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Biomedical subjects

O Matsuo

Publications and source records attributed to O Matsuo.

At least 55 records · Page 3Linked to original sources
PubMed1995

Fibrinolytic activity in liver tissues of stroke-prone spontaneously hypertensive rats.

1. Plasminogen activator activity was detected in the extract solution of the liver tissues of both stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats by the synthetic substrate assay. 2. The total PA activity in the liver extract of WKY (26.8 +/- 8.3 i.u.) was about 1.5-fold higher than that of SHRSP (18.5 +/- 4.1 i.u., n = 8, P < 0.005). 3. The enzymography of the liver extract revealed three lytic bands with a molecular weight of 67 kDa, 44 kDa and 38 kDa. 4. The inhibitor activity of the liver extract was detected by the reverse fibrin autography method with one lytic resistance band at 70 kDa. 5. Thus, fibrinolytic components exist in the liver tissue of both strains of rats, but their contribution to the stroke requires further study.

Amino Acid Sequence↗
PubMed1995

Identification of urokinase-type plasminogen activator receptor in human endothelial cells and its modulation by phorbol myristate acetate.

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulfate and plasminogen activator (PA). Using an established cell line, TKM-33, from human umbilical vein endothelial cells, the pericellular urokinase-type PA (u-PA) activity and expression of u-PA receptor (u-PAR) were investigated. The endothelial cells produced and secreted large amounts of u-PA and low levels of tissue-type PA (t-PA) and of PA inhibitor-1 (PAI-1), which were identified by immunohistochemical study and electrophoretic enzymography. Diisopropylfluoro-phosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 3.46 +/- 1.17 nM, and Bmax of (0.09 +/- 0.04) x 10(6) sites/cell. mRNA of u-PAR was detected by using Northern blot analysis. Thus, these endothelial cells express u-PAR which bounds u-PA specifically. Phorbol myristate acetate (PMA) stimulation to the endothelial cells altered the Kd value to 3.18 +/- 0.64 nM, and Bmax value to (0.19 +/- 0.10) x 10(6) sites/cell, respectively. PMA treatment of endothelial cells increased u-PAR mRNA. Similarly, H7-treated endothelial cells showed a dose-dependent increase of u-PAR mRNA. However, PMA and H7 did not stimulate the expression of u-PA and t-PA mRNAs significantly. The expression of PAI-1 mRNA was increased by both PMA and H7. These findings suggest that the established endothelial cell line, TKM-33, possesses the character of endothelial cells and expresses u-PAR on their cell surface which is occupied by intrinsic u-PA secreted from the cells. The pericellular u-PA activity and the expression of u-PAR were regulated by protein kinase pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Kinetic analysis of plasminogen activation by staphylokinase/plasminogen complex in the presence of fibrin.

Staphylokinase (SAK) expresses plasminogen activator activity by forming a complex with plasminogen. In order to elucidate the mechanism for the expression of enzymatic activity of the complex, a cross-linked staphylokinase/plasminogen (SAK/plg) complex was produced with disuccinimidyl suberate, and its enzymatic characteristics were compared with those of a streptokinase/plasminogen (SK/plg) complex. SAK/plg complex and SK/plg complex showed a band with a molecular weight of 110 kDa and 140 kDa by SDS-PAGE under non-reduced condition, respectively. Both complexes exhibited plasminogen activator activity in a concentration-dependent manner on fibrin film and synthetic chromogenic substrate assay. The kinetic analysis of enzymatic activity of both complexes was performed. The plasminogen activator activity of SAK/plg complex was enhanced about 5-fold in the presence of FCB-2. However, SK/plg complex showed only 1.7-fold increase in the presence of FCB-2. These findings indicate that the SAK/plg complex reacts with fibrin, and efficient plasminogen activation is induced on fibrin surface.

Amino Acid Sequence↗
PubMed1994

Effects of alpha 2-plasmin inhibitor on plasminogen activation by staphylokinase/plasminogen complex.

