Laboratory of Immunovirology, Department of Microbiology & Immunology, University of Montreal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada
Correspondence: Ali Ahmad, D.V.M., Ph.D., Research Center, Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, Quebec, H3T 1C5, Canada. E-mail: ahmada{at}justine.umontreal.ca
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Key Words: platelets • thrombocytes • Fas-L • apoptosis • tumor cells
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R)] on their
surface and can potentially play a role in immune regulation
[8
9
10
11
]. We show here, for the first time, that
activated human platelets express biologically active Fas-L on their
surface and can induce apoptosis in Fas-positive human tumor cells.
Furthermore, upon activation, they release this preformed Fas-L rapidly
into medium. Fas-L is a type II membrane glycoprotein that belongs to the tumor necrosis factor (TNF) family of death-inducing cytokines [12 ]. It is expressed on activated T cells, natural killer (NK) cells, and monocytes [13 14 15 ]. Its cognate receptor, Fas (Apo-1, CD95), is a type I membrane glycoprotein that belongs to the nerve growth factor (NGF) and TNF receptor superfamily and is expressed on a wide variety of normal and malignant human cells [reviewed in refs. 16 17 ]. Fas/Fas-L interaction induces the assembly of death-inducing signaling complex comprising Fas, Fas-associated death domain (FADD), and caspases and culminates in the apoptotic death of the Fas-positive cells [16 ]. The apoptosis medicated by the Fas/Fas-L interactions plays an important role in embryonic development, normal cellular homeostasis, and immune regulation [16 , 17 ]. The expression of Fas-L by activated human platelets, shown here, is yet another example of the role these blood elements play in biological processes beyond hemostasis and thrombosis.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Ethylenediaminetetraacetate (EDTA) was added to the PRP to a final concentration of 5 mM and was centrifuged at 2000 g for 15 min to pellet the platelets, which were then washed twice in the wash buffer containing 0.015 M Tris-HCl (pH 6.5), 0.145 M NaCl, 2.0 mM EDTA, 0.1% glucose, and 0.05% bovine serum albumin (BSA). The washes also contained apyrase (0.5 mg/ml; Sigma, St. Louis, MO) and hirudin (0.1 U/ml; Sigma). The platelets were counted with a hemocytometer after diluting in 1% ammonium oxalate. The platelet preparations used in this study contained 1 x 108 platelets per ml in RPMI 1640 containing 2% heat-inactivated fetal bovine serum (FBS) and always contained fewer than 104 white blood cells in 1 ml (unpublished results).
Activation of platelets
Two known platelet activators, thrombin and adenosine
5'-diphosphate (ADP), were used to induce platelet activation. Both of
these reagents were purchased from Boehringer Mannheim (Laval, Quebec,
Canada) and were used at 1 U/ml and 100 uM final concentration,
respectively. The platelet preparations were incubated at room
temperature with one or the other activator for 5 min unless specified
otherwise and then the activation process was terminated by adding an
equal vol of 2% paraformaldehyde in PBS. After further incubation at
room temperature for 30 min, the fixed platelets were washed further
with PBS to remove the activators and paraformaldehyde and then
resuspended in appropriate buffer/medium for further studies. The
platelet activation was monitored by determining the surface expression
of gp53 (CD63) using a monoclonal antibody (mAb; Bio/Can, Mississauga,
Ontario, Canada). This glycoprotein is known to be translocated to the
surface of platelets after activation [19
].
Determination of Fas-L expression on the surface of platelets
Fas-L expression on activated and nonactivated platelets was
determined by flow cytometry. For this purpose, the platelet
preparations (100 µl) were resuspended in equal vol of PBS containing
2% heat-inactivated FBS, 0.01% sodium azide, and then incubated on
ice for 45 min with a control or anti-Fas-L mAb (1 µg/sample; Catalog
#65321A; Pharmingen, San Diego, CA) and washed three times with PBS.
After 10 min preincubation with 2 µl normal mouse serum, the
platelets were incubated further on ice for 45 min with 100 µl of the
1:100 diluted fluorescein isothiocyanate (FITC)-conjugated goat
antimouse IgG (Becton Dickinson). After three final washes with PBS,
the stained cells were resuspended in PBS and analyzed by flow
cytometry using FACScan (Becton Dickinson). For this analysis,
platelets were gated using their forward- and side-scatter profiles. In
some experiments, platelets were fixed and double-stained for Fas-L and
CD63. For this purpose, they were first stained with anti-Fas-L mAb and
FITC-conjugated goat antimouse IgG, as described above, and then
incubated on ice with phycoerythrin (PE)-conjugated anti-CD63
mAb (Bio/Can) for 45 min. After this incubation, the stained platelets
were washed and analyzed by flow cytometry.
