CD36 regulates factor VIII secretion from liver endothelial cells

Coagulation factor VIII (FVIII) is essential for hemostasis,1-3 and elevated FVIII is a risk factor for venous thromboembolism (VTE).2-6 The liver is a major source of FVIII, and a subset of liver endothelial cells (ECs), marked by CD32+, produce high levels of FVIII.7-12 FVIII is secreted by vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane in a pathway that includes the proteins COPII, lectin mannose binding 1 (LMAN1), and the ER cargo receptor complex MCFD2.13,14 Lipopolysaccharide stimulates EC release of FVIII,15,16 but little is known about other agonists that induce FVIII release from ECs. Genome-wide association studies of FVIII have identified candidate genes that regulate FVIII levels in humans. A genome-wide association studies and whole-exome sequencing association study found 13 loci associated with plasma FVIII activity, of which 1 locus includes the CD36 gene.17-19 The lead genetic variant of CD36 associated with FVIII, designated rs3211938, is a loss-of-function variant.20 The minor allele frequency of this variant is 8% in individuals of African ancestry, and is not detected in individuals of European ancestry.17 These data identify CD36 as a candidate protein that regulates FVIII levels in humans. CD36 is a cell surface protein expressed in diverse cells, including ECs.21,22 CD36 is a scavenger receptor that interacts with ligands including long-chain fatty acids and oxidized low-density lipoprotein (oxLDL).21,22 We hypothesized that CD36 regulates FVIII levels by controlling its release from the ECs. We purified CD32+ human liver ECs (HLEC) from a commercial source of liver ECs, cultured HLEC in vitro, and measured FVIII release by enzyme-linked immunosorbent assay. We silenced CD36 in HLEC, added oxidized LDL and an inhibitor of p38, and measured FVIII release. Detailed methods are provided in supplemental Material. We first identified HLEC that express FVIII.10-12 We reanalyzed publicly available single cell RNA sequencing (scRNA-Seq) data of the human liver (GSE115469)10 and, consistent with previous reports, identified 3 subpopulations of ECs in the human liver (Figure 1A).10,11 A set of differentially expressed genes separated these 3 subpopulations (Figure 1B). One cluster of ECs (cluster 11) expressed high levels of CD36 and F8 (Figure 1C). This subpopulation also expressed high levels of cell surface protein CD32 (encoded by FCGR2B) (Figure 1C). We refer to these CD32-expressing HLEC as CD32+ HLEC.