Analysis of complex formation between plasmin(ogen) and staphylokinase or streptokinase.
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Binding Sites↗
Biomedical subjects
H Fukao
Publications and source records attributed to H Fukao.
Enhancement of tissue-type plasminogen activator (t-PA)-mediated plasminogen activation on the surface of endothelial cells by t-PA receptor.
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Cell Membrane↗
Effect of hyperthermia on the viability and the fibrinolytic potential of human cancer cell lines.
The effects of heat treatment on the viability and fibrinolytic potential of four cultured human carcinoma cell lines, fibrosarcoma cells (HT-1080), lung adenocarcinoma cells with highly metastatic potential (HAL-8), melanoma cells (Bowes) and osteosarcoma cells (NY), determined by measuring their levels of urokinase-type plasminogen activator (u-PA) and its specific receptor (u-PAR), were investigated by comparing them with those of human umbilical vein endothelial cells (HUVECs). HUVECs incubated at 43 degrees C for 120 min exhibited no decrease in viability but exhibited an increase in both u-PA and u-PAR. HT-1080 and HAL-8 showed a moderately high heat-resistance (viability, 60-90%) that correlated with the reduction of u-PAR but not u-PA. On the other hand, Bowes and NY cells, with poor heat-resistance (viability, 20-50%), exhibited stronger cell-associated u-PA activity when they survived at 43 degrees C for 120 min. Since the u-PA/u-PAR system is directly involved in the invasiveness and metastatic potential of carcinoma cells, hyperthermia would alter the biological activity of these carcinoma cells.
Adenocarcinoma↗
Binding of mutant tissue-type plasminogen activators to human endothelial cells and their extracellular matrix.
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.
Aminocaproic Acid↗
Antithrombotic regulation in human endothelial cells by fibrinolytic factors.
Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
The effect of argatroban on injured endothelial cells by thrombin.
When endothelial cells are exposed to thrombin, they become perturbed and acquire thrombogenic properties. Argatroban is an arginine derivative, synthetic small molecule that binds to the active site of thrombin and inhibits its catalytic activity. Therefore, the effects of argatroban on endothelial cells, which had been injured by thrombin, were investigated. The established endothelial cell line, TKM-33, which had been cloned from human umbilical vein endothelial cells, was used. Endothelial cells produce plasminogen activator (PA) to prevent thrombosis and maintain the blood flow. When the endothelial cells were injured by thrombin, secretion of plasminogen activator inhibitor-1 (PAI-1) increased and then the PA activity proportionally decreased. The treatment of endothelial cells with argatroban after thrombin injury did not restore their reduced PA activity. However, the treatment of endothelial cells with argatroban prior to thrombin injury resulted in inhibiting the induction of PAI-1 secretion. Thus, pretreatment of endothelial cells with argatroban suppresses the inhibition of their PA activity by thrombin. Since the effect of thrombolytic agent may be modified by the fibrinolytic factors produced by the endothelial cells, the activity of staphylokinase (SAK) was measured in the presence of endothelial cells that had been injured by thrombin. SAK is a newly developed thrombolytic agent. SAK activity in the presence of injured endothelial cells by thrombin was lower than that in the presence of endothelial cells without thrombin injury. However, treatment of endothelial cells with argatroban prior to thrombin injury revealed higher SAK activity than that after thrombin injury. These findings indicate that argatroban pretreatment prevents thrombin injury of endothelial cells, which may then maintain their physiological function.
Arginine↗
Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice.
Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin; this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP) in plasma. However, SAK's activity is protected against inhibition by alpha2-AP in the presence of fibrin because the plasmin-SAK complex binds to fibrin. In the present study, the interaction between SAK and murine plasminogen was investigated in the plasma of alpha2-AP-deficient (alpha2-AP-/-) mice or plasminogen-deficient (Plg-/-) mice. Although the human plasmin-SAK complex was formed in equimolar mixtures of plasmin and SAK, the murine plasmin-SAK complex was not formed. Human plasminogen was activated by the human plasmin-SAK complex, although equimolar mixtures of murine plasmin and SAK did not activate murine plasminogen. These findings suggest that SAK does not react with murine plasmin. However, the murine plasminogen was activated by the human plasmin-SAK complex, although this activation was approximately 100-fold weaker than human plasminogen. Human and wild-type mouse plasminogens were not activated by the human plasmin-SAK complex in their plasma. In alpha2-AP-/- mouse plasma, murine plasminogen was activated by the human plasmin-SAK complex. Human or murine plasminogen, which had been added to Plg-/- mouse plasma, was not activated by the human plasmin-SAK complex. However, plasma clot lysis by the human plasmin-SAK complex was observed in both human and murine plasma. These findings indicate that: (1) murine plasmin does not react with SAK, (2) human plasmin-SAK complex activates murine plasminogen, (3) this activation is inhibited by murine alpha2-AP, but (4) this activation is not inhibited by murine alpha2-AP in the presence of fibrin.
