Anticardiolipin Antibodies in Antiphospholipid Syndrome

描述(由申请人提供)：该项目的长期目标是鉴定和表征抗磷脂综合征(APS)中的致病性抗心磷脂抗体(ACL)。最近，我们发现4/5的患者来源的、血栓前状态的单抗Ig G-ACL与纤溶酶、凝血酶和活化蛋白C(APC)结合。在这4种纤溶酶反应性ACL中，CL15可抑制纤溶酶介导的纤溶和APC的抗凝活性，并导致妊娠小鼠流产。它与纤溶酶结合的相对Kd值为7×10/-10，比抗β2GPI的抗体对APS中的主要自身抗原β2GPI的亲和力高3-4倍。这些数据使我们假设纤溶酶是一种重要的自身抗原，在一些APS患者中驱动某些Ig G ACL，并且一些由纤溶酶驱动的Ig G ACL是前血栓形成的(如CL15)。为了检验这些假设，具体目标是： 1.研究APS患者纤溶酶反应性Ig G-ACL的发生率、特点及致病意义。免疫球蛋白将被提纯并分析抗CL和纤溶酶的抗体，并通过交叉抑制来确定纤溶酶反应性的免疫球蛋白ACL的存在。纤溶酶反应性Ig G ACL将被亲和纯化，并分析其与纤溶酶的结合亲和力、与凝血酶和APC的反应性以及狼疮的抗凝活性和致病性。 2.用人纤溶酶免疫小鼠，研究其诱导产生的抗体及妊娠结局。血样将分析抗人纤溶酶和CL的抗体，并通过交叉抑制来确定是否存在纤溶酶反应性的Ig G ACL。具有高滴度纤溶酶反应性Ig G ACL的雌性小鼠将进行交配，并将检查妊娠结果，以评估纤溶酶反应性Ig G ACL的致病性。 3.从免疫的小鼠中制备和研究单克隆性纤溶酶反应性ACL。将单抗单独或成对注射到怀孕小鼠体内，以研究它们的致病潜力，并分析其与小鼠纤溶酶、人凝血酶和APC的反应活性以及相关的功能活性。

DESCRIPTION (provided by applicant): The long-term objectives of this project are to identify and characterize pathogenic anticardiolipin antibodies (aCL) in the antiphospholipid syndrome (APS). Recently, we found 4/5 patient-derived, prothrombotic, monoclonal IgG aCL bind to plasmin, thrombin and activated protein C (APC). Of these 4 plasmin-reactive aCL, CL15 inhibits plasmin-mediated fibrinolysis and anticoagulant activity of APC, and causes fetal loss when injected into pregnant mice. It binds to plasmin with a relative Kd value of 7 x 10/-10 that is 3-4 logs higher than the affinity of IgG anti-beta2GPI antibodies (Ab) toward beta2GPI, the major autoantigen in APS. These data lead us to hypothesize that plasmin is an important autoantigen that drives certain IgG aCL in some APS patients, and that some of the plasmin-driven IgG aCL are prothrombotic (like CL15). To test these hypotheses, the specific aims are: 1. To study the prevalence, characteristics and pathogenic significance of the plasmin-reactive IgG aCL in APS patients. IgG will be purified and analyzed for Ab against CL and plasmin, and by cross inhibition to determine the presence of plasmin-reactive IgG aCL. The plasmin-reactive IgG aCL will be affinity purified and analyzed for binding affinity to plasmin, reactivity with thrombin an APC, and for lupus anticoagulant activity and pathogenicity. 2. To immunize mice with human plasmin and study the induced Ab and the pregnancy outcomes. Blood samples will be analyzed for IgG Ab against human plasmin and CL, and by cross inhibition to determine the presence of plasmin-reactive IgG aCL. Female mice with high titers of plasmin-reactive IgG aCL will be mated and the pregnancy outcomes will be examined to assess the pathogenicity of the plasmin-reactive IgG aCL. 3. To generate and study monoclonal plasmin-reactive aCL from immunized mice. The monoclonal Ab will be injected (individually or in pairs) into pregnant mice to study their pathogenic potentials, and be analyzed for reactivity with murine plasmin, and human thrombin and APC, and for relevant functional activity.