Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

Abstract Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

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Protein Cysteine Sulfenylation By CD36-Dependent Reactive Oxygen Species Signaling Promotes Platelet Activation

Blood ◽

10.1182/blood-2019-122465 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2338-2338

Author(s):

Moua Yang ◽

Calvin Harberg ◽

Wenjing Chen ◽

Sarah L. Wynia-Smith ◽

Kate S. Carroll ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Platelet Activation ◽

Arterial Thrombosis ◽

Reactive Oxygen ◽

Src Family Kinases ◽

Dependent Manner ◽

Oxygen Species ◽

H2o2 Formation ◽

Clinically Significant ◽

Specific Oxidation

Arterial thrombosis in the setting of dyslipidemia produces clinically significant events, including myocardial infarction and stroke. Oxidized lipids in circulating lipoproteins (oxLDL) are a risk factor for atherothrombosis in dyslipidemia and are recognized by platelet scavenger receptor CD36. OxLDL binding to CD36 promotes platelet activation and thrombosis by generating intracellular reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). Downstream signaling events initiated by ROS in this setting are largely unknown. We hypothesize that H2O2 generated by CD36 signaling promotes oxidative cysteine modification of cellular regulators of arterial thrombosis. Platelets isolated from healthy human donors were stimulated with oxLDL and H2O2 levels were measured by HPLC fluorescence quantification of 7-hydroxycoumarin generated by H2O2-specific oxidation of the coumarin boronic acid probe. OxLDL induced time- and concentration-dependent H2O2 formation (up to 80 ±13% greater than unstimulated platelets), while control "native" LDL showed negligible H2O2 formation. Pre-treatment of platelets with a CD36-blocking antibody or with PEG-catalase, an enzyme that degrades H2O2, normalized H2O2 formation by oxLDL to levels observed in unstimulated platelets. To mimic pathophysiologic conditions platelets were sensitized with oxLDL before stimulating with classic activators: adenosine diphosphate (ADP) and collagen-related peptide (CRP). Sensitization synergistically increased H2O2 formation through collagen receptors, but not ADP receptors. Since H2O2 can induce transient protein cysteine sulfenylation as a posttranslational oxidative modification, we assayed platelet cysteine sulfenylation using an alkyne-containing benzothiazine-based probe, BTD. BTD was loaded into platelets prior to exposure to oxLDL and detected by click-chemistry with biotin-PEG-azide followed by detection by western blot with streptavidin. OxLDL, but not LDL, induced two-fold increase in sulfenylation in the platelet proteome within 15 minutes. Src family kinases (SFK) are known to be recruited to and activated by CD36 in a ligand-dependent manner and SFK were also previously shown to be sulfenylated by H2O2 at Cys185 and Cys277, maintaining the kinase in an activated state. To determine if SFK cysteines are oxidatively modified by CD36 signaling, lysates from BTD-loaded, oxLDL-stimulated platelets were biotinylated as above, immunoprecipitated with anti-Src antibody, and then analyzed by immunoblot to detect BTD incorporation. SFK were sulfenylated in a time-dependent manner and this was blocked by a CD36 blocking monoclonal antibody or by treatment with PEG-catalase. We then showed by immunoblot that SFK cysteine sulfenylation by CD36/H2O2 was associated with phosphorylation at Y416, a signature activation motif in the kinase domain. OxLDL induced Src Y416 phosphorylation was prevented by PEG-catalase. Platelet aggregometry was used to determine the functional impact of protein sulfenylation and revealed that oxLDL-induced platelet aggregation was inhibited in a concentration-dependent manner (IC50 2.02 mM) by modifying sites of sulfenylation with BTD. BTD had no impact on aggregation induced by low- or high-concentration of ADP or CRP, suggesting these physiologic activators alone do not generate sufficient H2O2 to promote cysteine sulfenylation. OxLDL/CD36 induced procoagulant phosphatidylserine externalization, assessed by flow cytometry using fluorophore-tagged annexin V, was also prevented by BTD. In conclusion, our studies show that platelet CD36 signaling in response to oxLDL induces intracellular H2O2 generation which in turn induces cysteine sulfenylation of Src family kinases to promote platelet activation. Cysteine sulfenylation by CD36 could potentially be targeted to reduce the risk for clinically significant thrombotic events while maintaining hemostasis. Disclosures No relevant conflicts of interest to declare.

