Interaction of Anti-Phospholipid Antibodies With Late Endosomes of Human Endothelial Cells

Abstract

—Anti-phospholipid antibodies (APLAs) are associated with thrombosis and/or recurrent pregnancy loss. APLAs bind to anionic phospholipids directly or indirectly via a cofactor such as β₂-glycoprotein 1 (β₂GPI). The lipid target of APLA is not yet established. Recently, we observed that APLAs in vitro can bind lysobisphosphatidic acid (LBPA). The internal membranes of late endosomes are enriched in this phospholipid. The current study was undertaken to determine to what extent binding of APLA to LBPA is correlated with binding to cardiolipin and to β₂GPI and to determine whether patient antibodies interact with late endosomes of human umbilical vein endothelial cells (HUVECs) and thus modify the intracellular trafficking of proteins. Binding of patient immunoglobulin G (n=37) to LBPA was correlated significantly with binding to cardiolipin. Although LBPA binding was correlated to a lesser extent with β₂GPI binding, we observed that β₂GPI binds with high affinity to LBPA. Immunofluorescence studies showed that late endosomes of HUVECs contain LBPA. Patient but not control antibodies recognized late endosomes, but not cardiolipin-rich mitochondria, even when we used antibodies that were immunopurified on cardiolipin. Incubation of HUVECs with patient plasma samples immunoreactive toward LBPA resulted in an accumulation of the antibodies in late endosomes and led to a redistribution of the insulinlike growth factor 2/mannose-6-phosphate receptor from the Golgi apparatus to late endosomes. Our results suggest that LBPA is an important lipid target of APLA in HUVECs. These antibodies are internalized by the cells and accumulate in late endosomes. By modifying the intracellular trafficking of proteins, APLA could contribute to several of the proposed pathogenic mechanisms leading to the antiphospholipid syndrome.