cd40 ligand influences platelet release of reactive...

CD40 Ligand Influences Platelet Release of ReactiveOxygen Intermediates

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

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

Methods and Results—Platelet stimulation in the presence of recombinant sCD40L (rsCD40L) led to enhanced generationof RONS as measured by DCFHDA oxidation and confocal microscopy. Incubation with rsCD40L led to enhancedplatelet P-selectin expression, aggregation, and platelet-leukocyte conjugation. Platelets isolated from CD40L-deficientmice had decreased agonist-induced NO release as compared with wild-type mice. Incubation of platelets with rsCD40Lenhanced stimulation-induced p38 MAP kinase and Akt phosphorylation.

Conclusion—Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well asincreases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signalingpathways. These data suggest that sCD40L regulates platelet-dependent inflammatory and thrombotic responses thatcontribute to the pathogenesis of atherothrombosis. (Arterioscler Thromb Vasc Biol. 2005;25:0-0.)

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

CD40 ligand (CD40L, CD154) is a transmembrane pro-tein belonging to the tumor necrosis factor (TNF)

superfamily. Originally identified as a surface moleculeexpressed on activated T-cells, CD40L was shown to beimportant for the induction of B-cell development and pro-liferation, as well as for the IgM to IgG isotype switch.1–3

CD40L is expressed in a variety of other cell types includingmonocytes, macrophages, dendritic cells, mast cells, ba-sophils, eosinophils, B-cells, and platelets.3–6 CD40L isinvolved in the initiation of an inflammatory response at thevessel wall by inducing the expression of adhesion moleculesand the secretion of chemokines by vascular endothelialcells.7

In addition to the trimeric membrane-bound form, CD40Lalso exists in a soluble 18-kDa form (sCD40L) that is releasedfrom platelets after stimulation.5,8,9 In contrast to membrane-bound CD40L, sCD40L fails to induce an inflammatoryresponse in endothelial cells.5 Soluble CD40L has beenshown to stabilize arterial thrombi10 and is reportedly aplatelet GPIIb/IIIa ligand.11 In addition, sCD40L participatesin platelet outside-in signaling after binding via its KGDmotif.12

Platelets are the richest source of sCD40L in the circula-tion,13,14 and platelet aggregation and activation is associatedwith the release of sCD40L.5 A recent study suggests thatplatelet gp91phox contributes to the regulation of the release

of CD40 ligand from platelets.15 Platelet activation andaggregation also induce oxidative stress16,17 through thegeneration of reactive oxygen and nitrogen species(RONS)18–20; however, the role of CD40L in the generationof RONS by platelets is unknown. In the current study, usingCD40L-deficient mice and recombinant CD40 ligand(rsCD40L), we examined the effect of sCD40L on RONSregulation in platelets. The role of the stress response proteinkinases Akt and p38 MAP kinase, known to mediate RONSgeneration, was also examined.

Materials and MethodsFor detailed Materials and Methods, please see the data supplementavailable online at http://atvb.ahajournals.org.

ResultsProlonged Release of sCD40L AfterPlatelet StimulationPlatelets have recently been identified as a source of inflam-matory chemokines and cytokines.8,21,22 As shown in Figure1, platelet activation by thrombin results in the release ofsCD40L as a function of time, whereas unstimulated plateletsrelease only minor amounts of sCD40L. Recent data suggest-ing a role for sCD40L in platelet activation23 have importantimplications in atherosclerosis as unrestricted release ofsCD40L by activated platelets may serve as a positive

Original received June 13, 2005; final version accepted August 17, 2005.From the Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass.A portion of this work was presented at the American Heart Association Meeting, November 2004, New Orleans, La.Correspondence to Subrata Chakrabarti, Boston University School of Medicine, 715 Albany St, W507, Boston, MA 02118. E-mail [email protected]© 2005 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000184765.59207.f3

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feedback mechanism, amplifying activation of otherplatelets.24

The Functional Effects of sCD40LTo verify the functional effects of sCD40L, platelet aggrega-tion and P-selectin expression by using flow cytometry weremeasured after incubation with rsCD40L. As shown in theTable, rsCD40L enhances platelet P-selectin expression evenat ng/mL concentration. In addition, rsCD40L significantlyenhances platelet aggregation. Because P-selectin expressionmay lead to platelet–leukocyte interaction, the effects ofrsCD40L on platelet–neutrophil and monocyte conjugateformation in human blood were studied. As shown in Table I(available online at http://atvb.ahajournals.org), rsCD40Lenhances ADP induced platelet–neutrophil and monocyteconjugate formation. In addition, rsCD40L alone inducesplatelet–monocyte conjugation.

Recombinant Soluble CD40L Induces PlateletRelease of Reactive Oxygen IntermediatesAlthough the role of sCD40L in the generation of ROS hasbeen suggested in other settings,25,26 the role of sCD40L inthe modulation of RONS generation in platelets is unknown.Figure 2A depicts levels of platelet-derived RONS as mea-sured by oxidation of the redox sensitive probe, DCFHDA.The addition of rsCD40L significantly enhances generationof RONS in the TRAP-stimulated platelets in a dose-dependent fashion (Figure 2B).

Recently, it has been shown that CD40L is a glycoproteinIIb/IIIa ligand that induces platelet outside-in signaling.12 Todetermine the receptor specificity for CD40L dependent

generation of RONS, the effect of CD40L antibody orGPIIb/IIIa inhibition on the rsCD40L-induced platelet gener-ation of RONS was studied. As shown in Figure 2C, bothanti-CD40L antibody and GPIIb/IIIa inhibition with abcix-imab significantly attenuate generation of the RONS byplatelets. To define the specific source of RONS, plateletswere incubated with the NOS inhibitor, L-NAME, or theNADPH oxidase inhibitor, apocynin. Both L-NAME andapocynin inhibited rsCD40L-induced RONS generation (Fig-ure I, available online at http://atvb.ahajournals.org) suggest-ing that both eNOS and NADPH oxidase contribute to theobserved DCFHDA oxidation. Additionally, rsCD40L in-duces platelet superoxide in a dose dependent manner (FigureI) even at low concentrations (10 ng/mL to1 �g/mL).

The effect of rsCD40L on platelet generation of RONS wasconfirmed by confocal microscopy using 2 complementaryfluorescent probes to detect RONS generation: dihydrorho-damine (DHR), a reactive oxygen sensitive probe and Mito-Tracker red, a redox-sensitive reactive oxygen–sensitive mi-tochondrial probe. As shown in Figure 3A and 3B, TRAP-activated platelets generate RONS, and this was markedlyenhanced in the presence of rsCD40L.

