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Biomedical subjects

Yoshihiko Sakurai

Publications and source records attributed to Yoshihiko Sakurai.

18 recordsLinked to original sources
PubMed2007

Heparin-induced inhibitory effects of a prothrombin complex concentrate on global tests of haemostasis.

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.

Blood Coagulation Factors↗
PubMed2006

Successful use of recombinant factor VIII devoid of von Willebrand factor during multiple teeth extractions in a patient with type 3 von Willebrand disease.

We report a 55-year-old patient with type 3 von Willebrand disease who underwent multiple tooth extractions with successful hemostatic management using recombinant factor VIII. The patient was previously misdiagnosed and treated incorrectly then at 53 years old, he was diagnosed with type 3 von Willebrand disease. As he had avoided dental treatments for two decades due to severe bleeding after dental extraction, multiple severe caries and marginal periodontitis were revealed. The patient refused the use of blood products in hemostatic management because he was afraid of blood-borne diseases and development of anti-von Willebrand factor alloantibodies. After close consultation, we therefore decided to use recombinant factor VIII. Four teeth extraction procedures were executed twice. Before extraction, bolus recombinant factor VIII (50 IU/kg) was administered intravenously followed by continuous infusion (5-10 IU/kg per h) for approximately 48 h. The factor VIII:C level increased from about 1 to 20-32% 30 min after bolus infusion. During continuous infusion (10 IU/kg/h), factor VIII:C was maintained at more than 10%. Little bleeding occurred during and after the multiple teeth extractions and during suture removal. On frequent examinations during a 1-year follow-up, neither von Willebrand factor nor factor VIII inhibitors were detected.

Blood Loss, Surgical↗
PubMed2006

Molecular characterization of an arthritogenic collagen peptide interacting with I-Ar.

Collagen-induced arthritis (CIA) is an autoimmune arthritis that can be elicited by the immunization of genetically susceptible strains of mice with type II collagen (CII). We have analysed the molecular interactions that occur between an arthritogenic T-cell determinant CII (442-457) and the murine class II susceptibility allele I-A(r). To determine which amino acid residues within the CII (442-457) sequence are responsible for binding to I-A(r), a soluble I-A(r):IgG2aFc fusion protein-peptide binding assay was developed. Various concentrations of analogue peptides were tested for their ability to compete with biotinylated CII (607-622) for binding to I-A(r), thereby establishing a relative comparison of the binding affinities among these analogues. Analogue peptides with substitutions at positions 447 (Ala --> Val), 448 (Gly --> Ala) and 451 (Gly --> Ala) bound poorly to the I-A(r) molecule. These data suggest that positions 447, 448 and 451 on CII are the major anchor points to I-A(r) molecules. In cytokine assays, only substitutions within positions 445-454 decreased the interferon-gamma production by T cells. These data narrow the core of the arthritogenic T-cell determinant to CII (445-454). Identification of the molecular interactions involved in T-cell recognition of CII should lead to antigen-specific means of inhibiting autoimmune arthritis.

Animals↗
PubMed2006

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro.

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.

Blood Platelets↗
PubMed2006

Analog peptides of type II collagen can suppress arthritis in HLA-DR4 (DRB1*0401) transgenic mice.

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in diarthrodial joints. We have previously established that mice transgenic for the human DR genes associated with RA are susceptible to collagen-induced arthritis (CIA) and we have identified a determinant of type II collagen (CII(263-270)) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII(263-270) would suppress disease in DR1 transgenic mice. Because the immunodominant determinant is the same for both DR1 transgenic and DR4 transgenic mice, we attempted to determine whether the analog peptide that was suppressive in DR1 transgenic mice would also be effective in suppressing CIA in DR4 transgenic mice. We treated DR4 transgenic mice with two analog peptides of CII that contained substitutions in the core of the immunodominant determinant: CII(256-276) (F263N, E266D) and CII(256-270) (F263N, E266A). Mice were observed for CIA, and T-cell proliferative responses were determined. Either peptide administered at the time of immunization with CII significantly downregulated arthritis. Binding studies demonstrated that replacement of the phenylalanine residue in position 263 of the CII peptide with asparagine significantly decreased the affinity of the peptide for the DR4 molecule. In contrast, replacement of the glutamic acid residue in position 266 with aspartic acid or with alanine had differing results. Aspartic acid reduced the affinity (35-fold) whereas alanine did not. Both peptides were capable of suppressing CIA. With the use of either peptide, CII(256-276) (F263N, E266D) or CII(256-270) (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-gamma. We have identified two analog peptides that are potent suppressors of CIA in DR4 transgenic mice. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of HLA-DR4 that can suppress autoimmune arthritis.

Amino Acid Substitution↗
PubMed2005

Adenovirus-mediated transfer of human placental ectonucleoside triphosphate diphosphohydrolase to vascular smooth muscle cells suppresses platelet aggregation in vitro and arterial thrombus formation in vivo.

