研究者詳細 - 涌井　昌俊

論文 - 涌井　昌俊

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Application of clot-fibrinolysis waveform analysis to assessment of in vitro effects of direct oral anticoagulants on fibrinolysis

Oka S., Wakui M., Fujimori Y., Kuroda Y., Nakamura S., Kondo Y., Nakagawa T., Katagiri H., Murata M.

International Journal of Laboratory Hematology （International Journal of Laboratory Hematology） 42 （ 3 ） 292 - 298 2020年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 17515521

© 2020 John Wiley & Sons Ltd Introduction: Acceleration of fibrinolysis by direct oral anticoagulants (DOACs) has been reported by several groups, suggesting contribution of not only anticoagulant but also fibrinolytic effects to the therapeutic efficacy. The present study aims to evaluate the usability of clot-fibrinolysis waveform analysis (CFWA) for assessment of in vitro effects of DOACs on fibrinolysis. Methods: The experimental conditions were optimized according to how t-PA concentrations and a time length after t-PA adjustment affect parameters of CFWA. Addition of the activated partial thromboplastin time (APTT) reagent followed by that of calcium and t-PA was done to obtain clotting and fibrinolytic reaction curves which were mathematically differentiated for CFWA (APTT-CFWA). The positive and negative modes of waveforms were defined as the direction toward fibrin generation and that toward fibrin degradation, respectively. The maximum positive and negative values (Maxp1 and Maxn1) correspond to the maximum coagulation velocity and the maximum fibrinolysis velocity, respectively. Plasma spiked with each of DOACs (rivaroxaban, apixaban, edoxaban, and dabigatran) was subjected to APTT-CFWA. Results: Optimization of t-PA use was based on Maxn1. Roughly biphasic effects of rivaroxaban and dabigatran but not apixaban or edoxaban on fibrinolysis were observed through Maxn1 and the fibrinolysis peak time, which was defined as a time length from the time when Maxp1 (Maxp1 time) to the time when Maxn1 appears (Maxn1 time). Conclusion: The results suggest the usability of CFWA for assessment of DOAC effects and provide insights into relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis.

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CWAによる基礎的検討

涌井昌俊

Thrombosis Medicine （先端医学社） 10 （ 1 ） 45 - 54 2020年03月

研究論文（その他学術会議資料等）, 単著

Effect of refrigeration of blood samples in lithium-heparin tubes on QuantiFERON TB Gold Plus test result

Uwamino Y., Sakai A., Nishimura T., Noguchi M., Uno S., Fujiwara H., Mori M., Wakui M., Murata M., Hasegawa N.

Journal of Infection and Chemotherapy （Journal of Infection and Chemotherapy） 26 （ 3 ） 312 - 314 2020年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1341321X

© 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases The QuantiFERON TB Gold Plus (QFT-Plus) test is a newly approved interferon-gamma releasing assay test for detecting latent tuberculosis. Although blood samples for QFT test can be refrigerated for 48 h in lithium-heparin tubes according to package inserts, no published data are available on the effects of sample refrigeration on the test results. We conducted a clinical study that aimed to elucidate whether sample refrigeration for 48 h affects QFT-Plus test results. We collected 2 blood samples each from 40 participants for QFT-Plus; one sample was refrigerated before incubation for QFT-Plus assay, while the other sample was incubated soon after collection and treated as control. After comparing QFT-Plus test results of refrigerated samples and control samples, the concordance rate and kappa coefficient between them were 95% and 0.90, respectively. Thus, blood samples for QFT-Plus test can be refrigerated for 48 h in lithium-heparin tubes without influencing the test results.

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臨床検査における質量分析法の可能性と落とし穴

涌井昌俊、中川央充

臨床検査（医学書院） 64 （ 3 ） 262 - 271 2020年03月

研究論文（学術雑誌）, 共著

Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays

Wakui M., Fujimori Y., Nakamura S., Kondo Y., Kuroda Y., Oka S., Nakagawa T., Katagiri H., Murata M.

Journal of Clinical Pathology （Journal of Clinical Pathology） 72 （ 12 ） 817 - 824 2019年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00219746

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Aims Bivalent direct thrombin inhibitors (DTIs), hirudin and bivalirudin, bind to the active site and exosite 1 of thrombin irreversibly and reversibly, respectively. The present study aims to assess in vitro effects of hirudin and bivalirudin through clot waveform analysis (CWA) and enzyme kinetics in coagulation assays. Methods The pooled normal plasma and its dilutions were spiked with hirudin or bivalirudin. The activated partial thromboplastin time (APTT) assay and the Clauss fibrinogen assay were performed using the CS-5100 (Sysmex). The APTT-CWA data were automatically gained by the CS-5100 programme. Results In APTT-CWA, the maximum coagulation velocity, acceleration and deceleration were decreased dependently on the drug concentrations, demonstrating evidence for the blockade of thrombin-positive feedback by hirudin or bivalirudin. The Hill plot analysis was applied to the dose-dependent curves in bivalirudin. The Hill coefficients were greater than 1, showing positive anticoagulant cooperativity. Regarding the dose-dependent curves in hirudin, all the parameters dropped to almost zero without making an asymptotic line. In the Clauss fibrinogen assay, the Lineweaver-Burk plots demonstrated that both drugs exhibit mixed inhibition mimicking uncompetitive binding. The Dixon plots in bivalirudin were linear and supported the inhibition type described above. The Dixon plots in hirudin were non-linear and inappropriate to use for determination of the inhibition type. In addition, the inverse function of the clotting time appeared to drop to zero without making an asymptotic line, suggesting complete loss of thrombin activity by irreversible binding. Conclusions The results provide insights into anticoagulation with bivalent DTIs.

