Abstract

Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

Introduction

Reactive oxygen species (ROS) are partially reduced metabolites of oxygen that are generated during cellular homeostasis. During the last decade, considerable evidence has emerged supporting the concept that ROS regulate many normal physiological vascular responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis.1,2  The production and metabolism of ROS are tightly regulated by cellular redox switches.3  During pathologic states, excessive generation of ROS can overwhelm endogenous antioxidant systems, leading to dysregulated redox balance with adverse consequences on cellular function. For example, dysregulation of ROS has been observed in a variety of disease states associated with endothelial dysfunction.4 

Despite convincing evidence from in vitro studies and animal models implicating oxidative stress in the pathogenesis of vascular disease, clinical trials of systemic antioxidant therapy have suggested little or no benefit for the prevention of vascular or thrombotic events.5,6  One potential explanation for the lack of benefit of nonspecific antioxidant therapy is that such therapy might interfere with physiologic and pathologic redox mechanisms. There are numerous potential oxidative targets of ROS, including cellular proteins, lipids, and DNA. However, the specific molecular targets and signaling pathways linking ROS to vascular disease have remained elusive. This review focuses on the role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis.

Redox reactions in vascular cells

Figure 1 depicts the generation of some of the major forms of ROS in vascular cells. A principal intermediate form of vascular ROS is superoxide (O2•−), an oxygen anion that is formed through the univalent reduction of oxygen by enzymes such as reduced NAD phosphate (NADPH) oxidase, xanthine oxidase, and uncoupled endothelial nitric oxide synthase, or as a result of mitochondrial oxidative phosphorylation.7  Superoxide itself is relatively unreactive toward biomacromolecules under physiologic conditions.8  In many biological systems, superoxide acts as a stronger reductant than oxidant.9  In addition, it is a negatively charged free radical, making it unsuitable for crossing membranes. Despite its relatively short half-life, superoxide can be transformed into a variety of other biologically important ROS. For example, it can react with nitric oxide (NO) to form a highly reactive molecule, peroxynitrite (ONOO), or undergo dismutation to form hydrogen peroxide (H2O2) (Figure 1). H2O2 can in turn give rise to hydroxyl radical (OH) in a reaction catalyzed by Fe2+ or Cu2+ or to hypochlorous acid (HOCl) by serving as a substrate for myeloperoxidase. Both OH and HOCl are highly reactive ROS that can produce cellular damage and tissue injury.

Figure 1

Major pathways of ROS generation and elimination in vascular cells. One electron reduction of O2 can produce superoxide anion (O2•−). The main pathways for O2 transformation are via dismutation to hydrogen peroxide (H2O2) catalyzed by superoxide dismutase (SOD) or reaction with nitric oxide (NO) to form peroxynitrite (ONOO). H2O2 can be further metabolized to hypochlorous acid (HOCl) by myeloperoxidase (MPO) or hydroxyl radical (OH) through reactions catalyzed by metal ions (Fe2+ or Cu2+) via the Fenton reaction. Elimination of H2O2 is facilitated by antioxidant enzymes such as glutathione peroxidase, peroxiredoxin, and catalase.

Figure 1

Major pathways of ROS generation and elimination in vascular cells. One electron reduction of O2 can produce superoxide anion (O2•−). The main pathways for O2 transformation are via dismutation to hydrogen peroxide (H2O2) catalyzed by superoxide dismutase (SOD) or reaction with nitric oxide (NO) to form peroxynitrite (ONOO). H2O2 can be further metabolized to hypochlorous acid (HOCl) by myeloperoxidase (MPO) or hydroxyl radical (OH) through reactions catalyzed by metal ions (Fe2+ or Cu2+) via the Fenton reaction. Elimination of H2O2 is facilitated by antioxidant enzymes such as glutathione peroxidase, peroxiredoxin, and catalase.

Unlike superoxide, H2O2 has a relatively long half-life (up to milliseconds), is uncharged, and is capable of diffusing across cellular membranes.10  These characteristics make H2O2 suitable to act as an intracellular signaling molecule and a mediator of posttranslational oxidative amino acid modifications of proteins. Sulfur-containing amino acids such as cysteine and methionine are especially susceptible to oxidation by H2O2. Reversible oxidation and reduction of cysteine thiols can regulate protein function through disulfide exchange, glutathionylation, and formation of nitrosothiols, sulfenic acids, and sulfonic acids.11  Protein methionine residues also can undergo reversible redox reactions with H2O2, HOCl, and other ROS to form methionine sulfoxide (Figure 2). Protein methionine oxidation has emerged recently as a potential molecular mechanism of redox regulation of protein function in vascular biology.

Figure 2

Biochemistry of protein methionine oxidation and reduction. Oxidation of protein methionine residues produces 2 sulfoxide diastereomers, methionine-S-sulfoxide and methionine-R-sulfoxide, which can be stereospecifically reduced back to methionine by 2 classes of mammalian methionine sulfoxide reductases, MSRA and MSRB, respectively. Further oxidation of methionine sulfoxide to methionine sulfone is biologically irreversible.

Figure 2

Biochemistry of protein methionine oxidation and reduction. Oxidation of protein methionine residues produces 2 sulfoxide diastereomers, methionine-S-sulfoxide and methionine-R-sulfoxide, which can be stereospecifically reduced back to methionine by 2 classes of mammalian methionine sulfoxide reductases, MSRA and MSRB, respectively. Further oxidation of methionine sulfoxide to methionine sulfone is biologically irreversible.

Protein methionine oxidation

ROS-mediated protein methionine oxidation occurs via addition of a single oxygen molecule to the sulfur atom of protein methionine, forming methionine sulfoxide (MetSO).12  This reaction creates a chiral center with two diastereomers: methionine-S-sulfoxide and methionine-R-sulfoxide (Figure 2). MetSO contains an amino acid side chain that is more polar than that of methionine.13  This change can have profound structural and functional consequences on the target protein, such as altered conformation, solubility, protein function, or stability.14,15  Most higher organisms have evolved methionine sulfoxide reductase (MSR) enzymes that can reverse the oxidation of protein methionine residues.16  In mammals, the MSR system is composed of 2 groups of enzymes, MSRA and MSRB, which catalyze the stereospecific reduction of the S- and R- diastereomers, respectively, of MetSO (Figure 2). MSRA is primarily localized in the cytoplasm and mitochondria. There are multiple isoforms of mammalian MSRB that differ in subcellular localization.17  In the face of persistent oxidative stress, both methionine-S-sulfoxide and methionine-R-sulfoxide can undergo a second oxidation reaction to form methionine sulfone, which is not a substrate for MSR and is therefore considered to be an irreversible posttranslational modification.11 

The MSR reaction reduces MetSO to methionine, resulting in the formation of a disulfide bond in the active site of the MSR enzyme. Regeneration of the reduced thiol form of MSR requires the action of the thioredoxin/thioredoxin reductase system, which uses catalytic redox-active cysteine residues to reduce the active site disulfide of MSR through a series of thiol exchange reactions (Figure 3). This system of reversible methionine oxidation (by ROS) and reduction (by MSR) is emerging as a common biological mechanism for cellular regulation, analogous to protein phosphorylation/dephosphorylation and cysteine oxidation/reduction.

Figure 3

General mechanism of MSR and regeneration by the thioredoxin system. Reduction of methionine sulfoxide to methionine by MSR results in the transient formation of an intramolecular disulfide bond that inactivates the enzyme. Disulfide exchange reaction with thioredoxin (TRX) regenerates the active form of MSR and leads to inactivation of TRX. Regeneration of reduced TRX occurs through a transfer of electrons from NADPH in a reaction catalyzed by thioredoxin reductase (TR). The active reduced forms of the enzymes are depicted in yellow and the inactive oxidized forms of enzymes in gray.

Figure 3

General mechanism of MSR and regeneration by the thioredoxin system. Reduction of methionine sulfoxide to methionine by MSR results in the transient formation of an intramolecular disulfide bond that inactivates the enzyme. Disulfide exchange reaction with thioredoxin (TRX) regenerates the active form of MSR and leads to inactivation of TRX. Regeneration of reduced TRX occurs through a transfer of electrons from NADPH in a reaction catalyzed by thioredoxin reductase (TR). The active reduced forms of the enzymes are depicted in yellow and the inactive oxidized forms of enzymes in gray.

Protein methionine oxidation has been shown to be important in a variety of physiologic processes in several organisms. Studies in bacteria demonstrated a role for methionine oxidation and its regulation by MSR in bacterial viability. Bacteria lacking MSR had increased susceptibility to ROS-induced killing, which was rescued by restoration of MSR expression.18  Similarly, mice homozygous for a targeted deletion of MSRA (MsrA−/− mice) were reported to have enhanced susceptibility to oxidative stress and to accumulate higher levels of oxidized proteins than wild-type mice.19  MSR also may have a protective role in redox regulated processes associated with cancer, innate immunity, and neurodegeneration.20-22 

Protein methionine oxidation in vascular disease

There is evidence for a causal effect of ROS in vascular diseases such as atherosclerosis, ischemic heart disease, hypertension, and thrombosis,4  and emerging data suggest that protein methionine oxidation may play a pathogenic role. For example, cardiac myocytes from MsrA−/− mice are hypersensitive to oxidant stress.23  Conversely, overexpression of MSRA in cultured cardiac myocytes provided strong protection against injury in a hypoxia-reoxygenation model,24  and transgenic mice overexpressing a myristoylated isoform of MSRA were found to be protected against ischemia-reperfusion injury of the heart ex vivo.25  Moreover, 2 recent clinical studies have identified a single nucleotide polymorphism (rs10903323) in the human MSRA gene to be associated with increased risk of cardiovascular events and coronary artery disease.26,27  The impact of the MSRA rs10903323 polymorphism on MSRA protein expression or enzymatic activity is unknown.

Several proteins important in vascular biology have been shown to contain oxidation-sensitive methionine residues with potential regulatory roles in the pathogenesis of vascular or thrombotic diseases (Table 1). This list is likely to grow in the future as proteomic approaches to detect and identify modified methionine residues are increasingly used.28  The functional significance of many of these specific protein methionine oxidation reactions remains to be determined. In the remainder of this review, we will focus on some of the better characterized vascular proteins that contain redox-active methionine residues, including calcium/calmodulin-dependent protein kinase II (CaMKII), apolipoprotein A-I, thrombomodulin (TM), and von Willebrand factor (VWF), and highlight their potential roles in the pathogenesis of vascular disease.

Table 1

Partial list of proteins with oxidation-sensitive methionine residues

Target protein Oxidation-sensitive methionine(s)* Effect of MetSO on protein function† Reference(s) 
Proteins relevant to cardiovascular biology    
 CaMKII Met281, Met282 ↑ 31,32  
 Apolipoprotein A-I Met86, Met112, Met148 ↓ 43,45,46  
 Actin Met46, Met49 ↓ 21,93  
 IκBα Met45 ↑ 94,95  
 p53 Met340 ↓ 96  
 S100A9 Met63, Met83 ↓ 92  
Proteins involved in hemostasis and thrombosis    
 Thrombomodulin Met388 ↓ 57,60  
 Activated protein C Met59 ↓ 67  
 VWF Met1606 ↑ 75,76  
 ADAMTS13 Met249, Met331, Met496 ↓ 80  
 Fibrinogen Met78, Met367, Met476 ↔ 84,85  
 α-2-Antiplasmin 10 Met residues ND 90  
 Antithrombin III Met314, Met315 ND 90,91  
 Factor VII Met298, Met306 ↓ 89  
 Plasminogen activator inhibitor-1 Met347 ─ 87,88  
 Tissue plasminogen activator Met207 ─ 86  
Target protein Oxidation-sensitive methionine(s)* Effect of MetSO on protein function† Reference(s) 
Proteins relevant to cardiovascular biology    
 CaMKII Met281, Met282 ↑ 31,32  
 Apolipoprotein A-I Met86, Met112, Met148 ↓ 43,45,46  
 Actin Met46, Met49 ↓ 21,93  
 IκBα Met45 ↑ 94,95  
 p53 Met340 ↓ 96  
 S100A9 Met63, Met83 ↓ 92  
Proteins involved in hemostasis and thrombosis    
 Thrombomodulin Met388 ↓ 57,60  
 Activated protein C Met59 ↓ 67  
 VWF Met1606 ↑ 75,76  
 ADAMTS13 Met249, Met331, Met496 ↓ 80  
 Fibrinogen Met78, Met367, Met476 ↔ 84,85  
 α-2-Antiplasmin 10 Met residues ND 90  
 Antithrombin III Met314, Met315 ND 90,91  
 Factor VII Met298, Met306 ↓ 89  
 Plasminogen activator inhibitor-1 Met347 ─ 87,88  
 Tissue plasminogen activator Met207 ─ 86  

The listed proteins have been characterized by the presence of ≥1 methionine residue with a potential regulatory role or pathologic effect in cardiovascular biology or thrombosis.

*

The numbering scheme of the oxidation-sensitive methionine residues is based on the species and isoform of the proteins studied in the cited references.

The relative change in function as a consequence of methionine oxidation is indicated as follows: ↑, enhanced; ↓, suppressed; ↔, altered structure; ─, no effect; ND, not determined.

CaMKII: a prototypical methionine redox sensor

CaMKII is serine/threonine kinase that functions as a key mediator of intracellular calcium signaling by phosphorylating proteins involved in gene transcription, cell survival, excitation–contraction coupling, and calcium homeostasis.29  Like many kinases, CaMKII is subject to autoregulation. Following transient activation by calcium/calmodulin binding, the catalytic domain of CaMKII can autophosphorylate its C-terminal regulatory domain at Thr287.30  Autophosphorylation at Thr287 results in a conformational change in CaMKII that causes sustained activity that is no longer dependent on calcium/calmodulin (Figure 4). In addition to this mechanism of autoregulation via phosphorylation, CaMKII is also subject to redox regulation mediated by oxidation of a pair of tandem methionine residues (Met281/Met282) located within its regulatory domain.31  When oxidized, the 2 redox-active methionine residues block reassociation of the regulatory domain with the catalytic domain, resulting in sustained, calcium/calmodulin-independent kinase activity similar to that observed after Thr287 autophosphorylation (Figure 4). Oxidation of Met281/Met282 can be reversed by the action of MSRA, restoring CaMKII to an inactive state.31,32  In this way, CaMKII acts as a dynamic, reversible redox sensor during normal cellular homeostasis.

Figure 4

CaMKII domain structure and activation. (A) Each CaMKII monomer contains a catalytic kinase domain (blue), a regulatory domain involved (yellow), and an association domain (white). Dimerization of monomeric subunits is mediated via the association domains, followed by oligomerization to form a holoenzyme consisting of 2 stacked hexameric rings (not shown). The sequence of the regulatory domain containing 2 redox-active methionine residues (Met281/Met282) (red) and Thr287 (black) are indicated. (B) In the resting state, the kinase activity of CaMKII is autoinhibited by an interaction between its catalytic and regulatory domains. Binding of calcium/calmodulin (Ca2+/CaM) (green) to the regulatory domain causes disassociation and transient activation of the catalytic domain. Autophosphorylation at Thr-287 or oxidation at Met281/Met282 prevents autoinhibition and causes sustained activation of CaMKII that is independent of calcium/calmodulin binding. Adapted with permission from Scott et al.39 

Figure 4

CaMKII domain structure and activation. (A) Each CaMKII monomer contains a catalytic kinase domain (blue), a regulatory domain involved (yellow), and an association domain (white). Dimerization of monomeric subunits is mediated via the association domains, followed by oligomerization to form a holoenzyme consisting of 2 stacked hexameric rings (not shown). The sequence of the regulatory domain containing 2 redox-active methionine residues (Met281/Met282) (red) and Thr287 (black) are indicated. (B) In the resting state, the kinase activity of CaMKII is autoinhibited by an interaction between its catalytic and regulatory domains. Binding of calcium/calmodulin (Ca2+/CaM) (green) to the regulatory domain causes disassociation and transient activation of the catalytic domain. Autophosphorylation at Thr-287 or oxidation at Met281/Met282 prevents autoinhibition and causes sustained activation of CaMKII that is independent of calcium/calmodulin binding. Adapted with permission from Scott et al.39 

Excessive or dysregulated CaMKII activity in cardiac myocytes promotes downstream signaling pathways involved in the pathophysiology of heart failure and arrhythmias.30  Increased levels of oxidized CaMKII in mice have been shown to promote sinus node dysfunction33  and atrial fibrillation.34  Stimulation of cardiac myocytes with angiotensin II or aldosterone leads to oxidative activation of CaMKII, which can be reversed by MSRA.31,32  Treatment of MsrA−/− mice with aldosterone caused exaggerated CaMKII oxidation and myocardial apoptosis, leading to impaired cardiac function and cardiac rupture after myocardial infarction.32  Increased levels of oxidized CaMKII have been detected in atrial tissue from diabetic patients after myocardial infarction and in mouse models of type 1 diabetes.35  Interestingly, mice expressing an oxidation-resistant form of cardiac CaMKII (in which the tandem redox-active methionine residues are mutated to valines) are resistant to diabetes-attributable mortality after myocardial infarction, suggesting that oxidation of CaMKII may contribute to sudden cardiac death in diabetes. Together, these in vivo studies confirm a regulatory role for methionine oxidation of CaMKII in cardiac pathophysiology and suggest that therapeutic approaches to decrease the oxidation of CaMKII or inhibit the activity of oxidized CaMKII may prevent or reduce complications of heart disease.

The role of CaMKII oxidation in vascular disease and thrombosis remains less clear. CaMKII is expressed in vascular cells, including vascular smooth muscle and endothelial cells, as well as in blood cells such as platelets. Recent studies have suggested that activation of CaMKII regulates vascular muscle responses such as proliferation, migration, and vascular remodeling.36,37  Vascular injury is associated with elevated levels of ROS,38  and CaMKII oxidation was observed to be increased following vascular injury in mice, possibly contributing to vascular smooth muscle migration and phenotypic switching.39  Subsequent studies found that oxidized CaMKII not only promotes vascular muscle cell migration and apoptosis, but also induces ROS generation by NADPH oxidase, providing a positive pro-oxidant feedback loop.40  Thus, there is emerging evidence that CaMKII functions in the redox regulation of vascular function, at least in vascular smooth muscle cells. Analogously, oxidation of CaMKII in the bronchial epithelium of the lung was found to promote inflammation and airway hyperreactivity, and contribute to the pathogenesis of allergic asthma.41  The potential role of CaMKII protein methionine oxidation in vascular endothelial cells and platelets remains largely undefined and is a potentially interesting area for future investigation.

Apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoprotein (HDL).42  The primary sequence of apoA-I contains 3 oxidation-sensitive methionine residues (Met86, Met112, and Met148). These methionine residues were initially hypothesized to function as an endogenous antioxidant system by reducing lipid peroxides and cholesterol in oxidized lipoproteins43,44  and protecting other functional domains of apoA-I from oxidation.45  Subsequent studies found that oxidation of Met148 impairs the ability of apoA-I to activate lecithin-cholesterol acyltransferase (LCAT), an enzyme that converts cholesterol to cholesterol ester and is required for reverse cholesterol transport to the liver via HDL.46  apoA-I-mediated activation of LCAT is rescued when Met148 oxidation is reversed by MSR in vitro, and HDL prepared with a mutant apoA-I (M148L) is resistant to oxidative loss of LCAT activity.46  A decrease in LCAT activity can lead to increased atherogenesis, and it has been reported that patients with atherosclerosis have elevated levels of apoA-I oxidized at Met148.47  However, it remains unknown if apoA-1 protein methionine oxidation is reversible in vivo, and a causal relationship between apoA-I Met148 oxidation and atherosclerosis has not been established.

TM

TM is a transmembrane protein that is expressed on the luminal surface of vascular endothelium, where it functions as a high-affinity thrombin receptor.48  TM functions as an endogenous anticoagulant by modulating the substrate specificity of thrombin from that of a procoagulant to an anticoagulant protease.49  When bound to TM on the endothelial surface, thrombin is unable to convert fibrinogen to fibrin, activate factor V, or trigger platelet aggregation. Instead, thrombin becomes an efficient activator of protein C. The activated form of protein C (APC) is an anticoagulant protease that selectively inactivates coagulation factors Va and VIIIa, thereby inhibiting amplification of the coagulation cascade. APC also has potent anti-inflammatory effects on endothelial cells and monocytes.50 

The clinical importance of the TM/protein C anticoagulant pathway is underscored by the strong association between venous thromboembolism and resistance to APC caused by the factor V Leiden mutation51  and by the severe thrombotic phenotype of children born with homozygous or compound heterozygous forms of protein C deficiency.52  The TM/APC pathway also may modulate atherosclerotic plaque stability53  and susceptibility to ischemic stroke.54  Gene targeted mice with endothelium-specific loss of TM develop severe spontaneous thrombosis in both the arterial and venous circulations.55 

TM contains an epidermal growth factor (EGF) homology region consisting of 6 EGF-like domains (Figure 5). The high-affinity thrombin binding site is located within EGF-like domains 4 and 5, and the minimal region of TM that supports protein C activation consists of EGF-like domains 4, 5, and 6.56  An oxidation-sensitive methionine residue (Met388) is located in a short linker sequence connecting EGF-like domains 4 and 5. Oxidation of Met388 destabilizes the orientation between EGF-like domains 4 and 5, resulting in a fivefold decrease in the Kcat for protein C activation and a 90% decrease in TM anticoagulant activity.57,58  When Met388 is mutated to leucine, TM activity is maintained, even in the face of oxidative challenge.59,60  Using animal models, our group has demonstrated that the TM/protein C antithrombotic pathway becomes compromised by atherosclerosis and improves during regression of atherosclerosis.61-63  More recently, we demonstrated increased susceptibility to thrombosis and obtained evidence for inactivation of TM in hypercholesterolemic mice expressing human TM.64  Loss of TM-dependent protein C activation in mice is prevented by superoxide dismutase.65,66  These observations support the hypothesis that oxidation of TM Met388 may contribute to a prothrombotic phenotype. Additionally, APC itself has been shown to undergo protein methionine oxidation at Met59, which may directly contribute to loss of APC anticoagulant activity when it is exposed to oxidants such as H2O2 and HOCl.67  It remains to be definitively determined, however, whether oxidation of TM and/or APC occurs in vivo under pathophysiologic conditions of vascular oxidative stress and to what extent these redox reactions may affect susceptibility to thrombosis and vascular disease in humans.

Figure 5

Schematic representation of thrombomodulin domain structure. The amino terminal portion of TM, which projects into the vascular lumen, contains a lectin-like domain and 6 EGF-like domains. EGF-like domains 5 and 6 are required for thrombin binding, and EGF-like domains 4 to 6 (yellow) are necessary for efficient activation of protein C. The location of the oxidation-sensitive regulatory methionine (M388) is in the short linker region between EGF-like domains 4 and 5 (arrow). TM also contains a transmembrane domain and a cytoplasmic tail (CYTO).

Figure 5

Schematic representation of thrombomodulin domain structure. The amino terminal portion of TM, which projects into the vascular lumen, contains a lectin-like domain and 6 EGF-like domains. EGF-like domains 5 and 6 are required for thrombin binding, and EGF-like domains 4 to 6 (yellow) are necessary for efficient activation of protein C. The location of the oxidation-sensitive regulatory methionine (M388) is in the short linker region between EGF-like domains 4 and 5 (arrow). TM also contains a transmembrane domain and a cytoplasmic tail (CYTO).

VWF

VWF is a multimeric protein synthesized by endothelial cells as high-molecular-weight multimers that are released into circulation through both constitutive and regulated pathways.68  The primary function of VWF in hemostasis is to mediate initial platelet adhesion and promote clot formation at sites of vascular injury in a process that is dependent on large multimers of VWF. Deficiency of VWF or loss of high-molecular-weight VWF multimers causes the clinical bleeding disorder von Willebrand disease.69  In contrast, an overabundance of ultralarge multimers of VWF causes the microvascular thrombotic disorder, thrombotic thrombocytopenic purpura.70  The classic form of thrombotic thrombocytopenic purpura results from a hereditary or acquired deficiency of the VWF-cleaving metalloprotease A disintegrin and metalloproteinase with a thrombospondin type 1 motif, 13 (ADAMTS13).71  Partial deficiency of ADAMTS13 may predispose to thrombotic events such as acute coronary syndromes and stroke.72,73 

ADAMTS13 cleaves VWF at the Tyr1605-Met1606 peptide bond within its A2 domain.74  VWF Met1606 is susceptible to oxidation, which can affect its cleavage by ADAMTS13.75,76  Oxidation of Met1606 can be enhanced by shear stress77  or ristocetin,78  which induces a conformational change in VWF that unfolds the A2 domain. Met1606-oxidized VWF multimers are hyperactive in inducing platelet agglutination.77  Therefore, methionine oxidation of VWF Met1606 during vascular oxidative stress represents a potential prothrombotic mechanism, leading to the persistence of ultralarge VWF multimers that are resistant to proteolytic processing by ADAMTS13. Consistent with this notion, elevated plasma levels of large VWF multimers and oxidized VWF (including VWF with oxidized Met1606) have been detected in patients who have risk factors for thrombotic vascular disease, such as chronic kidney disease.79  Recent work suggests that ADAMTS13 also may be a target of methionine oxidation, leading to loss of its enzymatic activity and contributing to the accumulation of large VWF multimers during inflammation.80  Under such conditions, cleavage of VWF multimers may occur primarily through the proteolytic activity of leukocyte serine proteases rather than ADAMTS13.81,82  Interestingly, the activity VWF-cleaving proteases such as neutrophil elastase may be further regulated by the oxidative inactivation of α-1-antitrypsin, which itself contains several oxidation-sensitive methionine residues.83  Thus, the overall effect of protein methionine oxidation on VWF multimer structure and function is likely to be complex and may involve multiple redox regulated proteins. Future studies are needed to determine whether these oxidized methionines can be reduced by MSR and better define the impact of these redox reactions in vivo.

Other targets of protein methionine oxidation

Several additional proteins involved in vascular function and/or thrombosis contain oxidation-sensitive methionine residues (Table 1). Fibrinogen, the substrate for thrombin-mediated fibrin formation, contains three methionine residues (Met78, Met367, and Met476) that are highly susceptible to oxidation by HOCl.84  Oxidation of these methionine residues alters fibrin polymerization, which affects the structural and mechanical properties of the fibrin clot, potentially leading to delayed fibrinolysis.85  Interestingly, both tissue plasminogen activator and its major inhibitor, plasminogen activator inhibitor-1, also contain methionine residues that are subject to oxidation,86,87  but it is not yet known whether methionine oxidation of these proteins affects fibrinolysis in vivo.88  Several other hemostatic proteins, including factor VII, antithrombin, and α-2-antiplasmin, have been shown to contain oxidation-sensitive methionine residues that can regulate their function in vitro.89-91  It should be recognized that almost all of the work characterizing the effects of protein methionine oxidation on hemostatic protein function has been performed in vitro; therefore, the pathophysiological relevance of these reactions has not been established.

Macrophage function can be regulated by the stereospecific oxidation and reduction of methionine residues (Met46 and Met49) in actin.21,92,93  Methionine oxidation inhibits actin polymerization and actin-dependent processes such as cytokine release, which can be reversed by stereospecific reduction by MSRB. Additionally, there is some evidence that signaling pathways involved in inflammation and cell cycle regulation can be regulated by methionine oxidation.94-96  Thus, methionine oxidation and reduction might contribute to a variety of cellular signal transduction processes related to vascular disease.

Perspectives and clinical implications

It is now understood that redox biology is central to normal cellular homeostasis. ROS are generated widely as products of aerobic metabolism.97  ROS function not only as signaling molecules but also as redox regulators of proteins and other macromolecules via reversible reactions such as protein methionine oxidation. Because of their inherent reactivity, ROS are tightly regulated by a myriad of paired molecular oxidation and reduction reactions (redox switches). During pathologic states of oxidative stress, the normally tight regulation of cellular ROS flux can become disrupted. In light of the central role of redox reactions in normal vascular function, it is perhaps not surprising that the early trials of systemic antioxidant therapy failed to demonstrate a clinical benefit for the prevention of vascular or thrombotic events.5,6  One lesson from these trials is that a better basic understanding of specific vascular redox reactions is needed.

Certain protein methionine residues are particularly sensitive to reversible oxidation, especially those residing in accessible surface domains or domains that can become exposed to ROS through conformation changes induced by shear stress or protein-protein interactions. Like other reversible protein modification reactions such as protein phosphorylation/dephosphorylation and cysteine oxidation/reduction, protein methionine oxidation/reduction can regulate protein-protein interactions, enzymatic activity, and cellular function. These effects are highly dependent on subcellular localization, accessibility to regulatory enzymes such as MSR, and the redox environment of the specific intracellular or extracellular compartment in which the target protein functions. Some proteins with oxidation-sensitive methionine residues, including CaMKII, actin, inhibitor of κBα (IκBα), p53, and S100A9, are intracellular proteins that are likely to be protected from oxidation by an in vivo redox environment that is highly reducing and rich in MSR. Conversely, many vascular and hemostatic proteins containing oxidation-sensitive protein methionine residues, such as TM, VWF, and apoA-I, are plasma or endothelial cell surface proteins, which may be more susceptible to oxidation due to the oxidizing redox state of the extracellular environment.

None of the mammalian MSR isoforms are known to function extracellularly, but MsrA−/− mice have been reported to have higher levels of MetSO in serum proteins compared with wild-type mice,98  which suggests that MSRA can influence protein methionine oxidation of secreted proteins. For most of the proteins listed in Table 1, the identification and characterization of oxidation-sensitive methionine residues was accomplished using in vitro systems under nonphysiologic conditions. Therefore, with a few notable exceptions such as CaMKII and actin, the pathophysiologic consequences of these protein methionine oxidation reactions and whether or not they are regulated by MSR in a physiological environment have not been rigorously studied. Future studies using more physiological approaches, such as oxidation-resistant mutants, will be necessary to better delineate the role of methionine oxidation in vascular disease. Finally, it is noteworthy that several MSR isoforms are expressed at high levels in mitochondria,17  which are a major source of intracellular ROS. Defining the regulatory role of MSR on mitochondrial function will be an interesting topic for future investigation.

Future progress in defining the contribution of dysregulated protein methionine oxidation in vascular disease will depend on the development of quantitative methods to detect and measure MetSO in biological samples. Recently, there has been considerable effort directed toward developing methods for the large-scale detection and quantification of specific protein methionine residues using proteomics approaches.99  For example, adapting a combined fractional diagonal chromatography method100  to enrich for methionine-oxidized substrates shows promise for the high-throughput detection and identification of MetSO in specific proteins. A recent proteome-wide analysis identified >2000 specific protein methionine oxidation sites in a H2O2-treated T-cell line.28  This method also was applied to a mouse model of sepsis to demonstrate that it can be extended to identify protein MetSO in vivo.28  The functional significance of methionine oxidation in the vast majority of these target proteins remains to determined. Molecular dynamic simulations are now able to provide considerable insight into the potential effects of methionine oxidation on protein structure and function.67,85  However, new methods and techniques clearly are needed to better investigate the structural and biological effects of protein methionine oxidation.

As our understanding of the specific molecular targets of protein methionine oxidation improves, it is very likely that new candidate biomarkers of oxidative vascular disease will emerge.101,102  The prospects for the development of novel therapeutic approaches to target protein methionine oxidation are perhaps more daunting. For proteins such as CaMKII or VWF, whose function is upregulated by protein methionine oxidation, it might be more feasible to develop pharmacologic inhibitors that inhibit the function of the oxidized protein rather than attempting to prevent or reverse the formation of MetSO. Unfortunately, it is more often the case that protein methionine oxidation results in impairment of protein function rather than hyperactivation (Table 1). Potential therapeutic strategies in such cases might include the administration of an oxidation-resistant protein therapeutic. For example, to overcome the inhibitory effect of TM Met388 oxidation on its anticoagulant activity, a TM M388L analog has been designed that is insensitive to oxidative inactivation.103,104  This oxidation-resistant TM analog could potentially be used as an anticoagulant therapeutic agent in patients with acute thrombotic vascular disease.105  More general approaches might include targeting ROS-generating enzymes, such as NADPH oxidases, or developing selective ROS scavengers or antioxidant mimetics. A more specific method of controlling methionine redox state theoretically could be accomplished by manipulating MSR expression or activity. For example, new methods for nanocarrier-mediated targeted delivery of antioxidant enzymes such as MSR to vascular cells are being developed as potential therapeutic strategies in vascular disease.106 

Conclusions

A number of vascular and hematostatic proteins are recognized to undergo protein methionine oxidation. In some cases, such as the oxidation of Met281/Met282 of CaMKII or Met1606 of VWF, protein methionine oxidation leads to hyperfunctioning of the target protein. For many other target proteins, such as TM and APC, oxidation of specific protein methionine residues causes loss of protein function. Most of our current understanding of the specific protein targets and functional consequences of protein methionine oxidation are derived from in vitro or animal studies. Therefore, much remains to be accomplished to fully understand the role of methionine oxidation in vascular disease and thrombosis. New methods and models are needed to define the mechanisms that regulate specific methionine oxidation reactions and the molecular effects of methionine oxidation on protein and cellular function. Tissue-specific animal models with altered expression and/or activity of MSR or oxidation-resistant vascular proteins are needed to explore effects of defined protein methionine reactions in endothelial and vascular smooth muscle cells and in hematopoietic cells such as platelets and leukocytes. A better appreciation of methionine redox biology and its vascular targets has the potential to lead to novel biomarkers and therapies for vascular disease and thrombosis.

Acknowledgments

This work was supported by the American Society of Hematology; National Institutes of Health grants GM007337 from the National Institute of General Medical Sciences, HL063943 and HL062984 from the National Heart, Lung, and Blood Institute; and an American Heart Association Predoctoral Fellowship award 12PRE940065.

Authorship

Contribution: S.X.G. wrote the paper; J.W.S. and S.R.L. reviewed and revised the paper; and all authors approved the final version of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Steven R. Lentz, Department of Internal Medicine, C21 GH, The University of Iowa, 200 Hawkins Dr, Iowa City, IA 52242; e-mail: steven-lentz@uiowa.edu.

References

References
1
Taniyama
Y
Griendling
KK
Reactive oxygen species in the vasculature: molecular and cellular mechanisms.
Hypertension
2003
, vol. 
42
 
6
(pg. 
1075
-
1081
)
2
Kim
YW
Byzova
TV
Oxidative stress in angiogenesis and vascular disease.
Blood
2014
, vol. 
123
 
5
(pg. 
625
-
631
)
3
Dikalov
SI
Harrison
DG
Methods for detection of mitochondrial and cellular reactive oxygen species.
Antioxid Redox Signal
2014
, vol. 
20
 
2
(pg. 
372
-
382
)
4
Sugamura
K
Keaney
JF
Jr
Reactive oxygen species in cardiovascular disease.
Free Radic Biol Med
2011
, vol. 
51
 
5
(pg. 
978
-
992
)
5
Sesso
HD
Buring
JE
Christen
WG
, et al. 
Vitamins E and C in the prevention of cardiovascular disease in men: the Physicians’ Health Study II randomized controlled trial.
JAMA
2008
, vol. 
300
 
18
(pg. 
2123
-
2133
)
6
Cook
NR
Albert
CM
Gaziano
JM
, et al. 
A randomized factorial trial of vitamins C and E and beta carotene in the secondary prevention of cardiovascular events in women: results from the Women’s Antioxidant Cardiovascular Study.
Arch Intern Med
2007
, vol. 
167
 
15
(pg. 
1610
-
1618
)
7
Mueller
CF
Laude
K
McNally
JS
Harrison
DG
ATVB in focus: redox mechanisms in blood vessels.
Arterioscler Thromb Vasc Biol
2005
, vol. 
25
 
2
(pg. 
274
-
278
)
8
Thomas
SR
Witting
PK
Drummond
GR
Redox control of endothelial function and dysfunction: molecular mechanisms and therapeutic opportunities.
Antioxid Redox Signal
2008
, vol. 
10
 
10
(pg. 
1713
-
1765
)
9
Buettner
GR
The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate.
Arch Biochem Biophys
1993
, vol. 
300
 
2
(pg. 
535
-
543
)
10
Reczek
CR
Chandel
NS
ROS-dependent signal transduction.
Curr Opin Cell Biol
2015
, vol. 
33
 (pg. 
8
-
13
)
11
Burgoyne
JR
Oka
S
Ale-Agha
N
Eaton
P
Hydrogen peroxide sensing and signaling by protein kinases in the cardiovascular system.
Antioxid Redox Signal
2013
, vol. 
18
 
9
(pg. 
1042
-
1052
)
12
Kim
G
Weiss
SJ
Levine
RL
Methionine oxidation and reduction in proteins.
Biochim Biophys Acta
2014
, vol. 
1840
 
2
(pg. 
901
-
905
)
13
Black
SD
Mould
DR
Development of hydrophobicity parameters to analyze proteins which bear post- or cotranslational modifications.
Anal Biochem
1991
, vol. 
193
 
1
(pg. 
72
-
82
)
14
Chao
CC
Ma
YS
Stadtman
ER
Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems.
Proc Natl Acad Sci USA
1997
, vol. 
94
 
7
(pg. 
2969
-
2974
)
15
Hoshi
T
Heinemann
S
Regulation of cell function by methionine oxidation and reduction.
J Physiol
2001
, vol. 
531
 
Pt 1
(pg. 
1
-
11
)
16
Kim
HY
Gladyshev
VN
Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals.
Biochem J
2007
, vol. 
407
 
3
(pg. 
321
-
329
)
17
Lee
BC
Dikiy
A
Kim
HY
Gladyshev
VN
Functions and evolution of selenoprotein methionine sulfoxide reductases.
Biochim Biophys Acta
2009
, vol. 
1790
 
11
(pg. 
1471
-
1477
)
18
St John
G
Brot
N
Ruan
J
, et al. 
Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates.
Proc Natl Acad Sci USA
2001
, vol. 
98
 
17
(pg. 
9901
-
9906
)
19
Moskovitz
J
Bar-Noy
S
Williams
WM
Requena
J
Berlett
BS
Stadtman
ER
Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals.
Proc Natl Acad Sci USA
2001
, vol. 
98
 
23
(pg. 
12920
-
12925
)
20
De Luca
A
Sanna
F
Sallese
M
, et al. 
Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype.
Proc Natl Acad Sci USA
2010
, vol. 
107
 
43
(pg. 
18628
-
18633
)
21
Lee
BC
Péterfi
Z
Hoffmann
FW
, et al. 
MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation.
Mol Cell
2013
, vol. 
51
 
3
(pg. 
397
-
404
)
22
Butterfield
DA
Galvan
V
Lange
MB
, et al. 
In vivo oxidative stress in brain of Alzheimer disease transgenic mice: Requirement for methionine 35 in amyloid beta-peptide of APP.
Free Radic Biol Med
2010
, vol. 
48
 
1
(pg. 
136
-
144
)
23
Nan
C
Li
Y
Jean-Charles
PY
, et al. 
Deficiency of methionine sulfoxide reductase A causes cellular dysfunction and mitochondrial damage in cardiac myocytes under physical and oxidative stresses.
Biochem Biophys Res Commun
2010
, vol. 
402
 
4
(pg. 
608
-
613
)
24
Prentice
HM
Moench
IA
Rickaway
ZT
Dougherty
CJ
Webster
KA
Weissbach
H
MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death.
Biochem Biophys Res Commun
2008
, vol. 
366
 
3
(pg. 
775
-
778
)
25
Zhao
H
Sun
J
Deschamps
AM
, et al. 
Myristoylated methionine sulfoxide reductase A protects the heart from ischemia-reperfusion injury.
Am J Physiol Heart Circ Physiol
2011
, vol. 
301
 
4
(pg. 
H1513
-
H1518
)
26
García-Bermúdez
M
López-Mejías
R
González-Juanatey
C
, et al. 
Association of the methionine sulfoxide reductase A rs10903323 gene polymorphism with cardiovascular disease in patients with rheumatoid arthritis.
Scand J Rheumatol
2012
, vol. 
41
 
5
(pg. 
350
-
353
)
27
Gu
H
Chen
W
Yin
J
Chen
S
Zhang
J
Gong
J
Methionine sulfoxide reductase A rs10903323 G/A polymorphism is associated with increased risk of coronary artery disease in a Chinese population.
Clin Biochem
2013
, vol. 
46
 
16-17
(pg. 
1668
-
1672
)
28
Ghesquiere
B
Jonckheere
V
Colaert
N
, et al. 
Redox proteomics of protein-bound methionine oxidation.
Mol Cell Proteomics
2011
, vol. 
10
 
5
pg. 
M110006866
 
29
Anderson
ME
Brown
JH
Bers
DM
CaMKII in myocardial hypertrophy and heart failure.
J Mol Cell Cardiol
2011
, vol. 
51
 
4
(pg. 
468
-
473
)
30
Hudmon
A
Schulman
H
Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.
Biochem J
2002
, vol. 
364
 
Pt 3
(pg. 
593
-
611
)
31
Erickson
JR
Joiner
ML
Guan
X
, et al. 
A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation.
Cell
2008
, vol. 
133
 
3
(pg. 
462
-
474
)
32
He
BJ
Joiner
ML
Singh
MV
, et al. 
Oxidation of CaMKII determines the cardiotoxic effects of aldosterone.
Nat Med
2011
, vol. 
17
 
12
(pg. 
1610
-
1618
)
33
Swaminathan
PD
Purohit
A
Soni
S
, et al. 
Oxidized CaMKII causes cardiac sinus node dysfunction in mice.
J Clin Invest
2011
, vol. 
121
 
8
(pg. 
3277
-
3288
)
34
Purohit
A
Rokita
AG
Guan
X
, et al. 
Oxidized Ca(2+)/calmodulin-dependent protein kinase II triggers atrial fibrillation.
Circulation
2013
, vol. 
128
 
16
(pg. 
1748
-
1757
)
35
Luo
M
Guan
X
Luczak
ED
, et al. 
Diabetes increases mortality after myocardial infarction by oxidizing CaMKII.
J Clin Invest
2013
, vol. 
123
 
3
(pg. 
1262
-
1274
)
36
House
SJ
Singer
HA
CaMKII-delta isoform regulation of neointima formation after vascular injury.
Arterioscler Thromb Vasc Biol
2008
, vol. 
28
 
3
(pg. 
441
-
447
)
37
Singer
HA
Ca2+/calmodulin-dependent protein kinase II function in vascular remodelling.
J Physiol
2012
, vol. 
590
 
Pt 6
(pg. 
1349
-
1356
)
38
Papaharalambus
CA
Griendling
KK
Basic mechanisms of oxidative stress and reactive oxygen species in cardiovascular injury.
Trends Cardiovasc Med
2007
, vol. 
17
 
2
(pg. 
48
-
54
)
39
Scott
JA
Xie
L
Li
H
, et al. 
The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9.
Am J Physiol Heart Circ Physiol
2012
, vol. 
302
 
10
(pg. 
H1953
-
H1964
)
40
Zhu
LJ
Klutho
PJ
Scott
JA
, et al. 
Oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulates vascular smooth muscle migration and apoptosis.
Vascul Pharmacol
2014
, vol. 
60
 
2
(pg. 
75
-
83
)
41
Sanders
PN
Koval
OM
Jaffer
OA
, et al. 
CaMKII is essential for the proasthmatic effects of oxidation.
Sci Transl Med
2013
, vol. 
5
 
195
pg. 
195ra197
 
42
Fielding
CJ
Fielding
PE
Molecular physiology of reverse cholesterol transport.
J Lipid Res
1995
, vol. 
36
 
2
(pg. 
211
-
228
)
43
Garner
B
Witting
PK
Waldeck
AR
Christison
JK
Raftery
M
Stocker
R
Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that accompanies lipid peroxidation and can be enhanced by alpha-tocopherol.
J Biol Chem
1998
, vol. 
273
 
11
(pg. 
6080
-
6087
)
44
Garner
B
Waldeck
AR
Witting
PK
Rye
KA
Stocker
R
Oxidation of high density lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.
J Biol Chem
1998
, vol. 
273
 
11
(pg. 
6088
-
6095
)
45
Shao
B
Oda
MN
Bergt
C
, et al. 
Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.
J Biol Chem
2006
, vol. 
281
 
14
(pg. 
9001
-
9004
)
46
Shao
B
Cavigiolio
G
Brot
N
Oda
MN
Heinecke
JW
Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I.
Proc Natl Acad Sci USA
2008
, vol. 
105
 
34
(pg. 
12224
-
12229
)
47
Shao
B
Tang
C
Sinha
A
, et al. 
Humans with atherosclerosis have impaired ABCA1 cholesterol efflux and enhanced high-density lipoprotein oxidation by myeloperoxidase.
Circ Res
2014
, vol. 
114
 
11
(pg. 
1733
-
1742
)
48
Martin
FA
Murphy
RP
Cummins
PM
Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects.
Am J Physiol Heart Circ Physiol
2013
, vol. 
304
 
12
(pg. 
H1585
-
H1597
)
49
Esmon
CT
Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface.
FASEB J
1995
, vol. 
9
 
10
(pg. 
946
-
955
)
50
Danese
S
Vetrano
S
Zhang
L
Poplis
VA
Castellino
FJ
The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications.
Blood
2010
, vol. 
115
 
6
(pg. 
1121
-
1130
)
51
Kearon
C
Crowther
M
Hirsh
J
Management of patients with hereditary hypercoagulable disorders.
Annu Rev Med
2000
, vol. 
51
 (pg. 
169
-
185
)
52
Goldenberg
NA
Manco-Johnson
MJ
Protein C deficiency.
Haemophilia
2008
, vol. 
14
 
6
(pg. 
1214
-
1221
)
53
Seehaus
S
Shahzad
K
Kashif
M
, et al. 
Hypercoagulability inhibits monocyte transendothelial migration through protease-activated receptor-1-, phospholipase-Cbeta-, phosphoinositide 3-kinase-, and nitric oxide-dependent signaling in monocytes and promotes plaque stability.
Circulation
2009
, vol. 
120
 
9
(pg. 
774
-
784
)
54
Zlokovic
BV
Griffin
JH
Cytoprotective protein C pathways and implications for stroke and neurological disorders.
Trends Neurosci
2011
, vol. 
34
 
4
(pg. 
198
-
209
)
55
Isermann
B
Hendrickson
SB
Zogg
M
, et al. 
Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis.
J Clin Invest
2001
, vol. 
108
 
4
(pg. 
537
-
546
)
56
Wood
MJ
Sampoli Benitez
BA
Komives
EA
Solution structure of the smallest cofactor-active fragment of thrombomodulin.
Nat Struct Biol
2000
, vol. 
7
 
3
(pg. 
200
-
204
)
57
Glaser
CB
Morser
J
Clarke
JH
, et al. 
Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.
J Clin Invest
1992
, vol. 
90
 
6
(pg. 
2565
-
2573
)
58
Prieto
JH
Sampoli Benitez
BA
Melacini
G
Johnson
DA
Wood
MJ
Komives
EA
Dynamics of the fragment of thrombomodulin containing the fourth and fifth epidermal growth factor-like domains correlate with function.
Biochemistry
2005
, vol. 
44
 
4
(pg. 
1225
-
1233
)
59
Clarke
JH
Light
DR
Blasko
E
, et al. 
The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.
J Biol Chem
1993
, vol. 
268
 
9
(pg. 
6309
-
6315
)
60
Wood
MJ
Becvar
LA
Prieto
JH
Melacini
G
Komives
EA
NMR structures reveal how oxidation inactivates thrombomodulin.
Biochemistry
2003
, vol. 
42
 
41
(pg. 
11932
-
11942
)
61
Lentz
SR
Fernández
JA
Griffin
JH
, et al. 
Impaired anticoagulant response to infusion of thrombin in atherosclerotic monkeys associated with acquired defects in the protein C system.
Arterioscler Thromb Vasc Biol
1999
, vol. 
19
 
7
(pg. 
1744
-
1750
)
62
Lentz
SR
Miller
FJ
Jr
Piegors
DJ
, et al. 
Anticoagulant responses to thrombin are enhanced during regression of atherosclerosis in monkeys.
Circulation
2002
, vol. 
106
 
7
(pg. 
842
-
846
)
63
Wilson
KM
McCaw
RB
Leo
L
, et al. 
Prothrombotic effects of hyperhomocysteinemia and hypercholesterolemia in ApoE-deficient mice.
Arterioscler Thromb Vasc Biol
2007
, vol. 
27
 
1
(pg. 
233
-
240
)
64
Raife
TJ
Dwyre
DM
Stevens
JW
, et al. 
Human thrombomodulin knock-in mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis.
Arterioscler Thromb Vasc Biol
2011
, vol. 
31
 
11
(pg. 
2509
-
2517
)
65
Wilson
KM
Leo
L
Lentz
SR
Gene transfer of human ECSOD enhances protein C activation in hypercholesterolemic mice.
Arterioscler Thromb Vasc Biol
2009
, vol. 
29
 
7
pg. 
e8
 
66
Dayal
S
Gu
S
X.; Wilson, K.M., Hutchins, R.; Lentz, S.R. Endogenous superoxide dismutase protects from impaired generation of activated protein C and enhanced susceptibility to experimental thrombosis in mice.
Arterioscler Thromb Vasc Biol
2014
, vol. 
34
 
Suppl 1
pg. 
A34
 
67
Nalian
A
Iakhiaev
AV
Possible mechanisms contributing to oxidative inactivation of activated protein C: molecular dynamics study.
Thromb Haemost
2008
, vol. 
100
 
1
(pg. 
18
-
25
)
68
Denis
CV
Molecular and cellular biology of von Willebrand factor.
Int J Hematol
2002
, vol. 
75
 
1
(pg. 
3
-
8
)
69
Sadler
JE
New concepts in von Willebrand disease.
Annu Rev Med
2005
, vol. 
56
 (pg. 
173
-
191
)
70
Sadler
JE
Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura.
Blood
2008
, vol. 
112
 
1
(pg. 
11
-
18
)
71
George
JN
Nester
CM
Syndromes of thrombotic microangiopathy.
N Engl J Med
2014
, vol. 
371
 
19
(pg. 
1847
-
1848
)
72
Hanson
E
Jood
K
Nilsson
S
Blomstrand
C
Jern
C
Association between genetic variation at the ADAMTS13 locus and ischemic stroke.
J Thromb Haemost
2009
, vol. 
7
 
12
(pg. 
2147
-
2148
)
73
Miura
M
Kaikita
K
Matsukawa
M
, et al. 
Prognostic value of plasma von Willebrand factor-cleaving protease (ADAMTS13) antigen levels in patients with coronary artery disease.
Thromb Haemost
2010
, vol. 
103
 
3
(pg. 
623
-
629
)
74
Furlan
M
Robles
R
Lämmle
B
Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis.
Blood
1996
, vol. 
87
 
10
(pg. 
4223
-
4234
)
75
Chen
J
Fu
X
Wang
Y
, et al. 
Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13.
Blood
2010
, vol. 
115
 