BACKGROUND: Platelet-rich thrombus formation is a critical event in the onset of cardiovascular disease. Because ADP plays a significant role in platelet aggregation, its metabolism is important in the regulation of platelet activation and recruitment. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is a key enzyme involved in vascular ADP metabolism. We recently isolated 2 isoforms of E-NTPDase from the human placenta. The present study examined whether these isoforms suppress platelet aggregation and thrombus formation after adenovirus-mediated gene transfer to vascular smooth muscle cells (SMCs). METHODS AND RESULTS: We constructed adenovirus vectors expressing human placental E-NTPDase isoforms I (AdPlac I) and II (AdPlac II) or bacterial beta-galactosidase (AdLacZ). Vascular SMCs infected with AdPlac I expressed significant NTPDase activity and inhibited the platelet aggregation induced by ADP and collagen in vitro. In contrast, SMCs infected with AdPlac II and AdLacZ did not exert antiplatelet effects. To investigate the antithrombotic and antiproliferative effects of placental E-NTPDase isoform I in vivo, we generated thrombosis in rat carotid arteries by systemically administered rose Bengal and transluminal green light 5 days after gene transfer and examined neointimal growth 3 weeks after thrombus formation. Blood flow in AdLacZ-infected arteries rapidly deteriorated and vanished within 96+/-18 seconds of occlusive thrombus formation. In contrast, blood flow in AdPlac I-infected arteries was preserved for at least 10 minutes during irradiation. In addition, thrombus formation and subsequent neointimal growth were obviously suppressed. CONCLUSIONS: The local expression of placental E-NTPDase in injured arteries might prevent arterial thrombosis and subsequent neointimal growth.

Adenosine Triphosphatases↗
PubMed2005

Highly conserved antigenic structure of the factor VIII C2 domain in some mammals.

To elucidate differences in the antigenic structure of factor VIII (FVIII) among mammals, we evaluated cross-reactivities of well-defined antihuman FVIII antibodies with canine and other mammalian FVIII proteins. Monoclonal antibodies against human FVIII recognizing the A1 domain in the heavy chain and the A3 domain in the light chain showed 2.7% and 0% cross-reactivities, respectively, with canine FVIII. The cross-reactivities of 2 alloantibodies and a monoclonal antibody that recognized the A2 domain in the heavy chain were 10%, 38%, and 0%, respectively. On the other hand, 2 kinds of alloantibodies and a monoclonal antibody recognizing the C2 domain in the light chain showed 160%, 390%, and 130% cross-reactivity, respectively, with canine FVIII. The anti-C2 monoclonal antibody (NMC-VIII/5) showed a type 2 inactivating property when tested with canine and human plasma. Moreover, cross-reactivities of NMC-VIII/5 with simian and feline FVIII were 54.5% and 82.8%, respectively, while the cross-reactivities of the anti-A2 monoclonal antibody (JR8) with simian and feline FVIII were 1.3% and 0%, respectively. These findings suggest that the antigenic structure of the C2 epitope has remained relatively conserved throughout mammalian evolution in contrast to the A2 epitope and that a canine model of hemophilia A is useful for FVIII inhibitor experiments.

Animals↗
PubMed2004

Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome.

Early-onset sarcoidosis (EOS) and inheritable Blau syndrome (BS) share characteristic clinical features of juvenile-onset systemic granulomatosis syndrome that mainly affects skin, joints, and eyes. However, no direct evidence has been shown for the possible common origin of these 2 diseases. Recent discovery of CARD15 mutations in BS families encouraged us to investigate similar CARD15 mutations in EOS patients. Among 10 EOS cases retrospectively collected in Japan, heterozygous missense mutations were found in 9 cases; 4 showed a 1000C>T (R334W in amino acid change) that has been reported in BS, 4 showed novel 1487A>T (H496L), 1538T>C (M513T), 1813A>C (T605P), and 2010C>A (N670K), and 1 case showed double 1146C>G (D382E)/1834G>A (A612T) mutations on different alleles. All 6 of these variants of CARD15 showed increased basal nuclear factor (NF)-kappaB activity. These findings indicate that the majority of EOS and BS cases share the common genetic etiology of CARD15 mutations that cause constitutive NF-kappaB activation.

Age of Onset↗
PubMed2004

A critical role for thrombin in platelet aggregation under high shear stress.

