CYTOKINE mRNA EXPRESSION IN HUMAN PLATELETS AND A MEGAKARYOCYTIC CELL LINE AND CYTOKINE MODULATION OF PLATELET FUNCTION

CYTOKINE mRNA EXPRESSION IN HUMAN PLATELETS AND A MEGAKARYOCYTIC CELL LINE AND CYTOKINE MODULATION OF PLATELET FUNCTION Gerald Soslau,1,2

Doris A. Morgan,2 Jonathan S. Jaffe,3 Yihe Wang3

Isadore Brodsky,2

Platelet formation and function are regulated, in vivo, to varying degrees by cytokines in the micro-environment. While white blood cells are the major source of cytokines within the cardiovascular system, the question addressed in this study was whether platelets and the platelet precursor, the megakaryocyte, may also serve as a source of cytokines. Cytokines produced by or carried within platelets could be released at sites of vascular injury and participate in wound healing. Platelets and a human megakaryocyte-like cell line, HU3, were found to express message for interleukin 7 (IL-7), stem cell factor (SCF), transforming growth factor beta (TGF-b), cMpl, the IgE receptor subunits FceRIag and the transcription factor, NF-E2. Other cytokines expressed in HU3 cells but not in platelets included IL-1b, IL-6, IL-10, IL-13, TNF-a and the FceRIb subunit. The HU3 cell line seemed to be further along the maturation/differentiation pathway to platelet formation than a second blood derived bipotential cell line, MB02. The MB02 cell line did not express IL-6, IL-10, SCF, TNF-a nor cMpl. Furthermore, culturing the HU3 cells in TPO appeared to repress expression of FceRIb directing the cell closer to the platelet phenotype. In light of the presence of cytokine expression in platelets/megakaryocytes, agonist-induced platelet aggregation was measured in the presence of added cytokines as a means to evaluate potential cytokine modulation of platelet function. Collagen-induced aggregations were significantly enhanced by IL-6, SCF and TPO. Other cytokines tested significantly stimulated the thrombin receptor activating peptide, SFLLRNP-, U46619- and ADP-induced platelet aggregations with TPO being the most consistent activator. It is possible that cytokines released from platelets act in concert with cytokines released from other cellular sources to modulate haemostasis and thrombosis differentially depending upon the site of injury. 7 1997 Academic Press Limited

Platelets are anucleated cells that circulate in the blood with a life span of 7–10 days.1 Platelet production is regulated, in part, by a hormone/ cytokine (known as cMpl ligand, thrombopoietin or MGDF) in a multi-step differentiation process from haemopoietic stem cells.2 It appears that platelet formation is also dependent upon the expression of a transcription factor, NF-E2.3 Platelets are ultimately formed by the fragmentation of the cytoplasmic portion of the multinucleated megakaryocyte.4 As such, newly produced platelets contain all of the From the 1Depts of Biochemistry, 2Medicine Divisions of Hematology/Oncology and 3Allergy/Immunology, Allegheny University of the Health Sciences, Philadelphia, PA 19102, USA Correspendence to: Gerald Soslau, Department of Biochemistry/ IMS, Mail Stop 344, Allegheny University of the Health Sciences, Broad & Vine STS., Philadelphia, PA 19102, USA Received 27 August 1996; accepted for publication 27 December 1996 7 1997 Academic Press Limited 1043–4666/97/060405 + 11 $25.00/0/ck960182 KEY WORDS: cytokine expression/megakaryocute/platelet CYTOKINE, Vol. 9, No. 6 (June), 1997: pp 405–411

normal cytosolic components along with specialized components such as dense granules and a granules that participate in haemostasis.5,6 Platelet viability is maintained by normal metabolic processes,7,8 however, they possess only limited capacities to synthesize macromolecules.9,10 They contain some species of long-lived mRNA that presumably encode for selected protein synthesis.11 Recent reports describe the presence of GPIb,11 osteocalcin,12 chemokine and chemokine receptor mRNAs13 in human platelets. Also, a full-length cDNA encoding the neutrophilactivating peptide, ENA-78, has been cloned from human platelets.14 Platelets interact directly or indirectly with all cells within the cardiovascular system. During platelet activation, a plethora of bioactive molecules are released from platelets into the micro-environment.15 While the protein synthetic capacity of platelets may be quite low the release of newly synthesized products could play a significant role in normal or pathological processes. For example, platelet-associated growth 405

406 / Soslau et al.

factors may regulate endothelial and smooth muscle cell growth at sites of vascular injury or atherosclerotic plaques.16 Cytokines may also regulate these proliferative processes as well by regulating immune responses17 and platelet reactivity.18 These latter cytokine-induced responses are of great concern since platelet concentrates, used for platelet transfusions, often cause febrile non-haemolytic transfusion reactions, presumably due to released cytokines that accumulate during storage.19,20 The polypeptide cytokines produced by a multitude of cell types act as extracellular messengers/ modifiers that allow cells to communicate with each other.17 Little12 if anything is currently known about the capacity of platelets, or, their precursor, the megakaryocyte, to produce cytokines. This study focused on the potential expression of a wide variety of cytokines and other regulatory factors in human platelets and two human blood-derived cell lines, one with megakaryocytic properties.21,22 The expression of some mRNA species in platelets and one bi-potential cell line, HU3, but not the second bi-potential cell line, MB02, indicate that HU3 is further along the differentiation process of megakaryocyte maturation. Selected cytokines expressed and released from platelets and/or other cells within the cardiovascular system appear to modulate platelet function in vitro and may also impact on haemostasis in vivo.

RESULTS RNA was isolated from two megakaryocytelike cell lines (HU3 and MBO2) and platelets to determine if mRNA species for cytokines and/or platelet related factors were present. Table 1 lists the mRNA species searched for by RT-PCR and the species detected in platelets, HU3, MBO2 and peripheral blood mononuclear cells (PBMC). All positive samples contained a PCR product of the correct predicted molecular weight. The absence of most species in platelets that were present in PBMC confirmed the purity of the platelet preparations. The detection of two house-keeping gene products, GAPDH and b-actin, in all RNA preparations with equal intensities confirmed the intactness of the RNA and the equal concentration of RNA in the RT-PCR samples. The absence of two platelet/megakaryocyte gene products, cMpl and NF-E2, in PBMC preparations indicate little, if any, contamination of these cells with platelets. Only three of the 17 cytokines searched for were detected in platelets, IL-7, SCF and TGF-b. Messages for FceRI a and g, NF-E2 and cMpl were also present in the platelet preparations. Selected genes for cytokines and other factors were expressed in both

CYTOKINE, Vol. 9, No. 6 (June, 1997: 405–411)

TABLE 1. Factor IL-1a IL-1b IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-9 IL-10 IL-12 IL-13 GM-CSF SCF TNF-a TGF-b INF-g FceRIa FceRIb FceRIg NF-E2 cMpl GAPDH b-actin

Platelet

HU3

MBO2

PBMC

− − − − − − − + − − − − − + − + − + − + + + + +

− + − − − − + + − + − + + + + + − + + + + + + +

− + − − − − − + − − − + + − − + − + + + + − + +

+ + + + + + + + + + + + + + + + + + + + − − + +

HU3 and MBO2 cell line. The cytokine genes for IL-6, IL-10, SCF and TNF-a and the gene for the cMpl receptor were expressed in the HU3 cell line but not the MBO2 cell line. Message for the transcription factor, NF-E2, required for platelet formation,3 was present in platelets and both human cell lines but absent in PBMC. However, message for the cMp1 receptor, also required for platelet formation,2 was not present in the MBO2 cell line nor in PBMC. When HU3 cells were cultured in TPO (thrombopoietin) (100 units/ml for 30 days) in place of GM-CSF (granulocyte-macrophage colony-stimulating factor) expression of the Fce1RIb gene was absent/repressed and the FceRI abg mRNA pattern appeared to be the same as the mature platelet (Fig. 1). Culturing these HU3 PLT

GM

IL-3

TPO

α β γ GAPDH Figure 1. The agarose gel electrophoretic bands of the RT-PCR products synthesized from human platelet (plt) and HU3 cell total cellular RNA. The RT-PCR products for the IgE receptor subunits, FceRIa, b and g produced from plts and HU3 cells cultured in GM-CSF (GM), IL-3 or thrombopoietin (TPO) are depicted relative to the control GAPDH PCR product according to procedures described in Materials and Methods.

