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Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease
Atherosclerosis 238 (2015) 289e295
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease J. Patzelt a, b, 1, K.A.L. Mueller b, 1, S. Breuning b, A. Karathanos b, R. Schleicher a, P. Seizer b, M. Gawaz b, H.F. Langer a, b, *, 2, T. Geisler b, **, 2 a b
Section for Cardioimmunology, Eberhard-Karls University Tuebingen, 72076 Tuebingen, Germany University Hospital, Department of Cardiovascular Medicine, Eberhard-Karls University Tuebingen, 72076 Tuebingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 15 October 2014 Accepted 4 December 2014 Available online 18 December 2014
Objective: Inhibition of components of the complement system or of its receptors has been postulated as a concept for primary and secondary prevention in atherosclerosis and was applied in clinical trials. Although the anaphylatoxin-receptors C3aR and C5aR are commonly associated with inflammatory cells, in vitro studies suggested their expression also on platelets. Methods and Results: Expression levels of C3aR and C5aR were measured by flow cytometry in a collective of 302 patients with documented coronary artery disease (CAD) including patients with stable CAD (n ¼ 152), unstable angina (n ¼ 54), acute myocardial infarction (AMI; Non-ST elevation myocardial infarction, n ¼ 70, ST elevation MI, n ¼ 26) or healthy controls (n ¼ 21). Patients with stable CAD, unstable angina or AMI had significantly higher expression of C5aR on platelets in comparison to healthy controls (MFI 14.68 (5.2), 14.56 (5.18) and 13.34 (4.52) versus 10.68 (3.1)); p < 0.001). In contrast, the expression of C3aR on platelets was significantly enhanced in patients with stable and unstable CAD but not in patients with AMI compared to controls. While there was a strong correlation between the soluble ligands of these receptors C3a and C5a, we observed only a weak correlation with their receptors on platelets. Similarly, agonist induced aggregation (MEA, ADP, and TRAP) showed only a weak correlation with the expression level of anaphylatoxin e receptors on platelets. Of note, the expression of both anaphylatoxin-receptors on platelets strongly correlated with platelet activation as assessed with the surface activation marker P-selectin (r ¼ 0.47, p > 0.001 for C3aR, r ¼ 0.76 for C5aR, p < 0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1. Conclusion: In summary, we observed a positive correlation between the expression of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with CAD. Further investigations are needed to study the clinical and mechanistic relevance of these findings. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Anaphylatoxin receptors C3a C5a Platelets Coronary heart disease
1. Introduction Historically, platelets have been predominantly associated with late events in the course of atherosclerotic disease, for instance when the atherosclerotic plaque ruptures [1]. As a consequence, life threatening vascular thrombotic complications such as myocardial
* Corresponding author. Section for Cardioimmunology, Eberhard-Karls University Tübingen, Otfried-Mueller-Straße 10, 72076 Tübingen, Germany. ** Corresponding author. E-mail addresses: [email protected] (H.F. Langer), tobias. [email protected] (T. Geisler). 1 These authors contributed equally. 2 These authors share senior authorship. http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.002 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
infarction or stroke become apparent [1,2]. Accumulating evidence indicates that platelets may contribute to atherosclerosis by promoting diverse pro-inflammatory mechanisms even before the atherosclerotic plaque forms [3e5]. Platelet adhesion to the vasculature promotes endothelial inflammation [3], subsequent recruitment of leukocytes by platelets enforces accumulation of inflammatory cells [6] and secretion of paracrine effectors or upregulation of proinflammatory receptors on platelets such as CD40L or P-selectin then aggravate the inflammatory reaction [7e9]. Recently, novel platelet associated effectors including SDF-1 and its receptors on platelets have been reported to be upregulated in atherosclerotic patients and may contribute to the pathophysiology of the disease [10e12]. Although very early reports suggested a potential role of the complement system for platelet function
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[13,14], information on a potential relevance of the complement system in diseases featuring platelet activation are scarce. Recent evidence has highlighted a role of the complement system in atherosclerosis. For instance, complement components have been identified in relevant amounts in atherosclerotic plaques [15,16]. Inhibition of C5 in murine models of atherosclerosis resulted in reduced atherosclerosis [17,18]. Moreover, the efficacy targeting complement in coronary artery disease (CAD) was questioned in several clinical trials and was partially successful in patients with CAD. A trial [19] found significantly reduced mortality in STEMI patients undergoing treatment with an anti C5 antibody. Other trials that examined complement inhibition in patients undergoing coronary artery bypass graft surgery found positive effects on morbidity and mortality [20,21]. Although early reports showed that complement receptors may be expressed on platelets [22e24] and the relevance of platelet activation for the course of CAD is unquestionable, a functional or pathophysiological role of complement receptor expression on platelets in this setting still remains to be established. Here, we assessed the expression of complement receptors on platelets and their correlation with platelet activation markers in patients with symptomatic CAD. 2. Methods 2.1. Patient characteristics and blood sampling We included 302 consecutive patients with symptomatic coronary artery disease (CAD). 152 patients had stable angina pectoris (SAP), 54 unstable angina pectoris (UAP) and 96 patients presented with acute myocardial infarction (AMI). AMI was diagnosed by a rise and/or fall of cardiac biomarker values [cardiac troponin (cTn)] with at least one value above the 99th percentile upper reference limit and with at least one of the following: Symptoms of ischemia, new or presumed new significant ST-segment-T wave (STeT) changes or new left bundle branch block (LBBB), development of pathological Q waves in the ECG, imaging evidence of new loss of viable myocardium or new regional wall motion abnormality or identification of an intracoronary thrombus by angiography [25]. The study was approved by the institutional ethics committee (270/ 2011BO1) and complies with the declaration of Helsinki and the good clinical practice guidelines [26e28]. For the cohort study, blood samples were collected from the arterial sheaths of patients with symptomatic CAD before percutaneous coronary intervention (PCI) and were immediately analyzed for surface expression of C3aR, C5aR, P-Selectin, and SDF1 by flow cytometry. Soluble C5a and C3a in the plasma were analyzed by ELISA. Venous blood samples of n ¼ 21 healthy volunteers served as controls. Blood was carefully drawn from the antecubital vein to prevent platelet activation. Platelet function and aggregation was measured by agonist induced aggregometry using the Multiplate analyzer. All blood samples were processed within 1 h after blood withdrawal. All subjects gave written informed consent. Patients were admitted to the department of cardiology of the University of Tübingen, Germany.
