THE SOMATOMEDIN-B DOMAIN OF VITRONECTIN - STRUCTURAL REQUIREMENTS FORTHE BINDING AND STABILIZATION OF ACTIVE TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR

We recently localized the high affinity binding site for activated typ e plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e. amino acid (aa) 1-51) of vitronectin (Vn). In this study to furt her define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity. V n polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding acti vity and stabilized PAI-1 to a similar extent as intact Vn, but polype ptides containing aa 1-30 did not bind to PAI-1 nor stabilize its acti vity. The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 bindi ng was also determined. One mAb bound to Vn and blocked its ability to bind to PAI-1. It also dissociated pre-existing PAI-1.Vn complexes, a nd prevented the incorporation of PAI-1 into extracellular matrix of H T 1080 cells. This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30. A second randomly chosen mAb with similar affinity for Vn was inactive in these assays a nd bound to the region between aa 52 and 239. These results indicate t hat the high affinity binding site for active PAI-1 in Vn is between a a 1 and 40, and that this domain may also stabilize active PAI-1.