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We previously demonstrated that tissue-type plasminogen activator (t-PA) specifically bound to its receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC). In addition to analyses of t-PA binding to plasminogen activator inhibitor-1 (PAI-1) in the extracellular matrix (ECM) and to the t-PAR, we further evaluated the binding of three t-PA mutants, deltaFE1X t-PA lacking finger (F), epidermal growth factor-like (E) domains and one sugar chain at Asn177 thus comprising two kringles (K1 and K2) and protease (P) domains, deltaFE3X t-PA with three glycosylation sites deleted at Asn117, 184, and 448, and deltaFEK1 t-PA comprising K2 and P domains without glycosylation. Wild-type t-PA bound to ECM with high affinity, which was completely blocked by anti-PAI-1 IgG. Wild-type t-PA, deltaFE1X t-PA and deltaFEK1 t-PA bound to two classes of binding sites with high and low affinities on monolayer HUVEC. However, all t-PAs bound to a single class of binding site in the presence of anti-PAI-1 IgG. DeltaFEK1 t-PA bound t-PAR maximally among these t-PAs. These results suggested that the high affinity binding of t-PA mainly occurred with PAI-1 on ECM while the low affinity binding was with t-PAR. The deletion of F, E domains and sugar chains had no effect on binding with t-PAR. However, since only K1-missing t-PA (deltaFEK1) exhibited significantly increased binding sites among these t-PAs, it was suggested that the binding to t-PAR was mediated mainly by K2 domain and that the increase of binding was due to direct exposure of K2 domain.

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulphate and plasminogen activator (PA). Bacterial extract, such as lipopolysaccharide (LPS), damaged the blood vessels and destroyed the balance between the antithrombotic and thrombotic functions of endothelial cells. The fibrinolytic system is involved in antithrombotic functions. The TKM-33 cell line was established from human endothelial cells. In order to determine whether TKM-33 is a good fibrinolytic system endothelial cell expression model, the expression of fibrinolytic factors in TKM-33 cells treated with or without LPS was studied. The endothelial cells which had not been treated with LPS produced and secreted a large amount of urokinase-type PA (u-PA), and small amounts of tissue-type PA (t-PA) and PA inhibitor-1 (PAI-1), which were identified immunohistochemically and by electrophoretic enzymography. Diisopropylfluorophosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 2.83 +/- 0.61 nM, and Bmax of (0.11 +/- 0.01) x 10(6) sites/cell. u-PAR expression was detected in endothelial cells by Northern blot analysis. Thus, endothelial cells was shown to express u-PAR which binds u-PA specifically. In the binding assay, the stimulation of endothelial cells with 0.1, 1.0 and 10 micrograms/ml of LPS altered the Kd values to 6.04 +/- 0.71, 7.03 +/- 1.55 and 7.38 +/- 1.03 nM, respectively. However the Bmax values did not change significantly. Although LPS treatment increased u-PAR expression in endothelial cells in a dose-dependent manner, the expression of u-PA and t-PA mRNAs was not altered significantly. LPS stimulation (10 micrograms/ml) increased the expression of PAI-1 mRNA, significantly. The PA activity recovered from the cell surface fraction was not affected by LPS stimulation, but the PAI-1 activity was increased. These findings suggest that the established endothelial cell line, TKM-33, possesses the characteristics of endothelial cells and they express u-PAR on their cell surface, which is occupied by intrinsic u-PA secreted from the cells, and that treatment of endothelial cells with LPS changes the cell surface characteristics and inhibited the u-PAR expression thus promoting the prothrombotic function concomitantly with increased PAI-1 activity.

Aims We investigated the pathophysiological changes in mice lacking α2-antiplasmin (α2-AP) and plasminogen activator inhibitor type-1 (PAI-1) genes, and elucidated the involvement of these inhibitors for fibrinolysis in immune response. Main methods The pathophysiological changes induced by a lack of both α2-AP and PAI-1 were investigated using double knockout (KO) mice. The lung, liver, kidney and spleen tissues from α2-AP/PAI-1-double KO mice were compared with those from wild-type (WT) mice. Furthermore, the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, and then the effects of the transplantation were studied. Key findings Plasma IgE levels in the α2-AP/PAI-1-double KO mice increased with age and exceeded 1000 ng/mL after 6 months of age. The plasma cells that produced IgE were detected in perivascular assembled lymphocytes. In the α2-AP/PAI-1-double KO mice, perivascular lymphocyte infiltration was observed in the lung, liver, and kidneys and peribronchial lymphocyte infiltration was present in the lung. When the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, the phenotypes of the recipients were similar to those of α2-AP/PAI-1-double KO mice. Significance The simultaneous expression of both the α2-AP and PAI-1 genes contributes to the maintenance of immunological functions that are related to IgE. Moreover, it is suggested that both α2-AP and PAI-1 are involved in the recruitment of lymphocytes in the peripheral tissues.

We previously demonstrated that cultured human umbilical vein endothelial cells (HUVECs) overlaid with a fibrin clot induced a slight increase in tissue-type plasminogen activator (t-PA) secretion and marked reduction in plasminogen activator inhibitor-1 (PAI-1) secretion. In this study, the intracellular signal transduction after fibrin stimulation was further investigated by analyzing cyclic AMP (cAMP) and protein kinase C (PK-C). When HUVECs were stimulated by fibrin clots, t-PA mRNA increased to 130% but PAI-1 mRNA decreased to 42%. These changes concurred with the data on the protein levels of t-PA and PAI-1 as previously reported. The effect of fibrin on t-PA production in HUVECs was not significantly altered after the elevation of cAMP by either forskolin or dibutyryl cAMP. Furthermore, an effect of fibrin on t-PA production did not appear when the cells were treated by phorbol 12-myristate 13-acetate (PMA) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). The suppressive effect of fibrin on PAI-1 secretion from HUVECs was not altered by elevation of cAMP. Regarding the activation of PK-C by PMA, PAI-1 secretion was enhanced, but was suppressed by fibrin stimulation. H-7 suppressed PAI-1 secretion and further stimulation by fibrin almost completely abolished PAI-1 secretion. These changes were well associated with mRNA levels of t-PA and PAI-1. These results suggested that fibrin on HUVECs preferably down-regulates PK-C resulting in a decrease of PAI-1 in both the protein and mRNA levels and that effect of fibrin on t-PA secretion is neither involved in PK-C nor cAMP pathway.

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.