Autoantibodies against human prothrombin ; epitope and thrornbogeneicity

磷脂酰丝氨酸依赖性抗凝血酶原抗体（aPS/PT）与抗磷脂综合征（APS）和LA的临床表现密切相关，而aPS/PT在凝血酶生成中的体外和体内特性均未阐明。本研究的目的是研究体外aPS/PT在凝血酶生成中的作用。单克隆抗凝血酶原抗体对磷脂酰丝氨酸，磷脂酰丝氨酸结合凝血酶原，和凝血酶原的反应性照射/非照射板进行了检查，通过酶联免疫吸附试验（ELISA）。为了研究aPS/PT的表位，使用单克隆抗凝血酶原抗体和来自APS患者的纯化IgG进行抑制ELISA。用凝血酶原酶复合物[磷脂、CaCl_2、人纯化因子Va（FVa）]显色法测定了鼠单克隆磷脂酰丝氨酸依赖性抗人凝血酶原抗体（MoaPS/PT）对凝血酶生成的影响。 ...更多信息 人因子Xa（FXa）和人纯化凝血酶原]。使用凝血酶的特异性底物（S2238）通过定量分析测量凝血酶生成。我们建立了小鼠单克隆抗体231 D和51 A6。231 D仅与磷脂酰丝氨酸结合的凝血酶原结合，被认为是aPSIPT。51 A6在照射和未照射的板上结合磷脂酰丝氨酸结合的凝血酶原和单独的凝血酶原，但不结合单独的磷脂酰丝氨酸。APS患者的IgG与磷脂酰甘油/凝血酶原复合物的结合被231 D抑制35- 70%，但不被51 A6抑制。在低浓度FVa（0.1ng/ml）存在下，231 D以剂量依赖性方式增加凝血酶产生达87%。相比之下，当加入高浓度的FVa（1.0ng/ml）时，231 D使凝血酶生成降低高达35%。在一定浓度的FVa作用下，高浓度的FXa可增强231 D的作用。51 A6在任何条件下都显示出对凝血酶生成的轻微抑制。231 D具有与自身免疫性aPS/PT相似的特性，仅与磷脂酰丝氨酸结合的凝血酶原反应。231 D与APS患者的IgG可能共享磷脂酰二丝氨酸/凝血酶原复合物上的表位，提示231 D代表自身免疫性aPS/PT的特性。根据FVa和FXa平衡，231 D对凝血酶生成的体外作用是双因素的，因此作为LA悖论的线索。少

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