Tetsumei Urano

Increased procoagulant and antifibrinolytic activities in the lungs with idiopathic pulmonary fibrosis

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.

The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

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′: Arg346-Met347). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02–1.0 nm; half-maximal effect at ∼0.1 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.

PAI-1, progress in understanding the clinical problem and its aetiology

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.

Daily variations of platelet aggregation in relation to blood and plasma serotonin in diabetes

The circadian rhythms of platelet aggregation in the whole blood and platelet rich plasma-PRP and plasma serotonin were studied in healthy volunteers (n = 10) and diabetic patients (type II diabetes mellitus n = 12). Platelet aggregation in the whole blood induced by collagen (2 micrograms/ml), ADP (10 microM), arachidonic acid (0.5 mM) and epinephrine (10 microM), and in PRP induced by collagen (2 micrograms/ml), ADP (5 microM), arachidonic acid (250 microM), epinephrine (10 microM) and serotonin-5-HT (1 microM) was measured at 7:30, 11:30, 17:00, 23:00, 4:00 and 7:00. In healthy subjects collagen- and ADP-induced platelet aggregation in the whole blood was significantly lower at 23:00 and 4:00 when compared to values at 7:30. In PRP normal and diabetic platelet response was the lowest during the night. Diabetic platelets exhibited an enhanced response to 5-HT starting from 17:00 until 4:00 when compared to 7:30. 5-HT-induced platelet aggregation was found to be significantly higher throughout the study in DM patients over controls in parallel to plasma 5-HT. In healthy volunteers plasma 5-HT was higher at 17:00 when compared to baseline values, whereas in DM patients plasma 5-HT was elevated starting from 17:00 until 4:00. An enhanced response of diabetic platelets to 5-HT together with elevated plasma 5-HT levels may contribute, at least partly, to the pathogenesis of diabetic vasculopathy and 5HT2 receptor blockers may be of value in DM patients.

Rapid, specific and active site-catalyzed effect of tissue-plasminogen activator on hippocampus-dependent learning in mice.

In the present study we trained tissue-plasminogen activator (tPA)-knockout (tPA -/-) and wild-type (tPA +/+) male mice in step-down inhibitory avoidance learning, a hippocampus-dependent task. tPA -/- displayed significantly shorter latencies to step down at 90 min, one, two and seven days after training indicating the learning deficit in these animals (P < 0.05 vs tPA +/+). The locomotor activity, the level of anxiety in an elevated-plus maze, as well as the pain threshold did not differ between the two strains of mice. The learning disability of tPA -/- was overcome by more intense training. The learning deficit was also partially restored by limited intrahippocampal delivery of tPA (infused for 2 h before training; P < 0.05 vs control), but not by the delivery of urokinase plasminogen activator, indicating the acute need for tPA in learning. The beneficial effect of tPA was abolished by co-infusion of its inhibitor tPA-STOP, indicating that the facilitatory effect of tPA on learning requires a proteolytic step. However, tPA activity in the hippocampus was not indispensable for effective memory retrieval in tPA-infused tPA -/- mice. Thus, rapid, specific and proteolytic action of tPA facilitates hippocampus-dependent learning, but not retrieval of previously acquired information.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Neuronal degeneration and a decrease in laminin-like immunoreactivity is associated with elevated tissue-type plasminogen activator in the rat hippocampus after kainic acid injection

Tissue-type plasminogen activator (tPA) is a serine protease that converts the inactive precursor plasminogen to the active protease plasmin. In the central nervous system, tPA has been suggested to participate in plasticity, memory and the neuronal degeneration caused by excitotoxins, but its precise functions during these processes are still unclear. We show in this report that tPA antigen level and extracellular tPA activity increased in the hippocampus during the early stages of neuronal degeneration in the CA3 region following the injection of kainic acid (KA) into the lateral cerebral ventricles. The increase in tPA antigen level was transient and its peak was at 4 h after the injection. tPA activity was also increased 4 h after the injection, but it remained at a high level for more than 8 h. Histological zymography showed that the increase in tPA activity was mainly localized in the CA3 region. In the same region, the disappearance of interneuronal laminin-like immunoreactivity and atrophic changes in pyramidal neurons were observed 4 h after the injection of KA. These results suggested that such focal and transient increases in tPA synthesis and release, which result in the proteolysis of laminin through plasminogen activation, could be involved in the neuronal degeneration in the CA3 region after the injection of KA.

Larger and more invasive colorectal carcinoma contains larger amounts of plasminogen activator inhibitor type 1 and its relative ratio over urokinase receptor correlates well with tumor size

Considering recent findings that both urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitors (PAIs) are involved in tumor growth through an urokinase‐type plasminogen activator (uPA) activity‐independent mechanism, the relation between the presence of these factors in tumor tissue and the clinicopathologic variables in colorectal carcinoma was reevaluated.

Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery.

We have previously demonstrated that natto-extracts containing nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with natto-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary natto-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall.

Cancer cells overexpress mRNA of urokinase-type plasminogen activator, its receptor and inhibitors in human non-small-cell lung cancer tissue: analysis by Northern blotting and in situ hybridization.

The transcriptional localizations of urokinase‐type plasminogen activator (uPA), its receptor (uPAR) and its inhibitors (PAI‐1 and PAI‐2), which are possibly involved in cancer metastasis, have not been determined in human lung cancer. To identify their regulation in primary non‐small‐cell lung cancer, we assayed mRNA levels by Northern blot analysis in 25 cases and determined the localizations of mRNA by in situ hybridization in 10 cases. The amounts of uPA and PAI‐2 mRNA were significantly higher in cancerous relative to normal lung tissues. However, no significant difference was observed in uPAR and PAI‐1 mRNA levels. All transcripts were present in cancer cells and were predominantly located in tumor edges in several cases. In addition, PAI‐1 transcripts were more abundant in poorly and moderately differentiated carcinomas relative to well‐differentiated carcinomas and PAI‐2 transcripts were more abundant in squamous cell carcinomas than in adenocarcinomas. Thus, PAIs may be involved in modulation of malignant potency. Our results indicate that human non‐small‐cell lung cancer cells can autonomously express the mRNAs of uPA, uPAR and PAIs, which are possibly involved in metastasis. Int. J. Cancer 78:286–292, 1998.© 1998 Wiley‐Liss, Inc.