Background-CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. Methods and Results-CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O2-) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O2- and CD40L expression. Conclusions-These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid-mediated gp91phox activation

Pignatelli P., Sanguigni V., Lenti L., Ferro D., Finocchi A., Rossi P., Violi F.

Pignatelli P.

Lenti L.

Ferro D.

Violi F.

SANGUIGNI, VALERIO

FINOCCHI, ANDREA

ROSSI, PAOLO

English

gp91phox-dependent expression of platelet CD40 ligand

Archivio della Ricerca - Università di Roma Tor vergata

gp91phox-Dependent Expression of Platelet CD40 Ligand
P. Pignatelli, MD; V. Sanguigni, MD; L. Lenti, MD; D. Ferro, MD; A. Finocchi, MD;
P. Rossi, MD; F. Violi, MD
Background—CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still
unclear.
Methods and Results—CD40L expression was measured in platelets from healthy subjects both with and without the
addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited
deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets
showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet
CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects
expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic
acid-stimulated platelets showed an almost complete absence of superoxide anion (O2) and CD40L expression.
Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O2
and CD40L expression.
Conclusions—These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid–mediated
gp91phox activation. (Circulation. 2004;110:1326-1329.)
Key Words: CD40 ligand  NADPH oxidase  oxidative stress  platelets
CD40 ligand (CD40L), a member of the tumor necrosisfactor ligand family, is a transmembrane protein with
proinflammatory and prothrombotic properties on interaction
with its receptor CD40.1 CD40L was found primarily on the
immune system cells and subsequently on macrophages,
smooth muscle cells, endothelial cells, and platelets.1 On
agonist stimulation, platelets express CD40L, which is then
cleaved on the membrane surface and finally circulates in the
soluble form.2 It has been calculated that95% of circulating
soluble CD40L (sCD40L) originates from platelets.2 The
mechanism accounting for CD40L expression by activated
platelets is still unclear. Previous studies demonstrated that
platelets produce reactive oxidant species, which in turn act
as intracellular signaling molecules that amplify platelet
response to agonists.3 In this study, we sought to determine
whether reactive oxidant species play a role in platelet
CD40L expression.
Methods
Materials
Anti-CD40L Ab (CD154 immunoglobulin (Ig) G1 mouse, clone
TRAP-1), anti-CD62P (IgG1 mouse, clone CLBThromb/6), and
isotypic control (IgG1 mouse, clone 679.IMC7) were obtained from
Beckman Coulter. Anti-91phox Ab (polyclonal IgG goat) and a
normal goat IgG (sc 2028 polyclonal) as irrelevant antibody (IRR)
were obtained from Santa Crutz. Immunoprecipitation products were
obtained from Amersham Pharmacia. Any other nonspecified prod-
ucts were obtained from Sigma Aldrich.
Platelet Isolation From Whole Blood
Blood samples were mixed with 0.13 mol/L sodium citrate (9:1
ratio). Washed platelets and platelet-rich plasma were prepared as
previously described.3
Flow Cytometric Analysis
CD40L and CD62P expression on platelet membrane was analyzed
with specific fluorescein isothiocyanate conjugated-labeled mono-
clonal antibodies (Mab). An irrelevant isotype-matched antibody
(anti-IgG1) was used as a negative control.
Mab (20 L) was added to platelets (200 L, 2108/mL)
previously fixed with 2% paraformaldehyde in PBS (0.1% bovine
serum albumin) and incubated for 60 minutes at 4°C. The unbound
Mab was removed by addition of 0.1% bovine serum albumin PBS
and centrifugation at 500g for 3 minutes (twice). Fluorescence
intensity was analyzed 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 evaluate the percentage
of positive platelets. Antibody reactivity is reported as mean fluo-
rescence according to this formula: Mean fluorescence of specific
antibody minus mean fluorescence of control antibody. Platelets
were incubated 10 minutes at 37°C with the antioxidants, AACOCF3
(a PLA2 inhibitor), or control medium before stimulation.
Analysis of Superoxide Anion
Superoxide anion (O2) production was measured by lucigenin (5
mol/L)3 and expressed as stimulation index (mean level of stimu-
Received November 21, 2003; de novo received March 18, 2004; revision received May 20, 2004; accepted May 20, 2004.
From Divisione IV Clinica Medica (P.P., D.F., F.V.) and Dipartimento di Medicina Sperimentale e Patologia (P.P., L.L., F.V.), Università di Roma
“La Sapienza,” and Dipartimento di Medicina Interna (V.S.) and Dipartimento di Medicina Sperimentale (A.F., P.R.), Università di Roma Tor Vergata,
Rome, Italy.
Correspondence to Francesco Violi, MD, Divisione IV Clinica Medica, Dipartimento di Medicina Sperimentale e Patologia, Università di Roma “La
Sapienza,” Policlinico Umberto I, 00185, Rome, Italy. E-mail Francesco.Violi@uniroma1.it
© 2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000134963.77201.55
1326
lated platelet luminescence divided by average level of luminescence
in unstimulated platelets).
Analysis of sCD40L
After 10 minutes of stimulation with agonists, the reaction was
blocked by acidification of the medium with ACD (d-sodium
hydrogen citrate, dglucose, and citric acid), platelet-rich plasma
was centrifuged (10 minutes at 360g), and the supernatant was stored
at 80°C until use. sCD40L was measured with a commercial
immunoassay (Quantikine CD40 Ligand, R&D Systems).
Immunoprecipitation and Western Blotting
The 91phox was immunoprecipitated from platelets (25109 cells,
55 mg protein/0.5 mL) and polymorphonuclear neutrophils (PMN)
(5106 cells) as positive controls in denaturized condition using a
91phox Ab polyclonal, size separated on SDS-PAGE (12% gel),
blotted, and stained with 91phox polyclonal Ab. Negative controls
involved a similar procedure using a goat IRR.4
X-Linked Chronic Granulomatous Disease
Patient Description
X-linked chronic granulomatous disease (X-CGD), an inherited
disorder characterized by the absence or deficiency of phagocyte-
NADPH oxidase activity, was diagnosed in 2 male patients (33 and
38 years of age) by demonstrating the absence or manifest deficiency
of oxidase activity in stimulated neutrophils.5 X-CGD diagnosis was
subsequently confirmed by the mutation analysis of the CYBB gene
encoding the gp91 subunit of phagocyte-NADPH oxidase.6 The
mutation in patient 1 was identified as a single-base substitution of
guanosine to adenosine at residue 252 in exon 3, resulting in a
splicing defect. A deletion of thymine 184 in exon 3 was identified
for patient 2, resulting in a frame shift.
Platelet Aggregation
Collagen-induced platelet aggregation (Born’s method) was mea-
sured as previously described.3
Statistical Analysis
Data are reported as meanSD. Comparison between variables in
the in vitro study was analyzed by Student’s t test for unpaired data;
the correlation study between CD40L expression and O2 and H2O2
formation was evaluated by linear correlation analysis, followed by
ANOVA.
Results
The flow cytometric analysis showed CD40L expression on
the cell surface after agonist stimulation; conversely, CD40L
expression was minimally observed in unstimulated platelets
(Figure 1). We then tried to determine a possible relationship
between reactive oxidant species and platelet CD40L expres-
sion. Compared with unstimulated platelets, collagen and
thrombin increased both O2 production (stimulation index,
6.10.4 and 3.40.3 with collagen and thrombin, respec-
tively) and CD40L expression (Figure 1). Using a scalar
concentration of collagen (n3 for each determination) of 0.5
to 16 g/mL and thrombin of 0.02 to 0.72 U/mL, we detected
a significant correlation between O2 and CD40L (r0.97,
P0.001 with collagen; r0.95, P0.007 with thrombin).
Catalase and mannitol did not affect agonist-induced CD40L
expression, whereas superoxide dismutase (SOD) signifi-
cantly decreased it (Figure 1). Conversely, SOD (300 U/mL)
did not affect collagen-induced CD62P expression (n5;
mean fluorescence: unstimulated platelets, 0.070.03;
collagen-stimulated platelets, 5.430.75; collagen plus
SOD–treated platelets, 5.310.66).
To determine the role of arachidonic acid in the interaction
between O2 and CD40L, agonist-stimulated platelets from
healthy subjects were investigated with or without the addi-
tion of AACOCF3. Compared with controls, platelets treated
with AACOCF3 showed a significantly lower expression of
O2 (79% with collagen and 66% with thrombin;
P0.001; n10) and CD40L (Figure 1). Conversely, incu-
bation of platelets with aspirin reduced O2 by 17% (P0.05)
with collagen and by 13% (PNS) with thrombin but did not
change CD40L expression (Figure 1); the specific
thromboxane receptor inhibitor SQ 29548 did not change
either platelet O2 or CD40L (not shown).
To determine whether gp91phox is detectable in human
platelets, we performed an in vitro study in platelets from
healthy volunteers. Neutrophils were tested as positive con-
trols, and an IRR was tested as a negative control. In this
experiment, denaturized conditions were used to prevent
interference of gp22phox with gp91phox detection on ac-
count of their natural complex form (flavocytochrome b558).
Leukocyte contamination in platelet suspension was assessed
to be 0.1% (50 PMN per 50 000 platelets per 1 mL). A
leukocyte suspension (1103 PMN/mL diluted to obtain 50
PMN/mL) was immunoprecipitated, showing no gp91phox
expression. Immunoprecipitation of the platelet sample al-
lowed us to demonstrate that gp91phox is expressed by
human platelets (Figure 2A).
The role of gp91phox in CD40L expression was then
investigated in 2 patients affected by X-CGD and in male
control subjects matched for age. Compared with control
subjects, X-CGD patients showed almost complete suppres-
sion of CD40L and O2 by collagen- and thrombin- stimu-
lated platelets (Figure 2C and 2D). Similar findings were
observed with arachidonic acid, thus suggesting that, in
platelets, NADPH oxidase activation and subsequent CD40L
expression occur via arachidonic acid pathway (Figure 2C
and 2D). In the 2 patients with X-CGD, plasma levels of
sCD40L were much lower than those found in healthy
subjects and did not increase after platelet stimulation with
the agonist (Figure 2B). In patients with X-CGD, collagen (4
g/mL)-induced platelet aggregation (patient 1: lag phase, 30
seconds; light transmission%, 88; and patient 2: lag phase, 25
seconds; light transmission%, 91%) was comparable to that
Figure 1. CD40L expression of agonist-stimulated platelets.
Collagen- and thrombin- induced platelet CD40L (n10).
*P0.001 vs agonist- stimulated platelets.
Pignatelli et al gp91phox and Platelet CD40L 1327
of healthy subjects (lag phase, 255 seconds; light transmis-
sion%, 859, n10).
Discussion
This study provides the first evidence that platelet production
of O2 plays a key role in CD40L expression. We observed
that platelets from healthy volunteers incubated with SOD, a
scavenger of O2, significantly inhibited CD40L. Moreover,
in 2 patients with an inherited deficiency of gp91phox, the
catalytic core of NADPH oxidase, O2 expression and
CD40L expression by activated platelets were almost com-
pletely suppressed. It should be noted that X-CGD patients, in
addition to showing a reduced platelet CD40L expression,
also exhibited very low levels of sCD40L in unstimulated
platelets and no changes after platelet stimulation.
These findings imply that platelets express gp91phox, an
NADPH oxidase subunit that had not been previously de-
tected in platelets. In fact, only the gp22phox, gp47phox, and
gp67phox subunits have been found so far.7 In this study, we
provide the first evidence that platelets express gp91phox and
demonstrate that NADPH oxidase has a central role in
generating O2 in platelets, like in phagocytic and nonphago-
cytic cells.8,9
The intracellular signaling eliciting NADPH oxidase acti-
vation by agonist-activated platelets is still unclear. Arachi-
donic acid has been shown to be an important respiratory
burst activator.10
Previous studies have demonstrated that arachidonic acid
synergizes with p47phox phosphorylation in phagocytes to
facilitate interaction with p22phox and to induce activation of
phagocyte-NADPH oxidase.11 Consistent with these findings,
platelet O2 production by arachidonic acid was almost
completely suppressed in 2 patients with an inherited
gp91phox deficiency.
Consistent with previous findings,12 aspirin did not affect
platelet CD40L expression; in addition, aspirin marginally
influenced platelet O2 expression, suggesting that NADPH
oxidase activation mainly occurs through a COX1-
independent mechanism. Several lines of evidence suggest
that the CD40L-CD40 dyad is implicated in atherothrombo-
sis. Engagement of CD40L with its receptor stimulates the
synthesis of adhesion molecules, chemokines, and tissue
factor and activates metalloproteinases.1,2 The role of CD40L
in atherogenesis is confirmed by the fact that, in hyperlipid-
emic mice, anti-CD40L antibodies reduced the atheroscle-
rotic lesion.13 Oxidized LDL seems to play a key role in
pathogenesis of atherosclerosis; in a recent study,14 oxidized
LDL dose dependently increased CD40L in human vascular
endothelial cells and smooth muscle cells, suggesting that
oxidative stress is implicated in CD40L expression. The fact
that oxidative stress also plays a role in platelet CD40L
expression suggests that an increase in CD40L expression
might be another mechanism through which oxidative stress
elicits the atherosclerotic damage.
Figure 2. gp91phox and CD40L expres-
sion in X-CGD patients and control sub-
jects. A, 91phox immunoprecipitation (IP)
in healthy subjects (HS). Platelets and
neutrophils were precipitated with anti-
gp91phox and IRR. HCh and LCh indi-
cate heavy and light chains of immuno-
globulin. Result is representative of 5
separate experiments. B, sCD40L in HS
(n4) and X-CGD patients (4 different
determinations). *P0.001. C and D,
CD40L expression and O2 formation in
X-CGD patients (4 different determina-
tion) and HS (n10).
1328 Circulation September 7, 2004
In conclusion, this study demonstrates that platelet CD40L
expression occurs via arachidonic acid–mediated NADPH
oxidase activation and suggests that scavenging O2 or
reducing platelet release of arachidonic acid could represent a
novel approach to inhibit CD40L expression.
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Pignatelli et al gp91phox and Platelet CD40L 1329


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gp91phox-dependent expression of platelet CD40 ligand

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