Fibrinogen Is a Ligand for Integrin alpha 5beta 1 on Endothelial Cells*

(Received for publication, June 6, 1996, and in revised form, December 10, 1996)

Kazuhisa Suehiro Dagger , James Gailit § and Edward F. Plow Dagger

From the Dagger  Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the § Department of Dermatology, State University of New York, Stony Brook, New York 11794-8165

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn2+ and was suppressed by Ca2+. The Mn2+-supported interaction was completely inhibited by RGD peptides but not by alpha vbeta 3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two alpha 5beta 1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified alpha 5beta 1; and also was observed with a second alpha 5beta 1-bearing cell type, platelets. The binding of fibrinogen to alpha 5beta 1 on endothelial cells in the presence of Mn2+ was time-dependent, specific, saturable, and of high affinity (Kd = 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at Aalpha 572-574 was implicated in fibrinogen recognition by alpha 5beta 1. Two circumstances were identified in which alpha 5beta 1 interacted with fibrinogen in the presence of Ca2+: when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for alpha 5beta 1 on endothelial and other cells, an interaction which may have broad biological implications.


INTRODUCTION

The luminal surface of endothelial cells (EC)1 is continuously exposed to a high concentration of plasma fibrinogen (Fg). Disruption of the endothelium results in local thrombus formation, and Fg/fibrin accumulates at such sites of vascular injury. Based upon these proximal relationships, the molecular mechanisms and functional consequences of the interaction of Fg with EC have been topics of considerable interest and investigation (1-6). Indeed, it has been shown that Fg can induce EC attachment, spreading, and migration (1, 7) and can support an angiogenic response (8). Several distinct receptors have been implicated in mediating Fg binding to EC. These include alpha vbeta 3 (9, 10), a member of the integrin family of cell adhesion receptors, as well as several non-integrin binding sites (6, 11). Transglutaminase-mediated Fg cross-linking to EC also has been demonstrated (12).

alpha vbeta 3 interacts with Fg via an Arg-Gly-Asp (RGD) recognition specificity; i.e. RGD-containing peptides block Fg binding to this receptor (13). This tripeptide sequence is recognized not only by alpha vbeta 3 but also by several other integrins (14-16), including alpha 5beta 1, which serves as a fibronectin (Fn) receptor on EC and many other cell types (17-20). Fg contains two RGD sequences within its Aalpha -chain: RGDF at Aalpha 95-98 and RGDS at Aalpha 572-575. Previous studies have shown that EC adhesion to immobilized Fg is blocked by a monoclonal antibody (mAb) to the carboxyl-terminal peptide containing RGD sequence at Aalpha 572-574 (3). Moreover, this adhesion was blocked by mAbs against alpha vbeta 3 (9, 10). Soluble Fg also binds directly to EC in monolayers or in suspension in a specific, saturable (1) and RGD inhibitable interaction (2).

Recently, we (21) and others (22) have shown that the capacity of purified alpha vbeta 3 to bind Fg is regulated by divalent cations: Mn2+ supports Fg binding to the receptor, whereas Ca2+ does not; and, in fact, Ca2+ inhibits Fg binding to alpha vbeta 3 in the presence of Mn2+. This pattern of cation regulation is not unique to alpha vbeta 3; Mn2+ enhances and Ca2+ suppresses ligand binding to several integrins. For example, recognition of Fn (23, 24) and anti-beta 1 mAb 9EG7 (25) by alpha 5beta 1 is enhanced and/or induced by Mn2+ and is inhibited by Ca2+. In view of the pivotal role of cations in regulating integrin specificity, we have re-examined the binding of Fg to EC, anticipating an interaction that would be favored by Mn2+ and would be mediated by alpha vbeta 3. Surprisingly, while Mn2+ enhanced binding, the receptor mediating this interaction was not alpha vbeta 3 but was alpha 5beta 1. Thus, a novel ligand has been identified for this receptor which may have broad biological implications.


MATERIALS AND METHODS

Purification and Radioiodination of Proteins

Fg was purified from human fresh-frozen plasma by differential ethanol precipitation and ammonium sulfate fractionation (26). Human Fn was isolated (27) and provided by Dr. Tatiana Ugarova (Cleveland Clinic Foundation, Cleveland, OH). The 120-kDa chymotryptic Fn fragment containing the central cell-binding domain was purchased from Life Technologies Inc. Fg and 120-kDa chymotryptic Fn fragment were labeled with Na125I (Amersham Life Science Inc.) using a modified chloramine-T method (28). The specific activity of 125I-Fg ranged from 0.8 to 1.2 × 10-6 cpm/molecule. alpha 5beta 1 was purified from human placenta as described (23). Briefly, placental tissue extract was applied to an affinity column of the 120-kDa chymotryptic Fn fragment, coupled to Sepharose, and the bound receptor was eluted with GRGDSP.

