The Glycosaminoglycan Binding Site Governs Ligand Binding to the Somatomedin B Domain of Vitronectin*

(Received for publication, November 4, 1996, and in revised form, January 27, 1997)

Dietmar Seiffert Dagger

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The ligand binding functions of vitronectin (Vn) are regulated by its conformational state/degree of multimerization. In the native plasma form of Vn, the C-terminal glycosaminoglycan (GAG) binding domain is believed to be cryptic. Here, evidence is provided that the addition of fucoidan or dextran sulfate to unfractionated plasma results in the formation of covalently and non-covalently stabilized Vn multimers. These multimers express conformationally sensitive antibody epitopes and ligand binding sites located in the N terminus of the Vn molecule. While heparin forms complexes with monomeric plasma Vn and induces conformational changes, a reduction in ionic strength is required for induction of multimerization. In addition, heparin serves as a template for the assembly of type 1 plasminogen activator inhibitor-induced disulfide-linked Vn multimers. These results support a new model for the structure of native Vn. The C-terminal GAG binding domain is predicted to be exposed in the native conformation, whereas the N terminus is cryptic. Ligand binding to the GAG binding site unfolds the N terminus, thereby exposing cryptic ligand binding sites.


INTRODUCTION

Vitronectin (Vn),1 an adhesive glycoprotein present in the circulatory system and in a variety of tissues, belongs to a group of molecules that play key roles in the attachment of cells to their surrounding matrix (1, 2). In addition, Vn also regulates the complement, coagulation, and fibrinolytic system. During the last few years, it has been increasingly appreciated that Vn is a conformationally labile molecule. For example, only 2% of the Vn in plasma is in a conformation capable of binding to heparin-Sepharose, whereas the relative amount increases to 7% by the generation of serum (3). In addition, the formation of complexes of Vn with type 1 plasminogen activator inhibitor (PAI-1) (4), thrombin-antithrombin III (5), and complement C5b-C9 (6) induces conformational changes and heparin binding. Moreover, conformational changes in the Vn molecule can also be induced by denaturation with chaotropic agents, heat treatment, and acidification (1, 2). These changes are accompanied by the spontaneous formation of disulfide-linked Vn multimers (7). Plasma Vn and Vn purified under non-denaturing conditions (i.e. native Vn) are monomeric and lack exposure of conformationally sensitive epitopes (8). In contrast, Vn purified from serum by heparin affinity chromatography in the presence of 8 M urea (i.e. denatured Vn) is highly multimerized and conformationally altered (8, 9). A second circulatory pool of Vn is contained in platelet alpha -granules (10). Platelet Vn is present in both monomeric and multimeric forms, and the latter form expresses epitopes for conformationally sensitive monoclonal antibodies (mAbs, see Ref. 8). Thus, platelets provide a physiological correlate for the experimentally observed multimerization of Vn.

Defining the biological functions of Vn has been complicated, because a number of its ligand binding domains are differentially expressed according to the conformation of this molecule. A number of ligands were identified that interact preferentially with the conformationally altered, denatured form of Vn. These ligands include collagen (11), GAGs (12-14), beta -endorphin (15), PAI-1 (16), urokinase receptor-urokinase complex (17), and cells (18). In contrast, thrombin-antithrombin III complexes appear to preferentially interact with native Vn (19), and PAI-1 appears to bind to both forms of Vn (20).

The observations that binding of plasma Vn to heparin is increased upon ligand binding or treatment with protein denaturants led to the concept that the GAG binding domain (located between amino acids 341 and 379) is, at least partially, cryptic in the native conformation (1, 2). However, conformational changes in the Vn molecule upon denaturation are apparently not limited to the GAG binding domain, but also occurred in the somatomedin B (SMB) domain (amino acids 1-51) and the connecting region/first hemopexin-like repeat (amino acids 52-239) (9). Moreover, the epitope for a mAb that blocks the high affinity interaction of PAI-1 with the SMB domain of Vn (i.e. mAb 153) was not expressed in plasma Vn (8). These observations raise the possibility that both the GAG binding domain and N-terminal ligand binding sites are cryptic in the native Vn molecule. However, most ligand binding events in Vn have been mapped to either of these domains. Despite these observations, plasma Vn binds ligands that are reported to interact with the N terminus. How could these apparently conflicting observations be explained considering that both binding sites were not available in the native form? Here, we provide evidence that sulfated GAGs bind to native plasma Vn, resulting in Vn multimerization and conformational changes, thereby exposing cryptic N-terminal ligand binding sites. Taken together, these observations imply that plasma Vn is GAG binding-competent.


