no Enfarte Agudo do Miocárdio:
um Estudo Longitudinal [82]
PATRÍCIANAPOLEÃO1,4, M.CRISTINASANTOS2, MAFALDASELAS3, ANAM. VIEGAS-CRESPO1, TERESAPINHEIRO4, R. CRUZFERREIRA3 1Centro de Biologia Ambiental, Universidade Lisboa, Lisboa, Portugal; 2Centro de Química e Bioquímica, Universidade Lisboa, Lisboa,
Portugal; 3Serviço Cardiologia, Hospital Santa Marta, Lisboa, Portugal; 4Laboratório de Feixe de Iões, Instituto Tecnológico e Nuclear,
Sacavém & Centro de Física Nuclear, Universidade Lisboa, Lisboa, Portugal
Rev Port Cardiol 2007; 26 (12): 1357-1363
1357 RESUMO
Actualmente a inflamação é considerada uma componente importante na aterosclerose, desde o seu início até à ruptura da placa seguida de trombose e da progressiva obstrução do vaso. A ruptura da cápsula fibrótica da placa expõe factores de tecido presentes no seu núcleo necrótico que induzem o processo inflamatório, promovendo a adesão celular e a coagulação e que conduzem à formação do trombo. Por seu turno, várias citocinas e moléculas de adesão celular contribuem activamente para o desenvolvimento da placa. Em particular a
citocina TNF-α e a molécula de adesão intercelular (ICAM-1) poderão ser indicadoras de inflamação enquanto que as formas solúveis de P-selectina e de CD40 ligando (sCD40L) poderão dar a magnitude da activação
plaquetária. Neste trabalho foram estudados 17 doentes
com enfarte de miocárdio submetidos a angioplastia (grupo AMI) e 16 doentes com
confirmação angiográfica de ausência de doença coronária. Os doentes do grupo AMI foram seguidos nas primeiras 24 h de evolução do enfarte agudo de miocárdio antes da administração de medicação e da intervenção
angiográfica e ao longo do período de recuperação, 2 e 40 dias após enfarte. Foram
medidas no soro por imunoensaio as concentrações de TNF-α e das formas solúveis de CD40L, ICAM-1
e P-selectina. Foram observadas variações significativas de
sP-selectina relativamente aos controlos. Imediatamente após o enfarte de miocárdio verificou-se um aumento de sP-selectina, seguido de uma descida brusca dos seus níveis
ABSTRACT
Variations in inflammatory markers in acute myocardial infarction: a longitudinal study
Inflammation is now considered a key compo-nent of atherosclerosis, from fatty streak formation to plaque rupture, subsequent thrombosis, and progressive mechanical and dynamic obstruction. Rupture of the arterial plaque’s fibrous cap exposes tissue factors present in the necrotic core, triggering inflammatory signaling, cell adhesion, and the coagulation cascade that eventually leads to thrombus. Cytokines and adhesion molecules are key components of these events that contribute to the development of an
atherosclerotic plaque. The cytokine TNF-α and intercellular adhesion molecule-1 (ICAM-1) are indicators of basal inflammation, while the soluble forms of adhesion molecules such as CD40L and P-selectin indicate the extent of platelet activation. This study reports on the follow-up of 17 patients with confirmed acute myocardial infarction (AMI group) undergoing angioplasty and a matched control group of 16 patients without coronary artery disease as verified by coronary angiography. Patients from the AMI group were assessed at the onset of the acute coronary syndrome, within 24 h, before the administration of glycoprotein IIb/IIIa inhibitors and coronary angioplasty, and during the recovery period, two and 40 days after intervention. For both groups, clinical characteristics were documented and serum concentrations of soluble CD40L, P-selectin, ICAM-1, TNF-α, and conventional biochemical indicators were analyzed. For AMI
Recebido para publicação: Dezmbro de 2006 • Aceite para publicação: Setembro de 2007 Received for publication: December 2006 • Accepted for publication: Setember 2007
INTRODUCTION
Inflammation is now considered a key compo-nent of atherosclerosis, from fatty streak formation to plaque rupture, subsequent thrombosis, and progressive mechanical and dynamic obstruction. A growing body of literature shows that the progressive development of an atherosclerotic plaque and the local vascular events that surround an acute coronary syndrome involve cytokines
such as TNF-α and interleukins, and acute phase
reactants such as CRP, among others(1,2). These
mediators may establish or be surrogate markers for basal inflammation that leads to development of
