Engagement of Platelet Toll-Like Receptor 9 by Novel Endogenous Ligands Promotes Platelet Hyperreactivity and ThrombosisNovelty and Significance
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.
Pathophysiological conditions associated with oxidative stress, such as dyslipidemia, diabetes, and acute or chronic infections, are frequently associated with the prothrombotic state in which increased platelet responses to agonists play a significant role. The clinical importance of increased platelet reactivity is supported by the observation that subjects with elevations in various measures of platelet reactivity are at an increased prospective risk for coronary events and death.1–3 A number of recent reports indicate that platelets may sense “pathological ligands” accumulating in circulation via a specific set of receptors and translate an activation signal into a prothrombotic state.4 These receptors may include pattern recognition receptors such as scavenger receptors and Toll-like receptors (TLRs). Although the role of platelet scavenger receptors in sensing oxidative stress-generated ligands has been recently highlighted,5 studies on the function of platelet TLR are only starting to emerge. TLRs recognize a number of pathogen-associated molecular patterns and damage-associated molecular patterns of host origin that include altered self-ligands.6 The recognition contributes to innate immune defense and a state of noninfectious inflammation, respectively. TLR1, TLR2, TLR4, TLR6, TLR8, and TLR9 are expressed in megakaryocytes and in platelets; however, their role in platelet function is poorly understood.7–9 It is believed that sensing various pathogens via different sets of receptors leads to the production of different profiles of proinflammatory cytokines by platelets contributing to the innate immunity or modulates platelet function and directly contributes to cardiovascular pathology.10,11 Recent studies have shown that the presence of pathogen-associated ligands for TLR2 and TLR4 (and their coreceptors TLR1 and TLR6) in circulation can induce a thromboinflammatory response in platelets.12,13 Although evidences are accumulating that altered self-ligands can induce signaling via TLR in cells, such as endothelial and macrophages, there is a lack of data on the involvement of platelet TLR in the recognition of such ligands and translation of the signal into pathological responses. The role of platelet TLR9 is unknown.
Free-radical oxidation of targets containing polyunsaturated fatty acids generates a host of oxidative products, including the hydroxy-ω-oxoalkenoic acids (Online Scheme I). Hydrolysis by phospholipase A2 followed by the reaction of the resulting unesterified hydroxy-ω-oxoalkenoic acid with proteins, or the reaction of esterified hydroxy-ω-oxoalkenoic acid with proteins followed by hydrolysis, gives rise to a family of carboxy (alkylpyrrole) protein adducts (CAPs).14,15 CAPs belong to the so-called altered self class of molecules. CAPs immunoreactivity was detected in atherosclerotic plaques, in tumors, and in healing wounds. Plasma levels of CAPs are significantly elevated in diabetes, atherosclerosis, renal failure, and degenerative diseases.14–18 When incorporated into sn-2 position of oxidized phospholipids, hydroxy-ω-oxoalkenoic acid is recognized by scavenger receptors CD36 and SR-BI that belong to pattern recognition receptors but are best known for their role in lipid metabolism.19,20 However, CAPs have been previously shown to be ligands for a member of TLR, another family of pattern recognition receptors.16
In this study, the effects of CAPs on platelets and molecular mechanisms of effects were examined. We found that CAPs represent a novel and unconventional ligand for TLR9. Moreover, CAPs can promote platelet activation and aggregation in vitro and accelerate thrombosis in vivo in a TLR9/myeloid differentiation factor 88 (MyD88)-dependent manner. Thus, our findings suggest that TLR9 on platelets is a functional receptor that links oxidative stress, innate immunity, and thrombosis.
Detailed Methods are available in the Online Data Supplement.
Preparation of 2-(ω-Carboxyalkyl)pyrrole Protein Adducts and Anti-CEP Antibodies
The 2-(ω-carboxypropyl)pyrrole (CPP) bovine serum albumin adduct (CPP-BSA), 2-(ω-carboxyethyl)pyrrole (CEP) human serum albumin adduct (CEP-HSA), and other CAPs were generated by Paal-Knorr condensation reaction with γ-dicarbonyl compounds as described.15,21 The polyclonal anti-CEP antibody was generated in a Pasteurella-free New Zealand White rabbit by subcutaneous inoculation of CEP-keyhole limpet hemocyanin along with complete Freund adjuvant, and the IgG fraction of anti-CEP antibodies was purified using immobilized protein G.15
Isolation of Platelets
Platelet-rich plasma and gel-filtered platelets were isolated as described.5 Platelet counts were assessed by a Cellometer Auto-M10 (Nexcelom Bioscience).