MATERIALS AND METHODS

Proteins and Antibodies

Denatured Vn was purified by heparin affinity chromatography in the presence of 8 M urea (21). PAI-1 was isolated and activated with guanidinium hydrochloride (22), dialyzed against 5 mM sodium phosphate, 1 mM EDTA, 0.345 M NaCl, pH 6.5, to stabilize the biological activity of PAI-1 (23), and the specific activity was determined by titration against urinary-type plasminogen activator (Calbiochem) (22). Prior to each experiment, PAI-1 was dialyzed into ice-cold phosphate-buffered saline (PBS). The Vn polypeptides encompassing amino acids 1-51 and 52-239 were expressed in Escherichia coli strain BL 21 and purified by nickel affinity chromatography from sonicated cell lysates according to the manufacturer (Novagen). Briefly, fragments of the human Vn cDNA were polymerase chain reaction-amplified using the following polymerase chain reaction primer containing BamHI sites at both the 5' and 3' end and ligated into the BamHI-digested expression plasmid using standard cloning procedures (24). 5' primer 1-51: 5'-TGC ATC ATG GAT CCC GAC CAA GAG TCA TGC AAG-3'; 3' primer 1-51: 5'-GTT TCA GGA TCC TTA CAT AGT GAA CAC ATC CCC-3'; 5' primer 52-239: 5'-TGC ATC ATG GAT CCG GAG GAT GAG TAC ACG-3'; 3' primer 52-239: 5'-AAC GTT TCA GGA TCC TTA GTC CAG GAC ACC ATC CTC-3'. The orientations of the inserts were determined by polymerase chain reaction using two oligonucleotide primers based in the insert and the parental plasmid, respectively, and the sequences were confirmed by double-stranded DNA sequencing. mAbs 153 and 1244 were obtained using standard hybridoma technology (22), and IgG was produced in mice as ascites fluid and purified by using protein A-Sepharose. Protein concentrations were determined by the bicinchoninic acid method (Pierce). Dextran sulfate (average Mr 8,000), fucoidan (average Mr 50,000), heparin sodium salt from porcine intestinal mucosa (170 USP units/mg), low molecular weight heparin from porcine intestinal mucosa (average Mr 3,000), and chondroitin sulfate A were obtained from Sigma, dissolved at 50 mg/ml in PBS, and stored at -80 °C. Protamine sulfate was obtained from Calbiochem, dissolved at 25 mg/ml in PBS, and stored at -80 °C. Platelet-poor plasma (PPP) was prepared under conditions aimed at preventing platelet activation as described (8). The Vn concentration in PPP was estimated by semiquantitative immunoblotting (8).

Structural Characterization of Vn Multimers

PPP (2 µl) was incubated (37 °C) with the indicated concentration of GAGs and/or PAI-1, and the volume was adjusted to 50 µl with PBS. At the end of the incubation period (2 h), the samples were immediately fractionated by gel electrophoresis and analyzed by immunoblotting using mAb 1244 (22). For some experiments, the GAG-treated PPP was analyzed by gel filtration chromatography using a Sephacryl S-300 superfine column (125 × 1.5 cm). The Vn concentration in the column fractions was determined by sandwich ELISA as described (4). The mAb used in the ELISA is conformationally sensitive. The fractions were heat-treated (55 °C, 1 h) to completely denature Vn, thereby avoiding differential recognition of Vn conformers by the ELISA (4).

Immunological and Functional Characterization of Vn Multimers

Conformational changes in the Vn molecule were quantified by competitive ELISA essentially as described (9). As a modification, microtiter wells were coated (5 µg/ml) with recombinant Vn fragments encompassing the immunoepitope of the mAbs (i.e. Vn amino acids 1-51 for mAb 153, and Vn amino acids 52-239 for mAb 1244). Results are expressed as percentage binding of antibodies in the presence of PPP (10 µg/ml) in the absence of PAI-1 or GAGs. The binding of Vn to PAI-1 was determined by competitive ELISA (22). Briefly, SMB polypeptides coated (see above) and blocked wells were co-incubated (1 h, 37 °C) with activated PAI-1 (0.6 nM) and PPP (10 µg/ml Vn; preincubated (1 h, 37 °C) with the indicated concentration of GAGS). Bound PAI-1 was detected using rabbit anti-human PAI-1 IgG (22). This assay system detects only the binding of active forms of PAI-1, whereas latent PAI-1 or plasminogen activator·PAI-1 complexes fail to interact with the immobilized SMB polypeptide (not shown). Control experiments revealed that in the concentration range used, GAGs did not interfere with mAb or PAI-1 binding to immobilized ligands or remove the immobilized ligand from the microtiter wells.


RESULTS

Heparin Shifts the Electrophoretic Mobility of Plasma Vn

The effects of highly sulfated GAGs on the structure of plasma Vn was characterized. Under non-reducing conditions, plasma Vn migrated as a broad band between Mr 75,000 and 65,000, and heparin fails to alter the mobility of plasma Vn on SDS-PAGE (Fig. 1, lane 1; compare with Fig. 3, lane 1). However, analysis by native Vn reveals that heparin shifts the mobility on a native PAGE in a dose-dependent manner. The first shift in the mobility is observed at 1 µg/ml heparin, and at 100 µg/ml, all plasma Vn is shifted to the cathode (Fig. 1, lanes 2-5). Analysis by gel filtration chromatography reveals that plasma Vn incubated with 1 mg/ml heparin elutes close to albumin, indicating that heparin fails to induce higher molecular weight Vn forms (not shown). It should be noted that the resolution of the S-300 column is not sufficient to discriminate between Vn monomers (Mr 75,000) and Vn monomer-heparin complexes (expected average size 95,000). Analysis of the Vn elution fractions by native PAGE (Fig. 1, lane 6) reveals that the electrophoretic mobility of the Vn is similar to heparin-treated plasma (compare Fig. 1, lane 5). This observation raised the possibility that a heparin-Vn monomer complex is formed that is stable during size exclusion chromatography. Protamine sulfate dissociates heparin-protein complexes. To test whether the shift of Vn mobility on native PAGE was due to non-dissociated Vn monomer-heparin complexes, PPP was incubated with heparin alone (Fig. 1, lane 7), or with heparin followed by the addition of protamine sulfate (Fig. 1, lane 8). Protamine sulfate reverses the heparin-induced shift in Vn mobility. This observation suggested that Vn monomer-heparin complexes are formed that are sensitive to protamine sulfate. The shift in the Vn mobility on native PAGE is not limited to standard heparin, but is also observed in the presence of LMW heparin (Fig. 1, lane 9).


