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Abstract：
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.
摘要：
这项研究的目的是表征斑马鱼凝血辅因子fviii和fv突变鱼，并评估它们是否在人类中出现典型的血友病A和因子V缺乏症。通过ENU诱变产生的fviii和fv斑马鱼杂合子突变体的胚胎购自ZIRC库。他们被抚养到成年并进行基因分型。将雄性和雌性杂合子杂交得到纯合子，杂合子，和野生型鱼。对正常和突变成年鱼进行了功能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，以确认它们分别在外显子19和外显子2中是否有过早的终止密码子，在两个突变体中，氨基酸谷氨酰胺被替换为终止密码子。与野生型对照相比，fviii和fv缺陷突变体在受精后5天（dpf）的纯合和杂合幼虫在静脉激光损伤后表现出延长的闭塞时间。纯合和杂合fviii成年突变体在血浆的动力学部分凝血活酶时间（kPTT）测定中显示出适度的出血和延迟的纤维蛋白形成。fv纯合幼虫的存活率超过12dpf。然而,与野生型同胞相比，杂合子fv突变体在kPTT和kPT测定中表现出大量出血和纤维蛋白形成延长。我们的表征显示，来自ZIRC的fviii和fv突变体在人类中表现出相当大的经典血友病A和因子V缺乏症，分别。这些模型应该可用于研究和开发逆转表型的新药，以及产生抑制突变以鉴定弥补这些缺陷的新因素。
