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 (TLR). Platelets express a number of TLR, 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 (CAPs), an altered self-ligand generated in oxidative stress, on platelet function and thrombosis. Methods and Results In this study we show that CAPs represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that CAPs 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 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.

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 αβ and αβ Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by β-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for αβ- and αβ-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 αβ- and αβ-mediated migration/retention of macrophages during inflammation.

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 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 manuscript, 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 or TLRs, 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-MyD88-IRAK1/4-TRAF6, leading to integrin activation via the SFK-Syk-PLCγ2 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.

Specific oxidized phospholipids (oxPC CD36 ) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC CD36 by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC CD36 by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC CD36 is an sn-2 acyl group that incorporates a terminal ␥-hydroxy (or oxo)-␣,␤-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC CD36 , other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B. Specific oxidized phospholipids (oxPC CD36 )3 accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions and plasma in dyslipidemia (1, 2). They serve as high affinity ligands for scavenger receptors class B: CD36 and SR-BI (3, 4). Recognition of oxPC CD36 on the surface of cell membranes and lipoprotein particles by scavenger receptors class B plays an important role in several pathophysiological processes, including atherosclerosis and thrombosis. oxPC CD36 phospholipids mediate uptake of oxidized low density lipoprotein (oxLDL) by macrophages via CD36 and promote a pro-thrombotic state via platelet scavenger receptor CD36 (1, 2). oxPC CD36 phospholipids also prevent binding of high density lipoprotein by SR-BI because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC CD36 interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes (4). These data demonstrate that oxidative stress and accumulation of specific oxidized phospholipids may have a detrimental effect due to specific interaction with scavenger receptors class B. However, the exact molecular mechanism of the recognition of oxPC CD36 by scavenger receptors class B is poorly understood.Initial studies have demonstrated that the sn-2 acyl group of oxPC CD36 that incorporates a terminal ␥-hydroxy (or oxo)-␣,␤-unsaturated carbonyl is essential for high affinity binding to...

Rationale Oxidative stress is an important contributing factor in a number of human pathologies ranging from atherosclerosis to cancer progression; however, the mechanisms underlying tissue protection from oxidation products are poorly understood. Oxidation of membrane phospholipids, containing the polyunsaturated fatty acid DHA, results in the accumulation of an end product, 2-(ω-carboxyethyl)pyrrole (CEP), which was shown to have pro-angiogenic and pro-inflammatory functions. While CEP is continuously accumulated during chronic processes such as tumor progression and atherosclerosis, its levels during wound healing return to normal when the wound is healed, suggesting the existence of a specific clearance mechanism. Objective To identify the cellular and molecular mechanism for CEP clearance. Methods and Results Here we show that macrophages are able to bind, scavenge, and metabolize carboxyethylpyrrole derivatives of proteins but not structurally similar ethylpyrrole derivatives, demonstrating the high specificity of the process. F4/80hi and M2-skewed macrophages are much more efficient at CEP binding and scavenging compared to F4/80lo and M1-skewed macrophages. Depletion of macrophages leads to increased CEP accumulation in vivo. CEP binding and clearance are dependent on two receptors expressed by macrophages, CD36 and TLR2. While knockout of each individual receptor results in diminished CEP clearance, the lack of both receptors almost completely abrogates macrophages’ ability to scavenge CEP derivatives of proteins. Conclusions Our study demonstrates the mechanisms of recognition, scavenging, and clearance of pathophysiologically active products of lipid oxidation in vivo, thereby contributing to tissue protection against products of oxidative stress.