New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1 vitronectin complex

Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that t he physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin, The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation . Although the complex is clearly significant, information is vague regardi ng the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo, Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstr ated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin, T o address this issue, competition studies using monoclonal antibodies speci fic for separate epitopes confirmed that the two distinct PAI-1-binding sit es present on vitronectin can be occupied simultaneously. Analytical ultrac entrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predomin ant associating species observed was high in molecular weight (M-r similar to 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1, Moreover, the size of this higher order complex in dicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is prop osed to facilitate interaction with macromolecules on surfaces.