| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
|
|---|
| Research Abstract |
Phosphatidylserine dependent antiprothrombin antibodies (aPS/PT) are closely associated with the clinical manifestations of antiphospholipid syndrome (APS) and LA, whilst neither in vitro nor in vivo properties of aPS/PT in thrombin generation have been clarified. The purpose of this study is to investigate the in vitro roles of aPS/PT in thrombin generation. The reactivity of monoclonal antiprothrombin antibodies against phosphatidylserine, phosphatldylserine bound prothrombin, and prothrombin on irradiated/non-irradiated plates were examined by enzyme-linked immunosorbent assay (ELISA). To investigate the epitopes for aPS/PT, an inhibition ELISA using monoclonal antiprothrombin antibodies and purified IgG from APS patients was performed. The effects of mouse monoclonal phosphatidyserine dependent anti-human prothrombin antibody (MoaPS/PT) on thrombin generation were evaluated by a chromogenic assay, using the prothombinase complex [phospholipid, CaCl_2, human purified factor Va (FVa)
… More
, human factor Xa (FXa), and human purified prothrombin]. Thrombin generation was measured by a quantitative analysis using a specific substrate for thrombin (S2238). We established mouse monoclonal antibodies, 231D and 51A6. 231D only bound to phosphatidylserine bound prothrombin, and was considered as aPSIPT. 51A6 bound to both phosphatidylserine bound prothrombin and prothrombin alone on irradiated and non-irradiated plates, but not to phosphatidylserine alone. The binding of IgG from APS patients to phosphaydilserin/prothrombin complex was inhibited by 231D between 35-70%, but not by 51A6. In the presence of low concentration of FVa (0.1ng/ml), 231D increased thrombin generation up to 87% in a dose-dependent manner. In contrast, when high concentration of FVa (1.0 ng/ml) were added, 231D decreased thrombin generation up to 35%. Under a constant concentration of FVa, high concentration of FXa enhanced the effect of 231D. 51A6 showed minor inhibition of thrombin generation in any conditions. 231D had similar characteristic to autoimmune aPS/PT that react only with phosphatidylserine bound prothrombin. 231D and APS patients' IgG may share the epitope on phosphaydilserin/prothrombin complex, suggesting that 231D represents the properties of autoimmune aPS/PT. The in vitro effects of 231D on thrombin generation are dual-factorial according to the FVa and FXa balance, therefore serving as a clue fort the LA paradox. Less
|