3
(pg. 
706
-
712
)
76
Lancellotti
S
De Filippis
V
Pozzi
N
, et al. 
Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress.
Free Radic Biol Med
2010
, vol. 
48
 
3
(pg. 
446
-
456
)
77
Fu
X
Chen
J
Gallagher
R
Zheng
Y
Chung
DW
López
JA
Shear stress-induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region.
Blood
2011
, vol. 
118
 
19
(pg. 
5283
-
5291
)
78
Chen
J
Ling
M
Fu
X
López
JA
Chung
DW
Simultaneous exposure of sites in von Willebrand factor for glycoprotein Ib binding and ADAMTS13 cleavage: studies with ristocetin.
Arterioscler Thromb Vasc Biol
2012
, vol. 
32
 
11
(pg. 
2625
-
2630
)
79
De Filippis
V
Lancellotti
S
Maset
F
, et al. 
Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure.
Biochem J
2012
, vol. 
442
 
2
(pg. 
423
-
432
)
80
Wang
Y
Chen
J
Ling
M
López
JA
Chung
DW
Fu
X
Hypochlorous acid generated by neutrophils inactivates ADAMTS13: an oxidative mechanism for regulating ADAMTS13 proteolytic activity during inflammation.
J Biol Chem
2015
, vol. 
290
 
3
(pg. 
1422
-
1431
)
81
Raife
TJ
Cao
W
Atkinson
BS
, et al. 
Leukocyte proteases cleave von Willebrand factor at or near the ADAMTS13 cleavage site.
Blood
2009
, vol. 
114
 
8
(pg. 
1666
-
1674
)
82
Lancellotti
S
De Filippis
V
Pozzi
N
, et al. 
Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS-13.
J Thromb Haemost
2011
, vol. 
9
 
8
(pg. 
1620
-
1627
)
83
Taggart
C
Cervantes-Laurean
D
Kim
G
, et al. 
Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity.
J Biol Chem
2000
, vol. 
275
 
35
(pg. 
27258
-
27265
)
84
Weigandt
KM
White
N
Chung
D
, et al. 
Fibrin clot structure and mechanics associated with specific oxidation of methionine residues in fibrinogen.
Biophys J
2012
, vol. 
103
 
11
(pg. 
2399
-
2407
)
85
Burney
PR
White
N
Pfaendtner
J
Structural effects of methionine oxidation on isolated subdomains of human fibrin D and αC regions.
PLoS ONE
2014
, vol. 
9
 
1
pg. 
e86981
 
86
Stief
TW
Martín
E
Jimenez
J
Digón
J
Rodriguez
JM
Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin.
Thromb Res
1991
, vol. 
61
 
3
(pg. 
191
-
200
)
87
Lawrence
DA
Loskutoff
DJ
Inactivation of plasminogen activator inhibitor by oxidants.
Biochemistry
1986
, vol. 
25
 
21
(pg. 
6351
-
6355
)
88
Strandberg
L
Lawrence
DA
Johansson
LB
Ny
T
The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1′ methionine.
J Biol Chem
1991
, vol. 
266
 
21
(pg. 
13852
-
13858
)
89
Kornfelt
T
Persson
E
Palm
L
Oxidation of methionine residues in coagulation factor VIIa.
Arch Biochem Biophys
1999
, vol. 
363
 
1
(pg. 
43
-
54
)
90
Stief
TW
Aab
A
Heimburger
N
Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor.
Thromb Res
1988
, vol. 
49
 
6
(pg. 
581
-
589
)
91
Van Patten
SM
Hanson
E
Bernasconi
R
, et al. 
Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding.
J Biol Chem
1999
, vol. 
274
 
15
(pg. 
10268
-
10276
)
92
Sroussi
HY
Berline
J
Palefsky
JM
Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro.
J Leukoc Biol
2007
, vol. 
81
 
3
(pg. 
818
-
824
)
93
Hung
RJ
Spaeth
CS
Yesilyurt
HG
Terman
JR
SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.
Nat Cell Biol
2013
, vol. 
15
 
12
(pg. 
1445
-
1454
)
94
Kanayama
A
Inoue
J
Sugita-Konishi
Y
Shimizu
M
Miyamoto
Y
Oxidation of Ikappa Balpha at methionine 45 is one cause of taurine chloramine-induced inhibition of NF-kappa B activation.
J Biol Chem
2002
, vol. 
277
 
27
(pg. 
24049
-
24056
)
95
Midwinter
RG
Cheah
FC
Moskovitz
J
Vissers
MC
Winterbourn
CC
IkappaB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-kappaB activity by glycine chloramine through methionine oxidation.
Biochem J
2006
, vol. 
396
 
1
(pg. 
71
-
78
)
96
Nomura
T
Kamada
R
Ito
I
Chuman
Y
Shimohigashi
Y
Sakaguchi
K
Oxidation of methionine residue at hydrophobic core destabilizes p53 tetrameric structure.
Biopolymers
2009
, vol. 
91
 
1
(pg. 
78
-
84
)
97
Schieber
M
Chandel
NS
ROS function in redox signaling and oxidative stress.
Curr Biol
2014
, vol. 
24
 
10
(pg. 
R453
-
R462
)
98
Oien
DB
Canello
T
Gabizon
R
, et al. 
Detection of oxidized methionine in selected proteins, cellular extracts and blood serums by novel anti-methionine sulfoxide antibodies.
Arch Biochem Biophys
2009
, vol. 
485
 
1
(pg. 
35
-
40
)
99
Ghesquière
B
Gevaert
K
Proteomics methods to study methionine oxidation.
Mass Spectrom Rev
2014
, vol. 
33
 
2
(pg. 
147
-
156
)
100
Gevaert
K
Ghesquière
B
Staes
A
, et al. 
Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies.
Proteomics
2004
, vol. 
4
 
4
(pg. 
897
-
908
)
101
Seraglia
R
Sartore
G
Marin
R
, et al. 
An effective and rapid determination by MALDI/TOF/TOF of methionine sulphoxide content of ApoA-I in type 2 diabetic patients.
J Mass Spectrom
2013
, vol. 
48
 
1
(pg. 
105
-
110
)
102
Oggianu
L
Lancellotti
S
Pitocco
D
, et al. 
The oxidative modification of von Willebrand factor is associated with thrombotic angiopathies in diabetes mellitus.
PLoS ONE
2013
, vol. 
8
 
1
pg. 
e55396
 
103
Su
EJ
Geyer
M
Wahl
M
, et al. 
The thrombomodulin analog Solulin promotes reperfusion and reduces infarct volume in a thrombotic stroke model.
J Thromb Haemost
2011
, vol. 
9
 
6
(pg. 
1174
-
1182
)
104
Carnemolla
R
Greineder
CF
Chacko
AM
, et al. 
Platelet endothelial cell adhesion molecule targeted oxidant-resistant mutant thrombomodulin fusion protein with enhanced potency in vitro and in vivo.
J Pharmacol Exp Ther
2013
, vol. 
347
 
2
(pg. 
339
-
345
)
105
van Iersel
T
Stroissnig
H
Giesen
P
Wemer
J
Wilhelm-Ogunbiyi
K
Phase I study of Solulin, a novel recombinant soluble human thrombomodulin analogue.
Thromb Haemost
2011
, vol. 
105
 
2
(pg. 
302
-
312
)
106
Hood
ED
Chorny
M
Greineder
CF
S Alferiev
I
Levy
RJ
Muzykantov
VR
Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation.
Biomaterials
2014
, vol. 
35
 
11
(pg. 
3708
-
3715
)