A truncated plasminogen activator inhibitor-1 protein induces and inhibits angiostatin (kringles 1-3), a plasminogen cleavage product.
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.
Mulligan-Kehoe
M J
MJ
Division of Vascular Surgery, Department of Surgery, Dartmouth Medical School, Dartmouth College, Hanover, New Hampshire 03756, USA. mary.j.mulligan-kehoe@dartmouth.edu
Wagner
R
R
Wieland
C
C
Powell
R
R
D043168:Angiostatins; D000818:Animals; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors* / Q000502:physiology*; D010958:Plasminogen / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019096:Vitronectin; D043168:Angiostatins; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator