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(Journal of Leukocyte Biology. 2001;69:123-128.)
© 2001 by Society for Leukocyte Biology

Activated human platelets express Fas-L and induce apoptosis in Fas-positive tumor cells

Rasheed Ahmad, José Menezes, Laurent Knafo and Ali Ahmad

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In addition to their role in hemostasis and thrombosis, platelets are important modulators of immune and inflammatory responses. We provide evidence here that human platelets contain abundant quantities of Fas-L, and upon activation, they express it on their surface as well as release it into medium. This surface-expressed Fas-L is biologically active and can induce apoptosis in Fas-positive human tumor cells. Therefore, activated platelets may represent an important player in Fas/Fas-L-mediated apoptosis.

Key Words: platelets • thrombocytes • Fas-L • apoptosis • tumor cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Platelets or thrombocytes are the second most numerous formed elements of blood [the first are red blood cells (RBCs); reviewed in ref. 1 ]. They are derived from their precursor cells—i.e., megakaryocytes in bone marrow by an unusual mitotic process—circulate in blood for 7–10 days, and are then eliminated by cells of the reticulo-endothelial system [1 ]. The role of platelets in hemostasis, thrombosis, and wound healing is well-documented [2 ]. Their involvement in immune modulation and host defense from pathogens and malignancy is also increasingly being realized [3 4 5 ]. They serve as pockets of several immunologically important cytokines and chemokines and release them upon activation. They can also modulate the effector functions of several immunocytes [6 , 7 ]. Furthermore, activated platelets express several molecules [e.g., CD40L, P-selectins, Fc receptor for immunoglobulin G (IgG; Fc{gamma}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Platelet preparations
Human platelet preparations were made as described after some modifications [18 ]. Briefly, peripheral veinous blood was obtained from healthy normal donors in citrated blood tubes (Vacutainer, Becton Dickinson, San Jose, CA) without applying tourniquet to the arm. The first 2–3 ml of blood were discarded. For the platelets to be used as unactivated controls, an equal vol of 2% paraformaldehyde in phosphate-buffered saline (PBS) was added. The blood samples were centrifuged at 150 g for 15 min, and the platelet-rich plasma (PRP) was obtained.

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 ].


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The flow cytometric analysis of platelets stained for Fas-L revealed that activated platelets, but not unactivated ones, express this molecule on their surface (Fig. 1a ). Platelets, whether activated with thrombin or with ADP, showed similar results. The platelets obtained from different healthy donors showed a slight variation in the level of expression of Fas-L upon activation. Figure 1b shows a relative positivity of three different donors for Fas-L expression upon activation with thrombin. To confirm that activated CD63-expressing platelets were positive for Fas-L expression, we performed dual staining for these two markers. As shown in Figure 1c , activated CD63-expressing platelets were positive for Fas-L. When analyzed by SDS-PAGE, platelets showed abundant expression of this protein (Fig. 2 ) compared with other cell types. Western blots with anti-Fas-L antibodies revealed that platelets expressed uncleaved (~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.



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  		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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  		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).

 
The Fas-L expressed on the surface of activated platelets is biologically active. As shown in Figure 3a , coculture of fixed, activated platelets with Fas-positive CEM cells induced apoptosis in these cells. Anti-Fas-L as well as anti-Fas mAb that blocks Fas/Fas-L interaction significantly blocked this apoptosis, indicating specificity of the platelet-induced apoptosis to the Fas/Fas-L interaction. Figure 3b shows these apoptosing cells when cocultured with platelets under a UV-microscope. Activated platelets inhibited cell proliferation of the indicator cell line (Fig. 4 ) and also significantly (p<0.05) reduced the 3H-thymidine uptake (Fig. 5 ). Both of these effects were significantly (p<0.05) blocked by the presence of anti-Fas-L antibodies but not by isotype-matched control antibodies. It is interesting that cocultures of the activated platelets and CEM cells did not show an increase in the number of trypan blue-positive cells (unpublished results), suggesting further that decreased cell proliferation in these cultures was a result of apoptosis and not because of necrosis. Furthermore, unfixed-activated platelets and the supernatant from activated platelets were not as efficient as fixed ones in mediating apoptosis of Jurkat T cells (unpublished results). This may be a result of the release of Fas-L, which has been shown to be inefficient in mediating apoptosis of Fas-positive cells; sFas-L may, in fact, inhibit Fas/Fas-L-mediated apoptosis [24 ]. Alternatively, some growth-promoting factor released by unfixed platelets into the supernatant may be overcoming the Fas/Fas-L-induced apoptosis (see below also).



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  		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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  		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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  		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.

 
Platelets are known to kill tumor cells [28 ]; however, the role of Fas/Fas-L interaction in this killing has not been investigated. The apoptosis induced by Fas/Fas-L interactions plays an important role in immune regulation, control of malignancy, and the maintenance of immune privilege at certain sites in the body [16 , 17 ]. It also represents an important mechanism in the NK and cytolytic T lymphocyte (CTL)-mediated cytotoxicity of the target cells. The only normal cells known to express Fas-L outside the immune system are in the testes and the anterior chamber of eye (the so-called immune-privileged sites). Our results provide evidence that platelets contain abundant amounts of Fas-L and express biologically active Fas-L on their surface as well as release it into medium upon activation. These results may have important implications in the pathogenesis of the disease conditions in which circulating platelets are known to be constitutively activated, e.g., in cancer and AIDS patients [5 , 29 ]. In the latter disease, aberrant apoptosis of various immunocytes has been well-documented [30 ]. Further studies will be needed to determine whether constitutively activated platelets in AIDS patients play any role in the enhanced apoptosis of immunocytes in this disease.

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.


   ACKNOWLEDGEMENTS
 
This work was supported by a research grant from the Medical Research Council of Canada (MRC), and by the immunology program of Ste. Justine Hospital. A. A. is also a recipient of the MRC Scholar Award. We gratefully acknowledge the secretarial assistance of Ms. Micheline Patenaude and Sylvie Julien.

Received March 28, 2000; revised August 28, 2000; accepted August 29, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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