Therefore, PAI-1 targeting represents a promising approach to the development of reliable, safe, and efficacious IPFT amenable to testing in clinical trials. Footnotes Author Contributions: conception and designA.A.K. and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and M antigens. Targeting PAI-1 did not induce bleeding, Lisinopril and rendered otherwise ineffective doses of scuPA able to improve outcomes in tetracycline-induced pleural injury. PAI-1Cneutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development. and testing against rabbit PAI-1, and used as adjuncts in combination with scuPA IPFT. As a result, the minimal effective dose of scuPA was decreased eightfold: from 0.5 mg/kg (24) to 0.0625 mg/kg. Open in a separate window Figure 1. Protection of urokinase (uPA) (E) from inactivation by endogenous plasminogen activator inhibitor (PAI)-1 (I) by monoclonal antibody (mAb)-mediated redirection of the mechanism from the inhibitory (ki) to the substrate (ks) branch. An enzyme (the online supplement) (28, 29) was used in these analyses. SigmaPlot 12.0 (SPSS Inc., San Jose, CA) was used to calculate the values of area under the curve (AUC) for fibrinolytic activity analyses. Data Analysis and Statistics Levels of statistical significance were determined using Kruskal-Wallis one-way ANOVA on ranks and pairwise multiple comparison procedures (Holm-Sidak method and Turkey test). Data analysis was performed using SigmaPlot 12.0 for Windows, as previously described (25). Correlation coefficients (experiments (Figures E1A and E1B in the online supplement) demonstrated the additivity of the neutralizing effects of MA-33H1F7 and MA-8H9D4 (30) on the reaction between recombinant rabbit PAI-1 and human uPA. Although the affinity of MA-33H1F7 to rabbit PAI-1 was reported to be decreased due to a single amino acid substitution in the epitope (31), results of experiments (Figure E1C) have shown directly that mAbs added to the PFs of rabbits with TCN-induced pleural injury protect exogenous uPA from inactivation. Half (0.25 mg/kg) of the effective dose of scuPA (24) was initially selected to test whether or not adjunctive PAI-1Cneutralizing Lisinopril mAbs (0.5 mg/kg) affect the outcome of IPFT of TCN-induced pleural injury in rabbits. Rabbits treated with intrapleural mouse IgG (0.5 mg/kg) with and without scuPA (0.25 mg/kg) were used as controls. Pleural injury outcomes were assessed at 24 hours after IPFT. GLIS values (Figure 2A) indicate an increase in the efficacy of the IPFT in the presence of MA-33H1F7 and MA-8H9D4. In contrast, intrapleural treatment with mouse isotypic IgG did not improve IPFT outcomes and/or GLIS versus vehicle alone (18) and 0.25 mg/kg scuPA (Figures 2AC2C). Chest ultrasonography before killing of the animals (data not shown) supported the visual assessment of the pleural injury at 24 hours after IPFT (Figures 2BC2D). mAbs and IgG were detected in PFs throughout the experimental time course (Figure 2E), Lisinopril and there was no increase in bleeding complications in any of the animals that received IPFT consisting of scuPA with mAbs (Figure E2). Therefore, intrapleural neutralization of PAI-1 improved the therapeutic outcome (Figure 2A), but did not affect local hemostasis, and was otherwise well tolerated. Open in Lisinopril a separate window Figure 2. AntiCPAI-1 mAbs Teriparatide Acetate (0.5 mg/kg) improve the outcome of intrapleural fibrinolytic therapy (IPFT) with 0.25 mg/kg single-chain uPA (scuPA). Animals were killed 24 hours after administration of IPFT (72 h after initiation of tetracycline [TCN]-induced pleural injury), and the level of injury was assessed and documented as described in the Materials and Methods and previously (18, 24). (to = 6; = 6; = 2); and scuPA (0.25 mg/kg) with antiCPAI-1 mAbs (0.5 mg/kg each) (= 6; = 0.007) in the median values among the treatment groups (on the = 6 independent experiments for mAbs and IgG). AntiCPAI-1 mAbs Protect Intrapleural uPA and PA Activity and Promote the Formation of Endogenous M/uPA Complexes To test the effects of antiCPAI-1 mAbs on the processing of intrapleural scuPA, samples of PF withdrawn during IPFT (0C80 min and 24 h) were analyzed as previously described (18). Intrapleural levels of free uPA (free two chain [tc] uPA; Figure 3A), M/uPA complexes (Figure 3C), and PA activity (Figure E3A) were determined. The observed first-order rate constant (kobs) for.