substrate for cell adhesion and migration (3). The incorporation of FN into the clot markedly enhances cell attachment to the fibrin matrix (4-6). For example, FN is the essential component of plasma clots that facilitates fibroblast migration in vitro (5). Similarly, it appears to provide a conduit for cell movement from collagen gels into fibrin-rich matrices (6). Effective cell interaction with FN-fibrin matrices is dependent upon the formation of covalent cross-links between FN and fibrin molecules that are catalyzed by activated factor XIII (7, 8). For example, Grinnell et al. (9) showed that baby hamster kidney cells required FN and factor XIII for adhesion on fibrin substrates. More recently, using recombinant FN (recFN) molecules that could not be cross-linked to fibrin, we have demonstrated a 50% decrease in cell attachment and spreading on mutant recFN-fibrin matrices when compared with wild type (10). These data indicate that cross-linking may affect interactions between the adhesive sites of FN and integrin cell surface receptors.

Integrins are the principle mediators of cell interactions with the ECM (11, 12). They are noncovalently linked heterodimers composed of one  and one  subunit that together determine ligand binding specificity. Evidence suggests that integrins functionally link ECM molecules and cytoskeletal proteins (13-15). This allows for the transmission of force by cells to anchorage sites, leading to actin cytoskeletal rearrangements and matrix remodeling (15). Traction forces generated by nucleated cells can deform three-dimensional matrices composed of collagen or fibrin (16-18). The stable exertion of physical force required for gel contraction is dependent on the integrin  subunit and is presumed to reflect the strength of linkages between the integrin cytoplasmic tail and cytoskeletal components (19). Recently, Chen et al. (20) compared the capacities of two ₃ integrins to mediate fibrin gel retraction by nucleated cells. They demonstrated that _v3 but not _IIb3 could spontaneously support this process. Similarly, _v3 has been shown to induce fibrin clot retraction by a human melanoma cell line (21). The integrin ₅₁ has also been described as a receptor for fibrin clot retraction (22). Although FN is a ligand for both _v3 and ₅₁, a role for FN in clot retraction has not been described.

In the present work, we demonstrate that FN enhances retraction of fibrin matrices by nucleated cells and that this effect is mediated by the integrin ₅₁. Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. Finally, we define some of the structural requirements for FN in the process of clot retraction. These data provide strong evidence that in a three-dimensional clot matrix, the engagement of the integrin ₅₁ with FN promotes post-receptor occupancy events that include clot retraction and cell shape change. This suggests that ₅₁-FN interactions may play an important role in the early events required for wound remodeling.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- CHO K1 cells transfected with a cDNA to the human ₅ integrin subunit have been previously described (CHO-₅; Ref. 23). These cells express endogenous ₅₁ but do not have ₃. CHO K1 cells transfected with a cDNA to the human ₃ integrin subunit (CHO-_v3; Ref. 24) were a kind gift from M. H. Ginsberg (Scripps Research Institute). CHO B2 cells were kindly provided by Y. Takada (Scripps Research Institute). Cells were maintained in Dulbecco's modified Eagle's medium, 2 mM glutamine, 1% nonessential amino acids, 100 µg/ml Geneticin (CHO-₅ cells only) and 10% fetal calf serum (FCS; Hyclone Laboratories). For culture in three-dimensional matrices, cells were trypsinized, washed, and resuspended in 0.025 M HEPES, pH 7.4, 0.13 M NaCl at 1 × 10⁷ cells/ml. BTI-TN-5B1-4 (High Five) insect cell line (Invitrogen Co.) was maintained in TNM-FH (Life Technologies, Inc.) supplemented with 10% FCS. For purification of recombinant protein, High Five cells were grown in Express Five (Life Technologies, Inc.) serum-free medium supplemented with 18 mM L-glutamine.

Preparation of Mutants and Construction of FN cDNAs-- Construction of recombinant FN monomers (recFN_M) was performed in the baculovirus vector pVL1393. Construction of recFN_M(WT) and recFN_M(Q3) have been previously described (10). To construct recFN_M(RGD+), a termination codon was generated by adding an XbaI linker to a StuI site located at position 6860 in the FNIII_1-7 cDNA (23), and this fragment was inserted between the BamHI and XbaI sites in pVL1393. To construct recFN_M(RGD), a RsrII-SacI fragment of a full-length FN(RGD) (23) was inserted into recFN_M(RGD+). To generate recFN_M, a V0 alternatively spliced variant of recFN_M(RGD+), a SacI-XbaI fragment of rat cDNA III_8-15V0 (25) was cloned into a SacI-XbaI digest of recFN_M(RGD+)_. For recFN_M(syn+) and (syn), termination codons were added to the StuI sites of full-length wild type FN and FN(syn), respectively (26). Constructions were confirmed by restriction digests. Recombinant protein production was performed as described previously (10).

