Stem Cells, Vol. 14, No. 4, 439-444,
July 1996
© 1996 AlphaMed Press
Thrombopoietin Modulates Platelet Activation in Vitro through Protein-Tyrosine Phosphorylation
Yoshitsugu Kubotaa,
Takeshi Araia,
Terukazu Tanakab,
Genji Yamaokaa,
Hiroyuki Kiuchia,
Tatsushi Kajikawaa,
Koichi Kawanishia,
Hiroaki Ohnishib,
Masahiro Yamaguchib,
Jiro Takaharab,
Shozo Irinoc
a Department of Clinical Laboratory Medicine and
b First Department of Internal Medicine, Kagawa Medical School, Kagawa, Japan;
c Kagawa Medical School, Kagawa, Japan
Key Words. Thrombopoietin • Platelet aggregation • ADP • Genistein • Tyrosine phosphorylation • c-Mpl • Cytokine receptor
Dr. Yoshitsugu Kubota, Department of Clinical Laboratory Medicine, Kagawa Medical School, 1750-1, Miki-cho, Kita-gun, Kagawa, 761-07, Japan.
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Abstract
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To determine the roles of thrombopoietin (TPO) in platelet function in vitro, we examined the effects of TPO on platelet aggregation. Although several proteins in platelets were tyrosine-phosphorylated by TPO treatment, TPO alone was unable to induce platelet aggregation. However, the secondary wave of platelet aggregation induced by adenosine diphosphate (ADP) was enhanced by TPO in a dose-dependent manner. TPO in conjunction with ADP augmented tyrosine phosphorylation of platelet proteins, including tyrosine-phosphorylated proteins induced by TPO alone. Genistein inhibited protein-tyrosine phosphorylation in platelets induced by TPO with ADP and suppressed TPOenhanced platelet aggregation. Moreover, tyrosine phosphorylation of MAP-kinases induced by TPO alone and TPO with ADP was consistent with TPO-enhanced platelet aggregation. These findings in the present study suggest that signal transduction involved in TPO-enhanced platelet aggregation is mediated in part by tyrosine-phosphorylated proteins, including MAP-kinases, in platelets through TPO-stimulated c-Mpl, TPO receptor.
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Introduction
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Thrombopoietin (TPO) is an important cytokine which regulates megakaryocytopoiesis and thrombopoiesis [1 –5]. Recently, the gene encoding TPO has been cloned by five independent research groups and mapped on human chromosome 3q27 [1–5]. TPO stimulates megakaryocyte colony formation and increases megakaryocyte size, polyploidization and expression of megakaryocytic differentiation markers in vitro [1, 3, 6–8]. TPO stimulates platelet production in vivo by expanding megakaryocyte progenitor cells and by increasing in megakaryocytic polyploidization [2, 6, 8].
The proto-oncogene product c-Mpl, which is a cellular homolog of v-Mpl, is the receptor for TPO [1–3]. Although c-Mpl is a member of the class I cytokine receptor superfamily and possesses no consensus sequence of tyrosine kinase domain, binding of TPO to c-Mpl leads to tyrosine phosphorylation of a set of proteins in the cells transfected with a cloned c-mpl gene [9]. Therefore, TPO-induced tyrosine phosphorylation of cellular proteins appears to play an important role in c-Mpl-mediated signal transduction involved in biological functions of the cells in the megakaryocytic lineage. However, the precise roles of signaling molecules, including tyrosine-phosphorylated proteins, in c-Mpl-mediated signal transduction pathways are still unknown. c-Mpl is expressed in CD34+ hematopoietic pro-genitor cells, megakaryocytes and platelets [10]. Although TPO is proved to stimulate platelet production, the role of TPO in platelet function remains to be elucidated. We report here that TPO-enhanced platelet aggregation induced by adenosine diphosphate (ADP), and that genistein, a specific inhibitor of tyrosine kinases, interfered with TPO-enhanced platelet aggregation by means of suppression of TPO-induced protein-tyrosine phosphorylation in human platelets.
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Materials and Methods
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Reagents
Genistein was purchased from Wako Co. (Tokyo, Japan). Human recombinant TPO was kindly provided by Kirin Brewery Co. (Tokyo, Japan). Other chemicals were purchased from commercial sources. Genistein was used after dilution with dimethylsulphoxide (DMSO).
