A Truncated Plasminogen Activator Inhibitor-1 Protein Induces and Inhibits Angiostatin (Kringles 1-3), a Plasminogen Cleavage Product

Abstract

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80-265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-123, with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-123 inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-123 exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-123 have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-123 interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-123 cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-1 23 can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.