18 results on '"Morio Arai"'
Search Results
2. Evaluation of platelet function under high shear condition in the small-sized collagen bead column
- Author
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Kaneo Satoh, Yukio Ozaki, Makoto Kaneko, Morio Arai, Toshiro Takafuta, Yutaka Yatomi, and Olga Cuyun-Lira
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Blood Platelets ,Platelet Aggregation ,Platelet Function Tests ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet membrane glycoprotein ,Antibodies ,Pathology and Forensic Medicine ,chemistry.chemical_compound ,Thromboxane A2 ,Von Willebrand factor ,hemic and lymphatic diseases ,von Willebrand Factor ,Von Willebrand disease ,medicine ,Humans ,Platelet ,Platelet activation ,Ristocetin ,Blood Coagulation ,Platelet-poor plasma ,Chromatography ,biology ,Chemistry ,General Medicine ,Platelet Activation ,medicine.disease ,Platelet Glycoprotein GPIb-IX Complex ,Biochemistry ,Biophysics ,biology.protein ,Collagen ,Rheology ,Shear Strength - Abstract
We previously reported that platelet retention rates as measured with collagen-coated bead columns (the conventional column) reflect the processes of platelet adhesion and aggregation under low shear stress, and that this system could serve as an easy-to-use platelet aggregometry. With this column, platelet glycoprotein (GP) VI and GPIIb/IIIa, but not the GPIb-von Willebrand factor (VWF) interaction, play major roles in platelet activation. To develop a system that can better reflect the GPIb-VWF interaction under high shear stress, we designed a column containing small-sized beads (125-212 microm) coated with porcine collagen type I. As expected, the GPIb-VWF interaction played a crucial role in platelet retention rates at higher flow rates. Adenosine 5'-diphosphate, but not thromboxane A2, appears to support platelet activation in this system. The platelet retention rates among healthy individuals with the new columns are in the range wider than the conventional columns, and this diversity could be attributed to the broad range of the VWF antigen and/or its activity. It is suggested that this new column can serve as an easy-to-use method for evaluating the VWF antigen levels and its activity and for monitoring patients with thrombotic or bleeding disorders related to the VWF-GPIb interaction.
- Published
- 2005
3. Excision of pseudotumour in a patient with haemophilia A and inhibitor managed with recombinant factor VIIa
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H. Takedani, N. Kawasaki, Morio Arai, Akira Yoshioka, Hiroyuki Naka, Y. Abe, and S. Mikami
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Male ,medicine.medical_specialty ,Haemophilia A ,Factor VIIa ,Postoperative Hemorrhage ,Thigh ,Hemophilia A ,Granuloma, Plasma Cell ,Bolus (medicine) ,Muscular Diseases ,medicine ,Humans ,Adverse effect ,Genetics (clinical) ,Blood Coagulation Factor Inhibitors ,biology ,Coagulants ,business.industry ,Factor VIII inhibitor ,Hematology ,General Medicine ,medicine.disease ,Hemostasis, Surgical ,Recombinant Proteins ,eye diseases ,Surgery ,medicine.anatomical_structure ,Recombinant factor VIIa ,Anesthesia ,Hemostasis ,Granuloma ,biology.protein ,business - Abstract
We describe a patient with haemophilia A and factor VIII inhibitor who underwent surgical excision of a large pseudotumour in the left femoral region. Haemostasis was successfully maintained with bolus doses of recombinant factor VIIa at 2-h intervals and anti-fibrinolytic therapy, and the pseudotumour was removed. Subsequently, the dose interval was gradually prolonged to 8 h for a total of 18 days. Although a spontaneous haemorrhage was observed on postoperative day 8, haemostasis was achieved by reducing the dosage interval. No adverse event occurred during the course of treatment.
