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Cardiovascular Research Advance Access first published online on February 15, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn040
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Tumour necrosis factor {alpha} upregulates platelet CD40L in patients with heart failure

Pasquale Pignatelli, Roberto Cangemi, Andrea Celestini, Roberto Carnevale, Licia Polimeni, Alessandra Martini, Domenico Ferro, Lorenzo Loffredo and Francesco Violi*

IV Divisione di Clinica Medica, Department of Experimental Medicine and Pathology, University of Rome ‘La Sapienza’, Policlinico Umberto I, 00185 Rome, Italy

* Corresponding author. Tel: +39 0644 61933; fax: +39 0649 970893. E-mail address: francesco.violi{at}uniroma1.it

Received 9 November 2007; revised 12 February 2008; accepted 13 February 2008

Time for primary review: 25 days


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Patients with heart failure (HF) have elevated values of the pro-inflammatory protein CD40L but the underlying mechanism is unclear. This study was performed to evaluate the interplay between tumour necrosis factor {alpha} (TNF{alpha}) and CD40L in HF.

Methods and results: In patients with HF (n = 86) and healthy subjects (HS, n = 43), plasma levels of soluble CD40L (sCD40L), TNF{alpha}, soluble receptors of TNF{alpha} such as soluble TNF receptors I and II (sTNFR1 and sTNFR2), and 8OH-dG, a marker of oxidative stress, were determined. Also, an in vitro study was performed by determining platelet CD40L regulation upon platelet stimulation with TNF{alpha}. Compared with HS, HF patients had higher plasma values of sCD40L, TNF{alpha}, sTNFR1 and sTNFR2, and higher platelet expression of TNFR1 and TNFR2 with a progressive increase from NYHA I to NYHA IV classification. sCD40L significantly correlated with TNF{alpha}, sTNFR1, and sTNFR2; plasma levels of TNF{alpha} significantly correlated with sCD40L. Incubation of platelets from HF patients with a TNF{alpha} receptor inhibitor significantly decreased platelet CD40L expression. The in vitro study demonstrated that TNF{alpha} significantly increased CD40L expression, an effect weakly influenced by aspirin but significantly reduced by AACOCF3, an inhibitor of PLA2, apocynin, an inhibitor of NADPH oxidase, or staurosporine, an inhibitor of PKC.

Conclusion: The study shows that in HF patients, platelet CD40L is upregulated by TNF{alpha} via a cyclooxygenase-1-independent, arachidonic acid-mediated oxidative stress mechanism.

