Fractalkine Activates a Signal Transduction Pathway Similar to P2Y 12






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variety of chemokines including fractalkine (chemo-kine [C-X3-C motif] ligand 1, FKN) induce proinflam-matory and proatherosclerotic effects in addition to their primary immunologic actions.1–4 FKN differs from other che-mokines by a polypeptide chain that carries the chemokine domain on top of an extended mucine-like stalk and allows the molecule to exist either as a membrane-anchored or as a soluble glycoprotein.5 FKN is expressed in endothelial cells in response to proinflammatory agents,6 and its expression is enhanced in vascular injury, atherosclerosis, and coronary artery disease (CAD).7–9 Furthermore, it induces platelet activation and adhesion via a functional FKN receptor (che-mokine [C-X3-C motif] receptor 1 [CX3CR1]) expressed on the platelet surface.10 Importantly, platelet activation via the FKN/CX3CR1 axis triggers leukocyte adhesion to activated endothelium, and FKN-induced platelet P-selectin is mandatory for leukocyte recruitment under arterial flow conditions.11

CX3CR1 has been described as a Gαi protein–coupled receptor,10 but the exact signaling pathway has not yet been

explored. Bearing in mind that one of the most important platelet activating receptors, the ADP receptor P2Y12, also exerts its action via a Gαi protein–linked mechanism, a link between the receptors’ downstream signaling is suspected.

Clopidogrel, an irreversible P2Y12 receptor inhibitor, has been shown to be efficient to prevent ischemic events in selected patients with atherosclerotic disease,12 Although, impaired responsiveness to clopidogrel has been observed and described.13 Activation of the P2Y12 receptor inhibits cAMP production by adenylyl cyclase.14 The platelet reactivity index (PRI) using vasodilator-stimulated phosphoprotein (VASP) phosphorylation is a standardized P2Y12-specific assay that is based on the P2Y12 -specific inhibition of adenylyl cyclase by ADP and that assesses the difference in prostaglandin E1 (PGE1)–induced adenylyl cyclase–mediated VASP phosphorylation in the absence and presence of ADP using flow cytometry.14

There are many hypotheses about the causes of the above- mentioned impaired clopidogrel response (eg, polymor-phisms in metabolizing enzymes,15 drug–drug interactions,16 certain comorbidities). The Residual Platelet Aggregation Received on: March 15, 2011; final version accepted on: April 23, 2012.

From the Klinik für Kardiologie und Angiologie, Medizinische Hochschule Hannover, Hannover (U.F., D.F., J.B., A.S.); Deutsches Herzzentrum und 1. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich (C.S., S.M.); and Medizinische Klinik und Poliklinik I, Universitätsklinikum, Julius-Maximilians-Universität Würzburg, Würzburg (E.L., T.R.), Germany.

The online-only Data Supplement is available with this article at Correspondence to Andreas Schäfer, MD, Klinik für Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail

© 2012 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at DOI: 10.1161/ATVBAHA.112.250720 Arterioscler Thromb Vasc Biol

1079-5642 10.1161/ATVBAHA.112.250720 ATV201443 00 00 0 0

© 2012 American Heart Association, Inc.

26 August 2012

Arterioscler Thromb Vasc Biol

Flierl et al Fractalkine Counteracts P2Y12 Inhibition

Objective—Fractalkine (FKN) activates a Gαi protein–coupled signaling pathway similar to the one activated by ADP via P2Y12, which is the drug target of clopidogrel. FKN levels are increased under several disease conditions associated with impaired clopidogrel responsiveness.

Methods and Results—Blood samples were obtained from healthy volunteers and from 40 patients under chronic clopidogrel

treatment. FKN reduced prostaglandin E1–induced vasodilator-stimulated phosphoprotein phosphorylation by ≈25%

(P<0.01) at least partially mimicking the effect of ADP via P2Y12. In vitro, FKN increased platelet reactivity index in clopidogrel-treated patients indicating potential activation of downstream targets of P2Y12. When stratifying patients by their FKN levels, patients within the highest quartile of FKN (2042±25 pg/mL) had the weakest response to clopidogrel (platelet reactivity index, 68±4%), and patients within the lowest quartile (479±50 pg/mL) had the strongest response (platelet reactivity index, 48±7%; P=0.0106). FKN by itself induced phosphoinositide 3-kinase activation leading to Akt phosphorylation at Ser473 (P<0.01 versus basal).

Conclusion—In addition to desensitizing platelets to prostaglandin E1 via Gαi, FKN induces phosphoinositide 3-kinase– dependent Akt phosphorylation via a Gβγ protein similar to ADP signaling through P2Y12. FKN increased the platelet ADP response in clopidogrel-treated patients. Once released from an atherosclerotic lesion, this mechanism could contribute locally to impaired clopidogrel responsiveness at the vulnerable plaque. (Arterioscler Thromb Vasc Biol. 2012;32:1832-1840.)

