Shear Stress-Induced Unfolding of Von Willebrand Factor Accelerates Oxidation of Key Methionine Residues In the A1A2A3 Region

Abstract 2112

The interaction between von Willebrand factor (VWF) and the platelet glycoprotein Ib-IX-V complex mediates the first step of platelet adhesion to the vessel wall at sites of injury in the hemostatic response to blood loss. This interaction is also involved in pathologic thrombosis, the most extreme case being thrombotic thrombocytopenic purpura, but the interaction has been proposed to have important pathogenic roles in disparate syndromes such as sepsis, HELLP syndrome, antiphospholipid syndrome, acute lung injury, sickle cell anemia, and cerebral malaria. These syndromes have in common an association with severe inflammation, one of the consequences of which is production of oxidants, in particular by neutrophils. We recently showed that one of the most potent neutrophil oxidants, hypochlorous acid (HOCl), which is produced by the myeloperoxidase-catalyzed reaction of H₂O₂ with chloride ion, markedly reduces ADAMTS13 proteolysis of VWF by oxidizing M1606 at the ADAMTS13 cleavage site within the A2 domain of VWF (Blood, 115(3) 706-12, 2010). In that study, M1606 present in a substrate A2 peptide was readily oxidized by HOCl, but only minimally oxidized in multimeric plasma VWF, except in the presence of the denaturing agent urea. As this requirement resembled the requirement of urea for ADAMTS13 proteolysis of plasma VWF, we wondered whether the application of shear stress would similarly enhance M1606 oxidation by HOCl. Using a system containing 25 nM MPO (a plasma concentration often seem in inflammatory conditions) and varying concentrations of H₂O₂, we found that application of 0.6 dynes/cm² shear stress through a closed circuit of plastic tubing rendered M1606 much more sensitive to oxidation: 80% oxidized within 1 hr. This suggestion of shear-induced unfolding and enhanced oxidation was verified when we examined 7 other methionine residues in the A1A2A3 region of VWF, the region containing the binding sites for platelets and collagen and the ADAMTS13 cleavage site. The Met residues were variably sensitive to oxidation, but all became increasingly oxidized over time in the presence of shear stress. Although the shear stresses we used in this experiment are far below the shear stress considered necessary to unfold even very large VWF multimers, the VWF solution also experienced constant elongational flow generated by a peristaltic pump, necessitating flow acceleration through the region narrowed by the rollers. Elongational flow can impart up to 100-fold more tensile stress to suspended VWF than the constant shear stress (Biophys. J., 98 L35, 2010). Two other findings favor the interpretation that oxidation of the A1A2A3 region is facilitated by domain unfolding. First, we further separated the oxidized VWF by gel-filtration into large, intermediate, and small multimeric fractions and found that methionine oxidation was much more prevalent in the fraction with the largest multimers and rare in the fraction with the smallest multimers. Second, we found that ristocetin, a VWF modulator that simulates the effect of shear stress on VWF, also accelerated oxidation of M1606. In functional tests, we found that HOCl-oxidized plasma VWF agglutinated fixed platelets at concentrations of ristocetin that induced minimal agglutination using unoxidized VWF. These findings have several important clinical implications. First, inflammatory conditions will not only activate endothelial cells and induce release of VWF, especially the largest and most adhesive forms (ultralarge VWF), the oxidants produced from endothelial cells themselves and from the neutrophil respiratory burst will render the VWF resistant to proteolysis. Second, these same oxidants will also convert the largest preexisting plasma VWF multimers that were previously rendered quiescent by ADAMTS13, into hyperfunctional and uncleavable forms. All of these mechanisms converge to generate a highly prothrombotic state, perhaps initially evolved as a mechanism to trap and isolate microorganisms, but which also has the potential to cause tremendous harm to those affected by these inflammatory conditions.