Detection of vitronectin by ligand blotting with type 1 plasminogen activator inhibitor

Ligand blotting procedures were developed for the detection of type 1 plasminogen activator inhibitor (PAI-1) binding protein(s) (BPs) transferred to nitrocellulose sheets after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purified vitronectin (Vn), a well characterised PAI-1 BP, was employed to optimise this assay system. After blocking with casein, the sheets were washed and then incubated with either ¹²⁵I-labelled PAI-1 (direct assay), or with unlabelled PAI-1 followed by a polyclonal antiserum to PAI-1 (indirect assay). In the latter case, the bound antibody was detected by using an ¹²⁵I-labelled second antibody. Binding was dose-dependent with respect to both Vn and PAI-1, and only active PAI-1 bound to Vn (i.e. latent PAI-1 and PAI-1 in complex with tissue-type plasminogen activator (t-PA), did not bind to Vn in this system). Moreover, t-PA, arginine and acidic conditions dissociated PAI-1 from Vn adsorbed to nitrocellulose. Analysis of bovine plasma by these techniques revealed the presence of a single PAI-I BP, and this protein was recognised by antisera to Vn. These results indicate that Vn previously fractionated by SDS-PAGE and transferred to nitrocellulose, continues to bind to PAI-I in a manner that resembles its behaviour in plasma and on extracellular matrix. These ligand blotting procedures may thus represent useful new approaches for the detection of other SDS-stable PAI-1 binding proteins in biological samples.