Engagement of Anti-GRP78 Autoantibodies with Cell Surface GRP78 on Endothelial Cells Drive Tissue Factor Activation

Abstract

Atherothrombosis is the underlying contributor of cardiovascular disease whereby the thrombotic events following atherosclerotic plaque rupture are linked to tissue factor (TF) and its procoagulant activity (PCA). We have reported previously that the 78-kilodalton glucose-regulated protein (GRP78) observed on the cell surface of tumour cells acts as a novel signalling receptor to regulate TF PCA. Cell surface GRP78 also acts as a neoantigen known to promote an immune response that leads to the generation of anti-GRP78 autoantibodies. Given that the proinflammatory cytokine tumour necrosis factor α (TNFα) is known to contribute to atherosclerotic lesion development and promote TF expression, I investigated whether the anti-GRP78 autoantibodies against cell surface GRP78 can regulate TF PCA in TNFα-treated cultured endothelial cells. In this M.Sc. thesis, I demonstrated that TNFα treatment promotes TF PCA, an effect mediated by NF-κB activation. Additionally, treatment with TNFα was observed to elevate cell surface GRP78 expression levels. Further, anti-GRP78 autoantibodies enhanced TF PCA in endothelial cells pre-treated with TNFα. These effects of the anti-GRP78 autoantibodies were further enhanced in cells pre-treated with ER stress-inducing agents, also known to enhance cell surface GRP78 levels. I also showed that anti-GRP78 autoantibodies can also elevate intracellular Ca2+ levels, which is known to contribute to TF activation. Sequestering the anti-GRP78 autoantibody or blocking the anti-GRP78 autoantibody from cell surface GRP78 attenuated anti-GRP78 autoantibody-induced TF PCA. In this study, we identified small chemical compounds predicted to bind to cell surface GRP78 (termed GRP78 binders) that inhibited anti-GRP78 autoantibody-induced TF PCA. Together, these findings provide evidence that the anti-GRP78 autoantibodies can contribute to enhanced TF PCA and that disrupting the engagement of the anti-GRP78 autoantibody to cell surface GRP78 with these GRP78 binders may represent a potential therapeutic strategy for the treatment and management of atherothrombosis.