Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells.

Abstract

Abstract. von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by ceturifugation on a self-generated Percoll gradietu. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of WeibelPalade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: (a) establish that the Weibel-Palade body is the endothelial-specific storage organdie for regulated VWF secretion; (b) demonstrate that in cultured EC, the VWF concentrated in secretory organdies is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; (c) imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; (d) provide a basis for further studies of intracellular protein trafficking in EC.

Protein mobilities and P-selectin storage in Weibel–Palade bodies

“…This includes budding of clathrin-coated vesicles from immature WPBs (Zenner et al, 2007) providing a mechanism for removal of missorted proteins and components not destined for regulated secretion (Arvan and Castle, 1998;Katsumata et al, 2007;Klumperman et al, 1998) and organelle acidification (Erent et al, 2007). Dehydration of organelle content is indicated by a high buoyant density of mature WPBs (Ewenstein et al, 1987) and a membrane tightly apposed to the paracrystalline core (Berriman et al, 2009). WPBs therefore undergo processes common to RSO biogenesis in other cells but uniquely, their size and morphology provide an opportunity to quantitatively analyse protein mobilities in individual RSOs in live cells.…”

Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.

“…The regulated secretory pathway secretes the largest multimeric forms of the protein, composed of processed subunits; the predominantly small multimeric forms, containing some pro-vWf subunits, are secreted constitutively (Sporn et al, 1986;Ewenstein et al, 1987). The intracellular movement of Weibel-Palade bodies during the process of regulated secretion appears to involve the microtubular cytoskeleton, since regulated secretion is inhibited by microtubule-depolymerizing agents.…”

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

“…Weibel-Palade bodies are composed almost entirely of von Willebrand factor (VWF) (2,3), which is a multimeric plasma glycoprotein that can exceed 20 million Da in mass and 4 m in length. Megakaryocytes synthesize large VWF multimers and package them into platelet ␣-granules that are roughly spherical rather than cigar-shaped.…”