Platelet aggregation was monitored using a Chronolog Model 560VS aggregometer with AGGRRO/LINK version 5.1.9 software at a stirring speed of 1000 rpm. Aliquots (200 μL) of platelet rich plasma were placed in cuvettes containing magnetic stirrer bars, warmed at 37°C, and stirred for 90 s to obtain a stable baseline. Platelet concentration in platelet-rich plasma was adjusted to 2×108 platelets/mL using platelet-poor plasma of the same genotype.
Human and murine platelet suspensions (2.5×107/mL) were prepared by gel filtration in modified HEPES-Tyrode buffer. P-selectin expression and integrin αIIbβ3 activation were assessed as described previously.5 Data were acquired using a fluorescent activated cell sorter Calibur instrument (Becton Dickinson) and analyzed using the FlowJo 9.0.2 software (Tree Star).
Human TLR9 Activation Assay in HEK-Blue hTLR9 Cells
The HEK-Blue hTLR9 (InvivoGen, San Diego, CA) cells were cultured in a 96-well plate in the presence of indicated concentrations of agonists, and NF-κB-induced SEAP activity was assessed using QUANTI-Blue (InvivoGen) and a reading at OD 620 nm.
Surface Plasmon Resonance
Real-time protein-protein interactions were analyzed using a Biacore3000 (BIAcore AB; GE Healthcare). Functional-grade TLR9 peptides (Novus Biological) were bound to CM5 biosensor chips (Biacore) at pH 5.5. Analyte binding to the immobilized ligand was recorded by measuring the variation of the surface plasmon resonance angle, and the results were expressed in resonance units.
Intravital thrombosis study was performed using an acute carotid artery injury model as previously described.5
Values are expressed as means±SEM. The statistical significance was evaluated between 2 groups of data using 2-tailed unpaired Student t test. We used 2-tailed nonparametric Mann-Whitney U test for analyzing the results of “intravital carotid thrombosis” and “tail cut bleeding time” studies. P values less < 0.05 was considered as statistically significant.
2-(ω-Carboxyalkyl)pyrrole Protein Adducts Induce Platelet Activation
We first assessed the effects on platelets of CEP protein and CPP protein adducts of albumin as the most abundant target in plasma for modification by products of lipid peroxidation. In vitro studies demonstrated that these adducts induce significant integrin αIIbβ3 activation and P-selectin expression in human platelets (Figure 1A and 1B). In contrast, the sham-modified protein had no effect on platelets. To see whether the nature of the modified protein has any role in this effect, we studied adducts of a number of plasma proteins, including albumins from several species, IgG, as well as keyhole limpet hemocyanin, a protein that is phylogenetically distant from mammalian proteins. The 2-(ω-carboxyalkyl)pyrrole adducts of the tested proteins, but not native unmodified proteins, induced significant platelet activation responses (Figure 1C and 1D), indicating that the nature of the modified protein plays no critical role. To test whether this effect is specific to 2-(ω-carboxyalkyl)pyrrole, we used platelet activation assay to test multiple protein adducts formed by oxidized lipids, which resembled hydroxy-ω-oxoalkenoic acids but were not capable of forming pyrroles (Online Table I). None of these nonpyrrole protein adducts had the ability to activate platelets, suggesting that the effect is specific for 2-(ω-carboxyalkyl)pyrrole modification of the proteins. We then tested whether the number of modification per protein plays a role in the effect of CAPs. CAPs with an increasing molar ratio of pyrrole per mole of protein were synthesized and tested in a platelet integrin αIIbβ3 activation assay. We observed a direct correlation between the extent of protein modification by CAPs and the capacity to induce activation of human platelets (Online Figure I).
CAPs Activate Platelets Via Toll-Like Receptors
CAPs belong to altered self-ligands that are commonly recognized by pattern recognition receptors.5,14,16 Several pattern recognition receptors are expressed in platelets, including class B scavenger receptors CD36 and SR-BI.4 Correspondingly, effects of CAPs were assessed using platelets from wild-type (WT) mice, CD36-deficient (CD36−/−) mice, and SR-BI-deficient (SR-BI−/−) mice. Platelets isolated from WT mice were activated by CAPs similarly to human platelets (Figure 2A). Activation of platelets of CD36−/− and SR-BI−/− mice was comparable to that of WT platelets (Figure 2B and 2C), ruling out significant involvement of class B scavenger receptors in platelet activation by the CAPs. Platelets also express a number of TLR (Online Figure IIA) that can recognize various pathogen-associated molecular patterns and altered self-ligands.22 The adaptor protein MyD88 is a common mediator of TLR signaling in cells and is present in platelets (Online Figure IIA). Therefore, to test the general involvement of TLR in CAPs-induced platelet activation, we first used platelets from MyD88−/− mice. CAPs failed to induce platelet activation in MyD88−/− platelets, demonstrating an absolute requirement for functional TLR-MyD88 signaling. This requirement was specific for CAPs because integrin αIIbβ3 activation response of MyD88−/− platelets to the physiological agonists such as ADP (Figure 2D) and thrombin (not shown) was similar to that of WT platelets.
CAPs Induce Platelet Activation in a TLR9-Dependent Manner
We next tested the involvement of TLR2, TLR4, TLR6, and TLR9, known to be expressed in platelets,7,9 in platelet responses to CAPs. We used Fab fragments of anti-human TLR2, TLR4, TLR6, and TLR9 antibodies or the chemical inhibitor of TLR4 (CLI 095, data not shown) to block a specific receptor. Unexpectedly, anti-human TLR9 Fab inhibited CAPs-induced platelet activation, whereas no significant effects of Fab fragments of TLR2, TLR4, and TLR6 were observed (Figure 3A, 3D, and 3F). The effect of the anti-TLR9 Fab was specific because it did not affect platelet activation by physiological agonists, such as thrombin or ADP (Figure 3C). TLR9 expression on human platelets has been previously reported.7,8 Using reverse transcriptase polymerase chain reaction and Western blot analyses, we detected the expression of TLR9 mRNA and protein in purified murine and human platelets, in the human megakaryocyte cell line Meg-01, and in Meg-01-derived platelet-like particles (Online Figure IIA). Immunofluorescence microscopy revealed granular staining of permeabilized human and murine platelets with anti-TLR9 antibody and lack of staining of platelets from TLR9-deficient mice (Online Figure IIB). Low levels of TLR9 expression were detected on nonpermeabilized resting platelets; however, platelet activation by physiological agonists resulted in a significant increase of TLR9 expression in platelets (Online Figure IIC). To further demonstrate the specific involvement of TLR9 in response to CAPs, we used platelets from TLR9-deficient mice. Response of platelets from TLR9−/− mice to CAPs was negligible as compared with that of WT platelets (Figure 3G and 3J, Online Figure III), demonstrating that TLR9 is a major mediator of the effects of CAPs on platelets. As an additional control, we tested the effects of CAPs using platelets from TLR2−/− and TLR6−/− mice and observed no significant effect of respective TLRs on integrin αIIbβ3 activation by CAPs (Online Figure IV).
CAPs Are Novel and Unconventional Ligands for TLR9
To demonstrate that CAPs represent new noncanonical ligands for TLR9, we used several approaches. We first studied direct interaction of CAPs with TLR9 by surface plasmon resonance using human TLR9 immobilized on the surface of a CM5 biosensor chip. As shown in Figure 4A, CAPs (CEP-BSA) interact with the immobilized TLR9 in a concentration-dependent manner at high binding affinity (KA = 8.53 × 105) and low dissociation (KDA = 1.17 × 10–6) constants. Interaction increased with increase in the extent of protein modification by CAPs, ie, the number of pyrroles per protein molecule (Figure 4B). Pretreatment of immobilized TLR9 with anti-TLR9 antibody completely blocked the binding of CAPs to a TLR9-coated surface (Online Figure VA). To test whether TLR9 binding is specific for 2-(ω-carboxyalkyl)pyrrole modification, we used surface plasmon resonance assay to test control protein adducts formed by oxidized lipids, which resembled hydroxy-ω-oxoalkenoic acids but are not capable of forming a pyrrole. We observed very weak and completely reversible binding of control adducts in comparison with that of CAPs (Online Figure VB, data for OHdiA-BSA are shown), suggesting that the effect is specific for 2-(ω-carboxyalkyl)pyrrole modification of the proteins. To further demonstrate direct interaction of CAPs and TLR9 in platelets, we incubated platelet lysate with CAPs and performed coimmunoprecipitation assay as described in the Methods section. TLR9 was specifically immunoprecipitated with CAPs, demonstrating the direct interaction of platelet TLR9 and CAPs (Figure 4C). To demonstrate that CAPs can induce cellular responses via TLR9, even though this receptor is usually localized intracellularly, we used an alternative system, the HEK-Blue hTLR9 cell line that expresses the human TLR9 (Figure 4D) and an NF-κB-inducible reporter gene. CAPs induced strong, concentration-dependent, and TLR9-mediated signaling in this cell line. The magnitude of the response was comparable with that of established TLR9-specific ligand unmethylated CpG oligodeoxynucleotide (ODN2006) (Figure 4E and 4F). Interestingly, although CAPs colocalize with platelet TLR9, we observed a lack of colocalization with the early endosomal marker Rab4 in platelets and partial colocalization with the late endosomal marker Rab9 (Online Figure VIA–C). These data are consistent with the recent finding of TLR9 localization to a novel electron-dense tubular system–related compartment.23 In nucleated cells, TLR9 undergoes proteolytic cleavage after endosomal acidification, a step required for TLR9 signaling. We observed that an inhibitor of endosomal acidification chloroquine significantly inhibited activation of platelets by CAPs (Online Figure VID and VIE) but not by physiological platelet agonists ADP, thrombin, or convulxin, suggesting that the key event leading to activation via TLR9 is similar in platelets and nucleated cells. Taken together, these results demonstrate that CAPs are new and unconventional ligands for TLR9 that could reach the endosomal TLR9, initiating the downstream signaling cascade.
CAPs Induce Platelet Aggregation and Promote Platelet Hypereactivity In Vitro
We next tested whether CAPs can induce platelet aggregation in vitro. We observed that only protein with a high degree of modification (≥5 pyrroles per molecule of protein) can induce significant platelet aggregation (Figure 5A). Because TLR9 in platelets is expressed intracellularly in specialized granules, and because we observed that TLR9 expression can be induced by physiological agonists, we tested effects of proteins with level modification of < 5 pyrroles per molecule on platelet activation and aggregation using platelets primed by either suboptimal concentrations of thrombin receptor activating peptide (TRAP) or weak physiological agonist ADP. Threshold concentrations of TRAP and ADP induced only reversible or no aggregation and activation responses (Figure 5B, 5E, and 5H). We observed irreversible and significantly accelerated platelet aggregation in TRAP and ADP-primed human (Figure 5B and 5C) and murine (Figure 5C and 5F) platelets in response to proteins with lower-level modification by CAPs. Correspondingly, although CAPs with lower level of modification induced only modest activation of platelets, we observed a strong increase in P-selectin expression (Figure 5D) and integrin αIIbβ3 activation (Figure 5G) when platelets were primed with physiological agonist. This accelerated activation response was TLR9-dependent because preincubation of platelet-rich plasma with anti-human TLR9 Fab significantly reduced platelet aggregation induced by CAPs in ADP-primed platelets (Figure 5H and 5I). Taken together, these data suggest that presence of carboxyalkylpyrrole protein adducts in vivo may promote platelet hyperactivity and aggregation response, especially when physiological platelet agonists are present.
CAPs Accelerate Thrombosis In Vivo in a MyD88-Dependent and TLR9-Dependent Manner
To test whether the presence of CAPs in circulation accelerates thrombosis in vivo and whether MyD88/TLR9 pathway is involved, we compared vessel occlusion times using a ferric chloride–induced carotid artery thrombosis model on sex matched and age-matched groups of MyD88−/−, TLR9−/−, and corresponding WT mice that received intravenous injections of either CAPs or sham-modified proteins before vascular damage. Occlusion times were similar in MyD88−/−, TLR9−/−, and corresponding WT mice receiving sham-modified proteins. In addition, tail cut bleeding time was similar in TLR9−/− and WT mice (Online Figure VII). These results and normal activation responses of platelets from MyD88−/− and TLR9−/− to physiological agonists in activation assay indicate that there are no major deficiencies in thrombosis in these 2 knockout mice. Time to complete thrombotic occlusion was significantly shortened in the WT mice that received CAPs as compared with the WT mice that received sham-modified protein. In contrast, the presence of CAPs in circulation of MyD88−/− and TLR9−/− mice had no significant effect on the occlusion times (Figure 6A and 6B). These results demonstrate that the presence of CAPs in vivo can accelerate thrombosis. They also demonstrate that TLR9 modulates thrombosis in vivo when specific ligands for TLR9 are present.
