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Yoichi Sakata

Publications and source records attributed to Yoichi Sakata.

31 records · Page 2Linked to original sources
PubMed2004

Latex immunoturbidimetric assay for soluble fibrin complex.

BACKGROUND: Soluble fibrin complex (SFC), composed of fibrin monomer and fibrinogen derivatives, is known to exist in the circulating blood in patients with thrombosis. Its detection and quantification are useful for obtaining information about the condition and degree of intravascular coagulation in early-stage thrombosis, but there is no rapid method to measure SFC in plasma for clinical use. METHODS: We obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay (LIA) using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. RESULTS: The method had an analytical range of 3-300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (CVs <3.0% and <2.0%, respectively). The assay detection limit was <0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10%), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470 000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases [mean (SD), 48.9 (57.6) mg/L; n = 160) were significantly higher than those in plasma from healthy individuals [1.8 (2.1) mg/L; P <0.001; n = 304]. CONCLUSION: In terms of linearity, precision, and sensitivity, the LIA, performed on a Hitachi 911 automated analyzer, may be useful for measurement of SFC in plasma.

Antibodies, Monoclonal↗
PubMed2004

Expression profiles of fibrinolytic components in nasal mucosa.

Components of the fibrinolytic pathway contribute to diverse pathways in many tissues, in addition to their well-recognized role in degradation of fibrin clots. In this study of nasal mucosa, we investigated the presence of mRNA of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), and plasminogen activator inhibitor-2 (PAI-2) using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization, and compared these results with their localization in immunostained tissues. According to real-time RT-PCR results, t-PA, u-PA, PAI-1, and PAI-2 mRNA were noted in human nasal mucosa. Particularly, expression of u-PA and PAI-1 mRNA was significantly high in allergic nasal mucosa in comparison with normal mucosa. t-PA mRNA was detected in endothelial cells and epithelium in normal nasal mucosa. t-PA mRNA was detected in mucous cells of allergic submucosal glands, but not in normal glands. In allergic rhinitis, u-PA and PAI-2 mRNA were detected in mucinous cells and epithelium, and PAI-1 mRNA was detected in serous cells and epithelium. Expression of u-PA and PAI-1 mRNA in normal nasal tissues was decreased in contrast to that in allergic nasal tissues. u-PA staining was observed in mucous cells of allergic submucosal glands and the staining pattern of PAI-2 was similar to that of u-PA. PAI-1 was present in serous cells of submucosal glands from allergy samples, while epithelial cells were almost devoid of stain. In contrast, with allergy, immunohistochemical staining of t-PA was negative in submucosal glands, though positive in endothelial cells and epithelium. However, the expression of t-PA mRNA in allergic nasal mucosa was noted in mucous cells. In fibrin autography of nasal discharge, u-PA was markedly activated in the allergic patient. These results suggest that t-PA synthesized in mucous cells is promptly secreted and modifies watery nasal discharge in allergic rhinitis, and that u-PA activity may help the passage of large amounts of rhinorrhea by also reducing its viscosity. A lot of cellular infiltration (eosinophils in particular) was recognized in allergic nasal mucosa. It is most likely that the modifications in expression of PAs and PAIs are due to the local release of cytokines or growth factors from these inflammatory and immune cells.

Fibrinolysis↗
PubMed2004

Changes in activity of plasma thrombin activatable fibrinolysis inhibitor in pregnancy.

The thrombin activatable fibrinolysis inhibitor (TAFI) is a recently described inhibitor of fibrinolysis. Changes in the concentration of plasma TAFI molecules that are present and functional after activation by thrombin-thrombomodulin complex (TAFI activity) during pregnancy have not previously been studied, and we therefore conducted a present prospective study. Plasma TAFI activity was assayed in 15 healthy non-pregnant women and 176 healthy pregnant women at various stages of gestation. TAFI activity was higher in pregnant than in non-pregnant women. TAFI activity seemed to increase progressively until around 20 weeks of gestation, then maintained a moderately elevated level until delivery, and promptly decreased after delivery.

Adult↗
PubMed2004

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.

Adult↗
PubMed2004

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

BACKGROUND: Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). METHODS: CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. RESULTS: We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. CONCLUSIONS: Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.

Animals↗
PubMed2003

Defective sorting to secretory vesicles in trans-Golgi network is partly responsible for protein C deficiency: molecular mechanisms of impaired secretion of abnormal protein C R169W, R352W, and G376D.

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Animals↗
PubMed2003

Modified Bentall operation in a patient with hemophilia A.

