Protein Phosphatase 2B Inhibition Promotes the Secretion of Von Willebrand Factor from Endothelial Cells

Ultra-large von Willebrand factor (ULVWF) multimers secreted from the Weibel-Palade bodies (WPBs) of endothelial cells plays a crucial role in the development of thrombotic microangiopathies. Secretion of WPB contents is regulated, in part, by the phosphorylation of vesicle trafficking proteins that constitutes the endothelial exocytotic machinery. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate the exocytosis of WPB contents, a role for protein phosphatases in regulating endothelial exocytosis is currently undefined. In this study, we show that inhibition of serine/threonine protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus (FK506) or a cell-permeable PP2B autoinhibitory (AI) peptide promotes the secretion of hyper-adhesive ULVWF from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs induce the tethering of adhesive platelets. In support of a role for PP2B in VWF secretion, we demonstrated that the catalytic subunit of PP2B expressed as a GST fusion protein interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, was significantly increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis and secretion of VWF. Furthermore, plasma VWF antigen levels were also enhanced in CsA-treated mice, and siRNA-mediated knockdown of PP2Bb in HUVECs significantly enhanced VWF secretion. These observations suggest that CsA promotes VWF release, at least in part by inhibition of PP2B activity. These observations are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans, as well as the association between prolonged exposure to CsA and thrombotic microangiopathy in a subset of exposed patients.