Intracoronary platelet and monocyte activation status within platelet-monocyte complexes are determinants of inflammation in ST elevation myocardial infarction 1

Abstract

INTRODUCTION: Platelet Monocyte Complexes (PMCs) are commonly expressed in coronary artery disease but their pathologic significance in ST elevation myocardial infarction (STEMI) is unclear. This study evaluates the relationship between locally activated PMCs and intracoronary inflammation in stable and unstable coronary disease.

MATERIAL AND METHODS: Micro catheter aspirated blood samples of 15 STEMI and 7 stable angina patients are collected from the coronary artery (CA), aorta (AO) and right atrium (RA). Samples are labelled with monoclonal antibodies and prepared for flow cytometry. CD 14 and CD 61 double positive cells are identified as PMC. P-selectin expression is identified by additional CD62P positivity and TF expression by additional CD142 positivity. Plasma TNF-alpha and IL-6 are measured using ELISA and CRP is measured in plasma using a high sensitivity automated microparticle enhanced latex turbidimetric immunoassay.

RESULTS: No site-specific difference is seen in overall PMC expression in STEMI or stable angina. Surface P-selectin expression in STEMI [median (IQR)] is significantly higher in CA [35.01 (23.15–56.99)] compared with AO [15.99 (10.3–18.85)] or RA [14.02 (10.42–26.08)] (p = 0.003). Intracoronary PMC correlates significantly with intracoronary TNF-alpha (r = 0.87, p = 0.001) and intracoronary IL-6 (r = 0.76, p = 0.03). Bound monocytes within P-selectin positive and tissue factor positive complexes correlate positively with intracoronary TNF-alpha (r = 0.81, p = 0.008 & r = 0.80, p = 0.009 respectively) and IL-6 (r = 0.54, p = 0.16 & r = 0.71, p = 0.05 respectively). No such correlation is observed in the peripheral circulation of STEMI and stable angina patients.

CONCLUSION: Inflammation is not attributable to PMC formation per se. However, increased intracoronary P-selectin expression by activated platelets and tissue factor expression by activated monocytes within the complexes are determinants of local intracoronary inflammatory burden in STEMI.

Keywords

Platelet monocyte complex platelet and monocyte activation status intracoronary inflammation ST elevation myocardial infarction

1 Introduction

Formation of platelet monocyte complexes (PMCs) is well described in ST elevation myocardial infarction (STEMI). However, it is unclear whether this phenomenon is merely a marker of generalised platelet activation or whether these complexes play a more local intracoronary role in the pathogenesis of STEMI. PMC may act as a link between platelet mediated thrombogenesis and monocyte mediated inflammation in STEMI. Binding of platelets with monocytes has been demonstrated in whole blood samples of healthy volunteers and the proportion of platelet bound monocytes has been demonstrated to be higher in cases of unstable angina, myocardial infarction, coronary artery disease and post-angioplasty restenosis [9 , 20].

A previous study has shown that platelets bind to monocytes predominantly by a divalent cation dependant P-selectin- glycoprotein ligand –1 pathway [18]. In addition, other integrin dependant mechanisms may have been involved in the formation of PMCs [5]. One hypothesis is that binding of activated platelets may alter monocyte activation status resulting in an increase in tissue factor expression from the monocyte component acting as a trigger for the initiation of a cascade of thrombogenesis and the release of proinflammatory cytokines, including IL-6, IL-1β and IL-12. However, binding with unstimulated platelets may not be associated with a similar cellular response [3]. Therefore, in the absence of platelet activation PMC formation may just represent a physiological phenomenon. Depending upon the level of activation, the platelets within PMCs may actually be responsible for triggering a pro-inflammatory response in monocytes. It is unclear whether platelet activation and PMC formation is a systemic response or whether there is a direct role for intracoronary PMC formation in the pathophysiology of acute myocardial infarction via monocyte activation, tissue factor(TF) expression and inflammation.

There is evidence that levels of P-selectin positive PMC (% of CD62P+CD14+CD61+ events) underestimate total PMC formation (as measured by double positivity for CD14+ and a constitutive platelet marker such as CD61) due to a phenomenon of P-selectin “shedding”. As a consequence, activated platelets can circulate in the peripheral blood, either bound or unbound to monocytes, even if negative for P-selectin [15]. This could results into a reduced percentage of P-selectin positive PMC in the peripheral circulation if platelet activation and consequent aggregation with monocytes to form PMC begins and/or is maximum at the site of coronary artery lesion. Taking into consideration of the phenomenon of P selectin shedding we measured the soluble P selectin in all the vascular compartments of both these groups to see whether there is any significant difference in the plasma soluble P-selectin levels in these groups of patients.

In this study we have examined the hypothesis that a differential expression of platelet activation (in the form of P-selectin expression) on PMCs occurs locally within the coronary artery compared with the systemic circulation. In addition we have investigated the complex interplay between platelet activation, TF expression (on the platelet bound monocytes) and local intracoronary inflammation in patients with STEMI.

2 Material and methods

Group 1- Fifteen consecutive patients admitted with acute STEMI have been recruited prior to primary percutaneous coronary intervention (PCI). Informed consent is obtained before the procedure. Ethical approval for the study is granted from the local research and ethics committee. All patients are given 600 mg of Clopidogrel and 300 mg of Aspirin before the procedure as per protocol for performing primary PCI in our institution. Prior to PCI patients have also received weight adjusted unfractionated heparin to maintain the activated clotting time (ACT) between 200–250 sec.

Group 2- Fifteen patients with stable angina have also been conseneted as controls prior to elective coronary angiography and ad hoc PCI. Out of these 15 Patients 8 patients had to be excluded following coronary angiography as the clinicians decided to treat those patients medically rather than undergo PCI. Eventually 7 stable patients were considered for percutaneous treatment and were included in the study. All the stable patients received 75 mg of Aspirin and 75 mg of Clopidogrel for 7 days prior to the procedure according to the protocol for performing elective PCI in our hospital. Prior to PCI they also received weight adjusted unfractionated heparin to maintain the ACT between 200–250 sec.

2.1 Exclusion criteria

Patients with renal failure, prior coronary artery bypass grafts and who had already received Glycoprotein IIb/IIIa antagonists prior to sampling were excluded.

2.2 Sample collection and preparation

Samples were aspirated from the coronary artery distal to the culprit lesion (using a coronary aspiration, Medtronic^® Export catheter, internal diameter 0.10 cm), aorta using a Judkin’s diagnostic catheter (5F, Cordis^®, internal diameter 0.11 cm) and from the right atrium, using a pigtail cather (5F, Cordis^®, internal diameter 0.11 cm). It has been previously demonstrated that aspiration of blood through the Export catheter does not induce artefactual platelet activation [25]. To assess systemic venous PMC expression samples were aspirated from the right atrium through the catheter in a similar manner to that of the aorta and coronary artery. This was done to maintain a uniformity of sample collection from different sites. Near identical internal diameters of the aspiration catheters were used in order to maintain uniform shear force while aspiration of blood to prevent differnetial shear force induced platelet activation while collecting the samples. Four mililitres of blood from each site were collected into 3 separate sterile vacutainer tubes containing a combination of 3.2% sodium citrate and EDTA for flow cytometric analysis of PMC, P-selectin and TF expression. EDTA was added to sodium citrate to stop in vitro calcium dependent PMC formation. A further 2.7 ml sample was collected from each site into separate 3.2% sodium citrate vacutainer tubes and the samples were used to prepare double spun platelet poor plasma by centrifuging the samples initially at 800 g for 10 minutes at room temperature. The supernatant was collected into eppendorfs and recentrifuged at 2500 g for 5 minutes and 90% of the supernatant plasma aspirated was immediately stored at –80°C for later analysis of Interleukin–6 (IL-6), C reactive protein (CRP) and tumour necrosis factor (TNF)-alpha with enzyme linked immunosobent assay (ELISA) (R&D systems). We choose to measure TNF- alpha (n = 10) and IL-6 (n = 8) as following PMC formation monocyte activation can result in the secretion of TNF- alpha and IL-6 [10]. Similarly TNF alpha activates platelets to express P-selectin on their surface and promotes PMC formation [7]. Therefore, TNF alpha and IL-6 are intimately associated with platelet actvation, P-selectin expression and PMC formation.

