Protein Phosphatase 2B Inhibitor, Cyclosporine A, Stimulates Endothelial Cell Exocytosis of Ultra-Large VWF Multimeric Strings by Regulating Munc18c Phosphorylation.

Exocytosis of Weibel Palade bodies (WPBs) containing ultra-large Von Willebrand factor (ULVWF) plays an essential role in the processes of inflammation and thrombosis. The exocytosis of ULVWF involves the fusion of WPBs to the endothelial cell plasma membrane via vesicular trafficking families of proteins. Reversible post-translational modification, including the phosphorylation of tyrosine (Tyr) and/or serine/threonine (Ser/Thr) residues or S-nitrosylation of cysteine residues on one or more vesicular trafficking proteins tightly regulates the exocytotic process. The phosphorylation of any trafficking protein is determined by the interplay between protein kinases and protein phosphatases and any alteration in the activity of either enzyme can affect exocytosis. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate exocytosis in endothelial cells, there is scant information about any possible role for the regulation of protein phosphatases in this process. Inhibition of protein phosphatase 2B (PP2B) also called calcineurin by the immunosuppressive drug, cyclosporine (CsA), is associated with thrombotic microangiopathy in a subset of transplant patients. Calcineurin is the target of a cyclophilin-CsA complex. We detected both calcineurin and cyclophilin A in resting human umbilical vein endothelial cells (HUVECs). Because CsA is a potent inhibitor of PP2B, we hypothesized that CsA induces ULVWF secretion and thrombotic events at least in part, by facilitating the phosphorylation of vesicle trafficking proteins that promote the exocytosis of ULVWF from endothelial cells. In vitro, we found that nanomolar concentrations of CsA or a cell permeable PP2B auto inhibitory peptide stimulated the release of ULVWF from HUVECs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assays revealed that PP2B inhibitors did not damage the HUVECs. In vivo, plasma levels of VWF were increased after intraperitoneal injection of CsA. These studies suggest that PP2B negatively regulate ULVWF exocytosis. Microscopic studies revealed that the ULVWF strings secreted by HUVECs treated with CsA or PP2B auto-inhibitory peptide induced the adhesion of platelets. CsA-treated HUVECs exhibited an increased serine phosphorylation of Munc18c, a vesicle trafficking associated protein that promotes granule exocytosis. These findings support a mechanism whereby inhibition of PP2B by CsA enhances cell secretion, including endothelial cell secretion of ULVWF by facilitating Munc18c phosphorylation.