Lai Shiue-Wei, Chang Chia-Yau, Lee Hwei-Jen, Chen Yeu-Chin
Hemophilia Care and Research Center, Tri-Service General Hospital, Taipei, Taiwan.
Division of Hematology/Oncology, Department of Internal Medicine, Tri-service General Hospital, National Defense Medical Center, No. 325, Section 2, Chenggong Road, Neihu 114, Taipei, Taiwan.
Thromb J. 2024 Dec 3;22(1):107. doi: 10.1186/s12959-024-00677-6.
Antithrombin (AT) is a serine protease inhibitor which exerts its anticoagulant effect through binding to serine residues in the active centers of procoagulant serine proteases. Its deficiency is associated with increased risk of venous thrombosis. We aim to investigate the pathogenic mechanism of two natural mutants (W221C and M284R) in inherited AT deficiency.
We analyzed 9 unrelated patients with inherited AT deficiency by extracting peripheral blood DNA and sequencing the SERPINC1 gene after amplification by polymerase chain reaction. Enzyme-linked immunosorbent assay and heparin affinity chromatography were used to assess AT secretion and purification efficiency. The mutant AT models were evaluated via computational simulations.
Among the 9 patients with inherited AT deficiency, 8 patients had type I AT deficiency, and one patient had type II AT deficiency with subtype of reactive site mutation. Seven of them experienced venous thrombotic events and all patients were found genetic mutations including missense (n = 6), deletion (n = 2) and insertion (n = 1). Two point mutations, W221C and M284R, were identified and were hypothesized to affect AT by destabilizing the central β-sheet. Based on immunoassays and heparin purification, the W221C mutant may impair AT secretion, whereas M284R mutant decreased the total AT production (696.8 ± 151.6 ng/ml versus 3833.72 ± 315.4 ng/ml, p = 0.029). Both mutants delayed the peak of AT release in heparin affinity chromatography.
Our study demonstrates that two mutations in SERPINC1 gene altered the production and structure of AT by in vitro protein expression and functional studies, including protein secretion and production. These findings enhance our understanding of the genetic basis of AT deficiency and its possible clinical implications.
抗凝血酶（AT）是一种丝氨酸蛋白酶抑制剂，通过与促凝血丝氨酸蛋白酶活性中心的丝氨酸残基结合发挥抗凝作用。其缺乏与静脉血栓形成风险增加有关。我们旨在研究遗传性AT缺乏症中两种天然突变体（W221C和M284R）的致病机制。
我们通过提取外周血DNA并在聚合酶链反应扩增后对SERPINC1基因进行测序，分析了9例非亲缘关系的遗传性AT缺乏症患者。采用酶联免疫吸附测定和肝素亲和色谱法评估AT的分泌和纯化效率。通过计算模拟评估突变型AT模型。
在9例遗传性AT缺乏症患者中，8例为I型AT缺乏症，1例为II型AT缺乏症且为反应位点突变亚型。其中7例发生静脉血栓事件，所有患者均发现基因突变，包括错义突变（n = 6）、缺失突变（n = 2）和插入突变（n = 1）。鉴定出两个点突变W221C和M284R，推测它们通过破坏中央β-折叠来影响AT。基于免疫测定和肝素纯化，W221C突变体可能损害AT分泌，而M284R突变体降低了AT的总产量（696.8±151.6 ng/ml对3833.72±315.4 ng/ml，p = 0.029）。两种突变体在肝素亲和色谱中均延迟了AT释放的峰值。
我们的研究表明，通过体外蛋白质表达和功能研究，包括蛋白质分泌和产生，SERPINC1基因中的两个突变改变了AT的产生和结构。这些发现加深了我们对AT缺乏症遗传基础及其可能临床意义的理解。
