Neutrophil-Derived Oxidants Induce a Gain-Of-Function Phenotype In GPIbα

Abstract

Platelets play a key role in hemostasis and thrombosis but also act as effectors in the inflammatory process, serving as an important link between inflammation and thrombosis. A major characteristic of inflammation is oxidative stress. Oxidants produced by neutrophils, endothelial cells, and monocytes include superoxide, hydrogen peroxide (H2O2), and hypochlorous acid (HOCl). H2O2 which reacts with chloride ion in a reaction catalyzed by myeloperoxidase (released by activated neutrophils and monocytes) to produce HOCl. Superoxide reacts rapidly with nitric oxide to produce yet another oxidant, peroxynitrite. Both HOCl and peroxynitrite can alter protein function through oxidation of methionine, which is oxidized on sulfur to the sulfoxide form. Methionine oxidation converts the side chain from hydrophobic to hydrophilic, often changing protein structure and function. In hemostatic proteins, methionine oxidation has been associated with prothrombotic changes in several instances: by inactivating thrombomodulin, by rendering von Willebrand factor (VWF) uncleavable and increasing its adhesive potency, and by inactivating the VWF cleaving protease ADAMTS13. Because two of these modifications affect the VWF/platelet adhesion axis (VWF and ADAMTS13) we examined the functional effects of oxidation of the platelet receptor for VWF, glycoprotein (GP) Ibα. This polypeptide is a component of the platelet GPIb-IX-V complex, which also includes GPIbβ, GPIX, and GPV. Only GPIbα contains a binding site for VWF, located within approximately 300 amino acids at its N-terminus. This region contains 2 methionine residues, at positions 52 and 239, and a single nucleotide polymorphism that encodes Met in the minor allele at position 145. Interestingly, Met239, when mutated to Val, yields a protein capable of spontaneously binding VWF in the absence of shear stress or collagen binding, which are required for wild-type GPIbα to bind VWF. The mutation was originally found in patients with the rare bleeding disorder platelet-type von Willebrand disease (VWD). This association suggested to us that oxidation of Met239 to methionine sulfoxide could alter the ability of GPIbα to interact with VWF. We examined the effect of oxidation on GPIbα function in several ways. First, we assessed the ability of lyophilized platelets to agglutinate in the absence and presence of ristocetin. While untreated platelets agglutinated poorly in the absence of ristocetin, those treated with HOCl displayed 40% agglutination. In the presence of ristocetin, untreated and treated platelets agglutinated to a similar extent (44%). We next examined another GPIba-dependent process, shear-induced platelet agglutination, shearing untreated and HOCl-treated platelets at 10,000/s for 5 minutes in a cone-and-plate viscometer in the presence of VWF. Agglutination of HOCl-treated platelets was 4-fold higher than untreated platelets. We then assessed the effect of HOCl treatment on the ability of GPIbα to bind VWF under flow conditions. CHO cells expressing GPIbα, GPIbβ and GPIX were perfused over a VWF surface in a parallel-plate flow chamber over a range of shear rates. HOCl treatment decreased the rolling velocity (a sign of increased adhesion) of the cells by 50% or more at low shear rates (0.43, 1.17, and 1.66 dyne/cm2) but enhanced rolling by 10% at high shear rates (20 and 40 dyne/cm2). We also treated recombinant GPIbα (amino acids 1-300) with HOCl and evaluated its ability to bind VWF in human plasma in the absence or presence of ristocetin. In the absence of ristocetin, GPIbα did not bind VWF. However, HOCl treatment allowed GPIbα to bind VWF to a level similar as when ristocetin was added. Mass spectrometry was used to analyze the extent of oxidation of Met52 and Met239. HOCl treatment increased oxidation of Met52 and Met239 4-fold over baseline levels. In summary, HOCl treatment increased platelet agglutination, shear-induced platelet agglutination and VWF binding, and reduced cell velocity under flow conditions. Together our results demonstrate that HOCl treatment mimics platelet-type VWD by increasing the capacity of GPIbα to bind to VWF. Disclosures:No relevant conflicts of interest to declare.