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ORIGINAL RESEARCH ARTICLE

Platelet Expression of CD40/CD40 Ligand and Its Relation to Inflammatory Markers and Adhesion Molecules in Patients with Atrial Fibrillation

Matthias Hammwöhner^*, Annelore Ittenson, Jutta Dierkes, Alicja Bukowska, Helmut U. Klein^*, Uwe Lendeckel and Andreas Goette^*,1

^* Division of Cardiology, Department of Internal Medicine, Institute of Immunology, Department of Laboratory Medicine, and Institute of Experimental Internal Medicine, University Hospital Magdeburg, Magdeburg 39120, Germany

¹To whom requests for reprints should be addressed at Division of Cardiology, Department of Internal Medicine, University Hospital Magdeburg, Leipzigerstr. 44, 39120 Magdeburg, Germany. E-mail: andreas.goette{at}medizin.uni-magdeburg.de

	Abstract

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Recent studies suggest the importance of prothrombotic and proinflammatory cascades in vascular thrombus formation. However, the impact of platelet CD40 and CD40 ligand (CD40L) expression and its relation to inflammatory markers in atrial clot formation have not yet been determined. Therefore, we studied a total of 40 patients. A total of 20 patients with persistent atrial fibrillation (AF) and 20 matched patients with sinus rhythm (SR) were included to quantify platelet surface expression of CD40/CD40L, serum levels of intercellular adhesion molecule-1 (ICAM), vascular adhesion molecule-1 (VCAM), high-sensitivity C-reactive protein (hsCRP), and monocyte chemoattractant protein-1 (MCP-1). Using fluorescence-activated cell sorting analysis, baseline CD40 expression (antibody binding capacity [ABC]) was increased during AF (AF: 7776 ± 8.46 ABC vs. SR: 7753 ± 7.32 ABC; P < 0.05), whereas CD40L expression was not different. In contrast to the effect of adenosine diphosphate, ex vivo stimulation with thrombin receptor activating peptide (TRAP) increased CD40 and CD40L expression in both groups. MCP-1, hsCRP, ICAM, and VCAM levels were significantly increased during AF, reaching highest levels in patients with atrial thrombi. Importantly, VCAM and MCP-1 were independent predictors for atrial thrombi (P < 0.05) using multivariate analysis. In contrast to declining levels of hsCRP, levels of ICAM, VCAM, MCP-1, and platelet CD40 expression remained elevated 5 weeks after successful electrical direct current cardioversion (CV). In conclusion, prothrombogenic markers are substantially elevated in patients with AF, reaching highest levels in patients with AF and atrial thrombi. Interestingly, amounts of adhesion molecules and platelet CD40 levels remain elevated even 5 weeks after successful CV, which may imply a persistently increased risk for atrial thrombus formation. In addition to hsCRP, MCP-1 and VCAM may serve as new biomarkers, which may help to identify patients with an increased risk for thromboembolic events.

Key Words: adhesion molecules • CD40 • fibrillation • inflammation • remodeling • platelets

	Introduction

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Several studies have shown that atrial fibrillation (AF) is accompanied by a hypercoagulative state that may contribute to the development of atrial thrombi and thromboembolism (1–9). In addition to enhanced activation of the plasma coagulation system, AF also influences platelet aggregability. Sohara et al. showed an acceleration of platelet activity developing 12 hrs after the onset of AF (6). Increasing evidence has emerged that activated platelets play a pivotal role in inflammatory processes as a result of platelet–leukocyte and platelet–endothelium interactions; thus, platelets are important and abundant inflammatory cells per se (10–12).

There also is increasing evidence of the importance of cellular adhesion molecule monocyte chemoattractant protein-1 (MCP-1) and CD40/CD40 ligand (CD40L) interaction among platelets and endothelial cells, where they trigger, modulate, or abate inflammatory mechanisms, especially in patients with acute coronary syndromes (13–17).

