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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2428.)
© 2005 American Heart Association, Inc.

Thrombosis

CD40 Ligand Influences Platelet Release of Reactive Oxygen Intermediates

Subrata Chakrabarti; Sonia Varghese; Olga Vitseva; Kahraman Tanriverdi; Jane E. Freedman

From the Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass.

Correspondence to Subrata Chakrabarti, Boston University School of Medicine, 715 Albany St, W507, Boston, MA 02118. E-mail subrata{at}bu.edu

	Abstract

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Objective— Soluble CD40 ligand (sCD40L) has been recently implicated in the pathogenesis of atherosclerosis. Elevated levels of sCD40L in acute coronary syndrome patients suggests enhanced platelet function; however, the exact mechanism by which this occurs is unknown. In this study, we examined the effect of sCD40L on platelet function and reactive oxygen and nitrogen species (RONS) generation.

Methods and Results— Platelet stimulation in the presence of recombinant sCD40L (rsCD40L) led to enhanced generation of RONS as measured by DCFHDA oxidation and confocal microscopy. Incubation with rsCD40L led to enhanced platelet P-selectin expression, aggregation, and platelet-leukocyte conjugation. Platelets isolated from CD40L-deficient mice had decreased agonist-induced NO release as compared with wild-type mice. Incubation of platelets with rsCD40L enhanced stimulation-induced p38 MAP kinase and Akt phosphorylation.

Conclusion— Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well as increases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signaling pathways. These data suggest that sCD40L regulates platelet-dependent inflammatory and thrombotic responses that contribute to the pathogenesis of atherothrombosis.

The role of sCD40L in platelet function and RONS formation was examined. Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well as increases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signaling pathways. sCD40L influences platelet-dependent thrombotic and inflammatory responses contributing to atherothrombosis.

Key Words: platelets • reactive oxygen species • CD40 ligand • fluorescence

	Introduction

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CD40 ligand (CD40L, CD154) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily. Originally identified as a surface molecule expressed on activated T-cells, CD40L was shown to be important for the induction of B-cell development and proliferation, as well as for the IgM to IgG isotype switch.^1–3 CD40L is expressed in a variety of other cell types including monocytes, macrophages, dendritic cells, mast cells, basophils, eosinophils, B-cells, and platelets.^3–6 CD40L is involved in the initiation of an inflammatory response at the vessel wall by inducing the expression of adhesion molecules and the secretion of chemokines by vascular endothelial cells.⁷

In addition to the trimeric membrane-bound form, CD40L also exists in a soluble 18-kDa form (sCD40L) that is released from platelets after stimulation.^5,8,9 In contrast to membrane-bound CD40L, sCD40L fails to induce an inflammatory response in endothelial cells.⁵ Soluble CD40L has been shown to stabilize arterial thrombi¹⁰ and is reportedly a platelet GPIIb/IIIa ligand.¹¹ In addition, sCD40L participates in platelet outside-in signaling after binding via its KGD motif.¹²

Platelets are the richest source of sCD40L in the circulation,^13,14 and platelet aggregation and activation is associated with the release of sCD40L.⁵ A recent study suggests that platelet gp91phox contributes to the regulation of the release of CD40 ligand from platelets.¹⁵ Platelet activation and aggregation also induce oxidative stress^16,17 through the generation of reactive oxygen and nitrogen species (RONS)^18–20; however, the role of CD40L in the generation of RONS by platelets is unknown. In the current study, using CD40L-deficient mice and recombinant CD40 ligand (rsCD40L), we examined the effect of sCD40L on RONS regulation in platelets. The role of the stress response protein kinases Akt and p38 MAP kinase, known to mediate RONS generation, was also examined.

	Materials and Methods

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For detailed Materials and Methods, please see the data supplement available online at http://atvb.ahajournals.org.

	Results

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Prolonged Release of sCD40L After Platelet Stimulation
Platelets have recently been identified as a source of inflammatory chemokines and cytokines.^8,21,22 As shown in Figure 1, platelet activation by thrombin results in the release of sCD40L as a function of time, whereas unstimulated platelets release only minor amounts of sCD40L. Recent data suggesting a role for sCD40L in platelet activation²³ have important implications in atherosclerosis as unrestricted release of sCD40L by activated platelets may serve as a positive feedback mechanism, amplifying activation of other platelets.²⁴

View larger version (13K): [in this window] [in a new window]   Figure 1. Platelets release soluble CD40 ligand (sCD40L) after thrombin stimulation. Washed human platelets (2x108 per mL) were stimulated with 0.2 u/mL human alpha thrombin (0.2 u/mL) as a function of time (n=4).

The Functional Effects of sCD40L
To verify the functional effects of sCD40L, platelet aggregation and P-selectin expression by using flow cytometry were measured after incubation with rsCD40L. As shown in the Table, rsCD40L enhances platelet P-selectin expression even at ng/mL concentration. In addition, rsCD40L significantly enhances platelet aggregation. Because P-selectin expression may lead to platelet-leukocyte interaction, the effects of rsCD40L on platelet-neutrophil and monocyte conjugate formation in human blood were studied. As shown in Table I (available online at http://atvb.ahajournals.org), rsCD40L enhances ADP induced platelet-neutrophil and monocyte conjugate formation. In addition, rsCD40L alone induces platelet-monocyte conjugation.

View this table: [in this window] [in a new window]   Recombinant Soluble CD40L (rsCD40L) Induces Platelet P-Selectin Expression and Stimulates Platelet Aggregation

Recombinant Soluble CD40L Induces Platelet Release of Reactive Oxygen Intermediates
Although the role of sCD40L in the generation of ROS has been suggested in other settings,^25,26 the role of sCD40L in the modulation of RONS generation in platelets is unknown. Figure 2A depicts levels of platelet-derived RONS as measured by oxidation of the redox sensitive probe, DCFHDA. The addition of rsCD40L significantly enhances generation of RONS in the TRAP-stimulated platelets in a dose-dependent fashion (Figure 2B).

View larger version (24K): [in this window] [in a new window]   Figure 2. Soluble CD40L induced generation of reactive oxygen species in platelets. Platelets were incubated with rsCD40L for 5 minutes followed by 3 minutes stimulation with TRAP. Generation of reactive oxygen species was determined by flow cytometric monitoring of DCFHDA oxidation. A, Representative tracing shows rsCD40L induced enhancement of reactive oxygen species generation in TRAP-stimulated platelets (n=7). B, Increasing concentration of rsCD40L leads to a dose-dependent increase in platelet reactive oxygen species generation (n=5; P<0.001). C, The inhibitory effect of anti-CD40 ligand antibody (10 µg/mL, 95% blockage) or GPIIb/IIIa blocking antibody (abciximab; 20 µg/mL, 93% blockage) on rsCD40L (rs, 1 µg/mL) induced DCFHDA oxidation in the setting of TRAP stimulation (n=3; *P<0.05).

Recently, it has been shown that CD40L is a glycoprotein IIb/IIIa ligand that induces platelet outside-in signaling.¹² To determine the receptor specificity for CD40L dependent generation of RONS, the effect of CD40L antibody or GPIIb/IIIa inhibition on the rsCD40L-induced platelet generation of RONS was studied. As shown in Figure 2C, both anti-CD40L antibody and GPIIb/IIIa inhibition with abciximab significantly attenuate generation of the RONS by platelets. To define the specific source of RONS, platelets were incubated with the NOS inhibitor, L-NAME, or the NADPH oxidase inhibitor, apocynin. Both L-NAME and apocynin inhibited rsCD40L-induced RONS generation (Figure I, available online at http://atvb.ahajournals.org) suggesting that both eNOS and NADPH oxidase contribute to the observed DCFHDA oxidation. Additionally, rsCD40L induces platelet superoxide in a dose dependent manner (Figure I) even at low concentrations (10 ng/mL to1 µg/mL).

The effect of rsCD40L on platelet generation of RONS was confirmed by confocal microscopy using 2 complementary fluorescent probes to detect RONS generation: dihydrorhodamine (DHR), a reactive oxygen sensitive probe and MitoTracker red, a redox-sensitive reactive oxygen-sensitive mitochondrial probe. As shown in Figure 3A and 3B, TRAP-activated platelets generate RONS, and this was markedly enhanced in the presence of rsCD40L.

View larger version (67K): [in this window] [in a new window]   Figure 3. Recombinant sCD40L enhances platelet release of reactive oxygen species. A, Washed platelets were incubated with 20 µmol/L dihydrorhodamine for 10 minutes in presence and absence of 10 µg/mL rsCD40L and stimulated with 50 µmol/L TRAP. Images were captured after 10 minutes incubation. B, Washed platelets were incubated with redox-sensitive mitochondrial probe, Mito Tracker Red CM H2XROS for 10 minutes in presence and absence of 10 µg/mL rsCD40L and then stimulated with 50 µmol/L TRAP (n=3).

