Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers.

Abstract

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.

Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

“…The VWF multimers that form can then be folded, in a pH-sensitive fashion, into tubular structures 25,26 . While structural mutations in VWF can lead to defects in multimer formation or tubulation, relatively little is known about what other cellular processes regulate the number, pH or secretory potential of WPBs.…”

A Covalent Oxidoreductase Intermediate in Propeptide-dependent von Willebrand Factor Multimerization

“…The Golgi is a hostile environment for disulfide bond formation or rearrangement due to its acidic nature and its lack of chaperones and oxidoreductases. Nevertheless, the lower pH of the Golgi appears to be required for VWF multimerization because raising the pH of human umbilical vein endothelial cells with ammonium chloride, chloroquine, or monensin inhibits VWF multimerization (13,14). Using partially purified pro-VWF, multimerization in vitro was reconstituted at pH 5.8 but not at neutral or basic pH (15).…”

Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

“…A number of the steps of vWf multimer assembly by cultured EC have been characterized, and some have been blocked by pharmacological means (6)(7)(8)(9)(10)(11)(12) Sessions of the American Heart Association, November 19, 1986. 1To whom reprint requests should be addressed.…”