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Abstract 54: Differential Regulation of NADPH-oxidases 1 and 2 and a Common Syk/PLC/Calcium-dependent ROS Signaling Pathway Mediating Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.54 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Zheng Xu

Keyword(s):

Reactive Oxygen Species ◽

Platelet Aggregation ◽

Signaling Pathway ◽

Platelet Activation ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Reactive Oxygen ◽

Differential Regulation ◽

Ros Generation ◽

Oxygen Species ◽

Activation Pathways

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. This study investigates how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) mediates different platelet activation pathways. Approach and Results: ROS generation in different platelet activation pathways are mediated differentially by NOX1 and NOX2. NOX1 -/y platelets showed no defects in platelet aggregation and secretion induced by glycoprotein (GP) VI agonists, collagen-related peptide (CRP), but were partially defective in platelet aggregation and secretion induced by low doses of agonists of G protein coupled receptor (GPCR), thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619. In contrast, NOX2 -/- platelets showed significantly defective platelet aggregation and secretion induced by CRP, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1 -/- platelets stimulated with thrombin, but not CRP, whereas NOX2 -/- platelets were defective ROS generation induced by CRP or thrombin. These differential effects of NOX1 and NOX2 is likely due to upstream differential regulation of these different enzymes, as thrombin-stimulated NOX1-/y platelets and CRP-stimulated NOX2-/- platelets similarly showed defective activation of tyrosine kinase Syk, its downstream target phospholipase Cγ (PLCγ) and calcium mobilization, which is mediated by PLC. Furthermore, mitogen-activated protein kinase pathways, which is another important platelet activation pathway was not significantly affected in either NOX1-/y or NOX2-/- platelets. Finally, NOX-/- platelets is defective in mediating arteriolar thrombosis in vivo, although minimally affected tail bleeding time. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways. The differential roles of these enzyme are due to differential upstream regulation. Both NOX isoforms mediates platelet activation via a common ROS-dependent activation Src-PLC-calcium signaling pathway.

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Reactive Oxygen Species and Platelet Activation in Reperfusion Injury

Circulation ◽

10.1161/01.cir.95.4.787 ◽

1997 ◽

Vol 95 (4) ◽

pp. 787-789 ◽

Cited By ~ 41

Author(s):

Rosemarie C. Forde ◽

Desmond J. Fitzgerald

Keyword(s):

Reactive Oxygen Species ◽

Reperfusion Injury ◽

Platelet Activation ◽

Reactive Oxygen ◽

Oxygen Species

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Hydrogen peroxide-responsive AIE probe for imaging-guided organelle targeting and photodynamic cancer cell ablation

Materials Chemistry Frontiers ◽

10.1039/d1qm00328c ◽

2021 ◽

Author(s):

Qian Wu ◽

Youmei Li ◽

Ying Li ◽

Dong Wang ◽

Ben Zhong Tang

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Cancer Cell ◽

Reactive Oxygen ◽

Living Cells ◽

Oxygen Species ◽

Cell Ablation ◽

Selective Monitoring ◽

Organelle Targeting

Hydrogen peroxide (H2O2), as one kind of key reactive oxygen species (ROS), is mainly produced endogenously primarily in the mitochondria. The selective monitoring of H2O2 in living cells is of...

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Upconversion-based nanosystems for fluorescence sensing of pH and H2O2

Nanoscale Advances ◽

10.1039/d0na01045f ◽

2021 ◽

Author(s):

Chunning Sun ◽

Michael Gradzielski

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Excitation Energy ◽

Reactive Oxygen ◽

Oxygen Species ◽

Fluorescence Sensing ◽

Living Organisms

Hydrogen peroxide (H2O2), a key reactive oxygen species, plays an important role in living organisms, industrial and environmental fields. Here, a non-contact upconversion nanosystem based on the excitation energy attenuation...

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Hydrogen peroxide and salinity stress act synergistically to enhance lipids production in microalga by regulating reactive oxygen species and calcium

Algal Research ◽

10.1016/j.algal.2020.102017 ◽

2020 ◽

pp. 102017

Author(s):

Tengsheng Qiao ◽

Yongteng Zhao ◽

Du-bo Zhong ◽

Xuya Yu

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Salinity Stress ◽

Reactive Oxygen ◽

Oxygen Species ◽

Lipids Production

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Novel Antioxidant Properties of Doxycycline

International Journal of Molecular Sciences ◽

10.3390/ijms19124078 ◽

2018 ◽

Vol 19 (12) ◽

pp. 4078 ◽

Cited By ~ 9

Author(s):

Dahn Clemens ◽

Michael Duryee ◽

Cleofes Sarmiento ◽

Andrew Chiou ◽

Jacob McGowan ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Chronic Inflammation ◽

Antioxidant Properties ◽

Antibacterial Properties ◽

Reactive Oxygen ◽

Protein Adducts ◽

Oxygen Species ◽

Free System

Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.

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