- Published
- 2004
4. Mechanisms of platelet retention in the collagen-coated–bead column
- Author
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Toshiro Takafuta, Kayoko Ohtsuki, Katsue Suzuki-Inoue, Kaneo Satoh, Yukio Ozaki, Yutaka Yatomi, Morio Arai, Masatoshi Ohnishi, Makoto Kaneko, and Olga Cuyun-Lira
- Subjects
Platelet Membrane Glycoprotein IIb ,Prostacyclin ,Platelet Membrane Glycoproteins ,Pharmacology ,Pathology and Forensic Medicine ,Thromboxane A2 ,chemistry.chemical_compound ,Platelet Adhesiveness ,Von Willebrand factor ,Platelet adhesiveness ,von Willebrand Factor ,medicine ,Von Willebrand disease ,Humans ,Platelet ,biology ,Receptors, Purinergic P2 ,Chemistry ,General Medicine ,medicine.disease ,Adenosine Diphosphate ,Platelet Glycoprotein GPIb-IX Complex ,Biochemistry ,Platelet-rich plasma ,biology.protein ,Collagen ,GPVI ,Platelet Aggregation Inhibitors ,medicine.drug - Abstract
Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.
- Published
- 2003
5. Clinical Trial to Investigate the Pharmacokinetics, Pharmacodynamics, Safety, and Efficacy of Recombinant Factor VIIa in Japanese Patients With Hemophilia With Inhibitors
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Yoko Minamoto, Tetsuhito Kojima, Midori Shima, Shigeru Fujita, Hideji Hanabusa, Katsuyuki Fukutake, Morio Arai, Akira Shirahata, Hiroyuki Naka, Hidehiko Saito, Junki Takamatsu, H. Tagami, Tadashi Kamiya, Akira Yoshioka, and Junji Kamizono
- Subjects
Adult ,Adolescent ,Factor VIIa ,Hemophilia A ,Japan ,Pharmacokinetics ,Isoantibodies ,medicine ,Coagulopathy ,Humans ,Adverse effect ,Prothrombin time ,medicine.diagnostic_test ,biology ,business.industry ,Hematology ,Middle Aged ,medicine.disease ,Recombinant Proteins ,Therapeutic Equivalency ,Consumer Product Safety ,Recombinant factor VIIa ,Anesthesia ,Pharmacodynamics ,Hemostasis ,biology.protein ,business ,Partial thromboplastin time - Abstract
A multicenter and open-labeled clinical trial of human recombinant factor VIIa (rFVIIa) was conducted in Japanese patients with severe hemophilia A or B with inhibitors. The trial consisted of 2 parts. In study 1, the pharmacokinetics, pharmacodynamics, and safety of a single dose of 120 microg/kg of rFVIIa were investigated in 8 patients. In the subsequent study 2, the hemostatic effect and safety of rFVIIa were evaluated during a 24-week period in 10 patients. In study 1, the mean maximum FVII-coagulant activity (FVII:C) was found to occur after 10 minutes; activity then decreased rapidly and returned to the baseline within 24 hours after a single intravenous infusion of rFVIIa. The mean half-life of FVII:C was 3.5 hours. The activated partial thromboplastin time and prothrombin time in the patients were immediately shortened but returned to the baseline within 24 hours after dosing. In study 2, 86 microg/kg to 120 microg/kg of rFVIIa (mean, 97 microg/kg) was administered 1 to 85 times to 10 patients. A total of 58.0% (91/157) of bleeding episodes were treated excellently or effectively, with 5 (3.2%) ineffective episodes. There was no apparent trend in the relationship of the hemostatic effect with bleeding sites, mean dose, or number of injections. The efficacy rate, however, was significantly higher (90.0%) in bleeding episodes treated within 3 hours than in those treated at longer intervals (31.0%). No treatment-related adverse events were observed, and there was no evidence of antibody formation to rFVIIa. In conclusion. rFVIIa is an effective and well-tolerated option for treatment of bleeding episodes in hemophilia patients with inhibitors.
