Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Plasminogen activator inhibitor-1 (PAI-1) mechanism and approaches to modulating PAI-1 activity in vivo. The PAI-1 reaction includes inhibitory (ki), substrate (ks), and latent (klat) branches, which result in a loss of activity and formation of a stable inhibitory complex (E-PAI-1), cleaved serpin (PAI-1*), and latent serpin (PAI-1lat), respectively. PAI-1 in an active conformation (PAI-1) complexed with endogenous vitronectin (Vn) in the pleural space interacts with the plasminogen activator [wild-type urokinase (wt-uPA) or urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA); enzyme, E] and forms a transient Michaelis complex (E·PAI-1·Vn). Once the enzyme cleaves PAI-1 and forms an acyl-enzyme, conformational changes in the serpin result in its stabilization in an inhibitory complex (E-PAI-1). Under physiological conditions, >90% of the PAI-1 reaction follows the inhibitory branch (ki), resulting in mutual, stoichiometric inhibition of the enzyme and PAI-1. Active PAI-1 can also slowly, spontaneously transform (klat) into an inactive, latent conformation (PAI-1lat), losing the ability to bind Vn. The substrate branch (ks) yields an inactive, cleaved serpin (PAI-1*) and an active enzyme. The PAI-1 reaction was modulated by three distinct mechanisms (mechanisms I–III). In mechanism I, alanine mutations of positively charged residues in the 37-loop of uPA result in ΔDS-uPA, which interacts with PAI-1 via the inhibitory branch (ki) considerably more slowly than the wild-type enzyme. In mechanism II, ligands (L), such as inactive uPA with Ser195Ala substitution (S195A-uPA) or monoclonal antibody MA-56A7C10, compete with uPA and bind PAI-1/Vn with nanomolar affinity [Kd = koff/kon, where Kd is the dissociation constant and koff and kon are the first-order rate constants of dissociation and association, respectively, of PAI-1/S195A-two-chain uPA (tcuPA) or MA-56A7C10] forming nonproductive “molecular sandwich”-type complexes (L·PAI-1·Vn), which compete with the Michaelis complex for uPA (Kd << Km). L·PAI-1·Vn also stabilizes the active conformation of PAI-1, inhibiting the latent branch (klat). The rate of enzyme inactivation by L·PAI-1·Vn becomes limited by a low koff. In mechanism III, monoclonal antibody MA-33B8 accelerates the transition of active PAI-1 or its complex with Vn to inactive PAI-1 (PAI-1lat).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, In Vivo, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Effects of a 179RHRGGS184→179AAAAAA184 urokinase mutant (ΔDS-uPA) on the rate of interaction with plasminogen activator inhibitor-1 (PAI-1) and activation of Glu-plasminogen. A: dependence of the observed first-order rate constants (kobs) for the interaction of PAI-1 with wild-type urokinase (wt-uPA; ○) and ΔDS-uPA (△) on enzyme concentration. The data for wt- and ΔDS-uPA are shown in different scales. The values of kobs were determined as previously described (45, 49, 51). Linear equations were fit (solid lines; r2 > 0.95) to the data, and values of the second-order association rate constant (kass) were determined from the slopes. The ratio of kass for wt-uPA over ΔDS-uPA was 63.0 for inhibition by PAI-1. B: dependence of the rates of accumulation of plasmin due to activation of Glu-plasminogen by wt-uPA (○) and ΔDS-uPA (△) on enzyme concentration. The rates of plasmin accumulation were determined from the slopes of linear equations, which were fit (solid lines; r2 > 0.99) to the data as described previously (49). The ratio of slopes for wt-uPA over ΔDS-uPA for activation of Glu-plasminogen was 1.5. AU, arbitrary units.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Mutagenesis, Activation Assay, Concentration Assay, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Accumulation of intrapleural α-macroglobulin (αM)/urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA) “molecular cage” complexes during intrapleural fibrinolytic therapy (IPFT). A: time dependence of the formation of intrapleural αM/ΔDS-uPA during IPFT with ΔDS-scuPA (0.0625 mg/kg). ΔDS-uPA amidolytic activity was measured after samples of pleural fluid withdrawn at 0–40 min were supplemented with an excess (100–200 nM) of exogenous recombinant human plasminogen activator inhibitor-1 (PAI-1) to inhibit free enzyme. PAI-1-resistant ΔDS-uPA amidolytic activity, which represents intrapleural ΔDS-uPA in “molecular cage” complexes with αM (46, 50), was converted to concentrations (nM) and plotted against time. A single exponential equation was fit to the dependence of the concentration of αM/ΔDS-uPA (A) or αM/uPA (not shown) on time to obtain the values of the apparent first-order rate constants (kapp; B) as previously described (46).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Recombinant, Concentration Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Intrapleural plasminogen activator inhibitor-1 (PAI-1)-independent inactivation of free urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA) is twofold faster than wild-type urokinase (wt-uPA). A: time dependence of the amidolytic activity of intrapleural free uPA (△) during intrapleural fibrinolytic therapy (IPFT) with ΔDS-prourokinase (ΔDS-scuPA; 0.0625 mg/kg). Briefly, the total ΔDS-uPA amidolytic activity was measured in samples of pleural fluid withdrawn at 10–40 min after IPFT. ΔDS-uPA activity that is resistant to an excess of exogenous human recombinant PAI-1 [represents α-macroglobulin (αM)/ΔDS-uPA complexes] was subtracted from total amidolytic activity to estimate the level of free ΔDS-uPA in the sample ([free ΔDS-uPA] = [total ΔDS-uPA] − [αM/ΔDS-uPA]). B: time dependence of free (not complexed) intrapleural uPA amidolytic activity (○) during IPFT with wt-scuPA (0.0625 mg/kg). Amidolytic activity of free uPA was the difference between total uPA activity and activity of αM/uPA complexes. C: observed first-order rate constants (kobs) for the intrapleural inactivation of free ΔDS- and wt-uPA. Values of kobs are given for loss of intrapleural amidolytic (▽) and plasminogen-activating (△) activities of ΔDS-uPA, as well as the plasminogen-activating activity of wt-uPA (○) during IPFT with 0.0625 mg/kg (n = 6). Values of kobs were estimated from the changes in activity with respect to time, as described previously (42, 49). A single exponential equation was fit to the dependence of [free enzyme] on time to obtain kobs of PAI-1-independent inactivation of ΔDS- and wt-uPA as previously described (46). The rate of intrapleural inactivation of ΔDS-uPA was statistically (P < 0.05) higher than that for wt-uPA. There was no statistically significant difference (P > 0.05) between the kobs of ΔDS-uPA inactivation estimated from measurements of amidolytic and Glu-plasminogen-activating activities.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Recombinant