The Role of CD40L in Platelet Nitric Oxide Generation
To determine whether CD40L deficiency alters platelet-derived NO release, platelets were isolated from CD40L-deficient mice and stimulation-induced (thrombin, ADP, or collagen) NO generation was determined. As shown in Figure 4A, CD40L deficiency significantly impairs platelet release of NO. Notably, this attenuation in NO generation was apparent in response to all 3 agonists examined. We confirmed these results by carrying out platelet aggregation, with normal human platelets, in the presence of rsCD40L. The presence of varying concentration of rsCD40L enhanced platelet NO generation (Figure 4B). The rsCD40L-induced NO generation was attenuated by anti-CD40L antibody (Figure II, available online at http://atvb.ahajournals.org) indicating specificity of the rsCD40L induced effect.

View larger version (32K): [in this window] [in a new window]   Figure 4. Measurement of NO after platelet aggregation. A, Platelets were isolated from CD40L-deficient mice and stimulation-dependent NO generation was measured by a microelectrode. Decreased NO generation was seen using the platelet agonists as compared with wild-type mice (thrombin [1u/mL], ADP [5 µmol/L], and collagen [10 µg/mL]; n=4; *P<0.001, **P<0.05). B, Washed human platelets were incubated with vehicle control or varying concentrations of recombinant CD40L (rsCD40L) for 5 minutes, and then TRAP aggregation-induced NO was measured (n=3; *P<0.004).

The Effect of rsCD40L on Phosphorylation of Akt and p38 MAP Kinase
The Effect of rsCD40L on Akt Phosphorylation
Recent studies have identified Akt as an important stress-response protein kinase in endothelial cells responding to changes in the redox environment.^27,28 The observation that rsCD40L enhances oxidative stress in platelets led to the examination of the status of Akt phosphorylation in the presence and absence of platelet stimulation. As shown in Figure 5A, addition of rsCD40L clearly enhances Akt phosphorylation in thrombin- and TRAP-stimulated platelets. Moreover, this enhanced Akt activation is reversed in the presence of the PI3 kinase inhibitor, LY294002 (ELISA results subsequently confirmed by Western blot analysis; Figure III, available online at http://atvb.ahajournals.org). Interestingly, the addition of rsCD40L alone significantly increases Akt phosphorylation. These data demonstrate the ability of rsCD40L to induce Akt activation in unstimulated and stimulated platelets.

View larger version (26K): [in this window] [in a new window]   Figure 5. The effect of rsCD40L on platelet AKT phosphorylation (A) or p38 MAP kinase phosphorylation (B). i, The effect of rsCD40L on thrombin-induced AKT/p38 MAP kinase phsophorylation. Platelet protein phosphorylation was determined after 5 minutes preincubation at 37°C with rsCD40L (10 µg/mL) followed by thrombin (1 u/mL) stimulation. ii, Quantitative evaluation (ELISA) of the effect of rsCD40L on AKT/p38 MAP kinase phosphorylation after TRAP stimulation (50 µmol/L). Platelets were preincubated with rsCD40L (10 µg/mL) at 37°C for 5 minutes followed by TRAP stimulation (n=4; *P<0.05 compared with resting platelets; **P<0.002 compared with TRAP stimulation [Akt]; +P<0.002 compared with rsCD40L+TRAP stimulation [Akt]; **P<0.02 compared with TRAP stimulation [p38 MAP kinase]; +P<0.02 compared with rsCD40L+TRAP stimulation [p38 MAP kinase]). [LY29004]=25 µmol/L, [SB203580]=10 µmol/L.

The Effect of rsCD40L on p38 MAP Kinase Phosphorylation
p38 MAP kinase has also been reported to regulate the stress-response mechanisms of vascular cells such as endothelial cells,²⁹ smooth muscle cells,³⁰ and human macrophages.³¹ In addition, a recent study showed that rsCD40L is capable of inducing platelet p38 MAP kinase phosphorylation.²³ Therefore, we examined the role of p38 MAP kinase activation under conditions where rsCD40L induces platelet RONS generation. Platelets were stimulated with thrombin or TRAP, and p38 MAP kinase phosphorylation was determined. As shown in Figure 5B, like Akt, p38 MAP kinase phosphorylation was significantly enhanced in the presence of rsCD40L after thrombin stimulation. The phospho-ELISA plots shown in Figure 5B (confirmed also by Western blot; Figure IV, available online at http://atvb.ahajournals.org) indicate that the addition of rsCD40L alone, or in combination with TRAP, significantly enhances p38 MAP kinase phosphorylation. Phosphorylation of p38 MAP kinase was attenuated in the presence of specific MAP kinase inhibitor, SB203580 (Figure 5B and Figure IV).