- Published
- 2001
6. BIACORETM, a System for Biomolecular Interaction Analysis Using Surface Plasmon Resonance
- Author
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Morio Arai
- Subjects
chemistry.chemical_compound ,Von Willebrand factor ,biology ,chemistry ,biology.protein ,Phospholipid ,Biophysics ,Surface plasmon resonance ,Biosensor - Published
- 1997
7. Analysis of Free Factor VIII Antigen in Commercial Factor VIII Concentrates
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Kagehiro Amano, Katsuyuki Fukutake, Morio Arai, Jun Watanabe, and Kazuhiko Kagawa
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biology ,medicine.drug_class ,Chemistry ,Arbitrary unit ,Size-exclusion chromatography ,Monoclonal antibody ,Molecular biology ,Thrombin ,Von Willebrand factor ,hemic and lymphatic diseases ,Hemostasis ,medicine ,biology.protein ,Factor VIII antigen ,medicine.drug ,Peroxidase - Abstract
The association of factor VIII with von Willebrand factor (vWF) is important for protection of factor VIII against proteolytic degradation in plasma. Recently developed factor VIII preparations, namely monoclonal antibody-purified factor VIII (M-factor VIII) and recombinant factor VIII, contain free form of factor VIII that is expected to form complexes with endogenous vWF after venous injection in the patients with hemophilia A. To clarify the difference of free factor VIII and factor VIII-vWF complex in the commercial factor VIII concentrates, we developed a quantitative assay for free factor VIII in which the sample was added to the wells of vWF-coated microtiter plates. Bound factor VIII was detected by monoclonal antibody against factor VIII, followed by incubation with peroxidase conjugated anti-mouse IgG, then the substrate ABTS for measuring absorbance at 405nm. When the amount of free factor VIII antigen in one unit factor VIII activity of recombinant factor VIII was defined as one arbitrary unit, the ELISA detected free factor VIII as low as 0.016 unit/ml. Factor VIII was incubated with vWF or plasma from a patient with severe Hemophilia A for various concentrations, prior to the free factor VIII assay. At saturation, the stoichiometry was one factor VIII molecule per 50vWF monomers. Of the products of factor VIII-vWF complex concentrate, no free factor VIII was detected. While a M-factor VIII concentrate contained free factor VIII comprising 24% of total factor VIII activity. In a gel filtration experiment of M-factor VIII, 15% of the total factor VIII was eluted as free factor VIII and 77% of that was co-eluted with vWF in the void volume. The free factor VIII and the complex form of factor VIII were immunoisolated and were analyzed after SDS-PAGE by immunoblotting. The factor VIII molecular structure and the susceptibility of thrombin cleavage were indistinguishable between the two forms of factor VIII. These results suggested that free factor VIII present in the M-factor VIII concentrate forms complexes with vWF in hemophilic plasma and is involved in physiologic hemostasis.
- Published
- 1997
8. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts
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Kazuko Iida, Naomasa Yamamoto, Mizuho Kaneda, Akitada Ichinose, Kohji Kasahara, Masayoshi Souri, Yoshiko Ohno-Iwashita, Mitsuhiro Abe, Ikuo Kawashima, Toshihide Kobayashi, Motoyuki Shimonaka, Toshiaki Miki, Morio Arai, Soichi Kojima, Naoko Sekino-Suzuki, Toshiro Okazaki, and Hidenori Suzuki
- Subjects
Blood Platelets ,Immunology ,Clot Retraction ,Gene Expression ,Transferases (Other Substituted Phosphate Groups) ,macromolecular substances ,Clot retraction ,Platelet Glycoprotein GPIIb-IIIa Complex ,Myosins ,Fibrinogen ,Biochemistry ,Fibrin ,Cell membrane ,Mice ,Thrombin ,Membrane Microdomains ,medicine ,Animals ,Humans ,Platelet ,Blood Coagulation ,Mice, Knockout ,biology ,Factor XIII ,Cell Biology ,Hematology ,Impaired clot retraction ,Cell biology ,Sphingomyelins ,Protein Transport ,medicine.anatomical_structure ,biology.protein ,lipids (amino acids, peptides, and proteins) ,medicine.drug ,Signal Transduction - Abstract
Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.