mouse monoclonal anti human SDF-1 (R&D systems), and mouse anti human CD42b PE (Becton Dickinson) or their respective isotype controls (mouse IgG1-FITC, mouse IgG2bPE from R&D systems) for 30 min at room temperature. After staining, the cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry (FACS-Calibur flow cytometer BectoneDickinson, Heidelberg, Germany). In some experiments, protein synthesis was inhibited by puromycin (2,5 mg/ml, Invitrogen, 30 min prior to platelet activation with 20 mM ADP) as indicated in figure legends or platelet alphagranule release was blocked by 30 min preincubation with brefeldin a (Cell Signaling Technologies, 10 mg/ml). 2.3. Enzyme-linked immunosorbent assay(ELISA) Plasma levels of C3a and C5a were determined in plasma collected from enrolled patients (n ¼ 302) or healthy volunteers (n ¼ 21) using a commercially available enzyme-linked immunosorbent assay kit according to the manufacturer's guidelines (R&D Systems, Minneapolis, MN, USA). Blood was collected from the arterial sheaths of patients during heart catheterization to avoid platelet activation and processed immediately. Ethylenediamine tetraacetic acid plasma probes were centrifuged for 15 min at 10.000 g within 30 min of collection. Probes were aliquoted and stored at 80 C until analysis. 2.4. Platelet function tests and aggregometry ADP-induced platelet aggregation (PA) in whole blood was assessed with multiple electrode aggregometry (MEA) using the Multiplate© analyzer (Dynabyte, Munich, Germany). This assay is suitable for monitoring antiplatelet drug response in different settings [29e31] taking into account physiological and inflammatory blood compounds that might affect platelet function. ADP (6.4 mol/l) was added to a 1:1 dilution of whole blood anticoagulated with hirudine and 0.9% NaCl. Impedance with MEA was continuously recorded for 5 min plotting arbitrary aggregation units (AU) against time (AU min) after stirring for 3 min in the test cuvettes at 37 C [31,32]. Results were analyzed as area under the curve units (AU min). All material and consumables used in the aggregometry including agonist solution were provided by the manufacturer (Dynabyte, Munich, Germany). 2.5. Statistical analysis All statistical analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago IL). Normally distributed data were compared by using independent student's T-test. Non-parametric data, including MFIs were compared using the U-Test by Mann and Whitney. Correlations were assessed by Spearman's rank correlation coeffi~ ). MFIs are presented as median values and 25th- and 75th cient (n percentiles. KruskaleWallis was performed to test for differences between multiple groups of SAP, UAP, AMI and healthy controls. 3. Results
2.2. Surface expression of platelet receptors analyzed by whole blood flow cytometry Platelets in whole blood were analyzed for the surface expression of C3aR, C5aR, SDF-1, and P-selectin (CD62P) gating for the platelet specific marker CD42b. Blood collected in CPDA was diluted 1:50 with PBS (Gibco) and incubated with the respective conjugated antibodies-mouse monoclonal anti human C3aR-FITC (AbD Serotec), mouse monoclonal anti human C5aR-FITC (AbD Serotec), mouse monoclonal anti human p-Selectin-FITC (Beckman Coulter),
Although a potential relevance of both platelet activation and initiation of the complement cascade in the course of atherosclerosis and particularly in ACS were suggested, expression of proinflammatory complement receptors in a sufficient patient cohort has not been analyzed, so far. Here, we investigated platelet expression of anaphylatoxin receptors C3aR and C5aR on platelets and their corresponding ligands C3a and C5a generated during complement activation as soluble effectors in the blood of patients with symptomatic CAD and healthy volunteers in a pilot study.
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3.1. Platelet surface expression of C3aR and C5aR is associated with the presence of symptomatic CAD and ACS
3.2. Surface platelet expression of C3aR and C5aR correlates with platelet activation and aggregation
Patients' baseline characteristics (age, gender, cardiovascular risk factors, co-medication) of the cohort (n ¼ 302 patients) and the subgroups of stable CAD (n ¼ 152), UAP (n ¼ 54) and AMI (n ¼ 96) are depicted in Table 1. 21 healthy untreated volunteers served as controls. Expression levels of C3aR and C5aR were analyzed in whole blood by flow cytometry. We gated on platelets using the specific marker CD42b as shown in Fig. 1A. Since the data for C3a and C5a receptor measured by flow cytometry (MFI) showed a nonparametric distribution, we first performed a multigroup comparison using the KruskaleWallis test. The test revealed significant intra-group differences for C3aR and C5aR between groups of healthy volunteers, patients with SAP, UAP and AMI (p<0.001 for both C3aR and for C5aR). Therefore we compared individual groups using ManneWhitney tests. The surface expression of C5aR on platelets was significantly higher in patients with stable CAD, UA or AMI compared to the control group [stable CAD: median 4.68, IQR 5.2, UA: median MFI 14.56 IQR 5.18, AMI median MFI 13.34, IQR 4.52 vs. 10.68, IQR 3.1 healthy controls p < 0.001] C3aR surface expression was significantly increased in stable CAD patients and patients with UA but not in patients with AMI compared to healthy volunteers [stable CAD: median MFI 7.94IQR3.28, p > 0.001; UA: median MFI 7.74, IQR 3.7, p ¼ 0.049 AMI: median MFI 7.1, IQR 2.52 p ¼ 0.49 vs. healthy volunteers 7.16 IQR 1.47) (Fig. 1B, C).
Plasma levels of C3a and C5a were measured by ELISA in 191 patients. As expected, we could detect a strong correlation of the soluble plasma amounts of C3a and C5a as shown in Supplemental Fig. 1(spearman rank coefficient 0.46, p < 0.001). Platelet C3aR and C5aR only weakly correlated with plasma C3a and not with C5a levels, respectively (spearman rank coefficient r ¼ 0.24, p ¼ 0.001 and r ¼ 0.13, p ¼ 0.67, respectively), Fig. 2A, B. Recent studies reported that platelet activation as assessed by platelet P-selectin is associated with substantial increase in platelet expression of SDF-1 [33e35]. Therefore, we analyzed the correlation of platelet C3aR and C5aR and these established platelet activation markers. Interestingly, platelet C3aR and C5aR showed a strong and significant correlation with the degree of platelet activation measured by the expression of P-selectin (spearman rank coefficient r ¼ 0.48, p < 0.001 and r ¼ 0.76, p < 0.001, respectively), Fig. 3A, B. Interestingly, in the presence of puromycin inhibiting platelet protein synthesis C3aR expression induced by ADP was not apparent any more (Supplemental Fig. 2), whereas there was no difference regarding its expression in the presence or absence of brefeldin inhibiting platelet granula release (data not shown). We could also detect a significant correlation between C3aR, C5aR and platelet SDF-1 expression (spearman rank coefficient r ¼ 0.31, p < 0.001 and r ¼ 0.44, p < 0.001, respectively), Fig. 4A, B. Moreover, the surface expression of C3aR and C5aR showed a weak but significant correlation with platelet function analyzed by ADP-induced platelet aggregation (spearman rank coefficient r ¼ 0.17, p ¼ 0.003 and r ¼ 0.14, p ¼ 0.005, respectively) shown in Fig. 5A, B. There was no significant difference for platelet C3aR and C5aR expression whether patients were pretreated with aspirin or not (MFI 7.76 vs. 8.38; p ¼ 0.11 and 15.2 vs. 15.7; p ¼ 0.49, respectively). Similarly, there was no effect of P2Y12 inhibitor pretreatment on receptor expression (clopidogrel C3aR: 7.57 vs. 8.14; p ¼ 0.28; C5aR: 15.16 vs. 15.44; p ¼ 0.79; prasugrel C3aR: 6.66 vs. 8.14; p ¼ 0.16; C5AR: 16.72 vs. 15.44; p ¼ 0.51; ticagrelor C3aR: 7.93 vs. 8.14; p ¼ 0.83; C5aR: 14.26 vs. 15.44; p ¼ 0.46). In summary, we observed a significant correlation between both complement receptor expression with various platelet activation markers, but only a weak correlation with platelet function measured by conventional aggregometry.
