(Received for publication, April 28, 1995; and in revised form, June 13, 1995)
From the
The fibulins are an emerging family of extracellular matrix and
blood proteins presently having two members designated fibulin-1 and
-2. Fibulin-1 is the predominant fibulin in blood, present at a
concentration of 30-40 µg/ml (1000-fold higher than
fibulin-2). During the course of isolating fibulin-1 from plasma by
immunoaffinity chromatography, a 340-kDa polypeptide was consistently
found to co-purify. This protein was identified as fibrinogen (Fg)
based on its electrophoretic behavior and reactivity with Fg monoclonal
antibodies. Radioiodinated fibulin-1 was shown to bind to Fg
transferred onto nitrocellulose filters after SDS-polyacrylamide gel
electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound
to Fg (and fibrin) adsorbed onto microtiter well plastic, and
conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to
fibulin-1 was also observed in surface plasmon resonance assays, and a
dissociation constant (K
) of 2.9 ±
1.6 µM was derived. In addition, fluorescence anisotropy
experiments demonstrated that the interaction was also able to occur in
fluid phase, which suggests that complexes of fibulin-1 and Fg could
exist in the blood. To localize the portion of Fg that is responsible
for interacting with fibulin-1, proteolytic fragments of Fg were
evaluated for their ability to promote fibulin-1 binding. Fragments
containing the carboxyl-terminal region of the B
chain (residues
216-468) were able to bind to fibulin-1. In addition, it was
found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed
fibrin-containing thrombi associated with human atherectomy specimens.
The interaction between fibulin-1 and Fg highlights potential new roles
for fibulin-1 in hemostasis as well as thrombosis.
The fibulins are a family of extracellular matrix (ECM) ()proteins currently consisting of two related members
designated fibulin-1 and -2 (Argraves et al., 1989; Argraves et al., 1990; Pan et al., 1993a; Pan et al.,
1993b). The pattern of expression of the fibulins has been described in
tissues of the chicken (Spence et al., 1992), mouse (Kluge et al., 1990; Pan et al., 1993b; Zhang et
al., 1993, 1995) and human (Zhang et al., 1994; Roark et al., 1995). These studies showed that both fibulins are
widely expressed intercellular components of connective tissues present
in matrix fibers and basement membranes. The association of fibulins
with these ECM structures presumably involves their ability to bind to
any of a number of ECM proteins including fibronectin (Balbona et
al., 1992; Godyna et al., 1994a), laminin and nidogen
(Pan et al., 1993a; Brown et al., 1994).
In addition to the fibulins being ECM proteins they are also present in blood (Argraves et al., 1990; Kluge et al., 1990; Pan et al., 1993b). The concentration of fibulin-1 in blood is 30-50 µg/ml, whereas fibulin-2 is present at very low levels, 20 ng/ml. Fibulin-1 is representative of a small number of ECM proteins including fibronectin, vitronectin, and von Willebrand factor, whose concentration in blood exceeds that of other ECM proteins such as laminin, type IV collagen, or thrombospondin by several orders of magnitude. Numerous biological functions have been ascribed to plasma fibronectin, vitronectin, and von Willebrand factor, particularly with respect to hemostasis and thrombosis; however, the function of plasma fibulin-1 is not known. In this report we demonstrate that plasma fibulin-1 can bind to fibrinogen and incorporate into fibrin clots formed in vitro and in vivo.
To determine
the K for the binding of fibulin-1 and
Fg, a surface plasmon resonance-based method (reviewed in Fisher and
Fivash(1994); Chaiken et al.(1992)) was used. This methodology
involved conjugating fibulin-1 (100 µg/ml in 10 mM sodium
acetate, pH 4.1) to carboxymethyl-dextran on the surface of a sensor
chip. Solutions containing various amounts of Fg (0.15-4 mg/ml)
in phosphate-buffered saline, 0.05% Tween 20 were added, and using a
biosensor instrument (Fisons, model IAsys), changes in the optical
phenomenon of plasmon resonance (changes in mass concentration on the
chip surface) were measured as the two molecules bound. From the
resulting sensorgram the association of the fibulin-1
Fg complex
was followed in real time, and when a protein-free buffer was added the
dissociation of the complex was also monitored. The kinetic parameters k
and k
were both obtained and the dissociation constant was derived
from the equation K
=k
/k
.
Fluorescent anisotropy
measurements were performed in TBS at room temperature using an
SLM-8000C fluorometer in a T format with emission and excitation
wavelengths set at 494 and 524 nm, respectively. Fg or FN in TBS was
added to a 0.1 µM FITCfibulin-1, TBS solution using
a motorized syringe while continually stirring. The change in
anisotropy (
A) as a function of titrant concentration was fitted
to a single class of binding sites by using the following equation,
where K is the apparent
dissociation constant and [titrant] is the concentration of
free Fg or FN.
