N-linked glycan stabilization of the VWF A2 domain

Lynch, Christopher J.; Lane, David A.

doi:10.1182/blood-2015-09-672014

Cited by 22 publications

(30 citation statements)

References 32 publications

(58 reference statements)

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“…42 Furthermore, differential scanning fluorimetry has confirmed that glycosylation at N1574 plays an important role in stabilization of the A2 domain against unfolding. 44 However, in contrast to its marked effect upon clearance, loss of the VWF N1515 glycan did not significantly influence susceptibility to ADAMTS13 cleavage and did not have any significant effect on the thermostability of the A2 domain. 42,44 Further studies will be necessary to define the biological mechanisms through which the carbohydrate structures at N1515 and N1574 regulate macrophage-mediated clearance.…”

Section: Discussionmentioning

confidence: 85%

“…44 However, in contrast to its marked effect upon clearance, loss of the VWF N1515 glycan did not significantly influence susceptibility to ADAMTS13 cleavage and did not have any significant effect on the thermostability of the A2 domain. 42,44 Further studies will be necessary to define the biological mechanisms through which the carbohydrate structures at N1515 and N1574 regulate macrophage-mediated clearance. However, it is interesting that ABO(H) blood group determinants, which influence both VWF proteolysis by ADAMTS13 and VWF clearance, are expressed on both of these complex N-linked glycans within the A2 domain of pd-VWF.…”

Section: Discussionmentioning

confidence: 85%

“…In keeping with previous studies defining the biological significance of these glycans, [42][43][44] each asparagine residue was mutated to glutamine (N1515Q and N1574Q) to eliminate the N-linked glycans at these positions. Mutations were verified by DNA sequencing to ensure the absence of any other randomly introduced mutations.…”

Section: Expression and Purification Of Recombinant Vwfmentioning

confidence: 99%

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N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

Chion

O’Sullivan

Drakeford

et al. 2016

Blood

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Key Points• The A1 domain of VWF contains a cryptic binding site that plays a key role in regulating macrophage binding and clearance.• The N-linked glycans presented at N1515 and N1574 within the A2 domain of VWF modulate macrophage-mediated clearance.Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3.Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo. (Blood. 2016;128(15):1959-1968

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 85%

Section: Expression and Purification Of Recombinant Vwfmentioning

confidence: 99%

See 1 more Smart Citation

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

Chion

O’Sullivan

Drakeford

et al. 2016

Blood

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“…Thermostability measurements have been applied to study the folding/unfolding of different domains in VWF , but they are rarely used to study the interaction between the VWF A1 domain and its ligands. According to our findings, the T m of the A1 domain is ≈ 51 °C when it is deprotected.…”

Section: Discussionmentioning

confidence: 99%

Delimiting the autoinhibitory module of von Willebrand factor

Deng

Voos

Colucci

et al. 2018

Journal of Thrombosis and Haemostasis

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Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

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“…Plasma VWF levels are approximately 25%–30% lower in group O subjects than in non-O individuals (Franchini & Mannucci, 2014; Liumbruno & Franchini, 2013; O’Donnell et al, 2005; Orstavik et al, 1985; Sousa et al, 2007). Although the mechanisms between ABO blood group and VWF levels are not fully understood, the effects are mediated by the N-linked oligosaccharide chains of VWF, which are similar to the ABO antigens (carbohydrate structure) (Gallinaro et al, 2008; Lynch & Lane, 2016; McKinnon et al, 2008). Alternatively, H antigen expression could mediate the ABO effect on plasma VWF level.…”

Section: Introductionmentioning

confidence: 99%

Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population

Wang

Dou

et al. 2017

PeerJ

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BackgroundABO blood group is a hereditary factor of plasma levels of coagulation factor VIII (FVIII) and von Willebrand factor (VWF). Age and gender have been shown to influence FVIII, VWF, fibrinogen (Fbg), and ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 motif, 13). We investigated the effects of ABO type, age, and gender on plasma levels of FVIII, Fbg, VWF, and ADAMTS13 in a Chinese population.MethodsA total of 290 healthy volunteers were eligible for this study. ABO blood group was determined by indirect technique. FVIII:C and Fbg were measured by clotting assays. VWF antigen (VWF:Ag), collagen-binding activity (VWF:CBA), and ADAMTS13 antigen were assessed by ELISA, whereas VWF ristocetin cofactor activity (VWF:Rcof) was performed by agglutination of platelets with ristocetin.ResultsMean FVIII:C and VWF levels (VWF:Ag, VWF:CBA, and VWF:Rcof) were significantly higher in non-O than in O type subjects (p < 0.05 for all comparison). ADAMTS13 antigen decreased with increasing age, whereas the other parameters increased. Other than ADAMTS13 (p < 0.01), no gender-related variations were observed in the other parameters. Moreover, FVIII:C, Fbg, VWF:Ag, VWF:CBA, and VWF:Rcof showed significant and positive relationships with age (r = 0.421, 0.445, 0.410, 0.401, and 0.589, resp.; all p < 0.001), whereas a negative relationship was observed for ADAMTS13 antigen (r = 0.306; p = 0.006). Furthermore, FVIII:C were strongly correlated with VWF:Ag, VWF:CBA, and VWF:Rcof (r = 0.746, r = 0.746, and r = 0.576, resp.; p < 0.0001). VWF parameters were also strongly correlated with each other (r = 0.0.847 for VWF:Ag and VWF:CBA; r = 0.722 for VWF:Ag and VWF:Rcof; p < 0.0001).ConclusionsABO blood group, age, and gender showed different effects on plasma levels of FVIII:C, Fbg, VWF:Ag, VWF:CBA, VWF:Rcof, and ADAMTS13 antigen. These new data on a Chinese population are quite helpful to compare with other ethnic groups.

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N-linked glycan stabilization of the VWF A2 domain

Cited by 22 publications

References 32 publications

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

Delimiting the autoinhibitory module of von Willebrand factor

Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population

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