Collected blood samples were placed on ice and transferred to the laboratory for immediate flow cytometry. Analysis of all samples was completed within 2 hours of collection. Two round bottom ploystyrene tubes (BD Falcon 12×75 mm style) were taken. Five microlitre of anti CD61 FITC, anti CD14 PerCp and anti CD 142 PE were taken in the first tube (tube 1) and in the second tube anti CD142 PE was replaced by anti CD62P PE (tube 2). One hundred microlitres of whole blood was aliquoted into each of the tubes. The samples were incubated at room temperature for 20 minutes. After that erythrocytes were lysed by addition of 2 ml of easy lysetrademark solution (Dako) (1 in 20 dilution) for 12 minutes at room temperature. Cells were washed with addition of 1000 microlitres of FACS flow and centrifuged at 300 g for 5 minutes. After that the supernatant was discarded and the cells resuspended in 500 microlitres of FACS flow for immediate flow cytometric analysis (FACS Calibur equipped with Cell Quest^® soft ware –BD Biosciences, Oxford, UK).

2.3 Flow cytometry

Cells were acquired on a 2D dot plot arraying CD14 (logarhithmic scale abscissa) and SSC height (linear scale ordinate). The monocyte population was identified by CD14 expression and distinctive intermediate side scatter height. A minimum of 5000 monocytes are acquired from each sample. An analysis region (R1 in Fig. 1a) was drawn around the monocyte population and cells within R1 are plotted again on a 2D dotplot arraying CD 61 signal width (linear scale abscissa) and CD14 signal height (logarithmic scale abscissa). To exclude false-positive PMC arising from co-incident analysis of free platelets and monocytes, a region (R2 in Fig. 1b) was drawn around the cells with narrow CD61 width and events falling within both R1 and R2 were subsequently plotted onto another 2D dotplot arraying CD61 FITC signal height (logarithmic scale abscissa) and CD 142 PE or CD 62P PE (logarithmic scale ordinate) (Fig. 1c & d) depending on the presence of antibody in tube 1 or tube 2. Co- incident cells have longer time of flight to pass through the laser and can be distinguished by eliciting signals which last longer than the single complex. This is reflected in the wider width of the signal. CD14 & CD61 double positive true PMCs were expressed as the percentage of the total monocyte population. P-selectin expression on the complexes was identified by co-expression of CD62P and TF expression on the bound monocytes was identified by CD 142 PE positivity (Fig. 1c). The P-selectin and TF expression on the complex was expressed as percentage of the total monocyte population as well as a percentage of PMC. The process was standardised in our laboratory and the percentage of PMCs in the peripheral circulation of normal healthy individuals was found to be 2.57±0.31 (CV 13.96±8.30%).

2.4 Estimation of IL-6,TNF-alpha and CRP

Interleukin-6 and TNF-alpha levels were measured using a quantitive sandwich enzyme immunoassay technique using a kit from R&D systems (Europe). Lower levels of detection for IL-6 and TNF alpha were 3.12pg/ml and 15.6 pg/ml respectively. The intra-assay CV for IL-6 varied from 1.6–4.2%, interassay CV 3.3–6.4%. The intra assay CV for TNF-alpha varied from 4.2–5.2% and interassay CV 4.6–7.4%.

CRP was measured in plasma using a high sensitivity automated microparticle enhanced latex turbidimetric immunoassay (COBAS MIRA; Roche Diagnostics GmbH). The lower limit of detection was 0.2 mg/l with an inter assay CV of 4.2% at 4 mg/l and 6.3% at 1 mg/l.