Cytokines in platelets and megakaryocytes / 407

DISCUSSION Platelet formation and reactivity to bioactive molecules in the microenvironment play a central role in hemostasis and thrombosis. Great strides have been made in our understanding of the differentiation of stem cells to megakaryocytes and the ultimate formation of platelets.2,3 However, due to the lack of a stable progenitor megakaryocyte cell line, details of the cytokine requirements to induce differentiation and maturation of platelets are incomplete. Also deficient, is knowledge of events at the molecular level, that are triggered or repressed during these processes. The detection of genes encoding cytokines and other platelet inducing factors that are expressed in megakaryocytes and/or platelets could potentially indicate autocrine or paracrine actions of these factors associated with platelet formation and platelet function. Cytokines are generally considered to be products of mature leukocytes within the lymphatic system. Cytokines released from these cells are instrumental in haematopoietic growth regulation and amplifying

45 Change in aggregation (%)

40

A

*

35 30 25 20

*

*

15

*

10

*

5 0 –5 –10

± SE 7.5 8.7 7.3 6.2 8.4 8.8 2.5 4.9

Collagen

7.8 9.0 9.1 7.0 5.3 5.6 7.3 6.8

SFLLRNP

45 40 Change in aggregation (%)

cells in IL-3 (5 ng/ml for 6 mos) did not alter the expression of the FceRI subunits compared to the HU3 maintained in GM-CSF. Based upon the results described above and the sensitivity of platelet activation to selected cytokines18,23–27 we tested agonist-induced platelet aggregations in the presence and absence of selected cytokines. The cytokines tested were those that were present in the platelets and/or the HU3 cells. Unlike most of the previous studies that focused primarily on ADP-induced platelet activation, we employed four platelet agonists, the thromboxane A2 analogue, U46619, collagen, ADP and the peptide, SFLLRNP, that activates the thrombin receptor. Agonist concentrations were adjusted to achieve 50–70% aggregations in order to detect stimulation or inhibition. This contributed to greater variability in agonist-induced platelet aggregations from platelet-to-platelet preparation. PRP samples were preincubated with cytokines for at least 15 min prior to the addition of agonist. The results, in Figure 2, demonstrated that the cytokines had differential effects on the four agonist-induced platelet aggregations. Only SCF, TPO and TGF-b stimulated the platelet aggregation response to all of the agonists. The collagen-induced platelet aggregations were generally stimulated by the cytokines tested to a greater extent than with the other agonists. Due to the variability in sample aggregations results were analysed by a paired Student’s t-test comparing each cytokine-treated sample to its own control sample.

B

35 30 25 20 15

*

*

10 5 0 –5 –10

± SE 5.7 4.2 7.1 5.1 5.8 7.2 7.7 4.5

U46619

7.1 8.1 8.6 5.4 6.0 6.1 8.6 8.4

ADP

Figure 2. Cytokine-induced alteration of agonist-induced platelet aggregations. Control levels of agonist-induced aggregation is represented as zero on graph compared with the corresponding percent change in aggregation induced by each of the 8 cytokines tested. Each point represents the mean of 6–8 different samples with standard errors indicated below the base line. The asterisk (*) represents cytokine-treated samples that aggregated significantly differently than its corresponding control with a P Q 0.05 by a paired Student’s t-test. The agonists employed in (B) were U46619 and ADP and in (A) were collagen and SFLLRNP. Each agonist concentration was adjusted to yield control aggregations of 50–70% as described in the text. (q), IL-1b; (Q), IL-6; (;), IL-7; (<), IL-10; (+), SCF; 0 === ), TNF-a, (q 0 ), TPO; ( ), TGF-b. (q 0

immune processes. The current studies indicated that megakaryocytes and the mature platelet may also contain and release selected cytokine species. These cytokines alone or in conjunction with other cellderived cytokines, may regulate platelet activities in an autocrine and/or paracrine fashion. It must be noted that the detection of mRNA by RT-PCR in cell samples does not allow one to determine quantity of mRNA nor translation of protein product. The quantitation of mRNA and protein product is problematic due to potentially low levels present in these cell preparations and were not pursued at this time. However, ‘‘undetectable’’ quantities of these components (by Western or

408 / Soslau et al.

