Detao Gao

Engagement of Platelet Toll-Like Receptor 9 by Novel Endogenous Ligands Promotes Platelet Hyperreactivity and Thrombosis

Rationale: A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance of altered-self ligands in circulation that can be recognized by Toll-like receptors (TLRs). Platelets express a number of TLRs, including TLR9; however, the role of TLR in platelet function and thrombosis is poorly understood. Objective: To investigate the biological activities of carboxy(alkylpyrrole) protein adducts, an altered-self ligand generated in oxidative stress, on platelet function and thrombosis. Methods and Results: In this study we show that carboxy(alkylpyrrole) protein adducts represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that carboxy(alkylpyrrole) protein adducts promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and it is Src kinase—dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. Conclusions: Our study demonstrates that platelet TLR9 is a functional platelet receptor that links oxidative stress, innate immunity, and thrombosis.

TLR2 Plays a Key Role in Platelet Hyperreactivity and Accelerated Thrombosis Associated with Hyperlipidemia

Rationale: Platelet hyperreactivity, which is common in many pathological conditions, is associated with increased atherothrombotic risk. The mechanisms leading to platelet hyperreactivity are complex and not yet fully understood. Objective: Platelet hyperreactivity and accelerated thrombosis, specifically in dyslipidemia, have been mechanistically linked to the accumulation in the circulation of a specific group of oxidized phospholipids (oxPCCD36) that are ligands for the platelet pattern recognition receptor CD36. In the current article, we tested whether the platelet innate immune system contributes to responses to oxPCCD36 and accelerated thrombosis observed in hyperlipidemia. Methods and Results: Using in vitro approaches, as well as platelets from mice with genetic deletion of MyD88 (myeloid differentiation factor 88) or TLRs (Toll-like receptors), we demonstrate that TLR2 and TLR6 are required for the activation of human and murine platelets by oxPCCD36. oxPCCD36 induce formation of CD36/TLR2/TLR6 complex in platelets and activate downstream signaling via TIRAP (Toll-interleukin 1 receptor domain containing adaptor protein)-MyD88-IRAK (interleukin-1 receptor–associated kinase)1/4-TRAF6 (TNF receptor–associated factor 6), leading to integrin activation via the SFK (Src family kinase)-Syk (spleen tyrosine kinase)-PLC&ggr;2 (phospholipase C&ggr;2) pathway. Intravital thrombosis studies using ApoE−/− mice with genetic deficiency of TLR2 or TLR6 have demonstrated that oxPCCD36 contribute to accelerated thrombosis specifically in the setting of hyperlipidemia. Conclusions: Our studies reveal that TLR2 plays a key role in platelet hyperreactivity and the prothrombotic state in the setting of hyperlipidemia by sensing a wide range of endogenous lipid peroxidation ligands and activating innate immune signaling cascade in platelets.

Analysis of Covalent Modifications of Proteins by Oxidized Phospholipids Using a Novel Method of Peptide Enrichment

Free radical-induced oxidation of phospholipids contributes significantly to pathologies associated with inflammation and oxidative stress. Detection of covalent interaction between oxidized phospholipids (oxPL) and proteins by LC-MS/MS could provide valuable information about the molecular mechanisms of oxPL effects. However, such studies are very limited because of significant challenges in detection of the comparatively low levels of oxPL-protein adducts in complex biological systems. Current approaches have several limitations, most important of which is the inability to detect protein modifications by naturally occurring oxPL. We now report, for the first time, an enrichment method that can be applied to the global analysis of protein adducts with various naturally occurring oxPL in relevant biological systems. This method exploits intrinsic properties of peptides modified by oxPL, allowing highly efficient enrichment of oxPL-modified peptides from biological samples. Very low levels of oxPL-protein adducts (<2 ppm) were detected using this enrichment method in combination with LC-MS/MS. We applied the method to several model systems, including oxidation of high density lipoprotein (HDL) and interaction of human platelets with a specific oxPL, and demonstrated its extremely high efficiency and productivity. We report multiple new modifications of apolipoproteins in HDL and proteins in human platelets.

Analysis of relationship between oxidized phospholipid structure and interaction with the class B scavenger receptors.