Fig. 1. Effect of heparin on the structure of plasma Vn. PPP (2 µl) was incubated in the presence of the indicated concentration of heparin for 2 h at 37 °C in PBS in a total volume of 50 µl and analyzed by SDS-PAGE (lane 1) or native PAGE (lanes 2-9) followed by immunoblotting using mAb 1244. Lane 1, PPP and 10 mg/ml heparin; lane 2, PPP in the absence of heparin; lane 3, PPP and 1 µg/ml heparin; lane 4, PPP and 10 µg/ml heparin; lane 5, PPP and 100 µg/ml heparin; lane 6, PPP incubated with 1 mg/ml heparin and analyzed by gel filtration chromatography (see Fig. 2). A column fraction that co-eluted with albumin was analyzed. Lane 7, PPP and 50 µg/ml heparin; lane 8, PPP and 50 µg/ml heparin followed by 100 µg/ml protamine sulfate for 30 min at 37 °C; lane 9, PPP and 100 µg/ml LMW heparin. The mobility of Mr standards for SDS-PAGE is indicated to the left, and the interfaces between separating and stacking gels are marked by arrowheads.
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Fig. 3. Effects of fucoidan on the structure of plasma Vn. Panel A, PPP (2 µl) was incubated (37 °C, 2 h) in the absence (lanes 1, 4, and 6) or presence of 10 µg/ml fucoidan (lanes 2 and 7) or 100 µg/ml fucoidan (lanes 3, 5, and 8) in a total volume of 50 µl of PBS and fractionated by SDS-PAGE under non-reducing conditions (lanes 1-3), SDS-PAGE under reducing conditions (lanes 4 and 5), or by native PAGE (lanes 6-8). Vn was detected by immunoblotting using mAb 1244. The mobility of Mr standards for SDS-PAGE is indicated to the left, and the interfaces between stacking and separating gels are indicated by arrowheads. Panels B and C, PPP (50 µg/ml Vn) was incubated in the presence (closed symbols) or absence (open symbols) of fucoidan (1 mg/ml) in PBS, 0.1% polyethylene glycol 3350 for 2 h at 37 °C and layered on top of a 125 × 1.5-cm Sephacryl S-300 column equilibrated in the same buffer. The column was developed at a flow rate of 20 ml/h, and 6 min fractions were collected. Panel B (circles), absorbance at 280 nm. Panel C, (squares), the Vn concentration was determined by ELISA (see "Materials and Methods"). The column was calibrated with the indicated markers: blue dextran 2000 (V0), ferritin (Mr 440,000), aldolase (Mr 158,000), and albumin (Mr 67,000).
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Heparin Induces Vn Multimerization at Low Ionic Strength

We recently reported (25) that increases in ionic strength prevented heat-induced multimerization of native Vn. Here, we tested whether Vn is more prone to multimerize upon reduction of the ionic strength. Plasma Vn incubated in the absence of heparin remains monomeric (Fig. 2, lane 1). However, in the presence of heparin, reduction of the NaCl concentration results in the formation of higher molecular weight Vn forms (Fig. 2, lane 2). The higher molecular weight forms of Vn are sensitive to reducing agents, indicative of disulfide-linked Vn multimers (not shown). Heparin also induces the formation of higher Mr Vn forms that fail to enter the separating gel on native PAGE (not shown). To confirm and extend these observations by an independent assay system, PPP was incubated in the presence of heparin under reduced ionic strength (i.e. 75 mM NaCl) and fractionated by size exclusion chromatography (Fig. 2B). In initial experiments, the possibility that the combination of heparin and reduction of ionic strength shifts the overall protein elution profile was tested. The protein elution profile (A280) was unchanged under reduced ionic strength (Fig. 2B) or at physiological NaCl concentrations (compare Fig. 3). These initial experiments excluded a generalized effect of the experimental conditions on the mobility of plasma protein on size exclusion chromatography. In the following experiments, the effect of heparin and reduction in ionic strength on the elution of Vn was evaluated. The majority of the Vn immunoreactivity is shifted to higher Mr fractions (Fig. 2C, closed squares), whereas Vn co-elutes as a single symmetric peak with albumin in the absence of heparin (open squares). Selected gel filtration fractions were also analyzed by SDS-PAGE under non-reducing conditions. While the starting material contains both multimeric and monomeric Vn (Fig. 2A, lane 3), the high molecular weight fractions consist of SDS-stable Vn multimers (Fig. 2A, lane 4), whereas the lower molecular weight fractions contain Vn monomers (Fig. 2A, lane 5). Thus, the gel filtration experiments confirmed the gel electrophoretic results, indicating that in the presence of heparin and reduced ionic strength, higher molecular weight Vn forms are generated.