Using a stable cross-linked SAK/plg complex, the effects of alpha 2-plasmin inhibitor on plasminogen activation by SAK were investigated. alpha 2-Plasmin inhibitor inhibited dose-dependently plasminogen activation by the SAK/plg complex. When FCB-2 or EACA was added to the reaction mixture of SAK/plg complex and alpha 2-plasmin inhibitor, the inhibitory activity of alpha 2-plasmin inhibitor was abolished and the enzymatic activity of the complexes was restored. alpha 2-Plasmin inhibitor inhibited the activity of the SK/plg complex, but neither FCB-2 nor EACA restored the plasminogen activator activity in the mixture of SK/plg complex and alpha 2-plasmin inhibitor. Using 125I-labeled SAK/plg complex or SK/plg complex, the reaction of the complex with alpha 2-plasmin inhibitor was analyzed. The SAK/plg complex produced a new complex with alpha 2-plasmin inhibitor. The formation of a new high molecular weight complex with alpha 2-plasmin inhibitor was abolished by both EACA or FCB-2. With regard to the SK/plg complex, neither EACA nor FCB-2 suppressed the complex formation with alpha 2-plasmin inhibitor. These findings indicate that the SAK/plg complex binds to fibrin, and that this complex expresses plasminogen activator activity without being affected by alpha 2-plasmin inhibitor.

Amino Acid Sequence↗
PubMed1994

Synthetic dipeptide, N-stearoyl-D-Ser-L-Pro-OEt, induces release of tissue-type plasminogen activator in cultured cells and in experimental animals.

The tissue-type plasminogen activator (t-PA)-releasing action of synthetic dipeptides containing Gly, Ser or Pro was investigated. Among 10 dipeptides, Boc-L-Ser-L-Pro-OH and H-L-Ser-L-Pro-OH induced t-PA release in vitro, but the others were inactive. Since Boc-L-Ser-L-Pro-OH was more effective than H-L-Ser-L-Pro-OH, 7 related dipeptides with N-acylation were synthesized. Five of them enhanced the release of t-PA; N-stearoyl-L-Ser-L-Pro-OH (FK-5) had the greatest effect. Four compounds were further examined for activity to enhance the release of t-PA in rats. FK-5 produced a two-fold increase in fibrinolytic activity, and N-palmitoyl-L-Ser-L-Pro-OH (FK-4) also markedly enhanced the release of t-PA. Since FK-5 caused severe hemolysis, 7 analogues of FK-5 were synthesized. All of them enhanced the release of t-PA from melanoma (Bowes) cells. In rats, FK-5, N-stearoyl-D-Ser-L-Pro-OH (FK-8) and N-stearoyl-D-Ser-L-Pro-OEt (FK-10) enhanced the fibrinolytic activity two-fold. FK-5 and FK-8 also exhibited strong hemolytic activity, but FK-10 did not induce hemolysis. Therefore, FK-10 was examined in rabbits. After the injection of this compound, the fibrinolytic activity in the euglobulin fraction was markedly enhanced without accompanying hemolysis. Thus, FK-10 potently enhances fibrinolytic activity both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1994

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the fibronectin finger-like domain and the kringle 1 domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which is used to treat acute myocardial infarction. Pharmacokinetically, t-PA is characterized by a rapid clearance from the circulation. In a previous study, we constructed variant forms of t-PA with genetic modifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N37S.S38V.G39V.R40E. A41F.Q42S had about a 6.0-fold higher plasma half-life in vivo than wild-type t-PA. Two variants with modifications in the K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improved kinetic parameters and a 2.2-fold higher plasma half-life in vivo than wild-type t-PA, respectively. To create a recombinant variant of t-PA with a higher enzymatic activity and a further prolonged half-life in vivo, the genes containing each modifications were joined and expressed in animal cells. The two variants, t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N11 5P, were purified from conditioned media and their biochemical, pharmacokinetic and thrombolytic profiles were investigated. Although the variant t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild-type t-PA, the half-life of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, following intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Effect of bone resorbing factors on u-PA and its specific receptor in osteosarcoma cell line.

This study investigated the effect of bone resorbing factors on the pericellular fibrinolytic system of osteosarcoma NY cells. Parathyroid hormone (PTH), prostaglandin E2, (PGE2) or tumor necrosis factor alpha (TNF-alpha) enhanced the secretion of urokinase-type plasminogen activator (u-PA) antigen and suppressed the secretion of plasminogen activator inhibitor-1 (PAI-1) antigen to the conditioned medium. The former two factors also increased u-PA antigen in the cell surface. Transforming growth factor beta (TGF-beta) enhanced u-PA antigen, but its activity was suppressed due to the increased secretion of PAI-1. The binding assay of [125I]DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a Kd of 5.51 nM and Bmax of 0.92 x 10(5) binding sites/cell. PTH or PGE2 increased Bmax 1.4-fold and enhanced the u-PA receptor (u-PAR) mRNA level 1.4-fold or 2.4-fold, respectively. However, TGF-beta did not alter either the Kd or u-PAR mRNA level. Thus, pericellular fibrinolytic activity by u-PA/u-PAR and PAI-1 is modulated by bone resorbing factors.

