| Budget Amount *help |
¥3,850,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
We firstly evaluated the global clotting function by clot waveform analysis and thrombin generation test in this study. Furthermore, we also studied about regulation mechanism of clotting reaction focusing on the factor VIII activation and inactivation mechanism.
1) Clot waveform analysis and thrombin generation test: Clot waveform parameters such as clotting time, maximum coagulation velocity and maximum coagulation acceleration were well associated with thrombin generation parameters in factor VIII or factor IX deficient plasma. The clot waveform analysis is useful even in the presence of very low level of factor VIII. Furthermore, clotting function of severe hemophilia A (factor VIII <0.2 IU/dl) measured by clot waveform parameters were severely affected and associated with clinical phenotype. Thrombin generation test was heavily dependent on the trigger reagents. The test using both intrinsic trigger (ellagic acid) and tissue factor was sensitive and reproducibility was high.
2) Mech
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anism of factor VIII activation and inactivation : Factor VIII is transformed into its active form by limiting proteolysis at Arg 372. The clotting cascade reaction was markedly enhanced by the presence of activated factor VIII. The thrombin binding site was localized within the residues 484-509 in the A2 domain of factor VIII heavy chain. Furthermore, essential residues in the binding sites, R484, R489, R490, H497, and K499, were identified. We demonstrated for the first time that plasmin, which has been known to be the most important fibrinolytic enzyme, directly inactivate factor VIII by the same fashion as activated factor X. The cleavage sites were completely the same as those of factor Xa, however the cleavage at Arg 336 occurred more rapidly. We also identified plasmin binding sites on factor VIII. Lastly, we localized the binding sites for von Willebrand factor and phospholipid, which are both essential for factor VIII function, within the C2 domain of the factor VIII light chain. Less
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