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Clinical Chemistry 53: 1231-1234, 2007. First published May 10, 2007; 10.1373/clinchem.2007.085332
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(Clinical Chemistry. 2007;53:1231-1234.)
© 2007 American Association for Clinical Chemistry, Inc.

Hemostasis and Thrombosis

Increased Plasma Concentrations of Soluble CD40 Ligand in Acute Coronary Syndrome Depend on in Vitro Platelet Activation

Boris T. Ivandic1,a,2, Eberhard Spanuth2,2, Detlef Haase3, Heiko-Gundmar Lestin3 and Hugo A. Katus1

1 Department of Medicine III, University of Heidelberg, Heidelberg, Germany.
2 Roche Diagnostics GmbH, Mannheim, Germany.
3 Institute of Laboratory Medicine, Helios Kliniken, Schwerin, Germany.

aAddress correspondence to this author at: Innere Medizin, Abt. III, Universitaetsklinikum Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Fax 49-6221-56-5235; e-mail boris.ivandic{at}med.uni-heidelberg.de.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Soluble CD40 ligand (sCD40L) was suggested as a novel biomarker of cardiovascular risk. We examined the effect of preanalytical variation on the measurement of sCD40L concentration.

Methods: From healthy control individuals (n = 20) and patients with acute coronary syndrome (ACS) (n = 20) or sepsis (n = 20), we obtained blood drawn into 5 tubes containing citrate or a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD). The tubes were incubated for 30 min at room temperature or 0 °C before a single or double centrifugation (15 min, 2500g) at room temperature or 4 °C, respectively. sCD40L, ß-thromboglobulin (ßTG), and platelet factor 4 (PF4) concentrations were measured using immunoassays.

Results: Concentrations of sCD40L were very low in all CTAD and citrated samples maintained at 0 °C (median ≤0.076 µg/L). Although increased ßTG and PF4 confirmed disease-related in vivo platelet activation, sCD40L was not higher in patients than in controls. In contrast, if the samples were processed at room temperature, sCD40L was significantly higher in ACS patients than in controls (P <0.02 in CTAD and citrated plasma at room temperature). Moreover, the ßTG:PF4 ratio decreased in patient but not control CTAD samples, suggesting a greater susceptibility of patient platelets to in vitro activation.

Conclusions: Increased sCD40L concentrations resulted from in vitro platelet activation during sample preparation. Disease-related in vivo activation did not contribute to sCD40L concentrations in plasma. Therefore, published studies of sCD40L demand cautious interpretation, because their preanalytical conditions were not standardized.


   Introduction
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 Abstract
 Introduction
Materials and Methods
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 References
 
CD40 ligand (CD40L 1 ; CD154) is a homotrimeric type II transmembrane protein. Its C-terminus contains a tumor necrosis factor homology domain required for binding to its receptor CD40. Intracellular CD40L is expressed on platelet membranes in response to platelet activation (1). Subsequent cleavage by metalloproteases produces soluble CD40L (sCD40L), an 18-kDa soluble fragment (2). Many reports have suggested that sCD40L is a promising clinical biomarker of atherothrombotic risk. Increased concentrations of sCD40L were reported mostly in disorders associated with platelet activation such as acute and stable coronary artery disease (3)(4)(5). Coronary artery bypass graft surgery and percutaneous coronary intervention (PCI) were shown to increase sCD40L (3)(6). Conversely, increased sCD40L was also directly associated with a higher cardiovascular risk in patients undergoing PCI (7).

In spite of the great interest in this biomarker, sCD40L still awaits appropriate clinical validation (8). Although preanalytical conditions may influence sCD40L concentrations significantly, they have not been standardized for the measurement of this marker (9)(10)(11)(12)(13). This lack of standardization prompted us to examine the effects of preanalytical conditions on the measurement of sCD40L in samples collected from healthy control individuals and patients with acute coronary syndrome (ACS) and sepsis—both diseases are associated with in vivo platelet activation. Platelet activation was independently determined by measurement of ß-thromboglobulin (ßTG) and platelet factor 4 (PF4) concentrations. In response to activation, these proteins are rapidly secreted from platelet granules in a constant molar ratio. Decrease of the ßTG:PF4 ratio is a sensitive indicator of in vitro platelet activation (14).


