Activation of Endothelial Cells by β₂-Glycoprotein I (β₂GPI) Antibodies Is Mediated by Annexin II Cross Linking and May Involve TLR4.

The antiphospholipid antibody syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (APLA) in association with thrombosis and/or recurrent fetal loss. Although initially thought to directly recognize anionic phospholipids, most APLA actually recognize phospholipid binding proteins, most commonly β₂-glycoprotein I (ß₂GPI). β₂GPI binds with high affinity to annexin II on the surface of endothelial cells (Ma et al, JBC, 2000), and β₂GPI-dependent “APLA” activate endothelial cells in a β₂GPI-dependent manner (Simantov et al., JCI, 1995). Moreover, a preliminary report from our laboratory has demonstrated that cross-linking of annexin II bound β₂GPI by APLA/anti-β₂GPI antibodies leads to endothelial cell activation through a pathway involving NF-κB (Zhang et. al, Blood 2003). However, the mechanism by which annexin II cross-linking might induce signaling responses is uncertain, as annexin II is not a transmembrane protein. We thus have investigated the hypothesis that activation of endothelial cell signaling pathways by annexin II cross-linking might require a transmembrane “adaptor” protein that spans the plasma membrane, yet associates with cell surface annexin II. First, human umbilical vein endothelial cell (HUVEC) surface proteins were biotin labeled using the membrane impermeable biotinylation reagent NHS-LC-biotin, and labeled annexin II binding proteins were affinity purified on immobilized annexin II. Purified annexin II binding proteins were detected following 10% SDS-PAGE, transfer to PVDF and development of the membranes using streptavidinperoxidase and chemiluminescence. These studies revealed bands of ~83, ~79, ~62 and ~34 kD. None of these bands were affinity-purified on immobilized bovine serum albumin, suggesting that specific annexin II binding proteins were present on the surface of endothelial cells. To identify these proteins, affinity-purification of annexin II binding proteins from ~40 x 10⁶ HUVEC was undertaken using Affi-Gel HZ to which 10 mg of recombinant annexin II was coupled. Elution of bound proteins from this column followed by SDS-PAGE and staining with Coomassie brilliant blue revealed proteins of similar Mr as those identified using cell-surface labeled HUVEC. These proteins were excised from the gel, and analyzed by LC-MS following in gel tryptic digestion. Results of these studies revealed that the ~83 kD band was the Toll-like receptor 4 (TLR-4), the ~79 kD band was nucleolin, the ~62 kD band was calreticulin, and the ~34 kD band was annexin II. While we have not yet analyzed the role of each of these proteins in APLA/anti-β₂GPI antibody-mediated endothelial cell activation, we have performed preliminary studies to address the potential involvement of TLR4, as a recent report demonstrated potential involvement of MyD88, a downstream mediator of TLR4-dependent signaling, in the activation of endothelial cell lines by APLA/anti-β₂GPI antibodies (Raschi et al., Blood 2003). Preliminary studies suggest that TLR4 co-immunoprecipitates with annexin II, and that APLA/anti-β₂GPI antibody induced endothelial cell activation, but not that caused by TNF-α, was partially blocked following transfection of endothelial cells with TLR4 siRNA. In conclusion, these studies confirm that cross-linking of endothelial annexin II initiates APLA/anti-β₂GPI antibody-induced endothelial cell activation, and suggests the involvement of TLR4, and perhaps other endothelial cell surface proteins.