Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids

Abstract

Komissarov AA, Florova G, Azghani A, Karandashova S, Kurdowska AK, Idell S. Active ␣-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids. Am J Physiol Lung Cell Mol Physiol 305: L682-L692, 2013. First published August 30, 2013 doi:10.1152/ajplung.00102.2013.-Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0 -80 min and 24 h after IPFT with scuPA (0 -0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with ␣-macroglobulin (␣M) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10 -20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2 ␤ ϭ 40 Ϯ 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 Ϯ 70 nM of ␣M/uPA formed in vivo [second order association rate (kass) ϭ 580 Ϯ 60 M Ϫ1 ·s Ϫ1 ]. ␣M/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active ␣2M, and ␣2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of ␣M/uPA; and 3) slow intrapleural degradation of ␣M/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications. fibrinolytic therapy; rabbit model; pleural injury; urokinase; ␣-macroglobulin; human FIBRINOLYSINS, INCLUDING tissue type (tPA) and urokinase (active two-chain enzyme; tcuPA), are plasminogen activators (PAs) that have long been used to treat a variety of thrombotic conditions including acute myocardial infarction (23; 44), deep vein thrombosis (33, 40), ischemic stroke (1, 3), acute respiratory distress syndrome (ARDS) (20, 21), pulmonary emboli, and organizing pleural effusions (8,9,11,31,37,43). Although intrapleural fibrinolytic therapy (IPFT) has been in use for over 60 years, it has recently undergone reassessment in light of the disparate results seen in clinical trials (9, 11, 37). The efficacy of IPFT in adults remains a subject of ongoing debate. Intrapleural streptokinase was ineffective in patients with complicated parapneumonic pleural effusions and empyema (EMP), whereas ...

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

“…Such serpin-independent rapid inactivation likely contributed to the ineffectiveness of streptokinase IPFT in Multicenter Intrapleural Sepsis Trial 1 (1). Therefore, intrapleural fibrinolysis that is maintained by activation of endogenous plasminogen operates as long as the total level of plasminogen activators is higher than PAI-1's antiproteinase activity (18). Thus, we postulate that neutralization of endogenous PAI-1, resulting in an increased half-life of intrapleural PA activity, should increase the efficacy of IPFT.…”

“…Increments of PAI-1 antigen have been reported to occur in inflammatory pleural effusions and empyema (15,16). However, the contribution of PAI-1 activity to the severity of loculation and poor outcomes of IPFT in a preclinical model of tetracycline (TCN)-induced pleural injury in rabbits were only recognized recently (17,18). The mechanism of intrapleural processing of single-chain uPA (scuPA) (18) predicts the critical role of a combination of high levels (10-20 nM) of endogenous active PAI-1 and fast PAI-1-independent inactivation of uPA in the outcome of intrapleural fibrinolysis.…”

“…However, the contribution of PAI-1 activity to the severity of loculation and poor outcomes of IPFT in a preclinical model of tetracycline (TCN)-induced pleural injury in rabbits were only recognized recently (17,18). The mechanism of intrapleural processing of single-chain uPA (scuPA) (18) predicts the critical role of a combination of high levels (10-20 nM) of endogenous active PAI-1 and fast PAI-1-independent inactivation of uPA in the outcome of intrapleural fibrinolysis. During IPFT, the initial excess of uPA quenches active PAI-1, and the activation of endogenous plasminogen results in intrapleural fibrinolysis.…”

“…During IPFT, the initial excess of uPA quenches active PAI-1, and the activation of endogenous plasminogen results in intrapleural fibrinolysis. However, fast PAI-1-independent inactivation of uPA (0.018 6 0.005 min 21 [18]) results in the progressive loss of intrapleural plasminogen activating (PA) activity. Such serpin-independent rapid inactivation likely contributed to the ineffectiveness of streptokinase IPFT in Multicenter Intrapleural Sepsis Trial 1 (1).…”

“…Thus, we postulate that neutralization of endogenous PAI-1, resulting in an increased half-life of intrapleural PA activity, should increase the efficacy of IPFT. Moreover, scuPA has been shown to generate high levels of durable PAI-1-resistant a-macroglobulin (aM)/uPA complexes, which provide durable, low-level intrapleural PA activity (18). We therefore reasoned that targeting endogenous active PAI-1 would enhance scuPA-based IPFT.…”