©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interaction of Fibulin-1 with Fibrinogen
A POTENTIAL ROLE IN HEMOSTASIS AND THROMBOSIS (*)

(Received for publication, April 28, 1995; and in revised form, June 13, 1995)

Huan Tran(§) (1) Asashi Tanaka(§) (1) Sergei V. Litvinovich (1) Leonid V. Medved (1) Christian C. Haudenschild (2) W. Scott Argraves (1)(¶)

From the  (1)Departments of Biochemistry and (2)Experimental Pathology, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 Bbeta 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.


INTRODUCTION

The fibulins are a family of extracellular matrix (ECM) (^1)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.


MATERIALS AND METHODS

Proteins

Fibulin-1 was isolated from human placenta by immunoaffinity chromatography as described previously in Argraves et al.(1990). Human thrombin and Fg (having 95% clottability) were purchased from Enzyme Research Laboratories Inc. (South Bend, IN). Fg fragments, designated D(H), D(Y), D(L), E3, alphaC (residues 220-581), and TSD, were prepared from bovine Fg according to methods described previously (Medved et al. 1986, 1988; Litvinovich et al., 1995; Gorkun et al., 1994). Recombinant human alphaC domain of Fg (residues Gln-Val of alpha chain) was provided by Dr. Ken Ighman (American Red Cross, Rockville, MD). Fibronectin (FN) was purified as described by Miekka et al. (1982). Recombinant human factor XIII (activated form, FXIIIa) was obtained from ZymoGenetics Inc. (Seattle, WA). Thrombospondin-1 (TSP1) was purified from human platelets by adsorption to barium citrate followed by heparin-agarose chromatography according to Alexander and Detwiler(1984). Ovalbumin was obtained from Sigma. Bovine serum albumin was obtained from U.S. Biochemical Corp. Human immunoglobulin was purchased from Cappel (Gaithersburg, MD).

Antibodies

The anti-fibulin-1 monoclonal antibody 3A11 has been described previously (Argraves et al., 1990). 3A11 IgG was purified by protein G-Sepharose (Pharmacia Biotech Inc.) chromatography. Monoclonal antibody against human Fg was provided by Dr. Bryan Butman (PerImmune, Rockville, MD). Rabbit polyclonal antibody against human Fg was provided by Plasma Derivatives Laboratory, American Red Cross.

Radiolabeling

Fibulin-1, FN, bovine serum albumin, and TSP1 (100 µg of each) were radioiodinated in 100 µl of phosphate-buffered saline, 0.5 mCi of NaI (Amersham Corp.) using 20 µg of IODO-GEN (Pierce) and 0.25 mM of NaI as carrier. The radiolabeled proteins were separated from the free iodine by using gel filtration on Sephadex G-25M columns (Pharmacia). The typical specific activities obtained ranged from 1 to 10 µCi/µg.

Fibulin-1 Purification from Human Plasma

Human plasma (100 ml) was precipitated by slowly adding ammonium sulfate (saturated) with continual stirring at room temperature in order to reach a concentration that was 20% that of the saturated solution, as described by Green and Hughes(1955). The suspension was centrifuged for 30 min at 5000 g at 4 °C, and the pellet was dissolved in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer (TBS). The dissolved precipitate was applied to either fibulin-1 monoclonal (3A11) IgG-Sepharose or normal IgG-Sepharose at a flow rate of 1 ml/min. The columns were washed with 10 column volumes of TBS and then eluted with 8 M urea, 50 mM Tris, pH 7.4. The eluted fractions were dialyzed extensively against TBS, and aliquots were analyzed by electrophoresis on SDS-containing 4-12% polyacrylamide gradient gels and stained with Coomassie Blue.

Immunoblotting Assays

Samples separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes. Unoccupied protein binding sites on the membranes were blocked by incubation in 3% nonfat dried milk. Antibodies diluted in 3% nonfat dried milk, 0.05% Tween 20, TBS were incubated with the filters for 2 h at room temperature. The membranes were washed with TBS, 0.05% Tween 20 and incubated with goat or rabbit anti-mouse horseradish peroxidase conjugate (Bio-Rad) in TBS, 0.05% Tween 20 for 1 h at room temperature. Bound antibodies were detected by using the chromogenic substrate 3,3`-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories, Burlingame, CA).

Gel Blot Overlay Assay

Nitrocellulose membranes containing proteins transferred from SDS-PAGE were blocked with 3% nonfat dry milk, TBS, 0.05% Tween 20 and incubated for 18 h at 4 °C with I-fibulin-1 (20 nM) in the same buffer containing 5 mM CaCl(2). Following the incubation, the filters were washed with TBS, 0.05% Tween 20 and used to expose Kodak X-OMAT AR film (Rochester, NY) at -70 °C.

Solid Phase Binding Assays

Solid phase binding of fibulin-1 to Fg and its fragments or Fg to fibulin-1 were performed in 96-well microtiter plates (Corning Inc., Corning, NY) using an enzyme-linked immunosorbent assay (ELISA) as described by Balbona et al. (1992). Binding of fibulin-1 to Fg or Fg fragments was detected by using the fibulin monoclonal antibody (3A11). Binding of Fg to fibulin-1 was detected by using a polyclonal Fg antibody. Bound antibodies were detected by using either goat anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) and the substrate 3,3`,5,5`-tetramethylbenzidine (Kiekegaard & Perry, Gaithersburg, MD). For ELISAs used to examine the interaction of fibulin-1 with fibrin, microtiter wells were coated by diluting monomeric fibrin (10 µg/ml, kept at acidic pH) in microtiter wells containing 1 M Tris buffer, pH 8.5. The apparent dissociation constants (K) of fibulin-1 binding to Fg, Fg subfragments, or fibrin were estimated by fitting the binding data as described previously (Balbona et al., 1992).

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-1bulletFg 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.

Fluorescence Anisotropy

Fluorescein-labeled fibulin-1 was prepared according to the method of Busby and Ingham(1988) using fluorescein isothiocyanate (FITC) (Molecular Probes Inc., Eugene, OR). Briefly, fibulin-1 was dissolved in 0.1 M NaHCO(3) buffer, pH 9.5, and mixed with a 40-fold molar excess of FITC. The labeling reaction proceeded in the dark for 4 h at room temperature, after which the FITC-fibulin-1 was separated from the free FITC by gel filtration on a Sephadex G-25 M column (Pharmacia). The degree of labeling was determined optically as described by Ingham and Brew(1981). A typical labeling efficiency was 4-5 mol of FITC/mol of fibulin-1.

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 FITCbulletfibulin-1, TBS solution using a motorized syringe while continually stirring. The change in anisotropy (DeltaA) 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. DeltaA(max) is the maximum anisotropy change that would be achieved at saturating concentrations of titrant (Ingham et al., 1988).

Incorporation of Fibulin-1 into Fibrin Clots

Fibrin clots were formed in 1.5-ml microcentrifuge tubes using purified human Fg and thrombin as described by Wilson and Schwarzbauer(1992) and Bale et al.(1985). Human Fg (0.8 mg/ml) was mixed with I-fibulin-1 (64 nM), fibulin-1 (50 µg/ml, 641 nM), and 10 mM CaCl(2). A molecular mass of 78,842 daltons (determined from laser desorption mass spectroscopy) was used to calculate the molar concentration of fibulin-1 containing solutions in which the protein concentration was determined by using an extinction coefficient of A = 0.467. Radiolabeled TSP1, FN, and bovine serum albumin were used in parallel experiments. For all the proteins studied by this assay, a 1:10 molar ratio of radiolabeled to unlabeled protein was used. Clot formation was initiated by the addition of thrombin (0.2 units/ml), and the fibrin formation reaction was allowed to proceed for 30 min at 37 °C. After centrifugation for 15 min at 12,000 g the pelleted fibrin clots were washed three times with TBS and solubilized with an equal volume of 8 M urea, 10% SDS, 2% 2-mercaptoethanol, 0.16 M Tris-HCl, pH 6.8. The amount of radiolabeled protein incorporated into the fibrin clots was determined by using a -counter. In some experiments, factor XIIIa was added to a final concentration of 6.0 µg/ml prior to the initiation of fibrin formation.

Turbidity Measurements during Fibrin Polymerization

Turbidity measurements were made throughout the course of in vitro fibrin polymerization as described by Bale and Mosher(1986) using a Perkin Elmer (model Lambda 5) spectrophotometer. Fibrin clot formation was initiated by the addition of thrombin (0.02 NIH units/ml) to a 10-mm disposable cuvette containing purified Fg (270 µg/ml) in a solution of TBS, 3.2 mM CaCl(2) and various concentrations of fibulin-1 (50-150 µg/ml). To measure changes in turbidity of the polymerizing fibrin, light absorbance at 600 nm was monitored continuously for 60 min. The lateral aggregation rate of the reaction was characterized by determining the slope of a line drawn tangent to the absorbance curve such that a maximal slope value was derived. The lag time to turbidity rise was considered to be the x intercept of the tangent line. The final turbidity was measured 18-24 h after initiation of clot formation.