Table 1 Baseline characteristics of the Patient cohort. Characteristics
Patient collective
Stable CAD Unstable (SAP) angina (UAP)
Acute myocardial infarction (AMI)
n ¼ 302
n ¼ 152
n ¼ 54
n ¼ 96
68.4 ± 15.5 14 (25.9%)
63.9 ± 13.7 25 (26.0%)
46 (85.2%)
82 (86.3%)
14 (25.9%) 41 (75.9%)
37 (38.5%) 48 (51.1%)
13 (24.1%) 12 (22.2%)
32 (33.3%) 16 (16.7%)
1 (1.9%)
0 (0%)
Agea 67.6 ± 8.6 67.9 ± 10.4 Female gender 81 (26.8%) 42 (27.6%) Cardiovascular risk factors e no (%) Arterial 261 (86.4%) 133 Hypertension (87.5%) Diabetes 104 (34.4%) 53 (34.9%) Hyperlipidemia 194 (64.2%) 105 (69.1%) Tobacco use Active 74 (24.5%) 29 (19.1%) - Former smoker 62 (20.5%) 34 (22.4%) (>6 months) - Former smoker 5 (1.7%) 4 (2.6%) (<6 months) Acute coronary syndrome - UAP 54 (17.9%) - NSTEMI 70 (23.2%) - STEMI 26 (8.6%) LV-Function 52.2% ± 10.8% 53.6 ± 10.3 Comedication e no (%) at admission ASA 193 (63.9%) 113 (74.3%) Clopidogrel 42 (13.9%) 30 (19.7%) Prasugrel 10 (3.3%) 10 (6.6%) Ticagrelor 15 (5.0%) 12 (7.9%) ACE-Inhibitors 134 (44.4%) 83 (54.6%) AT1-Blockers 66 (21.9%) 38 (25%) Beta-Blockers 187 (61.9%) 116 (76.3%) Statins 164 (54.3%) 107 (70.4%) y
55.59 ± 10.3 48.7 ± 11.0 36 (66.7%)
44 (45.8%)
5 (9.3%) 0 (0%) 1 (1.9%) 19 (45.4%) 14 (25.9%) 33 (61.1%)
7 (7.3%) 0 (0%) 2 (2.1%) 32 (33.3%) 14 (14.6%) 38 (39.6%)
30 (55.6%)
27 (28.1%)
Hyperlipidemia was defined as triglycerides ¼ 175 mg/dl and/or LDLcholesterol ¼ 100 mg/dl and/or taking any of lipid lowering drugs, ASA: aspirin. a Mean value ± standard deviation.
4. Discussion Platelets play an important role in the genesis of atherosclerosis as well as in its late sequels, for instance thrombus formation resulting in ACS and myocardial infarction. Recent mouse studies highlighted a potential therapeutic relevance of complement activation in atherosclerosis [17]. Results from clinical trials with various complement inhibitors turned out ambiguous [19e21,36]. Since expression of complement receptors has been thought to be restricted to a defined cell pool of inflammatory cells, the contribution of platelet derived complement receptors was not considered in this context, so far. Here, we evaluated the expression of platelet surface markers (P-selectin, SDF-1) and their correlation with platelet expressed complement receptors (C3aR and C5aR) in a large cohort of ACS patients. In this study, we could demonstrate for the first time that both C3aR and C5aR were up-regulated in patients with coronary artery disease compared to healthy untreated controls and that the expression of the two anaphylatoxin receptors on platelets correlates with markers of platelet activation including agonist induced aggregation as well as P-selectin and SDF-1 surface expression. There was no effect of pretreatment neither with aspirin nor with a P2Y12 inhibitor on C3aR and C5aR
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Fig. 1. (A, B, C): Platelets in whole blood of healthy controls or patients with CAD were analyzed for the surface expression of receptors using flow cytometry. (A) The gating strategy and representative flow cytometry diagrams are depicted. Platelets were identified using CD42b, histograms show expression levels of C3aR and C5aR (the blue curve represents an example of a healthy individual, the red line shows an example of a patient with CAD).(B) Surface expression of C3aR on platelets in patients with stable CAD and ACS [stable CAD: median MFI 7.94 IQR 3.28, p > 0.001; UA: median MFI 7.74, IQR 3.7, p ¼ 0.049; AMI: median MFI 7.1, IQR 2.52 p ¼ 0.49 vs. healthy volunteers 7.16 IQR 1.47) (median MFI with 25th and 75th percentile)). (C) Surface expression of C5aR on platelets in patients with stable CAD and ACS [stable CAD: median 14.68, IQR 5.2, UA: median MFI 14.56 IQR 5.18, AMI median MFI 13.34, IQR 4.52 vs. 10.68, IQR 3.1 healthy controls, p < 0.001]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. In parallel to flow cytometric analysis, serum plasma was prepared from the patients' blood for analysis by ELISA.(A, B). Correlation of platelet C3aR and C5aR and plasma C3a and C5a levels (spearman rank coefficient r ¼ 0.24, p ¼ 0.001and r ¼ 0.13, p ¼ 0.67, respectively). Plasma levels of C3a and C5a were analyzed by ELISA. Spearman's rank correlation, r ¼ correlation coefficient).
expression. The expression of C3aR was enhanced in SAP and UAP not in AMI, whereas C5aR was enhanced in all three groups. This could be due to differential expression of the receptors in the time course after myocardial infarction, or the receptors may have differential functions after myocardial infarction such as mediating regenerative vs. inflammatory reactions. We recently demonstrated that C5a but not C3a modulates angiogenesis through binding to its receptor
on macrophages, which induced the release of anti-angiogenic factors [37]. Accordingly, the differential role of both anaphylatoxin receptors in atherosclerotic disease will have to be assessed thoroughly in further experimental and clinical studies. Our experiments showed that the concentration of soluble C3a and C5a was constant in all three groups, which may indicate a regulation on the receptor level, rather than on the level of the soluble ligand. Data currently available seem to support a dual role of the
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Fig. 3. (A, B)Correlation of platelet surface expression of C3aR (A) and C5aR (B)and the degree of platelet activation analyzed by expression of P-selectin (spearman rank coefficient r ¼ 0.48, p < 0.001 and r ¼ 0.76, p < 0.001, respectively).
complement system, i.e. proatherosclerotic or antiatherosclerotic effects of complement in atherosclerosis [15]. Platelet C3aR and C5aR having different ligands, namely C3a and C5a, could be responsible for those differential effects in atherosclerosis. One possible explanation for the differential expression of anaphylatoxin receptors on platelets in the different groups might be that anaphylatoxin-receptors become more and more internalized in the progress of CAD from SAP towards AMI. One other explanation could be that they are bound with anaphylatoxins and, thus, are not detectable via FACS-analysis any more. There are data suggesting that C3 has an antiatherosclerotic effect [38,39]. One hypothesis would be that C3a being a cleavage product of C3 is increasingly bound to platelet C3aR in UAP and AMI in an attempt to counteract the deleterious course of disease. C5a however, has been shown to have proatherogenic effects [17,18]. Taken together, future mechanistic in vitro, ex vivo and in vivo studies will have to address, whether the anaphylatoxins C3a and C5a and their corresponding receptors on platelets influence atherosclerosis, platelet activation and the diseases caused by atherothrombosis on different levels. Recently, it was shown that platelets missing a cell nucleus are capable of protein synthesis [40,41]. Interestingly, in the case of platelet C3aR increased surface expression after platelet activation was not apparent any more, when platelet protein synthesis was inhibited. There is emerging evidence that the activation of complement receptors on platelets may be of pathophysiological relevance in certain diseases such as atypical haemolytic uraemic syndrome (aHUS) [42]. Our results point in the same direction for patients with CAD as we found that in the prothrombotic environment in ACS patients, the up-regulation of anaphylatoxin receptors C3aR and C5aR on platelets correlates with platelet activation markers such as SDF-1 and P-selectin. It is well known that platelet Pselectin is associated with plateleteleukocyte interaction [43]as well as recruitment and aggregation of platelets at sites of vascular lesions [44]. P-selectin may also promote interaction between platelets and the complement system. In fact, del Conde et al. could show that P-Selectin binds C3b which led to both C3a generation and C5b-9 MAC complex formation [45]. The role of this interaction for patients with CAD will have to be addressed in future trials. We furthermore observed a correlation of anaphylatoxin receptors with other markers of platelet activation. SDF-1 is a major chemokine for stem cell mobilization from bone marrow to ischemic tissues [46,47] and has been associated with platelet activation in ACS patients but not stable angina [12,34]. It is involved in vascular inflammation and has been detected in significant amounts in
atherosclerotic plaques [48]. Here, we could demonstrate that in ACS patients a positive correlation exists between platelet surface bound SDF-1 and platelet expression of anaphylatoxin receptors C5aR and C3aR. We analyzed only a defined part of the complement cascade in the context of platelet activation e the anaphylatoxins C3a and C5a and their receptors. Mechanisms involved in activation of the classical pathway, the lectin pathway and the alternative pathway converging in cleavage of C3 were not evaluated towards their influence on platelet activation in this study, but may deserve future consideration and study. For example, it is known that a casein kinase released from activated platelets causes phosphorylation of C3 and thus amplifies the complementmediated binding of immune complexes to complement receptor 1 [49]. Also further downstream events of the complement cascade could influence platelet function and will therefore have to be considered in the context of platelet activation. For instance, it was already demonstrated that formation of the membrane attack complex (MAC) enhances platelet activation and consecutively the hemostatic response [50]. Given the positive results in first clinical trials with inhibitors in patients suffering from CAD [51,52] and the very potent drug pipeline for inhibitors of the complement cascade [53], potential translational applications of complement targeted therapeutics for CAD may become evident in the next years. As we found that the expression of anaphylatoxin receptors on platelets is increased in patients with a prothrombotic state presenting with ACS, and their expression on platelets correlates with platelet activation markers, a crosstalk of platelet derived molecules with the complement cascade should be taken into future experimental and therapeutic considerations. This study certainly has limitations, for instance the different patient characteristics regarding premedication. At admission and time of blood sampling 64% were on aspirin and 22% had a P2Y12 inhibitor, after PCI all patients were put on ASS and a P2Y12 inhibitor. Statistical analysis showed no influence of premedication with aspirin of P2Y12 inhibitor on platelet C3a or C5a receptor expression. Blood sampling in patients was performed via arterial sheaths whereas healthy controls had cubital blood sampling due to ethical reasons. The different methods of sampling could have influenced the degree of platelet activation. Future studies will have to address, whether the positive correlation of anaphylatoxin receptor expression on platelets with markers of their activation can be seen in the general context of inflammation with subsequent platelet activation, whether C3aR
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Fig. 4. Correlation of platelet surface expression of C3aR (A) and C5aR (B) and the degree of platelet activation analyzed by platelet SDF-1 expression (spearman rank coefficient r ¼ 0.31, p < 0.001 and r ¼ 0.44, p < 0.001, respectively). (Spearman's rank correlation, r ¼ correlation coefficient).
Fig. 5. Correlation between the surface expression of C3aR (A) and C5aR (B) and platelet function analyzed by ADP-induced platelet aggregation (spearman rank coefficient r ¼ 0.17, p ¼ 0.003and r ¼ 0.14, p ¼ 0.005, respectively). (Spearman's rank correlation, r ¼ correlation coefficient).
and C5aR on platelets contributes to this inflammation or whether there is a potential crosstalk of the described molecules with C3aR and C5aR on the platelet surface. Sources of founding This work was supported by grants from the Deutsche Forschungsgemeinschaft (KFO274) and the Tuebingen Platelet Investigative Consortium (TuePIC). This work was furthermore supported by the Volkswagen Foundation (Lichtenberg program), the Juniorprofessorenprogramm of the county BadenWuerttemberg, the German Heart Foundation and the Wilhelm Sander e Foundation (Project Nr. 2011.005.1). Disclosures The authors have nothing to disclose. Acknowledgments The authors acknowledge Sarah Gekeler for excellent technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.12.002.
