Nebulized antithrombin limits bacterial outgrowth and lung injury in Streptococcus pneumoniae pneumonia in rats

Abstract

Introduction: Disturbed alveolar fibrin turnover is a cardinal feature of severe pneumonia. Clinical studies suggest that natural inhibitors of coagulation exert lung-protective effects via anticoagulant and possibly also anti-inflammatory pathways. Intravenous infusion of the natural anticoagulants increases the risk of bleeding. Local administration may allow for higher treatment dosages and increased local efficacy while at the same time reducing the risk of bleeding. We evaluated the effect of nebulized anticoagulants on pulmonary coagulopathy and inflammation in a rat model of Streptococcus pneumoniae pneumonia.

Methods: In this randomized controlled in vivo laboratory study rats were challenged intratracheally with S. pneumoniae, inducing pneumonia, and randomized to treatment with normal saline (placebo), recombinant human activated protein C (rh-APC), plasma-derived antithrombin (AT), heparin or danaparoid, by means of nebulization.

Results: S. pneumoniae infection increased pulmonary levels of thrombin-antithrombin complexes and fibrin degradation products. All nebulized anticoagulants significantly limited pulmonary coagulopathy. None of the agents except danaparoid resulted in changes in systemic coagulopathy. Treatment with plasma-derived AT reduced outgrowth of S. pneumoniae and histopathologic damage in lungs. In vitro experiments confirmed outgrowth was reduced in bronchoalveolar lavage fluid (BALF) from rats treated with plasma-derived AT compared with placebo. Neutralizing of cationic components in BALF diminished the inhibitory effects on bacterial outgrowth of BALF, suggesting a role for cationic antimicrobial proteins.

Conclusions: Nebulization of anticoagulants attenuates pulmonary coagulopathy during S. pneumoniae pneumonia in rats while only danaparoid affects systemic coagulation. Nebulized plasma-derived AT reduces bacterial outgrowth and exerts significant lung-protective effects.

Figure 1

Pulmonary coagulation and fibrinolysis. The effects of anticoagulants nebulized into the lungs of rats on levels of (a) thrombin-antithrombin complexes (TATc), (b) antithrombin activity (AT), (c) fibrin degradation products (FDP), (d) plasminogen activator activity (PAA%), and (e) plasminogen activator inhibitor-1 (PAI-1) in bronchoalveolar lavage fluid (BALF), 40 hours after intra-tracheal bacterial challenge (Streptococcus pneumoniae, serotype 3, ATCC 6303). Dotted lines stipulate the normal values in healthy animals and untreated animals with pneumonia. Data represent mean ± standard deviation. * P < 0.01 vs. placebo. APC = recombinant-human activated protein C; AT = plasma = derived human antithrombin; Hep = heparin; Dan = danaparoid.

Figure 2

Systemic coagulation and fibrinolysis. The effects of anticoagulants nebulized into the lungs of rats on plasma levels of (a) thrombin-antithrombin complexes (TATc) and (b) systemic plasminogen activator activity (PAA), 40 hours after intra-tracheal bacterial challenge. Dotted lines stipulate the normal values in healthy animals and untreated animals with pneumonia. Data represent mean ± standard deviation. * P < 0.01 vs. placebo. APC = recombinant-human activated protein C; AT = plasma = derived human antithrombin; Hep = heparin; Dan = danaparoid.

Figure 3

The effects of anticoagulants on numbers of Streptococcus pneumoniae colony forming units in (a) lung homogenate and (b) bronchoalveolar lavage fluid (BALF), 40 hours after intra-tracheal bacterial challenge. Data represent median ± interquartile range. * P < 0.05 vs. placebo. APC = recombinant-human activated protein C; AT = plasma = derived human antithrombin; CFU = colony-forming units; Hep = heparin; Dan = danaparoid.

Figure 4

Lung wet weights (a) and total protein levels in (b) bronchoalveolar lavage fluid (BALF), 40 hours after intra-tracheal bacterial challenge. Dotted lines stipulate the normal values in healthy animals and untreated animals with pneumonia. Data represent median ± interquartile range. * P < 0.01 vs. placebo. APC = recombinant-human activated protein C; AT = plasma = derived human antithrombin; Hep = heparin; Dan = danaparoid.

Figure 5

(a) TNF-α, (b) IL-6, and (c) cytokine-induced neutrophil chemoattractant (CINC)-3, determined in lung homogenates 40 hours after intra-tracheal bacterial challenge. Dotted lines stipulate the normal values in healthy animals and untreated animals with pneumonia. Data represent median ± interquartile range. APC = recombinant-human activated protein C; AT = plasma-derived human antithrombin; Hep = heparin; Dan = danaparoid.

Figure 6

Histopathological changes in Streptococcus pneumoniae pneumonia. Shown are representative hematoxylin and eosin-stained photomicrographs (magnification, ×100) of lung tissue from rats treated with (a) saline, (b) recombinant human activated protein C (APC), (c) plasma-derived antithrombin (AT), (d) heparin (Hep), (e) danaparoid (Dan), at 40 hours after bacterial challenge. (f) Total histopathologic scores are presented as median with interquartile range. * P < 0.05 vs. placebo.

Figure 7

The effects of BALF from placebo treated rats (BALF-none) and BALF from rats treated with plasma-derived AT (BALF-AT) on survival of Streptococcus pneumoniae in vitro. (a) After five hours outgrowth of S. pneumoniae in bronchoalveolar lavage fluid from rats treated with plasma-derived antithrombin (BALF-AT) was reduced (closed symbols), compared with the outgrowth in BALF-none samples (open symbols). * P < 0.0001 vs. BALF-AT. (b) In vitro added plasma-derived AT did not affect the number of colony-forming units (CFU; for clarity only the highest concentrations are shown: 1.1 mg/mL, open symbols; 4.4 mg/mL, closed symbols). (c) Sodium polyanetholsulphonate (SPS) blocked the inhibitory effect of BALF-AT on outgrowth of S. pneumoniae (BALF-AT without SPS, closed symbols; BALF-AT with SPS, open symbols). Data represent mean ± standard deviation. * P < 0.0001 vs. BALF-AT without SPS.