Fig. 2. Heparin induces Vn multimerization at reduced ionic strength. Panel A, PPP (2 µl) was incubated in the absence (lane 1) or presence (lane 2) of 1 mg/ml heparin in 10 mM phosphate buffer, pH 7.4, containing 75 mM NaCl in a total volume of 50 µl for 2 h at 37 °C and analyzed by SDS-PAGE followed by immunoblotting using mAb 1244. Starting material (lane 3), fraction 40 (lane 4), and fraction 53 (lane 5) of the gel filtration experiment (see panel B) were analyzed by SDS-PAGE as in lanes 1 and 2. The mobility of Mr standards is indicated to the left and the interface between separating and stacking gels is marked by the arrowhead. Panels B and C, PPP (50 µg/ml Vn) was incubated in the presence (closed symbols) or absence (open symbols) of heparin (1 mg/ml) in 10 mM Tris-HCl, pH 7.4, containing 75 mM NaCl and 0.1% polyethylene glycol 3,350 for 2 h at 37 °C and layered on top of a 125 × 1.5-cm Sephacryl S-300 column equilibrated in the same buffer. The column was developed at a flow rate of 25 ml/h, and 6-min fractions were collected. Panel B (circles), absorbance at 280 nm. Panel C (squares), the Vn concentration was determined by ELISA (see "Materials and Methods"). The column was calibrated with the indicated markers: blue dextran 2000 (V0), ferritin (Mr 440,000), aldolase (Mr 158,000), and albumin (Mr 67,000).
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Dextran Sulfate and Fucoidan Induce Vn Multimerization

The results obtained with heparin were compared with fucoidan and dextran sulfate, two GAGs which have an increased sulfate content in comparison to heparin. Increasing amounts of fucoidan were added to a constant amount of plasma at physiological ionic strength, and after incubation for 2 h at 37 °C, the resulting samples were analyzed by SDS-PAGE under non-reducing (Fig. 3A, lanes 1-3) or reducing (Fig. 3A, lanes 4 and 5) conditions, followed by immunoblotting using mAb 1244. Addition of fucoidan to plasma (Fig. 3A, lane 1) results in a dose-dependent appearance of high molecular weight Vn immunoreactive bands (Fig. 3A, lanes 2 and 3) when analyzed by SDS-PAGE under non-reducing conditions. The molecular mass of the predominant high molecular weight Vn form was estimated to be 150 kDa. The size of this band is consistent with being a Vn dimer. The importance of covalent interactions for the formation of the high molecular weight Vn forms was evaluated. Under reducing conditions, plasma Vn migrates as a doublet of Mr 75,000 and 65,000 on SDS-PAGE (Fig. 3A, lane 4). The addition of fucoidan has no effect on the electrophoretic mobility of plasma Vn under reducing conditions (Fig. 3A, lane 5). Thus, fucoidan-induced high molecular weight Vn forms are sensitive to reducing agents. Electrophoresis under non-denaturing conditions was employed to understand whether the Mr 75,000-65,000 monomers detected in non-reducing SDS gels are actually Vn monomers or non-covalently associated SDS-sensitive Vn multimers. While plasma Vn is detected as a single major band that migrated to the anode (Fig. 3A, lane 6), the addition of fucoidan results in a dose-dependent disappearance of monomeric Vn and a shift of the immunoreactivity toward the cathode (Fig. 3A, lanes 7 and 8). Vn is completely shifted to the cathode on the native PAGE, but only a relatively small portion is present in SDS-stable disulfide linked forms even at the highest concentration of fucoidan employed. These observations raise the possibility that fucoidan induces the formation of non-covalently associated Vn multimers that are only partially stabilized by disulfide-exchange between Vn molecules. Alternatively, the change in electrophoretic mobility on native PAGE may be due to non-dissociated Vn-fucoidan complexes.

Molecular sieving chromatography was employed to discriminate between these two possibilities. Plasma was incubated for 2 h at 37 °C in the absence or presence of 1 mg/ml fucoidan and applied to a gel filtration column. The protein elution profiles (i.e. A280) are very similar in the presence or absence of fucoidan (Fig. 3B). The Vn content in the column fractions was determined by specific ELISA (Fig. 3C). The elution of plasma Vn is practically completely shifted to higher molecular weight fractions, and extended to the void volume (Mr 2 × 106) of the Sephacryl S-300 column. In contrast, plasma incubated in the absence of fucoidan eluted with the expected molecular weight of approximately 67,000, indicative of monomeric Vn (Fig. 3C). The size of the fucoidan-induced Vn multimers on size exclusion chromatography is higher than that expected for monomeric Vn-fucoidan complexes (i.e. Mr 120,000). Thus, fucoidan apparently induces non-covalently and covalently stabilized Vn multimers.

The induction of Vn multimerization under physiological ionic strength is not limited to fucoidan. Dextran sulfate induces the formation of higher molecular weight Vn forms on non-reducing SDS-PAGE in a dose-dependent manner (Fig. 4A, lanes 2 and 3). Again, these multimers are stabilized by disulfide bonds as revealed by sensitivity to reducing agents (not shown). Analysis by native PAGE reveals that dextran sulfate shifts in a dose-dependent manner the majority of the Vn to the cathode (Fig. 4A, lanes 4-6). Again, the majority of Vn is shifted to the cathode on native PAGE, whereas only limited amounts of SDS-stable Vn multimers were detectable. Analysis by gel filtration chromatography revealed that the overall protein elution profile is unchanged in the presence or absence of dextran sulfate (Fig. 4B). In contrast, the Vn elutes in two pools from the gel filtration column in the presence of dextran sulfate. The high molecular weight pool extends to the void volume, whereas the lower molecular weight peak co-incited with the albumin elution peak (Fig. 4C). Analysis of the high molecular weight peak by native PAGE reveals the presence of Vn multimers that failed to enter the separating gel (Fig. 4A, lane 7), whereas the lower molecular weight peak contains traces of Vn monomers and higher molecular weight Vn forms that enter the separating gel (Fig. 4A, lane 8).