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Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease J. Patzelt a, b, 1, K.A.L. Mueller b, 1, S. Breuning b, A. Karathanos b, R. Schleicher a, P. Seizer b, M. Gawaz b, H.F. Langer a, b, *, 2, T. Geisler b, **, 2 a b
Section for Cardioimmunology, Eberhard-Karls University Tuebingen, 72076 Tuebingen, Germany University Hospital, Department of Cardiovascular Medicine, Eberhard-Karls University Tuebingen, 72076 Tuebingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 15 October 2014 Accepted 4 December 2014 Available online 18 December 2014
Objective: Inhibition of components of the complement system or of its receptors has been postulated as a concept for primary and secondary prevention in atherosclerosis and was applied in clinical trials. Although the anaphylatoxin-receptors C3aR and C5aR are commonly associated with inflammatory cells, in vitro studies suggested their expression also on platelets. Methods and Results: Expression levels of C3aR and C5aR were measured by flow cytometry in a collective of 302 patients with documented coronary artery disease (CAD) including patients with stable CAD (n ¼ 152), unstable angina (n ¼ 54), acute myocardial infarction (AMI; Non-ST elevation myocardial infarction, n ¼ 70, ST elevation MI, n ¼ 26) or healthy controls (n ¼ 21). Patients with stable CAD, unstable angina or AMI had significantly higher expression of C5aR on platelets in comparison to healthy controls (MFI 14.68 (5.2), 14.56 (5.18) and 13.34 (4.52) versus 10.68 (3.1)); p < 0.001). In contrast, the expression of C3aR on platelets was significantly enhanced in patients with stable and unstable CAD but not in patients with AMI compared to controls. While there was a strong correlation between the soluble ligands of these receptors C3a and C5a, we observed only a weak correlation with their receptors on platelets. Similarly, agonist induced aggregation (MEA, ADP, and TRAP) showed only a weak correlation with the expression level of anaphylatoxin e receptors on platelets. Of note, the expression of both anaphylatoxin-receptors on platelets strongly correlated with platelet activation as assessed with the surface activation marker P-selectin (r ¼ 0.47, p > 0.001 for C3aR, r ¼ 0.76 for C5aR, p < 0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1. Conclusion: In summary, we observed a positive correlation between the expression of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with CAD. Further investigations are needed to study the clinical and mechanistic relevance of these findings. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Anaphylatoxin receptors C3a C5a Platelets Coronary heart disease
1. Introduction Historically, platelets have been predominantly associated with late events in the course of atherosclerotic disease, for instance when the atherosclerotic plaque ruptures [1]. As a consequence, life threatening vascular thrombotic complications such as myocardial
* Corresponding author. Section for Cardioimmunology, Eberhard-Karls University Tübingen, Otfried-Mueller-Straße 10, 72076 Tübingen, Germany. ** Corresponding author. E-mail addresses: [email protected] (H.F. Langer), tobias. [email protected] (T. Geisler). 1 These authors contributed equally. 2 These authors share senior authorship. http://dx.doi.org/10.1016/j.atherosclerosis.2014.12.002 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
infarction or stroke become apparent [1,2]. Accumulating evidence indicates that platelets may contribute to atherosclerosis by promoting diverse pro-inflammatory mechanisms even before the atherosclerotic plaque forms [3e5]. Platelet adhesion to the vasculature promotes endothelial inflammation [3], subsequent recruitment of leukocytes by platelets enforces accumulation of inflammatory cells [6] and secretion of paracrine effectors or upregulation of proinflammatory receptors on platelets such as CD40L or P-selectin then aggravate the inflammatory reaction [7e9]. Recently, novel platelet associated effectors including SDF-1 and its receptors on platelets have been reported to be upregulated in atherosclerotic patients and may contribute to the pathophysiology of the disease [10e12]. Although very early reports suggested a potential role of the complement system for platelet function
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[13,14], information on a potential relevance of the complement system in diseases featuring platelet activation are scarce. Recent evidence has highlighted a role of the complement system in atherosclerosis. For instance, complement components have been identified in relevant amounts in atherosclerotic plaques [15,16]. Inhibition of C5 in murine models of atherosclerosis resulted in reduced atherosclerosis [17,18]. Moreover, the efficacy targeting complement in coronary artery disease (CAD) was questioned in several clinical trials and was partially successful in patients with CAD. A trial [19] found significantly reduced mortality in STEMI patients undergoing treatment with an anti C5 antibody. Other trials that examined complement inhibition in patients undergoing coronary artery bypass graft surgery found positive effects on morbidity and mortality [20,21]. Although early reports showed that complement receptors may be expressed on platelets [22e24] and the relevance of platelet activation for the course of CAD is unquestionable, a functional or pathophysiological role of complement receptor expression on platelets in this setting still remains to be established. Here, we assessed the expression of complement receptors on platelets and their correlation with platelet activation markers in patients with symptomatic CAD. 2. Methods 2.1. Patient characteristics and blood sampling We included 302 consecutive patients with symptomatic coronary artery disease (CAD). 152 patients had stable angina pectoris (SAP), 54 unstable angina pectoris (UAP) and 96 patients presented with acute myocardial infarction (AMI). AMI was diagnosed by a rise and/or fall of cardiac biomarker values [cardiac troponin (cTn)] with at least one value above the 99th percentile upper reference limit and with at least one of the following: Symptoms of ischemia, new or presumed new significant ST-segment-T wave (STeT) changes or new left bundle branch block (LBBB), development of pathological Q waves in the ECG, imaging evidence of new loss of viable myocardium or new regional wall motion abnormality or identification of an intracoronary thrombus by angiography [25]. The study was approved by the institutional ethics committee (270/ 2011BO1) and complies with the declaration of Helsinki and the good clinical practice guidelines [26e28]. For the cohort study, blood samples were collected from the arterial sheaths of patients with symptomatic CAD before percutaneous coronary intervention (PCI) and were immediately analyzed for surface expression of C3aR, C5aR, P-Selectin, and SDF1 by flow cytometry. Soluble C5a and C3a in the plasma were analyzed by ELISA. Venous blood samples of n ¼ 21 healthy volunteers served as controls. Blood was carefully drawn from the antecubital vein to prevent platelet activation. Platelet function and aggregation was measured by agonist induced aggregometry using the Multiplate analyzer. All blood samples were processed within 1 h after blood withdrawal. All subjects gave written informed consent. Patients were admitted to the department of cardiology of the University of Tübingen, Germany.