In the setting of persistent AF, a marked increase in levels of inflammatory markers such as high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) has been identified (18, 19). However, the relationship between proinflammatory and prothrombogenic factors remains unclear, and platelet expression of CD40 and CD40L has not yet been assessed in patients with AF. Therefore, the purpose of the present study was to determine platelet surface expression of CD40 and CD40L before and after electrical direct current cardioversion (CV) of persistent AF. Furthermore, platelet activity was determined before and after ex vivo stimulation with thrombin receptor activating peptide (TRAP) and adenosine diphosphate (ADP). Activation of the CD40/CD40L system was correlated with systemic levels of intercellular adhesion molecule-1 (ICAM), vascular adhesion molecule-1 (VCAM), hsCRP, and MCP-1 in patients with and without persistent AF.

	Materials and Methods

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Patient Characteristics.
A total of 40 patients were included in our prospective study. A total of 20 patients with persistent AF (duration 4 months) were compared with 20 age- and gender-matched control patients in sinus rhythm (SR). Matching with regard to age was done within a limit of a 5-year difference. Detailed patient characteristics and medication used are listed in Table 1. In all patients, a 12-lead electrocardiogram and an echocardiogram were carried out at baseline. The study was approved by the Ethics Committee of the University Hospital Magdeburg, Germany, and all patients gave written informed consent to participate in the study.

Platelet CD40/CD40L Expression.
Blood samples collected in tubes containing 0.105 M sodium citrate were used for analysis of platelet CD40/CD40L expression. Platelet counts were determined by CELL-DYN 1600 Counter (Abbott, Wiesbaden, Germany). Platelets were diluted to 20 x 10⁹/l with buffered Hanks’ balanced salt solution + 1 mg/ml bovine serum albumin (BSA). A 36-µl volume of diluted blood was incubated for 10 mins at 37°C in: (i) a tube containing 4 µl buffer, (ii) a tube containing 4 µl of 50 µM ADP, and (iii) a tube containing 4 µl of 100 µM TRAP. For platelet identification, each sample was further incubated with 20 µl phycoerythrin (PE)–labeled monoclonal antibody against platelet glycoprotein IIb/IIIa (CD41a; Beckmann Coulter, Krefeld, Germany) and then with fluorescein isothiocyanate–labeled P-selectin (CD62P; Beckmann Coulter), as well as with monoclonal antibody against CD40 (FA CALTAG Laboratories; Karlsruhe, Germany) and CD40L (FA Bender MedSystems; Vienna, Austria) for 5 mins at 37°C. The process of activation and staining was stopped in each aliquot with 2 ml cold buffer (Hanks’ balanced salt solution, 4°C). The platelet fluorescence was determined by flow cytometry using a fluorescence-activated cell sorter (FACS) Calibur (Becton Dickinson, Heidelberg, Germany; Ref. 20). The quantification of the various antibody binding sites (CD62P, CD40, and CD40L) was performed by analysis of the fluorescence intensity using a Quantum Simply Cellular Microbeads Kit (Bangs Laboratories, Fishers, IN). After internal calibration, data were presented as antibody binding capacity (ABC).

Systemic Levels of hsCRP, ICAM, VCAM, and MCP-1.
Levels of hsCRP were determined using a random access analyzer (Hitachi 917; Roche Diagnostics, Mannheim, Germany) and CRP Dynamic Reagenz (BIOMED Labordiagnostik GmbH, Oberschleißheim, Germany); this is a latex particle-enhanced immunoturbidimetric assay. The range of the assay is from 0–30 mg/l. ICAM, VCAM, and MCP-1 levels were measured using a quantitative sandwich enzyme immunoassay technique (R&D Systems, Wiesbaden, Germany). The coefficients of variation of these assays were 4.5%, 6.1%, and 3.8%, respectively.

Electrical CV.
In patients with AF (six patients were excluded from CV due to atrial thrombi), direct current external CV was performed using a single monophasic 360-J shock under anesthetic with body weight–adapted doses of midazolam and etomidate. In 10 patients, who thereafter remained in stable SR, another set of blood samples was taken, as described above, at 24 and 48 hrs after CV and 5 weeks after CV.

