Akira Yoshioka
Nara Medical University
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Featured researches published by Akira Yoshioka.
Hepatology | 2005
Kazuo Ohashi; Jacob M. Waugh; Michael D. Dake; Takashi Yokoyama; Hiroyuki Kuge; Yoshiyuki Nakajima; Masaki Yamanouchi; Hiroyuki Naka; Akira Yoshioka; Mark A. Kay
Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole‐organ transplantation or liver‐directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth‐Holm‐Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth‐Holm‐Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naïve liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naïve liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders. (HEPATOLOGY 2005;41:132–140.)
Journal of Thrombosis and Haemostasis | 2006
Tomoko Matsumoto; Midori Shima; Masahiro Takeyama; Koichi Yoshida; Ichiro Tanaka; Yoshihiko Sakurai; Alan R. Giles; Akira Yoshioka
Summary.u2002 Background:u2002Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B.Materials and methods:u2002Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII‐ and FIX‐deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity‐purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height.Results:u2002Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0–0.1u2003IUu2003dL−1) of both factors.Conclusions:u2002These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.
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.
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
International Journal of Hematology | 2007
Keiji Nogami; Midori Shima; John C. Giddings; Masahiro Takeyama; Ichiro Tanaka; Akira Yoshioka
Some factor VIII (FVIII) inhibitor alloantibodies block FVIII binding to von Willebrand factor (VWF) and phospholipid (PL) and recognize a C2 domain epitope that overlaps both binding sites. We previously showed that FVIII peptide 2315-2330 neutralized FVIII inhibitors and that Cys2326 and Glu2327 contributed to the maximum neutralizing effect. In the present study, we investigated the relationship between the essential binding sites for VWF, PL, and anti-C2 inhibitors by means of competitive-inhibition assays with overlapping synthetic peptides that span the C terminus of the C2 domain (residues 2288-2332). We identified 2 peptides (residues 2303–2317 and 2315–2330) that specifically blocked FVIII binding to VWF or PL by approximately 80% (50%-inhibitory concentration [IC50], 9.0 μM) and 95% (IC50, 0.12 ?M), respectively. To examine in detail the residues responsible for PL binding, we prepared mutants of peptide 2315-2330 in which we sequentially substituted each residue with Gly. Two residues, Ile2317 and Met2321, were shown to be essential for PL binding. Their substitution with Gly reduced the inhibitory effect by >90%.The data suggest that the binding sites for VWF, PL, and anti-C2 inhibitors in the C2 domain are in very close proximity but are not identical.
Blood Coagulation & Fibrinolysis | 2007
Masahiro Takeyama; Yoshihiko Sakurai; Midori Shima; Tomoko Matsumoto; Keiji Nogami; Ichiro Tanaka; Tomohiro Takeda; John C. Giddings; Akira Yoshioka
Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.
Liver Transplantation | 2005
Saiho Ko; Ichiro Tanaka; Hiromichi Kanehiro; Hideki Kanokogi; Jun-ichi Ori; Midori Shima; Akira Yoshioka; Alan R. Giles; Yoshiyuki Nakajima
The cause of hemophilia is deficiency of coagulation factor VIII production in the liver, which can be cured by liver transplantation. Because the hepatic function of hemophilia patients is quite normal except for production of factor VIII, auxiliary partial orthotopic liver transplantation (APOLT) is beneficial in that patient survival is secured by preserving native liver even in the event of graft loss. However, it is not known whether the graft of APOLT would be enough to cure hemophilia. We evaluated the efficacy and feasibility of APOLT for hemophilia in a canine hemophilia A model that we established. Partial left liver graft was taken from the normal donor (blood factor VIII activity > 60%). The graft was transplanted to the hemophilia beagle dog (blood factor VIII activity < 5%) after resection of the left lobe preserving native right lobe. Changes in time of blood factor VIII activity and liver function parameters were observed after APOLT. APOLT and perioperative hemostatic management were successfully performed. The blood factor VIII activity increased to 30% after APOLT, and was sustained at least 6 weeks throughout the observation period without symptoms of bleeding. The result demonstrated sustained production of factor VIII in the hemophilia recipient after APOLT. Transplantation of approximately one third of whole liver resulted in cure of hemophilia. In conclusion, it is suggested that APOLT would be feasible as a curative treatment of hemophilia A to improve quality of life of the patients. (Liver Transpl 2005;11:579–584.)
Journal of Thrombosis and Haemostasis | 2006
Hiroshi Suzuki; Midori Shima; K. Nogami; Yoshihiko Sakurai; Katsumi Nishiya; Evgueni L. Saenko; Ichiro Tanaka; Akira Yoshioka
Summary.u2002 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 670u2003nmol 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.
International Journal of Hematology | 2007
Yoshihiko Sakurai; Hiroyuki Sugimoto; Koichi Yoshida; Ichiro Tanaka; Midori Shima; Yasuhito Tanaka; Maiko Takeda; Akitaka Nonomura; Akira Yoshioka
We present a 16-year-old boy with mild hemophilia A who developed superficial fibromatosis mimicking subcutaneous hematoma of the right upper dorsal region of the foot. This condition is very rare. Hematoma originally developed after the patient sprained the right ankle. Although cast immobilization and replacement therapy with factor VIII (FVIII) concentrates reduced the symptoms, the swelling and pain reappeared in the same region 4 weeks after the cast was removed. A clinical diagnosis of intramuscular hemorrhage was made. Despite readministration of regular FVIII concentrates and cast immobilization, the patient experienced a relapse after removal of the second cast. Magnetic resonance imaging demonstrated a tumor in the extensor digitorum brevis muscle of the right foot with high intensity on T2-weighted images. Marginal excision resulted in symptom resolution. A histopathologic examination of the tissue showed proliferation of fibroblastic cells with abundant collagen bundles. When a mass is found under the skin in hemophilia A patients, an etiology other than bleeding can be overlooked because subcutaneous hemorrhage in limited areas often results in a mass; however, the possibility of rare soft tissue tumors in hemophilia patients should be kept in mind.
International Journal of Hematology | 2007
Masahiro Takeyama; Shogo Kasuda; Yoshihiko Sakurai; Midori Shima; Tomohiro Takeda; Shoko Omura; Hiroyuki Naka; Akira Yoshioka
Although the efficacy of recombinant factor VIII (rFVIII) in the treatment of type 3 von Willebrand disease (VWD) has been reported, the mechanisms by which FVIII concentrates devoid of von Willebrand factor (VWF) induce improvements in hemostasis are poorly understood. To address the role of FVIII or intrinsic coagulation in the absence of VWF, we performed a hemostatic analysis. Blood samples were obtained before and after the administration of rFVIII to 2 patients with type 3 VWD. A rotating thromboelastometry assay was performed to examine global interactions in hemostasis. Studies of thrombin-and shear-induced platelet aggregation were also conducted to elucidate the effect on platelet activation. Furthermore, we assessed the rise in the thrombin-induced intracellular concentration of free calcium ([Ca2+]i). Addition of rFVIII to preinfusion blood in vitro corrected thromboelastometric parameters and thrombin-induced aggregation. In ex vivo studies, thromboelastometry analysis showed that rFVIII shortened the onset and progression of the coagulation process. Furthermore, rFVIII corrected low shear-induced and thrombin-induced platelet aggregation in platelet-rich plasma. In addition, rFVIII improved thrombin-induced [Ca2+]i flux in washed platelets. Our observations suggested that FVIII is incorporated into platelets to activate them, as well as to act directly in intrinsic coagulation in the absence of VWF. FVIII may play a critical role even in the absence of VWF.