KEYWORDS Heart failure; TNF{alpha}; Platelets; CD40L; Oxidative stress


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Heart failure (HF) is one of the most prevalent diseases in western countries and has a negative social impact, as patients with HF have frequent hospitalization and low survival.1 In the last decade, many studies focused on the possibility that inflammation may complicate the clinical course of HF via impairing cardiac contractility, promoting apoptosis and fibrosis and ultimately leading to myocardial remodelling.2,3 Several markers of inflammation, including interleukin-6, tumour necrosis factor {alpha} (TNF{alpha}), and C-reactive protein, have been found elevated in HF and predictive of HF risk.4,5 Also, activation of inflammatory pathways has been reported to predict poor outcome in this setting.6 More recently, CD40L, a protein of TNF{alpha} superfamily, has been found elevated in HF.7 This finding is potentially interesting because CD40L has not only inflammatory but also pro-coagulant properties via overexpression of tissue factor, a glycoprotein that converts factor X to Xa.8 CD40L may be measured in the peripheral circulation as soluble CD40L (sCD40L), which is prevalently dependent on CD40L release from platelet membrane upon agonistic stimulation.9 As a consequence, the increase of sCD40L in HF is likely to reflect platelet activation but the underlying mechanism is still unclear. We have previously shown that patients with HF have higher circulating levels of TNF{alpha} and platelet overexpression of TNF{alpha} receptors (TNFR);10 inhibition of TNFR 1 resulted in reduction of collagen-induced platelet aggregation, suggesting that in HF, TNF{alpha} was a stimulus for platelet activation. These findings prompted us to speculate that in HF CD40L upregulation could be mediated by TNF{alpha} via interaction with its platelet receptors. This hypothesis was based on previous studies indicating that (i) platelet CD40L expression is mediated by activation of NADPH oxidase, one of the most important cellular producer of reactive oxidant species (ROS),11 and that (ii) TNF{alpha} has been shown to enhance oxidative stress via NADPH oxidase activation.12,13 Therefore, the aim of the first study was to analyse the interplay among the systemic levels of TNF{alpha}, oxidative stress, as assessed by plasma levels of 8-hydroxy-2'-deoxyguanosine (8OH-dG),14 and sCD40L in a population with several degrees of HF. Such interplay was also analysed at cellular level, as platelets are known to produce ROS via activation of NADPH oxidase.11 It is of note that arachidonic acid is a stimulus for the activation of platelet NADPH oxidase,15 and TNF{alpha} enhances platelet ROS production via an arachidonic acid pathway.10 Hence, we also performed in vitro study to assess whether TNF{alpha} upregulated platelet CD40L via arachidonic acid-mediated oxidative stress.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Patients
Between September 2003 and October 2004, we analysed 86 consecutive Caucasian patients (54 males and 32 females, age 73.05 ± 11.5 years) with a diagnosis of HF, enrolled from the outpatient clinic of our institution. Patients were included in the study when clinical features of HF were present and ejection fraction, calculated with the modified Simpson method, was <45%. Exclusion criteria were the following: congenital heart disease, pericarditis or myocarditis, acute coronary syndrome and/or percutaneous coronary intervention in the last month, evidence of acute hepatitits (ALT more than two folds the normal range), other acute or chronic inflammatory disease, recent (last 3 months) surgery intervention, assumption of immuno-modulating drugs or antioxidants in the last 4 weeks. Structural heart disease responsible for HF was valvular (n = 8), idiopathic (n = 7), or due to coronary heart disease (n = 71). Idiopathic dilated cardiopathy was defined as the presence of a dilated cardiopathy in the absence of other clinical reasons of HF and in the presence of a negative coronary study. Coronary heart disease diagnosis was made when a patient had undergone a positive coronary angiography (at least 1 month before the enrolment) or a positive myocardial scintigraphy. No patients had current symptoms of acute ischaemia. According to New York Heart Association (NYHA) classification, patients were divided in two categories (NYHA class I–II and NYHA class III–IV, n = 36 and n = 50, respectively). A more detailed study of the population’s characteristics, including heart-related co-morbidities, is reported in Table 1. Concomitant medical treatment is reported in Table 2.


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  		Table 1 Clinical characteristics of healthy subjects and heart failure patients

 


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  		Table 2 Pharmacological treatment of heart failure patients

 
Forty-three healthy subjects (HS, n = 43, males 23 and females 20, age 68.1 ± 10.3 years), sex- and aged-matched with the HF patients, were enrolled as control population. Clinical history, physical evaluation, and ECG analysis excluded any cardiovascular disease in these subjects at the start of the present study.

Routine biochemical tests (glucose, creatinine, electrolytes, total cholesterol, HDL cholesterol, and triglycerides) were carried out in all patients and controls. All subjects gave informed consent to participate in the study.

The study was approved by the Ethical Committee of our institution and conducted according to the Declaration of Helsinki. Both patients and control subjects gave formal informed consent to the study.

2.2 Blood sampling protocol and platelet isolation
Patients had fasted for the last 12 h before blood sampling. Blood samples were drawn between 8 and 9 AM from an antecubital vein with a 21 gauge needle and then mixed in a tube with 0.13 mM/L sodium citrate (ratio 9:1). To obtain platelet-rich plasma (PRP), sample was centrifuged (15 min at 180 g). PRP was then recentrifuged (20 min at 800 g) to concentrate platelets, and the pellet was resuspended in Tyrode buffer to obtain a final platelet concentration of 2 x 108/mL.10

2.3 Plasma levels of tumour necrosis factor {alpha}, CD40L, soluble TNF receptors I and II, and 8-hydroxy-2'-deoxyguanosine
Blood samples were immediately centrifuged at 2000 r.p.m. for 20 min at 4°C, and the supernatant was collected and stored at –80°C until measurement. Plasma levels of sCD40L, TNF{alpha}, soluble TNF receptors I and II (sTNFR1 and sTNFR2), and 8OH-dG were measured with a commercial immunoassay (Quantikine R&D Systems). Intra-assay and inter-assay coefficients of variation were 5 and 7%, respectively, for sCD40L, 5.2 and 7.4% for TNF{alpha}, 4.7 and 8.8% for both TNFR1 and TNFR2, and 2.1 and 4.5% for 8-OHdG.

