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It is well recognized that activation of the coagulation system plays an important role in bleomycin (BLM)-induced lung injury and fibrosis. The protein C (PC) pathway is an important regulator of the coagulation system. In this study, we evaluated the bronchoalveolar lavage fluid (BALF) concentration of activated PC (APC) and the therapeutic effect of the intratracheal administration of APC on BLM-induced lung fibrosis in mice. APC levels in BALF were significantly lower in BLM-treated animals than in the saline-treated group. Fibrotic changes were progressive in mice treated with BLM and intratracheal instillation of vehicle (BLM/Veh) after 14 and 21 d of BLM infusion. Compared with the BLM/Veh group, histologic findings on Days 14 and 21 in mice treated with BLM and intratracheal instillation of APC (BLM/APC) showed less fibrotic lesions in the subpleural and central areas of the lung. The mean Aschcroft's fibrosis score in the BLM/Veh group was significantly (p < 0.05) higher than in the BLM/APC group. The lung hydroxyproline content on Day 21 was significantly higher (p < 0.05) in the BLM/Veh group (1.78 +/- 0.07 micromol/lung weight) than in the BLM/APC (1.30 +/- 0.06 micromol/lung weight) group. The ratio of plasminogen activator activity to thrombin level in BALF was significantly increased in the BLM/APC group compared with the BLM/ Veh group on Day 21. The expression of tumor necrosis factor-alpha and interleukin-1beta was significantly decreased in the lungs of the BLM/APC group compared with the BLM/Veh group on Day 14 after BLM infusion. These results showed that intratracheal APC administration inhibits the development of lung fibrosis in BLM-induced lung injury, giving further support to the important role that the PC pathway plays in the mechanism of lung fibrosis.

Inhalation of nitric oxide (NO) is useful for the treatment of patients with pulmonary hypertension. However, the potential toxicity of inhaled NO is still unclear. Coagulation activation plays an important role in lung injury. We assessed the effect of low- and high-dose inhaled NO on the coagulation system in the intraalveolar space of mice. The animals were assigned to five groups (n = 6): [RA] group, mice exposed to fresh air alone; [RA+2 ppm NO] group, fresh air and 2 ppm NO; [RA+40 ppm NO] group, fresh air and 40 ppm NO; [RA+2 ppm NO+O(2)] group, fresh air, 2 ppm NO and O(2); and [RA+40 ppm NO+O(2)] group, fresh air, 40 ppm NO and O(2). Each group was treated for 3 wk. Lung specimens of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups showed significant nitrotyrosine immunoreactivity. BALF concentrations of total protein, thrombin and soluble tissue factor were significantly increased in mice of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups compared with [RA] group. However, BALF concentrations of total protein, thrombin, and soluble tissue factor were not significantly increased in mice of [RA+2 ppm NO] and [RA+2 ppm NO+O(2)] groups compared with [RA] group. Lung tissue factor mRNA expression was higher in the high-dose NO group than in the low-dose NO group. NO donor increased significantly tissue factor activity on alveolar epithelial cells. This study has shown for the first time that long-term inhalation of high, but not low, concentration of NO may activate the clotting system by increasing the lung expression of tissue factor.

Excessive procoagulant activity in the alveolar space may play a relevant role in the pathogenesis of pulmonary fibrosis. Hypercoagulability results from the disruption of the balance between the procoagulant and anticoagulant factors. The aim of this study was to assess the levels of molecular markers of the anticoagulant protein C (PC) pathway in the bronchoalveolar lavage fluid (BALF) and plasma of 11 patients with idiopathic pulmonary fibrosis (IPF), 14 with sarcoidosis and 16 with collagen vascular disease (CVD)-associated interstitial lung disease (CVD-ILD). Six healthy nonsmoking volunteers served as control subjects. BALF concentrations of the marker of clotting activation, thrombin- antithrombin III complex (TAT), in patients with sarcoidosis and CVD-ILD were significantly greater than those in control subjects. PC levels in BALF were markedly higher in patients with IPF (610 +/- 150 ng/ml), sarcoidosis (680 +/- 170 ng/ml), and CVD-ILD (1,580 +/- 600 ng/ml) than in control subjects (230 +/- 140 ng/ml). BALF concentrations of activated PC-PC inhibitor (APC-PCI) complex were significantly decreased in IPF (0.46 +/- 0.16 ng/ml), sarcoidosis (0. 43 +/- 0.11 ng/ml), and CVD-ILD (0.50 +/- 0.15 ng/ml) patients as compared with control subjects (1.08 +/- 0.23 ng/ml). APC-PCI/PC ratios were significantly lower in patients with IPF (2.70 +/- 1.74 ng/microg), sarcoidosis (1.94 +/- 0.82 ng/microg), and CVD-ILD (1.89 +/- 0.68 ng/microg) than in control subjects (15.91 +/- 8.45 ng/microg). Plasma levels of APC- PCI and the APC-PCI/PC ratio were also significantly decreased in patients with CVD-ILD as compared with control subjects. Overall, these findings suggest that decreased PC activation with increased procoagulant activity occurs in patients with ILD.