Small molecule inhibitor screen to identify mechanisms controlling selective clearance of sickle hemoglobin by liver endothelial cells

• Liver endothelial cell mediated hemoglobin internalization predominantly occurs via fluid phase endocytosis.

• Presence of lipid droplets promotes endothelial HbS internalization while hypolipidemic condition inhibits it.

Intrahepatic accumulation of cell-free hemoglobin (Hb) is a significant pathology linked with hemolytic disorders such as sickle cell disease (SCD). In addition to hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) were recently reported to contribute to Hb clearance in SCD mice and patients via currently unknown endocytic mechanism. Using small molecule inhibitors of endocytic pathway components in primary human and mouse LSECs, we show that sickle-Hb (HbS) uptake by LSECs occurs predominantly through micropinocytosis or fluid-phase endocytosis. However, inhibiting clathrin-mediated endocytosis, receptor recycling, or drop in pH also significantly attenuated HbS uptake by LSECs. LSEC-driven HbS uptake was independent of haptoglobin. Finally, we found that presence of lipid droplets promotes endothelial HbS internalization while hypolipidemic condition inhibits it. In conclusion, the current study identifies previously unknown alternate mechanism of LSEC-mediated HbS internalization. Our findings also inform the need to evaluate the therapeutic potential of blocking these mechanisms to ameliorate hemolysis-associated liver damage in SCD and other hemolytic disorders.