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Control of VWF secretion in human endothelial cells by local production of NO

Endothelium‐derived nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS) exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP‐dependent signaling or through S‐nitrosylation of proteins. The respective contribution of eNOS subcellular localization to sGC dependent or independent pathways is not known. The ability of local production of NO to influence non‐sGC dependent mechanisms will be tested by monitoring the secretion of Von Willebrand factor (vWF). The secretion of vWF from Weibel‐Palade Bodies is tonically inhibited by NO which nitrosylates N‐ethylmaleimide sensitive factor (NSF). eNOS “knockdown” HAEC were reconstituted with eNOS fusion proteins targeted to PM or Golgi. vWF production was greatly attenuated in HAEC expressing PM versus Golgi localized eNOS. To further investigate the mechanisms underlying reduced vWF secretion, we incubated HAECs with the sGC inhibitor ODQ to block the sGC‐cGMP pathway. vWF secretion was not significantly modified by ODQ in cells expressing either PM or Golgi localized eNOS and PM eNOS induced greater S‐nitrosylation of NSF versus Golgi eNOS. Furthermore, reconstitution of HAEC with Golgi or PM –targeted Ca2+/CaM ‐insensitive eNOS constructs, which produce equal amounts of NO regardless of location, resulted in equivalent inhibition of vWF secretion.