Statistical Analysis.
Data are expressed as means ± SEM. Intergroup and intragroup differences were analyzed by ANOVA. The chi-square test, univariance analysis, and multinominal logistic regression analyses were used, where appropriate, to assess the association between the occurrence of AF, biomarkers (ICAM, VCAM, MCP-1, hsCRP) and clinical parameters (age, diabetes, New York Heart Association class, hypertension, smoking, left atrial diameter, atrial thrombus). The Pearson coefficient (r) was used to determine correlations between the various metric parameters. All statistical data were calculated using SPSS (SPSS Inc., Chicago, IL). A P value < 0.05 was considered to be statistically significant.

	Results

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Platelet CD40/CD40L Expression.
Platelet CD40 expression was increased during AF compared with patients with SR (AF: 7776 ± 8.46 ABC vs. SR: 7753 ± 7.32 ABC; P < 0.05), whereas baseline platelet CD40L levels were comparable in the two groups (AF: 7324 ± 5.03 ABC vs. SR: 7326 ± 4.50 ABC; P = ns). CD40/CD40L expression did not correlate with clinical variables such as age, gender, duration of AF, concomitant diseases, and medication (data not shown).

Ex vivo stimulation with TRAP induced a substantial increase in CD40 expression in both the AF and SR groups (AF: 7776 ± 8.46 ABC vs. 7813.7 ± 7.98 ABC, P < 0.001; SR: 7753 ± 7.32 ABC vs. 7791 ± 9.44 ABC, P < 0.01). CD40 expression remained higher in the AF group compared with the SR group after TRAP stimulation (Fig. 1A). Ex vivo TRAP stimulation also increased CD40L expression in both the AF and SR groups (AF: 7324 ± 5.03 ABC vs. 7376 ± 9.66 ABC, P < 0.01; SR: 7326 ± 4.50 ABC vs. 7366 ± 7.91 ABC, P < 0.001), with no difference between the two groups after stimulation (Fig. 1B). ADP stimulation did not show a significant effect on CD40 (AF: 7776 ± 8.46 ABC vs. 7774 ± 10.76 ABC, P = ns; SR: 7753 ± 7.32 ABC vs. 7771 ± 8.81 ABC, P = ns) or CD40L expression (AF: 7324 ± 5.03 ABC vs. 7330 ± 9.66 ABC, P = ns; SR: 7326 ± 4.50 ABC vs. 7328 ± 4.47 ABC, P = ns).

View larger version (11K): [in this window] [in a new window]   Figure 1. (A) Platelet CD40 expression before and after ex vivo TRAP stimulation in patients with AF (black columns) and SR (white columns). Values are means ± SEM. *P < 0.05 baseline AF vs. baseline SR; P < 0.01 baseline SR vs. SR after TRAP stimulation; P < 0.0001 baseline AF vs. AF after TRAP stimulation; P = 0.07 AF after TRAP stimulation vs. SR after TRAP stimulation. (B) Platelet CD40L expression before and after ex vivo TRAP stimulation in patients with AF (black columns) and SR (white columns). Values are means ± SEM. *P < 0.001 baseline AF vs. AF after TRAP stimulation; P < 0.01 baseline SR vs. SR after TRAP stimulation.

CD40 expression remained elevated after successful CV (before CV: 7776 ± 8.46 ABC; 24 hrs after CV: 7745 ± 36.22 ABC; 48 hrs after CV: 7760 ± 24.4 ABC; 5 weeks after CV: 7784 ± 9.56 ABC; P = ns; Fig. 2A). In contrast, CD40L levels declined progressively after successful CV (before CV: 7324 ± 5.03 ABC; 24 hrs after CV: 7320 ± 6.31 ABC; 48 hrs after CV: 7318 ± 10.91 ABC; 5 weeks after CV: 7296 ± 3.75 ABC; P < 0.05; Fig. 2B). Six patients were excluded from electrical CV because of left atrial thrombi detected by transesophageal echo. All atrial thrombi were located within the left atrial appendage. The clinical characteristics of these six patients were comparable to the remaining patients with AF (Table 2). In patients with atrial clot, levels of CD40 and CD40L and the inducible platelet activity after ex vivo stimulation were not different compared with the remaining patients in AF (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 2. (A) Platelet CD40 expression before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (square); SR (triangle). Values are means ± SEM. *P < 0.05 AF before CV vs. baseline SR; P < 0.01 AF after CV vs. baseline SR. (B) Platelet CD40L expression before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (square); SR (triangle). Values are means ± SEM. *P < 0.05 AF before CV vs. AF 5 weeks after CV.

Importantly, hsCRP, VCAM, ICAM, and MCP-1 levels were highest in the six patients with documented atrial thrombi (Fig. 3). Of note, MCP-1 (292.48 ± 23.24 pg/ml; P < 0.05) and hsCRP levels (15.13 ± 6.54 mg/l; P < 0.001) showed the most substantial differences compared with the remaining patients with AF. Successful CV caused a significant decline in hsCRP levels during follow-up (before CV: 5.44 ± 1.87 mg/l vs. 5 weeks: 2.08 ± 0.63 mg/l; P < 0.05). In contrast, ICAM, VCAM, and MCP-1 levels remained elevated after successful CV (VCAM before CV: 800.0 ± 29.13 ng/ml vs. 5 weeks: 779.68 ± 80.35 ng/ml, P = ns; MCP-1 before CV: 239.99 ± 13.6 pg/ml vs. 5 weeks: 221.75 ± 24.46 pg/ml, P = ns; ICAM before CV: 232 ± 12.6 ng/ml vs. 5 weeks: 204.08 ± 23.27 ng/ml, P = ns; Fig. 4). However, a linear correlation between CD40 and ICAM, VCAM, or MCP-1 levels could not be demonstrated (data not shown). In contrast, VCAM levels correlated with patient age (r = 0.49; P < 0.01). Using univariate analysis, the presence of diabetes mellitus was related to MCP-1 (P < 0.05) and VCAM (P =0.059). Smoking habits (pack years) correlated with baseline hsCRP levels (r =0.70; P < 0.01). Importantly, VCAM and MCP-1 levels were independent predictors for the presence of atrial thrombi on multinominal regression analysis (P < 0.05).

View larger version (8K): [in this window] [in a new window]   Figure 3. (A) Levels of hs-CRP in patients with SR (white columns), AF patients (striped bars), and AF patients with atrial thrombus (black columns). Values are means ± SEM. *P < 0.001 AF with atrial thrombus vs. SR; P < 0.001 AF with atrial thrombus vs. AF; P < 0.05 AF without atrial thrombus vs. SR. (B) MCP-1 levels: SR (white columns), AF (stripped bars), and AF with atrial thrombus (black columns). Values are means ± SEM. *P < 0.001 AF with atrial thrombus vs. SR; P < 0.05 AF with atrial thrombus vs. AF. (C) Serum ICAM (s-ICAM) levels: SR (white columns), AF (striped bars), and AF with atrial thrombus (black columns). Values are means ± SEM. *P < 0.05 AF with atrial thrombus vs. SR. P < 0.05 AF without atrial thrombus vs. SR. (D) Serum VCAM (s-VCAM) levels: SR (white columns), AF (striped bars), and AF with atrial thrombus (black columns). Values are means ± SEM. *P < 0.05 AF with atrial thrombus vs. SR; P < 0.05 AF without atrial thrombus vs. SR.

View larger version (7K): [in this window] [in a new window]   Figure 4. (A) Serum hsCRP levels before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (squares); SR (triangles). Values are means ± SEM. *P < 0.05 AF before CV vs. baseline SR; P < 0.05 AF before CV vs. AF after CV. (B) Serum ICAM levels before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (squares); SR (triangles). Values are means ± SEM. *P < 0.05 AF before CV vs. baseline SR. (C) Serum VCAM levels before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (squares); SR (triangles). Values are means ± SEM. *P < 0.05 AF before CV vs. baseline SR. (D) Serum MCP-1 levels before CV and 24 hrs, 48 hrs, and 5 weeks after successful electrical CV (squares); SR (triangles). Values are means ± SEM. *P < 0.05 AF before CV vs. baseline SR.