Peptides and Antibodies

The synthetic peptides GRGDSP (GRGESP) and the dodecapeptide (HHLGGAKQAGDV) from the extreme carboxyl terminus of Fg gamma -chain, referred to as H12, were prepared using an Applied Biosystems Model 430A Peptide Synthesizer (29). MAb 7E3 (30) which recognizes beta 3 integrins was provided by Dr. Barry Coller, Mount Sinai School of Medicine, New York. MAbs LM609 (10) directed to alpha vbeta 3, JBS5 (31) directed to alpha 5beta 1, CLB-706 (32) directed to alpha v, JB1a, originally designated J10 (33), directed to beta 1 were purchased from Chemicon International Inc. (Temecula, CA). MAb P1D6 (34) against alpha 5 was obtained from Life Technologies Inc. Two mAbs raised against RGD-containing synthetic peptides from Fg Aalpha -chain, anti-N directed to Aalpha 87-100 (TTNIMEILRGDFSS), and anti-C directed to Aalpha 566-580 (SSTAYNRGDSTFESK) (3) were provided by Dr. Zaverio Ruggeri, The Scripps Research Institute, La Jolla, CA. MAb 4A5 (35), to the C terminus of the gamma -chain of Fg, was a gift from Dr. Gary Matsueda, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. Anti-beta 1 mAb 8A2 (36) was provided by Dr. Nicholas Kovach, Washington University, Seattle, WA.

Cell Culture

Primary cultures of human umbilical vein EC (HUVEC) were provided by Dr. Paul DiCorleto (Cleveland Clinic Foundation) (37) and grown to confluence in 162-cm2 plastic flasks (Corning Costar Corp., Cambridge, MA) in Dulbecco's modified Eagle's medium/F-12 (BioWhittaker Inc., Walkersville, MD) supplemented with 15% fetal bovine serum (BioWhittaker Inc.), 150 µg/ml endothelial cell growth factor (Clonetics Corp., San Diego, CA) and 90 µg/ml heparin (Sigma). The cells were used within the second to fifth passage.

Fg Binding to Cells in Suspension

For binding of Fg to HUVEC in suspension, the adherent cells were washed twice with 10 ml of 10 mM HEPES, 150 mM NaCl, pH 7.5 (Buffer A), and detached by brief exposure (less than 1 min) to a 0.25 mg/ml trypsin, 0.01% EDTA solution (Clonetics Corp.). After neutralizing the trypsin, the cells were immediately centrifuged at 500 × g for 15 min. Cell pellets were suspended, washed twice with Buffer A, and resuspended in 1 ml of Buffer A containing 1% BSA. HUVEC (1 × 106/ml) were preincubated with or without mAbs or peptides for 30 min at 22 °C, and then 125I-Fg was added and incubated at 37 °C in the presence of a selected divalent cation in a final volume of 200 µl by continuously rotating the tubes end over end at approximately 6 rpm. All buffers were pretreated with Chelex 100 (Bio-Rad) to remove undesired cations. At selected times, 50 µl of the cell suspension were layered on 300 µl of 20% sucrose in Buffer A containing 1% BSA in conical polyethylene microcentrifuge tubes (38, 39). Bound ligand was separated from free by centrifugation for 2 min in a Beckman Microfuge 12. The tips of the tubes were cut off with a razor blade, and the radioactivity associated with the cell pellet was counted in a gamma -counter. Data were determined with triplicate measurements of each experimental point. The standard deviation of the triplicate measurements was less than 10% throughout the course of these studies. The binding of 125I-Fg to washed platelets was performed as described previously (38), using prostaglandin E1 (1 µg/ml) and theophylline (1 mM) to maintain the cells in a resting state during isolation and ligand binding assays.

SDS-Polyacrylamide Gel Electrophoresis and Autoradiography

Radioactivity associated with cells was extracted into buffer containing 10 mM Tris-HCl, 2 mM EDTA, 1.25% SDS, 1.5 mM phenylmethylsulfonyl fluoride, 5 mM o-phenanthroline, 0.1% NaN3, 1 µM leupeptin, 1 µM pepstatin, and 100 KIU/ml aprotinin, pH 7.4. Samples were boiled for 2 min and electrophoresed under reducing conditions using the buffer system of Weber and Osborn (40) with a 5% acrylamide gel. Gels were stained with Coomassie Brilliant Blue or dried and subjected to autoradiography.