Bone Resorption↗
PubMed1993

Regulation of scu-PA secretion and u-PA receptor expression in osteoblast-like cells.

The production of proteolytic enzymes by osteoblasts is considered important for initiating osteoclastic bone resorption. Using the established cell line NY as an example of osteoblast-like cells, the effect of intracellular cyclic AMP (cAMP) and protein kinase C (PKC) on plasminogen activator secretion and its specific binding to the cells were investigated. HT-1080 cells were used as the control. NY cells predominantly secrete single-chain urokinase-type plasminogen activator (scu-PA) and some two-chain u-PA. Both scu-PA and u-PA were present in the cell surface and cell lysate of NY cells, and their distribution in HT-1080 cells was quite similar to that of NY cells. Exposing cells to phorbol myristate acetate (PMA) or dibutyryl cyclic AMP (db cAMP) enhanced the secretion of scu-PA and two-chain u-PA, whereas 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) decreased scu-PA secretion, indicating that it is enhanced by protein kinase C (PKC) as well as by cAMP in NY cells. On the other hand, in HT-1080 cells, PMA decreased the level of two-chain u-PA secretion into the conditioned medium. The binding assay of 125I-DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a Kd of 2.23 nM and Bmax of 0.82 x 10(6) binding sites/cell. PMA however, altered neither the Kd nor the Bmax. Dibutyryl cAMP increased the Bmax 1.9 fold. Thus, NY cells secrete u-PA and express specific binding sites on the cell surface, which are modulated by cAMP and PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP↗
PubMed1993

[Molecular biology of tissue-type plasminogen activator (t-PA) and clinical application of recombinant t-PA].

Activation of blood fibrinolysis is initiated by the activation of plasminogen to plasmin by plasminogen activator (PA). The plasmin thus produced can degrade fibrin, the main component of thrombus. Tissue-type PA (t-PA) which is mainly secreted from vascular endothelial cells possesses a high affinity for fibrin in contrast to urokinase-type PA (u-PA). Enzymatic activity of t-PA is expressed in either single-chain form or two-chain form. Further, t-PA activity enhanced markedly in the presence of fibrin; 600-1,000-fold increase. Thus, the thrombolytic therapy is now being performed by using t-PA. The mechanism for thrombolysis by t-PA involves the activation of fibrinolytic system on the fibrin surface. The kringle 2 domain of t-PA molecule plays an important role on the binding to fibrin. But in single-chain t-PA, finger domain plays a role for its binding to fibrin. Recent studies have revealed that the t-PA specific receptor (t-PAR) is expressed on the endothelial cells which localizes t-PA activity by fixing t-PA around the cells.

Amino Acid Sequence↗
PubMed1993

[Mechanism of thrombolytic enzymes].

Thrombolytic enzymes belong to plasminogen activator (PA) in blood fibrinolysis and love been used for the efficient thrombolysis is achieved by the administration of PA which has high affinity for fibrin and induces plasminogen activation on the fibrin surface. PA with lower affinity for fibrin can activate plasminogen in circulating plasma, which causes bleeding tendency. At present, 11 PAs which are available for clinical usage or under investigation, are classified into two types: direct-type PA and indirect-type PA. The former which cleaved plasminogen directly and produces plasmin, includes urokinase-type PA, tissue-type PA, single-chain u-PA, bat PA, mutant PA, hybrid PA, IgG carrying PA and bispecific monoclonal antibody. The latter has no biological function by itself and expresses PA activity after forming a complex with plasminogen. This group includes streptokinase, anisoylated plasminogen streptokinase activator complex and staphylokinase. The PA activity of staphylokinase/plasminogen complex is inhibited by alpha 2-plasmin inhibitor, but can be expressed on the fibrin surface. Thus, PA activity of this complex is hardly observed in plasma. These feature of staphylokinase resembles to tissue-type PA.

Amino Acid Sequence↗
PubMed1992

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the finger domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7-9, 10-14, 15-19, 28-33, and 37-42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37-42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37-42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37-42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37-42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Amino Acids↗
PubMed1992

Tissue-type plasminogen activator, type 1 plasminogen activator inhibitor and their complex in plasma with disseminated intravascular coagulation.

