华法林抑制蛋白C的γ-羧基化致其滞留于内质网  

Warfarin inhibiting-carboxylation of protein C and retaining it in the endoplasmic reticulum

作　　者：李晓明[1,2] 沈滟 曹青[1,2] 沈国民 LI Xiaoming;SHEN Yan;CAO Qing;SHEN Guomin(College of Basic Medicine and Forensic Medicine,Henan University of Science and Technology,Luoyang 471023,China;Henan International Joint Laboratory of Thrombosis and Hemostasis,Luoyang 471023,China)

机构地区：[1]河南科技大学基础医学与法医学院,河南洛阳471023 [2]河南省血栓与止血国际联合实验室,河南洛阳471023

出　　处：《安徽大学学报(自然科学版)》2025年第2期84-90,共7页Journal of Anhui University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(81770140,82170133);河南省科技攻关项目(212102310629,212102310877)。

摘　　要：华法林是临床常用抗凝药,有研究表明华法林可以抑制蛋白C(PROC)的分泌,但机制不清.为探讨华法林导致PROC分泌水平降低的机制,笔者利用酶联免疫吸附试验检测PROC总抗原的分泌;通过免疫印迹实验检测细胞内和培养基上清中PROC的表达以及γ-羧基化水平;采用荧光成像以及荧光定量对PROC在胞内进行亚细胞定位分析.结果显示,华法林抑制PROC总抗原分泌,并存在浓度依赖效应.免疫印迹实验分析胞内和胞外分泌的PROC及其γ-羧基化水平,发现胞内PROC的γ-羧基化水平降低与PROC抗原分泌减少相关.通过亚细胞定位和荧光定量分析,发现华法林处理后的PROC会滞留在内质网,无法正常转移到高尔基体中;该结果还提示PROC的Gla结构域的γ-羧基化修饰是影响其分泌的关键因素.以上研究表明华法林通过抑制PROC的Gla结构域的γ-羧基化,导致其滞留于内质网并抑制其分泌.Warfarin is a commonly used clinical anticoagulant,and some studies have shown that Warfarin can inhibit protein C(PROC)secretion,but the mechanism is unclear.In this paper,to investigate the mechanism underlying the reduced secretion of PROC caused by warfarin,the authors employed an enzyme-linked immunosorbent assay(ELISA)to measure the levels of PROC antigen secretion;the expression of PROC and the level of-carboxylation in intracellular and culture medium supernatants were detected by immunoblotting assay;and the subcellular localization of PROC in the cell was analyzed by fluorescence imaging as well as fluorescence quantification.The results showed that Warfarin inhibited PROC total antigen secretion with a concentration-dependent effect.Immunoblot experiments analyzed the levels of PROC within cells and its secretion outside the cells,as well as the-carboxylation levels.The findings revealed that a decrease in the-carboxylation level of intracellular PROC was associated with a reduction in the secretion of PROC antigen.By cellular sublocalization and quantitative fluorescence analysis,it was found that Warfarin-treated PROC would be retained in the endoplasmic reticulum and could not be transferred to the Golgi apparatus normally;this result also suggested that the-carboxylation modification of the Gla structural domain of PROC was a key factor affecting its secretion.In conclusion,the above studies suggested that Warfarin affects the-carboxylation of the Gla domain of PROC,leading to its retention in the endoplasmic reticulum and inhibiting its secretion.

关 键 词：华法林 蛋白C γ-羧基化 内质网 维生素K循环 分泌 

分 类 号：Q28[生物学—细胞生物学]