Localization of Fibulin-1 in Clots Present in Human Atherectomy Specimens

Human atherectomy specimens were fixed postoperatively for 1 h at room temperature with 10% formalin. Following fixation, the tissues were placed in 70% ethanol and then embedded in paraffin and sectioned at 5-µm thickness. Tissue sections were deparaffinized with xylene and graded ethanol. Immunohistochemical staining was done as described in Roark et al.(1995) using either monoclonal antibodies to fibulin-1 or Fg and reagents supplied in a commercial staining kit (Elite, Sigma), which included horseradish peroxidase-conjugated anti-IgG and the chromogenic substrate DAB. Sections were subjected to Fraser-Lendrum staining (Lendrum et al., 1962) in order to chemically stain fibrin red.


RESULTS

Fibrinogen Co-purifies with Fibulin-1 Isolated from Plasma

Previous studies have shown that fibulin-1 is a blood glycoprotein present at a concentration of 30-50 µg/ml (Argraves et al., 1990; Kluge et al., 1990). To isolate fibulin-1 from blood, human plasma was fractionated by ammonium sulfate (AS) precipitation. Aliquots of the resulting AS fractions were evaluated for fibulin-1 content by immunoblotting. As shown in Fig. 1D, fibulin-1 was most abundant in the 10-20% saturated AS fraction. This fraction was applied to a monoclonal anti-fibulin IgG column, and bound fibulin-1 was eluted by using a buffer containing urea. As shown in Fig. 2A (lane2), in addition to the expected 80-kDa fibulin-1 polypeptide, a prominent polypeptide having a M(r) of 300,000 was also present in the eluate. Neither of the two polypeptides bound to plain Sepharose (data not shown) or to a column of bovine IgG (or mouse IgG) coupled to Sepharose (Fig. 2A, lane1). Under reducing conditions four polypeptides having M(r) values of 100,000, 66,000, 55,000, and 45,000 were apparent (Fig. 2B, lane2). The 100-kDa polypeptide under nonreducing conditions migrated with a M(r) value of 80,000 and corresponded to fibulin-1 as determined by its immunoreactivity with monoclonal fibulin-1 antibody (Fig. 2D, lane2). The M(r) values of the three other reduced polypeptides were consistent with their being the Fg Aalpha, Bbeta, and chains. Fg is a dimer having a mass of 345 kDa, composed of 2 pairs of disulfide-linked chains designated Aalpha, Bbeta, and that have mass values of 67.6, 54.7, and 46.4 kDa, respectively (Mckee et al., 1970; Mosesson, 1983). Immunoblot analysis confirmed that the three polypeptides seen in the reduced SDS-PAGE profiles of the anti-fibulin-1 IgG-Sepharose eluate were the Fg Aalpha, Bbeta, and chains (Fig. 2C, lane2). The results indicate that Fg co-purifies with fibulin-1 isolated from plasma.


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.



Fibulin-1 Binds to Fibrinogen in Solid Phase and Fluid Phase Conditions

To determine whether fibulin-1 could bind directly to Fg, several types of in vitro solid phase binding assays were used. As shown in Fig. 3B, I-fibulin-1 bound to human and bovine Fg that was immobilized on a nitrocellulose membrane after being electrophoresed in the absence of reducing agent on SDS-containing polyacrylamide gels. I-fibulin-1 did not bind to the individual Fg Aalpha, Bbeta, or chains after they were electrophoresed on SDS-containing polyacrylamide gels in the presence of reducing agent (data not shown). In addition, fibulin-1 did not bind to filter-immobilized myosin, beta-galactosidase, phosphorylase B, bovine serum albumin, or ovalbumin present in the molecular weight standards lane (Fig. 3B, lane1). The proteins contained within various AS fractions of plasma were also probed with I-fibulin-1 after their transfer to nitrocellulose from SDS-PAGE. I-fibulin bound to a polypeptide having a M(r) corresponding to that of Fg present in an AS fraction (Fig. 1B) that, based on immunoblotting analysis, was most enriched for Fg (Fig. 1C). The results indicate that I-fibulin-1 is capable of binding to Fg and that the disulfide-bonded structure of Fg is required for the binding.


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).