Involvement of PI3 Kinase/Akt and p38 MAP Kinase in Platelet RONS Generation
To understand the role of PI3 kinase/Akt or p38 MAP kinase in rsCD40L-induced platelet RONS generation, we carried out flow cytometric analysis monitoring DCFHDA oxidation (as described in Figure 2A) in presence of specific kinase inhibitors. Presence of the PI3 kinase inhibitor, Ly294002, and the p38 MAP kinase inhibitor, SB203580, eliminated platelet activation-induced RONS generation (Figures V and VI, available online at http://atvb.ahajournals.org).

	Discussion

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During inflammation, reactive oxygen species promote the adhesion of blood cells to the vascular endothelium by eliciting production of inflammatory mediators or activating nuclear transcription factors that bind to genes encoding adhesion molecules and cytokines.^32,33 CD40–40L signaling represents another pathway that enables blood cells to amplify the endothelial cell responses to inflammation and contribute to the regulation of hemostasis.⁷ Platelets can indirectly orchestrate (through CD40–40L interaction via the endothelium) changes in coagulation, leukocyte trafficking, and extracellular matrix modeling turnover.³⁴ CD40L has been associated also with numerous vascular diseases including diabetes and acute coronary syndromes.^35–41 In recent studies, the prognostic values of serum sCD40L in acute coronary syndromes⁴² and in unstable angina patients⁸ has been reported.

Reports of sCD40L release by platelets^5,7 have led to studies examining the mechanism of CD40L expression,¹⁵ release,^43,44 and its regulatory role in the inflammatory milieu of the vasculature.^10,45–48 Platelet activation is accompanied by release of both sCD40L and RONS such as superoxide^20,49 and NO¹⁹; however, the role of platelet-derived sCD40L in the generation of RONS is unknown. In the present study, we examined the effect of CD40L on the generation of RONS in platelets and determined the mechanism for the observed effects. Initially, we demonstrated a prolonged release of sCD40L by platelets for 6 hours after thrombin stimulation (Figure 1). Sustained release of platelet sCD40L⁹ might contribute to atherothrombotic disease^13,50 by forming platelet-platelet (CD40–40L⁵¹/CD40L-GPIIb/IIIA),¹⁰ platelet-leukocyte (CD40L-40/P-selectin-PSGL),⁵² and platelet-endothelial (CD40L-CD40) conjugates.²⁶ As shown in the Table and Table I, rsCD40L is capable of inducing platelet P-selectin expression and platelet-leukocyte conjugate formation in whole blood. Recombinant CD40L has also been recently reported to activate platelets,^23,51 resulting in secretion of P-selectin and suggesting the possibility of in vivo platelet-induced inflammation. Recent work by Wagner et al⁵³ has provided direct experimental evidence that CD154 (CD40L) can induce the expression of CD154 in endothelial cells and influence atherogenesis through activation of extravasating monocytes. Additionally, CD40L positive platelets have been shown to induce CD40L expression de novo in endothelial cells.⁵⁴ These observations support the proposed coordinated interactions among platelets and leukocytes with activated endothelium in diseased blood vessels through adhesion molecules and, particularly, CD40/40L.⁵⁵

Relevant to vascular inflammation is the effect of CD40L on RONS generation. Figures 2 and 3 show that rsCD40L increases stimulation-induced RONS generation by platelets and that this effect is reversible with specific antibodies. To determine whether platelet RONS generation is thrombin receptor-specific, we carried out further flow cytometric analysis with other agonists. In contrast to TRAP stimulation, we observed minimal generation of reactive oxygen species after ADP stimulation (data not shown). It is possible that the TRAP-induced RONS generation may be thrombin receptor-specific and independent of purinergic stimulation. Inhibition of the GPIIb/IIIa complex also decreased RONS generation, consistent with the previous observation showing that sCD40L is a GPIIb/IIIa ligand.¹⁰ Mechanistically, rsCD40L may ligate the platelet receptor CD40 or GPIIb/IIIa complex to initiate platelet signaling pathways that affect the generation of reactive oxygen and nitrogen species. In a clinical setting, recent studies have also shown that GPIIb-IIIa antagonists reduce circulating sCD40L and leukocyte-platelet aggregate formation in patients with acute coronary syndromes undergoing percutaneous coronary intervention.⁵⁶

The importance of CD40L in platelet generation of RONS was shown using CD40L-deficient mice (Figure 4A). The finding of impaired NO generation was complemented by the studies in human platelets where NO release was enhanced on addition of rsCD40L (Figure 4B). Additionally, CD40L-deficient mice had a lower extent of platelet DCFHDA oxidation as compared with wild-type mice platelets after thrombin stimulation (data not shown). It is possible that the combined production of NO mediated by Akt and/or p38 MAP kinase and superoxide mediated by p38 MAP kinase activation leads to the generation of peroxynitrite/RONS causing rsCD40L/TRAP-induced DCFHDA oxidation and fluorescence induction. The formation of peroxynitrite is further suggested by platelet fluorescence inhibition studies using NOS inhibitor, L-NAME, and NADPH oxidase inhibitor, apocynin (Figure I). p38 MAP kinase may also stimulate the PI3 kinase/Akt/eNOS pathway, enhancing NO generation.⁵⁷ TRAP-induced platelet DCFHDA oxidation study (Figures V and VI) indicates that both PI3 Kinase/Akt and p38 MAP kinase signaling pathways are involved in the rsCD40L-dependent RONS generation.

Some earlier studies have also documented that platelet generation of reactive oxygen intermediates²⁰ involves PI3 kinase/Akt and MAP kinase activation. To investigate whether rsCD40L-induced fluorescence is accompanied by Akt and p38 MAP kinase activation, we analyzed platelet proteins after stimulation in the presence or absence of rsCD40L. The results (Figure 5A) indicate that rsCD40L enhances Akt phosphorylation in both stimulated and unstimulated platelets. Akt is an endothelial cell stress-response protein^27,28 that counteracts external oxidative stress by enhancing NO generation after eNOS phosphorylation.^28,58 Enhanced Akt phosphorylation is also consistent with increased NO generation as observed during platelet aggregation (Figure 4B). These results suggest that, similar to endothelial cells,²⁸ rsCD40L may regulate pathways leading to enhanced NO formation.

In vascular cells, p38 MAP kinase is also known to regulate RONS-dependent pathways.^29,30,59,60 Similar to Akt, rsCD40L also induced p38 MAP kinase phosphorylation (Figure 5B). This observation is consistent with a recent report showing increased platelet p38 MAP kinase phosphorylation in response to rsCD40L.²³ MAP kinase may enhance the generation of superoxide by stimulating NADPH oxidase as inhibition of NADPH oxidase with apocynin⁶¹ decreased platelet generation of RONS (Figure I). We also examined whether involvement of p38 MAP kinase signaling is linked with platelet calcium mobilization during TRAP-induced fluorescence induction. Our findings (Figure VII, available online at http://atvb.ahajournals.org) suggest that p38 MAP kinase inhibition does not alter TRAP/rsCD40L-induced platelet calcium mobilization. We have also verified that rsCD40L/TRAP-induced platelet RONS generation is sensitive to calcium inhibition by dimethylbapta (data not shown).

In our findings there appears to be a discrepancy between the degree of enhancement of rsCD40L-induced Akt and p38 MAP kinase phosphorylation as compared with fluorescence induction (Figures 5 and 2 A). There are several possible explanations for this difference, including (1) the use of distinct experimental conditions, eg, temperature variations: whereas we carried out the TRAP-induced fluorescence induction study at room temperature, TRAP-stimulated phosphorylation studies were done at 37°C; (2) Measurement procedures: in flow cytometry we directly measure the fluorescence induction, whereas protein phosphorylation evaluation involves a multi step procedures. An approach involving simultaneous measurement of fluorescence induction and protein phosphorylation can only provide the exact stoichiometry for these 2 experiments.

There are some limitations to these studies. We carried out platelet stimulation-induced sCD40L release in washed platelets. However, in physiological conditions, platelet stimulation- and activation-induced sCD40L release may be confounded by numerous plasma proteins. Interestingly, recent studies⁴³ with platelet rich plasma reported iso-TRAP stimulation-induced sCD40L release in the range of 0.33 to 4.84 ng/mL in normal healthy volunteers only 20 minutes after stimulation time. This suggests that the presence of plasma proteins did not appear to have any apparent inhibitory effect on the stimulation-induced sCD40L release.

In summary, rsCD40L augments RONS generation in activated platelets after stimulation of kinase signaling pathways. These findings provide insight into the mechanisms by which CD40L regulates oxidative stress in platelets and may contribute to the atherothrombotic processes through further interaction with leukocytes and endothelial cells. Although additional studies will be necessary to characterize earlier signaling events involved in RONS generation, these observations provide new targets for studying and modifying platelet dependent inflammatory responses.

	Acknowledgments

 
We thank Dr Michael Kirber for his help and encouragement during confocal microscopic studies.

	Footnotes

 
A portion of this work was presented at the American Heart Association Meeting, November 2004, New Orleans, La.

Received June 13, 2005; accepted August 17, 2005.

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