Northern blot techniques), in small cell samples, may have significant physiological effects as evidenced by altered platelet responsiveness to different agonists by picogram to nanogram quantities of cytokines. The fact that any cytokine mRNA is present in the mature platelet may be highly significant. Cytokine mRNAs are usually tightly regulated, undergoing post-transcriptional regulation28 and are rapidly degraded as signaled by UAUUUA sequences in the 3' untranslated region.17 Their presence in freshly prepared platelets implies a half-life of hours to days and may, therefore, be required to sustain low levels of cytokine synthesis. While these studies did not quantitate the expression of selected genes in HU3 and MBO2 cells, the qualitative results indicated that the HU3 cell line was further along the differentiation pathway toward platelet production than the MBO2 cell line. This was based upon the presence of SCF and cMpl mRNA in HU3 and platelet preparations but not the MBO2 cells. The presence of SCF and cMpl mRNA in platelets implied that the mature megakaryocyte was actively expressing these two genes and that the transcripts were sufficiently stable to detect in platelets. Absence of both gene transcripts in MBO2 cells indicated that this bipotential cell line21 was further removed from the mature megakaryocyte than the HU3 cell line or belonged to a different lineage committed pathway. This was further borne out by the fact that q80% of the HU3 cells were positive (by FACS analysis) for GPIb and IIb/IIIa while the MBO2 cells were negative (unpublished results). FceRI is the tetrameric high affinity IgE receptor, consisting of an a-chain, a b-chain and two g-chain subunits (FceRIabg2 ). The Fc region of IgE is bound by the a-chain, while the b- and g-chains are involved in membrane trafficking and signal transduction.29 In vitro transfection studies have shown expression of the a- and g-chains are sufficient for membrane FceRI expression (FceRIag2 ).30,31 FceRI expression was believed to be a distinguishing characteristic specific for the mast cell/basophil lineage, but expression of a functional receptor has recently been detected on eosinophils from hypereosinophilic parasitized patients, monocytes of atopic dermatitis and normal epidermal Langerhans cells.32–35 Epidermal Langerhans cells have been reported to express a functional high affinity IgE receptor mediating tyrosine phosphorylation and endocytosis.35 PCR analysis of RNA derived from normal Langerhans cells have shown only FceRIa and FceRIg mRNA expression and confirms that trimeric FceRIag2 occurs naturally.35 The expression of mRNA for FceRIg by platelets is not unexpected, as this subunit also forms part of the low affinity IgG receptor FcRIII. Mature platelets express message for the a- and g-chains but not for the

CYTOKINE, Vol. 9, No. 6 (June, 1997: 405–411)

b-chain suggesting that mature platelets express the trimeric form of the IgE receptor. Moreover, the bipotential cell line HU-3 cultured with IL-3 or GM-CSF expresses messages for all FceRI subunits, but when stimulated toward further megakaryocyte differentiation with TPO, expression of FceRIb is no longer detected, while a- and g-chain mRNA is preserved. Expression of FceRIb was the only factor studied that was altered upon culturing HU3 cells for 30 days in TPO. Taken together, it appears that tetrameric FceRI is a characteristic of mast cells and pluripotential stem cells. The mRNA for the b-chain appears to be downregulated with further megakaryocytic differentiation. The expression of mRNAs for the a- and g-chains appears to be a feature of mature platelets and, therefore, must be posttranscriptionally regulated and stabilized in mature platelets. IgE, anti-IgE and allergenic peptides have been shown to alter platelet function, an effect previously presumed to be mediated through the monomeric low-affinity IgE-receptor FceRII.36–39 Our experiments suggest that the stimulation of platelets through ligation of antigen-specific IgE may be mediated through the high-affinity IgE-receptor, and an expanded role for the platelet in host defence and hypersensitivity. Others have shown that platelet activation may be regulated, in part, by IL-2,23 IL-6,18 GM-CSF and cMpl ligand (TPO).25–27 Our current studies indicated, for the first time, that platelet aggregation induced by four different agonists displayed differential sensitivities to added cytokines at a single concentration, presumed to represent physiological levels. Future studies with multiple cytokine concentrations are required to generate dose–response curves to evaluate the potential physiological effects on platelet function more acurately. Also, future studies with anti-cytokine antibodies should be conducted to demonstrate the specificity of action of the cytokine. Since the cytokine-altered levels of aggregation were small with many of the samples, one can only conclude that trends are evident. However, all agonists were significantly stimulated by TPO except U46619 (P = 0.075). The data is most conclusive with collagen-induced platelet aggregations. The different levels of cytokine-altered platelet aggregation with each agonist presumably reflects the fact that each agonist functions through a unique receptor–G-protein-coupled pathway. It is surprising that all of the cytokines tested altered most of the agonist-induced platelet aggregations. The duplication or opposing effects of some cytokines may reflect the need to differentially modulate platelet activation depending upon the type of localized injury and the agonist present within the microenvironment. It is interesting that collagen-induced platelet aggregations were stimulated to the greatest extent by most

Cytokines in platelets and megakaryocytes / 409

of the tested cytokines. This could play a significant role in platelet involvement in atherogenesis since collagen becomes exposed at sites of injury and chemokines and cytokines accumulate in regions of atherosclerotic plaques along with the generation of thrombin. It is also possible that combinations of cytokines may have opposing, additive or synergistic effects on platelet activation. This issue was not addressed in these studies.

