Iyer Neha, Al Qaryoute Ayah, Kacham Meghana, Jagadeeswaran Pudur
Department of Biological Sciences, University of North Texas, Denton, TX, USA.
J Thromb Haemost. 2021 Sep;19(9):2137-2150. doi: 10.1111/jth.15365. Epub 2021 Jun 6.
Coagulation factor IX (FIX) is a serine protease zymogen involved in the intrinsic blood coagulation pathway, and its deficiency causes hemophilia B. Zebrafish has three f9 genes, and the ortholog to human F9 is unknown.
To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology.
Gene and protein sequence analysis for three f9 genes, f9a, f9b, and f9l, present in the zebrafish genome was performed. In vivo and in vitro assays after knockdown of each gene and immunodepletion using specific antibodies were carried out.
Sequence analysis revealed that f9a and f9b are similar to human F9, whereas f9l is similar to human F10. RNA analysis showed an age-dependent increase in expression of all three genes. Zebrafish f9a gene knockdown and Fixa immunodepletion prolonged kinetic partial thromboplastin time (kPTT), whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kinetic prothrombin time, and kinetic Russell viper venom activation time. Laser-assisted venous thrombosis increased time to occlusion after f9a and f9l knockdown and antibody inhibition of Fixa and Fixl. Further, analysis of plasma proteins by mass spectrometry and immunohistochemistry detected all three proteins.
Our findings suggest that zebrafish f9a has functional activity similar to human F9. Fixl is functionally similar to Fx. The age-dependent increases of these factors are comparable to those observed in mice and humans. Thus, the zebrafish model could be used to study factors involved in increasing f9a expression during aging. It could also be used to test whether normal human Factor IX and Factor IX Leyden promoter work in zebrafish background.
凝血因子IX（FIX）是一种参与内源性凝血途径的丝氨酸蛋白酶原，其缺乏会导致B型血友病。斑马鱼有三个f9基因，其与人F9的直系同源基因未知。
利用序列分析和piggyback敲低技术鉴定斑马鱼中与F9直系同源的基因。
对斑马鱼基因组中存在的三个f9基因f9a、f9b和f9l进行基因和蛋白质序列分析。在每个基因敲低后进行体内和体外试验，并使用特异性抗体进行免疫耗竭。
序列分析显示f9a和f9b与人类F9相似，而f9l与人类F10相似。RNA分析显示所有三个基因的表达均随年龄增长而增加。斑马鱼f9a基因敲低和Fixa免疫耗竭延长了动态部分凝血活酶时间（kPTT），而f9l敲低和Fixl免疫耗竭延长了kPTT、动态凝血酶原时间和动态罗素蝰蛇毒激活时间。激光辅助静脉血栓形成在f9a和f9l敲低以及Fixa和Fixl抗体抑制后增加了闭塞时间。此外，通过质谱和免疫组织化学对血浆蛋白的分析检测到了所有三种蛋白。
我们的研究结果表明斑马鱼f9a具有与人类F9相似的功能活性。Fixl在功能上与Fx相似。这些因子随年龄增长的增加与在小鼠和人类中观察到的情况相当。因此，斑马鱼模型可用于研究衰老过程中f9a表达增加所涉及的因素。它还可用于测试正常人凝血因子IX和凝血因子IX莱顿启动子在斑马鱼背景下是否起作用。
