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• W1572927059 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 (Kd) 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 Bβ 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 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 (Kd) 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 Bβ 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. INTRODUCTIONThe fibulins are a family of extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixFgfibrinogenFNfibronectinTSP1thrombospondin-1FXIIIaactivated factor XIIIBSAbovine serum albuminTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresisDAB3,3′-diaminobenzidine tetrahydrochlorideELISAenzyme-linked immunosorbent assayFITCfluorescein isothiocyanateASammonium sulfate. proteins currently consisting of two related members designated fibulin-1 and −2 (2Argraves W.S. Dickerson K. Burgess W.H. Ruoslahti E. Cell. 1989; 58: 623-629Google Scholar; 3Argraves W.S. Tran H. Burgess W.H. Dickerson K. J. Cell Biol. 1990; 111: 3155-3164Google Scholar; 29Pan T-C. Kluge M. Zhang R-Z. Mayer U. Timpl R. Chu M-L. Eur. J. Biochem. 1993; 215: 733-740Google Scholar; 30Pan T-C. Sasaki T. Zhang R-Z. Fassler R. Timpl R. Chu M-L. J. Cell Biol. 1993; 123: 1269-1277Google Scholar). The pattern of expression of the fibulins has been described in tissues of the chicken (32Spence S.G. Argraves W.S. Walters L. Hungerford J.E. Little C.D. Dev. Biol. 1992; 151: 473-484Google Scholar), mouse (20Kluge M. Mann K. Dziadek M. Timpl R. Eur. J. Biochem. 1990; 193: 651-659Google Scholar; Pan et al., 1993b; 34Zhang H-Y. Kluge M. Timpl R. Chu M-L. Ekblom P. Differentiation. 1993; 52: 211-220Google Scholar, 35Zhang H-Y. Chu M -L. Pan T-C. Sasaki T. Timpl R. Ekblom P. Dev. Biol. 1995; 167: 18-26Google Scholar) and human (36Zhang R-Z. Pan T-C. Zhang Z-Y. Mattei M-G. Timpl R. Chu M-L. Genomics. 1994; 22: 425-430Google Scholar; 31Roark E.F. Keene D.R. Haudenschild C.C. Godyna S. Little C.D. Argraves W.S. J. Histochem. Cytochem. 1995; 43: 401-411Google Scholar). 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 (4Balbona K. Tran H. Godyna S. Ingham K.C. Strickland D.K. Argraves W.S. J. Biol. Chem. 1992; 267: 20120-20125Google Scholar; 12Godyna S. Mann D.M. Argraves W.S. Matrix Biol. 1994; 14: 467-477Google Scholar), laminin and nidogen (Pan et al., 1993a; 7Brown J.C. Wiedemann H. Timpl R. J. Cell Sci. 1994; 107: 329-338Google Scholar).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 METHODSProteinsFibulin-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 DH, DY, DL, E3, αC (residues 220-581), and TSD, were prepared from bovine Fg according to methods described previously (24Medved L.V. Litvinovich S.V. Privalov P.L. FEBS Lett. 1986; 202: 298-302Google Scholar, 25Medved L.V. Platonova T.N. Litvinovich S.V. Lukinova N.I. FEBS Lett. 1988; 232: 56-60Google Scholar; 22Litvinovich S.V. Henschen A.H. Krieglstein K.G. Ingham K.C. Medved L.V. Eur. J. Biochem. 1995; 229: 605-614Google Scholar; 14Gorkun O.V. Veklich Y.I. Medved L.V. Henschen A.H. Weisel J.W. Biochemistry. 1994; 33: 6986-6997Google Scholar). Recombinant human αC domain of Fg (residues Gln221-Val610 of α chain) was provided by Dr. Ken Ighman (American Red Cross, Rockville, MD). Fibronectin (FN) was purified as described by 27Miekka S.I. Ingham K.C. Menache D. Thromb. Res. 1982; 27: 1-14Google Scholar. 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 1Alexander R.J. Detwiler T.C. Biochem. J. 1984; 217: 67-71Google Scholar. Ovalbumin was obtained from Sigma. Bovine serum albumin was obtained from U.S. Biochemical Corp. Human immunoglobulin was purchased from Cappel (Gaithersburg, MD).AntibodiesThe 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.RadiolabelingFibulin-1, FN, bovine serum albumin, and TSP1 (100 μg of each) were radioiodinated in 100 μl of phosphate-buffered saline, 0.5 mCi of Na125I (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 PlasmaHuman 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 15Green A.A. Hughes W.L. Methods Enzymol. 1955; 1: 67-90Google Scholar. 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 AssaysSamples 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 AssayNitrocellulose 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 125I-fibulin-1 (20 nM) in the same buffer containing 5 mM CaCl2. 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 AssaysSolid 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 (Kd) 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 Kd for the binding of fibulin-1 and Fg, a surface plasmon resonance-based method (reviewed in Fisher and Fivash(1994); 9Chaiken I. Rose S. Karlsson R. Anal. Biochem. 1992; 201: 197-210Google Scholar) 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 ka and kd were both obtained and the dissociation constant was derived from the equation Kd=kd/ka.Fluorescence AnisotropyFluorescein-labeled fibulin-1 was prepared according to the method of 8Busby T.F. Ingham K.C. Biochemistry. 1988; 27: 6127-6135Google Scholar using fluorescein isothiocyanate (FITC) (Molecular Probes Inc., Eugene, OR). Briefly, fibulin-1 was dissolved in 0.1 M NaHCO3 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 18Ingham K.C. Brew S.A. Biochim. Biophys. Acta. 1981; 670: 181-189Google Scholar. 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 FITC•fibulin-1, TBS solution using a motorized syringe while continually stirring. The change in anisotropy (ΔA) as a function of titrant concentration was fitted to a single class of binding sites by using the following equation, ΔA=ΔAmax[titrant]Kd+[titrant](Eq. 1) where Kd is the apparent dissociation constant and [titrant] is the concentration of free Fg or FN. ΔAmax is the maximum anisotropy change that would be achieved at saturating concentrations of titrant (19Ingham K.C. Brew S.A. Isaacs B.S. J. Biol. Chem. 1988; 263: 4624-4628Google Scholar).Incorporation of Fibulin-1 into Fibrin ClotsFibrin clots were formed in 1.5-ml microcentrifuge tubes using purified human Fg and thrombin as described by 33Wilson C.L. Schwarzbauer J.E. J. Cell Biol. 1992; 119: 923-933Google Scholar and 5Bale M.D. Westrick L.G. Mosher D.F. J. Biol. Chem. 1985; 260: 7502-7808Google Scholar. Human Fg (0.8 mg/ml) was mixed with 125I-fibulin-1 (64 nM), fibulin-1 (50 μg/ml, 641 nM), and 10 mM CaCl2. 