Fig. 4. Effects of dextran sulfate on the structure of plasma Vn. Panel A, PPP (2 µl) was incubated (37 °C, 2 h) in the absence (lanes 1 and 4) or presence of 10 µg/ml dextran sulfate (lanes 2 and 5) or 100 µg/ml dextran sulfate (lanes 3 and 6) in a total volume of 50 µl of PBS and fractionated by SDS-PAGE under non-reducing conditions (lanes 1-3) or by native PAGE (lanes 4-6). Selected fractions from the gel filtration run (panels B and C) were also analyzed by native PAGE (lane 7, fraction 47; lane 8, fraction 61). Vn was detected by immunoblotting using mAb 1244. The mobility of Mr standards for SDS-PAGE is indicated to the left, and the interfaces between stacking and separating gels are indicated by arrowheads. Panels B and C, PPP (50 µg/ml Vn) was incubated in the presence (closed symbols) or absence (open symbols) of dextran sulfate (1 mg/ml) in PBS, 0.1% polyethylene glycol 3,350 for 2 h at 37 °C and layered on top of a 125 × 1.5-cm Sephacryl S-300 column equilibrated in the same buffer. The column was developed at a flow rate of 20 ml/h, and 6-min fractions were collected. Panel B (circles), absorbance at 280 nm. Panel C (circles), the Vn concentration was determined by ELISA (see "Materials and Methods"). The column was calibrated with the indicated markers: blue dextran 2000 (V0), ferritin (Mr 440,000), aldolase (Mr 158,000), and albumin (Mr 67,000).
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Sulfated GAGs Induce the Expression of Conformationally Sensitive Antibody Epitopes in Plasma Vn

Multimerization of Vn, as well as complex formation with molecules of the coagulation, complement, and fibrinolytic cascade, results in the exposure of immunoepitopes in the Vn molecule that are not present in the monomeric plasma form (8). To determine whether the Vn multimers induced by GAGs contain these neoepitopes, plasma Vn was compared with Vn incubated with GAGs by competitive ELISA using mAbs that are derived to these conformationally sensitive sites (Fig. 5). It should be noted that the competitive binding assays were performed with immobilized Vn fragments containing the respective antibody epitope. This modified procedure prevents interference of the assay by GAGs (presumably due to the binding of GAG·Vn complexes to the C-terminal GAG binding domain of intact immobilized Vn; not shown). Fucoidan (closed circles) and dextran sulfate (open circles) induce the exposure of the mAb 153 epitope (located between amino acids 1 and 51 (22)) in a dose-dependent manner (Fig. 5A). At higher concentrations, heparin (closed squares) and LMW heparin (closed triangles) also induce the exposure of this neoepitope, whereas chondroitin sulfate has little effects. In comparison to the mAb 153 epitope, GAGs have rather limited effects on the exposure of the mAb 1244 epitope (located between amino acids 52 and 239 (22)). For example, heparin (not shown) and LMW heparin (not shown) do not stimulate the exposure of the mAb 1244 epitope, and fucoidan (closed circles) and dextran sulfate (open circles) have a rather limited effect in comparison to the mAb 153 epitope (Fig. 5B; note the different scale on the y-axis in comparison to panel A). Thus, highly sulfated GAGs induce the exposure of N-terminal immunoepitopes in Vn that are not present in the native plasma form of this molecule.


Fig. 5. Effect of GAGs on the expression of conformationally sensitive antibody epitopes. PPP (20 µg/ml Vn) was incubated with the indicated concentration of GAGs for 2 h at 37 °C. The exposure of conformationally sensitive mAb epitopes was tested in competitive binding assays at 10 µg/ml Vn. Results are expressed as percentage antibody binding in comparison with untreated PPP. Panel A, mAb 153 (epitope located between amino acids 1 and 51). Panel B, mAb 1244 (epitope located between amino acids 52 and 239). Closed circles, fucoidan; open circles, dextran sulfate; closed squares, heparin; open squares, LMW heparin; closed triangles, chondroitin sulfate A.
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Sulfated GAGs Induce High Affinity PAI-1 Binding to the SMB Domain of Vn

To test whether the observation that highly sulfated GAGs induce the exposure of cryptic antibody epitopes in the SMB domain can be extended to physiological Vn ligands, the effects of different GAGs on the high affinity interaction of active PAI-1 and the isolated SMB domain was tested in competitive binding assays. Plasma was preincubated with a dose response of GAGs and tested for the competition of the binding of active PAI-1 to the immobilized SMB domain (Fig. 6A). Sulfated GAGs induce the exposure of the high affinity PAI-1 binding domain in plasma Vn in a dose-dependent manner, although to a different extent. While fucoidan (closed circles) and dextran sulfate (open circles) are approximately equally potent, heparin (closed squares) and LMW heparin (open squares) are significantly less effective in exposing the PAI-1 binding site in the SMB domain (Fig. 6A). In contrast, chondroitin sulfate (closed triangles) has no significant effect. Moreover, heparin (open triangle), or fucoidan, dextran sulfate, and LMW heparin (not shown) have no effect on the binding of denatured Vn to PAI-1 (Fig. 6A). These observations suggest that ligand binding to the C-terminal GAG binding domain regulates N-terminal ligand binding events.


