Katsumi Nishiya
Nara Medical University
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Featured researches published by Katsumi Nishiya.
Biochimica et Biophysica Acta | 2003
Yoshihiko Sakurai; Midori Shima; Tomoko Matsumoto; Hideo Takatsuka; Katsumi Nishiya; Shogo Kasuda; Yoshihiro Fujimura; Akira Yoshioka
One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.
Journal of Biological Chemistry | 2007
Keiji Nogami; Midori Shima; Tomoko Matsumoto; Katsumi Nishiya; Ichiro Tanaka; Akira Yoshioka
Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ∼2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ∼12- and ∼3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A137–336 and A2 subunits, by limited proteolysis at Lys36, Arg336, Arg372, and Arg740. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg1689 and Arg1721, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg336 proceeded faster than that at Arg372, in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg336 and Lys36 occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg336 was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg336 and Lys36 appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.
Journal of Biological Chemistry | 2009
Tetsuhiro Soeda; Keiji Nogami; Katsumi Nishiya; Masahiro Takeyama; Kenichi Ogiwara; Yoichi Sakata; Akira Yoshioka; Midori Shima
Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ∼95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182–2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182–2259, demonstrated that peptide 2228–2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228–2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
Haemophilia | 2006
T. Inoue; Midori Shima; Masahiro Takeyama; Tomoko Matsumoto; Katsumi Nishiya; Ichiro Tanaka; Yoshihiko Sakurai; John C. Giddings; Akira Yoshioka
The first line of therapy for acute bleeding in patients with low-responding factor VIII (FVIII) inhibitors is FVIII concentrates (1). Most inhibitors, recognizing the FVIII light chain, inhibit von Willebrand factor (VWF) and phospholipid binding to FVIII (2,3), and appears to be less active in vitro against plasmaderived FVIII concentrates containing VWF (pdFVIII/VWF) than VWF-free FVIII concentrates (4–7). These findings suggest that pdFVIII/VWF might be therapeutically more effective than recombinant FVIII (rFVIII) in patients with FVIII inhibitors. However, no clinical studies supporting this concept have been reported. In the present study, we have compared the recovery of FVIII activity (FVIII:C) after treatment with pdFVIII/VWF and rFVIII for massive intramuscular bleeding that occurred during regular infusion of FVIII for immune tolerance induction (ITI) therapy in a young male haemophilia A patient with an inhibitor. Immune tolerance induction therapy was commenced in our patient at the age of 9 years (18 October 1999) with the administration of 100 U kg of rFVIII (Recombinate; Baxter Healthcare Corp., Westlake Village, CA, USA) daily for 3 weeks, followed by infusions three to four times a week. Inhibitor levels fluctuated for 3 years after ITI therapy were initiated (maximum inhibitor level, 152.0 BU mL) and regular infusions of FVIII were continued. The number of bleeding episodes appeared to decline, and since January 2003, the inhibitor level has been kept constant within a low range from 0.9 to 2.1 BU mL. He was admitted into our hospital with severe pain in his right buttock and walking difficulties on 6 January 2004. He had suffered from painful swelling in his right buttock 2 weeks before admission without improvement in spite of daily infusions of FVIII (100 U kg). A subcutaneous haematoma (7 · 8 cm) was evident on the right buttock, with heat sensation, and impaired flexion and extension of the right hip joint. Computer tomography scanning demonstrated a massive intramuscular haematoma, measuring 10 cm in diameter, in the right gluteus maximus and gluteus medius muscles. On admission, 12 h after infusion of 4000 U (87 IU kg) of rFVIII, the FVIII inhibitor titre was 1.7 BU mL. As the bleeding manifestations had not responded to the infusion of rFVIII, 4000 U of activated prothrombin complex concentrate (Feiba Immuno; Baxter Healthcare Corp.) were administered. Nevertheless, the swelling and pain in the right buttock increased. Subsequently, replacement therapy with the same dose of rFVIII (4000 IU) was administered and continued (Fig. 1). Clinical symptoms gradually improved and the patient was discharged on 30 January. Regular prophylaxis in this patient is now maintained using FVIII/VWF concentrate. The inhibitor titre in this patient remained constant, within the range of 1.5–2.0 BU mL, throughout the present series of investigations. Therefore, it was possible to compare the recovery Correspondence: Midori Shima MD, Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara city, Nara 634-8522, Japan. Tel.: +81 744 29 8881; fax: +81 744 24 9222; e-mail: mshima@naramed-u.ac.jp
Blood Coagulation & Fibrinolysis | 2010
Kenichi Ogiwara; Keiji Nogami; Katsumi Nishiya; Midori Shima
Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (≥125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with a ∼two-fold increase of peak level of thrombin generation. Studies using α2-antiplasmin-deficient plasma revealed that much lower dose of Plm (≥16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.
