Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder resulting from mutations in Lman1. This gene encodes a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and factor VIII (FVIII) to the plasma. F5F8D is characterized by levels of both FV and FVIII reduced to ∼5-30% of normal. In contrast, Lman1 knockout mouse models of F5F8D exhibit FV and FVIII activities that are ∼50% of normal, relative to wildtype mice. Though FV and FVIII are synthesized at markedly different levels and potentially in different tissues, loss of the LMAN1 cargo receptor leads to parallel reductions in both FV and FVIII activity. FV is synthesized in hepatocytes (as well as megakaryocytes in the mouse). However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. We took advantage of the dependence of efficient FV and FVIII secretion on LMAN1 to examine the cellular source of each protein. FV and FVIII secretion profiles of conditional Lman1 knockout mice were characterized, relative to that of wildtype mice and ubiquitous Lman1 null mice (Lman1-/-). In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2-Cre or Albumin-Cre transgene was crossed into Lman1 conditional mice (Lman1fl). FV and FVIII activity levels were measured by functional coagulation activity assays. Though Lman1fl/fl/Tie2-Cre+ mice (endothelial-specific knockout) exhibit normal plasma FV activity (99.9%) relative to wildtype mice (set to 100%), FVIII activity is reduced to 53.5% (p < 2.5 x 10-6). In contrast, Lman1fl/fl/Alb-Cre+ (hepatocyte-specific knockout) mice demonstrate normal FVIII activity (89.0%) and reduced FV activity (37.0%) (p < 1.4 x 10-10). To confirm endothelial cells as the biosynthetic source of FVIII, we took advantage of the previously reported RiboTag mouse (Sanz et al., 2009. PNAS 106(33):13939-44) to isolate endothelial cell RNA for qPCR analysis from various murine tissues. RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre-recombinase expressing animal. qPCR analyses of endothelial cell RNA isolated from total liver lysates of five RiboTag/Tie2-Cre+ mice demonstrated 10-20 fold enrichment for gene transcripts that are known to be endothelial-specific, such as Cdhs (12.1 fold enrichment, p < 8.0 x 10-3), Vcam1 (13.4 fold enrichment, p <1.1 x 10-5), and Vwf (15.3 fold enrichment, p < 7.0 x 10-4), as well as for FVIII transcripts (11.4 fold enrichment, p < 4.0 x 10-5). In contrast, this analysis demonstrated a statistically significant depletion (5-10 fold) of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse. These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.