15-deoxy-Δ^12,14-PGJ₂ enhances platelet production from megakaryocytes

Platelets initiate clot formation and have important roles in both innate and adaptive immunity.^1,2  Loss of platelets either by their destruction in the periphery or their reduced production can occur in diseases such as immune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, human immunodeficiency virus (HIV) infection, aplastic anemia, and acute respiratory distress syndrome, and in approximately 1% to 5% of people receiving heparin therapy.³  In addition, cancer chemotherapy and radiation therapy are 2 of the most common causes of thrombocytopenia. Currently, platelet transfusions are the “gold standard” for treating the life-threatening complications of thrombocytopenia. However, platelet transfusions increase the risk of inflammation, disease transmission, and are costly and not always readily available.^4,5  A catastrophic event such as mass radiation exposure would leave many victims without treatment. Currently, recombinant human interleukin-11 (IL-11), the only clinically approved drug for treating thrombocytopenia, is used as an alternative to platelet transfusions to modestly raise platelet counts.⁶  Therefore, there remains a need for more efficacious and readily available treatments to increase platelet number.

Platelets are derived from megakaryocytes, which reside in the bone marrow.⁷  During megakaryocyte maturation, the polyploid cell undergoes a complex process of cytoskeletal rearrangement, followed by proplatelet elongation, and the release of cytoplasmic fragments as circulating platelets.^8,9  Proteomic studies have revealed that both megakaryocytes and platelets contain proteins of unknown function. Our laboratory reported that the ligand-activated transcription factor, peroxisome proliferator–activated receptor gamma (PPARγ), is present in both megakaryocytes and platelets.¹⁰  PPARγ functions as a heterodimer with the retinoid X receptor (RXR) to regulate adipogenesis, glucose metabolism, and inflammation.^11,12  We have also shown that the PPARγ ligands rosiglitazone and 15d-PGJ₂ dampen thrombin-induced human platelet activation and aggregation.¹³  Importantly, we recently determined that PPARγ is also found in platelet microparticles released during activation.¹⁴  These findings prompted us to study the effects of PPARγ ligands on megakaryocytes.

PGJ₂ and 15d-PGJ₂ are natural PPARγ ligands that are biologically active metabolites of PGD₂.^15,16  In addition to binding with high affinity to PPARγ, both PGJ₂ and 15d-PGJ₂ possess an electrophilic α,β-unsaturated carbonyl group in the cyclopentanone ring that reacts covalently with certain nucleophiles in some proteins.^17,18  This action accounts for many of the PPARγ-independent activities of J-type prostaglandins, which include the potentiation of apoptosis, reorganization of cytoskeletal proteins, and generation of reactive oxygen species (ROS). In this study, we determined the effect of key synthetic and natural PPARγ ligands on megakaryocyte function using human and mouse models of thrombopoiesis. We found that the electrophilic prostaglandin, 15d-PGJ₂, enhances platelet formation from megakaryocytes in vitro and in vivo.

15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) and rosiglitazone were purchased from Biomol (Plymouth Meeting, PA); 15-deoxy-Δ^12,14-PG2₂ (15d-PGD₂), PGJ₂, 9,10 dihydro-15d-PGJ₂ (CAY10410), PGE₂, PGI₂, PGF_2α, and GW9662 were purchased from Cayman Chemical (Ann Arbor, MI); N-acetylcysteine (NAC), glutathione reduced ethyl ester (GSH-EE), and fibrinogen were all purchased from Sigma-Aldrich (St Louis, MO); 5-(and-6-)-carboxy-2′,7′-dicholorodihydrofluorescein diacetate (carboxy-H₂DCFDA) and MitoSOX Red were purchased from Invitrogen (Carlsbad, CA); collagen from Chrono-log Corporation (Havertown, PA); CD61-FITC (GPIIIa), CD41-FITC (GPIIb), CD61-PE, and annexin V-FITC were purchased from BD Biosciences (San Jose, CA); GPIbβ and GPV were purchased from Emfret Analytics (Wurtzburg, Germany); recombinant human thrombopoietin (rhTPO) was purchased from R&D Systems (Minneapolis, MN); BIT9500 was purchased from StemCell Technologies (Vancouver, BC); and total actin (CP-01) was from Oncogene (Cambridge, MA).