Detection of Fas-L expression by Western blots
To confirm that platelets express Fas-L of the known molecular
weight, the cells were lysed in the lysis buffer containing
Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), and protease
inhibitors as described earlier [20
]. After sonication
for 20 sec, the lysates were clarified by centrifugation for 15 min at
14,000 g at 4°C. Protein contents of the lysates were
determined using a commercial protein determination kit (Bio-Rad,
Hercules, CA) using BSA as standard. Lysate proteins (50–70µg) were
resolved on 12% SDS-polyacrylamide gel electrophoresis (PAGE) under
reducing conditions. The resolved proteins were electroblotted onto
nylon membranes. The unbound sites on the membranes were blocked with a
blocking buffer (5% skim milk powder and 0.05% Tween 20 in PBS) as
described [20
]. The Fas-L protein bands were detected by
incubating membranes with a Fas-L-specific mAb (4 µg/ml; Pharmingen)
or with rabbit polyclonal antibodies (1:500 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA). The secondary antibodies used were
alkaline phosphatase-conjugated goat antirabbit or antimouse 1gG (both
from Promega, Madison, WI). The bands were revealed by using nitroblue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP;
Promega) as described earlier [20
]. To determine whether
Fas-L was released into medium from platelets upon activation,
supernatants from platelets were collected after their activation and
fixation by centrifugation. These supernatants were concentrated
fivefold using Microconcentrator 10 filters (Amicon Inc., Beverly, MA)
as recommended by the manufacturer. These supernatants were subjected
to Western blot analysis for Fas-L expression after determining their
protein concentrations as described above.
Determination of the biological activity of Fas-L
To determine whether Fas-L expressed on the surface of activated
platelets was biologically active, these cells were incubated, with and
without activation, with CEM cells. The latter cells are
derived from adult T cell leukemia, and are Fas-positive
[21
]. The ability of platelets to induce apoptosis and
inhibit the growth of CEM target cells was determined as below.
Induction of apoptosis
For this purpose, CEM cells were cultured in 24-well,
flat-bottomed plates (1x106/ml) in RPMI 1640 medium
containing 2% FBS. To each well, 1 x 108 platelets
were added. The platelets were activated with thrombin, ADP, or were
mock-treated and fixed with 2% paraformaldehyde and washed with PBS to
remove traces of activators and fixatives. Aliquots of the cultures
were stained with propidium iodide (PI) using a commercial kit (R&D
Systems, Minneapolis, MN). PI has been shown to stain cells undergoing
apoptosis [22
]. The stained cells were examined within
1 h under fluorescence microscope. The number of apoptotic cells
(red-stained, condensed nuclei) was counted by examining 200 cells. In
some cultures, Fas/Fas-L interactions were blocked using an anti-Fas-L
mAb (1 µg/ml; Pharmingen) or the same concentration of an
isotype-matched mouse immunoglobulin of irrelevant specificity as a
control.
Cell proliferation
The cocultures of platelets and the indicator cells (CEM) were
set up as described above. The number of viable cells per ml in each
coculture was determined by the trypan blue exclusion assay with a
hemocytometer at 12-h intervals.
3H-thymidine-uptake determination
The 3H-thymidine uptake by the indicator cells was
determined as described earlier [23
]. The cocultures of
CEM and platelets were essentially the same as described above except
that they were cultured in the wells of a round-bottomed, 96-well
microculture plate with 2 x 105 cells per well with
1 x 107 platelets in a total vol of 200 µl. Each
coculture had five replicates. These cocultures were also carried out
in the presence of anti-Fas-L mAb or an isotype-matched control
antibody of mouse origin as described in the above sections. After
16 h, the cultures were pulsed with 1 µCi
3H-thymidine (specific activity 20 Ci/mmole; ICN, Montreal,
Quebec, Canada) for 8 h and harvested, and the
3H-thymidine uptake was measured by liquid-scintillation
counting as detailed [23
].
Statistical analysis
Wherever needed, group means were compared using Student’s
two-tailed t-test. The differences were deemed significant
at 5% level-of-confidence as described earlier [23
].
