Native and Multimeric Vitronectin Exhibit Similar Affinity for Heparin

Abstract

For many years, the concept that the heparin-binding sequence is sequestered within vitronectin and exposed upon denaturation of the protein has guided experimental design and interpretation of related structurefunction studies on the protein. Within the circulation, complicated networks of interactions between proteins and other macromolecules are important for maintaining stasis. An example of the complex interplay that must exist among these biomolecules is provided by the human glycoprotein, vitronectin. Vitronectin interacts with a wide variety of ligands that are involved in control of diverse physiological processes including coagulation, fibrinolysis, tumor metastasis, the humoral immune response, and cellular migration (reviewed in Refs. 1-6). A partial list of target macromolecules that interact with vitronectin includes heparin, collagen, plasminogen, plasminogen activator inhibitor-1, serine protease inhibitor-protease complexes, and a subclass of integrin receptors on the surface of cells.Indeed, one of the first molecules shown to interact with vitronectin was heparin (7-11), and this interaction has since been widely investigated (12-16). Much of the early work on vitronectin was devoted to evaluating structural requirements for heparin binding, including tests of the effects of denaturation or proteolysis of vitronectin on heparin binding activity. Binding activity was evaluated from vitronectin interactions with a heparin column, estimated in terms of the salt concentrations required to elute bound vitronectin. From these studies came a most striking observation: vitronectin binding to heparin was enhanced upon treatment of the protein with urea, heat, or acid (9). This phenomenon was exploited by Yatohgo et al. (17) to develop an affinity-based purification scheme for vitronectin that invoked chemical denaturation of bulk plasma prior to chromatography on heparin-agarose. As early as 1984, a model emerged in which the heparin-binding region is buried within the native molecule, so that heparin binding is induced when this cryptic binding site is exposed upon denaturation of vitronectin (11,16). This model has been generally accepted, and more recent work has included attempts to correlate the heparin binding properties of native and denatured/renatured vitronectin with other functions of the protein (12, 18 -27). The two forms of the protein, which are sometimes denoted the "non-heparin-binding" (native) and "heparin-binding" (denatured/renatured) species, are characterized in much of the ongoing work in the field.Many years after the initial work examining effects of denaturation on vitronectin, it was recognized that denaturation is accompanied by self-association of the protein into a multimeric form (28,29). Multimerization of vitronectin ensues following unfolding of the protein to a structurally altered form that can be detected with a variety of conformationally sensitive monoclonal antibodies (29 -31). This partially folded intermediate form has a high propensity to self-associate int...

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

“…The concentrations of rVN and r⌬sBVN needed to inhibit heparin activity by 50% were 0.04 and 0.2 M, respectively. The corresponding value for multimeric vitronectin was 0.16 M, consistent with previous measurements using this assay (41,50). The K app of 0.04 M for rVN was comparable to other recombinant forms of vitronectin previously expressed in the baculovirus system, which demonstrated 50% inhibition at 0.05 M (41).…”

“…Binding of both the recombinant forms of vitronectin is similar to that of multimeric and much greater than that of native vitronectin, demonstrating the increased binding of the multimeric forms of vitronectin to heparin. This difference in the heparin-binding ability of monomeric and multimeric vitronectin has been attributed to the multivalent binding capacity of the multimeric form of vitronectin (50,51).…”

Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin

“…Furthermore, we cannot elucidate the mechanisms regulating the binding preferences, or the communication between sites from these data alone. An estimate of the average binding affinity, K 0.5 in the Hill equation, using these data gives a value of 1.5 nM, consistent with previously reported high affinity measurements for PAI-1-vitronectin binding interactions that were derived using an oversimplified 1:1 binding site model (25,31,(33)(34)(35)(36)(37). Although not equivalent to a true binding constant, this value of K 0.5 reflects a high affinity interaction that is in the range to give saturable binding of approximately nanomolar concentrations of PAI-1 under the conditions of this experiment, in which the vitronectin concentration was fixed at 0.5 nM.…”

“…The reported M r of circulating PAI-1⅐vitronectin complexes (19,20) is consistent with our results and raises the possibility that the complex, rather than the individual proteins, interacts with other macromolecules. Supporting this concept are studies demonstrating that vitronectin can bind PAI-1 and heparin simultaneously, indicating that PAI-1⅐vitronectin complexes can interact with other molecules (37).…”

Orientation of Heparin-binding Sites in Native Vitronectin

“…It was originally assumed that the heparin-binding sequence was encrypted in the native fold of the molecule (19), but more recent work from this laboratory has demonstrated that the heparinbinding site is fully exposed in native vitronectin (23). Confusion over the degree of exposure of sites within the conformationally labile protein had stemmed primarily from the application of varied preparation protocols that produce vitronectin forms that differ in oligomeric state.…”