The Role of CD40L in Platelet NitricOxide GenerationTo determine whether CD40L deficiency alters platelet-derived NO release, platelets were isolated from CD40L-deficient mice and stimulation-induced (thrombin, ADP, orcollagen) NO generation was determined. As shown in Figure4A, CD40L deficiency significantly impairs platelet releaseof NO. Notably, this attenuation in NO generation wasapparent in response to all 3 agonists examined. We con-firmed these results by carrying out platelet aggregation, withnormal human platelets, in the presence of rsCD40L. Thepresence of varying concentration of rsCD40L enhancedplatelet NO generation (Figure 4B). The rsCD40L-inducedNO generation was attenuated by anti-CD40L antibody (Fig-ure II, available online at http://atvb.ahajournals.org) indicat-ing specificity of the rsCD40L induced effect.

The Effect of rsCD40L on Phosphorylation of Aktand p38 MAP Kinase

The Effect of rsCD40L on Akt PhosphorylationRecent studies have identified Akt as an important stress-response protein kinase in endothelial cells responding tochanges in the redox environment.27,28 The observation thatrsCD40L enhances oxidative stress in platelets led to theexamination of the status of Akt phosphorylation in thepresence and absence of platelet stimulation. As shown in

Figure 1. Platelets release soluble CD40 ligand (sCD40L) afterthrombin stimulation. Washed human platelets (2�108 per mL)were stimulated with 0.2 u/mL human alpha thrombin (0.2 u/mL)as a function of time (n�4).

Recombinant Soluble CD40L (rsCD40L) Induces Platelet P-Selectin Expression and StimulatesPlatelet Aggregation

P-Selectin Positive Platelets (Fold Change) Aggregation (Fold Change)

None10 ng/mLrsCD40L

100 ng/mLrsCD40L

TRAP(0.2 �mol/L)

�1 �g/mLrsCD40L

�2 �g/mLrsCD40L

1 2.44 � 0.395* 4.01 � 1.41* 1 3.7 � 1.5** 4.9 � 1.2**

n�3; *P�0.02; **P�0.01.

2 Arterioscler Thromb Vasc Biol. October 2005


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Figure 5A, addition of rsCD40L clearly enhances Akt phos-phorylation in thrombin- and TRAP-stimulated platelets.Moreover, this enhanced Akt activation is reversed in thepresence 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 significantlyincreases Akt phosphorylation. These data demonstrate the

Figure 2. Soluble CD40L induced generation of reactive oxygen species in platelets. Platelets were incubated with rsCD40L for 5 min-utes followed by 3 minutes stimulation with TRAP. Generation of reactive oxygen species was determined by flow cytometric monitor-ing of DCFHDA oxidation. A, Representative tracing shows rsCD40L induced enhancement of reactive oxygen species generation inTRAP-stimulated platelets (n�7). B, Increasing concentration of rsCD40L leads to a dose-dependent increase in platelet reactive oxy-gen species generation (n�5; P�0.001). C, The inhibitory effect of anti-CD40 ligand antibody (10 �g/mL, 95% blockage) or GPIIb/IIIablocking antibody (abciximab; 20 �g/mL, 93% blockage) on rsCD40L (rs, 1 �g/mL) induced DCFHDA oxidation in the setting of TRAPstimulation (n�3; *P�0.05).

Chakrabarti et al CD40 Ligand and Platelet Reactive Oxygen Species 3


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ability of rsCD40L to induce Akt activation in unstimulatedand stimulated platelets.

The Effect of rsCD40L on p38 MAPKinase Phosphorylationp38 MAP kinase has also been reported to regulate thestress–response mechanisms of vascular cells such as endo-thelial cells,29 smooth muscle cells,30 and human macro-phages.31 In addition, a recent study showed that rsCD40L iscapable of inducing platelet p38 MAP kinase phosphoryla-tion.23 Therefore, we examined the role of p38 MAP kinaseactivation under conditions where rsCD40L induces plateletRONS generation. Platelets were stimulated with thrombin orTRAP, and p38 MAP kinase phosphorylation was deter-mined. As shown in Figure 5B, like Akt, p38 MAP kinasephosphorylation was significantly enhanced in the presenceof rsCD40L after thrombin stimulation. The phospho-ELISAplots 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 combina-tion with TRAP, significantly enhances p38 MAP kinasephosphorylation. Phosphorylation of p38 MAP kinase wasattenuated in the presence of specific MAP kinase inhibitor,SB203580 (Figure 5B and Figure IV).

Involvement of PI3 Kinase/Akt and p38 MAPKinase in Platelet RONS GenerationTo understand the role of PI3 kinase/Akt or p38 MAP kinasein rsCD40L-induced platelet RONS generation, we carriedout flow cytometric analysis monitoring DCFHDA oxidation(as described in Figure 2A) in presence of specific kinaseinhibitors. Presence of the PI3 kinase inhibitor, Ly294002,and the p38 MAP kinase inhibitor, SB203580, eliminatedplatelet activation–induced RONS generation (Figures V andVI, available online at http://atvb.ahajournals.org).

DiscussionDuring inflammation, reactive oxygen species promote theadhesion of blood cells to the vascular endothelium byeliciting production of inflammatory mediators or activatingnuclear transcription factors that bind to genes encodingadhesion molecules and cytokines.32,33 CD40–40L signalingrepresents another pathway that enables blood cells to am-plify the endothelial cell responses to inflammation andcontribute to the regulation of hemostasis.7 Platelets canindirectly orchestrate (through CD40–40L interaction via theendothelium) changes in coagulation, leukocyte trafficking,and extracellular matrix modeling turnover.34 CD40L hasbeen associated also with numerous vascular diseases includ-ing diabetes and acute coronary syndromes.35–41 In recentstudies, the prognostic values of serum sCD40L in acutecoronary syndromes42 and in unstable angina patients8 hasbeen reported.

Reports of sCD40L release by platelets5,7 have led tostudies examining the mechanism of CD40L expression,15

release,43,44 and its regulatory role in the inflammatory milieuof the vasculature.10,45–48 Platelet activation is accompaniedby release of both sCD40L and RONS such as superoxide20,49

and NO19; however, the role of platelet-derived sCD40L inthe generation of RONS is unknown. In the present study, weexamined the effect of CD40L on the generation of RONS inplatelets and determined the mechanism for the observedeffects. Initially, we demonstrated a prolonged release ofsCD40L by platelets for 6 hours after thrombin stimulation(Figure 1). Sustained release of platelet sCD40L9 mightcontribute to atherothrombotic disease13,50 by forming plate-let–platelet (CD40–40L51/CD40L–GPIIb/IIIA),10 platelet–leukocyte (CD40L–40/P-selectin–PSGL),52 and platelet–en-dothelial (CD40L–CD40) conjugates.26 As shown in theTable and Table I, rsCD40L is capable of inducing plateletP-selectin expression and platelet–leukocyte conjugate for-

Figure 3. Recombinant sCD40Lenhances platelet release of reactiveoxygen species. A, Washed plateletswere incubated with 20 �mol/L dihydror-hodamine for 10 minutes in presenceand absence of 10 �g/mL rsCD40L andstimulated with 50 �mol/L TRAP. Imageswere captured after 10 minutes incuba-tion. B, Washed platelets were incubatedwith redox-sensitive mitochondrial probe,Mito Tracker Red CM H2XROS for 10minutes in presence and absence of 10�g/mL rsCD40L and then stimulated with50 �mol/L TRAP (n�3).



