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Unconjugated bilirubin inhibits proteolytic cleavage of von Willebrand factor by ADAMTS 13 protease
Author(s) -
Lu R.N.,
Yang S.,
Wu H. M.,
Zheng X. L.
Publication year - 2015
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12901
Subject(s) - adamts13 , bilirubin , chemistry , von willebrand factor , heme , biochemistry , biliverdin , unconjugated hyperbilirubinemia , heme oxygenase , cleavage (geology) , medicine , enzyme , platelet , biology , paleontology , fracture (geology)
Summary 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 ADAMTS 13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor ( VWF ) and its analogs by ADAMTS 13. 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‐ rVWF 73‐H, D633‐ rVWF 73‐H, and GST ‐ rVWF 71‐11K by ADAMTS 13 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 ADAMTS 13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633‐ rVWF 73‐H and multimeric VWF , but not F485‐ rVWF 73‐H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS 13 activity in patients with hyperbilirubinemia increased after treatment with bilirubin oxidase. Conclusions Unconjugated bilirubin directly inhibits ADAMTS 13'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 ADAMTS 13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has prothrombotic effect in vivo remains to be determined in our future study.