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43 kDa;
membrane-bound form) as well as cleaved (26 kD; soluble form) Fas-L
(Fig. 2a
2b)
. These two forms of Fas-L were also detectable in several
other tested cell types and are consistent with previous findings that
Fas-L is cleaved by a matrix metalloprotease between Lys129 and gln130
into a soluble(s) form [24
25
26
]. The detection of
cleaved and uncleaved forms in cellular lysates may be a result of
anchorage of the cleaved forms with uncleaved partners, because Fas-L
occurs as trimers on cell surfaces [24
25
26
].
Surprisingly, the anti-Fas-L mAb reacted with platelets and HeLa cells
only but not with the Fas-L expressed by Jurkat cells (Fig. 2c)
. Fas-L
is heavily glycosylated, and because the glycosylation pattern for a
given protein is usually cell type-specific, these results suggest that
this antibody is probably recognizing a carbohydrate epitope on Fas-L.
Furthermore, membrane and sFas-L were readily detectable in the
supernatants of the activated but not unactivated platelets (Fig. 2c
and unpublished results), suggesting release of Fas-L from these blood
elements upon activation. This may also explain why a relatively low
percentage of platelets (compared with CD63 expression) expresses Fas-L
upon their surface despite abundant expression in lysates and the
medium. All these blots were negative when they were developed with
normal mouse or rabbit antibodies (unpublished results). Collectively,
these data suggest that platelets contain abundant quantities of Fas-L,
which they express on their surface upon activation and release into
medium. Fas-L is known to be expressed by activated T cells, NK cells,
and monocytes [16
, 17
]. Recent studies show
that these cells also contain Fas-L within their cytoplasmic granules
and express it on their surface as well as secrete it in a polarized
fashion upon activation [27
]. In the case of platelets,
several other immunologically important molecules/cytokines [e.g.,
transforming growth factor-ß (TGF-ß), P-selectin, CD40L, etc.] are
known to be stored preformed within cytoplasmic granules and become
translocated to the surface and secreted upon activation
[1
, 8
9
10
11
]. Our results suggest that Fas-L
behaves in a similar fashion in platelets.
 View larger version (33K): [in a new window] |
Figure 1. Expression of Fas-L on platelets by indirect membrane
immunofluorescence. Activated-fixed platelets were incubated with
anti-Fas-L or control antibodies, washed, and stained with
FITC-conjugated goat antirabbit Ig. The stained cells were examined by
FACScan. (a) Typical histograms of FITC-positive platelets for Fas-L
and CD63 expression at different time points after activation. The
numbers above each histogram represent %-positive platelets for the
relevant marker, whereas the numbers in parentheses show their mean
fluorescence intensities. (b) Mean % of Fas-L-expressing cells from
three different donors. The mean values of Fas-L-expressing activated
and nonactivated platelets for these three donors differed
significantly (p=0.0007). (c) Double-staining of activated
platelets for Fas-L (FL1 on x-axis) and for CD63 (FL2 on y-axis). A
shows gating for platelets using their forward and side-scatter
profiles. This gating eliminates any contaminating white blood cells
from analysis. B shows unactivated platelets (1.11% positive for the
two markers; upper right quadrant). C shows platelets fixed after
activation, which are 13.92% positive for both of these markers (upper
right quadrant). The activated platelets in C show a clear shift of
their fluorescence profile to the right (i.e., increase in FL1 or Fas-L
expression) compared with the unactivated platelets in B.
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 View larger version (24K): [in a new window] |
Figure 2. Western blot analysis of Fas-L expression on platelets. Equal amounts
of proteins from lysates of washed platelets or of the indicated cells
were resolved on 12% SDS-PAGE, transferred onto nylon membranes. The
membranes were developed using anti-Fas-L antibodies, alkaline
phosphate (AP)-conjugated secondary antibodies, and BCIP/NBT substrate.
(a) Fas-L protein bands detected by anti-Fas-L mAb. The lanes represent
platelets from donor I (1); platelets from donor II (2); E6.1 (3);
peripheral blood mononuclear cells (PBMC) after activation with
phytohemagglutinin (PHA) and interleukin (IL)-2 (4); and K562 (5). The
Fas-L bands of 42 kDa (uncleaved) and 27 kDa (cleaved) are evident. (b)
Fas-L protein bands detected by anti-Fas-L polyclonal antibodies. The
lanes represent K562 (1); Jurkat (2); and platelets (3). The upper
arrow shows different glycosylated, uncleaved forms, and the lower
arrow shows the cleaved form of Fas-L. (c) Fas-L protein detected by
the anti-Fas-L mAb in the medium of activated platelets. The lanes
represent lysate from Jurkat cells (1); lysate from HeLa cells (2);
supernatant from activated platelets (3); and lysate from COS-1
cells (4).