Haemophilia | 2004
Shogo Kasuda; Ichiro Tanaka; Midori Shima; Tomoko Matsumoto; Yoshihiko Sakurai; Katsumi Nishiya; Alan R. Giles; Akira Yoshioka
Summary. We report here the efficacy of factor VIII (FVIII) infusions in two haemophiliacs with inhibitors using clot waveform analysis on the MDA® II system, which was possible to detect very low levels of FVIII activity < 1.0 U dL−1. In the presence of type 1 inhibitors at the level of 6.2 (patient 1) and 14.4 (patient 2) Bethesda Units mL−1, 3.2 and 6.5 U dL−1 of FVIII:C remained 30 min after the infusion of FVIII (100 U kg−1), respectively. Moreover, 0.9 U dL−1 of FVIII:C remained 24 h after infusion in patient 2. In both cases, these changes were reflected by qualitative improvement in the aPTT clot waveform and quantitative changes in the minimum value of the second derivative of the aPTT waveform (Min2) that reflects clot acceleration. These results suggest that FVIII infusion may be continued with clinical benefit in some haemophiliacs with high responding inhibitors. Furthermore, the haemostatic response may be monitored accurately and efficiently by clot waveform analysis.
British Journal of Haematology | 2008
Masahiro Takeyama; Keiji Nogami; Evgueni L. Saenko; Tetsuhiro Soeda; Katsumi Nishiya; Kenichi Ogiwara; Akira Yoshioka; Midori Shima
Protein S functions as an activated protein C (APC)‐independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa‐dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17‐fold increase in Km for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in Km for factor X. Surface plasmon resonance‐based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (Kd; c. 10 nmol/l). Competition binding assays using Glu‐Gly‐Arg‐active‐site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg336 by factor IXa was c. 1·8‐fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down‐regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.
British Journal of Haematology | 2002
Keiji Nogami; Midori Shima; Katsumi Nishiya; Kazuya Hosokawa; Evgueni L. Saenko; John C. Giddings; Ichiro Tanaka; Akira Yoshioka
Summary. Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti‐FVIII C2 antibody (ESH8) with a Val‐2248–Gly‐2285 epitope inhibited FXa‐catalysed FVIII activation, and that a synthetic peptide designated EP‐2 (residues 2253–2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP‐2 on FXa‐catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP‐2 inhibited FXa‐catalysed activation in a clotting assay in a dose‐dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP‐2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose‐dependent manner, but not prothrombin time. Our results indicate that EP‐2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa‐catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.
Thrombosis and Haemostasis | 2009
Masahiro Takeyama; Keiji Nogami; Evgueni L. Saenko; Katsumi Nishiya; Kenichi Ogiwara; Midori Shima
We have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484-509) on A2, and synthetic peptide inhibited the A2 binding to protein S by approximately 60% and approximately 70%, respectively, in solid-phase binding assays. The 484-509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484-509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484-497 peptide, but not by the 498-509 peptide. Furthermore, N-terminal sequence analysis of the 484-509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated approximately four-fold lower affinity than wild-type A2. These results indicate that the 484-509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488-490, contributes to a unique protein S-interactive site.
Journal of Thrombosis and Haemostasis | 2006
Hiroshi Suzuki; Midori Shima; K. Nogami; Yoshihiko Sakurai; Katsumi Nishiya; Evgueni L. Saenko; Ichiro Tanaka; Akira Yoshioka
Summary. Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin‐catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the Kd values of anhydro‐thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L−1, respectively. FV activation was increased by approximately 9‐fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105‐kDa heavy chain and a 71/74‐kDa light chain by thrombin‐catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.