Solid-phase Ligand Binding Assay

The binding of Fg or the 120-kDa chymotryptic Fn fragment to purified and immobilized alpha 5beta 1 was performed as described (21). alpha 5beta 1 (70 µg/ml) was diluted 1:35 with Buffer A, immobilized in 96-well microtiter plates at 200 ng/well, and incubated overnight at 4 °C. After the plates were blocked with 20 mg/ml BSA in Buffer A, 125I-labeled ligands were added in Buffer A containing the selected divalent ions at 1 mM concentrations and incubated for 180 min at 37 °C. Nonspecific binding was measured by determining the ligand binding to BSA-coated wells at each cation condition, and these values were subtracted from the corresponding values for receptor-coated wells.

HUVEC Adhesion Assay

HUVEC were harvested as described and resuspended in 1 ml of Buffer A. Radiolabeling of HUVEC was performed by incubating with 0.5 mCi of Na251CrO4 (DuPont NEN) for 30 min at 22 °C. Fg (10 µg/ml in Buffer A) was immobilized in 96-well Immulon-3 microtiter plates (Dynatech Laboratories Inc., Chantilly, VA) at 1 µg/well and incubated overnight at 4 °C. The plates were blocked with 1% BSA (heat-inactivated for 1 h at 56 °C) in Buffer A for 90 min at 22 °C. 51Cr-labeled HUVEC cell suspension (7.5 × 105/ml in Buffer A containing 0.1% BSA), incubated with or without inhibitors in the presence of appropriate cations for 30 min at 22 °C, was applied to Fg-coated wells and incubated for 60 min at 37 °C. After washing four times, bound cells were lysed by adding 2% SDS, and radioactivity was measured in beta -counter.


RESULTS

Manganese Supports Fibrinogen Binding to HUVEC

To assess how divalent cations might affect the interaction of Fg with EC, 125I-Fg was incubated with HUVEC in suspension with 1 mM concentrations of different cations. Binding was measured after 45 min at 37 °C. As shown in Fig. 1A, Mn2+ supported Fg binding, whereas Ca2+ or Mg2+ failed to enhance binding above the level observed in EDTA. Since Ca2+ was unable to support binding, we considered whether it might interfere with Fg binding supported by 1 mM Mn2+ as reported for several integrin-ligand interactions (22, 24, 41). Indeed, Ca2+ did suppress Mn2+-supported Fg binding; 2 mM Ca2+ inhibited 125I-Fg binding by 64%. The effects of various Mn2+ concentrations on Fg binding is shown in Fig. 1B. No interaction was observed at Mn2+ concentrations below 20 µM, but higher concentrations stimulated binding. Ca2+ did not support binding in the 1 µM to 1 mM range. In subsequent experiments, 1 mM Mn2+ was chosen as a concentration which supported extensive Fg binding.


Fig. 1. A, effects of divalent cations on Fg binding to HUVEC. Radiolabeled Fg (40 nM) was incubated for 45 min at 37 °C with HUVEC (1 × 106 cells/ml) suspended in buffer containing 5 mM EDTA, 1 mM Ca2+, 1 mM Mg2+, or 1 mM Mn2+. To assess the effect of Ca2+ on Mn2+-supported binding of 125I-Fg to HUVEC, the Mn2+ concentration was 1 mM and Ca2+ was present at 0.2 or 2 mM. The data shown are means and standard deviation (S.D.) from three experiments. B, effect of divalent cation concentration on Fg-HUVEC interaction. Conditions are the same as in Panel A, and the results shown are the average of triplicate measurements performed at each experimental data point and are representative of three separate experiments.
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We sought to verify that the HUVEC-bound radioactivity was Fg. The radioactive ligand was bound to the cells for 10 or 45 min, and then the HUVEC lysates were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography (Fig. 2). The radiolabeled Fg gave three distinct bands, at the same positions as the Aalpha -, Bbeta -, and gamma -chains stained with Coomassie, and exhibited no detectable bands of higher or lower mobility. After a 10-min incubation, the 125I-Fg in the HUVEC lysate produced bands at these same positions although the intensity of Aalpha -chain was reduced relative to the Bbeta - and gamma -chains. This phenomenon also was reported by Dejana et al. (1). We found that the band corresponding to the Aalpha -chain became still less intense with the longer incubation time, whereas the Bbeta -chain remained unchanged. Coinciding with the loss of Aalpha -chain was the appearance of higher molecular weight bands. These results indicate that radioactivity which interacted with HUVEC in the presence of Mn2+ was Fg, although the ligand did undergo time-dependent modification.