2.5 Estimation of Soluble P-Selectin

Soluble P-Selectin was estimated as a soluble marker of platelet activation. Soluble P-selectin was assayed by the quatitative sandwich immunoassay technique using the R& D Systems kit. The CV of intra assay precision of this kit was between 4.9% and 5.6% and the CV of interassay precision of this test was between 7.9% and 9.9%. Minimal detectable dose of soluble P-selectin was less than 0.5 ng/ml. Mean value from healthy individual was 29 ng/ml (±1sd range 18–40 ng/ml).

2.6 Statistical methods

Normally distributed data were presented as mean±SD; data not normally distributed were presented as median and interquartile range. For comparison of continuous variables p values were calculated by using one way ANOVA for normally distributed data or a non parametric equivalent (Kruskal-Walis test) for non-normally distributed data. Correlation analysis is performed with Pearson’s or Spearman’s correlation method where appropriate. Two-tailed p values <0.05 were considered statistically significant. All calculations were performed using graphpad prism (version 5) software.

3 Results

The clinical and angiographic characteristics of the patients are summarized in Table 1. RCA was the most commonly affected vessel (9/15). All the patients underwent successful PCI and achieved TIMI-3 flow following the procedure.

3.1 Differential PMC expression

In the STEMI group % PMC (mean±sd) in the coronary artery, aorta and right atrium were 15.04±7.91, 12.33±6.92, 12.36±6.91 respectively. Although PMC expression was higher in the coronary compared with aorta and right atrium the difference was not significant (p = 0.85). In stable angina patients % PMC in the coronary artery, aorta and right atrium were 10.75±5.84, 9.78±6.36 and 12.57±4.86 respectively (p = 0.65).

In the STEMI group % PMC expressing P-selectin [median (IQR)] on their surface as a percentage of total PMC (P-selectin positive PMC, PMC) were found to be significantly higher in the coronary circulation [35.01 (23.15–56.99)] compared with the aorta [15.99 (10.3 –18.85)] and right atrium [14.02 (10.42–26.08)] (p = 0.003). No such difference was seen in the stable angina group (p = 0.60).

In the STEMI group % PMC expressing P-selectin [median (IQR)] on their surface as a percentage of the total monocyte population (P-selectin positive PMC, monocyte) was found to be significantly higher in the coronary circulation 5.42 (2.05–10.09) compared with the aorta [2.28 (0.84–3.69)] and right atrium [2.32 (0.90–3.42)] (P = 0.04). No such difference was seen in the stable angina group (p = 0.65).

3.1 PMC expression in STEMI versus stable angina

Percentage of P-selectin positive PMC (median IQR) in the coronary artery of the STEMI group was significantly higher [35.01 (23.15–56.99)] compared to the stable angina group [9.16 (7.42 –15.60)] (p = 0.004). However, in the aorta and right atrium no such difference was noted between these two groups (p = 0.56 in the aorta and p = 0.77 in the right atrium). Soluble P selectin levels were significantly higher in all the compartments of STEMI patients compared to the stable angina group (Table 2) though there were no significant differential increased levels of intracoronary P selectin level in the STEMI group (p = 0.91).

3.2 Inflammatory parameters concentration in STEMI versus stable angina

There was no significant difference in the TNF- alpha concentration in different vascular compartments between the STEMI and stable angina patients (Table 3). IL-6 concentrations were below detection levels in the coronary circulation of 3 patients with stable angina. The median (IQR) levels of IL-6 were 5.54 (2.6–12.6) pg/ml, 6.7 (2.8–10.2) pg/ml and 2.7 (1.5–6.9) pg/ml respectively in the coronary artery, aorta and right atrium of the STEMI patients. The median (IQR) of CRP level was 2.4 (0.8–5.9) mg/l, 3.0 (1.07–6.5) mg/l and 3.0 (1.07–6.5) mg/l respectively in the coronary artery, aorta and right atrium of the STEMI patients respectively (Table 4). There were no significant site specific difference in the TNF alpha, CRP and IL-6 concentrations in the STEMI patients (p = 0.81, p = 0.79 and p = 0.61 respectively).