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Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS

Nakagawa T., Wakui M., Hayashida T., Nishime C., Murata M.

Analytical and Bioanalytical Chemistry （Analytical and Bioanalytical Chemistry） 411 （ 27 ） 7221 - 7231 2019年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 16182642

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.

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古くて新しい凝固波形解析：抗凝固薬の試験管内評価への応用

涌井昌俊、藤森祐多、岡周作、片桐尚子

臨床病理（日本臨床検査医学会） 67 （ 10 ） 1046 - 1058 2019年10月

研究論文（学術雑誌）, 共著

血小板機能異常、凝固異常時に使用する検査とその解釈

片桐尚子、涌井昌俊

Hospitalist （メディカル・サイエンス・インターナショナル） 7 （ 3 ） 537 - 550 2019年09月

研究論文（その他学術会議資料等）, 共著, 査読有り

第65回日本臨床検査医学会学術集会シンポジウム2：質量分析検査診断学 2018 update－マーカー探索から検査室における活用まで－:司会のことば

野村文夫、涌井昌俊

臨床病理 67 （ 8 ） 820 - 822 2019年08月

研究論文（学術雑誌）, 共著

LC-MS/MSの臨床検査への可能性と課題

中川央充*、涌井昌俊

臨床病理 67 （ 8 ） 835 - 842 2019年08月

研究論文（学術雑誌）, 共著

GC-MSを用いた臨床検査における留意点　―尿ステロイドプロフィル検査の経験から―

本間桂子*、涌井昌俊.

臨床病理 67 （ 8 ） 824 - 833 2019年08月

研究論文（学術雑誌）, 共著

第65回日本臨床検査医学会学術集会Reversed Clinico-pathological Conference 2 (RCPC2) ：意識レベルが低下し入院した70歳代の女性

本田孝行松本剛、涌井昌俊、一色美和

臨床病理 67 （ 6 ） 625 - 632 2019年06月

研究論文（学術雑誌）, 共著

リンパ球サブセットにおけるCD3陽性とCD4陽性+CD8陽性の乖離に関する解析（2020年度日本臨床検査医学会優秀論文賞）

谷田部陽子、荒井智子、片桐尚子、清水長子、柴田綾子、三ツ橋雄之、涌井昌俊、村田満

臨床病理 67 （ 5 ） 443 - 448 2019年05月

研究論文（学術雑誌）, 共著, 査読有り

尿蛋白分画測定装置の比較検討

青木絵美、酒井昭子、堀井康司、太田敦美、野口昌代、大野明美、柴田綾子、涌井昌俊、菊池春人、村田　満

臨床病理 67 （ 4 ） 309 - 314 2019年04月

研究論文（学術雑誌）, 共著, 査読有り

Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis

Wakui M., Fujimori Y., Katagiri H., Nakamura S., Kondo Y., Kuroda Y., Nakagawa T., Shimizu N., Murata M.

Journal of Clinical Pathology （Journal of Clinical Pathology） 72 （ 3 ） 244 - 250 2019年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00219746

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Aims Clot waveform analysis (CWA) has been reported to extend the interpretation of clotting time measurement. The parameters obtained from successive derivatives of the clotting reaction curves reflect the rates of activation of individual coagulation factors, theoretically dissecting the cascade pathway. This study aims to assess the in vitro effects of direct thrombin inhibitors (DTIs) and activated factor X (FXa) inhibitors. Methods CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each drug. For CWA of APTT measurement curves (APTT-CWA), the positive mode of clotting reaction curves was defined as the direction towards fibrin generation. Results All the maximum positive values in the successive derivatives were decreased dependently on the concentrations of each drug. Moreover, the negative values in the second and third derivatives appeared putatively due to consumption of thrombin and factor FXa, respectively, to form complexes with plasma serine protease inhibitors. The decrease of the maximum negative values observed dependently on the concentrations of each drug appeared to be consistent with the decreased generation of thrombin and factor FXa. The analysis of Hill coefficients of each drug in the dose-response of changes in the APTT-CWA parameters revealed a difference in anticoagulant cooperativity between DTIs versus FXa inhibitors. Conclusions The APTT-CWA demonstrated evidence for the blockade of thrombin-positive feedback by DTIs and FXa inhibitors and that for the differences in anticoagulant cooperativity between them. The results demonstrate the usability of CWA for assessment of anticoagulation and provide insights into direct anticoagulants.

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Fatty acid β-oxidation-dependent and -independent responses and tumor aggressiveness acquired under mild hypoxia

Wakui M., Kawai K., Mizushima T., Nishime C., Serizawa A., Suemizu H., Asakura K., Yamauchi Y., Hayashida T., Suematsu M., Murata M.

Anticancer Research （Anticancer Research） 39 （ 1 ） 191 - 200 2019年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 02507005

© 2019 International Institute of Anticancer Research. All Rights Reserved. Background/Aim: The present study assessed whether and how tumor cells undergoing hypoxia contribute to disease progression after moving to areas with different oxygen conditions. Materials and Methods: Human colorectal carcinoma HCT116 cells cultured under mild hypoxia were subjected to in vivo experiments using transfer to immunodeficient murine recipients and to in vitro experiments using pharmacological inhibition of fatty acid β-oxidation (FAO). Results: Bone involvement and hepatic metastases were accelerated in transfer models of hypoxically cultured HCT116 cells. Hypoxic HCT116 cells exhibited FAO-dependent glycogen synthesis. FAO-dependent and -independent induction of gene expression also occurred under hypoxia. The distribution of glucose transporter 1 expression compared with heme oxygenase 1 expression in HCT116 cell spheroids seemed consistent with differential dependence of hypoxic expression of these molecules on FAO. Conclusion: These results provide insights into the contribution of hypoxia to tumor progression and the relevance of FAO.