Abstract:

ObjectiveThis study aims to investigate the mechanisms of thrombosis caused by gene mutations by analyzing the genotypic and phenotypic characteristics of thrombotic patients with mutations in the disintegrin-like domain encoded by a disintegrin and metalloproteinase with thrombospondin 1 motifs 13 (ADAMTS13).MethodsA thrombophilia gene panel for the Chinese population was established, which was based on next-generation sequencing and CNVplex® high-throughput copy number variation detection technology and included 35 thrombophilia-related genes commonly found in the Chinese population. Between July 2020 and August 2024, a total of 1 130 thrombotic patients attending the Thrombosis and Hemostasis Clinic at our hospital were continuously screened using this panel. Patients carryingADAMTS13mutations were identified, and probands with mutation sites located exclusively within the disintegrin-like domain encoded byADAMTS13were included for family investigation. For the probands, the coagulation function was evaluated using clotting tests. The activity and antigen levels of von Willebrand factor (vWF) were measured by immunoturbidimetry. The collagen-binding capacity of vWF was assessed using enzyme-linked immunosorbent assay. The distribution of vWF multimers with different molecular weights in patient plasma was analyzed using sodium dodecyl sulfate-agarose gel electrophoresis, followed by semi-quantitative grayscale value analysis. The activity of ADAMTS13 in the probands’ plasma was measured using the fluorescence resonance energy transfer method, and ADAMTS13 antigen levels were determined using enzyme-linked immunosorbent assay. The three-dimensional structures of wild-type and mutant ADAMTS13 were analyzed using PyMOL software to investigate the impact of the mutations on protein function.ResultsA total of 87 thrombotic patients carryingADAMTS13gene mutations were identified, among whom four probands (4/87) had mutations located exclusively within the disintegrin-like domain. These four probands with thrombophilia and their corresponding family members were included in the analysis. Both the probands and the family members carrying the same gene mutations experienced thrombotic events of varying severity, including cerebral venous sinus thrombosis, deep vein thrombosis of the lower extremities, and pulmonary embolism. All four probands carried heterozygous mutations in the gene encoding the disintegrin-like domain ofADAMTS13, which were located in exon 8 c. 901C>G (p. Pro301Ala) in proband 1, exon 8 c. 902C>G (p.Pro301Arg) in probands 2 and 3, and exon 9 c. 1045C>T (p. Arg349Cys) in proband 4. Verification with the Human Gene Mutation Database (HGMD) confirmed that the p. Pro301Ala and p. Pro301Arg mutations had not been previously registered. Coagulation function testing revealed that ADAMTS13 activity (Act: 57.42%-72.88%) and antigen levels (Ag: 66.94%-78.34%) were significantly reduced in all four patients, while vWF activity (Act: 15.2%-213.7%) and antigen levels (Ag: 167.2%-216.6%) were markedly increased. Electrophoretic analysis of vWF multimers showed that the proportion of high-molecular-weight multimers (HMWMs) in the patients’ plasma was significantly increased, indicating a marked elevation of HMWMs compared to normal individuals, with grayscale values ranging from 166. 6 to 218. 9 versus 117. 4. Analysis using PyMOL software indicated that the mutation sites were located in key regions of the disintegrin-like domain encoded byADAMTS13, which may reduce the stability of theADAMTS13protein and decrease its binding capacity to vWF.ConclusionsThis study reports, for the first time, two novel mutations (p. Pro301Ala and p. Pro301Arg) in the disintegrin-like domain encoded byADAMTS13. Similar to the previously reported p. Arg349Cys mutation, these two mutations have been confirmed to reduce the antigen levels and activity of the ADAMTS13 protein. This is manifested as impaired cleavage capacity of ADAMTS13 towards HMWMs of vWF, resulting in an abnormal increase in the proportion of vWF multimers, disruption of the dynamic balance of the ADAMTS13- vWF axis, and an elevated risk of thrombosis in these patients.

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