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.5 mg/kg) does not affect the rate of intrapleural inactivation of urokinase (uPA). A: time dependence of the amidolytic activity of intrapleural, free uPA during intrapleural fibrinolytic therapy (IPFT) with 0.5 mg/kg of S195A-tcuPA with (●; n = 5) and without (■; n = 3) 0.25 mg/kg of scuPA. The amidolytic activity of free uPA was determined in samples of pleural fluid withdrawn at 10–40 min after IPFT. The amidolytic activity of free uPA was calculated as the difference between total uPA activity and activity attributed to α-macroglobulin (αM)/uPA complexes, as previously described (46). There was a statistically significant difference (P < 0.05) between free uPA activity in animals treated with (○) and without (□) scuPA (0.25 mg/kg). B: time dependence of the amidolytic activity of free uPA during IPFT with scuPA (0.25 mg/kg, ○; n = 5) and vehicle control (PBS, □; n = 3). The amidolytic activity of free uPA was the difference between total uPA activity and activity of αM/uPA complexes. There was a statistically significant difference (P < 0.05) between free uPA activity in animals treated with (○) and without (□) scuPA (0.25 mg/kg). C: observed first-order rate constants (kobs) for the intrapleural inactivation of uPA with (●; n = 5) and without (○; n = 5) S195A-tcuPA (0.5 mg/kg). A single exponential equation was fit to the dependence of [free uPA] with respect to time to obtain the kobs of PAI-1-independent inactivation of uPA as previously described (46). There was a statistically significant difference (P < 0.05) between kobs in animals treated with (○) and without (□) scuPA (0.25 mg/kg). There was no statistically significant difference (P > 0.05) between the kobs observed for IPFT with scuPA (0.25 mg/kg) with (●) or without (○) S195A-tcuPA (0.5 mg/kg).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Effect of two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.5 mg/kg) on the accumulation of α-macroglobulin (αM)/urokinase (uPA) complexes during intrapleural fibrinolytic therapy (IPFT) with 0.25 mg/kg prourokinase (scuPA). A: time dependence of intrapleural levels of αM/uPA during IPFT with 0.5 mg/kg of S195A-tcuPA, with (●) and without (■) scuPA 0.25 mg/kg. Samples of pleural fluid withdrawn at 0–40 min after IPFT were supplemented with 100–200 nM of exogenous recombinant human plasminogen activator inhibitor-1 (PAI-1), and the amidolytic activity of uPA was measured as previously described (46). There was a statistically significant difference (P < 0.05) between αM/uPA in animals treated with S195A-tcuPA/scuPA and with S195A-tcuPA alone at 10, 20, and 40 min. B: values of the apparent first-order rate constants (kapp) for intrapleural accumulation of αM/uPA with (solid symbols) or without (open symbols) 0.5 mg/kg of S195A-tcuPA. A single exponential equation was fit to the dependence of [αM/uPA] for treatments with (A) or without (not shown) S195A-tcuPA on time as previously described (46). There was no statistically significant difference between the rates of accumulation of αM/uPA during IPFT with 0.25 mg/kg scuPA alone or in the presence of S195A-tcuPA (P > 0.05). C: levels of intrapleural αM/uPA “molecular cage”-type complexes at 24 h after IPFT with 0.25 mg/kg scuPA with (●) or without (○) S195A-tcuPA (0.5 mg/kg). Briefly, the amidolytic activity of uPA was measured after supplementation of samples of pleural fluid withdrawn at 24 h after IPFT with an excess (20–40 nM) of exogenous recombinant human PAI-1. There was no statistically significant difference between [αM/uPA] observed for treatment with 0.25 mg/kg scuPA with (●) or without (○) S195A-tcuPA (0.5 mg/kg; P > 0.05).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Recombinant, Activity Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Accumulation of active plasminogen activator inhibitor-1 (PAI-1) in pleural fluids 24 h after intrapleural fibrinolytic therapy (IPFT). A: changes in the activity of exogenous two-chain urokinase (tcuPA; 0.5 nM) added to the samples of pleural fluid collected 24 h after IPFT with 0.25 mg/kg prourokinase (scuPA) and MA-56A7C10 (dashed line) or MA-33B8 (dotted line). Thin, solid lines represent the best fit of a single exponential [first-order rate constant of intrapleural uPA inactivation (kobs) = 4.5 × 10−3 min−1] and linear equation to the data, respectively. The levels of PAI-1 activity in the pleural fluid are shown in the Table 1. B: Western blot analysis of PAI-1 complexed