Isolation of Human Platelets
Human blood was drawn from healthy volunteers and collected into a 0.1 volume of 3.8% citrate. Platelet rich plasma (PRP) was prepared by centrifuging the samples at 200 g for 10 min at room temperature (RT). After PRP was transferred to another tube, 0.5 µM prostaglandin E, (Sigma; St Louis, MO) was added to PRP, and it was centrifuged at 1,000 g for 10 min at RT to separate platelets from platelet poor plasma (PPP). The number of platelets in PRP was adjusted to 2.5 x 105/µl by dilution with PPP, and platelets in PRP were subjected to monitoring platelet aggregation. Platelets were gently resuspended in HEPES-Tyrode's buffer (pH 7.4). After centrifugation, the platelets were finally suspended in a small volume of the same buffer prior to their treatment with TPO and chemicals.
Platelet Aggregation
Platelets in PRP were prepared as described above. PRP was transferred into an aggregometer cuvette and incubated for 2 min at 37°C with stirring before addition of TPO and/or chemicals. After incubation with TPO at various concentrations at 37°C for 2 min, ADP was added to the samples. Then platelet aggregation was monitored at 37°C stirring at 1000 rpm in the lumiaggregometer (NBS Hema Tracer, MC Medical Inc.; Tokyo, Japan) using PPP as a reference. In the experiment using genistein, platelets were preincubated for 10 min at 37°C before TPO treatment.
Immunoprecipitation and Immunoblot Analysis
Isolated platelets were stimulated with 100 ng/ml TPO for 4 min at 37°C and with 100 ng/ml TPO for 3 min followed by incubation for 1 min after the addition of 2 µM ADP. In the experiments using genistein, it was added to the samples 10 min before TPO treatment. The reactions were terminated by adding the sample buffer to the samples followed by boiling for 3 min. Proteins were separated by using 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Micron Separations Inc.; Westboro, MA). Immunoblot analysis using antiphosphotyrosine monoclonal antibody (
PY mAb; Sigma) was performed as described previously [11]. Protein bands were visualized by using an enhanced chemiluminescence system (Amersham; Backinghamshire, England).
Platelets were lysed with the lysis buffer containing 1% Triton X-100 [11] after treatment with TPO, ADP and genistein as described above. Then the cell lysates were immunoprecipitated with anti-p44MAPI-kinase (ERK1) PcAb (Santa Cruz Biotechnology Inc.; Santa Cruz, CA). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with PY mAb or ERK1 PcAb.
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Results
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Effects of TPO on Platelet Aggregation in PRP
Treatment with TPO alone at various concentrations (0.01-200 ng/ml) was unable to induce platelet aggregation (data not shown). Therefore, we have examined whether TPO modulates platelet function in conjunction with stimuli which induce platelet aggregation. As shown in Fig. 1
, TPO enhanced the secondary wave of platelet aggregation induced by ADP in a dose-dependent manner. At a concentration of less than 0.01 ng/ml, TPO was unable to enhance ADP-induced platelet aggregation and 0.1 ng/ml TPO slightly enhanced it (data not shown). Enhancement of the secondary wave of platelet aggregation by TPO was clearly observed at more than 1 ng/ml TPO, which stimulates proliferation and differentiation of megakaryocytic progenitor cells [3, 7]. The maximal stimulating effect of TPO on ADP-induced platelet aggregation was obtained by using 10 ng/ml TPO.
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Figure 1. Effects of TPO concentrations on human platelet aggregation induced by 2 µM ADP. Human platelets in PRP were prepared as described under Materials and Methods. The platelets were incubated with indicated concentrations of TPO for 2 min at 37°C. After the addition of 2 µM ADP, platelet aggregation was monitored in the lumiaggregometer.
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Inhibition of TPO-Enhanced Platelet Aggregation by Genistein
It is known that genistein specifically inhibits protein tyrosine kinases (PTKs) and scarcely inhibits the serine- and threonine-specific kinases such as cAMP-dependent protein kinase, protein kinase C and phosphorylase kinase (IC50 > 500 µM). Thus, to disclose the role of protein-tyrosine phosphorylation in signal transduction pathways implicated in TPO-enhanced platelet aggregation, we examined whether genistein inhibited platelet aggregation enhanced by TPO treatment (Fig. 2). Genistein did not affect platelet aggregation induced by ADP alone. However, the percentage of maximal platelet aggregation induced by TPO in conjunction with ADP was decreased in the presence of genistein. Genistein inhibited the aggregation in a dose-dependent manner. The same volume of DMSO used as control did not affect platelet aggregation induced by TPO alone or TPO with ADP (data not shown).