The Fibulin-1 Binding Site Maps to the Carboxyl-terminal Region of the Bbeta Chain of Fg

To localize the regions on Fg that mediate the interaction with fibulin-1, various proteolytic fragments of bovine Fg were coated on microtiter wells and tested for their ability to promote binding of fibulin-1. As shown in Fig. 5B, the D region-derived fragments designated D(H), D(L), and D(Y) (see diagram in Fig. 5A) bound to fibulin-1 in a dose-dependent manner, but other fragments such as the E, alphaC (recombinant or proteolytically derived), or TSD did not bind (Fig. 5B). The coating efficiency of each of the Fg fragments was evaluated and found to be similar (data not shown). An apparent K of 2.3 ± 0.2 µM (n = 4) was determined for the binding of fibulin-1 to Fg fragment D(H). Since the smallest fibulin-1 binding fragment (D(Y)) differs from the nonbinding TSD fragment principally by the presence of the COOH-terminal portion of the Bbeta chain that includes residues 216-468 (Litvinovich et al., 1995), the fibulin-1 binding site is likely contained within this portion of the Bbeta chain. The binding of fibulin-1 to the D(Y) fragment was consistently of lower affinity than the binding to D(H) and D(L) (Fig. 5B) perhaps indicating that the conformation of the fibulin-1 binding site within the Bbeta chain might be stabilized by the and/or C domains.


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(H), D(L), D(Y), E, alphaC, 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.



Effect of Fibulin-1 on Polymerization of Fibrin

The portion of the Bbeta chain implicated in the binding of fibulin-1 also contains the fibrin polymerization site that is complementary to the Gly-His-Arg-containing site in the E domain (Doolittle and Laudano, 1980; Medved et al., 1993). Thus one can expect that binding of fibulin-1 could influence the fibrin polymerization process. We evaluated this possibility by comparing polymerization of fibrin in the presence and absence of fibulin-1 while monitoring changes in turbidity at 600 nm. As was shown by Hantgan and Hermans(1979) such changes reflect the course of fibrin assembly. A delay of turbidity increase (lag period) corresponds to the first polymerization step, during which two-stranded protofibrils are presumably formed, while the sharp turbidity increase corresponds to the second step of fibrin assembly, when the lateral aggregation of protofibrils into fibers occurs. As seen in Fig. 6, fibulin-1 increased the lag period and decreased the rate of turbidity rise; the final turbidity measured after 24 h was also decreased (from an A of 0.087 in the absence of fibulin-1 to 0.076 when fibulin-1 was present at 100 µg/ml). These results indicate that fibulin-1 can reduce the rate of both steps in the fibrin assembly process and possibly the thickness of fibrin fibers.


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(2) (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.



Fibulin-1 Can Incorporate into Fibrin Clots Formed in Vitro

In vitro clot-forming assays based on those performed by Wilson and Schwarzbauer(1992) were used to determine whether fibulin-1 could incorporate into a fibrin clot. As shown in Fig. 7A, 4% of the added I-fibulin-1 was able to incorporate into the fibrin clots to a similar extent as two other radiolabeled proteins known to incorporate into clots, TSP1 and FN (Bale et al., 1985; Wilson and Schwarzbauer, 1992). In contrast, only 0.5% of the I-BSA and 0.8% of the I-IgG added became incorporated into the fibrin clots formed in vitro. I-Fibulin-1 also incorporated into clots formed using human plasma instead of purified Fg (data not shown).


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(2) 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(r) 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.

Fibulin-1 Can Be Detected in Fibrin-containing Clots Formed in Vivo

Fibrin clots within human atherectomy specimens were immunohistochemically evaluated for the presence of fibulin-1. As shown in Fig. 8, fibulin-1 was consistently detected within newly formed fibrin-containing thrombi that were frequently associated with lumenal aspects of the extracted vascular tissue specimens. Typically, fibulin-1 was found deposited in a fibrous, mesh-like pattern within the thrombi with red cells often seen trapped within the fibrous mesh work. The findings indicate that fibulin-1 incorporation into fibrin clots occurs in vivo.


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.




DISCUSSION

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-1bulletFg 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-1bulletFg 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). (^2)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM42912. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These two authors contributed equally to this paper.

To whom correspondence should be addressed: J. H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0725; Fax: 301-738-0794; argraves{at}hlsun.red-cross.org.

(^1)
The abbreviations used are: ECM, extracellular matrix; Fg, fibrinogen; FN, fibronectin; TSP1, thrombospondin-1; FXIIIa, activated factor XIII; BSA, bovine serum albumin; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; DAB, 3,3`-diaminobenzidine tetrahydrochloride; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; AS, ammonium sulfate.

(^2)
S. Godyna, M. Diaz-Ricart, and W. S. Argraves, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Steingrimur Stefansson and Dudley K. Strickland for many helpful discussions and Elizabeth Smith and Clinton Lincoln for their immunohistological tissue-staining work.


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