with MA-56A7C10 and tcuPA with Ser195Ala substitution (S195A-tcuPA) isolated from pleural fluids of animals by immunoprecipitation. Complexes of endogenous active PAI-1 were precipitated with magnetic beads (Dynabeads M-280 with sheep anti-mouse IgG; Invitrogen by Thermo Fisher Scientific) per the manufacturer’s protocol, as described in experimental procedures. Western blot analysis detected rabbit PAI-1 in the precipitates obtained from pleural fluids of animals treated with MA-56A7C10 and scuPA (0.5 and 0.25 mg/kg, respectively; lane 4) or S195A-tcuPA (0.5 mg/kg; lane 2), but not in pleural fluids of animals treated with mouse IgG (0.5 mg/kg) or vehicle control [Dulbecco’s phosphate-buffered saline (DPBS); lanes 3 and 1, respectively]. Two pairs of lanes (lanes 1–4) represent parts of the same gel. The positioning of molecular weight markers is shown at right. Treatments are described in the table above the Western blot image. Bands on the Western blot other than rabbit PAI-1, which are present in every lane, represent nonspecific binding of secondary antibodies. *Pleural fluids of rabbits treated with DPBS and S195A-tcuPA (lanes 1 and 2, respectively) were supplemented with anti-human uPA monoclonal antibody (4–8 µg), 10 min before addition of magnetic beads. C: PAI-1 activity was precipitated by sheep anti-mouse IgG magnetic beads from pleural fluids of animals treated with MA-56A7C10 in combination with scuPA (0.5 and 0.25 mg/kg, respectively) and with S195A-tcuPA (0.5 mg/kg), but not from pleural fluids of animals treated with mouse IgG alone (0.5 mg/kg) or vehicle control. PAI-1 activity was measured by incubating an aliquot (5–10 µl) of the bead slurry with 0.2 nM uPA and fluorogenic substrate in DPBS with BSA (1 mg/ml). Amidolytic activity of uPA was measured using fluorogenic substrate as described in experimental procedures and elsewhere (42). Relative levels of active PAI-1 bound to the resin were estimated from decreases in the uPA activity. The levels of active PAI-1 (on average, 2 independent experiments) in pleural fluids were expressed in arbitrary units (AU).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Western Blot, Isolation, Immunoprecipitation, Magnetic Beads, Molecular Weight, Binding Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Changing plasminogen activator inhibitor-1 (PAI-1) activity affects intrapleural fibrinolytic therapy (IPFT) outcomes under conditions of slow fibrinolysis in the pleural space. Successful IPFT in tetracycline (TCN)-induced pleural injury in rabbits requires maintaining plasminogen-activating activity for 4–8 h (48). The minimal time necessary for effective fibrinolysis (4–8 h) is shown as a yellow zone between effective (green zone; >8 h) and ineffective (red zone; <4 h) IPFT outcomes. The rate of PAI-1-independent inactivation of uPA remains the same with different doses of prourokinase (scuPA; 46), shown as parallel solid lines, with the minimal effective dose in the middle. Fibrinolysis stops as soon as endogenous PAI-1 (black dashed line) inhibits the plasminogen activator(s) present (intercept of solid and dashed lines), which in turn determines outcomes for effective (yellow and green zones) and ineffective (red zone) IPFT. Neutralizing PAI-1 (blue arrow) decreases PAI-1 activity (blue dotted line). Consequently, an otherwise ineffective dose of scuPA (the lowest solid line) provides positive plasminogen-activating activity for >8 h (the intercept with the blue dotted line in the green zone), representing the increased efficacy of IPFT (decreasing the minimal effective dose). On the other hand, increasing the PAI-1 activity in the pleural space (red arrow) results in faster inhibition of intrapleural plasminogen activator (the intercept with the red dotted line in the red zone) and in ineffective IPFT with doses of scuPA that are normally effective. The efficacy of IPFT in tetracycline (TCN)-induced pleural injury was increased when PAI-1 was neutralized with MA-33B8 [Table 1; gross lung injury score (GLIS) = 3] or with monoclonal antibodies that redirect the PAI-1 mechanism toward the substrate branch (Fig. 1, ks; 25). The adverse effects of increased PAI-1 were observed during IPFT in the presence of MA-56A7C10 (Table 1; GLIS = 50), in animals subjected to serial computed chest tomography (48) and in rabbits with infectious pleural injury (empyema; 47). AU, arbitrary units; FT, fibrinolytic therapy.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Inhibition, Tomography