- Published
- 2013
9. A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease
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Hiroshi Inaba, Katsuyuki Fukutake, Morio Arai, Keiko Nagaizumi, Takeshi Hagiwara, Shinichi Yoshida, and Hideji Hanabusa
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Genetics ,biology ,Point mutation ,Nonsense mutation ,Hematology ,medicine.disease ,HaeIII ,Von Willebrand factor ,Polymorphism (computer science) ,hemic and lymphatic diseases ,Mutation (genetic algorithm) ,biology.protein ,Von Willebrand disease ,medicine ,Missense mutation ,medicine.drug - Abstract
SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.
- Published
- 1996
10. Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor
- Author
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K. Fukutake, Morio Arai, Kagehiro Amano, Kazuhiko Kagawa, and Suzuki T
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congenital, hereditary, and neonatal diseases and abnormalities ,animal diseases ,Immunoglobulin light chain ,Binding, Competitive ,Epitope ,law.invention ,Epitopes ,Von Willebrand factor ,Antibody Specificity ,law ,hemic and lymphatic diseases ,von Willebrand Factor ,Humans ,IC50 ,Autoantibodies ,C2 domain ,Factor VIII ,biology ,Chemistry ,Hematology ,Virology ,Molecular biology ,Recombinant Proteins ,Titer ,Immunoglobulin G ,biology.protein ,Recombinant DNA ,Antibody - Abstract
SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.
- Published
- 1996
11. Efficiency of intermittent prophylactic factor XIII subsitution in factor XIII a subunit deficiency
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Hidetaka Fukue, Yasuyuki Yamamoto, Masatoshi Hattori, Daisaku Sugimura, Katsumasa Koike, Tsuyoshi Oishi, Morio Arai, Shinichi Yoshida, and Katsuyuki Fukutake
- Subjects
Bleeding episodes ,biology ,business.industry ,Protein subunit ,Clinical manifestation ,Factor XIII ,Fibrin ,In vitro analysis ,Hemostasis ,Anesthesia ,biology.protein ,Medicine ,Every Four Weeks ,business ,medicine.drug - Abstract
Four patients with congenital factor XIII (FXIII) a subunit deficiency were treated with prophylatic FXIII substitution at a dose of 1, 000 or 1, 250 units every four weeks for 3 to 12 years to avoid the recurrence of severe bleeding, especially intracranial bleeding, which all had experienced. We evaluated the efficacy of this prophylactic therapy on the basis of both the clinical manifestation of hemostasis and of in vitro analysis of fibrin crosslinking in patient's plasma. Although the FXIII dosage was increased from 7, 837±4, 593units/yr to 12, 699±1, 336units/yr after the start of prophylaxis, the number of bleeding episodes were remarkably decreased from 4.15±1.54/yr to 0.23±0.19/yr with no life-threatening hemorrhage. Both γ-dimer and α-polymer formation was shown in fibrin obtained from patient's plasma taken 30 minutes after the administration of 1, 000units of FXIII, and γ-dimer crosslinking was still observed in fibrin from the plasma taken four weeks after the administration. These data suggest that intermittent prophylactic FXIII administration is valuable in the treatment of patients with congenital FXIII a subunit deficiency.