A
is the maximum anisotropy change
that would be achieved at saturating concentrations of titrant (Ingham et al., 1988).
Figure 1:
Detection of fibulin-1 in ammonium
sulfate precipitates of plasma. Plasma was fractionated by AS
precipitation. Aliquots of solubilized 0-10% saturated AS
precipitate (lane1), 10-20% saturated AS
precipitate (lane2), 20-30% saturated AS (lane3), and 30-40% saturated AS precipitate (lane4) were electrophoresed on SDS-containing
4-12% polyacrylamide gradient gels and stained with Coomassie
Blue (panel A) or transferred to nitrocellulose. Duplicate
membranes were incubated with I-fibulin (30 nM) (panelB), Fg polyclonal antibody (panelC), or fibulin-1 monoclonal antibody (panelD).
Figure 2: Fibrinogen co-purifies with fibulin-1 isolated from plasma by immunoabsorption on anti-fibulin-1 IgG-Sepharose. A 10-20% saturated ammonium sulfate precipitate of human plasma was solubilized in TBS and applied to either normal IgG-Sepharose or fibulin-1 monoclonal (3A11) IgG-Sepharose. Aliquots of proteins eluted from the normal mouse IgG-Sepharose (lane1) and mouse anti-fibulin-1 IgG-Sepharose (lane2) as well as the unbound material from the anti-fibulin-1 IgG-Sepharose (lane3) were electrophoresed on SDS-containing 4-12% polyacrylamide gradient gels under reducing (panelsB-D) or nonreducing conditions (panelA). PanelsA and B are Coomassie Blue-stained gels, panelC is an immunoblot using Fg monoclonal antibody, and panelD is an immunoblot using fibulin-1 monoclonal antibody.
Figure 3:
I-Fibulin-1 binds to
fibrinogen in gel blot overlay assay. Human Fg (lane2), bovine Fg (lane3) and molecular
weight standards (lane1) were electrophoresed on
SDS-containing 4-12% polyacrylamide gradient gels that were
subsequently stained with Coomassie Blue (panelA) or
electrophoretically transferred to nitrocellulose and the membrane
probed with
I-fibulin-1 (30 nM) (panelB).
ELISAs were also performed to evaluate the fibulin-1 interaction with Fg and fibrin. The results showed that fibulin-1 bound to microtiter wells coated with Fg in a dose-dependent manner but not to ovalbumin-coated wells (Fig. 4A). Conversely, Fg was found to bind in a dose-dependent manner to microtiter wells coated with fibulin-1 (Fig. 4C). EDTA at concentrations ranging from 0.002 to 50 mM did not inhibit the binding of fibulin-1 to Fg (data not shown), indicating that the interaction was not dependent on divalent cations. The ability of fibulin-1 to bind to immobilized fibrin was also examined, and as shown in Fig. 4B, fibulin-1 bound in a dose-dependent manner to wells coated with human fibrin.
Figure 4: Fibulin-1 binds to fibrinogen in both solid and fluid phase conditions. In panelsA-C, microtiter wells were coated with Fg, fibrin, fibulin-1, or ovalbumin (3 µg/ml). Increasing concentrations of fibulin-1 (0.0005-10 µM) (panelsA and B) or Fg (0.0015-30 µM) (panelC) were added to the coated wells and incubated for 18 h at 4 °C. Bound fibulin-1 was detected with a mouse monoclonal fibulin-1 antibody (3A11), goat anti-mouse IgG-peroxidase conjugate, and a peroxidase substrate. Bound Fg was detected with a rabbit polyclonal Fg antibody, goat anti-rabbit IgG-peroxidase conjugate, and a peroxidase substrate. The data shown in panelsA-C are mean values of duplicate determinations with the range indicated by bars and are representative of three experiments. In panelD, changes in fluorescence anisotropy are indicated as a function of the concentration of Fg that was added to a solution of FITC-labeled fibulin-1. The data shown in panelD are representative of three experiments. The curve represents the best fit of the data to a single class of sites using the equation described under ``Materials and Methods.''