Formation and Analysis of FN-Fibrin Clots-- Lyophilized human fibrinogen (98.8% clottable, American Diagnostica) and bovine thrombin were reconstituted as described previously (4). Endogenous human plasma FN contaminating the fibrinogen was removed by batch incubation with gelatin-agarose (4). SDS-polyacrylamide gel electrophoresis, and immunoblotting with an anti-human FN monoclonal antibody was used to monitor the efficiency of FN depletion as previously reported (4). Residual FN was estimated at less than 150 ng of FN/mg of fibrinogen (4). To assess the cross-linking efficiency of the recFNs versus pFN, the clotting reactions were composed of the following components mixed in a 10:1 physiologic ratio of fibrinogen to FN: fibrinogen 600 µg/ml, pFN/recFN 60 µg/ml, thrombin 2 units/ml, 0.02 M CaCl₂, 0.025 M HEPES, pH 7.4, 0.13 M NaCl. Human coagulation factor XIII (Calbiochem-Novabiochem Corp.) was added at 6 µg/ml, and the cross-linked products were analyzed as previously reported.

Clot Retraction Assays-- 24-well tissue culture dishes were coated with 1% bovine serum albumin overnight at 4 °C. To prepare fibrin or FN-fibrin clots for retraction assays, the clotting components as described above were mixed in a volume of 0.5 ml at room temperature. CHO-_v3 (4 × 10⁶/ml) or CHO-₅₁ (2 × 10⁶/ml) cells in 0.025 M HEPES, pH 7.4, 0.13 M NaCl were then added to clot components, quickly followed by thrombin. After the addition of thrombin, the mixture was rapidly pipetted into 24-well tissue culture dishes and incubated at 37 °C. Aprotinin was added to this system to inhibit fibrinolysis. After 30 min of incubation, 1 ml of Dulbecco's modified Eagle's medium plus 10% FN-depleted FCS was added to the dishes. The clots were then carefully detached from the dishes using a dissecting microscope. Clot retraction was visualized and measured over a 4-h time period using a ruler. The area of the clot was calculated, and the percentage of clot retraction was determined relative to the starting area using the following formula: % retraction = (starting area  measured area)/starting area × 100.

Cells in three-dimensional matrices were examined using inverted phase contrast optics. Photographic images were captured using an NEC video camera (NEC Corp.) connected to a Macintosh 7100/80 computer equipped with a Scion LG3 image capture board (Scion Corp).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fibronectin Enhances CHO-_v3 Retraction of Fibrin Matrices-- The retraction of fibrin matrices by nucleated cells is primarily mediated by the integrin _v3 (20, 21). Because plasma FN is also a ligand for _v3, we sought to determine whether the incorporation of FN into fibrin matrices would affect the ability of cells to retract these matrices. CHO K1 cell transfectants expressing _v3 (CHO-_v3) retract fibrin clots (20). As demonstrated in Fig. 1A, the extent of retraction of FN-fibrin matrices when compared with fibrin matrices was significantly greater at all time points ranging from 30 min to 4 h (p < .002 at 4 h, Student's unpaired t test). Clot retraction was dependent on cell number as has been demonstrated by others (data not shown).

View larger version (104K): [in this window] [in a new window]   Fig. 1.   CHO-v3 retraction of fibrin or FN-fibrin matrices. Fibrin () or FN-fibrin () matrices containing 4 × 106 cells/ml were incubated at 37 °C for 30 min following the addition of thrombin to allow clot gelation. Dulbecco's modified Eagle's medium plus 10% FN-depleted FCS was then added, and clots were either detached from the dishes to assay clot retraction (A) or left undisturbed to observe cell morphology (B). Clot retraction was visible and was measured at indicated times. Alternately, cell morphology was examined following overnight incubation, using inverted phase contrast optics. The data in A are expressed as the means ± S.E. of triplicate experiments.