MATERIALS AND METHODS The continuous megakaryocyte-like cell lines were cultured, in suspension, exactly as described previously.21 Peripheral blood was drawn from healthy volunteers who signed consent forms in accordance with the University Human Studies Committee and layered over Ficoll-hypaque to purify peripheral blood mononuclear cells (PBMC).40 Mononuclear cells thus obtained were viably frozen at −70°C in 90% fetal calf serum and 10% DMSO and stored in liquid nitrogen until use. PBMC were cultured in RPMI 1640 with 10 mM HEPES and 10% fetal calf serum (Fisher Scientific, Pittsburgh, PA). Cytokine mRNA expression by PBMC was induced by stimulation with anti-CD3 (OKT3 1 mg/ml) and PMA (10 ng/ml) for 6 h. Platelets, for RNA preparations only, were prepared from single units of blood that were therapeutically drawn from two patients with polycythemia vera (PV) and one kidney transplant-induced secondary PV. The advantage of employing samples from these individuals is the 3–4-fold increase of platelets per microlitre compared to controls. The patterns were the same for the two PV samples and the kidney transplant-induced secondary PV sample. The latter sample presumably represents a transient non-haematologi-

TABLE 2.

Oligonucleotide primers used to detect selected mRNA species by PCR Forward primer 5'

IL-1a IL-1b IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-9 IL-10 IL-12 IL-13 GM-CSF SCF TNF-a TGF-b FCEa FCEb FCEg NF-E2 cMpl GAPDH b-actin

cal disease. Therefore, it is likely that the detection of particular mRNA species in these cells are representative of corresponding expression in truly normal platelets. Units of blood were centrifuged at 400 × g for 7 min yielding platelet-rich plasma (PRP). Platelets were pelleted from the PRP at 1000 × g for 10 min and resuspended in a few ml of 0.1 M sodium citrate, pH 6.0. The platelets were then purified, as previously described, by gradient centrifugation through a 25–35% sucrose gradient.41 Platelets were free of PBMC contamination as monitored by light microscopy and as indicated by the absence of 15 out of 20 PBMC mRNA species detected by RT-PCR (see Results section) in each platelet preparation. Total cellular RNA (tcRNA) was isolated from samples by the guanidinium thiocyanate method.42 Reverse transcription (RT) and the polymerase chain reaction (PCR) were performed as described.43 To assay for mRNA expression 100 ng of tcRNA was reverse transcribed to cDNA in a 30 ml reaction volume and 1/6 volume of the RT reaction was amplified in each PCR reaction. Actin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were amplified for 25 cycles; all other reactions were 30 cycles. Specific cytokine oligonucleotide PCR primers (Table 2) were designed based on published cDNA sequences (Genbank, Los Alamos National Laboratory, Los Alamos, NM). The cMpl and NF-E2 primers were a gift from Dr Si Lok, ZymoGenetics, Seattle, WA. Whenever possible, amplified mRNA sequences spanned at least one intron in the genomic sequence to ensure any trace contamination with genomic DNA which would not yield a PCR product of the predicted size. The PCR products were electrophoresed in 2% agarose, ethidium bromide stained, visualized by UV transillumination and photographed. The size of the PCR product generated was compared to the expected product size (based on the known cDNA sequence) and was determined by electrophoretic migration relative to a

