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O-linked glycans protect VWF
with a shortened VWF half-life should be considered as a distinct type 1C (1-Clearance) subgroup.4-8 Interestingly, subsequent studies have highlighted that enhanced VWF clearance also contributes to pathogenesis in patients with low VWF, as well as type 2 and type 3 VWD.7,9-12
Given the importance of enhanced clearance in the pathogenesis of VWD, significant research has focused on defining the cellular and molecular clearance pathways involved. Potential roles for macrophages, liver sinusoidal endothelial cells and hepatocytes have been proposed.13-17 A number of specific clearance receptors have also been described.3 These include the low-density lipoprotein receptor-related protein-1 (LRP1), the scavenger receptor class A member I (SR-A1), sialic-acid-binding- immunoglobulin-like-lectins 5 (Siglec 5) and the macrophage galactose-type lectin (MGL) which are all expressed on macrophages.18-21 On liver sinusoidal endothelial cells, receptors that may play a role in VWF clearance include stabilin-2 (STAB2), scavenger receptor class A member 5 (SCARA 5) and C-type lectin domain family 4 member M (CLEC4M).17,22,23 Finally, the asialogly- coprotein receptor (ASGPR), predominantly expressed on hepatocytes and macrophages, has also been implicated.24
More than 30 different VWF sequence variations have been reported in patients with increased VWF clear- ance.3,25 The archetypal type 1C mutation is the VWD Vicenza variant which is characterized by an R1205H substitution in the D3 domain of VWF.15,26 VWF glycosy- lation also plays a critical role in determining the rate of clearance of the protein.27-30 For example, plasma VWF:antigen (VWF:Ag) levels are 20-30% lower in blood group O individuals compared to non-O subjects due to a significant reduction in plasma half-life.31,32 Enzymatic removal of terminal sialic acid residues from VWF also markedly enhances clearance.27,28 Moreover, genetic inac- tivation of the ST3Gal-IV sialyltransferase was associated with a significant reduction in plasma VWF half-life.33 These data are important from a clinical perspective because the majority of both the N- and O-linked glycans of VWF are normally capped by sialic acid residues.34-37 As glycoproteins age in plasma, there is a stepwise elimina- tion of saccharides from the termini of complex glycan chains.38 Glycan remodeling begins with loss of capping sialic acid, catalyzed by plasma neuraminadases 1 (Neu1) and 3 (Neu3) respectively. This time-dependent desialyla- tion is important in triggering clearance of senescent gly- coproteins.38 Significantly reduced VWF sialylation levels have also been observed in a number of pathological con- ditions including sepsis, pulmonary hypertension and liver cirrhosis.39-41 Importantly, several groups have report- ed reduced VWF sialylation in patients with type 1 VWD.10,33,40,42 Together, these data suggest that quantita- tive sialylation plays a critical role in regulating both physiological and pathological clearance of VWF in vivo.
Grewal et al. originally described a role for the ASGPR in regulating enhanced clearance of desialylated VWF (particularly in the context of sepsis).24 More recently, we identified MGL as another receptor involved in regulating hyposialylated VWF clearance.21 Critically, however, important questions remain unanswered regarding the roles played by VWF sialylation in regulating physiologi- cal and/or pathological clearance. These include: (i) the relative importance of N- versus O-linked sialylation in regulating VWF clearance; (ii) the relative contributions of the ASGPR and MGL clearance receptors; and (iii) the
molecular mechanisms through which hyposialylated VWF interacts with its clearance receptors.
Methods
A detailed description of the materials and methods can be found in the associated Online Supplementary Material.
Isolation and purification of human plasma-derived von Willebrand factor
Plasma-derived VWF (pdVWF) was purified from the VWF- containing concentrate Fandhi® (Grifols, Barcelona, Spain) as pre- viously described.21 Platelet-VWF was purified from lysed platelets as described elsewhere.43 Eluate fractions were then assessed for VWF antigen, multimer distribution, and purity.
Glycosidase digestion and quantitative analysis of glycan expression
To generate VWF glycoforms, pdVWF was treated with α2-3 neuraminidase, α2-3,6,8,9 neuraminidase, β1-3 galactosidase, peptide N-glycosidase F (PNGase F) and/or O-glycosidase under non-denaturing conditions overnight at 37°C.29,44 Following gly- cosidase digestion, changes in VWF glycans were assessed using specific lectin enzyme-linked immunosorbent assays (ELISA) as previously described.10
Expression and purification of recombinant von Willebrand factor variants
The expression vectors pcDNA-VWF encoding full length recombinant VWF, VWF-A1A2A3, VWF-A1, VWF-A2, VWF-A3, VWF-D’A3 or VWF-A3-CK fragments have previously been described.16 Additional VWF-A1 constructs containing either of the two O-linked glycan (OLG) clusters were also included; A1- OLG cluster 1 (T1248A, T1255A, T1256A, S1263A), and A1-OLG cluster 2 (T1468A, T1477A, S1486A, T1487A). All recombinant VWF variants were transiently expressed in HEK293T cells. Conditioned serum-free medium was harvested 72 h after trans- fection and concentrated via anion exchange chromatography as described before.16
In vitro von Willebrand factor binding studies
Solid phase plate-binding assays were used to evaluate VWF binding to MGL. Briefly, recombinant human MGL (Stratech, UK) was immobilized on a PolySorpTM 96-well plate (Nunc, Thermo ScientificTM), the wells were blocked, and VWF was incubated at 37°C for 1 h. Bound VWF was detected using horseradish peroxidase (HRP)-conjugated polyclonal anti-VWF (Dako, Agilent Technologies), high-sensitivity streptavidin-HRP (ThermoScientific, UK) or anti-His-HRP antibody (Qiagen, UK)
(seeOnlineSupplementaryMaterialfordetails).
von Willebrand factor clearance studies in MGL1-/-, VWF-/- and VWF-/-/Asgr1-/- mice
All clearance experiments were performed on mice 6 to 8 weeks old. All animal studies were approved by the Health Product Regulatory Authority, Ireland and an internal ethics com- mittee. VWF-/- and Asgr1-/- mice, both on a C57BL/6J background, were obtained from the Jackson Laboratory (Sacremento, CA, USA) and crossbred to obtain a dual VWF-/-/Asgr1-/- knockout model as previously described.21 MGL specific clearance studies were also performed after inhibition of murine MGL1/2 using a commercial polyclonal goat anti-mouse MGL1/2 antibody (2 mg/kg) (R&D Systems, UK) as previously described.21 For endoge- nous clearance studies, murine VWF was labeled with N-hydrox-
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