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Article: Oxidative Modification of von Willebrand Factor by Neutrophil Oxidants Inhibits its Cleavage by ADAMTS13

Oxidative Modification of von Willebrand Factor by Neutrophil Oxidants Inhibits its Cleavage by ADAMTS13

Oxidative Modification of von Willebrand Factor by Neutrophil Oxidants Inhibits its Cleavage by ADAMTS13

Blood Journal
January 21, 2010

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 Tyr1605-Met1606 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. Met1606 was oxidized to the sulfoxide in a concentration-dependent manner, with complete oxidation at 75μM HOCl, whereas only a miniscule percentage of Tyr1605 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.


During acute inflammation, endothelial cells are activated by inflammatory cytokines such as interleukin-1 and tumor necrosis factor-α. Activated endothelial cells release von Willebrand factor (VWF) and expose P-selectin on the cell surface, a result of exocytosis of Weibel-Palade bodies, the major endothelial storage granules., On a longer time scale, other adhesion molecules, such as E-selectin and intracellular adhesion molecule-1, are synthesized and exposed on the cell surface. These molecules provide an adhesive surface for neutrophils, which attach to and roll on the endothelium, then become activated and migrate through the vessel wall into the surrounding tissues.

Although a fraction of the VWF remains transiently attached to the activated endothelium, most is secreted into the plasma., Elevated VWF levels in plasma have been described in diseases associated with systemic inflammation, such as acute lung injury/acute respiratory distress syndrome, and sepsis. In each of these disorders, increased plasma VWF levels correlated independently with increased risk of death. The elevated plasma VWF level is primarily a consequence of endothelial cell activation, but other mechanisms may be involved. Several recent studies have shown that systemic inflammation is also associated with reduced activity of the VWF-processing enzyme ADAMTS13 activity in syndromes such as acute systemic inflammation caused by endotoxin, acute pancreatitis, severe sepsis and septic shock, sepsis-induced disseminate intravascular coagulation, and sepsis-induced organ dysfunction. It is therefore likely that in addition to enhanced release of VWF, delayed or reduced VWF processing by ADAMTS13 contributes to the elevated level and adhesiveness of VWF in these inflammatory disorders.

The only known substrate for ADAMTS13 is VWF. The most important function of this metalloprotease is to cleave the newly released very large and hyperadhesive forms of VWF (known as ultralarge VWF or ULVWF) to the smaller, less adhesive multimeric forms that are normally found in the plasma. Failure of this process leads to ULVWF accumulation and formation of systemic microvascular thrombi, the unique characteristic of the microangiopathic disease thrombotic thrombocytopenic purpura. ULVWF spontaneously binds platelets and induces their clumping, whereas plasma VWF requires a modulator such as ristocetin or botrocetin to do so. Physiologically, the interaction of plasma VWF with platelets is also induced by pathologically elevated fluid shear stress or VWF immobilization on subendothelial collagen. The hyperadhesive nature of ULVWF is not merely a consequence of its large size and increased content of VWF dimers (the protomer for making large multimers), as single molecule experiments showed that ULVWF could form spontaneous, high-strength bonds with glycoprotein Ibα (GPIIbα), its platelet receptor, whereas plasma VWF only recognized GPIbα in the presence of ristocetin or botrocetin. Thus, the VWF A1 domain, which contains the GPIbα binding site, appears to exist in a different conformation in ULVWF than in plasma VWF. This gain-of-function conformation is recognized by the llama nanobody AU/VWFa-11, which displays enhanced binding to VWF from patients with type 2B von Willebrand disease, thrombotic thrombocytopenic purpura, HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), malaria, and antiphospholipid syndrome. Therefore, an important function of ADAMTS13 is to convert hyperactive ULVWF to cryptic plasma VWF.

Because ADAMTS13 activity is often decreased in patients with acute systemic inflammation, the processing of ULVWF could also be impaired, especially if the secretion of ULVWF is increased over baseline because of endothelial cell activation. It therefore seems reasonable to speculate that systemic microvascular thrombosis caused by enhanced platelet aggregation could contribute to the pathology of systemic inflammation.

An important aspect of systemic inflammation is oxidative stress caused by the oxidative burst in activated neutrophils, which subsequently release several reactive oxygen species (ROS) that include superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions (Figure 1 reaction 1). HOCl oxidizes cysteine and methionine residues 100 times faster than it oxidizes tyrosine, converting cysteine to cysteine sulfinic or sulfonic acids and methionine to methionine sulfoxide (Figure 1 reaction 2). HOCl can also oxidize tyrosine to chlorotyrosine (Figure 1 reaction 3). In other proteins, these oxidative modifications have been shown to profoundly influence function. For example, HOCl activates matrix metalloproteinase-7 from its latent proform by oxidizing a cysteine to cysteine sulfinic acid in the regulatory domain known as the cysteine switch. Methionine oxidation in α1-antitrypsin and thrombomodulin markedly reduces the functions of both proteins. The capacity of α1-antitrypsin to inhibit elastase was dramatically reduced after oxidation of methionine residue 351 or 358 to the sulfoxides. Similarly, oxidation of Met388 in thrombomodulin decreased the ability of the thrombomodulin-thrombin complex to activate protein C, a vital anticoagulant function. Conversion of Tyr192 to chlorotyrosine in apolipoprotein A1 of high density lipoprotein (HDL) interferes with HDL's ability to remove cholesterol from macrophages. Chlorotyrosine has been identified in HDL isolated from atherosclerotic lesions.

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