   Materials and Methods
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 Materials and Methods
Results
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patients
The study was approved by the institutional review board in accordance with the Declaration of Helsinki. Blood samples were obtained from 3 groups: (a) 20 healthy volunteers (controls), who were not taking any antiplatelet medication within 7 days before sampling; (b) 20 patients admitted with ACS; and (c) 20 patients hospitalized with sepsis. All patients were consecutively recruited and gave informed consent.

preanalytical sample processing
Five samples of venous blood were drawn via a butterfly canula (21-gauge) into 2 plastic syringes (S-Monovette, Sarstedt AG) containing 1/10 of the syringe volume of 0.106 mol/L trisodium citrate, and into 3 plastic syringes containing citrate and the inhibitors of platelet activation theophylline (15 mmol/L), adenosine (3.7 mmol/L), and dipyridamole (0.198 mmol/L) (CTAD). One of the CTAD syringes was precooled (ice-water mix) to minimize in vitro platelet activation, degranulation, and formation of microparticles. We used each syringe to examine a different condition of preanalytical processing, i.e., a combination of anticoagulant (citrate or CTAD) and incubation temperature (0 °C or room temperature). After incubation of whole blood at room temperature or 0 °C (ice-water mix) for 30 min, we obtained plasma samples after a single centrifugation (2500g) at 15 °C for 15 min and stored them at –20 °C until analysis. Whole blood, which was drawn into the precooled CTAD tube, was incubated at 0 °C before centrifugation (2500g) at 4 °C for 15 min. The plasma was removed from the central portion of the supernatant, centrifuged again to obtain platelet-poor plasma (PPP), and analyzed immediately.

analytical methods
sCD40L was measured by use of an electrochemiluminescence immunoassay using the ElecSys® 2010 automated analyzer (Roche Diagnostics) (13). Within-run imprecision, reflected by CV, in pools of citrated plasma and serum (n = 21) was 1.8% at 0.1 µg/L and 1.2% at 6.9 µg/L; between-run imprecision, over 6 days, was 4.6% and 6.4%, respectively. Interference of CTAD with sCD40L analysis was negligible: sCD40L was comparable (6.81 and 6.96 µg/L) in a serum sample diluted 10:1 with CTAD and NaCl (0.154 mmol/L), respectively. Commercial ELISAs (Diagnostica Stago/Roche Diagnostics) were used to measure ßTG and PF4 concentrations.


   Results
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study participants
Twenty healthy individuals were studied as controls. A 57-year-old man was excluded from analysis because his sCD40L concentration was unexplainably very low (plasma 0.013 µg/L). The remaining controls included 9 men and 10 women. Their mean (SD) age was 41 (12) years. The ACS group included 15 men and 5 women, age 66 (10) years. In this group, 9, 7, and 4 patients presented with unstable angina, non–ST-segment elevation, and ST-segment elevation myocardial infarction, respectively. Eleven patients had increased cardiac troponin T (>0.03 µg/L), and PCI was performed in 15 ACS patients. The sepsis group included 13 men and 7 women, age 68 (11) years.

SCD40L, ßTG, and pf4 in the different sample preparations
All samples processed at 0 °C (and particularly CTAD PPP samples) exhibited very low concentrations of sCD40L (median ≤0.076 µg/L). There was no difference between controls and ACS patients, but values were lower in septic patients (Table 1 ). Samples from ACS patients processed at room temperature had increased sCD40L concentrations (Table 1 ). In contrast, median concentrations of ßTG were significantly higher in CTAD samples (processed at 0 °C) from ACS and sepsis patients compared with controls (Table 2 ; Mann–Whitney U, P = 0.0157 and P = 0.0388 for ACS and sepsis, respectively). Median concentrations of PF4 were also increased in patients with ACS and sepsis compared with controls (Table 2 ). Sample processing at room temperature decreased ßTG:PF4 ratios (Table 2 ). Interestingly, CTAD samples obtained from healthy controls maintained a high ßTG:PF4 ratio in spite of these conditions.


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  		Table 1. sCD40L concentrations (µg/L) in plasma.1


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  		Table 2. Median values of ß-TG, PF4, and ß-TG:PF4 ratio.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Accumulated evidence suggests that sCD40L is an important link between inflammation, atherosclerosis, and thrombosis. Numerous studies reported increased sCD40L in various clinical conditions and diseases associated with platelet activation but did not adequately describe the preanalytical conditions of sCD40L analysis (9). sCD40L concentrations are much higher in serum than in plasma, because platelet CD40L is released during clotting (11)(13). Moreover, sCD40L increases during preanalytical sample processing, suggesting that in vitro platelet activation may account for the release of sCD40L (11)(12)(13). This novel biomarker was suggested to require systematic validation before it could be applied for clinical studies (8).

