Subcellular Localization of Von Willebrand Factor Mutants Correlates with Particular VWF Multimer Patterns

Abstract 98

Von Willebrand disease (VWD) is a bleeding disorder caused by mutations of von Willebrand factor (VWF), a huge multimeric glycoprotein that is essential for platelet-dependent primary hemostasis. VWF undergoes a highly complex biosynthesis that starts with the production of the 250 kD monomers that are further post-translationally modified within the ER. Here, N-linked glycosylation occurs and dimerization is facilitated by the formation of interchain disulfide bonds. These dimers are being released from the ER to the Golgi apparatus where the high mannose glycans are processed to the complex form and O-linked oligosaccharides are added. The pH-dependent assembly of VWF High Molecular Weight Multimers (HMWM), up to 80 monomers in size, occurs in the trans-Golgi where they become organized in tubules that are subsequently stored in Weibel-Palade bodies (WPB) until stimulated release into the circulation. This process may partly or completely be disrupted, in particular in several VWD type 2A subtypes (IIC, IID, IIE) but also in some VWD type 3 mutants with missense mutations. Mutant VWF in such patients is characterized by lack or a significant decrease of VWF HMWM which can be due to impaired dimerization and multimerization or poor secretion. The phenotypes are ranging from a relative decrease of HMWM (IIE, IID) over a severe reduction (IIC) to their complete absence beyond the size of a dimer in some patients with VWD type 3. To identify the cause of VWF HMWM deficiency in type 2A (subtypes IIC, IID and IIE) and type 3 VWD patients with known or novel missense mutations, we overexpressed representative VWF mutants in HEK293 cells. Confocal immunofluorescence was employed to investigate their intracellular localization by parallel visualization of cellular sub-compartments using antibodies against marker proteins for the ER, Golgi, endosomes and pseudo-WPB.

The type IIC mutants (I94N, L526S, and G550R) as well as the IID mutants (C2771R, S2775C and G2752D) did not form any cigar-shaped pseudo-WPB and were almost completely localized within the ER where small granules were formed. The type 2A/IIE mutants W1144G, Y1146C and C1169W in contrast exhibited a massive increase in pseudo-WPB formation with most of their contents remaining intracellularly as a result of poor secretion. VWF Type 3 mutants (W377C, W1120S and C2304Y) are retained in the ER in a similar pattern as type IIC and IID mutants.

In conclusion, we show that type 2A (IIC, IID, IIE), and type 3 VWF mutations correspond to a discrete disruption in the VWF intracellular transport, resulting in a characteristic subcellular localization of the particular VWF mutants and correlating with a particular VWF multimer pattern. The respective localization is indicative for the processing and secretion defect of VWF which is ultimately responsible for the manifestation of a specific VWD phenotype.