Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13.

Elevated plasma von Willebrand factor (VWF) and low ADAMTS13 activity have been reported in several inflammatory states, including sepsis and acute respiratory distress syndrome. One hallmark of inflammation is neutrophil activation and production of reactive oxygen species, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). HOCl is produced from hydrogen peroxide and chloride ions through the action of myeloperoxidase. HOCl can oxidize methionine to methionine sulfoxide and tyrosine to chlorotyrosine. This is of interest because the ADAMTS13 cleavage site in VWF, the Tyr(1605)-Met(1606) peptide bond, contains both oxidation-prone residues. We hypothesized that HOCl would oxidize either or both of these residues and possibly inhibit ADAMTS13-mediated cleavage. We therefore treated ADAMTS13 substrates with HOCl and examined their oxidative modification by mass spectrometry. Met(1606) was oxidized to the sulfoxide in a concentration-dependent manner, with complete oxidation at 75muM HOCl, whereas only a miniscule percentage of Tyr(1605) was converted to chlorotyrosine. The oxidized substrates were cleaved much more slowly by ADAMTS13 than the nonoxidized substrates. A similar result was obtained with multimeric VWF. Taken together, these findings indicate that reactive oxygen species released by activated neutrophils have a prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13.

Characterization of a core binding site for ADAMTS-13 in the A2 domain of von Willebrand factor.

ADAMTS-13, a metalloprotease in plasma, specifically cleaves the Tyr-1605-Met-1606 bond in the A2 domain of von Willebrand factor (VWF) to regulate the polymer distribution of VWF in circulation, which is critical for primary hemostasis. A 73-aa peptide (VWF73) was previously identified as the minimal substrate cleavable by ADAMTS-13. In this study, VWF73 was enzymatically and chemically cleaved into shorter peptides, and the inhibition of cleavage of a VWF73-derived substrate by these purified peptides was measured in competition studies using a quantitative assay we recently reported. A 24-aa peptide encompassing Pro-1645-Lys-1668 (P'40-P'63) and situated 40 aa downstream from the cleavage site was the minimal peptide that could bind to and competitively inhibit ADAMTS-13 (K(i) = 12 microM). This peptide and longer peptides encompassing this core sequence also inhibited the cleavage of multimeric VWF by ADAMTS-13. These results suggest the presence of a complementary extended binding site, or exosite, on ADAMTS-13. Mutation of Asp-1653 and Asp-1663 to Ala in this region significantly reduced the rate of cleavage of the substrate peptide, whereas the Glu1655Ala mutation caused an enhanced rate of cleavage. These results suggest that ionic interactions of the Pro-1645-Lys-1668 region with the exosite on ADAMTS-13 play a significant role in mediating substrate recognition.