Key Words: fractalkine ◼ G protein–coupled receptors ◼ P2Y12 ◼ thienopyridines

Fractalkine Activates a Signal Transduction Pathway Similar

to P2Y


and Is Associated With Impaired Clopidogrel


Ulrike Flierl, Daniela Fraccarollo, Eva Lausenmeyer, Tim Rosenstock, Christian Schulz,

Steffen Massberg, Johann Bauersachs, Andreas Schäfer



after Deployment of Intracoronary Stent score was established to estimate the likelihood of impaired clopidogrel responsive-ness based on easily available patient details.17 Thus, among others, diabetes mellitus, renal failure, and chronic heart fail-ure are associated with impaired response to P2Y12 block-ade. Interestingly, elevated FKN levels were observed in the above-mentioned comorbidities.18–21 Therefore, a potential link between FKN levels and clopidogrel responsiveness was assumed.

We hypothesized that FKN induces a pathway similar to that of P2Y12, thus inhibiting cAMP production and desen-sitizing platelets to the endogenous platelet inhibitor PGE1. Furthermore, we evaluated a link between FKN levels and clopidogrel responsiveness in patients with CAD.



For in vitro experiments in the absence of clopidogrel pretreat-ment, blood samples were collected from healthy donors who gave informed consent and who had not taken any antiplatelet medication within the last 10 days.

To evaluate clopidogrel responsiveness and FKN levels, blood samples were obtained from 40 consecutive clopidogrel-treated patients with stable CAD. Clopidogrel responsiveness was deter-mined by the P2Y12-specific PRI with a cutoff value of 50% as previ-ously reported.22 All patients had been admitted to the Cardiology Division of the University Hospital of Würzburg and gave informed consent. The study was performed in accordance with the Declaration of Helsinki.

Blood Sample Collection

Blood samples were collected from an antecubital vein using a 21-gauge needle. The first 5 mL of blood was discarded to avoid spontaneous platelet activation. Platelet-rich plasma (PRP) was pre-pared from citrated blood by centrifugation at 180g for 10 minutes. PRP was kept at 37°C before use. Platelet-poor plasma was prepared by centrifuging PRP at 1000g for 5 minutes.

Platelet Aggregation

Platelet aggregation was performed with light transmittance aggregometry using a commercial 8-channel platelet aggregation profiler (PAP-8; Bio/Data Corp, Horsham, PA). Aggregation was induced by different concentrations of ADP in PRP. Some samples of PRP were preincubated with PGE1 and FKN (1 µg/mL and 2 µg/mL; 5 minutes) and were afterward stimulated with ADP. Light transmis-sion was adjusted to 0% with PRP and to 100% using platelet-poor plasma for each measurement. Curves were recorded for 6 minutes. Aggregation was measured at primary, secondary, and maximal aggregation. In some experiments, the maximum aggregation of samples after preincubation with PGE1 and FKN was calculated in percentage of the maximum aggregation of PRP not preincubated with FKN, which had been stimulated in parallel with the same dose of ADP.

Preparation of Washed Platelets

For experiments requiring washed platelet preparations, blood was collected into acid citrate dextrose (citric acid [3.8 mmol/L] and dex-trose [125 mmol/L]; 1-mL acid citrate dexdex-trose/4-mL blood). PRP was obtained by centrifugation at 320g for 10 minutes, and HEPES (N -2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid)–modified Tyrode solution buffer 1 (NaCl, 132 mmol/L; KCl, 4 mmol/L; NaHCO3, 11.9 mmol/L; NaH2PO4, 0.36 mmol/L; glucose, 10 mmol/L; pH 6.5; 1-mL PRP/3.5-mL buffer 1) was added. Plasma-free platelet suspen-sions were obtained by centrifugation (400g; 17 minutes) of PRP; the

resulting pellet was resuspended in HEPES-modified Tyrode solu-tion buffer 2 (buffer 1; pH 7.4; CaCl2, 1 mmol/L; MgCl2, 1 mmol/L) to achieve a final platelet density of 200 000/µL. When indicated, aspirin (20 µg/mL) and the α-adrenoreceptor inhibitor BRL 44408 (10 µmol/L) were added to platelet samples.

Western Blotting Analysis

Washed platelets were kept in a water bath at 37°C for 15 minutes before adding 100 µL of a 3× sodium dodecyl sulfate stop buffer (con-sisting of Tris/HCl, 200 mmol/L [pH 6.7]; 15% [vol/vol] glycerol; 6% [wt/vol] sodium dodecyl sulfate) per 100 µL of washed platelet suspension. Samples were boiled for 5 minutes at 95°C before stor-age at −20°C.

Platelet samples were mixed with sample loading buffer and were separated under reducing conditions on 10% sodium dodecyl sulfate polyacrylamide gel. Proteins were electrotransferred onto a poly-vinylidene fluoride membrane (0.2 µmol/L; Immun-Blot, Bio-Rad, Hercules, CA). After transfer, the membranes were blocked in blotting solution (Tris-HCl, 20 mmol/L; NaCl, 150 mmol/L; 0.05% Tween 20; pH 7.6) with 5% blocking agent (Amersham, Little Chalfont, United Kingdom) overnight at 4°C followed by incubation with the primary monoclonal rabbit antiphospho-akt (Ser473) or antiphospho-akt (Thr308) antibody (1:1000; Cell Signaling, Danvers, MA) in blot-ting solution with 0.5% blocking agent for 2 hours. After washing, the blots were incubated with horseradish peroxidase–labeled goat anti-rabbit IgG antibody (1:10 000; Amersham) for 1 hour. The bands were detected using the enhanced chemiluminescence assay (ECL-Plus kit; Amersham). After autoradiography (Kodak Biomax; Kodak, Rochester, NY), densitometric analysis was performed using National Institutes of Health Image computer software. Molecular weights were determined using prestained SDS-PAGE standard and precision protein standards (Bio-Rad). All reagents for electrophore-sis were purchased from Sigma.


PRI was determined by assessing VASP status using flow cytometry (FACSCalibur; Becton Dickinson, Heidelberg, Germany). VASP phosphorylation was quantified with labeled monoclonal antibod-ies using a commercially available kit (PLT VASP/P2Y12 Test kit; Biocytex, Marseille, France), for which platelets were stimulated with PGE1±ADP followed by fixation with formaldehyde. After per-meabilization with Triton X-100, VASP phosphorylation at Ser239 was determined using a monoclonal antibody (16C2) and a fluorescein isothiocyanate–labeled secondary antibody whereas platelets were counterstained with a PE-labeled anti-CD61 antibody, similar to the one described initially.23 The PRI was calculated after measurement of VASP phosphorylation after stimulation with PGE1 (0.5 µmol/L) by mean fluorescence intensity (MFI PGE1), as well as stimulation with PGE1 in the presence of ADP (20 µmol/L; MFI PGE1+ADP). PRI is defined as: ([MFI PGE1]−[MFI PGE1+ADP])/[MFI PGE1]×100%, whereby background fluorescence is subtracted from each measure-ment. The lower the P2Y12 PRI, the better the inhibition of P2Y12 activity by clopidogrel.


Human serum FKN was determined using the Human CX3CL1/ Fractalkine Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).


Unless stated otherwise, all chemicals were obtained from Sigma -Aldrich (Deisenhofen, Germany) in the highest purity avail-able. Recombinant human FKN (365-FR) was purchased from R&D Systems. Ticagrelor was provided by AstraZeneca (Mölndal, Sweden).



Data are presented as means±SEM and analyzed using 1-way ANOVA with a Tukey post hoc test. P<0.05 was considered statisti-cally significant.


Phosphorylation of VASP in Platelets

One particular P2Y12-specific activity of ADP signaling is Gαi-dependent inhibition of adenylyl cyclase leading to reduced sensitivity of platelets to prostanoids like PGE1.

FKN also induces platelet activation through a Gαi-coupled receptor.10 We wondered whether FKN would also desensitize platelets for endogenous platelet inhibitors in a Gαi-dependent mechanism similar to P2Y12. In PRP from healthy human donors, FKN significantly reduced PGE1-induced VASP phosphorylation (Figure 1A–1C; Figure I in the online-only Data Supplement).

In Vitro Effects of FKN Despite P2Y12 Blockade

Similar to the above-mentioned PRI (see Methods), blood samples were stimulated with PGE1 and treated with ADP

in the presence/absence of ticagrelor, a reversible P2Y12 inhibitor that directly interacts with the receptor without requiring metabolism to generate an active compound.24 The concentration of ticagrelor (1 µg/mL) was chosen to completely block P2Y12-mediated adenylyl cyclase inhibition and to guarantee that the FKN effect is assessed in the absence of P2Y12 signaling.

Thereafter, VASP phosphorylation was detected by flow cytometry. ADP significantly decreased PGE1-mediated VASP phosphorylation, which was blunted in the presence of ticagrelor. Interestingly, the addition of FKN significantly reduced PGE1-induced VASP phosphorylation in the pres-ence of ticagrelor indicating a CX3CR1-mediated mechanism, which mimics the ADP effect and is functionally independent from P2Y12 activation (Figure 2).

In Vitro Effects of FKN on Platelet Aggregation

Functionally, PGE1-induced cAMP formation suppresses the ADP-evoked, P2Y12-mediated secondary phase of plate-let aggregation. Pretreatment with FKN (1 µg/mL) abro-gated the PGE1-mediated reduction of platelet aggregation

Figure 1. Platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser157 after stimulation with prostaglandin E1 (PGE1) and fractalkine (FKN). PGE1-induced VASP phosphorylation was significantly attenuated by preincubation with FKN (1 μg/mL) in a time-dependent pattern (A) and consistently at different concentrations of PGE1 (B and C). n=7–11. MFI indicates mean fluorescence intensity; ns, nonsignificant.


after stimulation with ADP with impact on primary, second-ary, and final aggregation (Figure 3). FKN did not influ-ence ADP-induced calcium signaling nor did it have an effect of its own (data not shown). Moreover, neither shape change nor aggregation to FKN alone was observed (data not shown).

Exploration of FKN-Mediated Platelet Signaling Pathway

Because the aforementioned results indicate an FKN-induced pathway similar to the Gαi-mediated signaling of P2Y12 activation, we investigated whether there is an impact not only on Gαi-mediated but also on Gβγ-mediated P2Y12-like

Figure 3. Modulation of prostaglandin E1 (PGE1)-induced and cAMP-mediated inhibition of the secondary phase of ADP-induced aggregation by fractalkine (FKN, 1 μg/mL) in platelet-rich plasma from healthy volunteers. ADP concentrations, which ranged from 1.5 to 2.5 μmol/L as medium extent of aggregation, were intended for ADP induction. A, Representative aggregation response curves from a healthy donor were recorded in the presence of ADP only (trace 1), ADP+FKN (trace 2), ADP+PGE1 (trace 3), and ADP+FKN+PGE1 (trace 4). The quantitative analysis is shown for primary (B), secondary (C), and maximal (D) aggregation. *P<0.05 vs all; n=6.

