OC 45.2 - Knockdown of Zebrafish Tmem242 Enhances Production of ROS That Signals to Increase Foxa2 and Foxa3 Expression That May Increase Clotting Factors Resulting in Thrombosis and Bleeding

Background: Transmembrane proteins (TMEMs) are cell membrane-embedded proteins whose functions are elusive. RNAseq analysis of young and mature thrombocytes showed 62 tmems expressed in the thrombocytes. We hypothesized that the Tmems may play a role in hemostasis.

Aims: To decipher the role of Tmems in hemostasis.

Methods: Knockdown was by piggy-back method. Bleeding was monitored by gill bleeding assay. Thrombocyte aggregation was performed with ADP, collagen, and ristocetin. The mRNA and protein levels were quantified by qRT-PCR and Western blots, respectively. The kPT, kPTT, and laser thrombosis assays were used to measure clotting activity. ROS was measured using Dichlorofluorescin diacetate in larvae.

Results: Knockdown of 62 Tmems identified Tmem242 and Tmem183a as genes affecting bleeding compared to controls. Tmem242 knockdown showed no differences in thrombocyte aggregation in the presence of ADP and collagen; ristocetin showed less agglutination in knockdown samples, suggesting Vwf levels may be affected. We found increased Vwf mRNA levels. We tested for the expression of clotting factor genes F7, F8, F9a, F9b, F9l, F10, and TF. mRNAs for all these factors except F8 showed increased levels; F9a mRNA levels increased >10 fold. F9a also increased at the protein level. kPT and kPTT assays after Tmem242 knockdown showed slow clotting. Larval Tmem242 knockdown showed prolonged TTO and thrombi, suggesting thrombosis and bleeding. Since Tmem242 influences ATP synthase activity, it may control ROS levels. Knockdown of Tmem242 and oligomycin treatment showed ROS increases. Oligomycin also increased F9a levels. To test how ROS increases clotting factor levels, we quantified mRNA for hepatocyte transcription factors Hnf4a, Cebpa, Foxa1, Foxa2, Foxa3, Gata4, Gata6, and Prox1a. Foxa2 and Foxa3 transcripts increased but not other gene transcripts.

Conclusion(s): Tmem242 knockdown resulted in a lack of ATP synthase activity that enhances ROS production. This increases Foxa2 and Foxa3 expression, which may increase clotting factor transcripts resulting in thrombosis and bleeding.