THE CLUSTER OF BASIC-AMINO-ACIDS IN VITRONECTIN CONTRIBUTES TO ITS BINDING OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 - EVIDENCE FROM THROMBIN-CLEAVED, ELASTASE-CLEAVED AND PLASMIN-CLEAVED VITRONECTINS AND ANTIPEPTIDE ANTIBODIES

Derivatives of vitronectin obtained by specific cleavage at its cluste r of basic amino acids with thrombin, elastase and plasmin are shown t o have a decreased ability to bind plasminogen activator inhibitor-1 ( PAI-I). The identification and localization of the segment involved in the binding of PAI-1 (Lys(348)-Arg(379)) were carried out by purifica tion of these cleaved vitronectins and their subsequent structural cha racterization (sequence analysis, phosphorylation of Ser(378) with cAM P-dependent protein kinase and immunostaining with peptide-specific an tibodies), then measurement of the vitronectin-PAI-l interaction by (a ) a two-phase system (ELISA); (b) co-precipitation of the vitronectin- PAI-1 complex out of solution, and (c) analysis of the stereospecific interaction between the active conformation of PAI-1 and a peptide der ived from the above-mentioned cluster; this interaction occurs when th e peptide is composed of all-L-amino acids but not when it is composed of all-D-amino acids. Our results explain why workers who have used i mmobilized vitronectin to study this interaction could not have observ ed the involvement of the cluster of basic amino acids in PAI-1 bindin g, since the immobilization of vitronectin is shown to render this clu ster inaccessible for interaction. We propose that vitronectin binds a ctive PAI-I by interaction via amino acid residues that originate from distal locations in the N- and C-termini of vitronectin.