Animals↗
Co-localization of urokinase and its receptor on established human umbilical vein endothelial cell.
Vascular endothelial cells possess antithrombotic properties, which are determined by the balance between plasminogen activators (PAs) and PA inhibitors (PAls). A cell line, TKM-33, has been established and cloned from human umbilical vein endothelial cells, was previously reported to produce a large amount of urokinase-type PA (u-PA) and small amounts of tissue-type plasminogen activator (t-PA) and PA inhibitor-1 (PAI-1). Moreover, TKM-33 expressed the u-PA receptor (u-PAR) which plays an important role in the localization of fibrinolytic activity on cell surface. In the present study, we investigated the localization of u-PA, t-PA, PAI-1 and u-PAR in TKM-33 by using immunofluorescence staining technique. The endothelial cells were strongly stained with anti-PAI-1, anti-u-PA and anti-u-PAR IgGs, and slightly with anti-t-PA IgG. The double immunofluorescence staining with mouse anti-u-PA IgG and rabbit anti-u-PAR IgG followed by rhodamine-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG showed the co-localization of u-PA and u-PAR on the same section of endothelial cells. Although u-PA antigen also existed in the cytoplasm of endothelial cells, u-PAR antigen did not. The treatment of endothelial cells with phorbol-myristate-acetate (PMA) upregulated the expression of u-PA and u-PAR antigens. In this stimulation, u-PAR antigen was detected not only on the surface of the cells but also in the cytoplasm. Thus, the binding of u-PA to u-PAR was confirmed by double immunofluorescence staining.
Antibodies↗
Analysis of tissue-type plasminogen activator receptor (t-PAR) in human endothelial cells.
Vascular endothelial cells regulate the fibrinolytic system in blood by expressing cell-surface receptors for plasminogen and plasminogen activators (PAs) as well as secreting PAs and their inhibitors. Although several receptors for plasminogen and PAs have been identified in many cell types, little is known about tissue-type PA (t-PA)-specific receptor (t-PAR) on endothelial cells except a few reports. By using suspended human umbilical vein endothelial cells (HUVEC), which are free from the formation of extracellular matrix (ECM)--where type-1 plasminogen activator inhibitor (PAI-1) preferably accumulates and interacts with t-PA with high affinity--we demonstrated a relatively low affinity binding site for t-PA on the cells and identified a novel t-PAR. The isolation and characterization of HUVEC-derived t-PAR was performed in the present study. A 20-kDa t-PAR was successively isolated and purified by high performance liquid chromatography system from HUVEC which specifically binds t-PA and not plasminogen forming a 90-kDa complex with t-PA. When t-PA binds the immobilized t-PAR stoichiometrically 1:1, the enzymatic activity of t-PA was enhanced 90-fold. Thus, it is suggested that the t-PAR may function as a t-PA-enhancing molecule expressed on the surface of endothelial cells.
Cells, Cultured↗
Enhancement of tissue-type plasminogen activator (t-PA) activity by purified t-PA receptor expressed in human endothelial cells.