mouse monoclonal anti human SDF-1 (R&D systems), and mouse anti human CD42b PE (Becton Dickinson) or their respective isotype controls (mouse IgG1-FITC, mouse IgG2bPE from R&D systems) for 30 min at room temperature. After staining, the cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry (FACS-Calibur flow cytometer BectoneDickinson, Heidelberg, Germany). In some experiments, protein synthesis was inhibited by puromycin (2,5 mg/ml, Invitrogen, 30 min prior to platelet activation with 20 mM ADP) as indicated in figure legends or platelet alphagranule release was blocked by 30 min preincubation with brefeldin a (Cell Signaling Technologies, 10 mg/ml). 2.3. Enzyme-linked immunosorbent assay(ELISA) Plasma levels of C3a and C5a were determined in plasma collected from enrolled patients (n ¼ 302) or healthy volunteers (n ¼ 21) using a commercially available enzyme-linked immunosorbent assay kit according to the manufacturer's guidelines (R&D Systems, Minneapolis, MN, USA). Blood was collected from the arterial sheaths of patients during heart catheterization to avoid platelet activation and processed immediately. Ethylenediamine tetraacetic acid plasma probes were centrifuged for 15 min at 10.000 g within 30 min of collection. Probes were aliquoted and stored at 80 C until analysis. 2.4. Platelet function tests and aggregometry ADP-induced platelet aggregation (PA) in whole blood was assessed with multiple electrode aggregometry (MEA) using the Multiplate© analyzer (Dynabyte, Munich, Germany). This assay is suitable for monitoring antiplatelet drug response in different settings [29e31] taking into account physiological and inflammatory blood compounds that might affect platelet function. ADP (6.4 mol/l) was added to a 1:1 dilution of whole blood anticoagulated with hirudine and 0.9% NaCl. Impedance with MEA was continuously recorded for 5 min plotting arbitrary aggregation units (AU) against time (AU min) after stirring for 3 min in the test cuvettes at 37 C [31,32]. Results were analyzed as area under the curve units (AU min). All material and consumables used in the aggregometry including agonist solution were provided by the manufacturer (Dynabyte, Munich, Germany). 2.5. Statistical analysis All statistical analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago IL). Normally distributed data were compared by using independent student's T-test. Non-parametric data, including MFIs were compared using the U-Test by Mann and Whitney. Correlations were assessed by Spearman's rank correlation coeffi~ ). MFIs are presented as median values and 25th- and 75th cient (n percentiles. KruskaleWallis was performed to test for differences between multiple groups of SAP, UAP, AMI and healthy controls. 3. Results
2.2. Surface expression of platelet receptors analyzed by whole blood flow cytometry Platelets in whole blood were analyzed for the surface expression of C3aR, C5aR, SDF-1, and P-selectin (CD62P) gating for the platelet specific marker CD42b. Blood collected in CPDA was diluted 1:50 with PBS (Gibco) and incubated with the respective conjugated antibodies-mouse monoclonal anti human C3aR-FITC (AbD Serotec), mouse monoclonal anti human C5aR-FITC (AbD Serotec), mouse monoclonal anti human p-Selectin-FITC (Beckman Coulter),
Although a potential relevance of both platelet activation and initiation of the complement cascade in the course of atherosclerosis and particularly in ACS were suggested, expression of proinflammatory complement receptors in a sufficient patient cohort has not been analyzed, so far. Here, we investigated platelet expression of anaphylatoxin receptors C3aR and C5aR on platelets and their corresponding ligands C3a and C5a generated during complement activation as soluble effectors in the blood of patients with symptomatic CAD and healthy volunteers in a pilot study.
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3.1. Platelet surface expression of C3aR and C5aR is associated with the presence of symptomatic CAD and ACS
3.2. Surface platelet expression of C3aR and C5aR correlates with platelet activation and aggregation
Patients' baseline characteristics (age, gender, cardiovascular risk factors, co-medication) of the cohort (n ¼ 302 patients) and the subgroups of stable CAD (n ¼ 152), UAP (n ¼ 54) and AMI (n ¼ 96) are depicted in Table 1. 21 healthy untreated volunteers served as controls. Expression levels of C3aR and C5aR were analyzed in whole blood by flow cytometry. We gated on platelets using the specific marker CD42b as shown in Fig. 1A. Since the data for C3a and C5a receptor measured by flow cytometry (MFI) showed a nonparametric distribution, we first performed a multigroup comparison using the KruskaleWallis test. The test revealed significant intra-group differences for C3aR and C5aR between groups of healthy volunteers, patients with SAP, UAP and AMI (p<0.001 for both C3aR and for C5aR). Therefore we compared individual groups using ManneWhitney tests. The surface expression of C5aR on platelets was significantly higher in patients with stable CAD, UA or AMI compared to the control group [stable CAD: median 4.68, IQR 5.2, UA: median MFI 14.56 IQR 5.18, AMI median MFI 13.34, IQR 4.52 vs. 10.68, IQR 3.1 healthy controls p < 0.001] C3aR surface expression was significantly increased in stable CAD patients and patients with UA but not in patients with AMI compared to healthy volunteers [stable CAD: median MFI 7.94IQR3.28, p > 0.001; UA: median MFI 7.74, IQR 3.7, p ¼ 0.049 AMI: median MFI 7.1, IQR 2.52 p ¼ 0.49 vs. healthy volunteers 7.16 IQR 1.47) (Fig. 1B, C).
Plasma levels of C3a and C5a were measured by ELISA in 191 patients. As expected, we could detect a strong correlation of the soluble plasma amounts of C3a and C5a as shown in Supplemental Fig. 1(spearman rank coefficient 0.46, p < 0.001). Platelet C3aR and C5aR only weakly correlated with plasma C3a and not with C5a levels, respectively (spearman rank coefficient r ¼ 0.24, p ¼ 0.001 and r ¼ 0.13, p ¼ 0.67, respectively), Fig. 2A, B. Recent studies reported that platelet activation as assessed by platelet P-selectin is associated with substantial increase in platelet expression of SDF-1 [33e35]. Therefore, we analyzed the correlation of platelet C3aR and C5aR and these established platelet activation markers. Interestingly, platelet C3aR and C5aR showed a strong and significant correlation with the degree of platelet activation measured by the expression of P-selectin (spearman rank coefficient r ¼ 0.48, p < 0.001 and r ¼ 0.76, p < 0.001, respectively), Fig. 3A, B. Interestingly, in the presence of puromycin inhibiting platelet protein synthesis C3aR expression induced by ADP was not apparent any more (Supplemental Fig. 2), whereas there was no difference regarding its expression in the presence or absence of brefeldin inhibiting platelet granula release (data not shown). We could also detect a significant correlation between C3aR, C5aR and platelet SDF-1 expression (spearman rank coefficient r ¼ 0.31, p < 0.001 and r ¼ 0.44, p < 0.001, respectively), Fig. 4A, B. Moreover, the surface expression of C3aR and C5aR showed a weak but significant correlation with platelet function analyzed by ADP-induced platelet aggregation (spearman rank coefficient r ¼ 0.17, p ¼ 0.003 and r ¼ 0.14, p ¼ 0.005, respectively) shown in Fig. 5A, B. There was no significant difference for platelet C3aR and C5aR expression whether patients were pretreated with aspirin or not (MFI 7.76 vs. 8.38; p ¼ 0.11 and 15.2 vs. 15.7; p ¼ 0.49, respectively). Similarly, there was no effect of P2Y12 inhibitor pretreatment on receptor expression (clopidogrel C3aR: 7.57 vs. 8.14; p ¼ 0.28; C5aR: 15.16 vs. 15.44; p ¼ 0.79; prasugrel C3aR: 6.66 vs. 8.14; p ¼ 0.16; C5AR: 16.72 vs. 15.44; p ¼ 0.51; ticagrelor C3aR: 7.93 vs. 8.14; p ¼ 0.83; C5aR: 14.26 vs. 15.44; p ¼ 0.46). In summary, we observed a significant correlation between both complement receptor expression with various platelet activation markers, but only a weak correlation with platelet function measured by conventional aggregometry.