Recognition of specific oxidized phospholipids oxPCCD36 by scavenger receptors CD36 and SR-BI plays a critical role in several pathophysiological processes. The structural basis for the recognition of oxPCCD36 by CD36 and SR-BI is poorly understood. We describe here the design and synthesis of a series of model oxidized phospholipids having various functional groups at sn-1, sn-2, and sn-3 positions. Synthetic methodologies and experimental details for the preparation of specific examples of model oxidized phospholipids are presented. The correlation between their structure and their ability to serve as ligands for CD36 and SR-BI was determined using competitive binding assay on cells overexpressing scavenger receptors, direct binding assay to scavenger receptors expressed as GST-fusion proteins, and cholesterol ester synthesis assay using mouse peritoneal macrophages.

Oxidative modifications of extracellular matrix promote the second wave of inflammation via β2 integrins

Early stages of inflammation are characterized by extensive oxidative insult by recruited and activated neutrophils. Secretion of peroxidases, including the main enzyme, myeloperoxidase, leads to the generation of reactive oxygen species. We show that this oxidative insult leads to polyunsaturated fatty acid (eg, docosahexaenoate), oxidation, and accumulation of its product 2-(ω-carboxyethyl)pyrrole (CEP), which, in turn, is capable of protein modifications. In vivo CEP is generated predominantly at the inflammatory sites in macrophage-rich areas. During thioglycollate-induced inflammation, neutralization of CEP adducts dramatically reduced macrophage accumulation in the inflamed peritoneal cavity while exhibiting no effect on the early recruitment of neutrophils, suggesting a role in the second wave of inflammation. CEP modifications were abundantly deposited along the path of neutrophils migrating through the 3-dimensional fibrin matrix in vitro. Neutrophil-mediated CEP formation was markedly inhibited by the myeloperoxidase inhibitor, 4-ABH, and significantly reduced in myeloperoxidase-deficient mice. On macrophages, CEP adducts were recognized by cell adhesion receptors, integrin αMβ2 and αDβ2 Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by β2-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for αMβ2- and αDβ2-dependent macrophage migration. The presence of a carboxyl group rather than a pyrrole moiety on these adducts, resembling characteristics of bacterial and/or immobilized ligands, is critical for recognition by macrophages. Therefore, specific oxidation-dependent modification of extracellular matrix, aided by neutrophils, promotes subsequent αMβ2- and αDβ2-mediated migration/retention of macrophages during inflammation.

Characterization of covalent modifications of HDL apoproteins by endogenous oxidized phospholipids

ABSTRACT High density lipoprotein (HDL) is cardioprotective, unless it is pathologically modified under oxidative stress. Covalent modifications of lipid‐free apoA‐I, the most abundant apoprotein in HDL, compromise its atheroprotective functions. HDL is enriched in oxidized phospholipids (oxPL) in vivo in oxidative stress. Furthermore, oxidized phospholipids can covalently modify HDL apoproteins. We have now carried out a systematic analysis of modifications of HDL apoproteins by endogenous oxPL. Human HDL or plasma were oxidized using a physiologically relevant MPO−H2O2−NO2− system or AIPH, or were exposed to synthetic oxPL. Protein adduction by oxPL was assessed using LC‐MS/MS and MALDI‐TOF MS. The pattern of HDL apoprotein modification by oxPL was independent of the oxidation systems used. ApoA‐I and apoA‐II were the major modification targets. OxPL with a &ggr;‐hydroxy (or oxo)‐alkenal were mostly responsible for modifications, and the Michael adduct was the most abundant adduct. Histidines and lysines in helices 5–8 of apoA‐I were highly susceptible to oxPL modifications, while lysines in helices 1, 2, 4 and 10 were resistant to modification by oxPL. In plasma exposed to oxidation or synthetic oxPL, oxPL modification was highly selective, and four histidines (H155, H162, H193 and H199) in helices 6–8 of apoA‐I were the main modification target. H710 and H3613 in apoB‐100 of LDL and K190 of human serum albumin were also modified by oxPL but to a lesser extent. Comparison of oxPL with short chain aldehyde HNE using MALDI‐TOF MS demonstrated high selectivity and efficiency of oxPL in the modification of HDL apoproteins. These findings provide a novel insight into a potential mechanism of the loss of atheroprotective function of HDL in conditions of oxidative stress. Graphic abstract Figure. No caption available. HighlightsModification sites on HDL apoproteins, adduct types and oxPL involved are identified.Modification of HDL apoproteins by oxPL is fast, efficient and highly selective.The pattern of HDL apoprotein modification is independent of the HDL oxidation method.HDL apoA‐I residues in helices 5–8 are highly susceptible to modification by oxPL.Michael adduct of &ggr;‐hydroxy/oxo alkenal PL and apoA‐I histidine is prevalent in oxHDL.