Binding and release of factor VIII/von Willebrand's factor by human endothelial cells

The uptake and release of factor VIII/von Willebrand's protein by cultured human umbilical vein endothelial cells have been examined using highly purified 125I-factor VIII possessing von Willebrand's factor activity. 125I-factor VIII/vWF was taken up by the cells, reaching maximum binding within 4 h with a t1-2 of binding of 15 min. Endothelial cell binding of 125I-factor VIII/vWF reached saturation at a concentration of 1.5 mg/l. Binding was inhibited by coincubation of excess unlabelled factor VIII/vWF. Most of the cell-associated radioactivity was released by treatment of the cells with trypsin. Internalization of bound protein was evidenced by the incorporation into the cells of radioactivity which could not be released by trypsin. Human vascular smooth muscle cells did not bind 125I-factor VIII/vWF. Addition of 0.1 microM epinephrine to the 125I-factor VIII/vWF labelled endothelial cultures induced the release of cell bound, protein-associated radioactivity into the medium. Propranolol inhibited completely epinephrine-induced release, whereas phenylephrine had no effect. Endothelial cells maintained in medium partially depleted of factor VIII/vWF by tricalcium citrate cellulose treatment of plasma did not release factor VIII antigen into the culture medium during subsequent incubation. Although [3H]proline was incorporated into proteins released by endothelial cells under these experimental conditions, specific incorporation of label into factor VIII/vWF antigen was not detectable by a sensitive solid-phase immunoradiometric assay. We conclude that factor VIII/vWF binds to endothelial cells and that this cell-bound protein is mobilized by epinephrine through beta-adrenergic stimulation.