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Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

      Highlights

      • VWF string detection with GPIb-beads instead of platelets.
      • Assay suitable for fluorescent applications.
      • Semi-automated, quantitative assessment of shear-dependent ADAMTS13 function.
      • Kinetic assays possible for recombinant and plasma ADAMTS13 under flow conditions.
      • Assay could foster and complement VWD and TTP research and diagnostics.

      Abstract

      Introduction

      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.

      Material and 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.

      Abbreviations:

      VWF (von Willebrand factor), WPB (Weibel-Palade Body), ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), GPIb (glycoprotein Ib), USS (Upshaw-Schulman Syndrome), TTP (Thrombotic Thrombocytopenic Purpura), RT (room temperature), PFA (paraformaldehyde), ROI (region of interest)

      Keywords

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