Development and characterization of a tPA-hepatic transfection model that promotes chronic hyperfibrinolytic state in mice. (A) Schematic representations of the generated pLIVE-tPA constructs, including the complete tPA sequence (5 domains: Finger [F], epithelial growth factor [EGF], kringle 1 and 2 [K1, K2], and serine protease [SP]). tPA-GFP and tPA-Luc plasmids contain the whole tPA sequence with the fusion proteins green fluorescent protein (GFP) and luciferase (Luc), respectively. (B) Schematic representation of the experimental timeline including in vitro (zymography), in vivo (bioluminescence), and ex vivo (immunofluorescence [IF] and NIRF) experiments. (C) Fluorescence images of pLIVE-tPA-GFP-transfected animals showing tPA overexpression (GFP labeling) in the liver, and not the brain, at 48 hours after transfection. (D) Fibrin-agar zymography of liver homogenates of pLIVE-0- and pLIVE-tPA-transfected animals. (E) In vivo luminescence in pLIVE-tPA-Luc-transfected mice after intraperitoneal coelenterazine (luciferase substrate) injection (48 hours after transfection). (F) Representative ex vivo images of the liver and brain from pLIVE-0- and pLIVE-tPA-Luc-transfected mice, revealing significant luminescence only in the liver of pLIVE-tPA-Luc-transfected mice. (G) Fibrin-agar zymography from plasma samples of pLIVE-0- and pLIVE-tPA-transfected mice before and 24 hours after hepatic transfection. Only pLIVE-tPA mice presented significant levels of free circulating tPA. (H) Representative fibrin-agar zymography of a pLIVE-tPA-transfected mice plasma, which demonstrates the presence of free plasmatic tPA several days after the transfection. 4′,6-diamidino-2-phenylindole (DAPI, blue) was used as a nuclear marker; tPA, urokinase plasminogen activator (uPA), and Pln (plasmin) were used as standards in the zymographies. All the images presented are representative of at least 5 biological replicates of the experiment.