We demonstrated previously that tissue-type plasminogen activator (t-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC) in suspension and that t-PAR of mol wt. 20 kDa interacted only with t-PA to form 90 kDa complex (Fukao, H., Hagiya, Y., Nonaka, T., Okada, K., and Matsuo, O. (1992) Biochem. Biophys. Res. Commun. 187, 956-962). In the present study, 20 kDa t-PAR was purified from HUVEC and the function of the t-PAR was investigated by analyzing its effect on plasminogen activation by t-PA. About 2.2 microg t-PAR protein was purified from cell lysate of 1.0 X 10(9) HUVEC as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by gel filtration with TSK-3000SW and reversed phase separation with high performance liquid chromatography (HPLC). 125I-t-PA but not 125I-plasminogen specifically bound to the purified t-PAR in ligand blot assay. Plasminogen activation by t-PA in the presence of purified t-PAR in solution was increased. Furthermore, t-PA bound to immobilized t-PAR efficiently expressed its plasminogen activation activity. Kinetic analysis revealed that t-PA in the presence of soluble t-PAR and t-PA bound to immobilized t-PAR exhibited 34- and 90-fold increase in plasminogen activation, respectively. The t-PAR did not interact with anti-annexin II antibody. These findings indicate that the 20 kDa t-PAR is a novel molecule which immobilizes t-PA and enhances its proteolytic activity on the cell surface of endothelial cells.
Cells, Cultured↗
The role of the pericellular fibrinolytic system in angiogenesis.
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Animals↗
Enhanced urokinase-type plasminogen activator activity by extracellular matrix protein obtained from highly metastatic human lung adenocarcinoma cell line.
A protein which enhanced urokinase-type plasminogen activator (u-PA) activity was purified from the extracts of extracellular matrix of highly metastatic cell line HAL-8 derived from human lung adenocarcinoma. The protein showed a single band with molecular weight of 65 kDa after the purification by Sephadex G-150 and diethylaminoethyl-cellulose followed by reversed phase separation in a high performance liquid chromatography system. The purified protein in the immobilized conditions enhanced u-PA activity in both plasminogen activation and S-2444 amidolysis by 4.6- and 2.8-fold increases in the second order rate constants (Kcat/K(m)), respectively. This protein was related to neither plasminogen nor single-chain u-PA by the immunological studies and with respect to retention time on reversed phase analysis. These results suggest that the purified material acts as an enhancer of u-PA in extracellular matrix of the cancer cells, inducing an effective tissue destruction and cell invasion and possessing a highly metastatic potential.
Adenocarcinoma↗
Effects of fibrin and alpha2-antiplasmin on plasminogen activation by staphylokinase.
Staphylokinase obtains plasminogen activating activity by forming a complex with plasminogen. Although the enzymatic activity of staphylokinase is enhanced by fibrin, how fibrin enhances enzymatic activity has not been determined yet. The effects of fibrin, or fibrinogen fragments, on the activation of plasminogen by staphylokinase was investigated using CNBr-digested fibrinogen fragments (FCB-2 and FCB-5) and plasmin-degraded cross-linked fibrin fragments ((DD)E complex, DD fragments and E fragments). Kinetic analysis of the activity of staphylokinase revealed that its plasminogen activating activity, which was expressed as kcat/Km, was enhanced by FCB-2 (10-fold) and FCB-5 (5-fold). These fibrin fragments caused 38-, 30-, and 8.5-fold increases in activity for the DD fragment, (DD)E complex and E fragment, respectively. Although alpha2-antiplasmin inhibited the activation of plasminogen by staphylokinase, FCB-2 abolished its inhibitory effects, and the plasminogen activating activity of staphylokinase was restored. The inhibitory effects of alpha2-antiplasmin on the activation of mini-plasminogen by staphylokinase were less than for Glu- or Lys-plasminogen, and the inhibitory effect of alpha2-antiplasmin was not altered by fibrin or EACA. These findings indicate that the staphylokinase/plasmin(ogen) complex reacts with fibrin even in the presence of alpha2-antiplasmin, and efficient plasminogen activation takes place on the surface of fibrin.
Aminocaproic Acid↗
Effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells.
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.
Binding, Competitive↗
Effects of endothelial cells on activity of staphylokinase.
