Abstract：
Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC\'s anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.
摘要：
蛋白C(PC),血浆中维生素K依赖性丝氨酸蛋白酶酶原，可以被凝血酶-血栓调节蛋白（TM）复合物激活，导致活化蛋白C（APC）的形成。APC通过灭活活性凝血因子V（FVa）和VIII（FVIIIa）来下调凝血酶的产生。PC缺乏会增加静脉血栓栓塞（VTE）的风险。我们已经确定了两名具有相同杂合突变的无关VTE患者（c.1384T>C，P.Ter462GlnextTer17）在PROC中。为了理解这种突变在VTE发展中的作用，我们在哺乳动物细胞中表达了重组PC-Ter462GlnextTer17,并使用已建立的凝血测定系统评估了其特性.功能研究表明，凝血酶或凝血酶-TM复合物对突变体的激活有明显损害。此外，APC-Ter462GlnextTer17对显色底物S2366的水解活性降低。APTT和FVa降解实验表明，突变蛋白的抗凝血活性均明显受损，无论蛋白S是否存在。这些结果进一步得到使用纯化和基于血浆的系统进行的凝血酶生成测定的支持。总之,Ter462GlnextTer17突变在PC的C端引入了一个新的尾巴，导致PC酶原激活和APC抗凝血功能的活性受损。这种损害有助于携带这种杂合突变的个体的血栓形成,并且代表VTE的遗传风险因素。