Journal: Marine Drugs

Article Title: Undaria pinnatifida and Fucoxanthin Ameliorate Lipogenesis and Markers of Both Inflammation and Cardiovascular Dysfunction in an Animal Model of Diet-Induced Obesity

doi: 10.3390/md14080148

Figure Lengend Snippet: Effect of brown algae Undaria pinnatifida and fucoxanthin on ( A ) adiponectin (μg/mL); ( B ) leptin (ng/mL); ( C ) C reactive protein (μg/mL); ( D ) PAI-1 (pg/mL) measured on blood serum. The groups are abbreviated as: standard diet (SD); high-fat diet (HF); HF + Undaria pinnatifida (UP); HF + Fucoxanthin (FU). Values are shown as mean ± SE, ( n = 6 per group). Data was compared by one-way ANOVA with Holm-Sidak test for multiple comparisons. Means with different superscripts are significantly different ( p < 0.05).

Article Snippet: Quantitative estimations of leptin (cat. EZRL-83K; Millipore, St. Charles, MO, USA), adiponectin (cat. EZRADP-62K, Millipore, MO, USA), C-reactive protein (cat. CYT294; Millipore, St. Charles, MO, USA) and PAI-1 serum levels (cat. Ab198509, Abcam, Cambrige, UK) were performed following the manufacture’s protocols using specific ELISA kits.

Techniques: