Oxidative Modification of Von Willebrand Factor Inhibits Its Cleavage by ADAMTS13

Abstract

During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways:by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, andby liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product.We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.