Genetic induction of a releasable pool of factor VIII in human endothelial cells

Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):2689-95. doi: 10.1161/01.atv.20.12.2689.

Abstract

Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release 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 regulated secretion of stored FVIII, endothelial cells with intact agonist-stimulated release pathways were used. Human umbilical vein endothelial cells (HUVECs) were transduced with retroviral FVIII construct [hFVIII(V)] to create a FVIII/vWF storage pool. Immunofluorescent staining of transduced cells demonstrated FVIII in Weibel-Palade bodies. In contrast, the transduction of hFVIII(V) into HT-1080 and HepG2 cells displayed FVIII only in the cytoplasm. We studied the regulated release of both FVIII and vWF from endothelial cells after agonist-induced stimulation and demonstrated a parallel release of FVIII and vWF proteins. This released FVIII was functionally active. Hence, endothelial cells transduced with hFVIII(V) store FVIII together with vWF in Weibel-Palade bodies, creating a releasable storage pool of both proteins. Because FVIII secretion can be physiologically regulated by agonists in culture, this may explain the pharmacological agonist-induced release of FVIII by drugs such as desmopressin in vivo and suggests vascular endothelium as a reasonable target of gene therapy of hemophilia A.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Cytoplasm / metabolism
  • Deamino Arginine Vasopressin / pharmacology
  • Endothelium, Vascular / metabolism*
  • Factor VIII / biosynthesis
  • Factor VIII / genetics*
  • Fluorescent Antibody Technique, Indirect
  • Genetic Therapy / methods
  • Genetic Vectors
  • Hemophilia A / therapy
  • Hemostatics / pharmacology
  • Humans
  • Transduction, Genetic
  • von Willebrand Factor / biosynthesis
  • von Willebrand Factor / genetics

Substances

  • Hemostatics
  • von Willebrand Factor
  • Factor VIII
  • Deamino Arginine Vasopressin