Department of Biological Sciences, University of North Texas, Denton, Texas, USA.
Blood Coagul Fibrinolysis. 2024 Jul 1;35(5):238-247. doi: 10.1097/MBC.0000000000001308. Epub 2024 Jun 10.
The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.
本研究旨在对斑马鱼凝血因子 VIII 和因子 V 突变鱼进行特征描述，并评估它们是否在表型上类似于人类的经典血友病 A 和因子 V 缺乏症。通过ENU 诱变产生的 fviii 和 fv 杂合突变斑马鱼胚胎购自 ZIRC 库。将它们饲养至成年并进行基因分型。杂合子雌雄鱼交配以获得纯合子、杂合子和野生型鱼。对正常和突变成年鱼进行功能性动力学凝血测定和出血测定，并对幼虫进行静脉激光损伤测定。对 fviii 和 fv 突变体的 DNA 进行测序，以确认它们是否在第 19 外显子和第 2 外显子中分别具有过早的终止密码子，并且在这两个突变体中，谷氨酰胺氨基酸被终止密码子取代。与野生型对照相比，fviii 和 fv 缺陷突变体的 5 天受精后（dpf）的纯合和杂合子幼虫在静脉激光损伤后出现闭塞时间延长。fviii 纯合和杂合突变体的成年鱼的血浆在动力学部分凝血活酶时间（kPTT）测定中表现出轻微出血和纤维蛋白形成延迟。fv 纯合子幼虫在 12 dpf 后存活率较差。然而，杂合 fv 突变体在 kPTT 和 kPT 测定中表现出严重出血和纤维蛋白形成延长，与野生型兄弟姐妹相比。我们的特征描述表明，ZIRC 的 fviii 和 fv 突变体在很大程度上分别模拟了人类的经典血友病 A 和因子 V 缺乏症。这些模型应该有助于研究和开发逆转表型的新型药物，并产生抑制突变以鉴定补偿这些缺陷的新因子。