Figure 2. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser157 (A) and Ser239 (B) in the presence of P2Y

12 receptor inhibitor ticagrelor (Tica) was determined using flow cytometry. Samples were stimulated with prostaglandin E1 (PGE1, 1 μmol/L) and ADP (20 μmol/L) in the absence or presence of fractalkine (FKN, 1 μg/mL) and Tica (1 μg/mL). ADP significantly decreased PGE1-medi-ated VASP phosphorylation, which was abrogPGE1-medi-ated in the presence of Tica. In the presence of full P2Y12 blockade by Tica, FKN signifi-cantly reduced PGE1-induced VASP phosphorylation. n=5–6; MFI indicates mean fluorescence intensity.


signal transduction. Hence, immunoblotting experiments with washed platelets obtained from healthy volunteers were per-formed. Such a pathway would have to activate phosphoinosit-ide 3-kinase (PI3K) and lead to Akt phosphorylation.25 FKN by itself induced PI3K activation leading to significantly increased Akt phosphorylation at Ser473 compared with non-stimulated samples (Figure 4A and 4B). Moreover, FKN even potentiated PI3K activation when added to ADP compared with ADP stimulation alone (Figure 4A and 4C), alhough no significances after FKN incubation were observed for Akt- phosphorylation at Thr308 (Figure II in the online-only Data Supplement).

Furthermore, Akt phosphorylation was suppressed by pre-incubation of samples with TGX-221 (0.5 µmol/L; Cayman Chemical Corp, Ann Arbor, MI), a potent, selective, and cell permeable inhibitor of PI3K p110β (Figure III in the online-only Data Supplement). Four different isoforms of PI3Ks have been described; a key role for the p110β isoform was postulated in regulating thrombus formation by prevention of formation of stable integrin αIIbβ3 adhesion contacts.26 Using isoform-selective PI3K inhibitors and knockout mouse models, p110β was identified to be primarily responsible for Gi-dependent linkage to Akt activation in ADP-stimulated platelets.27 We also performed Western blotting analysis using the nonselective PI3K inhibitor Ly294002. As expected, FKN-induced signaling was completely inhibited by preincubation of samples with Ly294002 (data not shown). Taken together, these results once more indicate the impact of FKN on Gβγ activation.

Impact of FKN on Platelet Activation in P2Y12 Receptor Blocker-Treated Patients

To demonstrate the assumed FKN-mediated effect in the presence of a P2Y12 inhibitor, blood samples obtained from 5 patients with CAD who had been pretreated with clopidogrel were incubated with FKN in vitro. Thereafter, PRI was measured as described above. The PRI significantly increased in the presence of FKN implying reduced clopidogrel-mediated platelet inhibition in the presence of elevated concentrations of the chemokine (Figure 5A).

FKN Levels and P2Y12 Inhibition in CAD

If FKN actually interacts with clopidogrel responsiveness, an association between FKN levels and the extent of clopi-dogrel responsiveness should be observable in clopiclopi-dogrel- clopidogrel-treated patients. Therefore, the extent of P2Y12 inhibition was assessed in clopidogrel-treated CAD patients using the P2Y12-specific PRI.28,29 In parallel, FKN serum levels were assessed by ELISA. Indeed, when stratifying the patients’ FKN serum levels, higher FKN levels were associated with impaired clopidogrel responsiveness (Figure 5B and 5C). Interestingly, the disease conditions identified by the Residual Platelet Aggregation after Deployment of Intracoronary Stent score were also associated with increased FKN levels (Figure 5D). Patient characteristics are described in the Table.


Our data show analogies in intraplatelet P2Y12- and CX3CR1-coupled pathways. Similar to P2Y12, on stimulation, CX3CR1

Figure 4. Immunoblotting experiments with washed platelets obtained from healthy volunteers; (A) Western blotting demonstrating vary-ing extent of phosphoinositide 3 (PI3)-kinase activation after stimulation with ADP (5 μmol/L and 10 μmol/L), fractalkine (FKN, 1 μg/mL and 2 μg/mL), and their combination. FKN by itself induced PI3-kinase activation leading to significantly enhanced Akt phosphorylation at Ser473 compared with nonstimulated samples (B). FKN even potentiated ADP-induced PI3K activation compared with ADP stimula-tion alone (C) demonstrating the impact of FKN not only on Gαi-mediated but also on Gβγ-mediated signal transduction. n=9; ns indicates nonsignificant.


exerts Gαi-dependent inhibition of adenylyl cyclase and Gβγ-dependent activation of PI3K, whereby the extent of acti-vation by FKN is weaker than by ADP.