Fig. 6. GAG binding to Vn exposes the cryptic N-terminal PAI-1 binding domain. Panel A, plasma (containing 20 µg/ml Vn) or denatured Vn (2 µg/ml) was incubated (2 h, 37 °C) with the indicated concentration of GAGs and analyzed for their ability to compete with active PAI-1 for binding to immobilized SMB polypeptides. Closed circles, fucoidan; open circles, dextran sulfate; closed squares, heparin; open squares, LMW heparin; closed triangles, chondroitin sulfate A; open triangle, denatured Vn incubated with heparin. Results are expressed as a percentage of binding in the absence of GAGs at a Vn concentration of 10 µg/ml (plasma) or 1 µg/ml (denatured Vn). Panel B, PPP was incubated with heparin as described under "Materials and Methods" followed by a 30-min incubation period with NaCl. The ability of the resulting samples to compete with the binding of active PAI-1 for binding to immobilized SMB polypeptides was tested as in panel A. Panel B: A, PPP; B, PPP and heparin (10 mg/ml); C, PPP, heparin (10 mg/ml), and 0.25 M NaCl; D, PPP, heparin (10 mg/ml), and 0.5 M NaCl; E, PPP and 0.5 M NaCl. Results are expressed as a percentage of binding in the absence of GAGs at a Vn concentration of 10 µg/ml (plasma).
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The interaction of heparin with proteins is mainly due to electrostatic interactions and thus is reversible by an increase in ionic strength (26). This consideration suggests that NaCl should reverse the heparin-induced conformational changes in Vn. To test this hypothesis, unfractionated plasma was first incubated with heparin, followed by a dose response of NaCl, and the competition for the binding of active PAI-1 to immobilized SMB polypeptide was determined (Fig. 6B). Increases in ionic strength reverses heparin-induced PAI-1 binding in a dose-dependent manner, and at the highest concentration of NaCl, approaches that of untreated plasma. Control experiments reveal that the addition of GAGs or NaCl in the concentration range employed in this study has no effect on the binding of PAI-1 or mAbs to the immobilized polypeptides (not shown).

Heparin Functions as a Template for the Assembly of Covalently Linked PAI-1-induced Vn Dimers

We recently reported that the addition of active PAI-1 to unfractionated PPP induces the formation of disulfide-linked Vn dimers (4). Moreover, heparin also induces conformational changes in Vn, thereby promoting high affinity binding to active PAI-1 (Fig. 6). In some biological interactions (e.g. formation of thrombin-antithrombin III complexes), heparin not only induces conformational changes, but also serves as a template for the assembly of these complexes. The latter approximation mechanism due to the heparin binding function of both antithrombin III and thrombin is characterized by a biphasic heparin dose response. While at low and intermediate concentrations of heparin complex formation is stimulated, high concentrations result in reduced association between the molecules. The possibility that heparin may also serve as a template for the assembly of PAI-1-induced disulfide-linked Vn dimers was tested. Active PAI-1 was incubated with native Vn present in plasma and an extended dose response of heparin (Fig. 7). PAI-1 in the absence of heparin induces the formation of disulfide-linked Vn dimers (Fig. 7, lane 2) as previously reported (4). At high concentrations of heparin, the PAI-1 induced Vn dimer formation is practically completely blocked (Fig. 7, lane 3). Intermediate concentrations of heparin increase the formation of disulfide-linked Vn dimers (Fig. 7, lanes 4-7), whereas low concentrations of heparin are ineffective (Fig. 7, lane 8). This biphasic dose-response curve is typical for a template mechanism of heparin and thus is compatible with the idea that heparin functions as a template for the assembly of disulfide-linked Vn dimers.


Fig. 7. Heparin functions as a template for the assembly of covalently linked Vn dimers. Unfractionated plasma was preincubated (37 °C, 1 h) with the indicated concentration of heparin prior to the addition of active PAI-1 for an additional 2 h at 37 °C. The resulting samples were fractionated by SDS-PAGE under non-reducing conditions and analyzed by immunoblotting using mAb 1244. Lane 1, PPP incubated in the absence of heparin and PAI-1; lane 2, PPP incubated with PAI-1; lane 3, PPP incubated with 5 mg/ml heparin and PAI-1; lane 4, PPP incubated with 0.5 mg/ml heparin and PAI-1; lane 5, PPP incubated with 50 µg/ml heparin and PAI-1; lane 6, PPP incubated with 5 µg/ml heparin and PAI-1; lane 7, PPP incubated with 0.5 µg/ml heparin and PAI-1; lane 8, PPP incubated with 50 ng/ml heparin and PAI-1. The mobility of Mr standards is indicated to the left, and the interface between separating and stacking gels is denoted by the arrowhead.
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DISCUSSION

