2012 Fiscal Year Final Research Report
Development of new analytical method for antithrombin resistance
| Project/Area Number |
22659114
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Nagoya University |
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Principal Investigator |
TAKAGI Akira 名古屋大学, 医学系研究科(保健), 助教 (30135371)
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| Co-Investigator(Kenkyū-buntansha) |
KOJIMA Tetsuhito 名古屋大学, 医学系研究科(保健), 教授 (40161913)
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| Project Period (FY) |
2010 – 2012
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| Keywords | 臨床血液学 |
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| 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.
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Research Products
(6 results)
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[Journal Article] Thrombosis from a Prothrombin Mutastion Conveying Antithrombin Resistance2012
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
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Journal Title
The NEW ENGLAND JOURNAL of MEDICINE
Volume: 366巻
Pages: 2390-2396
Peer Reviewed
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[Presentation] A novel prothrombin gene mutation leads to an at-resistant thrombin in afamily with inherited thrombophilia2011
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
Year and Date
20110000
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