Fig. 2. Characterization of the ligand bound to HUVEC. 125I-Fg (40 nM) was incubated with HUVEC (1 × 106 cells/ml) for 10 min (lane 3) and 45 min (lane 4) at 37 °C in the presence of 1 mM Mn2+. At each time point, 0.5-ml aliquots of cell suspension were collected, separated by centrifugation, and lysed in SDS. The sample was then subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography. Lane 1 is Fg stained with Coomassie Brilliant Blue, and lane 2 is the autoradiogram of 125I-Fg. Aalpha , Bbeta , and gamma  indicate the constituent Fg chains.
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Fibrinogen Binds to alpha 5beta 1 on HUVEC

To establish the specificity of Fg binding to HUVEC in the presence of Mn2+, several competitors were tested for their ability to inhibit the interaction (Fig. 3A). A 100-fold excess of nonlabeled Fg inhibited the binding of the radiolabeled ligand by more than 80%. Furthermore, a RGD ligand peptide, GRGDSP, inhibited binding by 90%, while the control peptide, GRGESP, had no effect. H12, the dodecapeptide from the carboxyl terminus of the Fg gamma -chain (residues 400-411), which effectively inhibited Fg binding to alpha IIbbeta 3, had no effect on HUVEC binding of the ligand.


Fig. 3. Specificity of Fg binding to HUVEC in the presence of Mn2+. A, HUVEC were preincubated with excess unlabeled Fg (4 µM), GRGDSP (100 µM), GRGESP (100 µM), and H12, the Fg gamma -chain dodecapeptide HHLGGAKQAGDV (500 µM). B, HUVEC were preincubated with anti-beta 3 mAb 7E3 (20 µg/ml), anti-alpha vbeta 3 mAb LM609 (20 µg/ml), anti-alpha v mAb CLB-706 (1/100 dilution), anti-beta 1 mAb JB1a (1/100 dilution), anti-alpha 5 mAb P1D6 (1/100 dilution), and anti-alpha 5beta 1 mAb JBS5 (1/100 dilution). In both panels, the preincubations were for 30 min at 22 °C. 125I-Fg then was added at a final concentration of 40 nM, and the incubation proceeded for 45 min at 37 °C in the presence of 1 mM Mn2+. Values represent the means and S.D. of three to five determinations and are expressed as percent of a control lacking inhibitors.
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The stimulatory effect of Mn2+ and the inhibition by the RGD peptide is consistent with a role of alpha vbeta 3 in Fg-HUVEC interaction. However, the binding was not inhibited by two alpha vbeta 3 blocking mAbs (Fig. 3B). MAb 7E3 and mAb LM609, which block Fg binding to alpha vbeta 3 (10, 30), had no effect on 125I-Fg binding to HUVEC. The functionality of these mAbs was confirmed by inhibition in HUVEC adhesion to Fg (see below). In considering other candidate Fg-binding sites, we examined the role of alpha 5beta 1 as this integrin also exhibits RGD recognition specificity and its function is stimulated by Mn2+ (23, 24). Indeed, as shown in Fig. 3B, inhibition by anti-beta 1 mAb JB1a was significant (58%). Moreover, anti-alpha 5 mAb P1D6 and anti-alpha 5beta 1 mAb JBS5 inhibited this interaction by 88 and 91%, respectively. Thus, alpha 5beta 1 is centrally involved in the Mn2+ supported Fg binding to suspended HUVEC.

We sought to determine if alpha 5beta 1 could be implicated in Fg binding to another cell type. Platelets were used as a model. Little 125I-Fg bound to non-stimulated platelets in the presence of Ca2+ (Fig. 4). This limited binding was inhibited by anti-beta 3 mAb 7E3, and anti-alpha 5beta 1 mAb JBS5 was without effect. Fg binding in the presence of Mn2+ was three times higher than in the presence of Ca2+, and now anti-alpha 5beta 1 mAb inhibited this interaction by 47%. The anti-beta 3 mAb blocked binding by 65% and, in combination with anti-alpha 5beta 1 mAbs, almost totally inhibited the interaction (92%). The number of Fg molecules bound per platelet in the presence of Mn2+ is consistent with the reported number of alpha 5beta 1 receptors (42).


