Tanratana Pansakorn, Seanoon Karnsasin, Payongsri Panwajee, Kadegasem Praguywan, Chuansumrit Ampaiwan, Sirachainan Nongnuch
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Ramathibodi Hemostasis and Thrombosis Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Thromb Haemost. 2025 Jun;125(6):533-544. doi: 10.1055/a-2408-9529. Epub 2024 Sep 3.
Protein C (PC) deficiency is a well-established risk factor for thromboembolism (TE), commonly manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms of two novel PC mutations, C238G and R189W, identified in Thai children with both venous and arterial TE.The effects of wild-type (WT), C238G, and R189W PC variants were investigated through transient transfection of HEK293T cells. PC secretion levels were measured, and immunofluorescence analysis was performed to assess intracellular localization. ER stress-related gene expression and UPR activation were evaluated. Structural analysis was conducted to explore the significance of the C238 and R189W residue in PC functionality.The C238G mutation led to a severe 95% reduction in PC secretion, while R189W showed a 30% decrease compared with WT. Immunofluorescence revealed that C238G-PC was predominantly retained in the ER, indicating protein misfolding. C238G-expressing cells exhibited significant upregulation of ER stress-related genes and UPR activation. In contrast, R189W resulted in only a modest increase in UPR gene expression, suggesting a less pronounced impact on protein folding and secretion. Structural analysis demonstrated the critical role of the C238 residue in maintaining PC's disulfide bond and overall conformation.This study reveals distinct molecular mechanisms by which the C238G and R189W mutations contribute to PC deficiency and increased thrombotic risk. The findings emphasize the essential role of the C238 residue in preserving PC structure and secretion, enhancing the understanding of PC deficiency-associated TE in pediatric patients.
蛋白C（PC）缺乏是血栓栓塞（TE）公认的危险因素，常见于儿科患者。本研究旨在阐明在泰国患有静脉和动脉TE的儿童中鉴定出的两种新型PC突变C238G和R189W的致病机制。通过瞬时转染HEK293T细胞研究野生型（WT）、C238G和R189W PC变体的作用。测量PC分泌水平，并进行免疫荧光分析以评估细胞内定位。评估内质网应激相关基因表达和未折叠蛋白反应（UPR）激活。进行结构分析以探索C238和R189W残基在PC功能中的意义。C238G突变导致PC分泌严重减少95%，而R189W与WT相比减少30%。免疫荧光显示C238G-PC主要保留在内质网中，表明蛋白质错误折叠。表达C238G的细胞表现出内质网应激相关基因的显著上调和UPR激活。相比之下，R189W仅导致UPR基因表达适度增加，表明对蛋白质折叠和分泌的影响较小。结构分析证明了C238残基在维持PC二硫键和整体构象中的关键作用。本研究揭示了C238G和R189W突变导致PC缺乏和血栓形成风险增加的不同分子机制。这些发现强调了C238残基在维持PC结构和分泌中的重要作用，增进了对儿科患者PC缺乏相关TE的理解。
