Abstract：
BACKGROUND: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported.
OBJECTIVE: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC.
METHODS: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays.
RESULTS: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC.
CONCLUSIONS: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.
OBJECTIVE: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC.
METHODS: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays.
RESULTS: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC.
CONCLUSIONS: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.
摘要：
背景：蛋白C（PC）途径通过凝血酶-血栓调节蛋白（TM）复合物激活PC，并随后在蛋白S的帮助下失活活化的因子V（FVa）和因子VIII（FVIIIa），从而有助于止血平衡。我们确定了两名患有复发性血栓形成并携带相同杂合突变c.115.51A>G的无关患者，p。PROC基因中的Met343Val(M343V)。这种突变以前没有报道过。
目的：探讨PROC携带M343V突变患者抗凝缺陷的分子基础。
方法：我们在哺乳动物细胞中表达PC-M343V变体，并通过凝血试验表征其特性。
结果：我们的发现表明，虽然凝血酶TM对突变酶原的激活受到轻微影响，APC-M343V对显色底物的裂解明显受损。然而,Ca2+使切割效率提高了约50%。此外,APC-M343V和Na+之间的亲和力严重降低。此外，APC-M343V对FVa的抑制能力明显受损。结构和模拟分析表明，Val343可能会破坏Trp380的潜在氢键，并导致Trp380更接近His211，从而可能干扰底物结合并使APC的催化三联体不稳定。
结论：患者的M343V突变会对活性位点的反应性和/或折叠以及生理底物与蛋白酶的结合产生不利影响，导致蛋白C抗凝血活性受损，最终导致血栓形成。
目的：探讨PROC携带M343V突变患者抗凝缺陷的分子基础。
方法：我们在哺乳动物细胞中表达PC-M343V变体，并通过凝血试验表征其特性。
结果：我们的发现表明，虽然凝血酶TM对突变酶原的激活受到轻微影响，APC-M343V对显色底物的裂解明显受损。然而,Ca2+使切割效率提高了约50%。此外,APC-M343V和Na+之间的亲和力严重降低。此外，APC-M343V对FVa的抑制能力明显受损。结构和模拟分析表明，Val343可能会破坏Trp380的潜在氢键，并导致Trp380更接近His211，从而可能干扰底物结合并使APC的催化三联体不稳定。
结论：患者的M343V突变会对活性位点的反应性和/或折叠以及生理底物与蛋白酶的结合产生不利影响，导致蛋白C抗凝血活性受损，最终导致血栓形成。
