Thrombin Contributes to Anti-myeloperoxidase Antibody Positive IgG-Mediated Glomerular Endothelial Cells Activation Through SphK1-S1P-S1PR3 Signaling

Background: Activation of coagulation system plays an important role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. Thrombin, generated during coagulation could disrupt endothelial barrier integrity through protease-activated receptor 1 (PAR1). Our previous study found that sphingosine-1-phosphate (S1P) contributed to myeloperoxidase (MPO)-ANCA-positive IgG-induced glomerular endothelial cell (GEnC) activation through a S1P receptor (S1PR)-dependent route. In recent years, S1P signaling was reported to be involved in thrombin effects on endothelial cells. This current study investigated whether the interaction between thrombin-PAR and S1P-S1PR signaling contributed to MPO-ANCA-positive IgG-induced GEnC dysfunction. Methods: The effect of thrombin on GEnC activation was analyzed from three aspects. First, morphological alteration of GEnCs was observed. Second, permeability assay was performed to determine GEnC monolayer activation quantitatively. Third, endothelin-1 (ET-1) levels were measured. Expression levels of sphingosine kinases (SphKs) and S1PRs were detected. In addition, antagonists of PAR1 and S1PR3 were employed to determine their roles. Eventually, PAR1 and tissue factor (TF) expression levels as well as TF procoagulant activity were analyzed. Results: Thrombin induced further damage of tight junction, increase in endothelial monolayer permeability as well as upregulation of ET-1 levels in GEnCs stimulated with MPO-ANCA-positive IgG. Blocking PAR1 downregulated ET-1 levels in the supernatants of GEnCs treated by thrombin plus MPO-ANCA-positive IgG. Expression levels of SphK1, S1PR3 increased significantly in GEnCs treated with thrombin plus MPO-ANCA-positive IgG. S1P upregulated PAR1 and TF expression, and enhanced procoagulant activity of TF in MPO-ANCA-positive IgG-stimulated GEnCs. Conclusion: Thrombin synergized with SphK1-S1P-S1PR3 signaling pathway to enhance MPO-ANCA-positive IgG-mediated GEnC activation.