Bilirubin Directly Inhibits Proteolytic Cleavage of a Fluorogenic VWF73 Peptide and Plasma Von Willebrand Factor

Bilirubin is a yellow breakdown product of heme catabolism. Increased levels of plasma bilirubin are a condition commonly seen in neonates and adults with hemolysis. The unconjugated bilirubin consists of an open chain of four pyrrole-like rings. Previous studies have shown that bilirubin decreases the cleavage of a commercially available FRETS­­­­­-VWF73 assay, resulting in falsely low plasma ADAMTS13 activity. However, the mechanism underlying such a reduction of ­­­­ADAMTS13 activity is not fully understood. Plasma ADAMTS13 activity is critical for differential diagnosis of thrombotic microangiopathy in which low ADAMTS13 activity (less than 5-10%) is more consistent with thrombotic thrombocytopenic purpura (TTP), while normal to mildly reduced ADAMTS13 activity (greater than 10-20%) is the common findings of hemolytic uremic syndrome (HUS). Here, we show that bilirubin directly inhibits the proteolytic cleavage of a fluorogenic VWF73 peptides labeled with fluorescein-5-maleimide (FL485-VWF73) (ext. 485nm/emi.530nm) or DyLight-633-maleimide (DL633-VWF73) (ext.638nm/emi.658 nm) in a concentration-dependent manner (Fig. 1). The IC₅₀ for inhibiting the cleavage of FL485-VWF73 and DL633-VWF73 was estimated to be 13 micro mol/L and 70 micro mol/L, respectively. To confirm the direct inhibitory activity, bilirubin at various concentrations was incubated with plasma-derived multimeric VWF and recombinant ADAMTS13 in the presence of 1.5 M urea, 5 mM Tris-HCl, pH 8.0 for four hours. The cleavage of multimeric VWF was determined by 1% agarose and SDS-polyacrylamide gel electrophoreses, followed by Western blotting with rabbit anti-VWF IgG. The results demonstrated that bilirubin also inhibited recombinant ADAMTS13-mediated cleavage of VWF in a concentration-dependent manner with an IC₅₀ ~0.43 milli mol/L (Fig. 2). The inhibitory activity of bilirubin on the cleavage of a fluorogenic peptide or a multimeric VWF substrate was largely eliminated by addition of bilirubin oxidase that converts bilirubin to biliverdin (not shown). Our results demonstrate for the first time that unconjugated bilirubin directly inhibits plasma and recombinant ADAMTS13 activity in addition to its known interference with certain fluorogenic assays for ADAMTS13 activity. These findings are clinically important concerning diagnosis and differential diagnosis of thrombotic microangiopathy. Additionally, our findings may shed new light on the mechanism of thrombotic complications pertinent to acute hemolysis and liver diseases.

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