- Published
- 1996
12. Autoantibody to factor VIII that has less reactivity to factor Vlll/von Willebrand Factor Complex
- Author
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Kazuhiko Kagawa, Kagehiro Amano, Yasuharu Nishida, Morio Arai, Kimihito Koshihara, Katsuyuki Fukutake, and Takashi Suzuki
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Adult ,Hemolytic anemia ,Anemia, Hemolytic ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,animal diseases ,Immunoglobulin light chain ,law.invention ,Von Willebrand factor ,law ,hemic and lymphatic diseases ,Internal medicine ,von Willebrand Factor ,medicine ,Humans ,Autoantibodies ,Factor VIII ,biology ,Chemistry ,Autoantibody ,Hematology ,medicine.disease ,Endocrinology ,Monoclonal ,biology.protein ,Recombinant DNA ,Female ,Immunoglobulin Light Chains ,Antibody ,Autoimmune hemolytic anemia - Abstract
To determine the difference in reactivity of factor VIII (FVIII) inhibitor to FVIII/von Willebrand Factor (vWF) complex and FVIII free of vWF, an autoantibody to FVIII light chain was tested. A patient (1-3) suffered from autoimmune hemolytic anemia with autoantibody to FVIII. Epitope specificity of the patient's IgG (I-3 IgG) was shown to be the C2 domain of FVIII light chain (2170-2332) by Western blotting using recombinant FVIII deletions expressed in Escherichia coli. The inhibitory effect on FVIII procoagulant activity (VIII:C) of I-3 IgG was tested against a conventional FVIII concentrate; Haemate P, a monoclonal antibody-purified FVIII concentrate; Hemofil M, and a recombinant FVIII (rFVIII); Kogenate. I-3 IgG showed only 1.3 BU/mgIgG for Haemate P, in contrast to 20 BU/mgIgG for both Hemofil M and Kogenate. The ratio of VIII:C/vWF:Ag in Haemate P and Hemofil M was 1/3.43 and 1/0.01, respectively, while Kogenate did not contain vWF. The inhibitory effect of the I-3 IgG was then compared with Kogenate and its complex with vWF. The inhibitory effect was decreased against the rFVIII by forming a complex with vWF from 22 BU/mgIgG to 0.5 BU/mgIgG. Fab from the I-3 IgG had the same effect. In addition, vWF showed a protective effect on FVIII inactivation by the I-3 IgG in a dose dependent manner. Fifty-nine percent of residual VIII:C was retained in the presence of 8 U/ml of vWF after 1 hr incubation with I-3 IgG. These results suggested that vWF could compete with the I-3 IgG for binding to FVIII.
- Published
- 1995
13. Inhibitory Effect of Iodinated Contrast Media on Blood Coagulation System in vitro
- Author
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Tetsuya Yamagishi, Katsuyuki Fukutake, and Morio Arai
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medicine.medical_specialty ,biology ,medicine.diagnostic_test ,Chemistry ,Antithrombin ,Heparin ,Pharmacology ,Fibrin ,Iopamidol ,Surgery ,Thrombin ,Ioversol ,medicine ,biology.protein ,Iohexol ,circulatory and respiratory physiology ,medicine.drug ,Partial thromboplastin time - Abstract
This paper evaluates the effect of iodinated X-ray contrast media on plasma clotting time, plasma thrombin generation and thrombin activity. Investigated materials consisted of diatrizoate, iothalamate and ioxaglate as ionic contrast media (ICM), and iopamidol, iohexol, ioversol and iotrolan as nonionic contrast media (NICM). Both the prothrombin time and the activated partial thromboplastin time of normal pooled plasma were markedly prolonged in the presence of contrast media in a dose-dependent manner. All the contrast media also inhibited thrombin generation in plasma, as assessed in the def ibrinated plasma with a chromogenic substrate, S-2238. Although these contrast media did not affect the thrombin amidolytic activity, they interfered with thrombin-clotting of fibrinogen by affecting either thrombin-fibrinogen interaction or fibrin polymerization, or both. The anticoagulant activities of the contrast media were more evident in 3 ICM than in 4 NICM used in this study. These results suggested that NICM could not sufficiently suppress the thrombin generation in the catheter-syringe system because of this lesser degree of anticoagulant activity than that of ICM. In a separate set of experiment, heparin cofactor activity of antithrombin III was not impaired in the presence of these contrast media, suggesting that heparin would be an effective agent to prevent the thromboembolic complications during angiography using NICM.
