Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities

Preston, Roger J. S.; Rawley, Orla; Gleeson, Eimear M.; O’Donnell, James S.

doi:10.1182/blood-2012-10-415000

Cited by 67 publications

(77 citation statements)

References 96 publications

(64 reference statements)

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“…52,61 Nonetheless, it is interesting that in addition to influencing VWF clearance by macrophages, the N-linked glycans expressed within the A2 domain of VWF have also been shown to modulate susceptibility to proteolysis by ADAMTS13. 42,62 In particular, McKinnon et al showed that loss of the N-linked glycan at N1574 resulted in significantly enhanced VWF proteolysis by ADAMTS13. 42 Furthermore, differential scanning fluorimetry has confirmed that glycosylation at N1574 plays an important role in stabilization of the A2 domain against unfolding.…”

Section: Discussionmentioning

confidence: 99%

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: 99%

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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“…Changes in BHK manufacturing practices between Product B and Product D might have led to increased immunogenicity, 36 but the number of patients treated with Product B in both the RODIN study and in our cohort was too small for comparison with Product D. rFVIII molecules produced by BHK and CHO cells differ in several respects, such as the degree of tyrosine residue sulphation and type of glycosylation. 37 The presence of specific glycan chains might affect dendritic cell uptake and thereby modify rFVIII immunogenicity. 38 The amino acid sequence of the BHK rFVIII products (B and D) corresponds to the most frequent haplotype (H1) in the white population.…”

Section: Possible Biological Explanations and Implicationsmentioning

confidence: 99%

Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A

Calvez

Chambost

Claeyssens-Donadel

et al. 2014

Blood

137

171

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Key Points• A currently marketed rFVIII product is associated with a higher risk of inhibitor development in boys with severe hemophilia A.• This result, validated by extensive sensitivity analyses, confirms a recently published study and cannot be explained by identified biases.Six recombinant factor VIII (rFVIII) products have been marketed worldwide. In 2013, the Research of Determinants of Inhibitor Development (RODIN) study group reported an unexpectedly high risk of inhibitor development with a second-generation full-length rFVIII (Product D) in previously untreated patients (PUPs) with severe hemophilia A (HA). In 1994, French public health authorities established a prospective cohort to monitor hemophilia treatment safety. A PUP subgroup was designed to investigate inhibitor risk factors. We analyzed this subcohort in view of the RODIN findings. After excluding 50 patients who participated in the RODIN study, the primary analysis focused on 303 boys with severe HA first treated with a rFVIII product. A clinically significant inhibitor was detected in 114 boys (37.6%). The inhibitor incidence was higher with Product D vs the most widely used rFVIII product (adjusted hazard ratio [aHR], 1.55; 95% confidence interval [CI], 0.97-2.49). Similar results were found for high-titer inhibitors and in 10 sensitivity analyses. No heterogeneity was observed between RODIN and our results. Combined aHRs were 1.58 (95% CI, 1.17-2.14) for all inhibitors and 1.70 (95% CI, 1.15-2.52) for high-titer inhibitors. Our results confirm the higher immunogenicity of Product D vs other rFVIII products in PUPs with severe HA. (Blood. 2014;124(23):3398-3408)

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“…Within the hemostatic system, the presence of glycans on individual proteins has been demonstrated to alter expression, clearance, and catalytic activity. 15 O-glycans on von Willebrand factor (VWF) have been associated with changes in VWF plasma concentration, 16 platelet binding, 17 and response to shear stress. 18 O-glycosylation is also required for P-selectin-dependent leukocyte rolling, 19,20 and O-glycosylation of platelet glycoprotein Ib a (GP1BA) is important for binding to VWF.…”

Section: Introductionmentioning

confidence: 99%

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

et al. 2017

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Key Points• Human platelets, endothelial cells, and plasma proteins are extensively O-glycosylated, with .1123 O-glycosites identified in this study.• O-glycosites can be classified into functional subgroups; one important function includes the protection from proteolytic processing.The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (6 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans.Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies.

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Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities

Cited by 67 publications

References 96 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

Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

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