The results presented in this study suggest a new model for the folding of native plasma Vn and its multimerization upon ligand binding (summarized as a minimal model in Fig. 8). The first issue in this model is related to the exposure of the GAG binding domain in native Vn. Currently, it is believed that this positively charged domain is cryptic in the native conformation (reviewed in Refs. 1 and 2). However, this concept is already questioned by the published observations that heparin increases the mAb 8E6 epitope expression (located between amino acids 52 and 239 (22) in preparations of purified native Vn (5), that partially purified plasma Vn binds to heparin Sepharose under low ionic strength (7), and that plasma Vn functions as a non-competitive inhibitor of the heparin-stimulated reactions of antithrombin III (27). Furthermore, this study provides evidence that highly sulfated GAGs induce Vn multimerization at physiological ionic strength and that the electrophoretic mobility of plasma Vn on native PAGE is quantitatively shifted by heparin in a reversible fashion. Moreover, GAGs stimulates the expression of the mAb 153 epitope and high-affinity ligand binding events (i.e. PAI-1) to the SMB domain, again suggesting that plasma Vn is capable of interacting with GAGs. Taken together, these observations suggest that native plasma Vn has the potential to bind to GAGs.


Fig. 8. Ligand binding to plasma Vn. Panel A, native plasma Vn. The C-terminal GAG binding domain is ligand binding-competent (GAGs, PAI-1), whereas the SMB domain is cryptic. Plasma Vn contains at least one free sulfhydryl group (SH). Panel B, unfolded intermediate. Ligand binding to the GAG binding domain induces conformational changes in Vn, resulting in the exposure of the SMB domain. The resulting intermediate may be monomeric or multimeric. In the case of heparin, the unfolding of Vn appears to be reversible. Panel C, multimeric, conformationally altered Vn. The unfolded Vn molecules self-assemble to non-covalently stabilized multimers. Gel filtration studies suggest that the number of subunits (N) may be heterogenous (4). The non-covalently stabilized Vn multimers are partially stabilized by disulfide exchange between parallel or antiparallel assembled dimers.
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Four considerations may account at least in part for the lack of GAG binding of plasma Vn observed in a number of previous studies. First, heparin affinity chromatography experiments of unfractionated plasma could have been performed under conditions when heparin was the limiting factor. Under these conditions, the experiment determines the competition of plasma Vn with other heparin-binding proteins, like antithrombin III and histidine-rich glycoprotein, for heparin binding, rather than the affinity of heparin for Vn itself. Consistent with this assumption, reports vary between 2 and 20% binding of Vn present in unfractionated plasma to heparin-Sepharose (3, 12). Secondly, increased binding of Vn present in unfractionated urea-treated serum to heparin-Sepharose has been viewed as evidence that urea-dependent unfolding and multimerization of Vn is required for heparin binding. However, the apparent increased binding may be due to decreased or destroyed heparin affinity of many plasma proteins, thereby reducing the extent of competition for limited heparin binding sites on the column. Thirdly, urea treatment of serum induces formation of Vn multimers. Even under the assumption that the affinity of a single GAG binding domain within the Vn multimer remains unchanged in comparison to the monomer, the avidity of the multimer for a single heparin molecule, which is believed to contain more than one GAG binding site (28), should be drastically increased in comparison to the Vn monomer. This would result in selective retainment of Vn on the heparin column. Fourthly, Vn contains two consensus heparin binding sequences in the extended GAG binding domain located between residues 341 and 379. More specifically, heparin binding consensus sequences have been localized between amino acids 347-352 and 354-362 (26). Molecular modeling studies revealed that both elements in this domain form distinct areas of contact with heparin that wrap around and fold over a heparin octasaccharide (26). Thus, a single heparin molecule is expected to interact with more than one binding site in monomeric Vn, again increasing the avidity of the interaction. It is easily foreseeable that parts of the binding sites in heparin immobilized to a solid-phase support are blocked due to stearic hindrance or reduced flexibility, thus effectively reducing the avidity for Vn. It should be noted that GAG binding to native Vn apparently does not require a plasma cofactor, since similar shifts in the electrophoretic mobility of native Vn in the presence of heparin and fucoidan were also observed in a purified system (not shown).

The second issue in this model is related to the exposure of the SMB domain in plasma Vn. A number of studies suggest that this site is indeed cryptic in native Vn. Firstly, we recently reported that the mAb 153 binding site (epitope located between amino acids 1 and 51) is only weakly or not expressed in plasma Vn (8), but strongly expressed in urea-treated native Vn (9), denatured Vn (8), PAI-1-induced Vn multimers (4), or after GAG binding (this study). Secondly, kinetic studies revealed that the interaction of native Vn with active PAI-1 is at least one order of magnitude lower than that with denatured Vn (16). This observation suggests that the high affinity PAI-1 binding site in the SMB domain of Vn is not readily available in plasma Vn. Thirdly, the principal site for cell attachment in Vn (RGD sequence, amino acids 45-47) is located in the SMB domain, and multimeric conformationally altered Vn is more potent in promoting cell adhesion (18). Moreover, we provided evidence that native Vn fails to bind to purified glycoprotein IIb/IIIa and alpha vbeta 3, whereas multimerization of native Vn, induced either by heat treatment or binding to PAI-1, induces integrin binding competency.2 Taken together, these observations support the model depicted in Fig. 8A suggesting that the SMB domain is cryptic in native plasma Vn, whereas the GAG binding domain is readily available to solution-phase ligands.

