title: TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells.
abstract: BACKGROUND
  Elevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques.
  
  
  METHODS
  The effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy.
  
  
  RESULTS
  TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells.
  
  
  CONCLUSIONS
  The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.
authors.affiliation: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan. yassy19781119@yahoo.co.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Yasui
authors.forename: Yuko
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Hideto
authors.initials: H
authors.lastname: Sano
authors.forename: Takafumi
authors.initials: T
authors.lastname: Suda
authors.forename: Kingo
authors.initials: K
authors.lastname: Chida
authors.forename: Takashi
authors.initials: T
authors.lastname: Dan
authors.forename: Toshio
authors.initials: T
authors.lastname: Miyata
authors.forename: Tetsumei
authors.initials: T
authors.lastname: Urano
mesh_terms: D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D010879:Piperazines / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D062366:para-Aminobenzoates / Q000494:pharmacology*
chemical_list: C549556:5-chloro-2-(((2-(4-(diphenylmethyl)piperazin-1-yl)-2-oxoethoxy)acetyl)amino)benzoate; D005343:Fibrinolytic Agents; D010879:Piperazines; D017395:Plasminogen Activator Inhibitor 1; D062366:para-Aminobenzoates; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin
keywords: CLSM; HBS; HEPES-buffered solution; PAI-1; TIRF; TM5275; VEC; confocal laser scanning microscope; fibrinolysis; plasminogen activator inhibitor-1; plasminogen activator inhibitor-1 (PAI-1); plasminogen activator inhibitor-1 (PAI-1) inhibitor; tPA; tissue plasminogen activator; total internal reflection fluorescence; total internal reflection fluorescence (TIRF) microscopy; vascular endothelial cell