| Budget Amount *help |
¥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)
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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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