Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.
Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.
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DOI：
10.1021/bi030167f
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发表时间：
2004-04
期刊：
影响因子：
2.9
通讯作者：
A. Le;S. Dasgupta;S. Planque;S. Paul;P. Thiagarajan
中科院分区：
文献类型：
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作者：
A. Le;S. Dasgupta;S. Planque;S. Paul;P. Thiagarajan
凝血酶原自身抗体在系统性红斑狼疮患者中很常见。虽然它们的存在是血栓形成的危险因素，但它们的起源及其在诱导促凝状态中的确切作用尚不清楚。我们已经开发了一个噬菌体展示抗体库，从系统性红斑狼疮患者的抗凝血酶原抗体，我们已经选择了两个单链抗体片段（ScFvs）通过在凝血酶原包被的表面上淘选。在使用纯化组分的凝血原激活测定中，这些抗体促进凝血原激活。这些ScFvs，称为AN 78和AN 129，结合到固定化凝血酶原在一个浓度依赖性的特定方式，但不是其他阴离子磷脂结合蛋白，如β 2-糖蛋白I或膜联蛋白V。磷脂酰丝氨酸结合的凝血酶原，但不可溶性凝血酶原，抑制的结合表明，表位仅可用于固定化凝血酶原。为了定位表位，用凝血酶或因子Xa处理凝血酶原，随后分离各种凝血酶原活化片段并在ELISA中用ScFvs进行测试。AN 78和AN 129都与凝血酶前体I（缺少Gla结构域和第一kringle结构域的片段）、片段1.2（仅含有Gla和两个kringle结构域）和片段2结合，但不与凝血酶结合，从而将同源表位定位于凝血酶原中的kringle 2结构域。这些抗体的cDNA序列的分析显示在互补决定区中的成簇突变模式，表明可变结构域是抗原驱动的B细胞克隆成熟的产物。
Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.