Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier
Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier
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蛋白 C 中的 Gly197Arg 突变导致杂合子携带者复发性血栓形成
DOI：
10.1111/jth.14777
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发表时间：
2020-04-09
影响因子：
10.4
通讯作者：
Rezaie, Alireza R.
中科院分区：
文献类型：
--
作者：
Lu, Yeling;Giri, Hemant;Rezaie, Alireza R.
背景 活化蛋白 C (APC) 通过有限的蛋白水解使促凝血辅因子 Va 和 VIIIa 失活，从而下调凝血酶的生成。我们鉴定了两名蛋白 C 缺乏患者，他们都在 PROC 中携带杂合性 Gly197 至 Arg (G197R) 突变，并经历静脉血栓形成。 目的 本研究的目的是确定携带 G197R 突变的患者凝血缺陷的分子基础。 方法 我们在哺乳动物细胞中表达蛋白 C-G197R，并在已建立的凝血和抗炎测定系统中表征其特性。 结果 蛋白的激活凝血酶的C-G197R提高了10倍；然而，凝血酶对其的激活并不受血栓调节蛋白（TM）的促进。在组织因子介导的凝血酶生成测定中，向缺乏蛋白C的血浆中添加可溶性TM并补充蛋白C-G197R，对凝血酶生成参数没有显着的抑制作用。 APC-G197R 在纯化或血浆检测系统中均未表现出显着的抗凝血活性。 APC-G197R 基本上没有活性，因为它在 aPTT 测定中没有显示出活性。内皮细胞通透性测定表明，APC-G197R 的抗炎活性也显着受损。结构模型预测，Arg 的侧链不能容纳在 APC 的这个位点，除非局部结构发生重大变形，这种变形似乎会传播并对催化口袋的反应性/折叠产生不利影响。 结论 患者中的 G197R 突变似乎在功能上等同于杂合蛋白 C 敲除，其中一半蛋白没有显着活性，从而导致血栓形成。
Background Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis.Objective The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation.Methods We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems.Results The activation of protein C-G197R by thrombin was improved 10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket.Conclusion The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.