克隆人抗因子 XIII 单克隆抗体剖析了单个患者中多克隆抗体的作用机制。

Cloning of human anti-factor XIII monoclonal antibody dissects mechanisms of polyclonal antibodies in a single patient.

机构信息

Department of Molecular Patho-Biochemistry and Patho-Biology, Yamagata University School of Medicine, Yamagata, Japan; The Japanese Collaborative Research Group (JCRG) on Autoimmune Acquired Coagulation Factor Deficiencies supported by the Japanese Ministry of Health, Labor and Welfare, Tokyo, Japan; Department of Public Health and Hygiene, Yamagata University Graduate School of Medical Science, 2-2-2, Iida-Nishi, Yamagata, Japan.

Department of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan.

出版信息

J Thromb Haemost. 2023 Feb;21(2):255-268. doi: 10.1016/j.jtha.2022.11.019. Epub 2022 Dec 22.

DOI:10.1016/j.jtha.2022.11.019

PMID:36700504

Abstract

BACKGROUND

Coagulation factor XIII (FXIII) consists of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that cross-link and strengthen the hemostatic fibrin thrombus; thus, abnormal bleeding occurs when FXIII is significantly reduced. Autoimmune-acquired FXIII deficiency (AiF13D) is characterized by lethal bleeding secondary to the development of autoantibodies against FXIII. However, since anti-FXIII autoantibodies are polyclonal, the mechanism underlying FXIII dysfunction is unclear.

OBJECTIVES

The objective of this study was to dissect the inhibitory mechanisms of polyclonal anti-FXIII autoantibodies.

METHODS

In this study, we prepared the human monoclonal antibodies (hmAbs) from the peripheral blood of an 86-year-old man with AiF13D by using a new complementary DNA cloning method and analyzed the properties of each autoantibody.

RESULTS

Seventeen clones obtained from hmAbs were divided into the following 3 groups: dissociation inhibitors of FXIII-AB (6 clones), assembly inhibitors of FXIII-AB (3 clones), and nonneutralizing/inhibitory hmAbs (8 clones). Dissociation inhibitors strongly inhibited fibrin cross-linking and amine incorporation. Assembly inhibitors extracted FXIII-A from FXIII-AB, strongly inhibited binding of FXIII-A to FXIII-B, and activation peptide cleavage. However, the patient's plasma presented a strong inhibition of AB heterodimer assembly but only a slight inhibition of thrombin-Ca-dependent dissociation, suggesting that the assembly inhibitors concealed the effect of dissociation inhibitors in plasma. By contrast, nonneutralizing antibodies had little effect on the function of FXIII, suggesting that nonneutralizing hmAbs (and/or dissociation inhibitors and/or assembly inhibitors) promoted the clearance of FXIII-A from the blood.

CONCLUSION

Cloning of anti-FXIII autoantibodies enabled us to not only elucidate the mechanism and pathophysiology of AiF13D but also develop a completely new type of anticoagulant.

摘要

背景

凝血因子 XIII（FXIII）由 2 个 A 亚基（FXIII-A）和 2 个 B 亚基（FXIII-B）组成，可交联并增强止血纤维蛋白血栓；因此，当 FXIII 显著减少时，会发生异常出血。自身免疫获得性 FXIII 缺乏症（AiF13D）的特征是由于针对 FXIII 的自身抗体的发展而导致致命性出血。然而，由于抗 FXIII 自身抗体是多克隆的，因此 FXIII 功能障碍的机制尚不清楚。

目的

本研究旨在剖析多克隆抗 FXIII 自身抗体的抑制机制。

方法

本研究中，我们使用新的 cDNA 克隆方法从一名 86 岁患有 AiF13D 的男性的外周血中制备了人源单克隆抗体（hmAbs），并分析了每种自身抗体的特性。

结果

从 hmAbs 中获得的 17 个克隆分为以下 3 组：FXIII-AB 的解离抑制剂（6 个克隆）、FXIII-AB 的组装抑制剂（3 个克隆）和非中和/抑制性 hmAbs（8 个克隆）。解离抑制剂强烈抑制纤维蛋白交联和胺掺入。组装抑制剂从 FXIII-AB 中提取 FXIII-A，强烈抑制 FXIII-A 与 FXIII-B 的结合以及激活肽切割。然而，患者的血浆对 AB 异二聚体组装表现出强烈的抑制作用，但对凝血酶-Ca 依赖性解离仅有轻微的抑制作用，这表明组装抑制剂掩盖了解离抑制剂在血浆中的作用。相比之下，非中和抗体对 FXIII 的功能几乎没有影响，这表明非中和 hmAbs（和/或解离抑制剂和/或组装抑制剂）促进了 FXIII-A 从血液中的清除。