Novel aspects of urokinase function in the injured lung: role of α₂-macroglobulin

Abstract

(uPA) is decreased in lung fluids of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) whereas ␣2-macroglobulin (␣2-M), a plasma proteinase inhibitor, is a major component of these fluids. Since there have been reports describing the ability of ␣2-M to form complexes with uPA in vitro, we hypothesized that ␣2-M may interact with uPA in the lung to modulate its biological activity. Pulmonary edema fluids and lung tissues from patients with ALI/ARDS were evaluated for the presence of uPA associated with ␣2-M. Complexes between ␣2-M and uPA were detected in alveolar edema fluids as well as in lungs of patients with ALI/ARDS where they were located mainly in close proximity to epithelial cells. While uPA bound to ␣2-M retains its amidolytic activity towards low-molecularweight substrates, it is not inhibited by its main physiological inhibitor, plasminogen activator inhibitor 1. We also investigated the functional consequences of formation of complexes between uPA and ␣2-M in vitro. We found that when ␣2-M:uPA complexes were added to cultures of human bronchial epithelial cells (BEAS-2B), activation of nuclear factor-B as well as production of interleukin-6 and -8 was substantially suppressed compared with the addition of uPA alone. Our findings indicate for the first time that the function of uPA in patients with ALI/ARDS may be modulated by ␣2-M and that the effects may include the regulation of the fibrinolytic and signaling activities of uPA. epithelial cells; lung; inflammation; ␣ 2-macroglobulin; urokinase ␣ 2 -MACROGLOBULIN (␣ 2 -M), a high-molecular-weight (HMW; Ͼ700 kDa) plasma glycoprotein, forms a tetramer composed of identical subunits, linked in pairs by disulfide bonds. The major source of plasma ␣ 2 -M is the hepatocyte; however, other cells including monocytes and macrophages synthesize and secrete this protein. ␣ 2 -M is a "slow acting" proteinase inhibitor that binds proteinases from all major classes. The mechanism by which ␣ 2 -M inhibits proteinases is unique. When proteinases hydrolyze the specific peptide bond localized in the so called "bait" region of ␣ 2 -M, the resulting conformational change irreversibly traps the enzyme within the ␣ 2 -M molecule. One or two molecules of proteinase are bound per molecule of ␣ 2 -M depending on the association rate constant and possibly on the size of the proteinase. Further, among proteinase inhibitors, only ␣ 2 -M-entrapped enzymes can still interact with low-molecular-weight (LMW) substrates and inhibitors, which are able to reach the active site of the enzyme located in a "molecular cage" type complex with ␣ 2 -M.Conformational change of the ␣ 2 -M, which occurs upon reaction with proteinases, exposes binding sites for the ␣ 2 -M receptor/low-density lipoprotein receptor-related protein (LRP). LRP is present on the surfaces of many different cell types, including hepatocytes, macrophages, and dendritic cells. ␣ 2 -M in its native form does not bind LRP whereas ␣ 2 -M-proteinase complexes rapidly undergo endocytosis (6,18,...

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

“…Active ␣2M was determined in human PFs as previously described (35,36). Briefly, PFs were incubated with or without exogenous enzymes (tcuPA, 20 -50 nM) for 0 -18 h at 37°C in a volume of 100 -200 l of 50 mM HEPES/NaOH buffer with a pH of 7.4.…”

“…Reaction mixtures were sampled, with aliquots withdrawn at 0, 2, 7, and 18 h, and their activities were measured. To estimate the activity of ␣2M/enzyme complexes, amidolytic activity was measured in the presence of an excess of PAI-1 (final concentration in the reaction mixture, 80 -200 nM) (35,36).…”

“…␣2M/uPA were prepared from human ␣2M (Molecular Innovations), which contained 0.3 mol of active ␣2M per mole of dimer, and tcuPA (Abbott) as previously described (36). PLG activation by ␣2M/uPA was performed as described (32,34).…”

“…PAI-1 rapidly inhibits tPA and tcuPA [second order association rate (k ass ) Ͼ 10 6 M Ϫ1 ·s Ϫ1 ] (24, 53) but slowly inactivates single-chain uPA (scuPA) (35), making the latter a potentially advantageous agent for IPFT (27). In the airway and pleural fluids (PFs), uPA forms complexes with ␣-macroglobulins (␣M) (35,36). In humans, ␣ 2 M (5, 7, 48) is a major secondary endogenous proteinase inhibitor with broad specificity; its concentration in serum approaches 1-2 mg/ml, which is much higher than that of PAI-1 (6).…”

“…Although the inhibition of endogenous proteinases by ␣ 2 M is considered to be irreversible (14,45), residual activities toward high molecular weight (HMW) substrates and ligands have been reported for a number of complexes, including ␣ 2 M/uPA (50). Previously, we demonstrated the presence of ␣ 2 M/uPA complexes in edema fluids from patients with ARDS/acute lung injury (ALI) (36), observed the formation of similar complexes with exogenous urokinase ex vivo in rabbit PFs (35), and hypothesized that the endogenous activity of ␣M/uPA and their slow degradation could provide durable low-grade PA activity that might contribute to successful IPFT.…”