[Neurological symptoms associated with hypercortisolism].
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Animals↗
Biomedical subjects
A Yoshioka
Publications and source records attributed to A Yoshioka.
Observed decline in the rate of death among Japanese hemophiliacs infected with HIV-1.
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HIV Infections↗
Role of factor VIII C2 domain in factor VIII binding to factor Xa.
Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.
Amino Acid Sequence↗
Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions.
Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca(2+)]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca(2+) indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca(2+)]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca(2+)]i) to green (high [Ca(2+)]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s(-1), no change in [Ca(2+)]i was observed during translocation of platelets, but [Ca(2+)]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca(2+)]i elevation. The intracellular Ca(2+) chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca(2+)]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca(2+)]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.
Blood Platelets↗
Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow.
We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s(-1)). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (>/=1, 210 s(-1)) but normal thrombus formation under low shear (</=340 s(-1)). When shear rate was increased stepwise to 1,500 s(-1) during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s(-1). However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s(-1), whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s(-1) during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an "initiator and promoter," whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a "stabilizer" against heightened shear forces that could lead to peeling off of platelets from the surface.
Blood Coagulation Disorders↗
Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients.
Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope.
Absorption↗
Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow.
We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.
Animals↗
Tympanoplasty in a patient with hemophilia B.
We report the case of a 36-year-old hemophilia B who suffered from cholesteatoma and underwent tympanoplasty. Though the factor IX activity was less than 1% of normal before surgery, adequate replacement of factor IX led to the achievement of hemostasis during and after surgery. The cholesteatoma was completely extirpated with matrix, and a type I canal-up tympanoplasty was subsequently performed. Careful preoperative evaluation and close cooperation with the hematologist are required if surgery is to be successful. We also present here the use of continuous administration of monoclonal antibody-purified factor IX concentrate, Christmassin M.
Adult↗
Histocompatibility antigens and alleles in Japanese haemophilia A patients with or without factor VIII antibodies.
We carried out human leukocyte antigen (HLA)-A, B, Cw, DR and DQ serological typing and HLA-DQA1, DQB1, DRB1 and DPB1 genetic typing for 46 Japanese haemophilia A patients, including 20 who had developed an antibody to factor VIII. It appears that anti FVIII inhibitor formation is associated with the major histocompatibility complex in Japanese haemophilia A patients. Absence of HLA-A24 is a principal risk factor for inhibitor formation in Japanese haemophilia A patients. As supplemental risk factors, HLA-DR4.1, DQ4 and DQA1*0301=2 are positively associated with patients exhibiting inhibitor compared with normal subjects. This and previous studies show that the association between HLA antigens and the formation of inhibitor depends on race. Data of HLA typing may be useful for the recognition of groups at high risk for the possible formation of inhibitor among Japanese haemophilia A patients.
Alleles↗
Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments.
Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWE All these fragments completely inhibited botrocetin-induced VWF binding to GPIb alpha at a final concentration of 40-200 microM. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 microM in the following order: 315 kD Fr > or = 220 kD Fr >> 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 microM, and their IC50s were 13-39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1-loop fragment is important for optimal molecular interaction with GPIb alpha under high shear stress.
Humans↗
Identification of a factor VIII peptide, residues 2315-2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys2326 and Glu2327 for maximum effect.
Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3-C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy-terminal region of the C2 domain (residues 2288-2332). We found one peptide (2315-2330, L9) which neutralized the anti-FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173-2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218-2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315-2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg2320 and Cys2326 or Glu2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.
Amino Acid Sequence↗
Management of acute metabolic decompensation in maple syrup urine disease: a multi-center study.