The serine protease, thrombin, plays a crucial role in both coagulation and platelet activation. Anhydrothrombin (AhT) is a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine. AhT retains affinity for natural substrates of thrombin and may be a competitive inhibitor of thrombin-mediated coagulation and platelet reactions. In the present study, thrombelastography showed that AhT not only delayed the onset and the progress of the coagulation process but impaired clot strength, indicating that AhT may have both anticoagulant and antiplatelet activity. In addition, AhT prolonged the activated partial thromboplastin time dose-dependently, but had little effect on the prothrombin time, suggesting that its principal activity was mediated in the intrinsic coagulation pathway. AhT inhibited thrombin-induced aggregation of platelet-rich plasma. Complete inhibition of aggregation was evident at a concentration of 1.85 microM AhT. Furthermore, 3.7 microM of AhT almost completely abolished shear-induced platelet aggregation in PRP. Interpretation of this in vitro study requires confirmation in vivo, but the findings suggest that thrombin plays a critical role in shear related platelet mechanisms. AhT may be a useful tool for investigating platelet-based coagulation reactions and may provide the basis for a novel class of antithrombotic agents.

Anticoagulants↗
PubMed2004

An analog peptide that suppresses collagen-induced arthritis.

The authors undertook the identification of peptides capable of altering the immune response to type II collagen (CII) in the context of HLA-DR, as suppressing the immune response to CII could clarify the role of CII autoimmunity in the pathogenesis of disease. To produce synthetic peptides with the potential of disrupting the DR1-restricted immune response, synthetic analog peptides were developed that contain site-directed substitutions in critical positions. When these analog peptides were used to treat collagen-induced arthritis in DR1 transgenic mice, an analog peptide, CII 256-276 (N, D), was identified that inhibited T-cell responses in vitro. The data from studies with this analog peptide establish that CII 256-276 (N, D) is a potent suppressor of the DR-mediated immune response to CII and that its effect is mediated, at least in part, by interleukin-4. An analog peptide of CII recognized by T cells in the context of a human major histocompatibility complex molecule should have therapeutic significance for autoimmune arthritis.

Amino Acid Sequence↗
PubMed2004

Association of anti-idiotypic antibodies with immune tolerance induction for the treatment of hemophilia A with inhibitors.

BACKGROUND AND OBJECTIVES: Hemophilia A patients with inhibitors can be treated effectively with immune tolerance induction therapy (ITI). One of the underlying mechanisms of ITI is conceived to be a neutralizing activity of anti-idiotypic antibodies on inhibitors. The goal of the present study was to develop an uncomplicated method for assessing antiidiotypic antibodies and to prove the advent of anti-idiotypic antibodies in ITI. DESIGN AND METHODS: We studied a total of 26 plasma samples obtained from 9 hemophilic inhibitor patients who were treated with ITI. The samples were investigated with a novel method for detecting anti-idiotypic antibodies based on a liquid phase blocking immunoprecipitation. RESULTS: Plasma anti-factor VIII (FVIII) antibody titer was reduced by adding plasma from patients who had received completely successful ITI. This anti-FVIII antibody-neutralization activity of the plasma was impaired by treating the plasma with protein G beads. In addition, treating inhibitor plasma from patients in whom ITI had been unsuccessful with FVIII affinity beads resulted in the development of the anti-FVIII antibody-neutralization activity. Furthermore, the anti-FVIII antibody-neutralization activity of anti-FVIII antibody-depleted plasma obtained in a late period of ITI on inhibitor plasmas obtained during ITI increased over time. INTERPRETATION AND CONCLUSIONS: Our results suggest that; (i) plasma from patients in whom ITI was completely successful contained an anti-FVIII antibody-neutralization factor; (ii) the anti-FVIII antibody-neutralization factor was in the IgG fraction (i.e., the factor would be anti-idiotypic antibodies), and (iii) anti-idiotypic antibodies existed even in plasma from patients in whom ITI was unsuccessful. Our observations support the notion that the mechanism of ITI is associated with the development of anti-idiotypic antibodies.

Antibodies, Anti-Idiotypic↗
PubMed2003

Anticoagulant activity of M-LAO, L-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX.

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.

Agkistrodon↗
PubMed2003

Replacement therapy with plasma-derived factor VIII concentrates induces skew in T-cell receptor usage and clonal expansion of CD8+ T-cell in HIV-seronegative hemophilia patients.

Replacement therapy with factor VIII (FVIII) products causes immune abnormalities in human immunodeficiency virus (HIV)-seronegative hemophilia patients. However, the question remains why an absolute increase in the number of CD8+ T-cells and diminished proliferation responses of lymphocytes to antigen stimulation in vitro occurs in HIV-seronegative hemophilia patients. To examine whether the FVIII products induce skewing of T-cell receptor (TCR) repertoires, TCR variable region alpha-chain and beta-chain repertoires were analyzed for peripheral blood mononuclear cells (PBMCs) from 15 hemophilia patients treated with heated and/or non-heated plasma-derived FVIII concentrates and 10 age-matched healthy adults. Also, T-cell clonality was compared between these groups using complementarity-determining region 3 (CDR3) size spectratyping. The skewing of TCR repertoires was significantly greater for hemophilia patients than healthy controls. The extent of T-cell clonality was greater for hemophilia patients than the controls, indicating that clonal T-cells frequently expanded in hemophilia patients. The skew in TCR usage and clonal expansion were primarily observed in patients treated with non-heated plasma-derived products. The spectratyping and sequencing of CDR3 regions revealed that the clonal expansion of T-cells was observed for CD8+ T-cells, but not CD4+ T-cells. These results suggest that extensive expansion of CD8+ T-cells is induced by some viruses other than HIV present in FVIII preparations, and the resulting accumulation of CD8+ T-cells is responsible for changes in peripheral T-cell population in HIV-seronegative hemophilia patients.

