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-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 tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1(23) 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-1(23) interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1(23) 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.
Highlights
Plasmin, the end product of plasminogen activator-derived proteolysis, is a trypsin-like enzyme that functions in the degradation of fibrin and extracellular matrix (ECM)1 proteins
Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators at a reactive center loop located at the carboxyl-terminal amino acid residues 320 –351
The data we present show that when a truncated PAI-1 protein, rPAI-123, complexes with plasminogen or plasminogen and urokinase plasminogen activator (uPA), the following results occur: 1) Plasmin is cleaved to produce a proteolytic angiostatin-like cleavage product
Summary
The end product of plasminogen activator-derived proteolysis, is a trypsin-like enzyme that functions in the degradation of fibrin and extracellular matrix (ECM) proteins. PAI-1 is a secreted protein that is rapidly converted to a latent form at physiological temperatures It becomes stabilized and activated when it binds vitronectin (Vn) [5,6,7], a cell adhesion glycoprotein [6, 8] found primarily in the ECM at acute injury, in tissue repair [9], and in some malignant tumors [10]. The serine protease activity of plasmin degrades Vn such that one of the PAI-1 binding sites is mostly lost This results in destabilization of the Vn1⁄7PAI-1 complex and a loss of PAI-1 function as an inhibitor of plasmin formation through the plasminogen activator pathway. This same PAI-1 binding site on Vn overlaps a heparin binding site. Variations in Vn conformational states can convert PAI-1 inhibitory specific-
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