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 the 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 regarding 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 demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M r ϳ 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.Vitronectin is a versatile glycoprotein that is found in circulation, in the extracellular matrix of endothelial cells, in platelets, and within various tissues of the human body. Circulating at micromolar levels, vitronectin participates in the regulation of humoral responses such as coagulation, fibrinolysis, and the complement cascade (reviewed in Ref. [1][2][3][4]. Other functions of the protein that are confined to surfaces or tissues include cell-adhesion and regulation of pericellular proteolysis. Interactions with an assortment of biological molecules are responsible for the multiple functions exhibited by vitronectin. Defining the binding sites for these various biomolecules, along with determining the molecular mechanism of regulation, constitutes an active area of research on the protein. Work to date has focused on binding sites for several ligands, including heparin, PAI-1, 1 and integrins; a working model representing the domain structure of vitronectin has been recently reported from this laboratory (5).Arguably, one of the most important interactions known for vitronectin occurs with the serine protease inhibitor, PAI-1. PAI-1 is the physiological inhibitor of plasminogen activators, both tPA and uPA, the enzymes responsible for generating plasmin from...