Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of theF8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31^high, CD146^high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)⁺. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin⁻ and C-type lectin-like receptor-2 (CLEC-2)⁺.

Fig: F8 targeting strategy in F8 knock-in mice. (a) Schematic of F8 targeting construct. Diagrams show wild-type allele, targeting vector, targeting allele, floxed allele (F8^flox knock-in), and deleted allele (F8^Δ knock-in). The targeting vector was constructed by flanking the F8 exons 16–28 cDNA-SV40 polyA and PGK-Neo cassette with loxP sites, followed by linking 2A-EGFP cDNA-SV40 polyA. White boxes, F8 exon 16; dark gray boxes, F8 cDNA including exons 16–28 (stop codon); light gray boxes, 2A-EGFP cDNA; black boxes, SV40 poly A; white arrow, PGK-Neo cassette; black triangles, loxP sites; white triangles, FRT sites; black arrow, DTA. Restriction enzyme sites: Ap, ApaLI; S, SphI; A, ApaI. (b) Southern blotting of targeted embryonic stem cells. Genomic DNA was digested with ApaL I or Sph I, and probed with the 5′ or 3′ probe. Locations of probes are indicated in (a). (c) Confirmation of deletion of F8 cDNA (exons 16–28) in F8^Δknock-in mice. PCR was performed using the indicated F and R primers in (a).

In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31^high, CD146^high, Lyve1⁺, CLEC-2⁺, and podoplanin⁻ in liver sinusoidal endothelial cells.

Hayakawa, M., Sakata, A., Hayakawa, H. et al. Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.Sci Rep 11, 14824 (2021). https://doi.org/10.1038/s41598-021-94307-0Hayakawa, M., Sakata, A., Hayakawa, H. et al. Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.Sci Rep 11, 14824 (2021). https://doi.org/10.1038/s41598-021-94307-0