atherosclerotic plaque(2). Activation of endothelial
adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion mol-ecule-1 (VCAM-1), promoting monocyte adhesion and leukocyte migration into the extravascular space, increased oxidative stress promoting LDL às 48h, e de um incremento para valores
idênticos aos observados no grupo de controlo ao 40º dia. As variações observadas nas
concentrações de sCD40L não foram significativas relativamente aos controlos. No
entanto, verificou-se uma tendência de diminuição da concentração até 48h após o enfarte de miocárdio, seguindo-se um aumento que atingiu valores ligeiramente superiores ao do grupo controlo no 40º dia. As concentrações de TNF-α medidas foram sistematicamente superiores às verificadas no grupo controlo, tendo-se ainda observado uma subida gradual desde o enfarte de miocárdio até ao 40º dia, sendo este incremento significativo. Os valores de sICAM-1 não apresentaram quaisquer variações após o enfarte nem relativamente ao
grupo controlo. As variações observadas sugerem um papel
importante destes marcadores no processo inflamatório e na evolução do enfarte de miocárdio. O aumento brusco da concentração de sP-selectina após o enfarte de miocárdio evidencia a activação plaquetária e trombose. Na evolução do enfarte, e à medida que as variáveis hemodinâmicas retornam a valores
estáveis, devido à medicação aplicada, o aumento de sCD40L e TNF-α em circulação pode reflectir o papel destas moléculas na recuperação endotelial e do miocárdio.
Palavras-Chave
Síndromes coronárias agudas; Estudo longitudinal; Inflamação; P-selectina; CD40 ligando; TNF-α
patients, these indicators were recorded at study entry and during follow-up.
Concentrations of cytokines and adhesion molecules were measured using commercial immunoassay (ELISA) kits.
Significant variations in sP-selectin were observed relative to the control group. Immediately after myocardial infarction, sP-selectin levels rose markedly, followed by a sharp decrease two days later. After 40 days of recovery, sP-selectin levels rose again, returning to the initial values. Variations in sCD40L levels were not significant relative to controls. However, sCD40L concentrations tended to fall until the second day after infarction, followed by a rise, and by the 40th day of recovery levels were slightly higher than controls. Unlike sCD40L and sP-selectin, consistently higher levels of TNF-α relative to controls were observed, which were only significant after 40 days of recovery. No significant variations were observed for ICAM-1 serum concentrations in the AMI group. The variations observed demonstrate the role of inflammatory markers in AMI progression and highlight the importance of systemic
inflammation in disease evolution. The increased concentration of sP-selectin at infarction onset is evidence of thrombosis and platelet activation. Later, during the recovery period when hemodynamic variables are returning to stability in part due to medication, rises in circulating levels of sCD40L and cytokines such as TNF-α may reflect the role of these molecules in the recovery of
endothelial and myocardial tissues. Key words
Acute coronary syndromes; Patient follow-up; Inflammation; P-selectin; CD40 ligand; TNF-α
1359 oxidation and the development of foam cells,
and the degradation of the plaque fibrous cap by
metalloproteinase activity(3), are some of the events
which may predispose to plaque instability. Thus, inflammatory biomarkers may be used to predict atherothrombotic risk, such as rupture and/or erosion of unstable plaques and platelet acti-vation. Molecules such as ICAM-1, VCAM-1, CD40 ligand (CD40L), and P-selectin have been linked to
atherothrombotic events(4,5). On cell acti-vation these
molecules can be cleaved to a soluble form over the course of minutes to hours. Most soluble P-selectin appears to be derived from activated platelets, as its levels correlate with other established platelet
mark-ers but not with endothelial markmark-ers(5). The
expres-sion of ICAM-1 and P-selectin in endothelial cells mediates binding and rolling of leukocytes along the activated endothelium, whereas platelet P-selectin mediates formation of platelet aggregates and adhe-sion of circulating platelets and leukocytes in stress conditions.
