Oxidation of Key Methionine Residues in the N-Terminal Domains of ADAMTS13 Correlates with Loss of Its Proteolytic Activity,

Abstract 3276

Deficiency of ADAMTS13 is associated with a life-threatening thrombotic microangiopathic disease—thrombotic thrombocytopenic purpura (TTP), which is characterized by the accumulation of aggregates of hyper-reactive ultra-large von Willebrand factor (VWF) and platelets in the microvasculature. In addition to TTP, systemic inflammatory syndromes such as acute systemic inflammation caused by endotoxin, acute pancreatitis, and severe sepsis demonstrate reduced ADAMTS13 activity. The cause of low ADAMTS13 activity is not known. One possible cause is oxidative inactivation of the enzyme by hypochlorous acid (HOCl), a potent oxidant released from activated neutrophils that is known to damage proteins. In this study, we exposed ADAMTS13 to HOCl produced by a myeloperoxidase (MPO)-H₂O₂-Cl⁻ system and measured the proteolytic activity of oxidized ADAMTS13 using a small VWF A2 peptide and plasma VWFas substrates. ADAMTS13 activity decreased as a function of oxidant concentration. Treatment with 25 nM MPO plus 50 μM H₂O₂ reduced ADAMTS13 activity by over 85%. Such concentrations of MPO and H₂O₂ are routinely found in vivo at sites of inflammation.

ADAMTS13 contains a series of structural domains: a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat (TSP1,T), a Cys-rich domain (C), a spacer domain (S), 7 additional TSP1 repeats, and 2 CUB domains. The MDTCS domains are essential for its proteolytic activity, while the C-terminal TSP1 and CUB domains may act as modulators. The MDTCS region contains 10 Met residues, Met being the amino acid residue most vulnerable to oxidation by HOCl. Using mass spectrometry, we identified 7 Met-containing peptides in this region after proteolytic digestion of ADAMTS13. Three of these 7 methionines are highly sensitive to oxidation by HOCl, including M249, M331 and M496. M249 is situated in the ”Met-turn” at the catalytic center of the metalloprotease domain. M331 and M496 are located in the disintegrin-like domain and the Cys-rich domain, respectively. The extent of oxidation of these Met residues was proportional to the HOCl concentration, and strongly correlated with loss of enzymatic activity. The same three Met residues were also oxidized after exposure of ADAMTS13 to activated human neutrophils. These observations suggest an oxidative mechanism for ADAMTS13 inactivation in systemic inflammatory syndromes and that the oxidation-sensitive Mets may serve as biomarkers for this effect. Coupled with our earlier observation that HOCl oxidation of von Willebrand factor enhances its adhesive function and renders it resistant to cleavage by ADAMTS13, these findings indicate that pathological situations during which neutrophils are activated produce extremely prothrombotic conditions, perhaps explaining why many inflammatory syndromes are associated with thrombosis in small and large blood vessels.