ADP, one of the most important mediators of thrombosis, activates platelets through 2 G protein–coupled P2 receptors, P2Y1 and P2Y12.30 The P2Y

1 receptor mediates the initiation of ADP-induced aggregation through calcium mobilization, whereas the P2Y12 receptor is involved in the completion and amplification of the aggregation response (Figure IV in the online-only Data Supplement).30 P2Y

12 receptor activation Gαi- dependently inhibits cAMP production by adenylyl cyclase and reduces endogenous platelet inhibition.14 Clopidogrel blocks the platelet P2Y12 receptor and prevents the secondary ADP-triggered amplification of platelet activation. Patients undergoing elective percutaneous coronary intervention dis-play marked interindividual variability of platelet inhibition in response to clopidogrel.22,28,31 Impaired responsiveness to clopidogrel is associated with increased risk of stent throm-bosis29,32 and adverse cardiovascular events after coronary stenting.33

Interestingly, CX3CR1, the FKN receptor, is coupled to the same intracellular G proteins as P2Y12 , suggesting a potential overlapping downstream signaling, which could partially substitute during P2Y12 inhibition (Figure IV in the online-only Data Supplement). This is supported by the fact that FKN itself is only a weak stimulant for platelets without

affecting calcium flux requiring costimulation with agonists, such as ADP.10 Similar pathways have been described previously, in which alternative signaling through Gαi in platelets could at least partially substitute for inhibition of P2Y12.34

The intact endothelium releases prostanoids (eg, pros-tacyclin) among other substances, which serve as endog-enous platelet inhibitors by activating adenylyl cyclase. Patients with active CAD do not only have endothe-lial dysfunction resulting in reduced formation of these mediators, but also show decreased platelet responsive-ness to the antiaggregatory effects of NO and PGE1.35,36 Such impaired responsiveness is associated with a worse outcome.37 Especially, the activity of adenylyl cyclase is inhibited by Gαi-mediated signaling through P2Y12. When we assessed the effect of FKN on PGE1-mediated plate-let inhibition, we found that FKN desensitizes plateplate-lets to the endogenous platelet inhibitor prostaglandin by inhibit-ing adenylyl cyclase. Gi coupling of CX3CR1 has been described previously for Chinese hamster ovary cells38 and neurons.39

We could additionally show that FKN induces PI3K-dependent Akt phosphorylation via a Gβγ protein demonstrat-ing that not only the Gαi but also the Gβγ subunit is involved in FKN-mediated signaling similar to ADP signaling through P2Y12. Interestingly, an FKN-dependent PI3K activation has Figure 5. Increased platelet reactivity index (PRI) in blood samples from clopidogrel-treated patients with coronary artery disease (CAD) after in vitro incubation with fractalkine (FKN; 1 μg/mL; n=5) indicating impaired response to P2Y12 receptor inhibition in the presence of FKN (A). Analysis of FKN serum levels in patients. B, Patients within the highest quartile of FKN (2042±25 pg/mL) had the weakest response to clopidogrel (PRI, 68±4%), whereas patients within the lowest quartile of FKN (479±50 pg/mL) had the strongest response (PRI, 48±7%; P=0.0106). C, Scatter plot displaying the individual values for FKN vs PRI; n=40, r2=0.1862, P=0.0054. D, The Residual Platelet Aggregation after Deployment of Intracoronary Stent (PREDICT) scores were divided and associated with FKN levels of patients with CAD. A significant correlation occurred between FKN level and PREDICT score quartile 7 to 9 compared with quartile score 0.


been shown in reference to microglial activation in vivo.40 In general, different phosphorylation patterns of Akt phosphory-lation sites have been described previously. For full enzyme activity, Thr308, as well as Ser473, phosphorylation, was men-tioned to be required.41 Though, for tumor necrosis factor-α– induced activation of Akt, only phosphorylation at Ser473 not at Thr308 has been reported.42 Otherwise, phosphorylation at Thr308 but not at Ser473 correlates with Akt protein kinase activity in non-small cell lung cancer.43 However, in our exper-iments in human platelets, significances for Akt phosphoryla-tion only emerged for phosphorylaphosphoryla-tion site Ser473 but not for Thr308 indicating that phosphorylation at Ser473 seems to be the most important for FKN-induced Akt activation. The effect of FKN on platelet Ser473 phosphorylation was still detect-able in the presence of P2Y12 receptor inhibitors and the COX inhibitor acetylsalicylic acid (Figure V in the online-only Data Supplement).

In addition to acute myocardial infarction, where enhanced FKN levels could be explained by enhanced release from the ruptured atherosclerotic lesion, impaired clopidogrel respon-siveness has been observed in several comorbidities to CAD (eg, diabetes mellitus, renal failure, and congestive heart fail-ure). Interestingly, we and others have observed increased systemic FKN levels in patients with and experimental models of those diseases. Overexpression of FKN has been observed in patients with left ventricular dysfunction20 and in rats with heart failure.44 In diabetes mellitus, FKN levels are increased in animal models of type I,45 as well as type II,18 whereby induced FKN expression has been found in smooth muscle cells after high glucose conditions46 and in glomeruli after exposition to advanced glycation end products.45 FKN plays an important role in nephrogenesis and in the develop-ment of kidney diseases leading to chronic or acute renal fail-ure.21,47 It is remarkable that clinical disease conditions related to impaired clopidogrel responsiveness do display enhanced FKN formation. Therefore, it is tempting to speculate whether higher FKN levels might locally contribute to impaired plate-let inhibition under these conditions.

Of other weak agonists, epinephrine,48,49 macrophage-derived chemokine, or stromal cell–macrophage-derived factor-1α50 have also to be taken into account when postulating alternative signaling bypassing the inhibition of P2Y12. In our patient cohort, we assessed the potential interaction between stro-mal cell–derived factor-1 and macrophage-derived chemo-kine with the PRI; however, we found no correlation with PRI (Figure VI in the online-only Data Supplement). Another limitation to be considered is the fact that the PRI, which is based on modulation of VASP phosphorylation, only provides indirect evidence with regard to cAMP and adenylyl cyclase. However, it has been established as the only P2Y12-specific clinically usable test and was used in the present context to strengthen the potential clinical relevance.14

The analysis of clopidogrel-treated CAD patients revealed that patients with higher levels of FKN were more likely to have impaired clopidogrel responsiveness than patients with lower FKN levels. The bypassing of the clopidogrel-inhibited G protein–coupled P2Y12 receptor by FKN-mediated activa-tion of its specific receptor constitutes an important mecha-nism by which platelets remain hyperreactive despite using state-of-the-art antiplatelet therapy.

Hypothetically, this mechanism could be extremely rel-evant at the site of a ruptured plaque. First, FKN levels might be much higher at the site of release from an atheroscle-rotic lesion; second, platelet inhibition would be impaired at the one site in the circulation, where it is most needed. One potential impact could be a more important role of P2Y1 in alliance with weaker platelet agonists acting through Gz or Gi for thrombus formation. However, more studies are needed to establish the role of dynamic changes of FKN or other mediators acting through Gαi-coupled receptors on P2Y12 inhibition.

In conclusion, we show that increased FKN levels are associated with decreased endogenous platelet inhibition and impaired response to P2Y12 inhibition with clopidogrel. Furthermore, assumed similarities to the P2Y12 receptor con-cerning the signaling pathway could be verified. Thus, we

Table. Patient Characteristics


n 40

Sex, male/female 26/14

Age, y 66±12

NYHA class 1 (IQR, 0–3)

LVEF, % 54±15 BMI, kg/m2 27.3±3.6 Diabetes mellitus, % 24 Hyperlipoproteinemia, % 63 Hypertension, % 85 Current smoker, % 38

Family history for MI, % 33 No. of CVRFs 2 (IQR, 1–4) Total cholesterol, mg/dL 190.3±38.9

LDL-C, mg/dL 104.2±32.9

HDL-C, mg/dL 47.3±12.9

HbA1c, % 6.1±0.9

Estimated GFR, mL/min per 1.73 m2 83.3±28.1

C-reactive protein, mg/dL 1.61±3.11 Platelets, n*1000/µL 247±83 ASA, % 95 ACEi/ARB, % 85 β-Blocker, % 88 Statin, % 82

Hyperlipoproteinemia is total cholesterol>240 mg/dL or statin use for raised cholesterol.

Hypertension is blood pressure>140/90 mm Hg or on antihypertensive therapy for raised blood pressure.

GFR is estimated using the MDRD equation (Modification of Diet in Renal Disease Study Group).

Data are mean (±SD).

NYHA indicates New York Heart Association classification; IQR, interquar-tile range; LVEF, left ventricular ejection fraction; BMI, body mass index; MI, myocardial infarction; CVRF, cardiovascular risk factor; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin; GFR, glomerular filtration rate; ASA, acetylsalicylic acid; ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.


describe a novel pathomechanism by which alternative G pro-tein–coupled signaling induced by the proinflammatory and atherogenic mediator FKN might impede platelet inhibition by thienopyridines.

Sources of Funding

This work was supported by Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Würzburg (projects E-39 and Z-2/25 to Dr Schäfer).




1. Weber C. Platelets and chemokines in atherosclerosis: partners in crime.

Circ Res. 2005;96:612–616.

2. Hansson GK. Inflammation, atherosclerosis, and coronary artery dis-ease. N Engl J Med. 2005;352:1685–1695.

3. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004;95:858–866.

4. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354:610–621. 5. Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves

DR, Zlotnik A, Schall TJ. A new class of membrane-bound chemokine with a CX3C motif. Nature. 1997;385:640–644.

6. Harrison JK, Jiang Y, Wees EA, Salafranca MN, Liang HX, Feng L, Belardinelli L. Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart. J Leukoc Biol. 1999; 66:937–944.

7. Umehara H, Bloom E, Okazaki T, Domae N, Imai T. Fractalkine and vascular injury. Trends Immunol. 2001;22:602–607.

8. Damås JK, Boullier A, Waehre T, Smith C, Sandberg WJ, Green S, Aukrust P, Quehenberger O. Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, is elevated in coronary artery disease and is reduced during statin therapy. Arterioscler Thromb Vasc Biol. 2005;25:2567–2572.

9. Greaves DR, Häkkinen T, Lucas AD, Liddiard K, Jones E, Quinn CM, Senaratne J, Green FR, Tyson K, Boyle J, Shanahan C, Weissberg PL, Gordon S, Ylä-Hertualla S. Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and acti-vation-regulated chemokine, are expressed in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2001;21:923–929.