Fig. 4. Differential effects of mAbs on the binding of 125I-Fg to non-stimulated platelets. Washed platelets (5 × 108/ml), in the presence of 1 µg/ml prostaglandin E1 and 1 mM theophylline, were preincubated with anti-beta 3 mAb 7E3 (20 µg/ml), anti-alpha 5beta 1 mAb JBS5 (1/100 dilution), or both for 30 min at 22 °C, and 125I-Fg was added at a final concentration of 300 nM and incubated for 30 min at 22 °C in the presence of 1 mM Ca2+ or 1 mM Mn2+. The data shown are the average of triplicate measurements at each experimental data point and are representative of three experiments.
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Fibrinogen Binds to Purified alpha 5beta 1

Consistent with a role for alpha 5beta 1 in the interaction of Fg with HUVEC, binding of Fg to the purified receptor also was demonstrable. Purified alpha 5beta 1 was immobilized onto the wells of microtiter plates. Using the 120-kDa chymotryptic Fn fragment as a radiolabeled ligand, substantial binding was observed in the presence of Mn2+ but not Ca2+, entirely consistent with the data of Mould et al. (24), and this interaction was more than 97% inhibited by GRGDSP and an anti-alpha 5beta 1 mAb. Next, 125I-Fg binding to alpha 5beta 1 was performed in the same conditions. As shown in Fig. 5, 4.9-fold higher binding was obtained in the presence of Mn2+ than in the presence of Ca2+. This interaction in the presence of Mn2+ was also completely blocked by GRGDSP (94%) and anti-alpha 5beta 1 mAb (95%) (Fig. 5).


Fig. 5. Fg binding to purified alpha 5beta 1. 125I-Fg (20 nM) was added to microtiter wells coated with alpha 5beta 1 (200 ng/well) in the presence of 5 mM EDTA, 1 mM Ca2+, or 1 mM Mn2+ and incubated for 180 min at 37 °C. The effects of GRGDSP (200 µM), GRGESP (200 µM), anti-alpha 5beta 1 mAb JBS5 (1/100 dilution), or control mouse ascites (Ctrl) (1/100 dilution) were tested in the presence of 1 mM Mn2+. Binding in the presence of 1 mM Mn2+ without inhibitor was assigned a value of 100%. The data shown are means and S.D. from three experiments.
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Effect of Fibronectin on Fibrinogen Binding to HUVEC

Fg binds to Fn (43, 44), and secreted Fn may enhance HUVEC adhesion and spreading on Fg (45). Thus, Fn bound to alpha 5beta 1 might mediate the observed Fg binding to HUVEC. In this case, increased occupancy of alpha 5beta 1 by Fn should enhance Fg-HUVEC interaction. On the other hand, if Fg bound directly to the receptor, then occupancy by Fn should inhibit binding since both ligands bind via a RGD recognition specificity. To distinguish these possibilities, two sets of experiments were performed. First, the effect of Fn on 125I-Fg binding to HUVEC was assessed. When both were added simultaneously to the HUVEC, Fn produced marked (>85%) inhibition of Fg binding (Fig. 6). Second, HUVEC were preincubated with Fn, GRGDSP peptide, or both and then washed extensively before incubation with 125I-Fg. Preincubation with Fn inhibited but did not enhance 125I-Fg binding (Fig. 6). When the cells were preincubated with GRGDSP or Fn plus GRGDSP and then washed, only minor inhibition of Fg binding was observed (Fig. 6), suggesting that the GRGDSP and unbound Fn were effectively removed by the washing. Since Fn, either free or bound to the cells, did not enhance and did, in fact, inhibit 125I-Fg binding, Fg appears to interact directly with alpha 5beta 1 on HUVEC.


Fig. 6. Effect of Fn on Fg binding to HUVEC. HUVEC (1 × 106 cells/ml) were incubated with 125I-Fg (40 nM) by simultaneously adding excess unlabeled Fg (2 µM) or fibronectin (Fn) (2 µM) for 45 min at 37 °C in the presence of 1 mM Mn2+ (Without Washing). HUVEC (1 × 106 cells/ml) were preincubated with Fn (200 nM), GRGDSP (1 mM), or both for 30 min at 37 °C, and then washed three times in Buffer A containing 1 mM Mn2+ before incubation with 125I-Fg (With Washing). Binding was expressed as percent of the control without inhibitor. The data shown are means and S.D. from three experiments.
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Characterization of Fibrinogen Binding to alpha 5beta 1 on HUVEC

The time course of the binding of Fg to HUVEC via alpha 5beta 1 in the presence of Mn2+ was examined over a 2-h time course with 125I-Fg at 40 nM (not shown). Specific binding (inhibitable by 1 mM GRDGSDP) of 125I-Fg to HUVEC reached a plateau level in 45-60 min. In subsequent experiments, 45 min was selected as the incubation time. The specific 125I-Fg binding to alpha 5beta 1 on HUVEC was saturable (Fig. 7A). A Scatchard plot (Fig. 7B) of these data suggested that the interaction could be described by a single class of high affinity binding sites with an apparent dissociation constant (Kd) of 65.4 ± 6.1 nM and with a maximum of 3.15 ± 0.18 × 105 binding sites per cell (n = 3). This number of Fg-binding sites is similar to the previously reported number of Fn-binding sites per HUVEC (7.5 × 105) (20).


