O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions.

We have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D' to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor.

We examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3- to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression.