Impact of O‐linked glycosylation of the VWF‐A1‐domain flanking regions on platelet interaction

Abstract

This study investigated the functional impact of O-linked glycosylation of von Willebrand Factor (VWF) A1 domains on the interaction with platelet receptors. Native or mutant VWF-A1-domains were transiently overexpressed on COS-7 cells as membrane glycosylphosphatidylinositol (GPI)-anchored FLAG-tagged fusion proteins. Cytofluometric analysis assured comparable levels of A1-domain expression among native and mutant homologues as well as for different culture conditions. Expressing native VWF-A1-domains under O-linked glycosylation blocking conditions increased the platelet aggregatory responses observed for fully glycosylated forms. Utilizing a neuronal network for prediction of O-linked glycosylation of mammalian proteins, threonine (T) and serine (S) residues located in the VWF-A1-loop flanking regions - not in the loop itself - were determined to be glycosylated n-terminal at amino acids T485, S490, T492 and T493 and c-terminal at T705. Simultaneous selective charge-to-alanine mutation of S490, T492 and T493 led to gain in aggregatory responses. When compared with native forms, equivalent alterations of T485 did not dictate functional differences. Any alanine-substitution for T705 revealed a substantial loss in aggregatory effects - possibly as a result of structural desintegration of the VWF-A1-binding site for glycoprotein (GP) Ib. These data suggest specific O-linked glycosylation of the amino-terminal VWF-A1-loop-flanking region to have a negative regulatory impact on the A1-domain affinity of non-activated human VWF for human platelet-GPIb.

Conformational Stability and Domain Unfolding of the Von Willebrand Factor A Domains

“…A1-A2-A3 has two N-linked glycosylation sites and nine O-linked glycosylation sites, four of which are located within the linker region between domains A1 and A2. 22 The increase in secondary structure content from the predicted is probably due to an increased order of these linker regions between the domains and may also result from the glycosylation present in the triple domain structure. In the following urea denaturation studies, we monitor the unfolding of the A2 domain at 216 nm and domains A1 and A3 and the triple A1-A2-A3 domain at 222 nm.…”

Von Willebrand factor-A1 domain binds platelet glycoprotein Ibα in multiple states with distinctive force-dependent dissociation kinetics

“…2B and 3B), supporting a cooperative role of A3 or other structural elements of the VWF in regulating A1–GPIbα interaction against the high-shear forces. The glycosylation sites in or around Q1238-E1260 could also in part contribute to differences between bacterial expressed recombinant fragments and pVWF [9,38]. Of note, the collagen affects the 1261-A1 in a similar way as it does to 1238-A1 except that it switch the intrinsic slippery state for 1261-A1 to a catchy state.…”

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

“…This may also be true for the single O-glycan in the B1–3 domain, which contains 4 additional N-linked glycans. Taken together, we feel that our model is not less relevant than other heterologous models [9], [14], [15], [27] to study the effect of glycosylation mutations on VWF structure and function, despite the potential limitations of our in vivo model.…”

“…A first report described a decreased interaction between VWF lacking O-linked glycans and GPIb when ristocetin was used as a modulator but not when botrocetin was used, suggesting a potential role of O-linked glycans in the interaction with ristocetin rather than directly with GPIb [14]. A second more recent study used a mutagenesis approach to replace specific glycosylation sites with alanine residues [15]. However, this study was done on isolated A1 domain encompassing residues 1236–1476, limiting the evaluation to 4 O-glycosylation sites upstream of the A1 domain and to only 1 site downstream of the A1 domain.…”

“…In 1992, by producing recombinant VWF in Chinese Hamster Ovary (CHO) cells allowing selective suppression of O-linked glycosylations, Carew et al suggested that these structures were important for optimal binding of VWF to platelet glycoprotein (GP) Ib in the presence of ristocetin [14]. In 2005, another study using a number of isolated recombinant A1 domains carrying mutations at O-glycosylation sites, reported either enhanced or decreased platelet interaction according to the mutation examined [15].…”