The addition of sulfated GAGs to unfractionated plasma increased the expression of an immunoepitope (i.e. mAb 153) and high affinity active PAI-1 ligand binding events to the N-terminal SMB domain. Vn contains a single GAG binding domain (29). Thus, these observations suggest that ligand binding to the C-terminal GAG binding domain induces conformational changes in the native Vn molecule, resulting in the expression of the N-terminal SMB domain. Thus, interactions with C-terminal ligand binding sites modulate N-terminal ligand binding events. These observations raise the possibility that an unfolded intermediate is formed upon GAG interaction with the GAG binding domain. The predicted unfolded intermediate is depicted as a monomer in the cartoon (Fig. 8, panel B), but it remains unclear at present whether this intermediate is a conformationally altered monomer or reversibly associated multimer. The formation of this unfolded intermediate appears to be, at least in the case of heparin, reversible. This conclusion is based on the observations that increases in ionic strength after heparin binding to native plasma Vn resulted in reduced N-terminal high affinity PAI-1 ligand events. Also, neutralization of heparin by protamine sulfate reversed the shift in the electrophoretic mobility of Vn on native PAGE. In addition, Vn purified by heparin affinity chromatography under low ionic strength is present in the native conformation (7).

The third issue in this model is related to the temporal events of disulfide-linked versus non-covalently associated multimerization. We previously reported (4) that the non-covalently stabilized PAI-1-induced Vn multimerization is complete within 1 h of incubation, whereas the formation of disulfide bonds increased up to 24 h. Moreover, PAI-1 (4) and dextran sulfate and fucoidan (this study) completely shifted the mobility of Vn on the native PAGE, whereas the extent of disulfide-linked Vn multimers was rather limited. In addition, fucoidan completely shifted Vn immunoreactivity to higher molecular weight fractions on the size-exclusion chromatography, whereas again Vn was only partially present in disulfide-linked multimers. Taken together, these observations suggest that initially non-covalently associated Vn multimers are formed, whereas the covalent stabilization of Vn multimers is a relatively slow secondary event. However, the formation of disulfide bonds requires a close approximation between two Vn molecules within non-covalently associated Vn multimers. The easiest explanation for this observation is that Vn multimers present a higher order assembly of non-covalently associated Vn dimers that are then secondarily stabilized by intermolecular disulfide bonds. This model is depicted in Fig. 8, panel C. It remains unclear whether Vn dimers are formed by parallel or anti-parallel assembly of Vn monomers.

The fourth issue in this model is related to the question of how ligands with a high affinity for the SMB domain (e.g. PAI-1) can interact with native plasma Vn. At least two possibilities should be considered to account for this apparent discrepancy. Firstly, plasma Vn may be in a dynamic equilibrium between an N-terminal-folded and unfolded conformation. If this model is correct, mAb 153 should bind to the SMB domain in the unfolded state. The affinity of mAb 153 for Vn is relatively high (kd 10-9 mol/liter (22)), and thus the binding should shift the equilibrium between the folded and unfolded forms. This would predict that plasma Vn should compete to an appreciable extent with the binding of mAb 153 to immobilized denatured Vn. However, this was not observed experimentally (8) suggesting that this model is not valid. Secondly, a two-site interaction with Vn could account for the high affinity ligand binding to plasma Vn. For example, the GAG binding domain has been identified as a low affinity PAI-1 binding site (30). This observation raises the possibility that low affinity interactions of PAI-1 with the GAG binding domain can replace heparin, thereby inducing an unfolded intermediate and facilitating high affinity PAI-1 binding events to the N-terminal SMB domain. Further support for this concept is derived from the competition experiment using overlapping synthetic peptides derived from the GAG binding domain of Vn (30). These experiments indicated that the structural requirements in the GAG binding domain for binding to heparin and PAI-1 are very similar (30), pointing to the possibility that PAI-1 can replace heparin in this function.

Heparin is known to exert dual actions on the thrombin/antithrombin III system (28). First, it binds to antithrombin III and by that induces conformational changes resulting in increased reactivity with thrombin. Secondly, heparin serves as a template for the assembly of thrombin-antithrombin III complexes and also PAI-1·thrombin complexes (20). Here, evidence is provided that heparin exerts similar actions on the PAI-1/Vn interaction. It induces conformational changes in the Vn molecule, leading to the exposure of the N-terminal high affinity PAI-1 binding site. Moreover, heparin also serves as a template for the PAI-1-induced assembly of disulfide-linked Vn dimers. It should be noted that the template mechanism was already observed at pharmacological concentrations of heparin, suggesting that heparin therapy can modulate the PAI-1/Vn interaction in vivo.


FOOTNOTES

*   This work was supported by California Tobacco-related Disease Research Program Grant 4KT-0192 and National Institutes of Health Grant HL50704.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: E400/3438, Experimental Station, Dupont Merck Research Laboratories, P. O. Box 80400, Wilmington, DE 19809-0400. Tel.: 302-695-7069; Fax.: 302-695-4162; E-mail: seiffeda{at}a1.lldmpc.umc.dupont.com.
1   The abbreviations used are: Vn, vitronectin; GAG, glycosaminoglycan; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PAI-1, type 1 plasminogen activator inhibitor; PAGE, polyacrylamide gel electrophoresis; SMB, somatomedin B; PCR, polymerase chain reaction; PPP, platelet-poor plasma; ELISA, enzyme-linked immunosorbent assay; LMW, low molecular weight.
2   D. Seiffert and J. W. Smith, submitted for publication.

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