KINETIC-ANALYSIS OF THE INTERACTION BETWEEN TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR AND VITRONECTIN AND EVIDENCE THAT THE BOVINE INHIBITOR BINDS TO A THROMBIN-DERIVED AMINO-TERMINAL FRAGMENT OF BOVINE VITRONECTIN

The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (K(d)) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4-degrees-C (T1/2 > 24 h), but dissociated with a T1/2 of 4 h at 37-degrees-C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of M(r) 60 000, 54 000 and 38 000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (M(r) 38 000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule.