A new approach to vitronectin research : using molecular biology and biophysical chemistry to elucidate the contributions of the C-terminal domain of vitronectin to heparin binding, PAI-1 binding, and vitronectin self-association

Vitronectin is a multifunctional plasma protein that plays a regulatory role in humoral defense reactions that include coagulation and fibrinolysis. Circulating vitronectin is a mixture of a single-chain form and a two-chain form that is derived from proteolytic cleavage at position 379, near the C-terminus, just beyond a sequence that plays a major role in vitronectin function. This sequence, which spans residues 340-379, is reported to be the primary binding site for a number of ligands, including the anticoagulant drug, heparin, and the antifibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). However, binding sites for these ligands have also been located close to the N-terminus in vitronectin. The C-terminal domain has also been hypothesized to play a key role in stabilizing the multimeric form of vitronectin that endows it with "Velcro-" like interactions with target ligands. Most researchers assume that there are no major functional differences between cleaved and uncleaved vitronectin, but potential changes have not been previously investigated.

The purpose of this study was to use techniques of molecular biology and biophysical chemistry to elucidate the role of the C-terminal domain in heparin binding, PAI-1 binding, and in multimerization, paying close attention to functional differences that result from proteolytic processing at position 379. A T7 expression system was used to generate a recombinant C-terminal domain in Escherichia coli. The baculovirus system was also used to produce recombinant full-length vitronectin and a truncated form of vitronectin (residues 1-380), which represents the large fragment of two-chain vitronectin. Kinetic, immunologic and fluorescence assays were used to characterize the fold and function of the recombinant proteins. Also, fluorescently labeled vitronectin and PAI-1 were prepared, and the fluorescence of the two molecules was measured to evaluate conformational changes that occur as the two molecules interact.

Results of these studies indicate that the entire binding site for heparin is contained between residues 331-380 of vitronectin. PAI- binding alters the conformation of the heparin-binding site, such that the fluorescence of a probe attached to arginines in the site is quenched upon binding. The binding sites for heparin and PAI-1 are distinct, so vitronectin can bind both molecules simultaneously. There are no functional differences between full-length and truncated vitronectin, with respect to heparin binding or PAl-1 binding, indicating that proteolytic processing at position 379 in vitronectin is not a regulatory event for controlling coagulation or the plasminogen activation system. Furthermore both forms of the molecule self-associate, suggesting that the C-terminal 80 amino acids are not needed for adopting the multimeric conformation that stabilizes vitronectin-binding events via multivalent interactions.