Fig. 7. Saturation isotherm and Scatchard analysis of 125I-Fg binding to HUVEC. A, binding isotherms of Fg to HUVEC were constructed by incubating increasing concentrations of 125I-Fg with HUVEC (1 × 106 cells/ml) in the presence of 1 mM Mn2+. Specific binding (bullet ) was calculated by subtracting nonspecific binding (black-triangle), measured as the residual binding in the presence of 100 µM GRGDSP peptide, from the total binding (black-square). The data shown are the average of triplicate measurements at each experimental data point and are representative of three separate experiments. B, Scatchard plots of Fg binding to HUVEC. The apparent dissociation constant (Kd) estimated from the slope of the line was 65.4 ± 6.1 nM (n = 3).
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Localization of the alpha 5beta 1 Recognition Sequence in Fibrinogen

The RGD sensitivity of Fg binding to alpha 5beta 1 suggests that one of the two RGD sequences in the Aalpha -chain or the carboxyl terminus of the gamma -chain is involved in receptor recognition. MAbs which recognize these sequences were employed to distinguish these possibilities. As shown in Fig. 8, anti-C significantly inhibited (73%) Fg binding to HUVEC, whereas anti-N produced a minimal effect (13%). MAb 4A5 also had no inhibitory effect on this interaction (Fig. 8) although, at the same concentration, this mAb inhibited 125I-Fg binding to alpha IIbbeta 3 by 92% (data not shown). These results suggest that alpha 5beta 1 on HUVEC interacts with the carboxyl-terminal RGD sequence in its recognition of Fg.


Fig. 8. Effects of anti-peptide mAbs on Fg binding to HUVEC. MAbs anti-N (directed to the N-terminal RGD sequence residues 87-100 of Fg Aalpha -chain), anti-C (directed to the C-terminal RGD sequence residues 566-580 of Fg Aalpha -chain), and 4A5 (reactive to the C-terminal region of Fg gamma -chain residues 395-411) were tested. Binding of 125I-Fg to HUVEC (1 × 106 cells/ml) in suspension in the presence of 1 mM Mn2+ was measured by simultaneously adding the selected mAbs (1/20 dilution) and incubating for 45 min at 37 °C. The binding of Fg to HUVEC in the presence of control mouse ascites (1/20 dilution) was assigned a value of 100%. The data shown are means and S.D. from three experiments.
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Manganese-independent Recognition of Fibrinogen by alpha 5beta 1 on HUVEC

Two circumstances were identified in which Fg was recognized by alpha 5beta 1 in the absence of Mn2+. First, when Fg binding to HUVEC was tested in the presence of the stimulatory anti-beta 1 mAb 8A2, an interaction was observed in the presence of Ca2+. 8A2, at a concentration (5 µg/ml) reported to enhance Fn binding to alpha 5beta 1 (36, 46), increased specific 125I-Fg binding to HUVEC in the presence of 1 mM Ca2+ by approximately 2-fold (not shown). Second, a role of alpha 5beta 1 in HUVEC adhesion to Fg was demonstrable in the presence of Ca2+. Adhesion of 51Cr-labeled HUVEC to immobilized Fg was compared in the presence of Ca2+ or Mn2+ (Fig. 9). The number of cells adherent in the presence of Mn2+ was 1.4 ± 0.2-fold (n = 5) higher than in the presence of Ca2+. Morphological differences also were noted under the two cation conditions: at 60 min, spreading of HUVEC was more prominent in the presence of Mn2+, whereas most of HUVEC were attached but not spread in the presence of Ca2+. The effects of inhibitors were tested under each cation condition. In the presence of Ca2+, GRGDSP peptide inhibited the adhesion of HUVEC to immobilized Fg by 96% (Fig. 9). Anti-beta 3 mAb 7E3 inhibited this interaction by 75%, consistent with a reported role of alpha vbeta 3 in HUVEC adhesion to Fg (9, 47). The alpha vbeta 3 specific mAb LM609 also inhibited adhesion to a similar extent (not shown). However, anti-alpha 5beta 1 mAb inhibited the interaction by 27% and the combination of anti-beta 3 and anti-alpha 5beta 1 mAb completely blocked adhesion (Fig. 9). These results suggest that alpha 5beta 1 contributes to HUVEC adhesion to immobilized Fg in the presence of Ca2+. In the presence of Mn2+ (Fig. 9), anti-beta 3 mAb, anti-alpha 5beta 1 mAb, or their combination resulted in minor inhibition although the interaction remained RGD inhibitable.


