Novel antibody against a glutamic acid-rich human fibrinogen-like protein 2-derived peptide near Ser91 inhibits hfgl2 prothrombinase activity.
Novel antibody against a glutamic acid-rich human fibrinogen-like protein 2-derived peptide near Ser91 inhibits hfgl2 prothrombinase activity.
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DOI：
10.1371/journal.pone.0094551
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Wang Z
中科院分区：
文献类型：
--
作者：
Li WZ;Wang J;Long R;Su GH;Bukhory DK;Dai J;Jin N;Huang SY;Jia P;Li T;Fan C;Liu K;Wang Z
纤维蛋白原样蛋白 2 (fgl2) 在微循环障碍相关疾病的微血管内皮细胞中高表达，在微血栓形成中发挥着至关重要的作用。先前的研究表明，Ser89 残基是小鼠 fgl2 凝血酶原酶活性的关键位点。本研究的目的是研究 hfgl2 衍生肽抗体的凝血酶原酶抑制能力。该肽被称为 NPG-12，因为它位于膜结合 hfgl2 的 N 末端，包含 12 个氨基酸残基（对应于残基 76 至 87），并且富含 Glu。该肽被选为抗原决定簇，使用 DNAStar 和 HomoloGene 软件程序在免疫兔子中产生抗体。通过 TNF-α 处理，在人脐静脉内皮细胞中诱导大量 hfgl2 表达。通过 ELISA、Western Blot 和免疫染色测定，生成的抗 NPG-12 抗体特异性识别 fgl2。此外，一阶段凝血和凝血酶生成试验提供的证据表明，抗体可以降低 hfgl2 凝血酶原酶活性，而不影响贫血小板血浆凝血酶原时间 (PT) 或活化部分凝血活酶时间 (APTT)。此外，这些抗体对大量 T 细胞群的增殖或活化产生了不可检测的影响。总之，选定的肽序列 NPG-12 可能是 hfgl2 凝血酶原酶活性的关键结构域，针对该序列的抑制剂的开发可能有望用于 hfgl2 相关微循环障碍的研究或管理。
Fibrinogen-like protein 2 (fgl2) is highly expressed in microvascular endothelial cells in diseases associated with microcirculatory disturbances and plays a crucial role in microthrombosis. Previous studies have demonstrated that the Ser89 residue is a critical site for mouse fgl2 prothrombinase activity. The aim of this study was to investigate the prothrombinase inhibitory ability of antibodies against an hfgl2-derived peptide. The peptide was termed NPG-12 because it is located at the N-terminus of membrane-bound hfgl2, contains 12 amino acid residues (corresponding to residues 76 to 87), and is rich in Glu. This peptide was selected as an antigenic determinant to produce antibodies in immunized rabbits using the DNAStar and HomoloGene software program. Abundant hfgl2 expression was induced in human umbilical vein endothelial cells through treatment with TNF-α. The generated anti-NPG-12 antibodies specifically recognize fgl2, as determined by ELISA, Western Blot and immunostaining. Moreover, one-stage clotting and thrombin generation tests provide evidence that the antibodies can reduce the hfgl2 prothrombinase activity without affecting the platelet-poor plasma prothrombin time (PT) or the activated partial thromboplastin time (APTT). In addition, the antibodies exerted undetectable influence on the proliferation or activation of bulk T cell populations. In conclusion, the selected peptide sequence NPG-12 may be a critical domain for hfgl2 prothrombinase activity, and the development of inhibitors against this sequence may be promising for research or management of hfgl2-associated microcirculatory disturbances.
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