	Discussion

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Platelets, Biomarkers, and AF.
CD40 and its ligand have been described as expressed on endothelial cells, smooth muscle cells, and fibroblasts, where they play a pivotal role in inflammatory responses, such as the expression of adhesion molecules, cytokines, matrix-degrading enzymes, and apoptotic mediators (21–23). It was not until recently that CD40 was found to be constitutively present on platelets, whereas preformed CD40L might be expressed seconds to minutes after platelet activation by diverse stimuli (12, 24). Once expressed, CD40L can interact with membrane-bound CD40 on endothelial cells, triggering an inflammatory reaction leading to local or systemic release of ICAM, VCAM, and MCP-1 (10, 12, 25). Upon platelet activation and thrombus formation, CD40/CD40L interaction among platelets may lead to shedding of CD40L, producing its soluble form, sCD40L. Whether this soluble form of CD40L is capable of inducing an inflammatory reaction acting as a bona fide cytokine or is inactivated upon cleavage is still a matter of discussion (24–28). It is also not finally clarified whether platelet–platelet interaction of CD40 and CD40L abrogates thrombus formation and inflammation by cleavage of CD40L, or whether further thrombus activation is achieved due to prothrombogenic effects of CD40 ligation with sCD40L (24, 28).

To the best of our knowledge, the present study is the first to analyze platelet surface expression of CD40 and CD40L in patients with AF. Our study shows that the CD40 system is activated during AF. However, the pattern of activation appears different compared with findings in patients with coronary artery disease, in whom CD40 and CD40L levels are increased (13–17). During AF, platelet CD40/CD40L appears very sensitive to TRAP but not to ADP, which may be clinically relevant for platelet inhibitory therapy with ADP receptor antagonists in patients with AF (29). Nevertheless, the absolute changes in the platelet CD40/CD40L system during AF were modest compared with the massive changes in platelet P-selectin expression after TRAP stimulation. Thus, the overall impact of the CD40 activation in AF might be limited, since studies in patients with acute coronary syndromes have shown an increase in platelet CD40/CD40L expression of up to 50%. Of note, successful CV did not lower CD40 levels. Thus, CD40-dependent platelet–platelet interactions, as well as platelet–endothelium interactions remain activated for at least 5 weeks after cardioversion. This suggests that activation of this system does not solely depend on the arrhythmia. CD40 activation might also be maintained by long-lasting procoagulative alterations at the atrial endocardium.

In addition to CD40-dependent effects, platelet–leukocyte/monocyte interactions (characterized by activation of P-selectin and MCP-1) were enhanced during AF. The finding that MCP-1 levels are independent predictors for atrial thrombi is of particular interest (10). Nitric oxide (NO) is an important regulator for MCP-1 expression, and it has been shown that reduced availability of NO induces a substantial upregulation of MCP-1 expression (30). Thus, the progressive reduction in NO availability, which has been demonstrated to occur in fibrillating atrial tissue, might be one factor causing the increase of MCP-1 as well as adhesion molecules (30–33). Kamiyama showed that 8 hrs of rapid atrial pacing increases ICAM expression at the left atrial endocardium in rabbits (34). Interestingly, upregulation of ICAM was associated with leukocyte adherence to the atrial endocardium, which might be influenced by platelet P-selectin expression and MCP-1.