The levels of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1), and t-PA/PAI-1 complex antigens were analyzed in the plasma of disseminated intravascular coagulation (DIC) patients and healthy controls. Other fibrinolytic parameters such as the levels of plasminogen, alpha 2-antiplasmin (alpha 2-AP), plasmin/alpha 2-AP (PAP), and D-dimer were also estimated to clarify the fibrinolytic states in these plasmas. The antigens of t-PA, PAI-1, and t-PA/PAI-1 complex were found to increase from 8.5 +/- 4.3, 54.4 +/- 21.2, and 8.6 +/- 3.5 ng/ml in normal plasma to 36.4 +/- 25.1, 106.8 +/- 54.7, and 46.6 +/- 34.5 ng/ml in DIC plasma, respectively. The molar ratio of total t-PA to total PAI-1 was 1:6 and 1:3 in normal plasma and DIC plasma, respectively, indicating an enhanced fibrinolytic state in the DIC plasma. The DIC plasma revealed a significant consumption of plasminogen (62.1 +/- 27.8%), and alpha 2-AP (63.7 +/- 25.3%) and an increase in PAP (2.6 +/- 2.7 micrograms/ml) and D-dimer (3.9 +/- 10.7 micrograms/ml). These results suggest that the production and secretion of t-PA and PAI-1 from endothelial cells were enhanced in DIC, resulting in an increased t-PA/PAI-1 complex with dominant fibrinolytic activity.

Disseminated Intravascular Coagulation↗
PubMed1992

Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells.

The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.

Aminocaproic Acid↗
PubMed1992

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Tissue-type plasminogen activator and its inhibitor in human glomerulonephritis.

We carried out an immunohistochemical study of tissue-type plasminogen activator (PA) and urokinase-type PA, and their inhibitors, PA inhibitor-1 and PA inhibitor-2, using renal biopsy specimens obtained from 86 patients with various forms of glomerulonephritis. The controls were four normal renal tissue specimens. On immunofluorescent observation, granular staining for tissue-type PA was found to be distributed along the glomerular capillary walls. The fluorescence was weak in the normal renal tissue and occasionally intense in the tissues of patients with IgA nephritis, minimal change nephrotic syndrome, and lupus nephritis. PA inhibitor-1 was abundant in the glomerular epithelial cells and scarce in the mesangial area and glomerular capillary lumens of the normal renal tissues. This was confirmed by immunoelectron microscopy using gold staining. The fluorescence of PA inhibitor-1 was weaker in some specimens of nephritic tissues than in the normal renal tissues. Urokinase-type PA and PA inhibitor-2 were negative within the glomeruli in all the specimens. In the glomerulonephritic tissues which were fibrin deposition-positive, tissue-type PA expression in the glomeruli tended to be strong. An association between fibrin deposition and PA inhibitor-1 staining was not clear. These data suggest that expression of tissue-type PA in the glomeruli increases in association with fibrin deposition.

Fibrin↗
PubMed1992

Cerebral plasminogen activator activity in spontaneously hypertensive stroke-prone rats.

BACKGROUND AND PURPOSE: In the spontaneously hypertensive stroke-prone rat, it is unclear whether plasminogen activator plays a role in the development of stroke. The present study was undertaken to investigate brain levels of plasminogen activator activity in spontaneously hypertensive stroke-prone rats and Wistar-Kyoto rats. METHODS: Plasminogen activator was purified from the brains of rats of both strains. The purification involved used ammonium sulfate precipitation, gel filtration, a zinc chelate-Sepharose column, and a concanavalin A-Sepharose column. Fraction I (0.15 M KCl-soluble fraction) and fraction II (2 M KCl plus 6 M urea-soluble fraction) were purified from both strains. RESULTS: Total plasminogen activator activity in the original homogenates for fractions I and II derived from spontaneously hypertensive stroke-prone rats was increased to twice the level found in Wistar-Kyoto rats. The final product purified from fractions I and II in both strains of rats revealed single bands of plasminogen activator activity on enzymatic analysis with a molecular weight of 72,000. The purified product had stronger S-2288 amidolytic activity than S-2444 amidolytic activity, and it also displayed fibrin-binding ability. CONCLUSIONS: The study demonstrated that there is an increased content of plasminogen activator in the brains of spontaneously hypertensive stroke-prone rats that might be related to the development of stroke.

Animals↗