Targeting of Plasminogen Activator Inhibitor 1 Improves Fibrinolytic Therapy for Tetracycline-Induced Pleural Injury in Rabbits

Abstract

Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (single-chain [sc] urokinase [uPA]) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24 hours. Pleural fluid (PF) was collected at 0, 10, 20, and 40 minutes and 24 hours after IPFT and analyzed for plasminogen activating (PA), uPA, fibrinolytic activities, levels of total plasmin/plasminogen, a-macroglobulin (aM), mAbs/IgG antigens, free active uPA, and aM/uPA complexes. Anti-PAI-1 mAbs, but not mouse IgG, delivered with an eightfold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (P , 0.05). mAbs and IgG were detectable in PFs at 24 hours. Compared with identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of aM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and aM antigens. Targeting PAI-1 did not induce bleeding, and rendered otherwise ineffective doses of scuPA able to improve outcomes in tetracycline-induced pleural injury. PAI-1-neutralizing 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. Keywords: plasminogen activator inhibitor 1; fibrinolytic therapy; animal model; prourokinase; monoclonal antibodies Clinical RelevanceOrganizing pleural injury remains an important clinical problem for which fibrinolytic therapy has been used with variable results for children and adults. This study demonstrates, for the first time, that the targeting of active plasminogen activator inhibitor 1 enhances the ability of relatively low doses of intrapleural single-chain urokinase to clear pleural effusions after induction of organizing injury. This work defines a new, well-tolerated approach for intrapleural fibrinolytic therapy that is promising and tractable for clinical trial testing.The results of Multicenter Intrapleural Sepsis Trials 1 and 2 demonstrated that intrapleural fibrinolytic therapy (IPFT) with either streptokinase, or tissue-type plasminogen activator (tPA) alone were ineffective (1, 2). In contrast, there is a growing body of clinical reports demonstrating the successful use of IPFT, including tPA,. It is likely that the disparate results of IPFT trials, which are largely successful in children (2, 6) and variably effective in adults (6, 7), relate to the lack of formal toxicological and dose-escalation studies, resulting in empiric dosing. A further impediment to the field is an incomplete underst...

The time course of resolution of adhesions during fibrinolytic therapy in tetracycline-induced pleural injury in rabbits

“…We also found that the intrapleural level of active PAI-1 dramatically affects the half-life of fibrinolysins and outcome of IPFT and that in vivo PAI-1 neutralization improves IPFT outcomes and decreases the required effective dose of a fibrinolysin (14). In the present study, we used chest ultrasonography (US) and computed tomography (CT) imaging modalities to define the time course for effective intrapleural fibrinolysis after IPFT by using known minimal effective doses (MED) of sc tissue PA (sctPA) and scuPA in our well-characterized TCN-induced pleural injury in rabbits (14,24,31,35). We found that the time needed for effective fibrinolysis for sctPA and scuPA were similar and protracted over several hours.…”

“…Female New Zealand white rabbits, weighing 3.0 -3.6 kg, were used (n ϭ 27). Pleural injury was induced by intrapleural administration of a single dose of TCN, as previously reported (14,24). Single intrapleural doses of either scuPA (Abbott Laboratories) or tPA (Activase, Genentech), known to clear pleural organization (MEDs 0.5 mg/kg and 0.145 mg/kg, respectively) in the model (in n ϭ 12 and 10 rabbits, respectively), or a vehicle control (n ϭ 3) were administered 48 h after induction of pleural injury by TCN (26).…”

“…US was performed for all rabbits at 0, 2, 8, and 24 h (n ϭ 22), as previously described (14). Additional US imaging was performed at 4 h (n ϭ 16; scuPA/US n ϭ 3, scuPA/US/CT n ϭ 6, sctPA/US n ϭ 3, sctPA/US/CT n ϭ 4) and at 12 h (n ϭ 12; scuPA/US n ϭ 4, scuPA/US/CT n ϭ 3, sctPA/US n ϭ 3, sctPA/US/CT n ϭ 2).…”

“…Previously, we showed that the fibrinolytic activity in the first 1.5 h after IPFT does not determine outcome and hypothesized that the time of successful fibrinolysis in TCN-induced pleural injury in rabbits is longer than 2 h (14,35). We also found that the intrapleural level of active PAI-1 dramatically affects the half-life of fibrinolysins and outcome of IPFT and that in vivo PAI-1 neutralization improves IPFT outcomes and decreases the required effective dose of a fibrinolysin (14).…”

“…Mouse PA inhibitor 1 (PAI-1) notably differs from human PAI-1 (12), and human uPA possesses poor affinity to the murine uPA receptor (30). The TCN-induced model of pleural injury has therefore been suc-cessfully used to evaluate the intrapleural processing of singlechain uPA (scuPA) (35) and to improve the efficacy of IPFT by targeting active PAI-1 (14).…”