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 A0.1%280 nm = 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 γ-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 PolymerizationTurbidity measurements were made throughout the course of in vitro fibrin polymerization as described by 6Bale M.D. Mosher D.F. J. Biol. Chem. 1986; 261: 862-868Google Scholar 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 CaCl2 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 SpecimensHuman 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.RESULTSFibrinogen Co-purifies with Fibulin-1 Isolated from PlasmaPrevious 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 Mr 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 Mr 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 Mr value of 80,000 and corresponded to fibulin-1 as determined by its immunoreactivity with monoclonal fibulin-1 antibody (Fig. 2D, lane2). The Mr values of the three other reduced polypeptides were consistent with their being the Fg Aα, Bβ, and γ chains. Fg is a dimer having a mass of 345 kDa, composed of 2 pairs of disulfide-linked chains designated Aα, Bβ, and γ that have mass values of 67.6, 54.7, and 46.4 kDa, respectively (23McKee P.A. Mattock P. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 738-744Google Scholar; 28Mosesson M.W. Ann. N. Y. Acad. Sci. 1983; 408: 97-113Google Scholar). 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 Aα, Bβ, and γ chains (Fig. 2C, lane2). The results indicate that Fg co-purifies with fibulin-1 isolated from plasma.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.View Large Image Figure ViewerDownload (PPT)Fibulin-1 Binds to Fibrinogen in Solid Phase and Fluid Phase ConditionsTo 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, 125I-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. 125I-fibulin-1 did not bind to the individual Fg Aα, Bβ, or γ 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, β-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 125I-fibulin-1 after their transfer to nitrocellulose from SDS-PAGE. 125I-fibulin bound to a polypeptide having a Mr 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 125I-fibulin-1 is capable of binding to Fg and that the disulfide-bonded structure of Fg is required for the binding.Figure 3:125I-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 125I-fibulin-1 (30 nM) (panelB).View Large Image Figure ViewerDownload (PPT)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.”View Large Image Figure ViewerDownload (PPT)To estimate the apparent dissociation constants (Kd) 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 Kd 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 Kd 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 (Kd = 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 Kd value of 2.9 ± 1.6 μM (n = 4) for Fg binding to immobilized fibulin-1. This value is in good agreement with Kd 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 Kd 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 Bβ Chain of FgTo 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 DH, DL, and DY (see diagram in Fig. 5A) bound to fibulin-1 in a dose-dependent manner, but other fragments such as the E, αC (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 Kd of 2.3 ± 0.2 μM (n = 4) was determined for the binding of fibulin-1 to Fg fragment DH. Since the smallest fibulin-1 binding fragment (DY) differs from the nonbinding TSD fragment principally by the presence of the COOH-terminal portion of the Bβ chain that includes residues 216-468 (Litvinovich et al., 1995), the fibulin-1 binding site is likely contained within this portion of the Bβ chain. The binding of fibulin-1 to the DY fragment was consistently of lower affinity than the binding to DH and DL (Fig. 5B) perhaps indicating that the conformation of the fibulin-1 binding site within the Bβ chain might be stabilized by the γ and/or γC 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 (DH, DL, DY, E, αC, 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.View Large Image Figure ViewerDownload (PPT)Effect of Fibulin-1 on Polymerization of FibrinThe portion of the Bβ 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 (10Doolittle R.F. Laudano A.P. Peptid. Biol. Fluids. 1980; 28: 311-316Google Scholar; 26Medved L.V. Litvinovich S.V. Ugarova T.P. Lukinova N.I. Kalikhevich V.N. Arademasova Z.A. FEBS Lett. 1993; 320: 239-242Google Scholar). 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 17Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Google Scholar 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" @default.
• W1572927059 created "2016-06-24" @default.
• W1572927059 creator A5008424026 @default.
• W1572927059 creator A5009505570 @default.
• W1572927059 creator A5043624266 @default.
• W1572927059 creator A5057692069 @default.
• W1572927059 creator A5066051839 @default.
• W1572927059 creator A5079526803 @default.
• W1572927059 date "1995-08-01" @default.
• W1572927059 modified "2023-10-15" @default.
• W1572927059 title "The Interaction of Fibulin-1 with Fibrinogen" @default.
• W1572927059 cites W1486108017 @default.
• W1572927059 cites W1493393899 @default.
• W1572927059 cites W1557258457 @default.
• W1572927059 cites W1596384556 @default.
• W1572927059 cites W1604635179 @default.
• W1572927059 cites W1971156818 @default.
• W1572927059 cites W1971299102 @default.
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