We then tested whether CAPs are present in hyperlipidemic apolipoprotein E (ApoE)−/− mice. Our data show that after intravenous injection, CAPs are rapidly removed from circulation (Online Figure VIII). Nevertheless, concentration of CAPs (data for CEP adduct are shown) was increased in ApoE−/− mice fed chow diet as compared with WT mice (Figure 6C). Western diet feeding led to a significant accumulation of CAPs in the plasma of ApoE−/− mice (Figure 6C). Platelets are capable of binding CAPs; therefore, we tested whether platelets can accumulate CAPs in vivo. We found a dramatic increase in platelet-associated CAPs in ApoE−/− mice fed a Western-type diet (Figure 6D and 6E). Immunostaining of the atherosclerotic plaques of aortic root in hyperlipidemic ApoE−/− mice also revealed the presence of CEP in atherosclerotic plaque, but not in surrounding tissue (Figure 6F), indicating that CAPs can also accumulate locally in hyperlipidemia. Taken together, these data confirm that CAPs accumulate in vivo in oxidative stress and demonstrate that presence of CAPs in circulation can modify platelet responses to physiological agonists and thrombosis.
CAPs Induce TLR9/MyD88/IRAK1 Signaling and Require PI3K and Src Kinases for Platelet Activation
Platelets express many components of a TLR signaling pathway downstream of MyD88,24 including the interleukin-1 receptor-associated kinase (IRAK)4 (S. Panigrahi and E. Podrez, unpublished data). IRAK1 is one of the key mediators of the TLR signaling pathway downstream of MyD88 and IRAK4. CAPs alone and CAPs in ADP-primed human platelets induced phosphorylation of IRAK1 (Figure 7A). The connection between TLR9/MyD88/IRAK1 activation and platelet integrin activation is not known. MyD88 signaling may involve cGMP-dependent protein kinase. However, we found no effect of a PKG inhibitor DT2-oligopeptide in a concentration range of 50 to 500 nmol/L on CAP-induced platelet activation (Online Figure IX). The PI3K/AKT pathway plays a key role in platelet activation,25–27 and it has been linked to the TLR9 signaling in other cell types.27–29 We found that in human platelets, CAPs could specifically induce AKT (Ser473) phosphorylation (Figure 7B). PI3K/AKT kinase inhibitor Ly294-002, Syc kinase inhibitor Bay61, Src kinase inhibitor PP2, but not the respective control PP3, also specifically blocked AKT1 phosphorylation in human platelets induced by CAPs at very low concentrations (Figure 7C). Furthermore, CAPs-induced P-selectin expression and integrin-αIIbβ3 activation of human platelets could be almost completely blocked by the Src kinase inhibitor PP2 (Figure 7D). Three AKT isoforms are expressed in platelets. Deletions of specific AKT isoforms in the mouse suggest that they play distinct and redundant roles in platelet responses to physiological agonists.26,30,31 We observed that CAP-induced platelet activation was significantly reduced in AKT1 and in AKT2, but not in AKT3-deficient platelets (Figure 7E), suggesting that, at least in murine platelets, AKT1 and AKT2 are the major isoforms involved in signaling events downstream of TLR9 and MyD88. Taken together, these findings demonstrate the role of PI3K/AKT and Src family kinases in platelet signaling induced by new unconventional ligands via TLR9/MyD88.
In this study, we have made several important observations. We have found, for the first time, that TLR9, a receptor previously implicated only in immune cell responses to bacterial DNA, is a functional platelet receptor capable of modulating platelet function and thrombosis. Furthermore, although unmethylated CpG sequences of bacterial DNA are the only known ligands for TLR9, our study identifies 2-(ω-carboxyalkyl)pyrrole protein adducts, a product of phospholipid oxidation generated in oxidative stress, as a novel noncanonical ligand for TLR9, which induces signaling events downstream of TLR9/MyD88 and promotes thrombosis.
