Development of new analytical method for antithrombin resistance

• Project/Area Number: 22659114
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Laboratory medicine
• Research Institution: Nagoya University
• Principal Investigator: TAKAGI Akira 名古屋大学, 医学系研究科(保健), 助教 (30135371)
• Co-Investigator(Kenkyū-buntansha): KOJIMA Tetsuhito 名古屋大学, 医学系研究科(保健), 教授 (40161913)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥3,100,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥300,000); Fiscal Year 2012: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2011: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2010: ¥1,800,000 (Direct Cost: ¥1,800,000)
• Keywords: 臨床血液学 / プロトロンビン変異 / アンチトロンビン抵抗性 / 病態解析 / 変異プロトロンビン / 制御因子抵抗性 / 血栓症リスク / 検査法開発 / プロトロンビン / 遺伝子変異

Research Abstract

Introduction: We have reported a variant prothrombin (p.Arg596Leu) conveying antithrombin resistance in a patient with hereditary thrombosis. To detect antithrombin resistance in plasma, we devised a laboratory test analyzing kinetics of thrombin inactivation using antithrombin. Materials and Methods: After incubation with prothrombin activator components (phospholipids, CaCl2, and snake venom), samples were treated with excess antithrombin in the presence or absence of heparin for various time periods. Subsequently, H-D-Phe-Pip- Arg-p-nitoranilide was added and changes in absorbance/min (ΔA/min) were measured at 405 nm. Results and Conclusions: After inactivation for 5 min using antithrombin and heparin, relative residual thrombin activity of recombinant mutant prothrombin (40%) was higher than that of wild type (0.5%). In addition, after 30 min without heparin, relative residual thrombin activity of recombinant mutant prothrombin (99%) was higher than that of wild-type (20%), indicating that this assay was able to detect antithrombin resistance of the variant 596Leu prothrombin. For reconstituted plasmas with recombinant prothrombins, the assay with or without heparin showed that relative residual thrombin activity of mutant plasma was higher than that of wild-type plasma. For the warfarinized plasma containing 596Leu prothrombin, the assay with or without heparin demonstrated that final relative residual thrombin activity was high compared with that for normal pooled plasma, although its initial thrombin activity was low. In conclusion, we devised a laboratory test detecting antithrombin resistance in plasma by analyzing kinetics of thrombin inactivation using antithrombin. This assay may be useful to detect antithrombin resistance in plasma, even in warfarinized patients.

Research Products

• [Journal Article] Thrombosis from a Prothrombin Mutastion Conveying Antithrombin Resistance (2012)
  • Author(s): Yuhri Miyawaki, Atsuo Sizuki, Junko Fujita, Asuka Maki, Eriko Okuyama, Moe Murata, Akira Takagi, Takashi Murate, Shinji Kunishima, Michio Sakata, Kohji Okamoto, Tadashi Matsushita, Tomoki Naoe, Hidehiko Saito, Tetsuhito Kojima
  • Journal Title: The NEW ENGLAND JOURNAL of MEDICINE Volume: 366巻 Pages: 2390-2396
  • Peer Reviewed
• [Journal Article] Thrombosis from a prothrombin mutation conveying antithrombin resistance. (2012)
  • Author(s): Miyawaki Y, Suzuki A, Fujita J, Maki A, Okuyama E, Murata M, Takagi A, Murate T, Kunishima S, Sakai M, Okamoto K, Matsushita T, Naoe T, Saito H, Kojima T
  • Journal Title: N Engl J Med. Volume: 366 Issue: 25 Pages: 2390-2396
  • DOI: 10.1056/nejmoa1201994
  • Peer Reviewed
• [Presentation] 静脈血栓塞栓症リスク・アンチトロンビン抵抗性とそのスクリーニング検査法 (2012)
  • Author(s): 高木明
  • Organizer: 日本血栓止血学会
  • Place of Presentation: ハイアットリージェンシー東京(東京都)(発表確定)
  • Year and Date: 2012-06-08
• [Presentation] 静脈血栓塞栓症リスク・アンチトロンビン抵抗性とそのスクリーニング検査 (2012)
  • Author(s): 高木 明、 宮脇由理、 鈴木敦夫、藤田絢子、牧明日加、奥山恵理子、村田 萌、村手 隆、松下 正、 小嶋哲人
  • Organizer: 第34回日本血栓止血学会学術集会
  • Place of Presentation: 東京
• [Presentation] 静脈血栓症リスク・アンチトロンビン抵抗性とそのスクリーニング検査法 (2012)
  • Author(s): 高木明
  • Organizer: 第34回日本血栓止血学会
  • Place of Presentation: 東京都
• [Presentation] アンチトロンビン抵抗性解析法とその評価 (2011)
  • Author(s): 高木明
  • Organizer: 日本臨床検査医学会
  • Place of Presentation: 岡山コンベンションセンター(岡山県)
  • Year and Date: 2011-11-18
• [Presentation] A novel prothrombin gene mutation leads to an AT-resistant thrombin in a family with inherited thrombophilia (2011)
  • Author(s): Yuhri Miyawaki
  • Organizer: International Society on Thrombosis and Haemostasis
  • Place of Presentation: ICC Kyoto (Japan)
  • Year and Date: 2011-07-25
• [Presentation] アンチトロンビン抵抗性を検出するトロンビン不活化動態解析法の開発 (2011)
  • Author(s): 高木明
  • Organizer: 日本検査血液学会
  • Place of Presentation: 川崎医療福祉大学(岡山県)
  • Year and Date: 2011-07-18
• [Presentation] アンチトロンビン抵抗性を検出するトロンビン不活化動態解析法の開発 (2011)
  • Author(s): 高木 明、 宮脇由理、 鈴木敦夫、藤田絢子、牧明日加、奥山恵理子、村田 萌、村手 隆、松下 正、小嶋哲人
  • Organizer: 12回日本検査血液学会学術集会
  • Place of Presentation: 倉敷
• [Presentation] A novel prothrombin gene mutation leads to an at-resistant thrombin in afamily with inherited thrombophilia (2011)
  • Author(s): Miyawaki Y, Suzuki A, Fujimori Y, Fujita J, Maki A, Takagi A, Murate T, Sakata M, Okamoto K, Matsushita T, Kojima T
  • Organizer: XXIIIth CONGRESS OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS
  • Place of Presentation: KYOTO
• [Presentation] アンチトロンビン抵抗性解析法とその評価 (2011)
  • Author(s): 高木明、 宮脇由理、 鈴木敦夫、藤田絢子、牧明日加、奥山恵理子、村田萌、村手隆、松下正、小嶋哲人
  • Organizer: 第58回日本臨床検査医学会学術集
  • Place of Presentation: 岡山
• [Patent(Industrial Property Rights)] 凝固因子として作用する異常トロンビンのためのトロンビン不活化動態測定方法及び試験方法、並びに、ポリヌクレオチド (2010)
  • Inventor(s): 高木明, 小嶋哲人, 松下正
  • Industrial Property Rights Holder: 名古屋大学
  • Industrial Property Number: 2010-289686
  • Filing Date: 2010-12-27