title: Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.
abstract: BACKGROUND
  Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state.
  
  
  RESULTS
  Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin.
  
  
  CONCLUSIONS
  The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.
authors.affiliation: Department of Medicine, New York Medical College, Valhalla, NY 11595, USA.
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Brodsky
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Malinowski
authors.forename: Marc
authors.initials: M
authors.lastname: Golightly
authors.forename: Jolyon
authors.initials: J
authors.lastname: Jesty
authors.forename: Michael S
authors.initials: MS
authors.lastname: Goligorsky
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010316:Particle Size; D010743:Phospholipids / Q000096:biosynthesis; D017395:Plasminogen Activator Inhibitor 1 / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology*; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D019030:Receptors, Vitronectin / Q000378:metabolism; D013917:Thrombin / Q000096:biosynthesis; D013927:Thrombosis / Q000209:etiology*
chemical_list: D000906:Antibodies; C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D017077:Culture Media, Conditioned; D046911:Macromolecular Substances; C524898:PLAUR protein, human; D010743:Phospholipids; D017395:Plasminogen Activator Inhibitor 1; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D019030:Receptors, Vitronectin; D013917:Thrombin