Identification of Zebrafish Ortholog for Human Coagulation Factor IX and its Age-dependent Expression

Background: 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. Objective: To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology. Methods: 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. Results: 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. f9a knockdown and Fixa immunodepletion prolonged kPTT, whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kPT, and kRVVT. 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. Conclusions: 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.