BACKGROUND: Therapeutic modalities in acute metabolic decompensation in maple syrup urine disease (MSUD) are variable, and outcomes of each therapeutic measure have been known only individually. Factors that affect neurological outcome are not clear. METHODS: A questionnaire was sent throughout Japan to each pediatrician treating any of the 42 MSUD patients. RESULTS: Necessary information was available for 13 patients through the questionnaire, and through a publication for one patient. In nine of the 14 patients episodes of metabolic decompensation developed in the neonatal period. In the other five, the onset of disease was delayed until infancy or later. In the nine patients with neonatal onset, a pretreatment level of plasma leucine greater than 40 mg/100 mL or a duration of altered level of alertness longer than 10 days was associated with a poor neurological outcome. The therapeutic measures employed included intravenous infusion of glucose and electrolyte solution or hypertonic glucose and electrolyte solution, exchange transfusion, peritoneal dialysis, a large dose of thiamine and intravenous hyperalimentation. All patients had survived the episodes and were alive at the time of the survey. Five of the nine patients with neonatal onset have developed neurological sequelae to varying degrees. Episodes of metabolic decompensation in infancy or thereafter did not affect, or only minimally affected, the neurological outcome. CONCLUSION: Therapeutic goals to improve neurological outcome are to shorten the duration of the altered level of consciousness, and to minimize the peak plasma leucine level as much as possible.
Acute Disease↗
Four cases of bleeding diathesis in children due to congenital plasminogen activator inhibitor-1 deficiency.
Congenital plasminogen activator inhibitor-1 (PAI-1) deficiency is an extremely rare disorder characterized by a bleeding diathesis that begins in childhood due to hyperfibrinolysis as a result of decreased PAI-1 activity. We now present 4 unrelated pediatric cases of congenital PAI-1 deficiency. All 4 patients had a history of recurrent episodes of subcutaneous bleeding beginning in early childhood. These episodes were characterized by abnormal prolonged bleeding after trauma, tooth extraction, and surgical procedures, as well as by rebleeding following initial hemostasis. The 2 female patients both had symptoms compatible with hypermenorrhea. The family history was positive in 2 of the 4 patients. Hemostatic screening studies in all 4 patients revealed no abnormalities. Testing for factor XIII antigen, von Willebrand factor antigen, ristocetin cofactor activity, alpha(2)-plasmin inhibitor (alpha2PI) activity, and plasminogen activity was normal. The euglobulin lysis times were shortened in all cases as compared with those in normal control subjects. None of the patients had elevated tissue plasminogen activator (tPA) antigen levels, but PAI activity was markedly decreased in all cases. Three of the patients also had reduced levels of PAI-1 antigen. There tended to be a reduction in tPA-PAI-1 complex in all cases. In addition, 2 patients had elevated PIC (plasmin-alpha2PI complex). Tourniquet tests were performed in 2 patients, with no appreciable rise in PAI-1 activity or PAI-1 antigen levels. The administration of tranexamic acid clearly improved hemorrhagic symptoms in these patients. We considered PAI-1 deficiency to be the likely etiology of the congenital bleeding diatheses in these 4 cases.
Adolescent↗
Immunogold labelling of cytokines in glomeruli in children with various renal diseases.
Precise localization of cytokines such as transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and IL-6 was observed in glomeruli using immunogold electron microscopy in 21 children with various types of renal diseases. The distribution pattern of these cytokines, as well as immunoglobulins, C3c and fibrinogen (Fg), was essentially confined to the electron-dense deposits (EDDs) regardless of their location. Frequency of positive labelling of each cytokine was different among various types of renal disorder, that is, TGF-beta was found mainly in lupus nephritis (LN), membranous nephropathy and IgA nephropathy, TNF-alpha in LN, and IL-1 in Henoch-Schönlein purpura nephritis. IL-6 was detected only in 1 case of LN. TNF-alpha was also found in the cytoplasm of glomerular epithelial cells. Furthermore, in order to evaluate the relation of cytokines to mesangial expansion, extracellular matrix components such as type IV collagen, laminin and fibronectin were stained. The result was that there was no significant correlation between the signal intensity or distribution pattern of cytokines and that of extracellular matrix components. These findings indicate that these cytokines could be associated with the formation of EDDs together with immunoglobulins, C3c and Fg. The involvement of each cytokine in renal pathophysiology might also depend upon the type of renal disease. They also raise the possibility that the glomerular epithelial cells might produce or absorb TNF-alpha. However, these results did not show significant correlation between cytokine involvement and mesangial expansion.
Biopsy↗
Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome.