To estimate the apparent
dissociation constants (K) for the
binding of fibulin-1 to Fg or fibrin, the data from the microtiter well
binding assays were fit to a form of the binding isotherm as described
in Balbona et al.(1992). As has been experienced previously
with studies of in vitro binding of fibulin-1 to fibronectin
(Balbona et al., 1992), saturable binding of fibulin-1 to Fg
or fibrin was not achieved. This was attributed to the fact that
fibulin-1 can self-associate (Balbona et al., 1992). An
apparent K
of 4.5 ± 1.7 µM (n = 6) was obtained from the best fit of the
ELISA data for the binding of fibulin-1 to Fg. A K
of 3.2 ± 0.7 µM (n = 2)
was estimated for the binding of Fg to immobilized fibulin-1. In
contrast, the apparent affinity of fibulin-1 binding to fibrin was
higher (K
= 2.56 ± 0.99
µM, n = 3) than its binding to Fg.
Using the technique of measuring changes in plasmon resonance as two
proteins bind on the surface of a sensor chip, we determined a K value of 2.9 ± 1.6
µM (n = 4) for Fg binding to immobilized
fibulin-1. This value is in good agreement with K
values estimated from ELISA. In control experiments, no
changes in plasmon resonance occurred when BSA was incubated with the
fibulin-1-coated sensor chip.
To determine if the fibulin-1-Fg
interaction could occur in fluid phase, fluorescein-labeled fibulin-1
was titrated with Fg while monitoring the change in fluorescence
anisotropy. As shown in Fig. 4D, there was a
dose-dependent increase in the anisotropy with the addition of Fg,
whereas the change in anisotropy was negligible when FN (not shown) was
added. By fitting the data to the equation for a single class of
homogenous binding sites (Ingham et al., 1988) a K of 6.7 ± 0.7 µM (n = 3) was determined for the fibulin-1-Fg
interaction. The inability of FN to elicit a change in anisotropy was
consistent with previous results that showed the interaction of
fibulin-1 and FN to occur only when one or the other protein was
immobilized (Balbona et al., 1992).
Figure 5:
Fibulin-1 binding to subfragments of the D
region of fibrinogen. Shown in panelA is a schematic
diagram of the domain structure of Fg and the scheme for generation of
subfragments of the D region of Fg (see ``Materials and
Methods''). For the ELISAs shown in panelB,
microtiter wells were coated with Fg fragments (D,
D
, D
, E,
C, or TSD) or ovalbumin (3
µg/ml). Increasing concentrations of fibulin-1 (0.004-8
µM) were added and incubated for 18 h at 4 °C. The
wells were washed, and bound fibulin-1 was detected with a mouse
monoclonal fibulin-1 antibody (3A11), goat anti-mouse IgG-peroxidase
conjugate, and a peroxidase substrate.
Figure 6:
Fibulin-1 effects fibrin clot structure as
indicated by turbidity measurements. Fg (270 µg/ml in TBS) was
mixed with the indicated concentrations of fibulin-1 and CaCl (3.2 mM final concentration) in a 10-mm disposable
cuvette. Thrombin (0.016 units/ml) was added, and the absorbance at 600
nm was continuously measured for 60 min at room temperature. The data
presented are representative of four
experiments.
Figure 7:
Incorporation of I-fibulin-1, -FN, -TSP1, and -BSA into fibrin clots
formed in vitro. In panelA, fibrin clots
were formed by adding thrombin (final concentration 0.2 units/ml) to
solutions containing Fg (final concentration 80 µg/ml), fibulin-1
(final concentration 50 µg/ml),
I-fibulin-1 (final
concentration 5 µg/ml) in TBS, 3.5 mM CaCl
for
30 min at 37 °C. Similarly, clots were formed with Fg and the
radiolabeled and unlabeled forms of TSP1, FN, BSA, or IgG. The clots
were centrifuged at 12,000
g for 15 min, pellets were
washed with 1
TBS, and radioactivity was measured using a
counter. In panelB, the effect of fibulin-1 (or BSA)
concentration on the incorporation of fibulin-1 (or BSA) into fibrin
clots formed in vitro was examined. Fibulin-1-containing (or
BSA-containing) clots were formed as in panelA;
however, varying amounts of fibulin-1 (or BSA) were added, but the
ratio of radiolabeled to unlabeled fibulin-1 (or BSA) was kept
constant. In panelC, the effect of the addition of
FXIIIa (6 µg/ml) on clot incorporation of the various radiolabeled
proteins is shown. Data shown are mean values of duplicate
determinations with the range indicated by bars.
When in vitro clot forming assays were performed in
which the (mol/mol) ratio of fibulin-1 to Fg was increased (from 0.05:1
to 1.5:1), the incorporation of I-fibulin-1 into clots
increased and reached saturation at a ratio of 1.5/1 (Fig. 7B). Parallel experiments with
I-BSA showed that its low level of incorporation remained
constant at
0.3% even as the BSA:Fg ratio was increased (from
0.065:1 to 1.76:1, Fig. 7B). The results showed that
fibulin-1 incorporated into fibrin clots in a dose-dependent manner.