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Figure 2. Inhibitory effect of genistein on TPO-enhanced platelet aggregation induced by 2 µM ADP. Human platelets in PRP were incubated with indicated concentrations of genistein for 10 min at 37°C. Then the platelets were stimulated with 2 µM ADP after treatment with 100 ng/ml of TPO for 2 min. Platelet aggregation was monitored in the lumiaggregometer.
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Effect of Genistein on TPO-Induced Protein-Tyrosine Phosphorylation
The inhibitory effect of genistein on TPO-enhanced platelet aggregation indicates that proteintyrosine phosphorylation induced by TPO treatment plays a crucial role in transducing the signals implicated in platelet aggregation. To determine whether TPO treatment induces tyrosine phosphorylation of proteins in platelets and whether genistein inhibits protein-tyrosine phosphorylation induced by TPO, tyrosine-phosphorylated proteins in platelets were analysed by immunoblotting using PY mAb (Fig. 3A). Treatment with TPO alone was able to induce tyrosine phosphorylation of proteins in platelets with molecular weight (mw) 92, 66, 46, 42 and 36 kDa, in spite of inability of TPO by itself to induce platelet aggregation. Although four of these tyrosine-phosphorylated proteins (mw 92, 66, 46 and 42 kDa) were also detected in ADP-treated platelets after longer exposure to the ECL® film (Amersham), the protein bands were fainter compared to those in TPO-treated platelets. After treatment with TPO and ADP, three proteins with mw 46, 42 and 36 kDa, which were detected to be tyrosine-phosphorylated in the lysates of TPO-treated platelets by immunoblotting using PY mAb, were more intensely tyrosine-phosphorylated. Moreover, tyrosine phosphorylation of a protein with mw 60 kDa was newly enhanced, although the difference in tyrosine phosphorylation of this protein between the platelets treated with TPO alone and those treated with TPO in conjunction with ADP was clearly detected after shorter exposure to the ECL® film (data not shown). Genistein suppressed tyrosine phosphorylation of platelet proteins with mw 92, 46, 42 and 36 kDa after treatment with TPO alone and TPO in conjunction with ADP.
TPO-Induced Tyrosine Phosphorylation of MAP-Kinases (ERKs) in Platelets
Two tyrosine-phosphorylated proteins (46 and 42 kDa) induced by TPO, and TPO in conjunction with ADP, have the molecular weights similar to those of MAP-kinases, which were found to be tyrosine-phosphorylated in hematopoietic cells expressing high levels of c-Mpl in response to TPO [12]. Therefore, to determine whether they are identical to MAP-kinases, we examined the effects of TPO, ADP and genistein on tyrosine phosphorylation of MAP-kinases in platelets. As shown in Fig. 3B
, tyrosine phosphorylation of ERK1 with mw 44-46 kDa was detected in ERK1 immunoprecipitates from the lysates of TPO-treated platelets, although its phosphorylation was not induced by treatment with ADP alone. After treatment with TPO and ADP, tyrosine phosphorylation of p42MAP2-kinase (ERK2) was detected in addition to ERK1 phosphorylation. Genistein suppressed phosphorylation of MAP-kinases induced by TPO and TPO in conjunction with ADP. The same amounts of ERK1 and ERK2 were immunoprecipitated from all samples because ERK1 PcAb used in the present study reacts with both ERK1 and ERK2. Besides, 52 kDa but not 46 kDa species of Shc proteins proved to predominate in platelets [13]. Taken together with these findings, the predominant tyrosine-phosphorylated proteins with mw 46 and 42 kDa in platelets treated with TPO and ADP are likely to be MAP-kinases.
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Discussion
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Several lines of evidence that aggregating agents cause a rapid increase in cellular protein-tyrosine phosphorylation, and that inhibitors of PTKs neutralized some actions of the agents, including platelet aggregation and secretion, have indicated that protein-tyrosine phosphorylation plays an important role in signal transduction in activated platelets [14–16]. G-CSF receptor and stem cell factor (SCF) receptor, c-kit, were recently identified on platelets, and activation of these receptors by ligand binding proved to modulate platelet aggregation induced by ADP and epinephrine [17, 18]. These findings suggest that hematopoietic growth factors have pleiotropic effects on platelet function, and that proteintyrosine phosphorylation is essential to transduction of the platelet aggregation signals from c-kit because c-kit per se has protein-tyrosine kinase activity, and the increase in its activity is required to initiate the signal transduction from c-kit.
