Shear stress–induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region

Abstract

VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H 2 O 2 , we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-offunction conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13. (Blood. 2011; 118(19):5283-5291) IntroductionVWF is an enormous plasma glycoprotein synthesized in endothelial cells and megakaryocytes, the primary function of which is to attach platelets to sites of blood vessel injury. 1 VWF also chaperones coagulation factor VIII in plasma, protecting it from degradation and delivering it to the platelet surface, where it participates in blood coagulation. 2 VWF comprises homopolymers of a 2050-amino acid polypeptide synthesized through N-terminal disulfide bonding of C-terminal disulfide-bonded dimers. 3 Each monomer has many domains, including 3 tandem A domains (A1A2A3) containing, respectively, the platelet glycoprotein (GP) Ib-binding site, the cleavage site for ADAMTS13, and a collagen-binding site. 3 The polymers can reach a mass of Ͼ 20 million Da 4 and are either constitutively secreted or stored in granules, the Weibel-Palade bodies of endothelial cells, or the ␣-granules of platelets, from which they are released upon stimulation. 5 Newly released VWF multimers (termed ultra-large VWF or ULVWF) are hyperadhesive for platelets compared with the VWF normally found in plasma, 6 but are rapidly converted to smaller, less reactive plasma forms by cleavage by the metalloprotease ADAMTS13. 1 In addition to its hemostatic roles, VWF is also involved in thrombosis. The most obvious example is when ADAMTS13 fails to process ULVWF, resulting in the sometimes fatal microvascular clotting syndrome, thrombotic thrombocytopenic purpura. 7 VWF has also been implicated in thrombosis associated with disorders such as HELLP syndrome (hemolysis, elevated liver enzymes, a...

Clinical and prognostic implications of low or high level of von Willebrand factor in patients with Waldenström macroglobulinemia

“…VWF may amplify the consequences of the inflammatory process because oxidative stress induces structural changes that render multimers more resistant to cleavage by ADAMTS13. 39 Although we found increased plasma levels of VEGF in 75% of patients with WM as described previously, 40 no correlation was observed between plasma VEGF and VWF. Beside a possible role of VWF itself on angiogenesis 41 and the aforementioned potential mechanisms, plasma concentration of VEGF may only reflect a limited role in VWF production and clearance.…”

Simultaneous Exposure of Sites in von Willebrand Factor for Glycoprotein Ib Binding and ADAMTS13 Cleavage

“…We previously showed that the chaotropic agent urea and applied shear force are both able to increase oxidation of Met1606 by HOCl, which provides an independent measure of exposure of the ADAMTS13 cleavage site, without the necessity of having ADAMTS13 in the mix. 17,22 We, therefore, assessed the effect of ristocetin on HOCl-induced oxidation of Met1606. Multimeric VWF was incubated with an HOCl-generating system, myeloperoxidase plus H 2 O 2 at various concentrations, in the presence of ristocetin or buffer for 1 hour.…”

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

“…Shear stress could greatly increase the oxidation of residues Met 540 and Met 541 at the ␣1-helix that are deeply buried by the adjacent ␣2-helix in the static structures (59). It suggests that shear stress may enhance A1 affinity through a similar mechanism as the GOF mutations, because A1 itself exposes the buried methionines through its conformational change under shear stress.…”