A truncated plasminogen activator inhibitor-1 protein induces and inhibitsangiostatin (kringles 1-3), a plasminogen cleavage product

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor th at binds plasminogen activators (uPA and tPA) at a reactive center loop loc ated at the carboxyl-terminal amino acid residues 320-351. The loop is stre tched across the top of the active PAI-1 protein maintaining the molecule i n a rigid conformation, In the latent PAI-1 conformation, the reactive cent er loop is inserted into one of the beta sheets, thus making the reactive c enter 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 bin ding site. The region we maintained corresponds to amino acids 80-265 of ma ture human PAI-1 containing binding sites for vitronectin, heparin (partial ), up, tPA, fibrin, thrombin, and the helix F region. The interaction of "i nactive" PAI-1, rPAI-1(23), with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties, Increasing amounts of rPAI-1(23) inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1(23) exhibit reduced proliferation, reduced tub e formation, and 47% apoptotic cells within 48 h, Transfected endothelial c ells secreting rPAI-1(23) have a 30% reduction in proliferation, vastly red uced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1(23) interactions with uPA and p lasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1( 23) cleaves plasmin to form a proteolytic ansostatin-like protein. In a sec ond mechanism, rPAI-1(23) can bind uPA and/or plasminogen to reduce the num ber of uPA and plasminogen interactions, hence reducing the amount of plasm in that is produced.