(Received for publication, June 6, 1996, and in revised form, December 10, 1996)
From the 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
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
v
3 blocking monoclonal antibodies. In
contrast, the interaction was completely blocked by two
5
1 monoclonal antibodies. This
interaction was not mediated by fibronectin bound to the integrin;
could be demonstrated with purified
5
1;
and also was observed with a second
5
1-bearing cell type, platelets. The binding of fibrinogen to
5
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 A
572-574 was implicated in fibrinogen recognition by
5
1. Two circumstances were identified in
which
5
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
5
1 on endothelial and other cells, an
interaction which may have broad biological implications.
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 v
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).
v
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
v
3 but also by
several other integrins (14-16), including
5
1, which serves as a fibronectin (Fn)
receptor on EC and many other cell types (17-20). Fg contains two RGD
sequences within its A
-chain: RGDF at A
95-98 and RGDS at A
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 A
572-574 (3). Moreover, this
adhesion was blocked by mAbs against
v
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 v
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
v
3 in the presence
of Mn2+. This pattern of cation regulation is not unique to
v
3; Mn2+ enhances and
Ca2+ suppresses ligand binding to several integrins. For
example, recognition of Fn (23, 24) and anti-
1 mAb 9EG7
(25) by
5
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
v
3. Surprisingly, while Mn2+
enhanced binding, the receptor mediating this interaction was not
v
3 but was
5
1. Thus, a novel ligand has been
identified for this receptor which may have broad biological
implications.
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 × 106 cpm/molecule.
5
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.
The synthetic peptides GRGDSP
(GRGESP) and the dodecapeptide (HHLGGAKQAGDV) from the extreme carboxyl
terminus of Fg -chain, referred to as H12, were prepared using an
Applied Biosystems Model 430A Peptide Synthesizer (29). MAb 7E3 (30)
which recognizes
3 integrins was provided by Dr. Barry
Coller, Mount Sinai School of Medicine, New York. MAbs LM609 (10)
directed to
v
3, JBS5 (31) directed to
5
1, CLB-706 (32) directed to
v, JB1a, originally designated J10 (33), directed to
1 were purchased from Chemicon International Inc.
(Temecula, CA). MAb P1D6 (34) against
5 was obtained
from Life Technologies Inc. Two mAbs raised against RGD-containing
synthetic peptides from Fg A
-chain, anti-N directed to A
87-100
(TTNIMEILRGDFSS), and anti-C directed to A
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
-chain of Fg, was a gift from Dr. Gary Matsueda, Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, NJ.
Anti-
1 mAb 8A2 (36) was provided by Dr. Nicholas Kovach,
Washington University, Seattle, WA.
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 SuspensionFor 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 -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.
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 AssayThe binding of Fg or the
120-kDa chymotryptic Fn fragment to purified and immobilized
5
1 was performed as described (21).
5
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 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 -counter.
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.
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 A-, B
-, and
-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 A
-chain was reduced relative to the B
- and
-chains. This phenomenon also was reported by Dejana et
al. (1). We found that the band corresponding to the A
-chain became still less intense with the longer incubation time, whereas the
B
-chain remained unchanged. Coinciding with the loss of A
-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.
Fibrinogen Binds to
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 -chain (residues 400-411), which effectively
inhibited Fg binding to
IIb
3, had no
effect on HUVEC binding of the ligand.
The stimulatory effect of Mn2+ and the inhibition by the
RGD peptide is consistent with a role of
v
3 in Fg-HUVEC interaction. However, the
binding was not inhibited by two
v
3
blocking mAbs (Fig. 3B). MAb 7E3 and mAb LM609, which block
Fg binding to
v
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
5
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-
1 mAb JB1a was significant (58%).
Moreover, anti-
5 mAb P1D6 and anti-
5
1 mAb JBS5 inhibited this
interaction by 88 and 91%, respectively. Thus,
5
1 is centrally involved in the
Mn2+ supported Fg binding to suspended HUVEC.
We sought to determine if 5
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-
3 mAb 7E3, and
anti-
5
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-
5
1 mAb inhibited this interaction by
47%. The anti-
3 mAb blocked binding by 65% and, in
combination with anti-
5
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
5
1 receptors
(42).