10. Schäfer A, Schulz C, Eigenthaler M, Fraccarollo D, Kobsar A, Gawaz M, Ertl G, Walter U, Bauersachs J. Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion. Blood. 2004;103:407–412.

11. Schulz C, Schäfer A, Stolla M, Kerstan S, Lorenz M, von Brühl ML, Schiemann M, Bauersachs J, Gloe T, Busch DH, Gawaz M, Massberg S. Chemokine fractalkine mediates leukocyte recruitment to inflammatory endothelial cells in flowing whole blood: a critical role for P-selectin expressed on activated platelets. Circulation. 2007;116:764–773. 12. A randomised, blinded, trial of clopidogrel versus aspirin in patients

at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee.

Lancet. 1996;348:1329–1339.

13. Cattaneo M. Resistance to antiplatelet drugs: molecular mechanisms and laboratory detection. J Thromb Haemost. 2007;5 Suppl 1:230–237. 14. Geiger J, Brich J, Hönig-Liedl P, Eigenthaler M, Schanzenbächer P,

Herbert JM, Walter U. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Arterioscler Thromb Vasc Biol. 1999;19:2007–2011.

15. Hulot JS, Bura A, Villard E, Azizi M, Remones V, Goyenvalle C, Aiach M, Lechat P, Gaussem P. Cytochrome P450 2C19 loss-of-function polymor-phism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood. 2006;108:2244–2247.

16. Lau WC, Gurbel PA. Antiplatelet drug resistance and drug-drug interac-tions: role of cytochrome P450 3A4. Pharm Res. 2006;23:2691–2708. 17. Geisler T, Grass D, Bigalke B, Stellos K, Drosch T, Dietz K,

Herdeg C, Gawaz M. The Residual Platelet Aggregation after Deployment of Intracoronary Stent (PREDICT) score. J Thromb

Haemost. 2008;6:54–61.

18. Schäfer A, Schöpp C, Flierl U, Schulz C, Leutke M, Massberg S, Bauersachs J. Increased vascular fractalkine expression and enhanced platelet reactivity to fractalkine in type-II diabetic ZDF-rats. Arterioscler

Thromb Vasc Biol. 2006;26:P88.

19. Kikuchi Y, Imakiire T, Hyodo T, Kushiyama T, Higashi K, Hyodo N, Suzuki S, Miura S. Advanced glycation end-product induces fractalkine gene upregulation in normal rat glomeruli. Nephrol Dial Transplant. 2005;20:2690–2696.

20. Husberg C, Nygård S, Finsen AV, Damås JK, Frigessi A, Oie E, Waehre A, Gullestad L, Aukrust P, Yndestad A, Christensen G. Cytokine expres-sion profiling of the myocardium reveals a role for CX3CL1 (fractalkine) in heart failure. J Mol Cell Cardiol. 2008;45:261–269.

21. Koziolek MJ, Müller GA, Zapf A, Patschan D, Schmid H, Cohen CD, Koschnick S, Vasko R, Bramlage C, Strutz F. Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure. Nephrol Dial Transplant. 2010;25:684–698.

22. Schäfer A, Weinberger S, Flierl U, Eigenthaler M, Störk S, Walter U, Ertl G, Bauersachs J. ADP-induced platelet aggregation frequently fails to detect impaired clopidogrel-responsiveness in patients with coronary artery disease compared to a P2Y12-specific assay. Thromb Haemost. 2008;100:618–625.

23. Grossmann R, Sokolova O, Schnurr A, Bonz A, Porsche C, Obergfell A, Lengenfelder B, Walter U, Eigenthaler M. Variable extent of clopidogrel responsiveness in patients after coronary stenting. Thromb Haemost. 2004;92:1201–1206.

24. Teng R, Oliver S, Hayes MA, Butler K. Absorption, distribution, metabolism, and excretion of ticagrelor in healthy subjects. Drug Metab

Dispos. 2010;38:1514–1521.

25. Garcia A, Kim S, Bhavaraju K, Schoenwaelder SM, Kunapuli SP. Role of phosphoinositide 3-kinase beta in platelet aggregation and throm-boxane A2 generation mediated by Gi signalling pathways. Biochem J. 2010;429:369–377.

26. Schoenwaelder SM, Ono A, Nesbitt WS, Lim J, Jarman K, Jackson SP. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem. 2010;285:2886–2896.

27. Schoenwaelder SM, Ono A, Sturgeon S, Chan SM, Mangin P, Maxwell MJ, Turnbull S, Mulchandani M, Anderson K, Kauffenstein G, Rewcastle GW, Kendall J, Gachet C, Salem HH, Jackson SP. Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets. J Biol Chem. 2007;282:28648–28658.

28. Bonello L, Paganelli F, Arpin-Bornet M, Auquier P, Sampol J, Dignat-George F, Barragan P, Camoin-Jau L. Vasodilator-stimulated phos-phoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascu-lar events. J Thromb Haemost. 2007;5:1630–1636.

29. Blindt R, Stellbrink K, de Taeye A, Müller R, Kiefer P, Yagmur E, Weber C, Kelm M, Hoffmann R. The significance of vasodilator-stimulated phosphoprotein for risk stratification of stent thrombosis. Thromb

Haemost. 2007;98:1329–1334.

