Characterization of a complex between active plasminogen activator inhibitor-1 and N-terminal fragments of vitronectin from human placenta

Abstract

Abstract Two different forms of active plasminogen activator inhibitor-1 (PAI-1) were purified from human placenta and characterized. One form with Mr>230 000 was identical to the complex between PAI-1 and multimeric vitronectin in plasma as evidenced by gel filtration and immunological analysis. The other form was a complex between PAI-1 and N-terminal fragments of vitronectin as demonstrated by a combination of amino acid sequence analysis and mass spectrometry of polypeptides obtained from the purified complex after reverse phase high pressure liquid chromatography (HPLC). The vitronectin fragments were 42 to 67 amino acids long, the dominant fragment being residues 1–49. This fragment of vitronectin encompasses the binding sites for both PAI-1, the urokinase receptor (uPAR) and integrins. Consistent with the primary binding site for PAI-1 being localized to the N-terminal domain of vitronectin, the complex between PAI-1 and N-terminal vitronectin fragments did not bind intact vitronectin. PAI-1 bound to N-terminal vitronectin fragments or to multimeric vitronectin showed the same functional stability and comparable second order rate constants for the inhibition of single-chain tissue-type plasminogen activator (tPA). The proteolytic cleavages producing the complex between PAI-1 and N-terminal vitronectin fragments may take place in vivo and lead to modulation of thrombotic and tissue remodelling processes.