title: The O-linked glycans of human von Willebrand factor modulate its interaction with ADAMTS-13.
abstract: BACKGROUND
  O-linked glycans (OLGs) are clustered on either side of the von Willebrand factor (VWF) A1 domain and modulate its interaction with platelets; however, their influence on the VWF interaction with ADAMTS-13 is unknown.
  
  
  OBJECTIVE
  To assess the role of the OLGs in VWF susceptibility to ADAMTS-13 proteolysis, which would help to explain their specific distribution.
  
  
  METHODS
  OLG sites were mutated individually and as clusters on either and both sides of the A1 domain, and expressed in HEK293T cells. First, their proteolysis by ADAMTS-13 was assayed in the presence of urea. Next, a parallel-flow chamber was used to analyze VWF-mediated platelet capture on collagen in the presence and absence of ADAMTS-13 under a shear stress of 1500 s(-1) . The decrease in platelet capture in the presence ADAMTS-13 was used as a measure of VWF proteolysis.
  
  
  RESULTS
  Initially, we found that, under denaturing conditions, the C-terminal S1486A and Cluster 2 and double cluster (DC) variants were less susceptible to ADAMTS-13 proteolysis than wild-type VWF. Next, we showed that addition of ADAMTS-13 diminished VWF-mediated platelet capture on collagen under flow; surprisingly, this was more pronounced with the S1486A, Cluster 2 and DC variants than with wild-type VWF, indicating that these are proteolyzed more rapidly under shear flow.
  
  
  CONCLUSIONS
  OLGs provide rigidity to peptide backbones, and our findings suggest that OLG in the A1-A2 linker region regulates VWF conformational changes under shear. Importantly, the impact of OLGs on ADAMTS-13 cleavage under shear stress is the opposite of that under denaturing conditions, highlighting the non-physiologic nature of in vitro cleavage assays.
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College, London, UK.
authors.forename: A A
authors.initials: AA
authors.lastname: Nowak
authors.forename: T A J
authors.initials: TA
authors.lastname: McKinnon
authors.forename: J M
authors.initials: JM
authors.lastname: Hughes
authors.forename: A C K
authors.initials: AC
authors.lastname: Chion
authors.forename: M A
authors.initials: MA
authors.lastname: Laffan
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D057809:HEK293 Cells; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13 protein human; collagen; glycosylation; hemostasis; proteolysis; von Willebrand factor