Adolescent↗
PubMed2002

A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C-catalyzed inactivation.

The protective effect of von Willebrand factor (VWF) toward activated protein C (APC)-catalyzed inactivation of factor VIII (FVIII) has been attributed mainly to inhibition of FVIII binding to phospholipid. In the present study, we demonstrated that VWF-mediated FVIII protection from APC also results from direct inhibition of FVIII binding to APC. Inhibition of FVIII binding to anhydro-APC by VWF would be consistent with partial or complete overlap of the FVIII binding sites for APC and VWF. We examined, therefore, the inhibitory effects of 6 synthetic peptides spanning residues 1996 to 2028 around the previously localized APC binding region (FVIII residues 2009-2018). Peptide 2009 to 2018 inhibited FVIII binding to anhydro-APC by 83% (50% inhibition, 55 microM). Similarly, peptide 2013 to 2022 inhibited FVIII binding to VWF by 84% (50% inhibition, 25 microM). It was also found that peptides 2009 to 2018 and 2013 to 2022 optimally bound to anhydro-APC and VWF, respectively. A rabbit antipeptide IgG, raised against peptide 2009 to 2022, blocked the binding of both anhydro-APC and VWF to FVIII. This immunoglobulin G inhibited proteolytic cleavage of FVIII by APC. Our results indicate that the essential regions for the binding of APC and VWF to FVIII overlap and that the protective effect of VWF on APC-catalyzed FVIII inactivation includes competitive inhibition of APC binding to FVIII by VWF.

Amino Acid Sequence↗
PubMed2002

Peptide-induced suppression of collagen-induced arthritis in HLA-DR1 transgenic mice.

OBJECTIVE: To identify peptides capable of altering the immune response to type II collagen (CII) in the context of HLA-DR. METHODS: Immunizing mice transgenic for the human HLA-DRB1*0101 immune response gene with CII elicits an arthritis (collagen-induced arthritis [CIA]) that resembles rheumatoid arthritis. We have previously identified an immunodominant determinant of CII, CII (263-270), recognized by T cells in the context of DR1. To produce synthetic peptides with the potential of disrupting the DR1-restricted immune response, synthetic analog peptides were developed that contain site-directed substitutions in critical positions. These peptides were used to treat CIA in DR1 transgenic mice. RESULTS: An analog peptide, CII (256-276, N(263), D(266)), that inhibited T cell responses in vitro, was identified. When DR1 mice were coimmunized with CII and CII (256-276, N(263), D(266)), the incidence and severity of arthritis were greatly reduced, as was the antibody response to CII. Moreover, CII (256-276, N(263), D(266)) was effective in down-regulating the immune responses to CII and arthritis, even when administered 2 weeks following immunization with CII. Spleen and lymph node cells from CII-immunized mice cultured with CII (256-276, N(263), D(266)) in vitro produced increased amounts of interleukin-4 (IL-4) compared with cells cultured with the wild-type peptide, CII (256-276). Furthermore, CII (256-276, N(263), D(266)) was incapable of preventing arthritis in DR1 IL-4(-/-) mice (genetically deficient in IL-4). CONCLUSION: These data establish that CII (256-276, N(263), D(266)) is a potent suppressor of the DR-mediated immune response to CII. Its effect is mediated, at least in part, by IL-4. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of a human major histocompatibility complex molecule that can suppress autoimmune arthritis.

Animals↗
PubMed2002

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

Antibody Specificity↗
PubMedDate not supplied

Ecthyma gangrenosum-like lesions in a healthy child after infection treated with antibiotics.

Ecthyma gangrenosum is a cutaneous infection associated most commonly with pseudomonal sepsis in the immunocompromised patient. We describe a previously healthy 4-year-old boy who developed ecthyma gangrenosum-like lesions secondary to antibiotic treatment for possible streptococcal infection. The skin, ears, and extremities were involved. This presentation emphasizes the importance of awareness of the rare complication of ecthyma gangrenosum-like lesions associated with non-Pseudomonas bacterial infection treated with antibiotics, even in a previously healthy child.

Acute Disease↗