Fig. 9. Adhesion of HUVEC to immobilized Fg. Radiolabeled HUVEC were allowed to adhere to Fg-coated microtiter wells (1 µg per well) for 60 min at 37 °C in the presence of 1 mM Ca2+ or 1 mM Mn2+. Prior to adhesion, cells were incubated with GRGDSP peptide (1 mM), anti-beta 3 mAb 7E3 (20 µg/ml), anti-alpha 5beta 1 JBS5 (1/100 dilution), or control mouse ascites (Ctrl) (1/40 dilution) for 30 min at 22 °C. After washing, bound cells were recovered by adding 2% SDS, and radioactivity was measured in beta -counter. Adhesion was expressed as percent of the control without inhibitor. The data shown are means and S.D. from three to five experiments.
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DISCUSSION

In the present study, we have characterized the binding of Fg to HUVEC and have examined how divalent cations regulate this interaction. The following conclusions are drawn from these analyses. First, the binding of Fg to HUVEC in suspension is selectively supported by Mn2+ and is suppressed by Ca2+. Second, the receptor on HUVEC which mediates this interaction is alpha 5beta 1, and the capacity of this integrin to recognize Fg is not restricted to HUVEC. Third, the binding of Fg to purified alpha 5beta 1 also was demonstrable. Fourth, the functional region in Fg to interact with alpha 5beta 1 on HUVEC is the carboxyl-terminal RGD sequence of the Aalpha -chain. Fifth, alpha 5beta 1 can recognize Fg in the presence of Ca2+ when activated with mAb 8A2 or when Fg is presented as an immobilized substrate.

Integrin alpha 5beta 1 is a major receptor for Fn (17, 48). Mould et al. (24) found that Mn2+ supported Fn binding, that Ca2+ did not, and that Ca2+ strongly inhibited Mn2+-supported ligand binding. These investigators suggested that Ca2+ and Mn2+ recognize different cation-binding sites. This concept is endorsed in a study of osteopontin binding to alpha vbeta 3 by Hu et al. (41), who showed that Ca2+ decreased the association rate of Mn2+-supported ligand binding but had no effect on the dissociation rate, again suggesting that Mn2+ and Ca2+ bind to different sites in this integrin. Moreover, recognition of Fg by alpha vbeta 3 exhibits a similar pattern of cation-controlled recognition; i.e. Mn2+ supports binding and Ca2+ suppresses the interaction (21, 22). Thus, the pattern of cation regulation of the Fg-alpha 5beta 1 interaction is consistent with the binding of this ligand to other integrins (e.g. Fg-alpha vbeta 3) and of other ligands to this integrin (e.g. Fn-alpha 5beta 1). Although this interpretation is supported by our data and the literature, a differential effect of Mn2+ and Ca2+ on the conformation of Fg cannot be entirely excluded as Fg contains three calcium-binding sites (49, 50).

Several Fg receptors on HUVEC have been identified, but our data appear to be the first to implicate alpha 5beta 1. Transglutaminase activity has been implicated in Fg binding to HUVEC (12), and we did find evidence of Aalpha -chain cross-linking in the bound ligand. However, Ca2+ suppressed Fg binding but optimally supports transglutaminase activity (12), suggesting that transglutaminase activity is not sufficient for Fg binding. Based upon our gel analyses, it is reasonable to propose that Fg binds to the cells and then its Aalpha -chains become cross-linked to one another or to other EC-associated proteins such as Fn. HUVEC adhesion to immobilized Fg involves alpha vbeta 3 (9, 10); our data support these previous studies but we also observed a contribution by alpha 5beta 1 in this adhesion. It is unclear why alpha vbeta 3 did not contribute to soluble Fg binding in the present study. Ligand binding to alpha vbeta 3 is controlled by the activation state of the receptor (51, 52) which may vary among HUVEC preparations. With respect to other Fg receptors on HUVEC, the expression of intercellular adhesion molecule 1, also shown to be a Fg receptor (6), is also dependent upon activation of EC and is expressed at very low levels on our unstimulated HUVEC preparations. Moreover, the nature of the ligand also may be influential. alpha vbeta 3 on HUVEC will only recognize soluble vitronectin when the ligand is presented in multivalent form (53). Fg also may exist in multiple forms which may influence its recognition by alpha 5beta 1 and alpha vbeta 3. It will be important to consider whether alpha 5beta 1 can distinguish soluble Fg, immobilized Fg, or fibrin and plasmic degradation products of Fg/fibrin. Precedence exists for differential recognition of various forms of Fg and other ligands by integrins (54). Thus, the availability of specific receptors on the luminal surface, the activation states of the EC and these receptors, the occupancy of these receptors by competing ligands, and the nature of the ligands may determine which receptors will mediate Fg recognition by HUVEC.

