Factor VIII originates primarily from anatomically distinct subsets of liver sinusoidal endothelial cells

Despite decades of research, the precise cellular origins of coagulation factor VIII (FVIII), deficient in the inherited bleeding disorder hemophilia A (HA), have remained controversial. Early studies proposed that FVIII comes from hepatocytes, similar to most other coagulation factors. More recent data pointed to liver sinusoidal endothelial cells (LSECs) as the primary source of FVIII, but conflicting reports and methodological limitations have obscured a clear understanding of hepatic and extrahepatic FVIII expression. Discordance between natural FVIII biosynthesis sites and cellular targets of adeno-associated viral (AAV) vectors has been implicated in efficacy limitations of current gene therapies for hemophilia A. Here, we developed and employed a novel gene-edited F8 promoter-reporter mouse model, humanized mice, and human tissue samples to definitively map FVIII-producing cells. We found that FVIII originates primarily from LSECs, not hepatocytes, corroborating most recent reports. Additionally, we detected the F8 reporter in renal endothelial cells. Further, we identified a striking microanatomical variation in FVIII expression between LSECs situated in different hepatic lobular zones, revealing that FVIII synthesis is predominantly localized to LSECs in Zones 2 and 3 of hepatic lobules in both mice and humans, with minimal expression in Zone 1. This zonal pattern was maintained even in the context of steatohepatitis. Our work clarifies the primary cellular source of FVIII and unravels the microheterogeneity of FVIII expression within the liver, providing insights for optimizing gene therapy strategies for hemophilia A that aim to induce FVIII production in its natural endogenous expression sites.