- Published
- 1995
14. Heterogenous Expression of Glycoprotein lb, IX and V in Platelets from Two Patients with Bernard-Soulier Syndrome Caused by Different Genetic Abnormalities
- Author
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Toshiro Takafuta, Kenjiro Tanoue, T Fujimoto, Morio Arai, Naomasa Yamamoto, Kingo Fujimura, Akira Suehiro, Eizo Kakishita, Masaaki Noda, Atsushi Kuramoto, Takeshi Shimomura, and A. Shimasaki
- Subjects
Genetics ,biology ,Point mutation ,Nonsense mutation ,Glycoprotein IX ,Hematology ,medicine.disease ,Molecular biology ,Bernard–Soulier syndrome ,Frameshift mutation ,Transmembrane domain ,Glycoprotein Ib ,biology.protein ,medicine ,Coding region - Abstract
SummaryBernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder, which is caused by deficiency or decrease of the platelet GPIb/IX/V complex. Analysis of two patients with BSS by How cytometry of the blood revealed different expression patterns of the components of the GPIb/IX/V complex. In case 1, GPIX was completely absent but residual amounts of GPIbα and GPV were detectable; in case 2, GPIbα was completely absent. We amplified the coding regions of GPIbα, GPIbß, GPV, and GPIX from the patients’ genomic DNA with the polymerase chain reaction (PCR) and sequenced the PCR products. In case 1, we identified a point mutation in the GPIX coding region that changes the codon for tryptophan-126 (TGG) to a nonsense codon (TGA). In case 2, we found a deletion of nucleotide within seven adenine repeats at the position of 1932 to 1938 in the coding region of GPIbα, which causes a frame shift that results in 58 altered amino acids and a premature stop codon. These genetic changes alter the transmembrane domain of GPIX or GPIbα and, therefore, would prevent proper insertion of the proteins in the plasma membrane. Thus, abnormality of a single component protein (GPIX or GPIbα) alters the assembly of the GPIb/IX/V complex and causes heterogenous surface expression of GPIbα, GPV and GPIX.
- Published
- 1995
15. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions
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Noriko Tamura, Shunnosuke Handa, Morio Arai, Kumi Kodama, Hiroshi Takayama, and Shinya Goto
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Adult ,Blood Platelets ,Male ,Platelet Membrane Glycoproteins ,Platelet membrane glycoprotein ,chemistry.chemical_compound ,Von Willebrand factor ,Physiology (medical) ,von Willebrand Factor ,Medicine ,Humans ,Platelet ,Platelet activation ,Thrombus ,Phosphorylation ,Ristocetin ,Whole blood ,biology ,business.industry ,Receptors, IgG ,Thrombosis ,medicine.disease ,Platelet Activation ,Molecular biology ,chemistry ,Immunology ,biology.protein ,Female ,Collagen ,Stress, Mechanical ,Cardiology and Cardiovascular Medicine ,business ,Type I collagen - Abstract
Background — We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Methods and Results — Whole blood obtained from 2 patients with GP VI–deficient platelets and the effects of the Fab of anti–GP VI antibody (Fab/anti–GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor γ-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s −1 ) or high (1500 s −1 ) wall shear rate, either in the case of blood containing GP VI–deficient platelets or in the presence of Fab/anti–GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI–associated Fc receptor γ-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI–deficient platelets. Conclusions — GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.