Table 1 Baseline characteristics of the Patient cohort. Characteristics
Patient collective
Stable CAD Unstable (SAP) angina (UAP)
Acute myocardial infarction (AMI)
n ¼ 302
n ¼ 152
n ¼ 54
n ¼ 96
68.4 ± 15.5 14 (25.9%)
63.9 ± 13.7 25 (26.0%)
46 (85.2%)
82 (86.3%)
14 (25.9%) 41 (75.9%)
37 (38.5%) 48 (51.1%)
13 (24.1%) 12 (22.2%)
32 (33.3%) 16 (16.7%)
1 (1.9%)
0 (0%)
Agea 67.6 ± 8.6 67.9 ± 10.4 Female gender 81 (26.8%) 42 (27.6%) Cardiovascular risk factors e no (%) Arterial 261 (86.4%) 133 Hypertension (87.5%) Diabetes 104 (34.4%) 53 (34.9%) Hyperlipidemia 194 (64.2%) 105 (69.1%) Tobacco use Active 74 (24.5%) 29 (19.1%) - Former smoker 62 (20.5%) 34 (22.4%) (>6 months) - Former smoker 5 (1.7%) 4 (2.6%) (<6 months) Acute coronary syndrome - UAP 54 (17.9%) - NSTEMI 70 (23.2%) - STEMI 26 (8.6%) LV-Function 52.2% ± 10.8% 53.6 ± 10.3 Comedication e no (%) at admission ASA 193 (63.9%) 113 (74.3%) Clopidogrel 42 (13.9%) 30 (19.7%) Prasugrel 10 (3.3%) 10 (6.6%) Ticagrelor 15 (5.0%) 12 (7.9%) ACE-Inhibitors 134 (44.4%) 83 (54.6%) AT1-Blockers 66 (21.9%) 38 (25%) Beta-Blockers 187 (61.9%) 116 (76.3%) Statins 164 (54.3%) 107 (70.4%) y
55.59 ± 10.3 48.7 ± 11.0 36 (66.7%)
44 (45.8%)
5 (9.3%) 0 (0%) 1 (1.9%) 19 (45.4%) 14 (25.9%) 33 (61.1%)
7 (7.3%) 0 (0%) 2 (2.1%) 32 (33.3%) 14 (14.6%) 38 (39.6%)
30 (55.6%)
27 (28.1%)
Hyperlipidemia was defined as triglycerides ¼ 175 mg/dl and/or LDLcholesterol ¼ 100 mg/dl and/or taking any of lipid lowering drugs, ASA: aspirin. a Mean value ± standard deviation.
4. Discussion Platelets play an important role in the genesis of atherosclerosis as well as in its late sequels, for instance thrombus formation resulting in ACS and myocardial infarction. Recent mouse studies highlighted a potential therapeutic relevance of complement activation in atherosclerosis [17]. Results from clinical trials with various complement inhibitors turned out ambiguous [19e21,36]. Since expression of complement receptors has been thought to be restricted to a defined cell pool of inflammatory cells, the contribution of platelet derived complement receptors was not considered in this context, so far. Here, we evaluated the expression of platelet surface markers (P-selectin, SDF-1) and their correlation with platelet expressed complement receptors (C3aR and C5aR) in a large cohort of ACS patients. In this study, we could demonstrate for the first time that both C3aR and C5aR were up-regulated in patients with coronary artery disease compared to healthy untreated controls and that the expression of the two anaphylatoxin receptors on platelets correlates with markers of platelet activation including agonist induced aggregation as well as P-selectin and SDF-1 surface expression. There was no effect of pretreatment neither with aspirin nor with a P2Y12 inhibitor on C3aR and C5aR
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Fig. 1. (A, B, C): Platelets in whole blood of healthy controls or patients with CAD were analyzed for the surface expression of receptors using flow cytometry. (A) The gating strategy and representative flow cytometry diagrams are depicted. Platelets were identified using CD42b, histograms show expression levels of C3aR and C5aR (the blue curve represents an example of a healthy individual, the red line shows an example of a patient with CAD).(B) Surface expression of C3aR on platelets in patients with stable CAD and ACS [stable CAD: median MFI 7.94 IQR 3.28, p > 0.001; UA: median MFI 7.74, IQR 3.7, p ¼ 0.049; AMI: median MFI 7.1, IQR 2.52 p ¼ 0.49 vs. healthy volunteers 7.16 IQR 1.47) (median MFI with 25th and 75th percentile)). (C) Surface expression of C5aR on platelets in patients with stable CAD and ACS [stable CAD: median 14.68, IQR 5.2, UA: median MFI 14.56 IQR 5.18, AMI median MFI 13.34, IQR 4.52 vs. 10.68, IQR 3.1 healthy controls, p < 0.001]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. In parallel to flow cytometric analysis, serum plasma was prepared from the patients' blood for analysis by ELISA.(A, B). Correlation of platelet C3aR and C5aR and plasma C3a and C5a levels (spearman rank coefficient r ¼ 0.24, p ¼ 0.001and r ¼ 0.13, p ¼ 0.67, respectively). Plasma levels of C3a and C5a were analyzed by ELISA. Spearman's rank correlation, r ¼ correlation coefficient).
