维生素K依赖性凝血因子缺乏1型(VKCFD1)是一种罕见的遗传性出血性疾病,由γ-谷氨酰羧化酶(GGCX)突变引起。VKCFD1患者终身接受高剂量维生素K治疗,以纠正出血表型。然而,所有VKCFD1患者不能实现凝血因子活性的正常化。
本研究旨在调查所有报道的GGCX突变对维生素K的反应性,以优化治疗。
这项研究开发了一种使用基因工程GGCXs-/-细胞的检测方法,其中通过ELISA分析了GGCX突变对维生素K依赖性的促凝血因子和抗凝血因子的γ-羧酸能力。此外,测量因子VII活性以证明蛋白质的功能性。对于特异性GGCX突变,进行免疫荧光染色以评估凝血因子相对于GGCX野生型和突变的细胞内定位。
所有GGCX突变都分为反应者和低反应者突变,从而确定补充维生素K的效率。大多数VKCFD1患者具有至少一个能够γ-羧酸凝血因子的维生素K反应性GGCX等位基因。在少数患者中,出血表型不能通过维生素K的给药逆转,因为两个等位基因上的GGCX突变影响结构或催化重要位点,从而导致γ-羧酸凝血因子的残留能力.
通过这些新的功能数据,我们可以预测每个VKCFD1基因型的出血结果,因此,建议使用维生素K或凝血酶原复合物浓缩物进行治疗。
Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-glutamyl carboxylase (
GGCX). VKCFD1 patients are treated life-long with high doses of vitamin K in order to correct the bleeding phenotype. However, normalization of clotting factor activities cannot be achieved for all VKCFD1 patients.
The current study aims to investigate the responsiveness to vitamin K for all reported
GGCX mutations with respect to clotting factors in order to optimize treatment.
This study developed an assay using genetically engineered
GGCX-/- cells, in which
GGCX mutations were analyzed with respect to their ability to γ-carboxylate vitamin K dependent pro-coagulatory and anti-coagulatory clotting factors by ELISA. Additionally, factor VII activity was measured in order to proof protein functionality. For specific GGCX mutations immunofluorescent staining was performed to assess the intracellular localization of clotting factors with respect to
GGCX wild-type and mutations.
All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. Most VKCFD1 patients have at least one vitamin K responsive
GGCX allele that is able to γ-carboxylate clotting factors. In few patients, the hemorrhagic phenotype cannot be reversed by vitamin K administration because GGCX mutations on both alleles affect either structural or catalytically important sites thereby resulting in residual ability to γ-carboxylate clotting factors.
With these new functional data we can predict the hemorrhagic outcome of each VKCFD1 genotype, thus recommending treatments with either vitamin K or prothrombin complex concentrate.