We detail a successful modified Bentall operation in a patient with hemophilia A. A 53-year-old man with mild hemophilia A and a history of few bleeding episodes was diagnosed with annuloaortic ectasia. Surgical repair was planned. Simple bolus infusions of factor VIII concentrate were given before and after cardiopulmonary bypass to achieve 100% blood levels and postoperatively every 12 hours for 7 days to maintain 50% levels. The patient received no transfusion other than 10 units of platelet concentrate. We used routine operative and cardiopulmonary bypass techniques. The patient recovered well postoperatively with no excessive bleeding despite warfarin therapy.

Aortic Aneurysm↗
PubMed2003

In vitro platelet activation by an echo contrast agent.

OBJECTIVE: We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. METHODS: Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5-75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 microL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. RESULTS: The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). CONCLUSIONS: In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.

Adult↗
PubMed2002

A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma.

BACKGROUND: Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF. METHODS: We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody. RESULTS: The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the alpha-chain N-terminal region exposed upon removal of the A peptide from the Aalpha-chain because F405 was found to bind to the alpha-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1-300 microg/ml. The assay detection limit was < 0.5 microg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (< 9.4% and < 10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean +/- S.D. of the SF concentration in the DIC and control samples were 63.4 +/- 65.3 microg/ml and 1.9 +/- 1.0 microg/ml, respectively. CONCLUSIONS: The ELISA using F405 is useful for the diagnosis of DIC.

Animals↗
PubMed2002

Perioperative changes in plasma antithrombin activity and platelet counts in patients undergoing gynecologic surgery.

The relationships among perioperative changes in plasma antithrombin (AT) activity, platelet counts, and clinical risk factors for postoperative thromboembolism has not been studied extensively in gynecologic patients. AT activity and platelet counts were determined preoperatively and on postoperative days 1 and 7 in 74 patients who underwent gynecologic surgery. The relationships between each clinical risk factor for postoperative thromboembolism and the decrease in AT activity and platelet count were analyzed using a univariate logistic regression model. No clinically significant thromboembolism occurred in any patient. AT activity and platelet counts decreased significantly on postoperative day 1 and exceeded preoperative levels on day 7. Postoperative patients with malignancy were more likely to exhibit decreased AT activity. AT activity decreased significantly with increases in operating time and blood loss. Known risk factors for postoperative thromboembolism did not appear to be associated with postoperative decreases in platelet counts. Transient hyperconsumption of AT and platelets occurs immediately after gynecologic surgery. Surgery-induced AT deficiency may contribute to postoperative thromboembolism in patients with long operating times and significant blood loss.

Adult↗
PubMed2002

Structural alterations in hereditary dysfibrinogens.

Dysfibrinogens can be grossly divided in two groups: (1) defective thrombin-catalyzed conversion of fibrinogen molecules to fibrin monomers, and (2) defective fibrin polymerization due to structural alterations in polymerization sites, that include "A" and "a" sites, end-to-end D:D abutment surfaces, and lateral association sites involving the carboxyl terminal region of the fibrin alpha-chain. Recently, a number of mutations in the fibrinogen genes have been identified, and many of these encode changes that occur in regions of fibrinogen that have been elucidated by high-resolution structural studies. Here we focus on the structure-function relationships of fibrinogen that can be inferred from studies involving these abnormal molecules.

Afibrinogenemia↗
PubMed2002

[Acute promyelocytic leukemia accompanied by retinoic acid syndrome with complications of acute myocardial infarction and cerebral infarction during treatment with all-trans retinoic acid].

A 71-year-old man visited our hospital complaining of fever and a bleeding tendency. The peripheral blood WBC count was 10,400/microliter with 90% promyelocytes. The bone marrow was hypercellular with 88% promyelocytes. Disseminated intravascular coagulation was recognized. The patient was diagnosed as having acute promyelocytic leukemia and was treated with daily oral administration of all-trans retionic acid (ATRA) (45 mg/m2/day) and cytarabine (160 mg/day, intravenous drip infusion for the initial five days). The ATRA treatment induced leukemic cells to undergo mature myeloid differentiation. On day 24 after the start of treatment, the WBC count rapidly increased and acute myocardial infarction appeared, with consciousness disturbance and bilateral Babinski reflex appearing three hours later. Magnetic resonance imaging showed a fresh lacunar infarction of the right lenticular nucleus, and serum levels of IL-6 and PAI-1 were found to be elevated at the onset of infarction. Since there was a possibility that the retinoic acid syndrome (RAS) might have helped bring about the infarctions, we stopped the ATRA treatment and started administration of methyl-prednisolone (500 mg/body/day for 3 days) and gabexate mesilate. The WBC count decreased immediately and the consciousness disturbance improved. In this case, ATRA treatment might have initiated the RAS and resulted in some endothelial damage, thus causing the infarctions.

Aged↗