Staphylokinase (SAK), produced by Staphylococcus aureus, induces fibrinolytic activity in circulation without systemic fibrinolytic activation. Since the effect of blood vessels on the activity of SAK has not yet been clarified, plasminogen activator (PA) activity of SAK in the presence or absence of endothelial cells was analyzed. The endothelial cells used in this experiment were of a cloned established cell line (TKM-33). In the expression of PA activity by SAK or streptokinase (SK), the kinetic constants revealed as Vmax/km were increased about 1.5-fold in the presence of endothelial cells. Furthermore, an initial lag phase which was observed during the plasminogen activation by SAK was markedly shortened in the presence of endothelial cells. In the case of SK, an initial lag phase was not observed in the absence or presence of endothelial cells. Although PA activity of SAK was inhibited by alpha 2-antiplasmin (alpha 2-AP), the inhibitory effect of alpha 2-AP in the presence of endothelial cells was weaker than in the absence of endothelial cells. The cyanogen bromide digested fibrinogen fragment-2 (FCB-2) distinctly enhanced the PA activity of SAK in the absence and the presence of endothelial cells. However, alpha 2-AP and FCB-2 did not cause a significant alteration of PA activity of SK even in the absence or presence of endothelial cells. These findings suggest that PA activity of SAK is enhanced by endothelial cells, but inhibited by alpha 2-AP. Moreover, PA activity of SAK is further enhanced by fibrin clot in the presence of endothelial cells.
Cell Line↗
Effect of heat shock on the expression of urokinase-type plasminogen activator receptor in human umbilical vein endothelial cells.
We investigated the effect of heat shock on the fibrinolytic potential of human umbilical vein endothelial cells (HUVECs) in culture. When cultured at 43 degrees C, the mRNA for heat shock protein 70 (HSP70) was dramatically induced within 120 min with a maximal induction of more than 90-fold compared with that in HUVECs cultured at 37 degrees C. The level of urokinase-type plasminogen activator (u-PA) receptor (u-PAR) mRNA increased up to 2.2-fold in response to heat shock, which was associated with the increased u-PA binding and cell-surface u-PA activity determined by adding exogenous u-PA to acid-treated HUVECs. The increased u-PAR mRNA returned to normal level when HUVECs were further incubated at 37 degrees C for 180 min, and this decline was not affected in the presence of actinomycin D. Though the secreted antigens for tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in the conditioned medium (CM) of HUVECs were simultaneously increased at 43 degrees C during this period, the increase in the levels of t-PA (about 26.6-fold at 120 min) was greater than that of PAI-1 (1.8-fold at 120 min). The fibrinolytic activity of CM obtained from HUVECs at 43 degrees C was significantly enhanced up to 3-fold, indicating that heat shock induced hyperfibrinolytic states in HUVECs. The secretion of u-PA into CM was also enhanced by heat shock. These results suggested that human endothelial cells respond to hyperthermia by inducing HSP70 followed by hyperfibrinolytic states with the enhanced expression of u-PAR as well as that of t-PA and u-PA.
Base Sequence↗
Effect of cyclic AMP on urokinase-type plasminogen activator receptor and fibrinolytic factors in a human osteoblast-like cell line.
We investigated the effect of cyclic AMP (cAMP) on the pericellular fibrinolytic system in NY cells. Dibutyryl cAMP (dbcAMP) or forskolin increased the level of urokinase-type plasminogen activator (u-PA) mRNA and enhanced the secretion of u-PA antigen into the conditioned medium. These agents also increased u-PA antigen on the cell surface. PA inhibitor-1 (PAI-1) antigen was inhibited by dbcAMP or forskolin. Butyrate had no effect on the production and secretion of u-PA and PAI-1. A binding assay of 125I-DFP-u-PA to NY cells revealed a single class of binding sites with a Kd of 3.85 nM and Bmax of 0.89.10(5) binding sites/cell. The Bmax was increased by dbcAMP (1 mM or 10 mM), forskolin (2 microM or 20 microM) of 1.0-, 1.4-, 1.2- and 1.8-fold, respectively. However, the Kd value was not changed. Furthermore, the level of mRNA for the u-PA receptor (u-PAR) was increased by these agents 1.2-, 1.7-, 1.8- and 2.5-fold, respectively. However, butyrate did not alter either the Bmax or the u-PAR mRNA level. These results indicated that the pericellular fibrinolytic activity induced by u-PA/u-PAR is modulated by cAMP in osteoblast-like cells.
Bucladesine↗
Effects of fibrin on the secretion of plasminogen activator inhibitor-1 from endothelial cells and on protein kinase C.
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.
Cells, Cultured↗