Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

中文翻译：

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人因子XII的kringle结构域中的突变会引起自发炎症，干扰酶原的静止并加速激活。

凝血因子XII（FXII）驱动炎性肽缓激肽的产生。编码FXII的F12基因中的病理突变引起遗传性血管性水肿（HAE）引起的急性组织肿胀。有趣的是，最近发现的导致W268R取代的F12突变与HAE不相关。取而代之的是，FXII-W268R携带者会遭受感冒引起的荨麻疹，关节痛，发烧和疲劳。在这里，我们旨在研究FXII-W268R变体的分子特征。我们在HEK293自由式细胞中表达了野生型FXII（FXII-WT），FXII-W268R和FXII-T309R（导致HAE）以及其他FXII变体。使用生色底物测定，免疫印迹和ELISA，我们分析了表达培养基，细胞裂解液和纯化的蛋白的FXII激活作用。重组FXII-W268R形成增加量的细胞内裂解产物，所述裂解产物也存在于表达培养基中并显示酶活性。活性位点丧失能力的变体FXII-W268R / S544A显示细胞内片段化很大程度上取决于自激活。与FXII-WT或FXII-T309R相比，纯化的FXII-W268R对血浆激肽释放酶和纤溶酶的激活高度敏感。此外，结合研究表明，FXII-W268R变体导致暴露于FXII-WT中隐秘的纤溶酶原结合位点。在血浆中，重组FXII-W268R自发触发高分子量激肽原的裂解。我们的发现表明，W268R取代会影响FXII-WT中隐藏的FXII蛋白构象和激活环的暴露。这导致活化的FXII的细胞内自激活和组成型低度分泌。这些发现有助于解释FXII-W268R变异体的载体中接触激活的长期增加。