We measured the plasma levels of adrenomedullin (AM), a novel vasodilating peptide, in 89 patients with various forms of systemic inflammatory response syndrome (SIRS) and 13 healthy volunteers serving as controls. Plasma levels of AM in SIRS (burns: 20.5 +/- 3. 2 fmol/ml [mean +/- SEM]; pancreatitis: 13.8 +/- 3.8 fmol/ml; trauma: 14.9 +/- 2.5 fmol/ml; traumatic shock: 41.1 +/- 7.8 fmol/ml; severe sepsis: 59.9 +/- 11.2 fmol/ml; septic shock: 193.5 +/- 30.1 fmol/ml) were significantly increased over those of controls (5.1 +/- 0.2 fmol/ml). The patients with traumatic shock or septic shock especially had higher levels of plasma AM than those with trauma or severe sepsis, respectively. These data showed that in patients with SIRS, plasma AM levels increased in proportion to the severity of illness. Subsequently, we measured the plasma levels of mediators such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, plasminogen activator inhibitor (PAI)-1, and thrombomodulin (TM) in patients with traumatic shock and septic shock. A significant correlation was observed between plasma AM and TNF-alpha levels in patients with septic shock, suggesting an important role for AM as well as of TNF-alpha in the pathophysiology of inflammation. Plasma AM and IL-8 levels correlated positively with Acute Physiology and Chronic Health Evaluation (APACHE) II score, peak multiple organ failure (MOF) score during the first month and prognosis in patients with septic shock, as did plasma IL-6 levels in patients with traumatic shock. The plasma AM level might serve as a useful marker for evaluating the severity of disease and as an early predictor of subsequent organ failure and outcome in septic shock.
APACHE↗
[Ibudilast prevents oxygen-glucose deprivation-induced oligodendroglial injury].
Previously we have demonstrated that ibudilast, which is used clinically for treating patients with asthma and cerebrovascular diseases, prevents excitotoxicity of oligodendroglial lineage mediated by Ca2+ influx via non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) channels. We here present a finding that ibudilast prevents oxygen-glucose deprivation (OGD)-induced oligodendroglial injury. The oligodendrocyte-like cells (OLC), differentiated from the CG-4 cell line established from rat oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, were exposed to hypoxia in the absence of glucose for 12 h and subsequent reoxygenation for 2 h. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. OGD for 12 h induced 30 to 50% LDH release into the medium. OLC damage induced by deprivation of oxygen and glucose was prevented by ibudilast at concentrations of > or = 50 microM. The protection given by ibudilast against OGD-induced injury was enhanced by prostacyclin (PGI2). OGD-induced OLC injury was prevented by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), an inhibitor of non-NMDA GluR or deprivation of Ca2+ from culture medium. While ibudilast increased intracellular cAMP at concentrations of > or = 10 microM, at least 100 microM concentrations were needed to increase intracellular cGMP. Therefore, we concluded that ibudilast prevented OGD-induced oligodendroglial injury possibly by increasing intracellular cAMP which modulates Ca2+ influx via non-NMDA GluR channels.
Animals↗
Utilization of previously treated patients (PTPs), noninfected patients (NIPs), and previously untreated patients (PUPs) in the evaluation of new factor VIII and factor IX concentrates. Recommendation of the Scientific Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis.
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Factor IX↗
Identification of the binding site for an alloantibody to von Willebrand factor which inhibits binding to glycoprotein Ib within the amino-terminal region flanking the A1 domain.
An alloantibody to von Willebrand factor (vWF) which developed in a Japanese boy with type 3 von Willebrand disease has been characterized. The antibody was non-precipitating IgG and the main subclasses were IgG2 and IgG4. The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its predominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryptic fragment of vWF (residues 449-728). No reaction was seen, however, with either a 39/34 kDa dispase fragment (480-718) or a recombinant vWF fragment (residues 465-728). These findings suggested that the essential epitope resided in the amino-terminal flanking region of the Al domain. We synthesized overlapping peptides corresponding to the region containing D3/A1 boundary. A peptide, residues 458-472, bound to the antibody and dose-dependently blocked the antibody binding to the 52/48 kDa fragment. The same peptide neutralized the inhibitory effect of the alloantibody on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which reacted with the 52/48 kDa fragment. Furthermore, the specific component of the antibody, directed against residues 458-472, blocked vWF binding to GPIb in absence of exogenous agonist. Our results suggest that the region flanking the Al domain plays an important role in regulating vWF binding to GPIb.
Antibody Specificity↗