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mation in whole blood. Recombinant CD40L has also beenrecently reported to activate platelets,23,51 resulting in secre-tion of P-selectin and suggesting the possibility of in vivoplatelet-induced inflammation. Recent work by Wagner etal53 has provided direct experimental evidence that CD154(CD40L) can induce the expression of CD154 in endothelialcells and influence atherogenesis through activation of ex-travasating monocytes. Additionally, CD40L positive plate-lets have been shown to induce CD40L expression de novo inendothelial cells.54 These observations support the proposedcoordinated interactions among platelets and leukocytes withactivated endothelium in diseased blood vessels throughadhesion molecules and, particularly, CD40/40L.55

Relevant to vascular inflammation is the effect of CD40Lon RONS generation. Figures 2 and 3 show that rsCD40Lincreases stimulation-induced RONS generation by plateletsand that this effect is reversible with specific antibodies. Todetermine whether platelet RONS generation is thrombinreceptor–specific, we carried out further flow cytometricanalysis 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 theTRAP-induced RONS generation may be thrombin receptor–specific and independent of purinergic stimulation. Inhibitionof the GPIIb/IIIa complex also decreased RONS generation,consistent with the previous observation showing thatsCD40L is a GPIIb/IIIa ligand.10 Mechanistically, rsCD40Lmay ligate the platelet receptor CD40 or GPIIb/IIIa complexto initiate platelet signaling pathways that affect the genera-tion of reactive oxygen and nitrogen species. In a clinicalsetting, recent studies have also shown that GPIIb-IIIa antag-onists reduce circulating sCD40L and leukocyte–plateletaggregate formation in patients with acute coronary syn-dromes undergoing percutaneous coronary intervention.56

The importance of CD40L in platelet generation of RONSwas shown using CD40L-deficient mice (Figure 4A). Thefinding of impaired NO generation was complemented by thestudies in human platelets where NO release was enhanced onaddition of rsCD40L (Figure 4B). Additionally, CD40L-deficient mice had a lower extent of platelet DCFHDAoxidation as compared with wild-type mice platelets afterthrombin stimulation (data not shown). It is possible that thecombined production of NO mediated by Akt and/or p38MAP kinase and superoxide mediated by p38 MAP kinaseactivation leads to the generation of peroxynitrite/RONScausing rsCD40L/TRAP-induced DCFHDA oxidation andfluorescence induction. The formation of peroxynitrite isfurther suggested by platelet fluorescence inhibition studiesusing NOS inhibitor, L-NAME, and NADPH oxidase inhib-itor, apocynin (Figure I). p38 MAP kinase may also stimulatethe PI3 kinase/Akt/eNOS pathway, enhancing NO genera-tion.57 TRAP-induced platelet DCFHDA oxidation study(Figures V and VI) indicates that both PI3 Kinase/Akt andp38 MAP kinase signaling pathways are involved in thersCD40L-dependent RONS generation.

Some earlier studies have also documented that plateletgeneration of reactive oxygen intermediates20 involves PI3kinase/Akt and MAP kinase activation. To investigatewhether rsCD40L-induced fluorescence is accompanied byAkt and p38 MAP kinase activation, we analyzed plateletproteins after stimulation in the presence or absence ofrsCD40L. The results (Figure 5A) indicate that rsCD40Lenhances Akt phosphorylation in both stimulated and un-stimulated platelets. Akt is an endothelial cell stress–responseprotein27,28 that counteracts external oxidative stress by en-hancing NO generation after eNOS phosphorylation.28,58

Enhanced Akt phosphorylation is also consistent with in-creased NO generation as observed during platelet aggrega-tion (Figure 4B). These results suggest that, similar toendothelial cells,28 rsCD40L may regulate pathways leadingto enhanced NO formation.

In vascular cells, p38 MAP kinase is also known toregulate 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 recentreport showing increased platelet p38 MAP kinase phosphor-ylation in response to rsCD40L.23 MAP kinase may enhancethe generation of superoxide by stimulating NADPH oxidaseas inhibition of NADPH oxidase with apocynin61 decreasedplatelet generation of RONS (Figure I). We also examined

Figure 4. Measurement of NO after platelet aggregation. A,Platelets were isolated from CD40L-deficient mice andstimulation-dependent NO generation was measured by amicroelectrode. Decreased NO generation was seen using theplatelet agonists as compared with wild-type mice (thrombin[1u/mL], ADP [5 �mol/L], and collagen [10 �g/mL]; n�4;*P�0.001, **P�0.05). B, Washed human platelets were incu-bated with vehicle control or varying concentrations of recombi-nant CD40L (rsCD40L) for 5 minutes, and then TRAP aggrega-tion–induced NO was measured (n�3; *P�0.004).



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whether involvement of p38 MAP kinase signaling is linkedwith platelet calcium mobilization during TRAP-inducedfluorescence induction. Our findings (Figure VII, availableonline at http://atvb.ahajournals.org) suggest that p38 MAPkinase inhibition does not alter TRAP/rsCD40L-inducedplatelet calcium mobilization. We have also verified thatrsCD40L/TRAP-induced platelet RONS generation is sensi-tive to calcium inhibition by dimethylbapta (data not shown).

In our findings there appears to be a discrepancy betweenthe degree of enhancement of rsCD40L-induced Akt and p38MAP kinase phosphorylation as compared with fluorescenceinduction (Figures 5 and 2A). There are several possibleexplanations for this difference, including (1) the use ofdistinct experimental conditions, eg, temperature variations:whereas we carried out the TRAP-induced fluorescenceinduction study at room temperature, TRAP-stimulated phos-phorylation studies were done at 37°C; (2) Measurementprocedures: in flow cytometry we directly measure thefluorescence induction, whereas protein phosphorylationevaluation involves a multi step procedures. An approachinvolving simultaneous measurement of fluorescence induc-tion and protein phosphorylation can only provide the exactstoichiometry for these 2 experiments.

There are some limitations to these studies. We carried outplatelet stimulation–induced sCD40L release in washedplatelets. However, in physiological conditions, platelet stim-ulation– and activation-induced sCD40L release may beconfounded by numerous plasma proteins. Interestingly, re-

cent studies43 with platelet rich plasma reported iso-TRAPstimulation–induced sCD40L release in the range of 0.33 to4.84 ng/mL in normal healthy volunteers only 20 minutesafter stimulation time. This suggests that the presence ofplasma proteins did not appear to have any apparent inhibi-tory effect on the stimulation-induced sCD40L release.

In summary, rsCD40L augments RONS generation inactivated platelets after stimulation of kinase signaling path-ways. These findings provide insight into the mechanisms bywhich CD40L regulates oxidative stress in platelets and maycontribute to the atherothrombotic processes through furtherinteraction with leukocytes and endothelial cells. Althoughadditional studies will be necessary to characterize earliersignaling events involved in RONS generation, these obser-vations provide new targets for studying and modifyingplatelet dependent inflammatory responses.

AcknowledgmentsWe thank Dr Michael Kirber for his help and encouragement duringconfocal microscopic studies.

References1. van Kooten C, Gaillard C, Galizzi JP, Hermann P, Fossiez F, Banchereau

J, Blanchard D. B cells regulate expression of CD40 ligand on activatedT cells by lowering the mRNA level and through the release of solubleCD40. Eur J Immunol. 1994;24:787–792.

2. Spriggs MK, Armitage RJ, Strockbine L, Clifford KN, Macduff BM, SatoTA, Maliszewski CR, Fanslow WC. Recombinant human CD40 ligandstimulates B cell proliferation and immunoglobulin E secretion. J ExpMed. 1992;176:1543–1550.

Figure 5. The effect of rsCD40L on platelet AKT phosphorylation (A) or p38 MAP kinase phosphorylation (B). i, The effect of rsCD40Lon thrombin-induced AKT/p38 MAP kinase phsophorylation. Platelet protein phosphorylation was determined after 5 minutes preincu-bation at 37°C with rsCD40L (10 �g/mL) followed by thrombin (1 u/mL) stimulation. ii, Quantitative evaluation (ELISA) of the effect ofrsCD40L on AKT/p38 MAP kinase phosphorylation after TRAP stimulation (50 �mol/L). Platelets were preincubated with rsCD40L (10�g/mL) at 37°C for 5 minutes followed by TRAP stimulation (n�4; *P�0.05 compared with resting platelets; **P�0.002 compared withTRAP stimulation [Akt]; �P�0.002 compared with rsCD40L�TRAP stimulation [Akt]; **P�0.02 compared with TRAP stimulation [p38MAP kinase]; �P�0.02 compared with rsCD40L�TRAP stimulation [p38 MAP kinase]). [LY29004]�25 �mol/L, [SB203580]�10 �mol/L.



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3. Kroczek RA, Graf D, Brugnoni D, Giliani S, Korthuer U, Ugazio A,Senger G, Mages HW, Villa A, Notarangelo LD. Defective expression ofCD40 ligand on T cells causes “X-linked immunodeficiency withhyper-IgM (HIGM1)”. Immunol Rev. 1994;138:39–59.

4. Gauchat JF, Aubry JP, Mazzei G, Life P, Jomotte T, Elson G, BonnefoyJY. Human CD40-ligand: molecular cloning, cellular distribution andregulation of expression by factors controlling IgE production. FEBS Lett.1993;315:259–266.

5. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA. The inflam-matory action of CD40 ligand (CD154) expressed on activated humanplatelets is temporally limited by coexpressed CD40. Blood. 2001;98:1047–1054.

6. Hermann A, Rauch BH, Braun M, Schror K, Weber AA. Platelet CD40ligand (CD40L)–subcellular localization, regulation of expression, andinhibition by clopidogrel. Platelets. 2001;12:74–82.

7. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, Kroczek RA. CD40 ligand on activated platelets triggers aninflammatory reaction of endothelial cells. Nature. 1998;391:591–594.

8. Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, SolumNO, Forfang K, Froland SS, Gullestad L. Enhanced levels of soluble andmembrane-bound CD40 ligand in patients with unstable angina. Possiblereflection of T lymphocyte and platelet involvement in the pathogenesisof acute coronary syndromes. Circulation. 1999;100:614–620.

9. Jin Y, Nonoyama S, Morio T, Imai K, Ochs HD, Mizutani S. Character-ization of soluble CD40 ligand released from human activated platelets.J Med Dent Sci. 2001;48:23–27.

10. Andre P, Prasad KS, Denis CV, He M, Papalia JM, Hynes RO, PhillipsDR, Wagner DD. CD40L stabilizes arterial thrombi by a beta3 inte-grin–dependent mechanism. Nat Med. 2002;8:247–252.

11. Prasad KS, Andre P, Yan Y, Phillips DR. The platelet CD40L/GP IIb-IIIaaxis in atherothrombotic disease. Curr Opin Hematol. 2003;10:356–361.

12. Prasad KS, Andre P, He M, Bao M, Manganello J, Phillips DR. SolubleCD40 ligand induces beta3 integrin tyrosine phosphorylation and triggersplatelet activation by outside-in signaling. Proc Natl Acad Sci U S A.2003;100:12367–12371.

13. Viallard JF, Solanilla A, Gauthier B, Contin C, Dechanet J, Grosset C,Moreau JF, Praloran V, Nurden P, Pellegrin JL, Nurden AT, RipocheJ. Increased soluble and platelet-associated CD40 ligand in essentialthrombocythemia and reactive thrombocytosis. Blood. 2002;99:2612–2614.

14. Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derivedCD40L: the switch-hitting player of cardiovascular disease. Circulation.2002;106:896–899.

15. Pignatelli P, Sanguigni V, Lenti L, Ferro D, Finocchi A, Rossi P, Violi F.gp91phox-dependent expression of platelet CD40 ligand. Circulation.2004;110:1326–1329.

16. Amer J, Fibach E. Oxidative status of platelets in normal and thalassemicblood. Thromb Haemost. 2004;92:1052–1059.

17. Krotz F, Sohn HY, Pohl U. Reactive oxygen species: players in theplatelet game. Arterioscler Thromb Vasc Biol. 2004;24:1988–1996.

18. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, MichelsonAD. Nitric oxide released from activated platelets inhibits plateletrecruitment. J Clin Invest. 1997;100:350–356.

19. Freedman JE, Keaney JF Jr. Nitric oxide and superoxide detection inhuman platelets. Methods Enzymol. 1999;301:61–70.

20. Wachowicz B, Olas B, Zbikowska HM, Buczynski A. Generation ofreactive oxygen species in blood platelets. Platelets. 2002;13:175–182.

21. Aukrust P, Ueland T, Muller F, Andreassen AK, Nordoy I, Aas H,Kjekshus J, Simonsen S, Froland SS, Gullestad L. Elevated circulatinglevels of C-C chemokines in patients with congestive heart failure. Cir-culation. 1998;97:1136–1143.

22. Aukrust P, Waehre T, Damas JK, Gullestad L, Solum NO. Inflammatoryrole of platelets in acute coronary syndromes. Heart. 2001;86:605–606.

23. Danese S, de la Motte C, Reyes BM, Sans M, Levine AD, Fiocchi C.Cutting edge: T cells trigger CD40-dependent platelet activation andgranular RANTES release: a novel pathway for immune response ampli-fication. J Immunol. 2004;172:2011–2015.

24. Aukrust P, Damas JK, Solum NO. Soluble CD40 ligand and platelets:self-perpetuating pathogenic loop in thrombosis and inflammation? J AmColl Cardiol. 2004;43:2326–2328.

25. Lee JR, Koretzky GA. Production of reactive oxygen intermediates fol-lowing CD40 ligation correlates with c-Jun N-terminal kinase activationand IL-6 secretion in murine B lymphocytes. Eur J Immunol. 1998;28:4188–4197.

26. Urbich C, Dernbach E, Aicher A, Zeiher AM, Dimmeler S. CD40 ligandinhibits endothelial cell migration by increasing production of endothelialreactive oxygen species. Circulation. 2002;106:981–986.

27. Niwa K, Inanami O, Yamamori T, Ohta T, Hamasu T, Kuwabara M.Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cellsexposed to hydrogen peroxide. Antioxid Redox Signal. 2003;5:713–722.

28. Thomas SR, Chen K, Keaney JF Jr. Hydrogen peroxide activates endo-thelial nitric-oxide synthase through coordinated phosphorylation anddephosphorylation via a phosphoinositide 3-kinase-dependent signalingpathway. J Biol Chem. 2002;277:6017–6024.

29. van Gorp RM, Heeneman S, Broers JL, Bronnenberg NM, van Dam-Mieras MC, Heemskerk JW. Glutathione oxidation in calcium- and p38MAPK-dependent membrane blebbing of endothelial cells. BiochimBiophys Acta. 2002;1591:129–138.

30. Blanc A, Pandey NR, Srivastava AK. Synchronous activation of ERK1/2, p38mapk and PKB/Akt signaling by H2O2 in vascular smoothmuscle cells: potential involvement in vascular disease (review). Int J MolMed. 2003;11:229–234.

31. Scholz H, Yndestad A, Damas JK, Waehre T, Tonstad S, Aukrust P,Halvorsen B. 8-isoprostane increases expression of interleukin-8 inhuman macrophages through activation of mitogen-activated proteinkinases. Cardiovasc Res. 2003;59:945–954.

32. Grisham MB, Granger DN, Lefer DJ. Modulation of leukocyte-endothe-lial interactions by reactive metabolites of oxygen and nitrogen: relevanceto ischemic heart disease. Free Radic Biol Med. 1998;25:404–433.

33. Cooper D, Stokes KY, Tailor A, Granger DN. Oxidative stress promotesblood cell-endothelial cell interactions in the microcirculation. Car-diovasc Toxicol. 2002;2:165–180.

34. Warkentin TE, Aird WC, Rand JH Platelet-endothelial interactions:sepsis, HIT, and antiphospholipid syndrome. Hematology (Am SocHematol Educ Program). 2003;497–519.

35. Jinchuan Y, Zonggui W, Jinming C, Li L, Xiantao K. Upregulation ofCD40–CD40 ligand system in patients with diabetes mellitus. Clin ChimActa. 2004;339:85–90.

36. Heeschen C, Dimmeler S, Hamm CW, van den Brand MJ, Boersma E,Zeiher AM, Simoons ML. Soluble CD40 ligand in acute coronary syn-dromes. N Engl J Med. 2003;348:1104–1111.

37. Garlichs CD, Eskafi S, Raaz D, Schmidt A, Ludwig J, Herrmann M,Klinghammer L, Daniel WG, Schmeisser A. Patients with acute coronarysyndromes express enhanced CD40 ligand/CD154 on platelets. Heart.2001;86:649–655.

38. Garlichs CD, John S, Schmeisser A, Eskafi S, Stumpf C, Karl M,Goppelt-Struebe M, Schmieder R, Daniel WG. Upregulation of CD40 andCD40 ligand (CD154) in patients with moderate hypercholesterolemia.Circulation. 2001;104:2395–2400.

39. Garlichs CD, Kozina S, Fateh-Moghadam S, Handschu R, Tomandl B,Stumpf C, Eskafi S, Raaz D, Schmeisser A, Yilmaz A, Ludwig J,Neundorfer B, Daniel WG. Upregulation of CD40-CD40 ligand (CD154)in patients with acute cerebral ischemia. Stroke. 2003;34:1412–1418.

40. Koutroubakis IE, Theodoropoulou A, Xidakis C, Sfiridaki A, Notas G,Kolios G, Kouroumalis EA. Association between enhanced soluble CD40ligand and prothrombotic state in inflammatory bowel disease. Eur JGastroenterol Hepatol. 2004;16:1147–1152.

41. Ludwiczek O, Kaser A, Tilg H. Plasma levels of soluble CD40 ligand areelevated in inflammatory bowel diseases. Int J Colorectal Dis. 2003;18:142–147.

42. Yan JC, Zhu J, Gao L, Wu ZG, Kong XT, Zong RQ, Zhan LZ. The effectof elevated serum soluble CD40 ligand on the prognostic value in patientswith acute coronary syndromes. Clin Chim Acta. 2004;343:155–159.

43. Furman MI, Krueger LA, Linden MD, Barnard MR, Frelinger AL 3rd,Michelson AD. Release of soluble CD40L from platelets is regulated byglycoprotein IIb/IIIa and actin polymerization. J Am Coll Cardiol. 2004;43:2319–2325.

44. Otterdal K, Pedersen TM, Solum NO. Release of soluble CD40 ligandafter platelet activation: studies on the solubilization phase. Thromb Res.2004;114:167–177.

45. Schonbeck U, Mach F, Libby P. CD154 (CD40 ligand). Int J BiochemCell Biol. 2000;32:687–693.

46. Schonbeck U, Mach F, Sukhova GK, Herman M, Graber P, Kehry MR,Libby P. CD40 ligation induces tissue factor expression in humanvascular smooth muscle cells. Am J Pathol. 2000;156:7–14.

47. Schonbeck U, Libby P. The CD40/CD154 receptor/ligand dyad. Cell MolLife Sci. 2001;58:4–43.

48. Damas JK, Otterdal K, Yndestad A, Aass H, Solum NO, Froland SS,Simonsen S, Aukrust P, Andreassen AK. Soluble CD40 ligand in pulmo-



Dow

nloaded from


nary arterial hypertension: possible pathogenic role of the interactionbetween platelets and endothelial cells. Circulation. 2004;110:999–1005.

49. Seno T, Inoue N, Gao D, Okuda M, Sumi Y, Matsui K, Yamada S, HirataKI, Kawashima S, Tawa R, Imajoh-Ohmi S, Sakurai H, Yokoyama M.Involvement of NADH/NADPH oxidase in human platelet ROS produc-tion. Thromb Res. 2001;103:399–409.

50. Vishnevetsky D, Kiyanista VA, Gandhi PJ. CD40 ligand: a novel targetin the fight against cardiovascular disease. Ann Pharmacother. 2004;38:1500–1508.

51. Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ. CD40 isconstitutively expressed on platelets and provides a novel mechanism forplatelet activation. Circ Res. 2003;92:1041–1048.

52. Harding SA, Sarma J, Josephs DH, Cruden NL, Din JN, Twomey PJ, FoxKA, Newby DE. Upregulation of the CD40/CD40 ligand dyad and plate-let-monocyte aggregation in cigarette smokers. Circulation. 2004;109:1926–1929.

53. Wagner AH, Guldenzoph B, Lienenluke B, Hecker M. CD154/CD40-mediated expression of CD154 in endothelial cells: consequences forendothelial cell-monocyte interaction. Arterioscler Thromb Vasc Biol.2004;24:715–720.

54. Danese S, Scaldaferri F, Papa A, Pola R, Gasbarrini A, Sgambato A,Cittadini A. CD40L-positive platelets induce CD40L expression de novoin endothelial cells: adding a loop to microvascular inflammation. Arte-rioscler Thromb Vasc Biol. 2004;24:e162.

55. Granger DN, Vowinkel T, Petnehazy T. Modulation of the inflammatoryresponse in cardiovascular disease. Hypertension. 2004;43:924–931.

56. Furman MI, Krueger LA, Linden MD, Fox ML, Ball SP, Barnard MR,Frelinger AL 3rd, Michelson AD. GPIIb-IIIa antagonists reduce throm-boinflammatory processes in patients with acute coronary syndromesundergoing percutaneous coronary intervention. J Thromb Haemost.2005;3:312–320.

57. Anter E, Thomas SR, Schulz E, Shapira OM, Vita JA, Keaney JF Jr.Activation of endothelial nitric-oxide synthase by the p38 MAPK inresponse to black tea polyphenols. J Biol Chem. 2004;279:46637–46643.

58. Cai H, Li Z, Davis ME, Kanner W, Harrison DG, Dudley SC Jr. Akt-dependent phosphorylation of serine 1179 and mitogen-activated proteinkinase kinase/extracellular signal-regulated kinase 1/2 cooperativelymediate activation of the endothelial nitric-oxide synthase by hydrogenperoxide. Mol Pharmacol. 2003;63:325–331.

59. Brown GE, Stewart MQ, Bissonnette SA, Elia AE, Wilker E, Yaffe MB.Distinct ligand-dependent roles for p38 MAPK in priming and activationof the neutrophil NADPH oxidase. J Biol Chem. 2004;279:27059–27068.

60. Djordjevic T, Pogrebniak A, BelAiba RS, Bonello S, Wotzlaw C, AckerH, Hess J, Gorlach A. The expression of the NADPH oxidase subunitp22phox is regulated by a redox-sensitive pathway in endothelial cells.Free Radic Biol Med. 2005;38:616–630.

61. Chlopicki S, Olszanecki R, Janiszewski M, Laurindo FR, Panz T,Miedzobrodzki J. Functional role of NADPH oxidase in activation ofplatelets. Antioxid Redox Signal. 2004;6:691–698.



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FreedmanSubrata Chakrabarti, Sonia Varghese, Olga Vitseva, Kahraman Tanriverdi and Jane E.

CD40 Ligand Influences Platelet Release of Reactive Oxygen Intermediates

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CD40 Ligand Influences Platelet Release of Reactive Oxygen Intermediates

Subrata Chakrabarti, Sonia Varghese, Olga Vitseva,

Kahraman Tanriverdi, and Jane E. Freedman

Materials and Methods

Recombinant CD40 ligand (rsCD40L) was purchased from R & D systems

(Minneapolis, MN). Dichlorodihydrofluorescein diacetate (DCFHDA) and

MitoTracker Red CM H2XROS, Fluo3-AM and Dimethylbapta-AM were

purchased from Molecular Probes (Eugene, OR). Apocynin (Acetovanillone)

was purchased from Aldrich Chemical Company (Milwaukee, WI). Thrombin

receptor activating peptide (TRAP) was purchased from Peninsula

Laboratories (San Carlos, CA). Dihydrorhodamine was purchased from

Cayman Chemicals (Ann Arbor, MI). Human alpha thrombin was obtained

from Enzyme Research Laboratories (South Bend, IN). Anti-p38 MAP kinase

antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-

phospho-p38 MAP kinase (Thr180/Tyr180), anti-Akt and anti-phospho-Akt

(ser473/472) antibodies were purchased from BD Biosciences (San Jose,

CA). HRP-conjugated anti-mouse antibodies were purchased from Upstate

Biotechnology (Lake Placid, NY). Anti-rabbit IgG-HRP antibodies were

procured from Cell Signaling Technology (Beverley, MA). ELISA kits for

1

phospho-p38 MAP Kinase and phospho-Akt were obtained from Biosource

(Camarillo, CA). Blocking anti-CD40 ligand antibody was purchased from

Coulter (Immunotech, Fullerton, CA). Total amounts of sCD40L released by

activated platelets were measured by ELISA (Bender Med Systems,

Burlingame, CA). Anti-β-actin monoclonal antibody was purchased from

Novus Biologicals (Littleton, CO). PE-labeled anti P-selectin antibody

(CD62P-PE), CD14-FITC and CD41-PE-Cy5 was obtained from Pharmingen-

B D Biosciences (San Diego, CA). Homozygous CD40L (-/-) and C57BL6

mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Platelet activation and release of soluble CD40 ligand

Peripheral blood was drawn from healthy adult volunteers1 who had not

consumed acetylsalicylic acid or any other platelet inhibitor for at least seven

days. The protocol was approved by the Boston Medical Center Ethics

Committee on Human Research.

Briefly, blood was drawn into a 10% sodium citrate containing syringe using a 21-

gauge needle. The citrated blood was centrifuged (150 g, 15 min, 22°C) and the

supernatant (PRP) was separated. Platelet pellets were obtained by a second

centrifugation (392 g, 20 min, 22°C) of the PRP after mixing with equal volume of

platelet wash buffer (10mM sodium citrate, 150mM NaCl, 1mM EDTA, 1%

dextrose, pH 7.4) supplemented with 0.2µM prostaglandin E1. Platelet pellets

were briefly washed twice with HEPES buffer (140mM NaCl, 6mM KCl, 2mM

MgSO4, 7H2O, 2mM Na2HPO4, 6mM HEPES, pH 7.4) before its final

2

resuspension in the same buffer. Resuspended platelets were kept in room

temperature and utilized within 2 hours of isolation.

Platelets were treated with 0.2 u/ml thrombin to induce platelet activation,

and the amount of sCD40L released from thrombin-activated platelets was

determined as a function of time. Samples were collected by (micro)

centrifugation at 11000g at 4o C for 5 minutes after the specified time of

incubation. The supernatant was stored at -20oC prior to measurement of

sCD40L levels by ELISA. The sCD40L levels from the platelet supernatant were

measured by a commercially supplied kit (High Sensitivity ELISA kit, Bender Med

Systems). Briefly, 20µl samples (mixed with 80µl standard diluent buffer) and

100µl standards in duplicate were loaded in the ELISA plate wells and processed

according to manufacturer’s instruction. The color developed was measured at

450nm using 650nm as the reference wavelength. The concentration of the

sCD40L released from the samples was determined from the linear portion of the

standard curve.

Platelet P-selectin Expression, Aggregation and Platelet—Leukocyte

Conjugate Formation

To study the functional effect of sCD40L on platelets, activation induced

expression of P-selectin and platelet aggregation were examined. By flow

cytometry, we also verified the effect of rsCD40L on platelet-leukocyte conjugate

formation in whole blood.

3

P-selectin expression study

Washed platelets in presence and absence of recombinant CD40L

(rsCD40L) were incubated in HEPES buffer for 8 minutes at 37oC. For

comparison purposes, selected samples were also stimulated with 20µM TRAP.

Samples were fixed with 1% fresh formaldehyde for 10 minutes at room

temperature. Platelets were collected by centrifugation (2700 g, 1 min),

resuspended in PBSG (0.5 % BSA and 5.5 mM D-glucose in PBS) and then

incubated (20 min, 22°C, in dark) with PE-labeled monoclonal CD62P antibody or

corresponding PE-labeled isotype controls. Platelet samples were washed and

resuspended in PBSG and analyzed in a FACScan flow cytometer (Becton

Dickinson) or in a Fluorescence Activated Cell Sorter (Dako Cytomation). At least

ten thousand platelet-specific events were collected for each set of data points.

Flow cytometric data were analyzed by histogram statistics (CellQuest software,

Becton Dickinson or Summit V3.1 Build 844).

Platelet Aggregation

Washed platelets were incubated in presence and absence of rsCD40L for

5 minutes at 37oC. Platelets supplemented with fibrinogen were subjected to

aggregation in a four channel Platelet Aggregation Profiler (Biodata Corporation).

Thrombin receptor activation peptide was used as an initiator of aggregation after

a stable baseline was obtained.

4

Analysis of Platelet-Leukocyte Conjugates

Human blood was incubated with rsCD40L (10µg/ml) in presence and

absence of ADP (4µM) for 15 minutes at room temperature. Platelet-leukocyte

conjugates were analyzed by using two-color flow cytometry in a similar manner

as described earlier 2 where CD14-FITC was used as a monocyte marker and

CD41-PE-Cy5 as a marker for platelets. EDTA (10mM) was used as a negative

control for conjugate formation.

Induction of platelet aggregation and measurement of nitric oxide

A NO selective microelectrode was used to monitor platelet aggregation-

induced NO production as described previously.3,4 Briefly, platelets (2x108/ml)

were washed and incubated with rsCD40L or vehicle control for 5 minutes at

room temperature. Aggregation was induced by appropriate agonists as

described previously.3

Assessment of reactive oxygen species by flow cytometry

Freshly isolated, washed platelets were incubated with the fluorescent

probe, DCFHDA, or vehicle control for 5 minutes. Samples were then stimulated

for 3 minutes with TRAP (20µM) and immediately analyzed using a Fluorescence

Activated Cell Sorter (Dako Cytomation). At least ten thousand platelet-specific

events were collected for each set of data points. The TRAP-induced mean

fluorescence induction was calculated after gating the stimulated platelet

5

population. Unstimulated platelets not receiving any fluorophore were used as a

negative control.

Measurement of Platelet Aggregation induced Superoxide Release

Platelet aggregation induced superoxide generation was determined as

described earlier.5 Briefly, washed platelets were incubated with varying

concentration of rsCD40L or control buffer for 5 minutes at 37oC. Superoxide

released following PMA (150nM) induced platelet aggregation was measured in a

lumiaggregometer using lucigenin as a luminescent agent.5

Assessment of reactive oxygen species and calcium mobilization by

confocal microscopy

Fluorescence confocal images were obtained using a two-photon laser-

scanning microscope. Platelets, at a concentration of 2x108 /ml, were incubated

for 10 minutes with 20µM dihydrorhodamine or 0.5µM of the redox sensitive

MitoTracker Red probe. A baseline reading was obtained prior to platelet

activation. Images were recorded at different time points following stimulation,

and the fluorescence data was analyzed using NIH image software after

background subtraction.

Platelet calcium mobilization was measured using calcium sensitive probe Fluo3-

AM. Washed platelets in HEPES buffer were incubated with 2.5µM Fluo3-AM for

45 minutes at room temperature. In order to verify the specificity of the calcium

response, some samples were treated with the calcium chelator Dimethylbapta-

6

AM (Molecular Probes). For other studies, specific samples were treated for 10

minutes with rsCD40L and/or p38 MAP kinase inhibitor SB203580 (10µM).

Following the capture of baseline fluorescence, samples were stimulated with

TRAP (50µM) and fluorescence induction was continuously monitored by

confocal microscopy. Data were analyzed using NIH image software after

background subtraction.

Analysis of proteins following platelet stimulation

Washed platelets (2x108 /ml) were pre-incubated at 370 C in presence or

absence of rsCD40L for 5 minutes and then stimulated with thrombin for 3

minutes. The reaction was terminated by the addition of 3XSDS sample buffer.

Protein samples were subjected to western blot analysis using standard

procedures. Protein blots were probed with anti-phospho-Akt (1:1000) and anti-

phospho-p38 MAP kinase (1:1000) antibodies in TBST containing 1% BSA. Total

Akt (1:500) and p38 MAP kinase (1:2500) or β-actin (1:5000) was probed in

TBST supplemented with 5% milk. The blots were developed with HRP-

conjugated anti-mouse (1:4000) or anti-rabbit (1:10000) secondary antibodies.

In other experiments, a commercially available ELISA kit (Biosource) was

employed to quantify Akt and p38MAP Kinase phosphorylation. Briefly, washed

platelets (7.5 x108 /ml) were pre-incubated at 370 C with vehicle control, PI3

kinase inhibitor (LY29004, 25µM), or p38 MAP kinase inhibitor (SB203580,

10µM) for 5 minutes in presence or absence of rsCD40L. Afterwards, platelets

were stimulated with TRAP (50µM) for an additional 3 minutes at 370 C. The

7

reaction was terminated by the addition of lysis buffer (Biosource) supplemented

with protease and phosphatase inhibitors (Sigma). The samples were lysed on

ice for 30 minutes with occasional stirring and centrifuged at 13,000 rpm for 10

minutes at 40 C. The supernatant was stored in aliquots at -700 C.

Statistical analysis

Data are expressed as mean ± standard deviation. Analysis of differences

between group means was evaluated with one-way analysis of variance

(ANOVA). P<0.05 was accepted as statistically significant.

References

1. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest. 1997;100:350-6.

2. O'Sullivan BP, Linden MD, Frelinger AL, 3rd, Barnard MR, Spencer-Manzon M, Morris JE, Salem RO, Laposata M, Michelson AD. Platelet activation in cystic fibrosis. Blood. 2005;105:4635-41.

3. Freedman JE, Loscalzo J, Benoit SE, Valeri CR, Barnard MR, Michelson AD. Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. J Clin Invest. 1996;97:979-87.

4. Freedman JE, Farhat JH, Loscalzo J, Keaney JF, Jr. alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation. 1996;94:2434-40.

5. Freedman JE, Keaney JF, Jr. Nitric oxide and superoxide detection in human platelets. Methods Enzymol. 1999;301:61-70.

8

Table I: Recombinant soluble CD40L (rsCD40L) enhances platelet-leukocyte

conjugate formation in human blood.

Platelet-leukocyte conjugates

None

EDTA

rsCD40L

ADP

ADP + rsCD40L

Platelet-monocyte (%)

19.64 ± 0.53

5.46 ± 1.23

27.4 ± 3 *

56.43 ± 5.6

80 ± 7 **

Platelet-neutrophil (%)

6.93 ± 1.48

5.93 ± 1.4

8 ± 1

17 ± 2

51 ± 9 **

n=3, *P<0.05 vs unstimulated, **P<0.01 vs ADP. [rsCD40L] =10µg/ml;

[ADP] =4µM, EDTA (negative control) =10mM

9

Figure I

10

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

A *

**

*

+ Apocyn in

+ L-N

AME

rsCD40L +

TRAP

+ Apocyn in

+ L-N

AME

P late

lets

+ T

RAP

P late

lets

+/-

rsCD40L

Mea

n F

luo

resc

ence

Inte

nsi

ty

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 00

4

8

1 2 B*

*

*

*

% S

up

ero

xid

e G

en

era

tio

n(A

U)

[ r s C D 4 0 L ] ( n g /m l )

Figure I: (A) TRAP and rsCD40L (10ng/ml) induced platelet DCFHDA oxidation in

the presence of a NOS inhibitor (L-NAME; 300µM) or NADPH oxidase inhibitor

(apocynin; 200µM) (n=3, *P<0.05) (B) The effect of low concentrations of

rsCD40L (10ng/ml to 1µg/ml) on platelet aggregation induced (150nM PMA)

superoxide generation as measured by lucigenin chemiluminescence (n=3,

*P<0.02).

10

Figure II

10

10

20

30

40

50

60

70

++*

rsCD40

L +

anti C

D40L

anti

body

+ anti

CD40

L

an

tibod

y

+ rsC

D40L

Contro

l

Plat

elet

Der

ived

NO

(AU

)

Figure II: Washed human platelets were pre-incubated with 1.8µg/ml rsCD40L in

presence and absence of 3.6µg/ml anti-CD40 ligand antibody for 5 minutes and

then TRAP aggregation induced NO generation was measured.

(n=3; *P<0.01, +P< 0.004).

11

Figure III

Platelet + + + + + + + + + rsCD40L (µg /ml) - 2 10 - 10 - 10 - 10 TRAP - - - + + + + + + SB 203580 - - - - - + + - - Ly29004 - - - - - - - + +

P-Akt

Figure III: Recombinant CD40 ligand (rsCD40L) induces platelet Akt phosphorylation.

Washed platelets were pre-incubated at 370 C with vehicle control, or PI3 kinase inhibitor,

(LY29004, 25µM), p38 MAP kinase inhibitor (SB203580, 10µM), for 5 minutes in

presence or absence of rsCD40L. Platelets were stimulated with TRAP (50µM) for an

additional 3 minutes at 370 C, lysed, and analyzed by western blot as described in the

methods section (a representative blot from 3 independent experiments).

12

Figure IV

Phospho- p-38 MAPK Platelet + + + + + + + + + rsCD40L - 2 10 - 10 - 10 - 10 (µg /ml) TRAP - - - + + + + + + SB 203580 - - - - - + + - - Ly29004 - - - - - - - + +

P-p38

Figure IV: Recombinant CD40 ligand (rsCD40L) induces platelet p-38 MAP kinase

phosphorylation. Washed platelets were pre-incubated at 370 C with vehicle control, or

p38 MAP kinase inhibitor (SB203580, 10µM), PI3 kinase inhibitor (LY29004, 25µM) for

5 minutes in presence or absence of rsCD40L. Platelets were stimulated with TRAP

(50µM) for an additional 3 minutes at 370 C, lysed, and analyzed by western blot as

described in the methods section (a representative blot from 3 independent

experiments).

13

Figure V

0

50

100

150

200

250**

*

TRAP + rsC

D40L

+ Ly

TRAP + rsC

D40L

TRAP + Ly

TRAP

Contro

l

Mea

n Fl

uore

scen

ce (%

)

Figure V: Inhibition of Akt phosphorylation significantly attenuates TRAP/rsCD40L

induced platelet reactive oxygen and nitrogen species generation. Platelet stimulation

induced RONS generation was determined using TRAP (20 µM) and rsCD40L (1 µg/ml)

with flow cytometric monitoring of DCFHDA oxidation. Enhanced RONS generation are

attenuated by PI3 kinase inhibitor Ly294002 (25 µM; n=3; *P<0.005; **P<0.001).

14

Figure VI

0

20

40

60

80

100

120

140

160

180

200**

*

TRAP +rsC

D40L

+

SB

TRAP + rs

CD40L

TRAP + SB

TRAPCon

trol

Mea

n Fl

uore

scen

ce (%

)

Figure VI: Inhibition of p38 MAP kinase phosphorylation attenuates

TRAP/rsCD40L induced platelet reactive oxygen and nitrogen species

generation. Platelet stimulation induced RONS generation was determined using

TRAP (20 µM) and rsCD40L (1 µg/ml) with flow cytometric monitoring of

DCFHDA oxidation in presence and absence of p38 MAP kinase inhibitor

SB203580 (10µM, n=3; *P<0.004; **P<0.001).

15

Figure VII Platelets (a) + TRAP (b) TRAP + BAPTA (c)

Platelets + SB (d) +TRAP + SB (e) TRAP + SB + BAPTA (f)

Platelets (g) +TRAP (h) TRAP + rsCD40L (i) + rs CD40L + rsCD40L + BAPTA

Platelets + SB (j) TRAP + SB (k) TRAP + SB (l) + rs CD40L + rsCD40L + rsCD40L+ BAPTA

16

Figure VII: Recombinant soluble CD40 Ligand (rsCD40L, 1µg/ml) enhances

stimulation-induced platelet calcium mobilization. Fluorescence from fluo3-

labeled platelets was inhibited by calcium chelator, Dimethyl bapta (10µM) but

not by p38 MAP Kinase inhibitor SB203580 (10µM). Confocal images are

presented at baseline (a, d, g, j) and after stimulation with 50µM TRAP in

presence and absence of different additives (only 30 second time point is

displayed; representative images from n=2).

17

cd40 ligand influences platelet release of reactive...

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