Nevertheless, AF is not an absolute prerequisite for the development of such prothrombogenic alterations at the atrial endocardium, because it was demonstrated that pressure overload ("stretch") per se causes increased expression of various endocardial proteins (35). Therefore, in many patients with AF it has to be considered that the development of the arrhythmia might be a secondary phenomenon related to preexisting structural and thereby prothrombogenic alterations of the atrial myocardium and endocardium ("endocardial remodeling"). Thus, in some patients a prothrombogenic milieu persists or is imminent, whereas the presence of AF on the surface electrocardiogram may serve only as a marker for such alterations (Fig. 5). This may help to explain the present finding that VCAM and MCP-1 levels remained elevated for weeks after successful CV of AF, because the underlying structural abnormalities of the atria are not instantaneously normalized by restoration of SR. Of note, MCP-1 and VCAM levels are independent predictors for atrial thrombi, which suggest a clinical importance for our findings. MCP-1 and VCAM appear as biomarkers, which may help identify patients with an increased risk for thromboembolic events. Further studies are warranted to assess the predictive value of these parameters in a larger cohort of patients with AF.

View larger version (18K): [in this window] [in a new window]   Figure 5. Hypothesis on causes and mechanisms of inflammation, AF, and clot formation. Preexisting comorbidities and risk factors lead to structural changes of the atrial tissue (structural remodeling) and, thereby, directly induce the expression of prothrombogenic markers (endocardial remodeling). In addition, structural alterations of atrial myocardium favor the occurrence of AF, which, in a positive feedback loop, enhances structural atrial remodeling. Recent data have already demonstrated that structural atrial changes are long lasting or may even be irreversible. This may encompass induced endocardial changes characterized by increased adhesion molecule (VCAM and ICAM) and MCP-1 expression as well as platelet activation, which appear as prerequisites for the development of atrial thrombi at the endocardium. However, hsCRP seems to be linked to the presence or absence of AF and atrial thrombi. Thus, in contrast to adhesion molecules and increased platelet activation, elevated CRP might be a consequence rather than a cause of thrombus development. This is supported by previous studies showing that CRP levels per se do not predict the development of vascular thrombi or thromboembolic events, although CRP levels increase after manifestation of vascular thrombi (37, 38).

Limitations.
The number of patients included in the study is limited, one reason being that fluorescence-activated cell sorting analyses of cell surface proteins are complex and therefore difficult to perform in a large series of patients. Analyses of large cohorts of patients usually are based on stable soluble blood markers (e.g., IL-6, CRP), whereas in our study cell surface markers, ex vivo stimulation techniques, and soluble serum markers were determined. It is known that coronary artery disease and other factors substantially influence systemic inflammatory markers. Thus, matched cohorts of patients with no or minimal concomitant diseases were included only to determine the impact of AF on these systemic factors. Nevertheless, the limited number of patients does not allow one to draw definite conclusions regarding all analyzed parameters. For example, the pathophysiologic significance of the fall of CD40L after CV below the SR group remains unclear and should therefore be reproduced in larger cohorts of patients.

Angiotensin-converting enzyme inhibitors and AT1 antagonists were unequally present in the two groups, with an increased use in AF patients. Nevertheless, both drugs may lower ICAM and VCAM levels (39, 40). Therefore, the different distribution is less likely to explain the increased ICAM and VCAM levels in the patients with AF. Nevertheless, we cannot rule out that the use of these drugs in patients with AF might have contributed to a reduction of these factors and, thereby, potential differences to patients in SR might have been mitigated. In addition to angiotensin-converting enzyme inhibitors, coumadin therapy was used more often in patients with AF. The specific effect of coumadin on inflammatory markers and adhesion molecule expression has not been studied in detail. The data available so far are very limited and give conflicting results (41, 42). However, medical therapy was not changed throughout the study in any patient, and thus intraindividual effects induced by medication were excluded.

Conclusions.
Prothrombogenic markers are substantially elevated in patients with AF, reaching highest levels in patients with AF and atrial thrombi. Interestingly, amounts of adhesion molecules and platelet CD40 levels remain elevated even 5 weeks after successful CV, which may imply a persistently increased risk for atrial thrombus formation. In addition to hsCRP, MCP-1 and VCAM may serve as new biomarkers, which may help to identify patients with an increased risk for thromboembolic events.

	Footnotes

 
This work was supported by a grant from the Bundesministerium für Bildung und Forschung, Germany (Kompetenznetz Vorhofflimmern, 01GI0204).

Received for publication July 18, 2006. Accepted for publication November 24, 2006.

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