2.4 Flow cytometric analysis of platelet tumour necrosis factor receptors I and II, CD40L, and p38 MAP kinase phosphorylation
In a subgroup of HS (n = 20) and HF patients (NYHA I–II n = 10 and NYHA III–IV n = 10), who were representative of the entire population, we performed the analysis of CD40L, TNFR-1, and TNFR-2 platelet surface expression. CD40L expression on platelet membranes was analysed with specific fluorescein isothiocyanate-labelled monoclonal antibodies (mAbs; anti-CD40L antibody by Beckman Coulter). TNFR-1 and TNFR-2 expressions on platelet membranes were analysed with an affinity-purified polyclonal antibody unconjugated (anti-TNFR-1 and anti-TNFR-2 antibodies by Santa Cruz Biotechnology, Inc.). The expression of antibodies was detected with a secondary antibody (anti-goat IgG-FITC by Santa Cruz Biotechnology, Inc.). In all assays, an irrelevant isotype-matched antibody was used as a negative control.

Twenty microlitres of mAb was added to 200 µL of platelet suspension (2 x 108/mL) previously fixed with tromboFix platelet stabilizer (Beckman Coulter) and incubated for 60 min at 4°C. The unbound mAb was removed by addition of 0.1% bovine serum albumin–phosphate-buffered saline (PBS) and centrifugation at 5000 g for 3 min (twice). p38 MAP kinase phosphorylation was analysed by flow cytometry, using specific antibodies (anti-p38 MAPkinase IgG goat polyclonal). Twenty microlitres of antibody was added to 200 µL of platelet suspension (2 x 108/mL) previously fixed with 2% paraformaldehyde (0.1% BSA) for 60 min at room temperature and then permeabilized with digitonin (100 µM) for 30 min at room temperature. Unbound antibody was removed by addition of 0.1% BSA PBS and subsequent centrifugation at 3000 g for 3 min (twice).

Fluorescence intensity was analysed on an Epics XL-MCL cytometer (Coulter Electronics) equipped with an argon laser at 488 nm. For every histogram, 50 000 platelets were counted to determine the proportion of positive platelets. Antibody reactivity is reported mean fluorescence.10

2.5 In vitro study
Platelets taken from patients and controls (NYHA I–II n = 10, NYHA III–IV n = 10, and HS n = 10) were washed and suspended in Tyrode’s buffer. Platelet suspensions were incubated with TNF{alpha} (30 pg/mL) for 15 min at 37°C in the presence or not of WP9QY (1 µM), an inhibitor of both TNFR1 and TNFR2; platelet expression of CD40L was measured as reported earlier.

Also, in another subgroup of NYHA patients (n = 10) not treated with aspirin and with physiological positive platelet response to arachidonic acid (aggregometric test by Born methods,13 data not shown), the COX-1 inhibitor aspirin (100 µM), the PLA2 inhibitor AACOCF3 (10 µM), NADPH-oxidase inhibitor apocynin (100 µM), the ETYA inhibitor lipoxygenase (10 µM), and the staurosporine inhibitor PKC (10 µM) were added before (15 min, 37°C) TNF{alpha} stimulation. The samples were then treated as described earlier to detect platelet CD40L expression and p38 MAP kinase phosphorylation.

2.6 Statistical analysis
Comparisons between groups were carried out by Student’s t-test; independence of categorical variables was tested by {chi}2 test. The correlation analysis was carried out by Pearson’s test. Multiple linear regression analysis was performed using a stepwise selection method and was performed to determine the independent parameters of sCD40L and TNF{alpha}. Statistical significance was defined at P < 0.05. Statistical analysis was performed with SPSS 13.0 software for Windows.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Funding
 References
 
Table 1 reports clinical characteristics of HS and HF patients. Compared with NYHA I–II patients, those with NYHA III–IV had higher prevalence of atrial fibrillation and diabetes; hypertension was more prevalent in NYHA I–II compared with those with NYHA III–IV.

Table 2 reports the pharmacological treatment of HF patients; 82% of patients were treated with ACE-inhibitors or angiotensin receptor blockers, whereas beta-blockers were used in 35% of total patients; in both cases, no statistically significant difference between the two groups (NYHA I–II and NYHA III–IV) was found. Loop diuretic drugs were equally distributed between NYHA I–II and III–IV, whereas only patients in NYHA class III–IV (12%) were treated with aldosterone inhibitors. Equally distributed between the two HF subgroups was the therapy with digoxin, antiplatelet drugs, oral anticoagulants, and statins. Conversely, antidiabetic therapy was slightly more prevalent in NYHA III–IV group (P < 0.045).

3.1 Plasma levels of tumour necrosis factor {alpha}, soluble CD40L, soluble tumour necrosis factor receptors I and II, and 8-hydroxy-2'-deoxyguanosine
TNF{alpha} was significantly higher in HF patients than in control subjects and progressively increased from NYHA I–II to NHYA III–IV (Figure 1A, P < 0.001); a similar behaviour was found for sCD40L (Figure 1B, P < 0.001), TNFR1 (Figure 1C, P < 0.001), TNFR2 (Figure 1D, P < 0.001), and 8OH-dG (Figure 1E, P < 0.001).


Figure 1
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  		Figure 1 Plasma levels of TNF{alpha} (A), sCD40L (B), sTNFR1 (C), sTNFR2 (D), and 8-OHdG (E) in healthy subjects (HS) and heart failure patients (NYHA I–II and NYHA III–IV). *P < 0.001.

 
Plasma levels of TNF{alpha} positively correlated with sCD40L plasma levels (r = 0.73, P < 0.001) in HF patients, whereas in HS, the correlation was weaker (r = 0.3, P < 0.04) (Figure 2A); moreover, TNF{alpha} positively correlated with sTNFR only in HF patients (sTNFR1: r = 0.69, P < 0.001; sTNFR2: r = 0.58, P < 0.001) but not in controls (sTNFR1: r = 0.11 and sTNFR2: r = –0.61) (Figure 2B and C); a correlation between TNF{alpha} and 8OH-dG was also found in HF patients (r = 0.68, P < 0.001) but not in HS (r = 0.07) (Figure 2D).


Figure 2
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  		Figure 2 Correlation between TNF{alpha} values and plasma levels of sCD40L (A), sTNFR1 (B), sTNFR2 (C), and 8-OHdG (D); HF, heart failure patients; HS, healthy subjects; NS, not significant.

 
In order to identify the potential independent predictors of sCD40L and TNF{alpha}, a multiple regression analysis (including as independent variables age, gender, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol, blood pressure, coronary heart disease, NYHA classes, sTNFR1 and sTNFR2, creatinine, and 8OH-dG) was performed (Table 3); this analysis showed that sCD40L, NYHA classes III–IV, and sTNFR1 were associated with TNF{alpha}, whereas 8OH-dG and TNF{alpha} were associated with sCD40L (Table 3).


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  		Table 3 Stepwise multiple linear regression analysis with tumour necrosis factor {alpha} and CD40L as the dependent variable in patients with heart failure

 
3.2 Platelet expression of tumour necrosis factor receptors I and II and CD40L
Figure 3 shows the expression on platelet surface of TNFR1 and TNFR2 in HF and control subjects. The platelet expression of the two receptors was higher in patients compared with controls and progressively increased from NYHA I–II to NHYA III–IV (NYHA I–II vs. controls P < 0.001, NYHA III–IV vs. controls P < 0.001; Figure 3). Moreover, platelet expression of TNFR1 and TNFR2 correlated with serum levels of the soluble form of each molecule (platelet TNFR1 vs. sTNFR1: r = 0.66, P < 0.05; platelet TNFR2 vs. sTNFR2: r = 0.63, P < 0.01).


Figure 3
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  		Figure 3 Flow cytometric expression of TNFR-1 and TNFR-2 on platelet surface in healthy subjects (HS) and patients with heart failure (NYHA I–II n = 10, NYHA III–IV n = 10, and HS n = 10). Results are expressed as mean fluorescence. *P < 0.001.

 
Platelet stimulation with TNF{alpha} resulted in a higher expression of platelet CD40L. Such effect was observed essentially in patients with HF and, within HF population, was more marked in patients with NYHA III–IV (NYHA I–II vs. control P < 0.001, NYHA III–IV vs. controls P < 0.001) (Figure 4). The upregulation of platelet CD40L by TNF{alpha} was abrogated when TNF{alpha}-stimulated platelets were treated with the inhibitor of TNF{alpha} receptors. Such effect was observed essentially in HF (Figure 4).


Figure 4
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  		Figure 4 Flow cytometric expression of CD40L in unstimulated platelets and platelets stimulated with TNF{alpha} (30 pg/mL) added or not with WP9QY (1 µM). Platelets were taken from patients with heart failure ( NYHA I–II n = 10, NYHA III–IV n = 10, and HS n = 10); results are expressed as mean fluorescence. *P < 0.001.

 
3.3 In vitro study
Apocynin, AACOCF3, and staurosporine significantly inhibited TNF{alpha}-induced platelet CD40L by 69% (P < 0.001), 74% (P < 0.001) and 68% (P < 0.001), respectively; conversely, ASA reduced it by only 21% (P > 0.05) (Figure 5), whereas the lipoxygenase inhibitor ETYA did not induce any change of CD40L expression (Figure 5).


Figure 5
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  		Figure 5 Flow cytometric expression of CD40L in unstimulated platelets and platelets stimulated with TNF{alpha} (30 pg/mL) added or not with apocynin (100 µM), AACOCF3 (10 µM), ASA (100 µM), ETYA (10 µM), and staurosporine (10 µM). Platelets were taken from heart failure patients ( NYHA I–II n = 10 and HS n = 10); results are expressed as mean fluorescence. *P < 0.001.

 
In order to further analyse the mechanism through which TNF{alpha} elicits platelet CD40L upregulation, the phosphorylation of p38 MAP kinase was investigated. This experiment showed that TNF{alpha} induced p38 MAP kinase phosphorylation, which was significantly inhibited by the TNF{alpha} receptor inhibitor, WP9QY (Figure 6).


Figure 6
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  		Figure 6 Flow cytometric analysis of p38 MAP kinase phosphorylation in unstimulated platelets and platelets stimulated with TNF{alpha} (30 pg/mL) added or not with Wp9qy (1 µM). Platelets were taken from patients with heart failure ( NYHA I–II n = 10); results are expressed as mean fluorescence. *P < 0.001.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Funding
 References
 
The study provides evidence that in patients with HF, both TNF{alpha} and platelet CD40L are overexpressed and that TNF{alpha} upregulates platelet CD40L with an arachidonic acid-mediated oxidative stress mechanism.

4.1 CD40L in heart failure patients
Previous studies investigated the behaviour of platelet CD40L in patients with HF but the results are not concordant. Ueland et al.7 found increased serum levels of sCD40L that correlated with the degree of HF, and Stumpf et al.16 described an upregulation of platelet CD40L; conversely, Chung et al.17 failed to show change in platelet CD40L regulation. Our study showed that both platelet CD40L and sCD40L were upregulated and significantly correlated. As sCD40L essentially stems from platelet activation,18 these data suggest that in HF patients, the increased circulating levels of sCD40L could be platelet-mediated. Our data also shows the existence of a link between the expression of CD40L and the degree of HF, as patients of NYHA III–IV had higher values of sCD40L compared with those of NYHA I–II.

4.2 Tumour necrosis factor {alpha} and its receptors in heart failure patients
There is a growing evidence that TNF{alpha} is upregulated in HF patients and seems to be involved in the pathophysiology of this disease.4,5 The present study confirms and extends the previous studies as it shows that both TNF{alpha} and the soluble form of its receptors, sTNFR1 and sTNRF2, are elevated in HF patients. As in the case of CD40L, the increase of these variables was associated with the severity of HF.

The two TNF{alpha} receptors, TNFR1 and TNFR2, have been found in different cells such as T lymphocytes and hepatocytes19 and more recently on platelets.20 Their soluble form is elevated in patients with HF21 but the cellular origin is still unclear. In the present study, the platelet receptors TNFR1 and TNFR2, even if in a different degree, were upregulated in HF patients and significantly correlated with the soluble forms of TNFR1 and TNFR2. This would suggest that the increase of the soluble form of the two receptors could reflect platelet TNFR1 and TNFR2 upregulation but this hypothesis needs to be further investigated.

4.3 Tumour necrosis factor {alpha}, oxidative stress, and CD40L
The coexistence of the upregulation of TNF{alpha}, platelet TNFR1 and TNFR2, and platelet CD40L prompted us to speculate that TNF{alpha} interaction with its platelet receptors10 could result in platelet CD40L upregulation. Consistent with this hypothesis, we observed that TNF{alpha}, at concentration observed in the peripheral circulation of HF patients, upregulated platelet CD40L; co-incubation of TNF{alpha}-treated platelets with a TNFR receptor inhibitor blunted such effect, indicating that TNF{alpha} elicited platelet CD40L upregulation via interaction with its platelet receptors. These data are apparently at variance with a previous study showing no effect of a TNF{alpha} inhibitor on platelet CD40L expression.22 However, differently from our study, thrombin but not TNF{alpha} was used to stimulate platelet function, therefore the interaction between TNF{alpha} and its receptors on platelet surface could unlikely be blunted.

As previous study showed that oxidative stress is implicated in platelet CD40L upregulation,23 we investigated the behaviour of oxidative stress in HF patients and its interplay with TNF{alpha}-mediated CD40L upregulation. In accordance with previous data,21 we found a significant increase of oxidative stress, which correlated with the circulating levels of TNF{alpha}. On the basis of previous data indicating that TNF{alpha} elicits the platelet production of ROS,24 we investigated whether a relationship among TNF{alpha}, platelet ROS formation, and CD40L expression does exist.

We observed that activation of NADPH oxidase was relevant in TNF{alpha}-induced CD40L, as its inhibition was associated with platelet CD40L downregulation. In order to elucidate the underlying mechanism, we focused our attention on the metabolism of arachidonic acid inasmuch as we have previously demonstrated that (i) TNF{alpha} enhances platelet ROS production via PLA224 and that (ii) arachidonic acid is able to activate NADPH oxidase.15 In vitro experiments showed that neither COX-1 nor lipoxygenase seemed to be involved in such phenomenon as in the fact that aspirin minimally influenced TNF{alpha}-induced CD40L expression and ETYA was ineffective. Conversely, PKC, which is activated by arachidonic acid and in turn stimulates NADPH oxidase,15,25 seemed to have a major role, as its inhibition blunted TNF{alpha}-induced platelet CD40L upregulation. Finally, we observed that TNF{alpha} enhanced p38 MAP kinase phosphorylation, suggesting that PLA2 activation by TNF{alpha} could be p38 MAP kinase-mediated.26

Together these data suggest that TNF{alpha} upregulated CD40L with a mechanism involving arachidonic acid-induced NADPH oxidase generating ROS. This effect seems to be independent from COX-1 and lipoxygenase activation but occurs via PKC pathway.

4.4 Implications and limitations
This study has potential implication. HF represents an important cause of morbidity and mortality in western populations.27 HF predisposes to thrombo-embolism, in particular, to ischaemic stroke, in a relatively high percentage of patients.28 The association between HF and thrombo-embolic stroke is higher in patients with severe HF compared with patients with mild-to-moderate HF,28 but the mechanisms favouring thrombo-embolism have not been fully clarified.27 Herewith we provide a novel mechanism linking inflammation and thrombosis. Thus, upregulation of platelet CD40L by TNF{alpha} could favour thrombo-embolism in virtue of the role played by C40L in activating platelets9 and in generating thrombin via the tissue factor pathway.8 Assuming that arachidonic acid has a central role in this phenomenon, the increase of n-3 on platelet membrane could represent an interesting future prospective to reduce platelet activation in HF patients.29

The study has some limitations that should be acknowledged. Even if the in vitro study suggests a cause–effect relationship between TNF{alpha} and platelet CD40L, we have only indirect evidence that this occurs in vivo. Therefore, further study is necessary to investigate whether, in HF patients, TNF{alpha} actually promotes platelet CD40L upregulation. Interventional trials with drugs that specifically reduce TNF{alpha} should be done to support such relationships.

Also, we have only indirectly shown that TNF{alpha} activates platelet NADPH oxidase; therefore, direct measurement of the activity of such enzyme is necessary to support the hypothesis that TNF{alpha} upregulates CD40L with an oxidative stress-mediated mechanism.

In conclusion, the study shows that in HF patients, there is a concomitant increase of TNF{alpha} and platelet CD40L and suggests that the upregulation of CD40L by TNF{alpha} is oxidative-stress mediated. This finding could provide a novel link between inflammation and thrombosis in the setting of HF.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
References
 
Ateneo 2006 No. 8.1.1.1.21.29, University of Rome ‘La Sapienza’.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 

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