title: Unconjugated bilirubin inhibits proteolytic cleavage of von Willebrand factor by ADAMTS13 protease.
abstract: BACKGROUND
  Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood.
  
  
  OBJECTIVE
  The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13.
  
  
  METHODS
  Fluorogenic, surface-enhanced laser desorption/ionization time-of-flight mass spectrometric assay, and Western blotting analyses were used to address this question.
  
  
  RESULTS
  Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration of ~13, ~70, and ~17 μmol L(-1) , respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia increased after treatment with bilirubin oxidase.
  
  
  CONCLUSIONS
  Unconjugated bilirubin directly inhibits ADAMTS13's ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has prothrombotic effect in vivo remains to be determined in our future study.
authors.affiliation: Division of Laboratory Medicine, Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL, USA.|Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China.
authors.forename: R-N
authors.initials: RN
authors.lastname: Lu
authors.affiliation: Department of Pathology, Ohio State University, Columbus, OH, USA.
authors.forename: S
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Pathology, Ohio State University, Columbus, OH, USA.
authors.forename: H M
authors.initials: HM
authors.lastname: Wu
authors.affiliation: Division of Laboratory Medicine, Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: X L
authors.initials: XL
authors.lastname: Zheng
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001663:Bilirubin / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006932:Hyperbilirubinemia / Q000097:blood / Q000175:diagnosis / Q000201:enzymology*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D011480:Protease Inhibitors / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D014841:von Willebrand Factor; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D001663:Bilirubin
keywords: ADAMTS13 protein, human; hyperbilirubinemia; thrombosis; thrombotic microangiopathies; von Willebrand factor