Although it is known that factor VIII (FVIII) plasma levels increase rapidl
y in response to a number of stimuli, the biological stimuli behind this re
lease is less clear. Previously, we showed that FVIII can traffic together
with von Willebrand factor (vWF) into storage granules in a pituitary tumor
cell line, AtT-20; however, AtT-20 cells could not be used to address the
release or functional activity of released FVIII. To investigate the regula
ted secretion of stored FVIII, endothelial cells with intact agonist-stimul
ated release pathways were used. Human umbilical vein endothelial cells (HU
VECs) were transduced with retroviral FVIII construct [kFVIII(V)] to create
a FVIII/vWF storage pool. Immunofluorescent staining of transduced cells d
emonstrated FVIII in Weibel-Palade bodies. In contrast, the transduction of
hFVIII(V) into HT-1080 and HepG2 cells displayed FVIII only in the cytopla
sm. We studied the regulated release of both FVIII and vWF from endothelial
cells after agonist-induced stimulation and demonstrated a parallel releas
e of FVIII and vWF proteins. This released FVIII was functionally active. H
ence, endothelial cells transduced with hFVIII(V) store FVIII together with
VWF in Weibel-Palade bodies, creating a releasable storage pool of both pr
oteins. Because FVIII secretion can be physiologically regulated by agonist
s in culture, this may explain the pharmacological agonist-induced release
of FVIII by drugs such as desmopressin in vivo and suggests vascular endoth
elium as a reasonable target of gene therapy of hemophilia A.