In order to determine whether fibulin-1 could become covalently
cross-linked into fibrin clots formed in vitro, the
incorporation assays were performed in the presence of the activated
transglutaminase, factor XIII (FXIIIa). As shown in Fig. 7C, I-fibulin-1 incorporation into
the clot did not increase in the presence of FXIIIa. In contrast, there
was an increase in the level of
I-FN (and to a lesser
extent
I-TSP1) that incorporated into clots formed in the
presence of FXIIIa. When the resulting clots were analyzed by SDS-PAGE
followed by autoradiography, both
I-TSP1 and
I-FN were seen to migrate at the top of the gel
corresponding to the migration position of cross-linked fibrin.
However,
I-fibulin-1 was not found co-migrating with
fibrin, but rather it migrated with an apparent M
of 100,000 (data not shown). The results indicate that fibulin-1
does not become covalently cross-linked to fibrin clots by FXIIIa as do
TSP1 and FN.
Figure 8: Immunohistological staining of fibulin-1 within fibrin-containing thrombi associated with human atherectomy specimens. Shown in each panel are parallel tissue sections of a thrombus formed on vascular connective tissue that were stained with fibulin-1 monoclonal antibody (A), Fg monoclonal antibody (B), the chemical staining technique of Lendrum et al.(1962) to stain fibrin (C), and normal mouse IgG (D). Bound primary antibodies were detected using goat anti-mouse IgG-horseradish peroxidase and the chromogenic substrate DAB.
In this report we have presented evidence demonstrating that fibulin-1 binds to Fg. We have shown that fibulin-1 bound to Fg in several types of solid phase binding assays, that Fg co-purified with fibulin-1 isolated from plasma, and that fibulin-1 was incorporated into fibrin clots formed both in vitro and in vivo. Furthermore, we have mapped the fibulin-1 binding site of Fg to the D region, within the carboxyl-terminal portion of the B chain that comprises residues 216-468. Consistent with the fact that this region of Fg is known to be involved with the lateral aggregation of fibrin fibers (Litvinovich et al., 1995), we found that fibulin-1 could interfere with the process of in vitro fibrin assembly.
Based on the dissociation constant determined for the
fibulin-1-Fg binding interaction (3-6 µM) and
the plasma con-centrations of fibulin-1 (0.42 µM) and Fg
(6-13 µM) (Ham and Curtis, 1938), it can be
predicted that circulating complexes of fibulin-1 and Fg exist in
blood. In support of this is our data showing that fibulin-1 and Fg can
indeed bind to one another in solution phase, that fibulin-1
co-purifies with Fg, and that commercial polyclonal Fg antisera that we
have tested have substantial titers to fibulin-1 (data not shown). If
one assumes that fibulin-1 and Fg form a 1:1 molar complex, it can be
calculated that
60-80% of fibulin-1 circulating in blood is
in complex with Fg, while only 3% of the Fg is complexed with
fibulin-1. The observed presence of fibulin-1 within newly formed
thrombi may therefore be the result of the incorporation of
fibulin-1
Fg complexes into the clot as opposed to incorporation
of free fibulin-1. The ability of fibulin-1 to self-associate (Balbona et al., 1992) may also enable circulating fibulin-1
Fg
complexes to bind not only to clot-incorporated fibulin-1 but also to
ECM-incorporated fibulin-1 that becomes exposed to blood at sites of
vascular injury. Considering that the local concentration of fibulin-1
within ECM may be higher as compared with its level in blood, the
interaction between ECM-incorporated fibulin-1 and circulating Fg may
be more favorable than that between fluid phase fibulin-1 and Fg.
The in vivo significance of the fibulin-1-Fg interaction
remains to be determined. We postulate that the interaction of plasma
Fg with fibulin-1 in vascular or connective tissue ECM that becomes
exposed to blood following injury may be important to the process of
thrombus formation. Fg bound to ECM-incorporated fibulin-1 could
support platelet adhesion leading to the formation of a platelet plug.
This possibility is consistent with our recent findings showing that
fibulin-1 can promote in vitro platelet adhesion via a bridge
of Fg (Godyna et al., 1994b). ()The finding that
fibulin-1 can be detected throughout fibrin-containing thrombi, within
regions of the clot apparently distal to its interface with ECM,
suggests that fibulin-1 may have additional roles in coagulation.
Further studies are required to determine the role of fibulin-1 in the
formation, organization, and/or stability of fibrin-containing thrombi.