expression. The expression of C3aR was enhanced in SAP and UAP not in AMI, whereas C5aR was enhanced in all three groups. This could be due to differential expression of the receptors in the time course after myocardial infarction, or the receptors may have differential functions after myocardial infarction such as mediating regenerative vs. inflammatory reactions. We recently demonstrated that C5a but not C3a modulates angiogenesis through binding to its receptor
on macrophages, which induced the release of anti-angiogenic factors [37]. Accordingly, the differential role of both anaphylatoxin receptors in atherosclerotic disease will have to be assessed thoroughly in further experimental and clinical studies. Our experiments showed that the concentration of soluble C3a and C5a was constant in all three groups, which may indicate a regulation on the receptor level, rather than on the level of the soluble ligand. Data currently available seem to support a dual role of the
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Fig. 3. (A, B)Correlation of platelet surface expression of C3aR (A) and C5aR (B)and the degree of platelet activation analyzed by expression of P-selectin (spearman rank coefficient r ¼ 0.48, p < 0.001 and r ¼ 0.76, p < 0.001, respectively).
complement system, i.e. proatherosclerotic or antiatherosclerotic effects of complement in atherosclerosis [15]. Platelet C3aR and C5aR having different ligands, namely C3a and C5a, could be responsible for those differential effects in atherosclerosis. One possible explanation for the differential expression of anaphylatoxin receptors on platelets in the different groups might be that anaphylatoxin-receptors become more and more internalized in the progress of CAD from SAP towards AMI. One other explanation could be that they are bound with anaphylatoxins and, thus, are not detectable via FACS-analysis any more. There are data suggesting that C3 has an antiatherosclerotic effect [38,39]. One hypothesis would be that C3a being a cleavage product of C3 is increasingly bound to platelet C3aR in UAP and AMI in an attempt to counteract the deleterious course of disease. C5a however, has been shown to have proatherogenic effects [17,18]. Taken together, future mechanistic in vitro, ex vivo and in vivo studies will have to address, whether the anaphylatoxins C3a and C5a and their corresponding receptors on platelets influence atherosclerosis, platelet activation and the diseases caused by atherothrombosis on different levels. Recently, it was shown that platelets missing a cell nucleus are capable of protein synthesis [40,41]. Interestingly, in the case of platelet C3aR increased surface expression after platelet activation was not apparent any more, when platelet protein synthesis was inhibited. There is emerging evidence that the activation of complement receptors on platelets may be of pathophysiological relevance in certain diseases such as atypical haemolytic uraemic syndrome (aHUS) [42]. Our results point in the same direction for patients with CAD as we found that in the prothrombotic environment in ACS patients, the up-regulation of anaphylatoxin receptors C3aR and C5aR on platelets correlates with platelet activation markers such as SDF-1 and P-selectin. It is well known that platelet Pselectin is associated with plateleteleukocyte interaction [43]as well as recruitment and aggregation of platelets at sites of vascular lesions [44]. P-selectin may also promote interaction between platelets and the complement system. In fact, del Conde et al. could show that P-Selectin binds C3b which led to both C3a generation and C5b-9 MAC complex formation [45]. The role of this interaction for patients with CAD will have to be addressed in future trials. We furthermore observed a correlation of anaphylatoxin receptors with other markers of platelet activation. SDF-1 is a major chemokine for stem cell mobilization from bone marrow to ischemic tissues [46,47] and has been associated with platelet activation in ACS patients but not stable angina [12,34]. It is involved in vascular inflammation and has been detected in significant amounts in
atherosclerotic plaques [48]. Here, we could demonstrate that in ACS patients a positive correlation exists between platelet surface bound SDF-1 and platelet expression of anaphylatoxin receptors C5aR and C3aR. We analyzed only a defined part of the complement cascade in the context of platelet activation e the anaphylatoxins C3a and C5a and their receptors. Mechanisms involved in activation of the classical pathway, the lectin pathway and the alternative pathway converging in cleavage of C3 were not evaluated towards their influence on platelet activation in this study, but may deserve future consideration and study. For example, it is known that a casein kinase released from activated platelets causes phosphorylation of C3 and thus amplifies the complementmediated binding of immune complexes to complement receptor 1 [49]. Also further downstream events of the complement cascade could influence platelet function and will therefore have to be considered in the context of platelet activation. For instance, it was already demonstrated that formation of the membrane attack complex (MAC) enhances platelet activation and consecutively the hemostatic response [50]. Given the positive results in first clinical trials with inhibitors in patients suffering from CAD [51,52] and the very potent drug pipeline for inhibitors of the complement cascade [53], potential translational applications of complement targeted therapeutics for CAD may become evident in the next years. As we found that the expression of anaphylatoxin receptors on platelets is increased in patients with a prothrombotic state presenting with ACS, and their expression on platelets correlates with platelet activation markers, a crosstalk of platelet derived molecules with the complement cascade should be taken into future experimental and therapeutic considerations. This study certainly has limitations, for instance the different patient characteristics regarding premedication. At admission and time of blood sampling 64% were on aspirin and 22% had a P2Y12 inhibitor, after PCI all patients were put on ASS and a P2Y12 inhibitor. Statistical analysis showed no influence of premedication with aspirin of P2Y12 inhibitor on platelet C3a or C5a receptor expression. Blood sampling in patients was performed via arterial sheaths whereas healthy controls had cubital blood sampling due to ethical reasons. The different methods of sampling could have influenced the degree of platelet activation. Future studies will have to address, whether the positive correlation of anaphylatoxin receptor expression on platelets with markers of their activation can be seen in the general context of inflammation with subsequent platelet activation, whether C3aR
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Fig. 4. Correlation of platelet surface expression of C3aR (A) and C5aR (B) and the degree of platelet activation analyzed by platelet SDF-1 expression (spearman rank coefficient r ¼ 0.31, p < 0.001 and r ¼ 0.44, p < 0.001, respectively). (Spearman's rank correlation, r ¼ correlation coefficient).
Fig. 5. Correlation between the surface expression of C3aR (A) and C5aR (B) and platelet function analyzed by ADP-induced platelet aggregation (spearman rank coefficient r ¼ 0.17, p ¼ 0.003and r ¼ 0.14, p ¼ 0.005, respectively). (Spearman's rank correlation, r ¼ correlation coefficient).
and C5aR on platelets contributes to this inflammation or whether there is a potential crosstalk of the described molecules with C3aR and C5aR on the platelet surface. Sources of founding This work was supported by grants from the Deutsche Forschungsgemeinschaft (KFO274) and the Tuebingen Platelet Investigative Consortium (TuePIC). This work was furthermore supported by the Volkswagen Foundation (Lichtenberg program), the Juniorprofessorenprogramm of the county BadenWuerttemberg, the German Heart Foundation and the Wilhelm Sander e Foundation (Project Nr. 2011.005.1). Disclosures The authors have nothing to disclose. Acknowledgments The authors acknowledge Sarah Gekeler for excellent technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.12.002.
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