title: A novel flow-based assay reveals discrepancies in ADAMTS-13 inhibitor assessment as compared with a conventional clinical static assay.
abstract: BACKGROUND
  Several static Bethesda-type assays are routinely used to determine ADAMTS-13-neutralizing autoantibodies in acquired thrombotic thrombocytopenic purpura (TTP), but the inhibitory activity of these antibodies has not been thoroughly evaluated under the more physiologic condition of flow.
  
  
  OBJECTIVE
  We investigated whether ADAMTS-13 inhibitor assessment with the FRETS-VWF73 assay is predictive for evaluation under flow.
  
  
  METHODS
  Anti-ADAMTS-13 autoantibodies were purified from patients with acquired TTP by chromatography involving an ADAMTS-13 affinity matrix and/or protein G. ADAMTS-13 activity was measured with the FRETS-VWF73 assay and a novel flow assay determining the ADAMTS-13-mediated decrease in platelet aggregate surface coverage, caused by perfusion of a suspension containing platelets, erythrocytes and von Willebrand factor (VWF) over a surface coated with extracellular matrix components. The neutralizing activities of ADAMTS-13 inhibitors were compared under static conditions and under flow by use of the two assays.
  
  
  RESULTS
  The suitability of the flow-based ADAMTS-13 activity assay for quantification of ADAMTS-13 inhibitors could be demonstrated by reversibility of the ADAMTS-13-dependent decrease in surface coverage upon addition of goat ADAMTS-13 antiserum. Testing the neutralizing activity of purified autoantibodies from six patients in the flow assay according to their FRETS-VWF73-based inhibitor titers gave rise to vastly different inhibitory effects, indicating a discrepancy in inhibitor assessment between static and flow conditions.
  
  
  CONCLUSIONS
  Anti-ADAMTS-13 autoantibodies may show inhibitory properties in vivo that are not consistent with the ADAMTS-13 inhibitor levels determined in routine static assays, possibly because certain epitopes are selectively exposed under shear. Consequently, the course of disease and treatment efficacy may vary among TTP patients, despite common inhibitor titers.
authors.affiliation: Baxter Innovations GmbH, Vienna, Austria.
authors.forename: R
authors.initials: R
authors.lastname: Grillberger
authors.forename: B
authors.initials: B
authors.lastname: Gruber
authors.forename: S
authors.initials: S
authors.lastname: Skalicky
authors.forename: G
authors.initials: G
authors.lastname: Schrenk
authors.forename: P
authors.initials: P
authors.lastname: Knöbl
authors.forename: B
authors.initials: B
authors.lastname: Plaimauer
authors.forename: P L
authors.initials: PL
authors.lastname: Turecek
authors.forename: F
authors.initials: F
authors.lastname: Scheiflinger
authors.forename: H
authors.initials: H
authors.lastname: Rottensteiner
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000276:immunology*; D000071120:ADAMTS13 Protein; D001323:Autoantibodies / Q000737:chemistry*; D001780:Blood Coagulation Tests / Q000295:instrumentation* / Q000379:methods; D003598:Cytoskeletal Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006403:Hematologic Tests / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D060588:LIM Domain Proteins / Q000737:chemistry; D010974:Platelet Aggregation; D011485:Protein Binding; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood* / Q000175:diagnosis; D016601:RNA-Binding Proteins; D033081:Shear Strength; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000737:chemistry
chemical_list: D001323:Autoantibodies; D003598:Cytoskeletal Proteins; D007074:Immunoglobulin G; D060588:LIM Domain Proteins; D016601:RNA-Binding Proteins; C429688:TES protein, human; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13 protein, human; autoantibodies; mechanical stress; thrombotic thrombocytopenic purpura; von Willebrand factor