3.3 Tissue Factor (TF) expression

In the STEMI patients TF expression (mean±SD) by platelet bound monocytes was found to be significantly higher compared to the free monocytes in the coronary artery, aorta and right atrium. No significant difference was noted in the coronary circulation (p = 0.33) and right atrium (p = 0.06) of stable angina patients (Table 5).

3.4 PMC correlates with inflammatory cytokines

In the STEMI group intracoronary PMC expression significantly correlates positively with intracoronary TNF-alpha (r = 0.87, p = 0.001) (Fig. 2) and intracoronary IL-6 (r = 0.76, p = 0.03) (Fig. 2). There was a non-significant positive correlation with intracoronary CRP (r = 0.32, p = 0.14). Similarly, bound monocytes within the P-selectin positive complexes have been found to correlate positively with intracoronary TNF-alpha and IL-6 (r = 0.81, p = 0.008, r = 0.54, p = 0.16 respectively) (Fig. 3). Bound monocytes within the TF positive complexes also demonstrated positive correlations with TNF-alpha and IL-6 (r = 0.80, p = 0.009 & r = 0.71, p = 0.05 respectively) (Fig. 4). No such correlation was observed in the peripheral circulation of the STEMI and in the stable angina patients (data not shown).

4 Discussion

This study demonstrates less measured % PMC compared with previous studies. In this study of STEMI patients % PMC expression (mean±sd) in the coronary artery, aorta and right atrium was 15.04±7.91, 12.33±6.92, 12.36±6.91 respectively. In the peripheral venous circulation of patients with acute coronary syndrome PMC formation has been shown to be 36.5±12.2 % by Wang et al. [21] and 70.1±15.4 % by Sarma et al. [19]. This discrepancy may be explained by co-incident events. As platelet concentration is much higher than monocytes the chances of having one or multiple platelets close to but not attached to a monocyte is higher. When slightly diluted samples are analysed by flow cytometry for platelet-monocyte complexes, the phenomenon of co-incidence cannot be neglected. These are incidents when a non-conjugated monocyte transits the laser adjacent to (but not conjugated to) one or more platelets. Some non interacting cells can come so close that they become indistinguishable and they are detected as one event. In our study we have excluded coincident events carefully by using the doublet discriminator strategy previously used to differentiate doublets containing G _0/1 phase DNA from single cell containing G2/M phase DNA [22]. We have also added EDTA to sodium citrate as an additional anticoagulant in order to reduce the divalent cation dependent ex-vivo PMC formation [3, 14]. These two additional measures during PMC assay have resulted in much lower values in our study. Our results compare favourably with the findings of Furman et al who have reported 11.6±11.4 % PMCs in the circulation of acute myocardial infarction patients [8].

Increased PMC expression is well described in coronary artery disease. Previous studies have demonstrated increased PMC in patients with acute coronary syndrome compared with stable angina patients [19]. Botto et al have demonstrated a local increase of platelet-leukocyte functional interaction at the site of coronary occlusion in STEMI patients [2]. The role of PMC formation locally inside the coronary artery in the pathogenesis of acute coronary event is unclear. It has even been suggested that binding of monocytes with the inactivated platelets can be a physiological phenomenon [3]. Therefore, the assumption that mere PMC formation has proinflammatory consequences has to be taken with caution. In our study though intracoronary PMC expression is higher in the STEMI group; we did not find any statistically significant site specific difference in PMC expression. Neither did we find any significant difference between the STEMI and stable angina patients. We also demonstrated that 35 % of complexes are associated with P-selectin expression by activated platelets in the coronary circulation of the STEMI patients. The percentage of complexes with P-selectin expression are lower in the peripheral circulation and in stable angina patients. This differential P-selectin expression suggests that PMC formation is a systemic phenomenon but the level of platelet activation in the form of P-selectin expression is maximum in the coronary circulation of STEMI patients.