The ability of cells to exert physical force on three-dimensional substrates has been correlated with cell shape change. For example, cells that have increased capacity to contract collagen matrices display an increased number of cell processes when placed into these gels (19). To determine whether CHO-_v3 cells display changes in morphology when cultured in FN-fibrin matrices, cells were seeded in fibrin or FN-fibrin gels and maintained in culture at 37 °C. After 18 h, there was a marked increase in the number of spread cells in the FN-fibrin matrices (Fig. 1B). When clot retraction was measured at 18 h, a significant difference between FN-fibrin and fibrin matrices was observed, comparable with the short time course experiments (data not shown).

Retraction of FN-Fibrin Matrices Is Mediated by Endogenous ₅₁-- The addition of FN to fibrin matrices increases the extent of clot retraction by CHO-_v3 cells. One possible explanation for this is that FN-_v3 interactions promote clot retraction. To investigate the contribution of _v3 to retraction of FN-fibrin matrices, CHO-_v3 cells were incubated with a cyclic RGD peptide inhibitor that is specific for _v3 at low concentrations (27). We used the peptide inhibitor rather than function blocking antibodies because others have shown antibodies to be less effective in this context (20). The effective concentrations of the inhibitor were determined through cell adhesion assays. The peptide at 1 µM blocked CHO-_v3 adhesion to fibrin- or fibrinogen-coated dishes while allowing adherence to FN-coated wells (data not shown). CHO-_v3 cells in suspension were then incubated in the presence or absence of the peptide inhibitor (1 µM) for 30 min at 37 °C prior to culture in fibrin matrices. When compared with untreated cells, the presence of the cyclic RGD peptide inhibited clot retraction by an average of 50% (Fig. 2A). However, when the CHO-_v3 cells were cultured in FN-fibrin matrices, there were no significant differences in clot retraction in the presence or absence of the inhibitor (Fig. 2B). These data strongly suggest that another integrin receptor may have a role in FN-mediated clot retraction.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effects of inhibitors on clot retraction by CHO-v3 cells. Cells (4 × 106/ml) were untreated () or preincubated with an v3-specific cycRGD peptide (), the hamster-specific anti-51 monoclonal antibody, PB-1 (), or both the peptide and PB-1 () for 30 min at 37 °C. Cells were then cultured in fibrin (A) or FN-fibrin (B) matrices in the continued presence of the inhibitors, and clot retraction was measured at indicated times as described in the legend to Fig. 1. The data are expressed as the means ± S.E. of duplicate experiments.

CHO-_v3 cells also express endogenous ₅₁, a major FN integrin receptor. To determine whether the integrin ₅₁ may be involved in the retraction of FN-fibrin matrices, cells were preincubated with the hamster-specific anti-₅₁ antibody PB-1 and then cultured in fibrin or FN-fibrin gels as described above. Fig. 2B demonstrates that incubation with PB-1 decreases the retraction of FN-fibrin matrices by CHO-_v3 cells when compared with untreated or cyclic RGD peptide-treated cells. When both the cyclic RGD inhibitor and the PB-1 antibody were present, significant inhibition of clot retraction was achieved for both fibrin and FN-fibrin matrices. These data indicate that nucleated cells can use the ₅₁ integrin to promote the retraction of FN-fibrin matrices.

FN-Fibrin Matrices Support ₅₁-mediated Postreceptor Occupancy Events-- To establish a role for ₅₁ in FN-mediated clot retraction by nucleated cells, CHO K1 cells expressing the human integrin ₅ subunit were used (CHO-₅). These cells have the specific advantage of producing little endogenous FN when compared with the parent cells (23). CHO-₅ cells were cultured in fibrin or FN-fibrin matrices as described above. CHO-₅ cell retraction of FN-fibrin clots was significantly greater than retraction of fibrin clots (Fig. 3, p  .0001 at 4 h; Student's unpaired t test). The extent of retraction was dependent on the concentration of FN. No increase in CHO-₅ cell retraction of fibrin clots was observed when added FN was less than 1.0 µg/ml. CHO B2 cells that do not express the ₅ integrin subunit do not retract fibrin or FN-fibrin matrices (data not shown). These data demonstrate that the addition of FN to the fibrin clot markedly improves ₅₁-mediated generation of tractional forces.

View larger version (59K): [in this window] [in a new window]   Fig. 3.   CHO-5 retraction of fibrin, FN-fibrin, or recFNM-fibrin matrices. Fibrin (), FN-fibrin (), or recFNM-fibrin matrices () containing 2 × 106 cells/ml were incubated at 37 °C for 30 min following the addition of thrombin. Clot retraction was examined as described in the legend to Fig. 1.

