To elucidate the pathophysiology of idiopathic pulmonary fibrosis (IPF), we examined procoagulant (tissue factor:TF), fibrinolytic (tissue type plasminogen activator:t-PA and urokinase type plasminogen activator:u-PA) and antifibrinolytic (plasminogen activator inhibitor-1:PAI-1 and PAI-2) activities in bronchoalveolar lavage (BAL) supernatant fluids and BAL cell lysates obtained from IPF patients. The results indicated that TF levels in BAL supernatant fluids from IPF patients were higher than those of normal subjects, especially in patients with progressive disease, suggesting that TF levels in the lung correlate with disease activity. PAI-1 levels in BAL supernatant fluids were significantly higher in IPF patients than in normal subjects (1.7 +/- 4.1 vs 0 ng/mg protein). PAI-2 levels in BAL cell lysates were also significantly higher in IPF patients than those in normal subjects (14.4 +/- 12.2 vs 3.0 +/- 3.0 ng/mg protein). However, u-PA levels in both BAL supernatant fluids and BAL cell lysates did not differ between the two groups. These observations suggest that u-PA inhibition exceeded u-PA activity in alveolar lining fluid resulting in an antifibrinolytic condition. Immunohistochemical analysis showed that TF was intensely stained in cuboidal epithelial cells and PAIs were positively stained in alveolar macrophages (AMs) and cuboidal epithelial cells, suggesting that cuboidal epithelial cells as well as AMs contribute to the increased procoagulant and antifibrinolytic activities in the lungs of IPF patients.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1: Arg 346 -Met 347 ). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nM; half-maximal effect at ϳ0.1 nM). Subtilisin NAT dose dependently (0.06 -1 nM) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 ؎ 1.4% at 1 nM) and especially in the presence of rpPAI-1 (78 ؎ 2.0% at 1 nM). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.Subtilisin NAT (1) (formerly designated BSP, or nattokinase), a serine proteinase from Bacillus subtilis, has been reported to have potent fibrinolytic activity (1, 2). The enzyme is composed of 275 amino acids with a molecular mass of 27.7 kDa in its mature form (1). DNA sequence analysis showed that subtilisin NAT was 99.5 and 99.3% homologous to subtilisins E and Amylosacchariticus, respectively (3). It is also homologous to other members of the subtilisin family (BPNЈ 86% and Carlsberg 72%), and sequences are conserved especially around the three amino acids (serine 221, histidine 64, and aspartic acid 32) essential for the catalytic center of serine proteinases.The mechanism for this enzyme to potentiate fibrinolysis is not fully understood. Subtilisin NAT is reported not to possess plasminogen activator activity but appears to directly digest fibrin by limited proteolysis (4). However, this direct cleavage of fibrin does not seem to account for all of the enhancement of the fibrinolytic activity that has been observed without affecting the fibrinolytic cascade. To explore other possible mechanisms, we have looked for interactions between subtilisin NAT and the physiological inhibitors of fibrinolysis, plasminogen activator inhibitor type 1 (PAI-1) 1 (5) and ␣ 2 -antiplasmin (␣ 2 -AP) (6). These inhibitors are both members of the serine protease inhibitor superfamily (SERPINs). The SERPINs are proteolytically cleaved and inactivated by a variety of proteases including members of the subtilisin family (7).PAI-1 is the primary inhibitor of tissue-type plasminogen activator (tPA) and regulates fibrinolytic activity in the vasculature at the initia...

Although abnormalities of alveolar fibrin turnover have been reported to play a role in the development of idiopathic pulmonary fibrosis (IPF), the pathophysiological relevance remains unclear. We therefore investigated the localization of tissue factor (TF) and fibrin deposition in patients with IPF using immunohistochemistry and compared the results with those from patients who had interstitial pneumonia associated with systemic sclerosis (IP-SSc) and idiopathic bronchiolitis obliterans with organizing pneumonia (BOOP). Expression of TF-mRNA was also assessed, using in situ hybridization with a digoxigenin-labeled cRNA probe. In patients with IPF, IP-SSc, and idiopathic BOOP, the TF antigen was positively stained in type II pneumocytes and in some alveolar macrophages. The fibrin antigen was stained in the type II pneumocytes and the adjacent area. Tissue factor-mRNA was expressed in the type II pneumocytes and in some alveolar macrophages. Neither TF antigens nor TF-mRNA were detected in the normal lung. These results indicate that type II pneumocytes are a major source of TF, suggesting that TF production in these cells is closely related to fibrin deposition in the lungs of people with these diseases.

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SummaryThe relationships between tissue plasminogen activator (tPA), its fast acting inhibitor (PAI-1) and euglobulin clot lysis time (ELT) were investigated with healthy volunteers’ plasma. Turbidimetric clot lysis assay by the microtiter plate reader was utilized for ELT with a slight modification. Both tPA and PAI-1 showed the significant correlation with ELT. tPA had a significantly positive, not negative, correlation with ELT (R = 0.387, p <0.001). Higher correlation coefficients (R = 0.580, p <0.001 and R = 0.599, p <0.001) were obtained between ELT and total PAI-1 or free PAI-1 than tPA or tPA-PAI-1 complex (R = 0.427, p <0.001). The positive correlation was also obtained between tPA and PAI-1. These data suggest that PAI-1 is a highly important factor for ELT, especially, the amounts of free PAI-1 being the key factor to determine the ELT, which can represent the potential activity of the fibrinolytic system.

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A positive correlation between age and the occurrence of thrombosis has been suggested. We studied the relationship between age and fibrinolytic activities; namely levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PA inhibitor) activity, and plasminogen activator activity (PA activity). A dramatic increase in both t-PA antigen and PA inhibitor was shown in persons with increasing age. PA activity decreased with age. Therefore it is suggested that the tendency of decreased PA activity with increasing age may be related to the high incidence of thrombosis in older persons.