Intracoronary PMC correlated significantly with intracoronary TNF alpha and IL-6 in STEMI patients. This phenomenon is not observed in the peripheral circulation or in stable angina patients. Heightened intracoronary P-selectin expression represents platelet activation within the bound complex which may trigger the proinflammatory response in monocytes. This finding supports the observation by Bournazos et al. [3] that monocyte binding with unstimulated platelets do not affect receptor expression or cytokine production; where as bound monocytes with stimulated platelets demonstrates inflammatory cytokine production. The source of intracoronary TNF alpha and IL-6 is not clear from this study but previous studies have demonstrated monocytes as a source of these cytokines [1, 23].

Increased TF expression on platelelet bound monocytes compared with free monocytes demonstrates heightened monocyte activation within this complex. Kopp et al. have demonstrated significant correlation of monocyte TF expression with constitutive platelet marker CD42b and the platelet activation marker CD62P in unstable angina and NSTEMI [13]. They have also demonstrated TF-activity of isolated mononuclear cells (MNC) was elevated in unstable angina and NSTEMI in comparison to stable angina as determined by chromogenic assay; and TF mRNA expression in isolated MNC was more frequent in unstable angina/NSTEMI than in stable angina. Their findings of increased monocyte TF activity and PMC formation in unstable angina and NSTEMI compared to stable angina suggest an association between PMC and TF activity in the pathogenesis of acute coronary syndrome. The positive correlation of PMC, P-selectin expression and TF expression with intracoronary TNF alpha suggests PMC may play a contributory role in the pathogenesis of intracoronary inflammation in STEMI patients through heightened P-selectin and TF expression. Celi et al have demonstrated [6] platelet surface P-selectin induces the expression of TF on monocytes and promotes fibrin deposition within the growing thrombus at the site of the vascular injury [16].

In conclusion PMC formation is found to be a systemic phenomenon in both STEMI and stable angina patients but differential increased intracoronary P-selectin expression on these complexes in STEMI suggests heightened platetlet activation within these complexes locally in the coronary artery of STEMI patients. We have also demonstrated Increased tissue factor expression by the platelet bound monocytes compared to the free monocytes in STEMI patients. Significant positive correlation of intracoronary P-selectin and TF expression with TNF-alpha suggests that heightened intracoronary platelet activation and local monocyte activation within the complex are related to intracoronary inflammation in STEMI. Submicron vesicles called microparticles derived from activated endothelial cells and platelets modulate inflammation and are particlularly elevated in patients presenting with STEMI due to occlusion of the left anterior descending artery. Furthermore, elevated endothelial and platelet derived microparticles correlated with the myocardium at risk [11].

We can conclude that by expressing P-selectin and TF, PMC may play a role in the pathogenesis of local intracoronary inflammation in STEMI patients. Mere formation of PMCs does not contribute to systemic inflammation as evidenced by the lack of correlation between PMC and inflammatory markers in the peripheral circulation of the STEMI and stable angina patients. Increased intracoronary expression of P-selectin by activated platelets and TF expression by activated monocytes within the complex may determine the local intracoronary burden of inflammation in STEMI patients. This adds to the evidence that in patients with CAD the number of circulating activated platelets are clearly increased [4, 17].

4.1 Study limitation

This is an observational study with small sample volume. As TNF-alpha and IL-6 are measured from plasma, so it is not possible to determine the exact source of these markers. Measurements of these markers on the cell surface of the PMCs could have been a more appropriate approach. All patients are treated with clopidogrel and some studies have shown that clopidogrel reduces PMC in atherosclerotic disease and reduces P-selectin expression [12, 24]. However,in the setting of acute STEMI it is unlikely that clopidogrel, with its weak and slow onset of action would have enough time to be responsible for PMC reduction.

Disclosures

None.

Footnotes

Acknowledgments

Authors acknowledge the contribution of Dr John Hurst and Mr Ray Sapsford (Academic Respiratory Medicine department of Royal Free Hospital) for kindly helping with the ELISA analysis. We acknowledge the contribution of Ms Janet North for her support with the flow cytometry technique. We also acknowledge the valuable opinion and support of Dr. Man Fai Shiu and Dr. John G. Coghlan during this study.

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