Phorbol Ester-Stimulated VWF Secretion from Human Umbilical Vein Endothelial Cells

Abstract

Von Willebrand factor (VWF) and the VWF-propeptide (proregion) are stored for secretion within specialized endothelial cell secretory organelles called Weibel-Palade bodies (WPBs). Because phorbol esters are potent VWF secretagogues and cause extensive WPB degranulation, it is generally assumed that PMA-evoked VWF secretion arises solely from WPB exocytosis (the VWF storage compartment). Here we present evidence that a significant fraction of the early phase of phorbol myristate acetate (PMA)-evoked VWF secretion from human endothelial cells arises from a cycloheximide (CHX)-sensitive (ie, nascent, non-storage) compartment. Stimulation for 15 minutes with either PMA (160nM) or histamine (100μM) caused similar amounts of VWF secretion. However, prior inhibition of protein synthesis (10μM CHX for 24 hours) reduced PMA-evoked secretion by ∼70% while histamine-evoked secretion was unaffected. Optical analysis of live cells expressing a fluorescent protein targeted to WPBs showed that the first 15 minutes of PMA stimulation was associated with fusion of 8.4 ±2.1% (mean±sem, n=11 cells) of fluorescent WPBs, compared to 41.4±4.0 % (mean±sem, n=12) for histamine. For both stimuli, the percentage of fluorescent WPB fusion events was unaffected by CHX treatment. Our data suggest that in addition to driving WPB exocytosis, PMA also causes acute release of non-stored VWF and proregion, most likely from the trans-Golgi network. The route and mechanisms regulating PMA-evoked release of non-stored VWF are under investigation.