Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population
Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population
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DOI：
10.1160/th12-10-0760
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发表时间：
2013-04-01
影响因子：
6.7
通讯作者：
Rezaie, Alireza R.
中科院分区：
文献类型：
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作者：
Ding, Qiulan;Yang, Likui;Rezaie, Alireza R.
蛋白 C 是血浆中维生素 K 依赖性丝氨酸蛋白酶原，在被凝血酶激活为活化蛋白 C (APC) 后，通过促凝血辅助因子 Va 和 Villa 的有限蛋白水解下调凝血级联。除了抗凝活性外，APC 还具有有效的细胞保护和抗炎活性。虽然 APC 的抗凝活性通过膜磷脂上蛋白 S 的辅因子功能得到增强，但 APC 的细胞保护性细胞内信号传导活性需要与血管内皮上表达的内皮蛋白 C 受体 (EPCR) 形成复合物。 APC 的两种天然变体 [Arg-147 至色氨酸取代 (R147W) 和 Lys-150 缺失 (K150del)] 在中国人群中已被鉴定为热点突变体，在 36 名蛋白 C 缺陷受试者中，出现频率分别为 27.8% 和 13.9%。受影响的个体表现出不同的血栓形成倾向。为了了解这些患者血栓表型的根本原因，我们在哺乳动物细胞中表达了这两种蛋白 C 变体，并使用已建立的体外和细胞测定来表征它们的抗凝和抗炎特性。我们的结果表明，R147W 和 K150del 变体在缺乏辅因子的情况下都具有正常的酰胺分解和蛋白水解活性。然而，在蛋白 S 存在的情况下，R147W 突变体与 EPCR 的结合亲和力降低了 3 倍，K150del 突变体的抗凝活性降低了 2-3 倍。这些结果为携带这些突变的中国患者中蛋白 C 缺乏的可能致病机制提供了一些见解。
Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the procoagulant cofactors Va and Villa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and, cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits similar to 3 times lower affinity for binding to EPCR and the K150del variant has similar to 2-3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.