The CAPs are present in circulation in a number of pathological conditions associated with oxidative stress, such as atherosclerosis, end-stage renal disease, and age-related macular degeneration.14,32,33 In the extracellular matrix of humans and animals, CAPs accumulate with age.33 We also detected increased levels of CAPs in circulation and in the atherosclerotic plaques in hyperlipidemic ApoE−/− mice. We observed significant accumulation of CAPs in platelets in hyperlipidemia, suggesting that effects of CAPs can be disproportional to circulating levels.
One pathway of CAPs formation is via hydrolysis of oxidized phospholipids containing hydroxy-ω-oxoalkenoic acid by PLA2. Unesterified hydroxy-ω-oxoalkenoic acids are chemically active and readily modify proteins, thus forming CAPs. When incorporated in the sn-2 position of oxidized phospholipids, hydroxy-ω-oxoalkenoic acids are recognized by scavenger receptor CD36 on platelets and contribute to platelet hyperreactivity in hyperlipidemia.5 Our finding demonstrates that the release of hydroxy-ω-oxoalkenoic acids from oxidized phospholipids also generates a product that promotes platelet activation, but via a different receptor and a different signaling pathway.
We demonstrated that CAPs can activate both platelets and HEK-293 cells that overexpress TLR9. In platelets, TLR9 is found in a recently described new electron-dense tubular system–related compartment.23 How CAPs initially interact with TLR9 is not clear. It is possible that a small amount of TLR9 found on the resting platelet surface is responsible for this initial response. We observed that CAPs can induce an expression of TLR9 on platelets similar to thrombin, thus promoting its own binding to platelets. Alternatively, CAPs may enter the platelets via endocytosis or pinocytosis known to be active in platelets34 with subsequent activation of TLR9. Although the early steps of CAPs interaction with TLR9 are not clear, our finding of inhibition of CAPs-induced activation by chloroquine suggests that the later steps are similar in platelets and nucleated cells.
Because proteins modified by CAPs are rapidly removed from circulation, it is not surprising that CAPs concentration in murine plasma is low. Nevertheless, this concentration is increased in conditions of mild oxidative stress present in ApoE−/− mice fed chow diet and is further increased in conditions of severe oxidative stress when ApoE−/− mice are fed a Western-type diet. We have previously shown that factors such as specific oxidized phospholipids that are present in circulation in hyperlipidemic ApoE−/− mice can directly activate platelets via receptors expressed on the surface of resting platelets.5 Although we did not show in this study that endogenously produced CAPs contribute to platelet activation and thrombosis, our multiple in vitro and in vivo data suggest that CAPs are likely to potentiate in vivo platelet activation, aggregation, and thrombosis induced by other agonists.
Ligand binding induces TLR9 dimerization and subsequent recruitment of adaptor protein MyD88 via cytoplasmic Toll/interleukin-1 receptor domains, leading to an association with the interleukin-1 receptor-associated kinase (IRAK) via the death domains.35 Platelets are shown to have several components of signaling pathway downstream of TLR, including MyD88, IRAK-1, and others.24 We observed that CAPs induce IRAK1 phosphorylation, suggesting that this pathway contributes to platelet activation. However, the molecular mechanism that links the TLR9/MyD88 pathway on one side and platelet integrin-αIIbβ activation and accelerated aggregation response on the other side needs further investigation. The PI3K/AKT pathway plays a key role in platelet activation25,26 and is a downstream signaling cascade that has been linked to the TLR9 in other cell types.27–29 Platelets contain both p110 catalytic and p85 regulatory subunits of PI3K involved in the inside-out signaling,25 and MyD88 has a binding motif for the p85 that can make a functional association in lipopolysaccharides-treated macrophages.36 We found that CAPs are a strong inducer of AKT phosphorylation and that the specific inhibition of the PI3K/AKT kinase pathway can partially block CAPs-induced activation of human platelets. Moreover, response to CAPs was notably reduced in murine AKT1-deficient and AKT2-deficient platelets. We also have found that the inhibition of the Src family kinases prevented platelet activation by CAPs and AKT phosphorylation, demonstrating critical involvement of Src family kinases in response to CAPs. TLR9-dependent activation of the Src family kinase, Lyn, has been shown in other cell types, but not in platelets.37,38 Whether Lyn is involved in signaling pathway induced by CAPs needs to be established.