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 View larger version (29K): [in a new window] |
Figure 3. Induction of apoptosis in CEM cells by activated platelets.
Fixed-activated or nonactivated platelets were added to CEM cultures
with or without anti-Fas-L or control antibodies. (a) Percentage of
apoptotic cells determined by PI positivity after 24 and 48 h, as
described in Materials and Methods. The letters represent CEM cells
growing in the culture medium with 2% FBS as negative control for
PI-negative cells (A); in the presence of activated platelets (B); with
activated + anti-Fas-L antibodies (C); with activated
platelets + control antibodies (D); in the culture medium without
FBS as positive control for PI-positive cells (E); and in the presence
of nonactivated platelets (F). All cultures except E were in the
culture medium containing 2% FBS. Each point represents mean number of
PI + cells ± SE from three replicates. The mean
values for A, C, and F differed significantly (p<0.05) from
those of B and D at the 24-h time point, except between D and F
(p=0.223). (b) Photomicrographs of the PI-positive CEM cells
in various cultures taken 24 h after the addition of platelets.
The panels in column 1 show cells under phase contrast, and in column
2, the same cells are photographed under UV light. These panels
represent CEM cells growing without FBS as positive control for
apoptosis or PI positivity (A); cells growing in the presence of FBS as
negative control (B); CEM cells in the presence of platelets (C), and
CEM cells as in C but with the addition of anti-Fas antibody (D).
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 View larger version (15K): [in a new window] |
Figure 4. Effect of platelets on cell proliferation. CEM cells were cultured with
or without platelets and anti-Fas-L or control antibodies, as described
in the legend to Figure 3
. The number of viable and dead cells in the
coculture was counted at 12-h intervals after staining with trypan
blue. Each point represents mean ± SE of the number
of viable cells in the culture. The letters represent cells growing in
the culture medium with 2% FBS (A); in the presence of activated
platelets (B); in the presence of activated platelets and anti-Fas-L
antibodies (C); in the presence of activated platelets and control
antibodies (D); and in the presence of 0% FBS (E). The cultures B–D
were in the presence of 2% FBS. The mean values of A and C each
differed significantly (p<0.05) from those of B, D, and E
at the 24-h time point.
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 View larger version (11K): [in a new window] |
Figure 5. Effect of activated platelets on 3H-thymidine uptake. CEM
cells (50,000) were cultured in 2% RPMI 1640 with or without activated
platelets (100 µl). After 16 h, microcultures were pulsed for
8 h with 1 µCi 3H-thymidine, and the cells were
harvested. Each bar in the figure represents mean
3H-thymidine uptake ± SE from five
replicate wells. The letters below the bars represent counts from cells
without platelets or antibodies (A); in the presence of platelet (B);
in the presence of platelets and anti-Fas-L antibodies (C); in the
presence of platelets and control antibody (D); in the presence of
anti-Fas-L antibody (E); and in the presence of control antibody (F).
The mean values of B and D each differed significantly
(p<0.05) from C and A.
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Finally, it may be relevant to point out that although our results provide evidence that activated platelets express biologically active Fas-L, these cells express/secrete several other molecules/cytokines that may provide growth-promoting signals and over-ride the apoptosis-inducing effects of the Fas/Fas-L interaction. For example, activated platelets also express CD40L, and CD40/CD40L interaction has been known to overcome the Fas/Fas-L-induced apoptosis in certain human cell types [8 , 31 ]. Platelets are also known to convert and secrete sphingosine into sphingosine 1-phosphate, which acts as a survival factor for vascular endothelial cells [32 ]. Thus, the ultimate outcome of the Fas/Fas-L interaction mediated by platelets would depend on the interacting cell type and may not necessarily result in the apoptosis and/or growth inhibition of the interacting cells.
In conclusion, we show here that human platelets contain abundant quantities of Fas-L and upon activation, rapidly release into medium as well as express it on their surface. The surface-expressed Fas-L is biologically active and can induce apoptosis of the Fas-positive human cells. Further studies should be forthcoming to determine whether activated platelets can also induce apoptosis in vivo by Fas/Fas-L interactions and whether they play any role in the pathogenesis of diseases, such as AIDS, in which circulating platelets are constitutively activated, and enhanced apoptosis of immunocytes has also been well-documented.
Received March 28, 2000; revised August 28, 2000; accepted August 29, 2000.
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