Engagement of the ₅₁ integrin receptor leads to "post-receptor occupancy" events that have been well described in two-dimensional culture systems (28, 29). These include cell spreading and intracellular protein tyrosine phosphorylation (30). However, the cellular events following ₅₁ ligation of FN in a three-dimensional culture system have not been described. To examine cell morphology, CHO-₅ cells were seeded in fibrin or FN-fibrin gels and observed in culture at 37 °C. By 18 h, it was evident that the incorporation of plasma FN into the fibrin matrix significantly improved cell spreading (Fig. 4). CHO-₅ cells cultured in FN-fibrin matrices displayed a unique, bipolar cell morphology, whereas those in fibrin matrices remained rounded. Further, CHO-₅ cell retraction of FN-fibrin matrices at 18 h remained significantly greater than retraction of fibrin matrices (data not shown).

View larger version (138K): [in this window] [in a new window]   Fig. 4.   FN-fibrin matrices support 51-mediated postreceptor occupancy events. Fibrin or FN-fibrin matrices containing 2 × 106 cells/ml were incubated at 37 °C for 30 min following the addition of thrombin to allow clot gelation. Dulbecco's modified Eagle's medium plus 10% FN-depleted FCS was then added, and the clots were left undisturbed overnight at 37 °C. Cell morphology was observed as described in Fig. 1.

Both Plasma FN and Recombinant FN Monomers Promote ₅₁-mediated Clot Retraction-- The ₅₁ integrin receptor is the primary FN receptor required for the assembly of FN into a fibrillar matrix. Matrix assembly is a process that requires that the FN molecule be in a dimeric form (31). In a clot, dimeric plasma FN is incorporated into the fibrin matrix. To determine whether the dimer structure is required for FN-mediated clot retraction, CHO-₅ cells were cultured in clots formed from fibrin and plasma FN or a recFN_M. As indicated in Fig. 3, the dimeric form of FN is not a requirement for clot retraction.

₅₁-mediated Clot Retraction Requires Both the RGD and Synergy Sites-- Our previous work suggests that the covalent incorporation of FN into a fibrin matrix may affect the availability or conformation of integrin-binding sites (4). The ligand requirements for ₅₁-FN interactions have been well described in a two-dimensional system and include the RGD sequence in repeat III₁₀ and the synergy site in repeat III₉ (11, 12, 32-34). The engagement of both sites by the ₅₁ integrin receptor is a requirement for full cell adhesion and spreading. To determine the role of these sites in ₅₁-FN interactions in a three-dimensional system, recFN molecules were expressed that contained either a deletion (RGD) or mutation (syn) in the important ligand-binding regions. Functional analyses of recFN_M polypeptides and equivalent wild type recombinant proteins were performed by assaying clot retraction. In these experiments, CHO-₅ retraction of fibrin matrices served as the negative control, and this background value for clot retraction was subtracted at each time point. When CHO-₅ cells were cultured in FN-fibrin matrices containing recFN_M (RGD), clot retraction was decreased by 90% when compared with the wild type recFN_M(RGD+) (Fig. 5A). These data demonstrate that the increase in CHO-₅ retraction of FN-fibrin matrices when compared with fibrin matrices is mediated by cell binding to FN. Although CHO-₅ cell retraction of recFN_M(syn)-fibrin matrices was significantly less than wild type recFN_M(syn+) at all time points (Fig. 5B), there was still measurable clot retraction. These data suggest that ₅₁ engagement of the RGD site can partially support the development of tension by cells that use the ₅₁ receptor but that full clot retraction requires the synergy site.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   CHO-5 retraction of recFNM-fibrin matrices requires both the RGD and the synergy site for maximal efficiency. RecFNM-fibrin matrices formed from wild type recFNM () or mutant recFNM () containing an RGD deletion (A) or a synergy site mutation (B) were compared for their ability to support clot retracton by CHO-5 cells. Clot retraction was measured at indicated times as described above. CHO-5 retraction of fibrin matrices served as the negative control, and this background value for clot retraction was subtracted at each time point. Data are expressed as the means ± S.E. of triplicate experiments.

Cross-linking of FN to Fibrin Is Required for Maximal Clot Retraction-- Cross-linking of FN to fibrin is required for efficient cell attachment to FN-fibrin matrices (9, 10). To determine whether cross-linking is also required for ₅₁-mediated clot retraction, fibrin clots containing CHO-₅ cells were prepared with either a wild type FN or a mutant recFN lacking the amino-terminal glutamine residues (Gln³, Gln⁴, and Gln¹⁶) that are important for covalent FN-fibrin interactions (10). CHO-₅ retraction of fibrin matrices served as the negative control, and this value was subtracted at each time point. Cells cultured in clots composed of the cross-linking-deficient recFN_M(Q³) were significantly less efficient in retracting the clot matrices despite the presence of the appropriate ligand-binding site (Fig. 6). These data demonstrate that covalent cross-linking of FN to fibrin is required for the maximal promotion of force generation by CHO-₅ cells within the clot.

