PATHOPHYSIOLOGY OF ANTIPROTHROMBIN AUTOANTIBODIES

抗凝血酶原自身抗体的病理生理学

• 批准号: 13670442
• 负责人: ATSUMI Tatsuya
• 金额: $ 2.56万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670442/
• 关键词: PROTHROMBIN; LUPUS ANTICOAGULANT; ANTIPHOSPHOLIPID SYNDROME; THROMBOSIS; MONOCLONAL ANTIBODY; EPITOPE; PHOSPHATIDYLSERINE; ELISA

Antiphospholipid antibodies (Apl) are immunoglobulins associated with a variety of clinical phenomena, including arterial and venous thrombosis. The term "antiphospholipid syndrome" (APS) is used to link these clinical manifestations to the persistence of Apl. Autoantibody against prothrombin is one of the most common and potent aPLs associated with vascular diseaeses. It has been shown that phosphatidylserine dependent antiprothrombin antibodies (Aps/PT) is a specific marker of APS and highly correlate with the presence of lupus anticoagulant. I raised mouse monoclonal Aps/PT (231D), which had high binding to phosphatidylserine-prothrombin complex but little binding to immobilized prothrombin directly on irradiated ELISA plates. This property was similar to autoimmune Aps/PT found in patients with APS. Normal plasma mixed with 231D had prolonged clotting time in a dose dependent fashion, and the excess of phospholipid reduced the prolongation of clotting time, thus 231D had a strong LA activity. In this study, I established a semiquantitative LA assay using 23ID as a standard. I showed that our semi-quantitative LA assay is simple and useful for the diagnosis of LA with very high sensitivity, thus this method is practical for the first screening for APS.In addition, I investigated the epitope of aPS/PT on prothrombin molecule. I digested andpurified prothrombin fragments (F1 and F1+2) and studies the binding between phosphatidylserine-fragments complex and aPS/PT. No binding of aPS/PT was found and the epitope may be on prethrombin side or comformational. In the next experiments, I shall prepare recombinant prothrombin and its mutant to investigate the importance of structure of prothrombin molecule.

抗磷脂抗体(APL)是一种与多种临床现象相关的免疫球蛋白，包括动脉和静脉血栓形成。术语“抗磷脂综合征”(APS)用于将这些临床表现与APL的持久性联系起来。抗凝血酶原自身抗体是与血管疾病相关的最常见和最有效的APL之一。研究表明，磷脂酰丝氨酸依赖的抗凝血酶原抗体(APS/PT)是APS的特异性标志物，并与狼疮抗凝剂的存在高度相关。我制备了与磷脂酰丝氨酸-凝血酶原复合体高度结合的小鼠单抗APS/PT(231D)，但在辐照的EL ISA平板上直接与固定化的凝血酶原结合很少。这一特性类似于APS患者中发现的自身免疫性APS/PT。正常血浆与231D混合后凝血时间延长呈剂量依赖关系，磷脂过量可使凝血时间延长幅度减小，231D具有较强的LA活性。在本研究中，我建立了以~(23)ID为标准的半定量LA检测方法。我们的半定量LA方法对LA的诊断具有很高的灵敏度，是一种简便、实用的方法，可用于AP的首次筛查。此外，我还研究了凝血酶原分子上的APS/PT表位。我消化纯化了凝血酶原片段F1和F1+2，并研究了磷脂酰丝氨酸片段复合体与APS/PT的结合。未发现APS/PT结合，表位可能位于凝血酶前侧或构象。在接下来的实验中，我将制备重组凝血酶原及其突变体，以研究凝血酶原分子结构的重要性。

项目成果

Atsumi T, Bertolaccini ML, Koike T.: "Genetics of antiphospholipid syndrome"Rheum Dis Clin N Am. 27. 565-572 (2001)

Atsumi T、Bertolaccini ML、Koike T.：“抗磷脂综合征的遗传学”Rheum Dis Clin N Am。

Tsutsumi A: "Anti-phosphatidylserine/prothrombin antibodies are not frequently found in patients with unexplained recurrent miscarriages"Am J Reprod Immunol. 46. 242-244 (2001)

Tsutsumi A：“在不明原因的复发性流产患者中并不经常发现抗磷脂酰丝氨酸/凝血酶原抗体”Am J Reprod Nutrition。

Amengual O: "Specificities, properties and clinical significance of antiprothrombin antibodies"Arthritis Rheum. 48. 886-895 (2003)

Amengual O：“抗凝血酶原抗体的特性、特性和临床意义”关节炎大黄。

Yasuda S, Tsutsumi A, Atsumi T, Bertolaccini ML, Ichikawa K, Khamashta MA, Hughes GRV, Koike T.: "Gene polymorphisms of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with antiphospholipid antibodies"J Rheumatol. 29. 1192-1

Yasuda S、Ttsutsumi A、Atsumi T、Bertolaccini ML、Ichikawa K、Khamashta MA、Hughes GRV、Koike T.：“抗磷脂抗体患者中组织纤溶酶原激活剂和纤溶酶原激活剂抑制剂-1 的基因多态性”J Rheumatol。

Amengual O, Atsumi T, Koike T.: "Specificities, properties and clinical significance of antiprothrombin antibodies"Arthritis Rheum. 48. 886-895 (2003)

Amengual O、Atsumi T、Koike T.：“抗凝血酶原抗体的特性、特性和临床意义”关节炎大黄。