Biogenesis of Weibel-Palade bodies in von Willebrand disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

AbstractBackground: Mutations of cysteine residues in von Willebrand factor are known to reduce its storage and secretion, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in type 2A(IID) von Willebrand disease patients who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies.Design and Methods: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection in human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced with phorbol 12-myristate 13-acetate. Results: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser was stored normally. As expected, wild type von Willebrand factor formed proteinaceous tubules that displayed as longitudinal striations in pseudo Weibel-Palade bodies under electron microscopy. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but it formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect the storage in Weibel-Palade bodies and secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds will lead to reduced von Willebrand factor storage and secretion due to ER retention.Thrombosis and Hemostasi