View larger version (62K): [in this window] [in a new window]   Fig. 6.   Effective CHO-5 retraction of recFNM-fibrin matrices requires covalent cross-linking of recFNM to fibrin. RecFNM-fibrin matrices formed from wild type recFNM () or a recFNM () containing mutations in key glutamine residues required for cross-linking were compared for their ability to support clot retracton. CHO-5 retraction of fibrin matrices served as the negative control as described above. Data are expressed as the means ± S.E. of triplicate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we have examined whether the covalent incorporation of FN into three-dimensional fibrin clots influences the retraction of these clots by nucleated cells. We demonstrate that FN enhances the retraction of fibrin clots by nucleated cells and that this effect is mediated by the integrin ₅₁. Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin clots. FN-fibrin clot retraction by ₅₁-expressing cells does not require a dimeric FN molecule. However, retraction does depend on both the RGD and the synergy sites in the cell-binding domain of FN and also requires covalent cross-linking of FN to fibrin. These data provide strong evidence that in a three-dimensional system, the engagement of the integrin ₅₁ with FN can contribute to the generation of mechanical force required for clot retraction, leading to distinct changes in cell shape. This suggests that FN may play an important early role in the process of tissue remodeling.

Fibrin clot retraction by both platelets and nucleated cells using receptors of the ₃ integrin family has been described (20, 21, 35). Integrin _IIb3 is essential for platelet-mediated clot retraction but cannot spontaneously support clot retraction in nucleated cells without the presence of an activating antibody (20, 35). However, nucleated cells that express _v3 can retract fibrin matrices (20). These data support a role for the integrin  subunit cytoplasmic domain in modulating the generation of mechanical force in three-dimensional substrates. This has also been observed in a collagen gel system using chimeric ₂ integrin subunits composed of the external and transmembrane domains of ₂ coupled to the cytoplasmic domains of ₅ (19). The chimeric ₂-₅₁ complex had similar function to ₂₁ in mediating collagen gel retraction, demonstrating that the integrin ₅ cytoplasmic tail can influence the generation of tension.

Plasma FN is a key adhesive component of the fibrin-rich matrices typically found at injury sites, and with a plasma concentration of 330 µg/ml, it seems likely that FN is deposited wherever fibrinous exudates occur (3). Further, the release of FN from activated platelets may contribute to the dispersal of FN throughout the clot. Although a role for FN in cell adhesion and migration has been well described, a contribution to clot retraction has not been determined. Our results demonstrate a consistent increase in the ability of the CHO-_v3 cells to retract FN-fibrin matrices when compared with fibrin matrices. This response resulted from an interaction between endogenous hamster ₅₁ and FN. One explanation for these observations could be that the engagement of additional integrin receptors strengthens the mechanical link between the ECM and the cytoskeleton by increasing the total number of receptor-ECM connections. In this instance, ₅₁ and _v3 would be functionally equivalent in their ability to mediate gel retraction. However, our data suggest that ₅₁ plays the major role in mediating FN-dependent clot retraction. This is indicated by the fact that the cyclic peptide has a limited ability to inhibit FN-mediated clot retraction in the presence of functioning ₅₁. Previous work has suggested that ₅₁ can exclude other FN integrins from particular functions. For example, Wennerberg et al. (36) showed that ₁-null cells that express _v3 were capable of assembling a FN matrix. However, when these cells were transfected with a ₁ subunit and expressed functional ₅₁, _v3 was excluded from sites of FN matrix assembly, suggesting that ₅₁ assumed a preferential role in this process. Similarly, the presence of ₅₁ can exclude _v1 from sites of cell-FN attachment (37).