In the present study, we demonstrated that TPO enhanced the secondary wave of platelet aggregation induced by ADP, and that genistein inhibited TPO-enhanced platelet aggregation. Because the inhibitory effect of genistein on TPO-enhanced platelet aggregation correlated with suppression of protein-tyrosine phosphorylation in platelets induced by TPO in conjunction with ADP, tyrosine-phosphorylated proteins induced by c-Mpl activation are likely to be involved in signal transduction pathways in TPO-enhanced platelet aggregation. These tyrosine-phosphorylated proteins, however, have not yet been identified and their function is still unknown. Recently, Drachman et al. [9] reported that proteins with mw 45, 52, 65, 75, 95 (doublet), 115-125 and 140 kDa were phosphorylated on tyrosine residues in BaF3 murine hematopoietic cells transfected with a cloned c-mpl gene in response to TPO, and that the proteins with mw 52, 95 (doublet) and 125 kDa corresponded to Shc, c-Mpl per se and Jak2, respectively. Other proteins, including Tyk2 (120 kDa), Cbl (120 kDa), Vav (93 kDa), PI3K (85 kDa), Shc (46 kDa) and MAP-kinase (44 kDa) were also found to be tyrosine-phosphorylated in hematopoietic cells expressing high levels of c-Mpl in response to TPO [12]. Besides, it has been demonstrated that treatment with TPO induced prominent increases in tyrosine phosphorylation of proteins with mw 130, 95 and 85 kDa and weak tyrosine phosphorylation of other minor proteins in human blood platelets, and that the proteins with mw 130 and 52 kDa were identical to Jak2 and Shc, respectively [19]. Taken together with these findings, a human platelet protein with mw 92 kDa shown to be phosphorylated on tyrosine residues in the present study may be c-Mpl. The findings in the present study indicate that tyrosine-phosphorylated proteins with mw 46 and 42 kDa are likely to be MAP-kinases, but not Shc. Therefore, MAP-kinases may play an important role in signal transduction implicated in TPO-enhanced platelet aggregation because tyrosine phosphorylation of MAP-kinases is consistent with the ability of TPO to enhance ADP-induced platelet aggregation. The difference in the TPO-induced protein-tyrosine phosphorylation pattern between c-Mpl-transfected BaF3 cells and human platelets treated with TPO may be attributed to differences in the cell origin and PY antibodies used for immunoblot analysis.
c-Mpl activation by TPO-binding induces tyrosine phosphorylation of a set of proteins in the cells transfected with c-Mpl which does not encode tyrosine kinase domains. Unidentified nonreceptor tyrosine kinases, therefore, should play a crucial role in c-Mpl-mediated signaling transduction. Recently, Jak2 and Tyk2 were found to be tyrosine-phosphorylated and activated in human platelets and hematopoietic cells transfected with the c-Mpl gene [12, 13]. Although the amount of both protein-tyrosine kinases is low in platelets, they may function in c-Mpl-mediated signal transduction pathways implicated in TPO-enhanced platelet aggregation. The ability of TPO to enhance ADP-induced platelet aggregation indicates that the glycoprotein IIb/IIIa (GPIIb/IIIa) complex is activated by treatment with TPO and ADP, and acts as a receptor for fibrinogen because platelet aggregation is mainly mediated by binding of fibrinogen to the activated GPIIb/IIIa complex. Findik et al. [20] have reported that the GPIIb/IIIa complex is tyrosine-phosphorylated and activated by pp6Oc-src. Furthermore, platelets contain abundant pp6Oc-src [21], and the proteins with mw 60 and 66 kDa in human platelets were found to be tyrosine-phosphorylated in response to TPO, as shown in the present study. Taken together with these findings, modulation of pp6Oc-src kinase activity in platelets induced by TPO treatment may lead to tyrosine phosphorylation of the GPIIb/IIIa complex followed by its activation, and ADP may enhance pp6Oc-src kinase activity stimulated by TPO. However, the mechanisms of cytokine-enhanced platelet aggregation have not yet been well-defined. Further experiments are required to disclose signal transduction pathways implicated in TPO-enhanced platelet aggregation and the role of each intracellular signal transduction molecule, including tyrosine-phosphorylated proteins induced by TPO.
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Acknowledgments
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We thank Kirin Brewery Co. (Tokyo, Japan) for providing recombinant human thrombopoietin and Ms. Mariko Takeuchi for assistance with immunoblot analysis.
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Footnotes
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Provisionally accepted December 12, 1995.
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References
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Received November 11, 1995;
accepted for publication April 8, 1996.
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Abstract
Introduction
