Regulated von Willebrand factor (vWf) secretion is restored by pro-vWf expression in a transfectable endothelial cell line.
Von Willebrand factor (vWf) is a glycoprotein involved in primary hemostasis and synthesized in endothelial cells (EC). vWf is stored in secretory granules specific for EC called Weibel-Palade bodies (WPb). Studies on the molecular mechanisms of vWf storage and acute release are hampered by the limitations of the available endothelial cell culture models. We created a suitable model by stable transfection of the vWf-negative ECV304 endothelial cell line with pro-vWf cDNA. Pro-vWf was normally cleaved to mature vWf and stored in WPb. Acute vWf release occurred in response to the calcium ionophore A23187. Thus, vWf expression is sufficient to restore functional secretory granules in ECV304 cells. We used this model to study the role of WPb in the storage of tissue-type plasminogen activator (t-PA), a key fibrinolytic enzyme that is acutely released by EC, but whose intracellular storage compartment is still a matter of debate. We observed that restoration of WPb in ECV304 cells results in the targeting of t-PA to these storage granules.
Rosnoblet
C
C
Division of Angiology and Hemostasis, Geneva University Hospital, 1211, Geneva, Switzerland.
Ribba
A S
AS
Wollheim
C B
CB
Kruithof
E K
EK
Vischer
U M
UM
D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D014841:von Willebrand Factor; C067003:von Willebrand factor propolypeptide; D010959:Tissue Plasminogen Activator