- Published
- 2002
16. Factor VIII Ise (R2159C) in a patient with mild hemophilia A, an abnormal factor VIII with retention of function but modification of C2 epitopes
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Shogo Morichika, Morio Arai, Edward G. D. Tuddenham, Masakazu Inoue, Seiki Kamisue, Midori Shima, K. Fukutake, Kazuhiko Kagawa, Akira Yoshioka, Hiroaki Nakai, koren Gale, Hiroshi Suzuki, and Ichiro Tanaka
- Subjects
Male ,congenital, hereditary, and neonatal diseases and abnormalities ,Antigenicity ,Adolescent ,medicine.drug_class ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,Arginine ,Hemophilia A ,Polymerase Chain Reaction ,Epitope ,Isoantibodies ,Epitopes ,Antigen ,hemic and lymphatic diseases ,von Willebrand Factor ,Medicine ,Humans ,Point Mutation ,Deamino Arginine Vasopressin ,Amino Acid Sequence ,Cysteine ,Polymorphism, Single-Stranded Conformational ,DNA Primers ,Factor VIII ,biology ,Base Sequence ,business.industry ,Antibodies, Monoclonal ,Hematology ,Exons ,Molecular biology ,Titer ,Monoclonal ,Immunology ,biology.protein ,Antibody ,business - Abstract
SummaryWe found a patient with mild hemophilia A who had no detectable factor VIII antigen (FVIII:Ag), as shown by two-site ELISA using inhibitor alloantibodies (TK). We then analyzed A2, A2/B, and C2 antigen of the patient's DDAVP-induced FVIII using several anti-FVIII monoclonal antibodies. Factor VIII activity (FVIII : C) was increased from 12 to 42 Uldl by the administration of DDAVP. The DDAVPinduced increases in the A2 and A2/B antigens were 40 and 36 Uldl, respectively. However, the increase in the C2 antigen was only 7.5 Uldl. SSCP analysis and subsequent sequencing demonstrated an Arg to Cys transition at codon 2159. The anti-FVII1:C titer of monoclonal antibody, NMC-VIII15 which recognized the C2 domain, against normal plasma was 450 Bethesda Ulmg of IgG. However, the titer against DDAVP-treated patient's plasma was only 15 Bethesda Ulmg. We also tested DDAVP-induced increase in the FVIII : Ag in another mild hemophilia A patient with the same mutation at Arg2159. Increase in his C2 antigen levels was only 19% of those in the A2 and A2/B antigen. We designate this abnormal FVIII as FVIII Ise. Our results show that a missense mutation at Arg2159 to Cys modifies the antigenicity of the C2 domain.
- Published
- 1997
17. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI
- Author
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Morio Arai, Hiroshi Takayama, Naomasa Yamamoto, Tatsuo Ichinohe, Yasuharu Ezumi, Minoru Okuma, and Hoyu Takahashi
- Subjects
Adult ,Integrins ,Receptors, Collagen ,Platelet Aggregation ,Integrin ,Proto-Oncogene Proteins pp60(c-src) ,Syk ,Cell Cycle Proteins ,macromolecular substances ,Platelet Membrane Glycoproteins ,Platelet membrane glycoprotein ,Biochemistry ,Collagen receptor ,chemistry.chemical_compound ,Proto-Oncogene Proteins ,Humans ,Syk Kinase ,Phosphotyrosine ,Proto-Oncogene Proteins c-vav ,Molecular Biology ,Cells, Cultured ,Enzyme Precursors ,biology ,Chemistry ,Phospholipase C gamma ,Microfilament Proteins ,Intracellular Signaling Peptides and Proteins ,Tyrosine phosphorylation ,Cell Biology ,Protein-Tyrosine Kinases ,Phosphoproteins ,Cell biology ,Enzyme Activation ,Isoenzymes ,Focal Adhesion Kinase 1 ,Focal Adhesion Protein-Tyrosine Kinases ,Type C Phospholipases ,Cancer research ,biology.protein ,Phosphorylation ,Collagen ,GPVI ,Cell Adhesion Molecules ,Cortactin ,Proto-oncogene tyrosine-protein kinase Src ,Signal Transduction - Abstract
Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha2 beta1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha2 beta1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha2 beta1. In response to collagen, these platelets exhibited alpha2 beta1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.
- Published
- 1997
18. Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid
- Author
-
D Scandella, L W Hoyer, and Morio Arai
- Subjects
Macromolecular Substances ,Immunoblotting ,Phospholipid ,Enzyme-Linked Immunosorbent Assay ,Phosphatidylserines ,Immunoglobulin light chain ,Binding, Competitive ,Epitope ,chemistry.chemical_compound ,Antigen ,Isoantibodies ,hemic and lymphatic diseases ,Humans ,Antigens ,Binding site ,Phospholipids ,Autoantibodies ,C2 domain ,Blood coagulation test ,Factor VIII ,biology ,Chemistry ,General Medicine ,Biochemistry ,biology.protein ,Binding Sites, Antibody ,Blood Coagulation Tests ,Antibody ,Research Article - Abstract
Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (less than 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid.
- Published
- 1989
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