Fibrinogen Binds to Purified
Consistent with a role for
5
1 in the interaction of Fg with HUVEC,
binding of Fg to the purified receptor also was demonstrable. Purified
5
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-
5
1 mAb. Next, 125I-Fg
binding to
5
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-
5
1 mAb (95%) (Fig. 5).
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
5
1 might mediate the observed Fg binding
to HUVEC. In this case, increased occupancy of
5
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
5
1 on HUVEC.
Characterization of Fibrinogen Binding to
The time course of the
binding of Fg to HUVEC via 5
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
5
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).
Localization of the
The RGD sensitivity of Fg binding to
5
1 suggests that one of the two RGD
sequences in the A
-chain or the carboxyl terminus of the
-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
IIb
3 by 92% (data not shown). These
results suggest that
5
1 on HUVEC
interacts with the carboxyl-terminal RGD sequence in its recognition of
Fg.
Manganese-independent Recognition of Fibrinogen by
Two circumstances were
identified in which Fg was recognized by
5
1 in the absence of Mn2+.
First, when Fg binding to HUVEC was tested in the presence of the
stimulatory anti-
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
5
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
5
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-
3 mAb 7E3 inhibited this interaction by 75%, consistent with a reported role of
v
3 in HUVEC adhesion to Fg (9, 47). The
v
3 specific mAb LM609 also inhibited adhesion to a similar extent (not shown). However,
anti-
5
1 mAb inhibited the interaction by
27% and the combination of anti-
3 and
anti-
5
1 mAb completely blocked adhesion
(Fig. 9). These results suggest that
5
1
contributes to HUVEC adhesion to immobilized Fg in the presence of
Ca2+. In the presence of Mn2+ (Fig. 9),
anti-
3 mAb, anti-
5
1 mAb,
or their combination resulted in minor inhibition although the
interaction remained RGD inhibitable.
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
5
1, and the capacity of this integrin to
recognize Fg is not restricted to HUVEC. Third, the binding of Fg to
purified
5
1 also was demonstrable.
Fourth, the functional region in Fg to interact with
5
1 on HUVEC is the carboxyl-terminal RGD
sequence of the A
-chain. Fifth,
5
1 can
recognize Fg in the presence of Ca2+ when activated with
mAb 8A2 or when Fg is presented as an immobilized substrate.
Integrin 5
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
v
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
v
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-
5
1 interaction is consistent with the
binding of this ligand to other integrins (e.g.
Fg-
v
3) and of other ligands to this
integrin (e.g. Fn-
5
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 5
1.
Transglutaminase activity has been implicated in Fg binding to HUVEC
(12), and we did find evidence of A
-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 A
-chains become cross-linked to one another or to
other EC-associated proteins such as Fn. HUVEC adhesion to immobilized
Fg involves
v
3 (9, 10); our data support
these previous studies but we also observed a contribution by
5
1 in this adhesion. It is unclear why
v
3 did not contribute to soluble Fg
binding in the present study. Ligand binding to
v
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.
v
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
5
1 and
v
3. It will be important to consider
whether
5
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 5
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 A
-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
5
1 using a solid-phase ligand binding
assay. These results indicate that Fg can bind directly to
5
1 on HUVEC.
Two circumstances were identified in which Fg interacted with
5
1 in the absence of Mn2+:
when the receptor was activated by 8A2 or when Fg was immobilized. MAb
8A2 is directed to the
1 subunit (55) and stimulates the binding of multiple ligands to multiple
1 integrins (36,
46). The adhesion of HUVEC to immobilized Fg was partially blocked by
anti-
5
1 and completely inhibited by the
combination of anti-
5
1 and
anti-
v
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
5
1 and
v
3
may become involved in Fg recognition.
v
1
and
v
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
5
1 and
v
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
5
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 5
1 bearing
cells come into close contact. An illustrative example is wound
healing. In vascular injury,
5
1-bearing
EC come in direct contact with Fg/fibrin, and
5
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),
5
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
5
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
5
1 demonstrated in this study may have broad biological implications and may be subject to complex control mechanisms.
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.