Our finding that TLR9 is a receptor for such disparate ligands as unmethylated CpG oligonucleotide and CAPs is surprising, but it is not unparalleled. TLRs are known for their promiscuity. For example, a recent report showed that TLR4 could functionally bind to a nonconventional TLR ligand Ni2+.39 Our study demonstrates that TLR9 activation also is not restricted to a specific interaction with its classic ligand, but it has the additional function of sensing of a specific group of lipid peroxidation end products. Thus, TLR9 joins a growing number of pattern recognition receptors that modulate cell function in response to bacterial ligands and oxidative stress–derived ligands, including the scavenger receptor CD36, TLR2, TLR4, and TLR6.40–43 In endothelial cells, hydroxy-ω-oxoalkenoic acids associated with protein are recognized by TLR2 without the involvement of scavenger receptors.16 Surprisingly, we did not find a contribution of TLR2 to platelet activation by CAPs, even though platelets do express low levels of functional TLR2. It is not clear why TLR2 does not apparently participate in CAPs-induced platelet activation, but it has been shown that even such a specific and potent TLR2 ligand as Pam3CSK4 can induce platelet activation only at very high concentrations.12,44 Thus, a possibility is that the TLR9/MyD88 pathway in platelets is more sensitive to this type of ligand. Another possibility is that microvascular endothelial cells express coreceptor that are not expressed in platelets.
In conclusion, our study has demonstrated a novel connection between oxidative stress, innate immunity, and thrombosis.
The authors thank Dr Niladri Kar, Dr Sudipta Biswas, and V. Verbovetskaya for technical assistance. The authors thank Dr Alejandro Zimman and Emelye Crehore for critical reading of the manuscript. The authors sincerely thank Dr Satya P. Yadav for his assistance with surface plasmon resonance studies.
Sources of Funding
This work was supported in part by National Institutes of Health grants HL077213, 3RO1HL077213-05S1, 2P01HL073311-06, HL073311, HL071625, HL053315, GM021249, and SIG-RR016789-01-A1.
In September 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.5 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.274241/-/DC1.
Non-standard Abbreviations and Acronyms
- Carboxy(alkylpyrrole) protein adducts
- Fluorescent Activated Cell Sorter
- Platelet rich plasma
- Platelet poor plasma
- Received May 23, 2012.
- Revision received October 10, 2012.
- Accepted October 15, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Platelets may sense “pathological ligands” accumulating in circulation via a specific set of receptors and translate an activation signal into a prothrombotic state.
Toll-like receptor 9 (TLR9), the canonical receptor for unmethylated CpG sequences of pathogenic DNA, is expressed at low levels in platelets; however, its role in platelet function is not known.
What New Information Does This Article Contribute?
TLR9 is a functional platelet receptor capable of modulating platelet function and thrombosis.
2-(ω-carboxyalkyl)pyrrole protein adducts (CAPs), a product of phospholipids oxidation, is generated in oxidative stress. It is a novel noncanonical ligand for TLR9, which induces platelet activation and promotes thrombosis via TLR9.
Control of platelet reactivity is regarded as critical for the prevention of coronary artery disease. Although increased platelet reactivity and thrombogenic potential are common in pathophysiological conditions associated with oxidative stress, the mechanisms responsible are still poorly understood. We investigated the biological activities of CAPs, an alteredself-ligand generated in oxidative stress, on platelet function and thrombosis. Using multiple complimentary approaches, we demonstrated that CAPs is a novel and unconventional ligand for TLR9. CAPs levels are increased in circulation in hyperlipidemic apolipoproteinE-/- mice, leading to pronounced accumulation of CAPs in platelets. CAPs can promote platelet activation and aggregation in vitro and accelerate thrombosis in vivo in a TLR9/MyD88-dependent manner. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. These findings suggest that TLR9 is a functional platelet receptor capable of modulating platelet function and thrombosis in response to oxidative stress and establishes a novel connection between oxidative stress, innate immunity, and thrombosis.