Phosphatidylinositol-3,4,5-triphosphate-dependent rac exchange factor 1 regulates epinephrine-induced exocytosis of weibel-palade bodies

Background: Weibel-Palade bodies (WPBs) function as storage vesicles for von Willebrand factor (VWF) and a number of other bioactive compounds, including angiopoietin-2 and insulin-like growth factor-binding protein 7. WPBs release their content following stimulation with agonists that increase the level of intracellular Ca²⁺, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Objective: Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Methods: Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Results: Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory β-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Conclusion: Our findings show that PREX1 regulates epinephrine-induced release of WPBs.