Dejana et al. (45) have shown that Fn secreted from HUVEC enhances HUVEC adhesion and spreading on Fg. This finding may suggest that the interaction of Fg with HUVEC is indirect and Fn mediates Fg-HUVEC interaction as a bridging molecule. We concluded that Fg binding to alpha 5beta 1 on HUVEC in suspension was not mediated by Fn because Fg-HUVEC interaction was not enhanced but was suppressed by Fn. We also found that mAb directed to the carboxyl-terminal RGD sequence of the Aalpha -chain of Fg significantly inhibited Fg binding to HUVEC. This mAb is specific for Fg and does not cross-react with other adhesive proteins, including Fn (3). Moreover, we showed direct binding of Fg to purified alpha 5beta 1 using a solid-phase ligand binding assay. These results indicate that Fg can bind directly to alpha 5beta 1 on HUVEC.

Two circumstances were identified in which Fg interacted with alpha 5beta 1 in the absence of Mn2+: when the receptor was activated by 8A2 or when Fg was immobilized. MAb 8A2 is directed to the beta 1 subunit (55) and stimulates the binding of multiple ligands to multiple beta 1 integrins (36, 46). The adhesion of HUVEC to immobilized Fg was partially blocked by anti-alpha 5beta 1 and completely inhibited by the combination of anti-alpha 5beta 1 and anti-alpha vbeta 3 mAb in the presence of Ca2+, implicating both receptors in HUVEC adhesion to Fg in the presence of Ca2+. This interaction was only partially inhibited by these mAbs in the presence of Mn2+. Since this interaction remained fully inhibited by RGD, one interpretation of this observation is that integrin(s) other than alpha 5beta 1 and alpha vbeta 3 may become involved in Fg recognition. alpha vbeta 1 and alpha vbeta 5 are potential candidates as both are known to be expressed on HUVEC (56). An alternative explanation is that Mn2+ may increase the affinity of alpha 5beta 1 and alpha vbeta 3 for Fg to an extent that the mAbs cannot effectively compete for the receptors. The greater spreading of HUVEC on Fg in the presence of Mn2+ is consistent with either explanation. Thus, in addition to physiologic and pathophysiologic conditions that elevate Mn2+ concentrations (57), Fg may interact with the receptor when alpha 5beta 1 is present in an appropriate activation state and/or when Fg is presented in an appropriate conformation.

There are a wide variety of physiologic and pathophysiologic circumstances in which Fg and alpha 5beta 1 bearing cells come into close contact. An illustrative example is wound healing. In vascular injury, alpha 5beta 1-bearing EC come in direct contact with Fg/fibrin, and alpha 5beta 1-Fg interactions may facilitate re-establishment of a nonthrombogenic EC monolayer on the luminal wall of the blood vessel. In a healing cutaneous wound, granulation tissue gradually accumulates as fibroblasts migrate into the fibrin clot that initially fills the wound. Since the clot matrix also contains Fn (58), alpha 5beta 1 may facilitate migration into the wound through interactions with two ligands: Fn, perhaps the principal ligand, and Fg/fibrin, a new potential ligand identified here. Furthermore, the alpha 5beta 1-Fg interaction, in addition to providing another method of attachment to Fg, may trigger unique intracellular signaling pathways. Thus, the recognition of Fg by alpha 5beta 1 demonstrated in this study may have broad biological implications and may be subject to complex control mechanisms.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants HL-38292 and HL-54924. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Joseph J. Jacobs Center for Thrombosis and Vascular Biology/FF20, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, Ohio 44195. Tel.: 216-445-8200; Fax: 216-445-8204.
1    The abbreviations used: EC, endothelial cells; BSA, bovine serum albumin; Fg, fibrinogen; Fn, fibronectin; HUVEC, human umbilical vein endothelial cells; mAb, monoclonal antibody; RGD, Arg-Gly-Asp.

Acknowledgments

We thank Drs. Barry Coller, Nicholas Kovach, Gary Matsueda, and Zaverio Ruggeri for providing monoclonal antibodies. We also thank Vicky Byers-Ward for technical assistance and Jane Rein for secretarial assistance.


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