P-selectin and particularly CD40L were found to
be highly expressed in platelets (4,5). CD40L is also
expressed in a number of cells, including T lympho-cytes, vascular endothelial cells, and smooth muscle
cells(6). CD40L signaling, through the related CD40
and TNF-α, mediates the expression of other adhe-sion molecules, chemokines and cytokines, amplify-ing inflammation and contributamplify-ing to the progression
of atherosclerosis and plaque instability(4,6). While
increased levels of soluble P-selectin have been consistently reported in acute coronary syndromes, variations in CD40L are still controversial. Both
increased(7,87)and decreased(9)serum levels of soluble
CD40L have been reported in the literature. The discrepancies may arise from evaluation of patients in different clinical states and the influence of medi-cation. Nevertheless, the P-selectin and CD40L domains appear to be pivotal links between throm-bosis and inflammation, making platelets important mediators in vascular inflammation and adhesion to
circulating monocytes and neutrophils(3).
The main objective of this study was to assess the inflammatory condition of AMI patients at the onset of the thrombotic event prior to intervention and to monitor changes over the recovery period. The study initiates a clinical registry of inflammation in coro-nary syndromes for the acute phase and for the recovery period, by measuring serum levels of TNF-α, and soluble forms of CD40L, P-selectin and ICAM-1.
METHODS
Study groups
From September 2005 to May 2006 a total of 33 subjects (10 women and 23 men), aged 25 to 75 years, were recruited from the inpatients of the Cardiology Department in Santa Marta Hospital. Of these, 17 patients with acute myocardial infarction constituted the myocardial infarction (AMI) group, while 16 patients without coronary artery disease as verified by coronary angio-graphy constituted the control group. The controls were matched to the AMI group for age and had no history and no clinical signs of myocardial infarction, peripheral artery disease or carotid artery disease. Patients aged >75 or with signifi-cant comorbidities such as peripheral artery disease or carotid artery disease, known antecedents of malignancy or infectious diseases, and chronic renal insufficiency, were excluded.
The subjects in the AMI group all had ST-elevation myocardial infarction in the first 24 hours of evolution and underwent primary percut-aneous transluminal coronary angioplasty as reperfusion therapy. The demographic and clinic-al data of the two groups are listed in Table I.
After angioplasty, AMI patients received antiplatelet and antiaggregant drugs such as aspirin, clopidogrel, or glycoprotein IIb/IIIa receptor antagonists, as well as β-blockers, ACE inhibitors and statins, according to international guidelines.
Study protocol
Percutaneous transluminal coronary angio-plasty of AMI patients was performed in the first 24 h of infarction evolution. The time from onset of chest pain to intervention was less than 6 h. A longitudinal follow-up of the AMI group was car-ried out on three different occasions. For all patients blood samples were obtained from the right femoral artery before the administration of IIb/IIIa receptor antagonists and coronary angio-plasty (day 0). During the recovery phase, blood samples were obtained by venous puncture two (day 2) and 40 (day 40) days after the initial angioplasty.
Assays
Blood samples were drawn from all patients into pyrogen-free blood collection tubes without additives and immediately centrifuged at 2500
rpm for 10 minutes. The serum was collected after centrifugation and placed on ice. Aliquots were stored at -80 ºC until analysis (no longer than 6 months). The samples were thawed only once.
All the assays were performed on serum according to the manufacturer’s recommenda-tions. Soluble concentrations of CD40L, ICAM-1 and P-selectin (sCD40L, sICAM-ICAM-1 and sP-selectin, respectively) were measured by com-mercially available enzyme-linked
immunosor-bent assays (ELISA) (R&D Systems, Minnea-polis, USA). The concentrations of TNF-α were assessed using a high sensitivity ELISA com-mercial kit (R&D Systems, Minneapolis, USA). Each sample were measured in duplicate; the intra-assay variation between the duplicates for all samples was <10%.
Statistical analysis
Continuous variables are expressed as mean and standard error (SE). Median values of measured quantities in the different groups were compared using the Student’s t test and differ-ences considered significant when p<0.05. Non-continuous variables were analyzed using a 2x2 table and Fisher’s exact test. The calculations were performed using SPSS 10.0 software.
RESULTS
Baseline characteristics of patients
As shown in Table I, there were no differences in baseline characteristics between patients in the AMI group and control subjects, except in terms of smoking status. Fifty-three percent of the AMI patients were smokers, contrasting with 6% of the controls. The two groups were matched in terms of age but not in sex ratio.
There were no significant differences in previ-ous medication between the groups.
Inflammatory markers
The results obtained for concentrations of the soluble cellular adhesion molecules sP-selectin and sICAM-1 and for sCD40L and the cytokine TNF-α in controls and AMI patients are present-ed in Figures 1 and 2, respectively.
At the onset of AMI (day 0), sudden variations in the inflammatory markers measured were observed relative to the control group, although only the increase in the concentration of sP-selectin was significant. TNF-α showed a similar trend to sP-selectin, while sCD40L showed a marked decrease on day 0. Although major differ-ences were observed in the mean concentrations of the two groups, the significance level was not reached, probably due to the considerable vari-ability found in these two parameters.
By day 2, levels of sP-selectin had fallen rapidly and significantly, probably reflecting the effect of medication. During this period the 1360 Controls (n=16) AMI (n=17) p Sex, F/M 8/8 2/15 0.026 Age, y 59±8 54±14 0.280 BMI 29±6 27±3 0.279 Waist circumference, cm 98±13 91±13 0.197
Risk factors and comorbidity:
- Hypertension, n (%) 10 (63) 6 (35) 0.169 - Smoking, n (%) 1 (6) 9 (53) 0.007 - Dyslipidemia, n (%) 12 (75) 20 (59) 0.465 Diabetes: - without insulin, n (%) 3 (19) 6 (35) 0.438 - with insulin, n (%) 0 (0) 1 (6) 1.000
Family history of CAD,
n (%) 0 (0) 3 (18) 0.227
Ejection fraction, % 68±7 53±12 0.000
No. of diseased vessels 02
-Culprit vessel: - LAD, n (%) 0 (0) 11 (65) -- RCA, n (%) 0 (0) 3 (18) -- LCx, n (%) 0 (0) 2 (12) -Previous AMI, n (%) 0 (0) 0 (0) -Coronary stents - coated, n (%) 0 (0) 15 (88) -- non--coated, n (%) 0 (0) 0 (0) -Previous medication: - Aspirin, n (%) 5 (31) 6 (35) 1.000 - IIb/IIIa, n (%) 0 (0) 0 (0) 1.000 - ACE-I, n (%) 3 (19) 2 (12) 0.656 - Statins, n (%) 6 (38) 3 (18) 0.259 − β-blockers, n (%) 5 (31) 2 (12) 0.225 Table I
Baseline clinical characteristics of the study population
Values are expressed as mean ± standard error, except otherwise indicated.
BMI: body mass index (weight in kilos divided by height in meters squared); CAD: coronary artery disease; LAD: left anteri-or descending canteri-oronary artery; RCA: right canteri-oronary artery; LCx: left circumflex artery; IIb/IIIa: glycoprotein IIb/IIIa receptor antagonists; ACE-I: angiotensin-converting enzyme inhibitors.
concentrations of sCD40L remained relatively steady and the smaller but continuous increase of TNF-α was found to be marginally significant (p=0.055) compared to the baseline value of the control group.
Forty days after the onset of AMI a marked increase in the concentrations of sP-selectin (p=0.01) and TNF-α (p=0.01) was observed rela-tive to the levels measured in the control group. sCD40L concentrations also increased in AMI patients to values similar to those of the control group. The increase of sCD40L relative to day 0 and 2 status was only marginally significant (p=0.06), again due to the considerable variability observed for this parameter, especially at day 40.
No significant variations were observed for sICAM-1 serum concentrations in the AMI group (Fig. 1).
DISCUSSION
The increased concentration of sP-selectin at infarction onset was clear evidence of thrombosis
and platelet activation. Shimomura et al.(10)
examined serial changes in sP-selectin levels (at admission, 1, 4, 24, and 48 hours, and 7 days after initiation of reperfusion therapy) and report-ed similar results. Levels of soluble CD40L, which is also expressed in activated platelets, were markedly depressed at infarction onset, suggesting a delayed action for this molecule and/or a slow rate of release of the CD40L soluble form. Recently published data indicate a pathogenic role for CD40L in acute myocardial syndromes. Increased expression of this molecule in platelets has been reported during acute coronary syndromes and cardiovascular disease
in general(5), but in the first 24 hours of
myocar-dial infarction sCD40L levels fell and were found to be negatively correlated to their expression in
platelets(5,6). Lip et al.(9) reported lower levels of
sCD40L in patients at higher risk of thromboem-bolic stroke.
The rise in soluble TNF-α protein 40 days after AMI, as determined in our study, is also described in the literature and variations in levels of this molecule alone or in combination with other cytokines such as IL-10 have been linked to
depressed cardiac function(11). The role of TNF-α
in cardiac recovery is still unclear, although beneficial effects of this cytokine when adminis-tered after AMI have been reported, leading to a significant reduction in myocardial injury. By contrast, increases in cytokine levels associated with alterations in TNF receptor expression in the cardiac microvasculature appear to contribute
to increased cardiovascular pathology(8). The
cytotoxic versus cytoprotective roles of TNF-α signaling during acute myocardial infarction
therefore remain unclear(12), since the available
data, including the results reported in this study, are not consensual.
After 40 days of recovery, increasing levels of TNF-α, P-selectin and sCD40L in the circulation may indicate an alteration in inflammatory status that could also derive from compensatory mecha-nisms or healing of damaged myocardium and epithelium. Following infarction due to coronary stenosis, the rapid fall in oxygen pressure in atrial myocytes and the vascular endothelium, among
other cells involved, triggers various events and 1361
Figure 1
Adhesion molecules sP-selectin (gray) and sICAM-1 (black) in subjects of control and AMI groups.
Control Day 2 Day 40
0 20 4 0 60 8 0 1 00 1 20 AM I Day 0 0 5 0 1 00 1 50 20 0 2 50 30 0 * sP -s el ec tin (n g/ m l) S ic am -1 (n g/ m l)
* significantly different from control group (p<0.05); + significantly different from day 0 (p<0.05).
Figure 2
* significantly different from control group (p<0.05).
sP -s el ec tin (n g/ m l) S ic am -1 (n g/ m l) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.5 1.0 1.5 2.0 2.5
Control Day 2 Day 40 AMI
Day 0
*
compensatory mechanisms such as those
regulat-ing blood pressure(4) that are necessary for the
recovery and survival of reversibly injured cells. A growing inflammatory response in cardiac and skeletal myocytes in the microvasculature, as well as upregulation of adhesion molecules in leukocytes, platelets and endothelium, may occur
as a consequence of the thrombotic event(2) and
may become obvious later during recovery. The ICAM-1 molecule mediates adhesion of leukocytes to activated endothelium, inducing arrest of inflammatory cells at the vascular surface, and participates in extravasation of leukocytes. Soluble levels of ICAM-1 could therefore reflect greater expression of ICAM-1 at
the endothelial surface(13-15)in patients with acute
myocardial infarction. Li et al.(13)reported higher
levels of sICAM-1 after 6 h of AMI onset and no further significant changes until 48 h, relative to controls. These results were similar to ours, as we found no major variations in sICAM-1 concentra-tions, and may indicate the importance of platelet-related thrombosis compared to endothe-lial activation, at least at the onset of infarction.
The origin of blood from both the femoral artery and the peripheral venous system cannot explain the variability observed, as previously
pointed out by Brueckmann et al.(8), who did not
observe variations in absolute concentrations of various inflammatory mediators whether measured in arterial or venous blood. There is also some
con-troversy regarding blood evaluation of sCD40L
(5,16-18). It is accepted that serum can yield higher
sCD40L concentrations than plasma, as post-hoc platelet activation can induce the release of
sCD40L(16-18). Nevertheless, accurate serum
meas-urements of sCD40L are possible as long as platelet activation is controlled and media temper-ature is kept close to 4 ºC during sample
process-ing(18), as was done in the present study.
The variations observed in the present study demonstrate the role of inflammatory markers in AMI progression and highlight the importance of systemic inflammation in disease evolution. However, the results obtained in the present study should be treated with caution, as a limited num-ber of patients were assessed and a healthy refer-ence group not subjected to medication was not included, which could have provided important information on baseline values and the role of these markers in AMI.
The increased concentration of sP-selectin at infarction onset is evidence of the functional state of thrombocytes and the endothelial cells that play an important role in the rupture or erosion of ath-erosclerotic plaque. Later, during the recovery period when hemodynamic variables are returning to stability in part due to synergistic physiological and pharmacological effects, rises in circulating levels of sCD40L and cytokines such as TNF-α may reflect the role of these molecules in the recovery of endothelial and myocardial tissues.
ACKNOWLEDGEMENTS
This work was financially supported by Fun-dação para a Ciência e Tecnologia (SFRHI/BD/ 18822/2004) and by Liga dos Amigos do Hospital de Santa Marta.
Pedido de separatas para: Address for reprints: Patrícia Napoleão
Laboratório de Feixes de Iões, Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2685–953 Sacavém, Portugal. Tel: +351219946250 Fax: +351 219941525 Telem: +351919673550 e-mail: pnapoleao@itn.pt 1362
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