Recently, the first immunoassay was developed for the automated analysis of sCD40L. When we used this assay, we measured only very low concentrations of sCD40L in carefully processed samples (CTAD PPP) from ACS and sepsis patients, although increased ßTG and PF4 confirmed in vivo platelet activation in these patients. Apparently, disease-related in vivo platelet activation did not contribute significantly to the increased ranges of sCD40L reported in the literature. Concentrations of sCD40L were influenced by the temperature of preanalytical incubation and centrifugation and the presence of CTAD, which inhibits platelet function by increasing intracellular cyclic adenosine monophosphate. Interestingly, sCD40L was significantly increased in ACS patients if the samples were processed at room temperature. Previous studies of sCD40L in ACS patients also applied these sample-processing conditions (15).

We also examined the ßTG:PF4 ratio to measure in vitro platelet activation in the different sample preparations. In vivo, this ratio is ~5.0, because PF4 has a short half-life (<20 min) compared with ßTG (100 min). In vitro, the ßTG:PF4 ratio decreases, because PF4 is no longer removed from circulation by rapid binding to the endothelial surface (14). The ßTG:PF4 ratio decreased significantly in CTAD samples from ACS and sepsis patients if the preanalytical processing temperature was increased to room temperature. In comparison, the ratio remained high in control CTAD samples, suggesting that platelets from healthy individuals may be more resistant to temperature-related in vitro activation than platelets from patients. Temperature-dependent formation of microparticles or specific enzymatic processes, or both, may explain this finding.

We conclude that previously reported plasma concentrations of sCD40L demand cautious interpretation, because they may result from in vitro platelet activation. For the time being, the role of sCD40L as a marker of atherothrombotic risk should be considered doubtful awaiting further evaluation.


   Acknowledgments
 
Grant funding/support: This study was funded by a grant from Roche Diagnostics, Germany.

Financial disclosures: None declared.

Acknowledgments: We are indebted to Prof. Georg Hess for helpful comments and discussions. We thank Christa Dewald for excellent technical assistance.


   Footnotes
 
2 These authors contributed equally to this work.

1 Nonstandard abbreviations: CD40L, CD40 ligand; sCD40L, soluble CD40L; PCI, percutaneous coronary intervention; ACS, acute coronary syndrome; ßTG, ß-thromboglobulin; PF4, platelet factor 4; CTAD, citrate, theophylline, adenosine, and dipyridamole; PPP, platelet-poor plasma.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998;391:591-594.[CrossRef][Medline] [Order article via Infotrieve]
  2. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA. The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40. Blood 2001;98:1047-1054.[Abstract/Free Full Text]
  3. Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, et al. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes. Circulation 1999;100:614-620.[Abstract/Free Full Text]
  4. Garlichs CD, John S, Schmeisser A, Eskafi S, Stumpf C, Karl M, et al. Upregulation of CD40 and CD40 ligand (CD154) in patients with moderate hypercholesterolemia. Circulation 2001;104:2395-2400.[Abstract/Free Full Text]
  5. Marx N, Imhof A, Froehlich J, Siam L, Ittner J, Wierse G, et al. Effect of rosiglitazone treatment on soluble CD40L in patients with type 2 diabetes and coronary artery disease. Circulation 2003;107:1954-1957.[Abstract/Free Full Text]
  6. Nannizzi-Alaimo L, Rubenstein MH, Alves VL, Leong GY, Phillips DR, Gold HK. Cardiopulmonary bypass induces release of soluble CD40 ligand. Circulation 2002;105:2849-2854.[Abstract/Free Full Text]
  7. Heeschen C, Dimmeler S, Hamm CW, van den Brand MJ, Boersma E, Zeiher AM, et al. Soluble CD40 ligand in acute coronary syndromes. N Engl J Med 2003;348:1104-1111.[Abstract/Free Full Text]
  8. Jaffe AS, Babuin L, Apple FS. Biomarkers in acute cardiac disease: the present and the future. J Am Coll Cardiol 2006;48:1-11.[Abstract/Free Full Text]
  9. Conde ID, Kleiman NS. Soluble CD40 ligand in acute coronary syndromes. [Letter]N Engl J Med 2003;348:2575-2577.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Bereczki D, Nagy E, Pal A, Magyar MT, Balla J. Should soluble CD40 ligand be measured from serum or plasma samples. [Letter]?Arterioscler Thromb Vasc Biol 2003;23:1129-1130.[Free Full Text]
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