STUDIES ON THE ROLE OF TRANSMEMBRANE PROTEINS IN HEMOSTASIS

Abstract

Transmembrane proteins (TMEMs) are a unique group of cell membrane-embedded proteins with largely undefined functions. Since we found 89 tmems in zebrafish thrombocytes by our RNAseq analysis, we hypothesized that they may play a role in thrombocyte biology and hemostasis. To verify this hypothesis, we screened these tmem genes by knockdown in zebrafish and assaying for gill bleeding. We found tmem242 and tmem183a knockdowns led to greater bleeding suggesting their roles in hemostasis. Therefore, we investigated Tmem242 and Tmem183a roles by knocking them down in zebrafish followed by assessing thrombocyte production, thrombocyte aggregation, fibrin formation, in vivo thrombus formation, and coagulation factors levels. We found that tmem242 knockdown did not affect thrombocyte production and aggregation, but it affected coagulation pathways observed by delaying fibrin formation and in vivo thrombusformation, which indicate bleeding phenotype. However, tmem242 knockdown led to increased mRNA levels of coagulation factors, especially f9a. Further investigations into Tmem242 role in hemostasis and F9a were done by conducting various assays including, western blot, microthrombi detection, ROS detection, and qRT-PCR to detect mRNA levels of hepatocyte transcription factors, such as sirt6 and nrf2. We found that tmem242 knockdown increased ROS production which signals to increase sirt6 transcription. This subsequently raises nrf2 expression, which in turn elevates f9a transcripts, leading to DIC-like conditions. On the other hand, tmem183a knockdown in zebrafish led to reduced thrombocyte counts, diminished aggregation, delayed fibrin generation, and prolonged in vivo thrombus formation. These findings were explained by the observed reduction in mRNA levels of thrombocyte producing genes, thpo and fli1 and by the decreased mRNA levels for several coagulation factors after tmem183a knockdown. Taken together, the knockdown of tmem183a in zebrafish resulted in bleeding phenotype linked to the downregulation of key genes involved in thrombopoiesis and decreased mRNA levels of coagulation factors. In summary, these studies highlight the crucial roles of Tmem242 and Tmem183a in maintaining normal hemostasis.