ATG GCC AAA GTT CCA GAC ATG TTTG ATG GCA GAA GTA CCT AAG CTC GC ATG TAC AGG ATG CAA CTC CTG TCTT GGG AAG ACC AAG ACA TTC TG TGC CTC CAA GAA CAC AAC TG CGT TTC AGA GCC ATG AGG ATG CTTC TCA ATG AGG AGA CTT GCC TG GCG CGG GAA GCA GGT GCC CTG CCC TGC TCC TGT GCT CCG AAA TTT GGT TCT AGG CCG GG CAT TGA GGT CAT GGT GGA TGC CG CTG CCC GTC TTC AGC CTA GCCG AGC ATG TGA ATG CCA TCCA G GCG CTG CGG GAA GCA GGG ATG AGC ACT GAA AGC ATG ATC CGG GAA AGC CCT CAA TTT CCC CTC CACG GTG ATG GAG GGC CAG CCCT CGG CCT CAT CCC CAC CAC TG CAC GGG CCT GAG CAC CAG GA ATG TCC CCG TGT CCT CCC CAG CAGA GCA CAC TAC AGG AGA CTG AGG CAT AGA AGG TGG TGA AGC AGG CGT CG GGG TCA GAA GCA TTC CTA TG

Reverse primer 5' GGT TTT CCA GTA TCT GAA AGT CAGT ACA CAA ATT GCA TGG TGA AGT CAG TT GTC ATG GTT GAG ATG ATG CTT TGAC TCA AAG TCG TCT GTT GAG CC AAC GTA CTC TGG TTG GCT TC TCT TTC CAC AGT ACC CCC TTG CACA GAT GAG TTG TCA TGT CCT GC CAT GGT CTG CGG GAG GCGC TGC CTG CCG TGG TTT GGT TGC GAG TAC AGG GGC ATG ATA TC CCC GCA CGC TAA TGC TGG CA CGA GGC CCC AGG ACC CCAG ATA GTC TGG GTT GCA CAG GA TGA CTT GGC AAA ACA TCC ATC CCG GCA ATG ATC CCA AAG TAG ACC TGC CC CCC GCG TGC TAA TGG TGG AAAC CAG CTG CCA CAC TTT GCC CG TCC TTT GAG TTC TTC CCC AGC TCCA TTG GGA ATG GGG AAG AGA AGA AGGG TCA GTC TGT GGC CTC CAT CTT GGT CCC TCA AGGCTGCTG CCA ATA GCT TAG TGGT CCT TGG AGG CCA TGT GGG CC GGT CTC AAA CAT GAT CTG GG

410 / Soslau et al.

commercial molecular weight DNA standard (bacteriophage FX174-HaeIII restriction fragments). The PCR products obtained from tcRNA were identical in size to those obtained by PCR amplification of the cDNA contained in plasmids. The authenticity of the products was further checked with restriction enzyme digestion for appropriate cleavage products. An additional RT reaction in which all reagents were present, but RNA was omitted, served as a negative control for PCR contamination. Platelet aggregations were conducted with a ChronologLumi aggregometer (Havertown, PA) as previously described44 with PRP derived from healthy donors who signed consent forms as above. None of these aggregation studies were conducted with PV patient platelets. Agonist concentrations were varied such that the level of platelet aggregation remained in the 50–70% range. The concentration of agonists were: 4.2–8.4 mg collagen/ml; 0.5–2 mM U46619; 1–10 mM ADP and, 3.8–7.6 mM SFLLRNP (a thrombin receptor activating peptide). To determine the effect of cytokines on agonist-induced platelet aggregation PRP was incubated with the following cytokines for 15–35 min prior to the addition of agonist: 10 ng IL-1b/ml; 20 ng IL-6/ml; 10 ng IL-7/ml; 10 ng IL-10/ml; 25 ng SCF/ml; 50 ng TNF-a/ml; 100 U TPO/ml (a gift from Dr Si Lok) and; 5 ng TGF-b/ml. All of the cytokines employed were human recombinant species obtained from R & D systems (Minneapolis, MN). To minimize total time of analysis with an individual platelet preparation each agonist was studied with only four cytokines at a time. Aggregations were followed for 3–4 min depending upon the agonist and when the reaction plateaued. Due to the expected variability of responses to agonists, plus or minus the cytokine, with platelet preparations from different individuals, a separate control was run for each cytokine-treated sample. Therefore, results with each cytokine represents a separate experiment and cannot be statistically evaluated by analysis of variance. The experiments were repeated 6–8 times with different platelet preparations and the data analysed by a paired Student’s t-test. A P value Q0.05 was taken as significant.

NOTE ADDED IN PROOF Our finding of IL-7 mRNA in platelets is corroborated by the recent report of IL-7 secreted from activated platelets45 and may indicate that platelets synthesize IL-7.

Acknowledgements We would like to thank Dennis Rudderow and Patrick Alcasid for their outstanding technical assistance.

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