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.

Summary Plasminogen activator inhibitor‐1 (PAI‐1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) activity. Although the principal function of PAI‐1 is the inhibition of fibrinolysis, PAI‐1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI‐1 functions in vivo and in vitro. The use of Serpine1‐deficient mice has particularly enhanced our understanding of the functions of PAI‐1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI‐1 and its role in clinical conditions are discussed.

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.

The activation of human [Glul]plasminogen ([Glu1]Pg) by high-molecular-weight two-chain human urinary urokinase [EC 3.4.21.31) and low-molecular-weight two-chain human urinary urokinase is inhibited by Cl-at physiological concentrations and stimulated by e-aminohexanoic acid (EAhx; E-aminocaproic acid). The inhibition by Cl-does not occur in the presence of concentrations of EAhx that saturate the acid's weak binding sites on [Glul] [Glu']Pg and HPm contain functionally important binding sites for agents such as L-lysine and e-amino hexanoic acid (EAhx). At least one strong binding site for these agents exists (6), which is responsible for the interaction of the proteins with fibrin(ogen) (7) and for the initial strong interaction of HPm with its major circulating inhibitor, a2 antiplasmin (8).[Glut]Pg also contains at least four weaker binding sites for eAhx (6), which are responsible for the dramatic conformational alteration (9) and concomitant increase in the activation rate of [Glut]Pg by UKase (10).Previous work from this laboratory has shown that monovalent anions, typified by physiologically relevant concentrations of Cl-, dramatically inhibit the activation rate of human [Glu1]Pg by SKase (11). We believed it important to ascertain whether the anionic inhibition of this process was also a property of the activation of [Glut]Pg by another important activator, UKase, and whether any relationship existed between the Cl-and eAhx effects on this process; this manuscript represents a report of the results of these investigations. MATERIALS AND METHODSProteins.[Glu1]Pg, affinity chromatography forms 1 and 2, were purified by our previously described modification (12) of the Deutsch and Mertz affinity chromatography technique (13). The lyophilized protein was dissolved in a buffer consisting of 10 mM 4-(2-hydroxyethyl)piperazinemethanesulfonic acid (Hepes)/NaOH, pH 7.4, and exhaustively dialyzed against that same buffer. For spectrophotometric determination of the concentrations of [Glu']Pg, an extinction coefficient (1%, 1 cm, 280 nm) of 17.0 and a Mr of 94,000 per mol were used (14).Low-molecular-weight urokinase (lMrUKase) was a gift of Abbott and high-molecular-weight urokinase (hMrUKase) was purchased from Calbiochem. Both proteins displayed the expected bands on reduced and nonreduced NaDodSO4/ PAGE and were judged to be at least 95% pure. Each protein was dialyzed against a buffer of 10 mM Hepes/NaOH, pH 7.4. The concentration of active urokinase in each stock solution was determined by spectrofluorimetric titration with 4-methylumbelliferyl p-guanidinobenzoate (15). The stock solution oflMrUKase contained -3.5 ,AM active enzyme, and for hMrUKase the active UKase concentration was -0.04 ,uM.[Glu']Pg Activation Assays. Continuous coupled assays for evaluating the activation rate of [Glut]Pg under differing conditions of Cl-and EAhx concentrations were based upon the temporal development of amidolytic activity of the plasmin that is generated as a result ofthe activation (16). The assays were perf...

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca2؉ channels eliciting Ca 2؉ influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca 2؉ influx alone can activate PKC. The present study was conducted to investigate the Ca

SummaryThe fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma hemostasis cofactors. Vascular endothelial cells (VECs) retain tissue‐type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI‐1), regulates fibrinolytic potential via inhibition of the VEC surface‐bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C‐terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin‐activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI‐1, TAFI, TM, and the fibrin cross‐linking enzyme, factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed.

Recombinant plasminogen-activator-inhibitor type 1 (PAI-1) purified in an activc form from Escherichiu coli and eucaryotic cells was found to contain a mixture of three functionally distinct forms: an active form that forms coinplexcs with plasminogen activators (PAS), an inactive (latent) form that remains intact after incubation with PAS, and a substrate-like form which is easily cleaved by PAS. Since active PAL-1 purified from bacteria (rpPAI-1) contains only trace amounts of the inactive latent and the substrate-like forms, this material was used to study the effect of sodium dodecyl sulphate (SDS) on the structure and function of active PAI-1. After treatment with 0.01 % SDS, active rpPAI-1 was converted to an inactive form that did not form complexes with PAS, but exhibited characteristics similar to those of latent PAI-1. After treatment with 0.1% SDS, PAI-1 lost its inhibitory activity and was cleaved as a substrate in the reactive center. Circular dichroism spectral analysis reveals that SDS changed the conformation of PAI-1 dramatically, mainly by increasing its a-helical content.Plasminogen activation provides an important source of proteolytic activity in a variety of physiological processes [I -61. In mammals there are two types of plasminogen activators (PAS), tissue-type PA ( P A ) and urokinase-type PA (uPA), that both convert the proenzyme plasminogen into the active protease plasmin [2]. The PA system is regulated at many different levels including the direct blockage of PA activity by specific PA inhibitors (PAIs) [7 -151. Among these inhibitors only PA-inhibitor type 1 (PAI-1) efficiently neutralizes all forms of PAS including the single chain of tPA [3, 36, 171. Like the PAS, the synthesis of PAI-1 is highly regulated in many tissues [is] and increased levels of PAI-1 in patients, as well as in transgenic animals are correlated to thrombotic disease [19, 201, suggesting their target proteascs by providing a so-callcd 'bait' residue (P1 residue), that mimics the normal substrate of the target protease [24,25, 271. The mature form of PAI-1 is composed of 379 amino acids and contains no cysteine residues [22, 281. An interesting feature of PAI-1 is that it exists in two forms. An activc form, produced by cells and secreted into the culture media, and an inactive or latent form, with an apparently smaller Stokes' radius [29, 301. PAI-1 coiiverts spontaneously to the latent form [29, 301 and can subsequently be reactivated by treatment with denaturants or negatively charged phospholipids [31, 321. Because active PAT-1 rapidly converts to the latcnt form in culture media, most of the biochemical and functional studies of this inhibitor have so far been performed on inhibitor preparations which have been purified in the latent form and subsequently activated by denaturants like guanidinium-HCl, urea or SDS [29-3331. The fraction of active inhibitor in such preparations has varied greatly from less than 5% up to 55% [29-341. In one study it was found that PAL-1 activated by SDS did not function a...