三种ADAMTS13去整合素样结构域突变致蛋白功能缺陷及其与血栓关联的研究

doi:10.16150/j.1671-2870.2025.04.010

摘要/Abstract

摘要：

目的：分析4例携带血管性血友病因子裂解酶13（a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13，ADAMTS13）去整合素样结构域杂合突变的血栓患者其基因型和表型特征，探讨ADAMTS13功能缺陷与血栓形成之间的关联。方法：通过易栓症基因Panel检测筛查血栓患者的易栓症相关基因突变，纳入4个携带ADAMTS13去整合素样结构域突变的易栓症家系。采用凝固法检测患者的凝血功能，使用免疫比浊法测定血管性血友病因子（von Willebrand factor， vWF）抗原和活性，酶联免疫吸附法检测vWF的胶原结合能力。通过十二烷基硫酸钠-琼脂糖凝胶电泳分析vWF多聚体的分布情况并进行灰度值半定量分析。采用荧光共振能量转移法测定血浆中ADAMTS13活性，酶联免疫吸附法检测ADAMTS13抗原水平。使用PyMOL软件对野生型及突变型ADAMTS13蛋白的三维结构进行对比分析。结果：4个家系的先证者均经历不同程度的血栓事件，包括脑静脉窦血栓、肺栓塞及下肢深静脉血栓。ADAMTS13蛋白去整合素样结构域突变约占ADAMTS13单杂合突变的4/87。遗传分析显示，4例先证者均携带ADAMTS13基因去整合素样结构域单杂合突变（p.Pro301Ala、p.Pro301Arg、p.Arg349Cys），其中p.Pro301Ala和p.Pro301Arg为首次报道突变。凝血功能检测结果表明，4例患者ADAMTS13活性和抗原水平显著降低（Act为57.42%~72.88%，Ag为66.94%~78.34%），vWF活性和抗原水平升高（Act为158.2%~213.7%，Ag为167.2%~216.6%）。vWF多聚体电泳分析显示，患者血浆中高分子量多聚体（high-molecular-weight multimers， HMWMs）比例显著增加（灰度值166.6~218.9比117.4），提示HMWMs较正常人明显增多。结构分析进一步表明，突变位点位于ADAMTS13蛋白去整合素样结构域的关键区域，可能破坏蛋白稳定性及与vWF的结合能力。结论：本研究首次报道了位于ADAMTS13蛋白去整合素样结构域上的2个新突变（Pro301Ala 和 Pro301Arg），并再次验证了已知突变Arg349Cys的致病特性。结果证实这些突变可导致ADAMTS13蛋白表达水平下降，并进一步引起其酶活性的显著降低，表现为对vWF高分子量多聚体的裂解能力减弱，功能试验证实患者体内存在异常增多的vWF多聚体，破坏了ADAMTS13‐vWF轴的动态平衡，从而增加血栓形成的风险。

关键词: 血管性血友病因子裂解酶13, 血管性血友病因子, 去整合素样结构域, 基因突变, 血栓形成

Abstract:

Objective To analyze the genotypic and phenotypic characteristics of four thrombotic patients carrying heterozygous mutations in the disintegrin-like domain of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13), and to explore the association between ADAMTS13 deficiency and thrombosis. Methods Thrombophilia-related gene mutations were screened in thrombosis patients using a thrombophilia gene panel, identifying four thrombophilia pedigrees carrying mutations in the ADAMTS13 disintegrin-like domain. Coagulation function was assessed using clotting tests. Antigen and activity of von Willebrand factor (vWF) were measured using immunoturbidime-try, and its collagen-binding capacity was measured using enzyme-linked immunosorbent assay (ELISA). The distribution of vWF multimers was analyzed by SDS-agarose gel electrophoresis with gray value semi-quantitative analysis. ADAMTS13 activity in plasma was measured by fluorescence resonance energy transfer assay, and its antigen levels were quantified via ELISA. Comparative analysis of three-dimensional structures between wild-type and mutant ADAMTS13 proteins was performed using PyMOL software. Results The probands from four pedigrees experienced thrombotic events of varying seve-rity, including cerebral venous sinus thrombosis, pulmonary embolism, and lower extremity deep vein thrombosis. Mutations in the ADAMTS13 disintegrin-like domain accounted for approximately 4/87 of ADAMTS13 heterozygous mutations. Genetic analysis showed that all four probands carried heterozygous mutations in the ADAMTS13 disintegrin-like domain (p.Pro301Ala, p.Pro301Arg, p.Arg349Cys), with p.Pro301Ala and p.Pro301Arg being newly reported. Coagulation tests demonstrated significantly reduced ADAMTS13 activity and antigen levels (Act: 57.42%-72.88%, Ag: 66.94%-78.34%), and elevated vWF activity and antigen levels (Act: 158.2%-213.7%, Ag: 167.2%-216.6%). Electrophoretic analysis of vWF multimers showed a significantly increased proportion of high-molecular-weight multimers (HMWMs) in patient plasma (Gray value: 166.6-218.9 vs 117.4), indicating markedly elevated HMWMs compared to normal controls. Structural analysis further suggested that the mutation sites were located in critical regions of the ADAMTS13 disintegrin-like domain, potentially disrupting protein stability and its binding capacity with vWF. Conclusion This study first reports two novel missense mutations—Pro301Ala and Pro301Arg—located in the disintegrin-like domain of the ADAMTS13 protein, and further verifies the pathogenic characteristics of the known Arg349Cys mutation. The results demonstrate that these mutations result in reduced ADAMTS13 protein expression levels and significantly decreased enzymatic activity, as evidenced by impaired cleavage capacity of high-molecular-weight vWF multimers. Functional tests confirm abnormally increased vWF multimers in patients, disrupting the dynamic balance of the ADAMTS13-vWF axis and consequently increasing thrombotic risk.

Key words: ADAMTS13, von Willebrand factor, Disintegrin-like domain, Gene mutation, Thrombosis

中图分类号:

R554
R446.11

林莉亚, 吴希, 毛胤祺, 陈光明, 武文漫, 戴菁, 王学锋, 丁秋兰. 三种ADAMTS13去整合素样结构域突变致蛋白功能缺陷及其与血栓关联的研究[J]. 诊断学理论与实践, 2025, 24(04): 431-440.

LIN Liya, WU Xi, MAO Yinqi, CHEN Guangming, WU Wenman, DAI Jing, WANG Xuefeng, DING Qiulan. Three disintegrin-like domain mutations of ADAMTS13： functional deficiency and association with thrombosis[J]. Journal of Diagnostics Concepts & Practice, 2025, 24(04): 431-440.

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Proband	Gene Analysis	ADAMTS13: Act（%）	ADAMTS13: Ag（%）	VWF:Act （%）	VWF:Ag （%）	VWF:CB /VWF:Ag（Ratio）	ADAMTS13:Act /VWF:Ag（Ratio）
1	ADAMTS13 c.901C>G:p.Pro301Ala*	62.03	70.35	211.6	214.9	1.47	0.289
2	ADAMTS13 c.902C>G:p.Pro301Arg*	67.35	75.46	213.7	216.6	1.28	0.311
3	ADAMTS13 c.902C>G:p.Pro301Arg*	72.88	78.34	158.2	167.2	1.39	0.436
4	ADAMTS13 c.1045C>T:p.Arg349Cys	57.42	66.94	179.5	188.9	1.33	0.304
	Normal range	60.00-150.00	40.00-120.00	50.0-150.0	50.0-150.0	0.70-1.20	0.500-2.000