title: Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.
abstract: BACKGROUND
  The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage.
  
  
  METHODS
  VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13.
  
  
  RESULTS
  To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity.
  
  
  CONCLUSIONS
  Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Emma
authors.initials: E
authors.lastname: Kraus
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kraus
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Obser
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Oyen
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Klemm
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schneppenheim
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address: m.brehm@uke.de.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Brehm
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000502:physiology*; D000071120:ADAMTS13 Protein; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D017022:Flow Injection Analysis / Q000379:methods*; D033081:Shear Strength / Q000502:physiology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000502:physiology*
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13; VWF strings; shear flow assay; von Willebrand factor