It is possible that the ₅₁-FN interactions leading to the generation of mechanical force in a three-dimensional matrix may be comparable with those required for FN matrix assembly. In this context, therefore, the ₅₁ integrin may preferentially trigger post-receptor occupancy events that promote force generation with greater efficiency than _v3 and thus exclude it from the process. Differences in the abilities of individual integrins to support tractional forces have been demonstrated. Interestingly, an inverse correlation has been reported between the ability of cells expressing specific integrins to generate tension and their rates of cell migration (19, 38). For example, transfectants that expressed chimeric ₂ integrin subunits composed of the external and transmembrane domains of ₂ coupled to the cytoplasmic domains of ₂, ₅, or ₄ were examined for their migratory rates on collagen (19). Cells that expressed the ₂/₄ chimera had the fastest migratory rate but the least capacity to contract collagen matrices. In contrast, the ₂/₅ chimera had a relatively low migratory rate but an increased capacity to retract collagen gels. Because the _v3 integrin is also thought to play a key role in cell migration events (40, 41), this integrin may be less effective than ₅₁ in FN-mediated clot retraction.

Recently, ₅₁ has been shown to bind to fibrin, both in two- and three-dimensional systems (22, 42). CHO-₅ transfectants retract fibrin matrices, but the addition of FN even at low concentrations increases clot retraction 2- to 3-fold. Cross-linking of FN to fibrin increases fibrin fibril thickness and strength (43, 44). These changes could alter fibrin structure, making it a more efficient ligand for ₅₁. When the retraction of recFN-fibrin matrices composed of recFN_M(RGD+) and recFN_M (RGD) are examined, however, CHO-₅ retraction of the recFN_M(RGD)-fibrin matrix is comparable with fibrin alone, demonstrating that the increased clot retraction results from the ligation of ₅₁ by FN specifically. Furthermore, FN-fibrin matrices support distinct cell shape change. It is possible that the ₅₁-mediated promotion of clot retraction may occur via the establishment of a functional link between FN and the cytoskeleton mediated by ₅₁ that supports the generation of mechanical force. In two-dimensional systems, ₅₁-FN interactions result in the assembly of FN into a fibrillar matrix, organization of the cytoskeleton into stress fibers, and distinct intracellular signaling events (23, 32). However, it is also possible that ₅₁-FN interactions could influence clot retraction via "inside-out" signaling events that may impact other integrin receptors.

The differences in the ability of ligands to promote post-receptor occupancy events may depend on the mechanism through which they occupy the ligand-binding pocket of ₅₁. Recent evidence suggests that cells can sense the rigidity of the ECM and respond by localized strengthening of cytoskeletal linkages (45). This process of reinforcement required occupancy of FN-binding sites and could not be duplicated by antibody cross-linking of the integrin receptors. Thus, it is possible that a ligand that does not effectively occupy the ₅₁-binding site permits only limited strengthening of the cytoskeletal attachments and incomplete force generation. In a three-dimensional matrix, therefore, fibrin may not effectively occupy the ligand-binding site of ₅₁. Thus, integrin-cytoskeletal linkages are not reinforced limiting the strength of cell-ECM contacts that are required for clot retraction and cell shape change. This model also provides an attractive explanation for why the synergy site is required for efficient FN-fibrin matrix retraction, because in its absence, the ligand-binding pocket of ₅₁ is only partially occupied.

There also may be a relationship between ECM rigidity and the generation of force at anchorage sites. This suggests a mechanism that explains the importance of cross-linking in FN-mediated clot retraction. In the absence of cross-linking, we observed a nearly 75% decrease in clot retraction. The generation of traction forces by cells requires that the ECM resist cell tension at attachment sites (45, 46). Perhaps, if FN molecules are not fixed in position by the cross-linking process, they "give" in response to applied mechanical stress. As a result, cells maintain a more rounded configuration, and the cytoskeletal rearrangements required for force generation are not achieved.

In summary, we have demonstrated that the incorporation of FN into fibrin matrices significantly improves clot retraction by nucleated cells and that this effect is mediated by the integrin ₅₁. Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. Finally, we have determined that both the RGD and synergy sites in FN, as well as the covalent incorporation of FN into the fibrin matrix are required for ₅₁-mediated promotion of clot retraction. These data provide strong evidence that ₅₁-FN interactions in a three-dimensional clot matrix may contribute to the events required for early tissue remodeling.

	ACKNOWLEDGEMENT

We thank Jan Sechler for helpful discussions.

	FOOTNOTES

^* This work was supported by National Institute of Health Grant CA-44627 (to J. E. S.).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.

^§ American Heart Association-Genentech Clinician Scientist Awardee. To whom correspondence is to be addressed. Tel.: 732-235-6096; Fax: 732-235-6003; E-mail: corbetsi@umdnj.edu.

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; recFN, recombinant fibronectin; recFN_M, recFN monomer; FCS, fetal calf serum; CHO, Chinese hamster ovary.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES