CD40-CD40L and Platelet Function - Circulation Research

CD40-CD40L and Platelet Function : Beyond Hemostasis
Jane E. Freedman
Circ Res. 2003;92:944-946
doi: 10.1161/01.RES.0000074030.98009.FF
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CD40-CD40L and Platelet Function
Beyond Hemostasis
Traditionally, platelets have been thought of as
anuclear, subcellular fragments derived from
megakaryocytes circulating in blood as small discs
and mitigating hemorrhage. This hemostatic process requires
platelet activation, a complex chain of events involving rapid
structural changes that activate adhesion receptors, remodel
the cytoskeleton, and lead to the eventual synthesis and
secretion of various platelet-derived factors. The result of this
cascade is the formation of platelet-dependent thrombosis
that primarily attenuates bleeding but, in pathophysiological
settings, contributes to occlusive vascular events. These
clinical realities have led to the bias that platelets are
primarily involved in thrombosis and hemostasis. This bias
has been reflected in the predominant use of platelet aggregometry
as the standard method for quantifying platelet
activation ex vivo. This technique, however, only identifies
platelet-platelet binding (homotypic aggregates) and, in clinical
settings, has promoted a relatively simplistic vision of
platelet function. The focus on platelet-dependent thrombosis
has made the platelet aggregate a common therapeutic target
in syndromes involving vascular occlusion. However, merely
preventing the process of platelet-platelet binding may not
always translate into clinical efficacy as seen in the recent
disappointing results associated with the use of the oral
IIb/IIIa inhibitors. 1
Recently, studies have suggested that platelets also participate
in inflammatory reactions. Platelets are known to
produce inflammatory mediators including platelet-derived
growth factor, platelet factor 4, and transforming growth
factor-. Platelets are also known to bind, via P-selectin
(CD62P) expressed on the surface of activated platelets to the
leukocyte receptor, P-selectin glycoprotein ligand-1 (PSGL-
1). The relevance of this binding is supported by a study
demonstrating that the infusion of recombinant soluble human
form of P-selectin glycoprotein ligand-1 in an animal
model of vascular injury reduced myocardial reperfusion
injury and preserved vascular endothelial function. 2 The
clinical implications of heterotypic aggregates are shown by
studies demonstrating that after acute myocardial infarction,
circulating monocyte-platelet aggregates are both increased
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Whitaker Cardiovascular Institute and Evans Department of
Medicine, Boston University School of Medicine, Boston, Mass.
Correspondence to Jane E. Freedman, MD, Boston University School
of Medicine, 715 Albany St, W507, Boston, MA 02118. E-mail
freedmaj@bu.edu
(Circ Res. 2003;92:944-946.)
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000074030.98009.FF
See related article, pages 1041–1048
Jane E. Freedman
and are a more sensitive marker of in vivo platelet activation
than platelet surface P-selectin. 3 Plaque rupture promotes
activation of the inflammatory responses, and the consistent
finding of heterotypic aggregates highlights the close interaction
between inflammation and thrombosis in vascular
disease.
Contributing to our understanding of the role of platelets in
inflammation are CD40-CD40 ligand (CD40L) interactions.
CD40 is a membrane glycoprotein belonging to the tumor
necrosis family receptor superfamily and its ligand CD40L
(CD154) is a glycoprotein from the tumor necrosis factor
family. CD40-CD40L interactions are central in immune
responses and inflammation. Ligation of CD40 on various
vascular cells contributes to the pathogenesis of atherosclerotic,
thrombotic, and inflammatory processes. 4,5 On endothelial
cells or monocytes, the engagement of CD40 leads to
the synthesis of adhesion molecules, chemokines, and tissue
factor and causes the activation of matrix metalloproteinases.
CD40L has also been detected in platelets 4 where, after
stimulation, it is translocated to the platelet surface (Figure).
The surface-expressed CD40 ligand is then cleaved from the
platelet over a period of minutes to hours subsequently
generating a soluble fragment (soluble CD40 ligand or
sCD40L). 6 It is estimated that 95% of the circulating
sCD40L is derived from platelets. 6 Found to be increased on
platelets in fresh thrombus, 4 sCD40L has been shown to be
elevated in cardiovascular disease 7,8 and associated with
increased cardiovascular risk in apparently healthy women. 9
Although sCD40L has been characterized as a marker of
thrombotic diseases, much less is known about its direct role
in platelet function. It has been suggested that CD40L is an
IIb 3 ligand, a platelet agonist, and contributes to stability of
arterial thrombi. 6 In this issue of Circulation Research,
Inwald and colleagues 10 add to the limited information
concerning the effects of sCD40L on platelet function and
demonstrate that CD40 ligation may be a mechanism for
platelet activation. After incubation of platelets with the
trimeric form of sCD40L (sCD40LT), they showed enhanced
CD62P expression as well as dense and -granule release.
Interestingly, the presence of a glycoprotein IIb/IIIa blocking
agent did not alter sCD40LT-induced CD62P expression. In
addition, -thromboglobulin and [ 14 C]-5-HT were shown to
be present after ligation reflecting - and dense granule
release, respectively. An intriguing part of this study is the
confirmatory data using subjects with X-linked hyper-IgM
syndrome and known absence of CD40L expression. The
ability to utilize these subjects is particularly interesting in
platelet-dependent studies as genetic manipulation in cell
culture is not possible.
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At issue is the form and concentration of sCD40L used in
the studies. In the experiments, the concentration of
sCD40LT used was 1 g/mL although platelet activation was
present at concentrations as low as 100 ng/mL. Although
these concentrations are similar to those reported in the
literature for CD40L activation of other cell types, it is still
greater than the levels reported in patients (50 ng/mL).
Although this discrepancy may question the clinical relevance
of the findings of Inwald et al, 10 it is possible that local
concentrations in active areas of thrombotic or inflammatory
disease are higher. This would allow a dose-dependent
mechanism for targeting the effects of sCD40L. Further
kinetic studies and assessment of in vivo concentrations are
needed to answer the question of relevance in relation to the
levels used in these studies. In addition, the trimeric form of
sCD40L was used in the studies and it has been shown that
the specific activity of sCD40L may depend on its form as a
trimer or monomer. Although the membrane-bound CD40L
may be trimeric, 11 there is debate concerning the soluble
form. 4,11 Further studies, specifically in platelets, would be
required to clarify this question.
In the studies by Inwald et al, 10 platelet activation via
sCD40LT occurred in the presence of a IIb 3 inhibitor. This
appears to be in contrast to previous work that demonstrates
that sCD40L is an IIb3 ligand. 6 However, a more intriguing
interpretation is that this is not only a novel means of platelet
activation but that the platelet may be activated in distinct
ways in different settings. In the present study by Inwald et al,
incubation with sCD40LT resulted in significantly less
CD62P expression compared with platelet activation with
thrombin receptor agonist peptide 1-6 (SFLLRN). In addition,
the increase in Pac-1 is minimal with sCD40L compared
with SFLLRN. Differences in dense granule release were also
detected with thrombin receptor stimulation leading to 5
times [ 14 C]-5-HT levels compared with sCD40LT incubation.
Similar results were not shown for -thromboglobulin and
further analysis of -granules as well as the effects of other
Freedman CD40-CD40L and Platelet Function 945
After platelet stimulation, CD40L is translocated
to the platelet surface. Surfaceexpressed
CD40 ligand is then cleaved
from the platelet over a period of minutes
to hours, subsequently generating a
soluble fragment, sCD40L. Soluble
CD40L can then promote inflammatory
or thrombotic response by causing further
platelet activation or, in high-shear
settings, may enhance the formation of
homotypic aggregates.
agonists would be of interest. However, it may be possible
that in areas of relevant concentrations, the presence of
sCD40L leads to selective activation and granule release but
excludes the formation of homotypic aggregates. This possibility
is strengthened by the finding that sCD40LT enhanced
platelet-neutrophil formation occurs in the setting of minimal
IIb 3 activation and that platelets from subjects deficient in
CD40L aggregated normally. However, in the setting of high
shear, IIb 3 may be engaged leading to thrombus formation.
Thus, these differences could allow for a partial separation of
the thrombotic and inflammatory roles of platelets. The
mechanism for the sCD40L-induced cascade of platelet
activation is not known. In the present study, the experiments
suggesting that there was no change in calcium flux after
sCD40L stimulation are too limited to draw conclusions or to
infer that protein kinase C signaling is responsible. However,
the possibility that ligation of CD40, as well as binding to
IIb 3, can lead to outside-in signaling in the absence of
inside-out signaling is intriguing. However, not all means of
inside-out stimulation in platelets are calcium-dependent. To
conclude that CD40-CD40L interactions are resulting in
outside-in signaling or G protein–independent activation
requires further examination.
Although many questions remain, these findings help to
clarify if sCD40L is released to further drive plateletdependent
thrombus formation or inflammatory reactions in
the vasculature. The partial and selective activation that
appears after engagement of CD40 suggests that the platelet
is involved in both functions and that it may have the capacity
to be differentially regulated. Continuing to define the role of
platelets in inflammation and the extent to which its thrombotic
functions are complementary will be challenging. The
anuclear platelet is being rediscovered as an intriguing link
between thrombosis and inflammation, and future research
should provide great insight into platelet-dependent mechanisms
leading to the regulation of vascular disease.
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946 Circulation Research May 16, 2003
Acknowledgments
This work was supported in part by NIH grants NIH RO1AG08226
(J.F.), NIH RO1HL62267 (J.F.), and an Established Investigator
Award from the American Heart Association.
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KEY WORDS: platelets CD40/CD40L thrombosis inflammation
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