30. Gachet C. Regulation of platelet functions by P2 receptors. Annu Rev

Pharmacol Toxicol. 2006;46:277–300.

31. Gurbel PA, Bliden KP, Samara W, Yoho JA, Hayes K, Fissha MZ, Tantry US. Clopidogrel effect on platelet reactivity in patients with stent thrombosis: results of the CREST Study. J Am Coll Cardiol. 2005;46:1827–1832.

32. Wenaweser P, Hess O. Stent thrombosis is associated with an impaired response to antiplatelet therapy. J Am Coll Cardiol. 2005;46:CS5–CS6. 33. Geisler T, Langer H, Wydymus M, Göhring K, Zürn C, Bigalke B,

Stellos K, May AE, Gawaz M. Low response to clopidogrel is associ-ated with cardiovascular outcome after coronary stent implantation. Eur

Heart J. 2006;27:2420–2425.

34. Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl

Acad Sci USA. 1998;95:8070–8074.

35. Chirkov YY, Chirkova LP, Sage RE, Horowitz JD. Impaired respon-siveness of platelets from patients with stable angina pectoris to anti-aggregating and cyclicAMP-elevating effects of prostaglandin E1.

J Cardiovasc Pharmacol. 1995;25:961–966.

36. Willoughby SR, Stewart S, Holmes AS, Chirkov YY, Horowitz JD. Platelet nitric oxide responsiveness: a novel prognostic marker in acute coronary syndromes. Arterioscler Thromb Vasc Biol. 2005;25:2661–2666. 37. Chirkov YY, Holmes AS, Willoughby SR, Stewart S, Wuttke RD, Sage

PR, Horowitz JD. Stable angina and acute coronary syndromes are


associated with nitric oxide resistance in platelets. J Am Coll Cardiol. 2001;37:1851–1857.

38. Davis CN, Harrison JK. Proline 326 in the C terminus of murine CX3CR1 prevents G-protein and phosphatidylinositol 3-kinase-depen-dent stimulation of Akt and extracellular signal-regulated kinase in Chinese hamster ovary cells. J Pharmacol Exp Ther. 2006;316:356–363. 39. Lauro C, Catalano M, Trettel F, Mainiero F, Ciotti MT, Eusebi F,

Limatola C. The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit. J Immunol. 2006;177:7599–7606.

40. Lyons A, Lynch AM, Downer EJ, Hanley R, O’Sullivan JB, Smith A, Lynch MA. Fractalkine-induced activation of the phosphatidylinositol-3 kinase pathway attentuates microglial activation in vivo and in vitro.

J Neurochem. 2009;110:1547–1556.

41. Kim S, Jin J, Kunapuli SP. Akt activation in platelets depends on Gi signaling pathways. J Biol Chem. 2004;279:4186–4195.

42. O’toole A, Moule SK, Lockyer PJ, Halestrap AP. Tumour necrosis fac-tor-alpha activation of protein kinase B in WEHI-164 cells is accompa-nied by increased phosphorylation of Ser473, but not Thr308. Biochem J. 2001;359(Pt 1):119–127.

43. Vincent EE, Elder DJ, Thomas EC, Phillips L, Morgan C, Pawade J, Sohail M, May MT, Hetzel MR, Tavaré JM. Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer. Br J Cancer. 2011;104:1755–1761. 44. Schäfer A, Schulz C, Fraccarollo D, Schöpp C, Pfrang J, Ertl G, Massberg

S, Bauersachs J. The CX3C chemokine fractalkine enhances vascular

dysfunction and aggravates platelet activation in rats with chronic heart failure. J Heart Dis. 2008;6:29.

45. Kikuchi Y, Ikee R, Hemmi N, Hyodo N, Saigusa T, Namikoshi T, Yamada M, Suzuki S, Miura S. Fractalkine and its receptor, CX3CR1, upregulation in streptozotocin-induced diabetic kidneys. Nephron Exp

Nephrol. 2004;97:e17–e25.

46. Dragomir E, Manduteanu I, Calin M, Gan AM, Stan D, Koenen RR, Weber C, Simionescu M. High glucose conditions induce upregulation of fractalkine and monocyte chemotactic protein-1 in human smooth muscle cells. Thromb Haemost. 2008;100:1155–1165.

47. Koziolek MJ, Vasko R, Bramlage C, Müller GA, Strutz F. The CX(3) C-chemokine fractalkine in kidney diseases. Mini Rev Med Chem. 2009;9:1215–1228.

48. Jackson CE, Dalzell JR, Hogg KJ. Epinephrine treatment of anaphy-laxis: an extraordinary case of very late acute stent thrombosis. Circ Cardiovasc Interv. 2009;2:79–81.

49. Nakamura T, Ariyoshi H, Kambayashi J, Ikeda M, Kawasaki T, Sakon M, Monden M. Effect of low concentration of epinephrine on human platelet aggregation analyzed by particle counting method and confocal microscopy. J Lab Clin Med. 1997;130:262–270.

50. Gear AR, Suttitanamongkol S, Viisoreanu D, Polanowska-Grabowska RK, Raha S, Camerini D. Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function. Blood. 2001;97:937–945.





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