Meg-01 and M07e cells were purchased from ATCC (Rockville, MD), and Dami cells were a generous gift from Dr Patricia J. Simpson-Haidaris (University of Rochester). All cells were cultured in RPMI-1640 tissue culture medium (Invitrogen) supplemented with 5% fetal bovine serum (FBS; Invitrogen), 10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; Sigma-Aldrich), 2 mM l-glutamine (Invitrogen), 4.5 g/L glucose (Invitrogen), and 50 μg/mL gentamicin (Invitrogen). M07e cells were also supplemented with 100 ng/mL of granulocyte monocyte–colony stimulating factor (GM-CSF, R&D Systems). All treatments were performed in normal growth media. Dimethyl sulfoxide (DMSO) was used as a vehicle (control) in the following experiments. Animal protocols used in this study were approved by the University of Rochester Committee on Animal Resources.

Bone marrow was isolated from the femora of male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and plated at a concentration of 10⁶ cells per well in 12-well plates in Iscove modified Dulbecco media (IMDM; Invitrogen) with 20% BIT 9500 (bovine serum albumin [BSA], insulin, transferrin) and supplemented with 100 ng/mL of rhTPO. Cells were cultured for 5 days, after which 15d-PGJ₂ (10 μM) was added to the culture medium for 24 hours. On the sixth day, cells were examined by phase-contrast microscopy using an inverted Olympus IX81 microscope (Olympus, Center Valley, PA), and images were captured using SPOT RT software (Diagnostic Instruments, Sterling Heights, MI). Megakaryocytes were identified by preincubating with anti–mouse CD16/32 (FcγR-blocking antibody) and then incubating with phycoerythrin (PE)-conjugated hamster anti–mouse CD41 antibody.

Human CD34+ cord blood cells were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Cells were plated at 2.5 × 10⁵ cells per well in a 12-well plate and cultured in serum-free medium as previously described¹⁹  and supplemented with 100 ng/mL rhTPO. After 14 days in culture, megakaryocytes were identified by staining with a CD61-FITC antibody and analyzed on a BD Biosciences FACSCalibur flow cytometer. Data were analyzed using FlowJo software (TreeStar, Ashland, OR). Cell images were captured using an Olympus IX81 inverted microscope.

Platelets were isolated from megakaryocytes by centrifugation at 150g for 10 minutes. Supernatants underwent sequential centrifugation (500g for 10 minutes and 1000g for 10 minutes). The remaining platelet pellet was fixed then permeabilized with a CD61 or CD41 antibody. 7-AAD (BD Pharmingen) was added for 10 minutes before analyzing the cells by flow cytometry. Platelets were identified using 3 well-described parameters^7,20-22 : size (using normal human platelets as a control), presence of CD61 or CD41, and lack of DNA as determined by the absence of 7-AAD staining. Ten thousand platelet events were collected, and platelets were quantified by rate (platelet events/second).²³ 

Whole blood was obtained under University of Rochester Institutional Review Board approval from male and female donors by venipuncture into a citrate phosphate dextrose adenine solution containing collection bag (Baxter Fenwal, Round Lake, IL) or vacutainer tubes containing buffered citrate sodium (BD Biosciences, Franklin Lakes, NJ). Platelets were then isolated as described.^13,14  On average, 5.5 × 10¹⁰ platelets/unit of blood were obtained, and the platelet purity was greater than 99%. Informed consent was obtained from all donors in accordance with the Declaration of Helsinki.

Platelets were isolated from the Meg-01 cells and human blood. Both normal human platelets and Meg-01–derived platelets were stained with annexin V-FITC then treated with 10 μg/mL of collagen for 15 minutes. Cells were analyzed for collagen-induced annexin V binding and change in size and shape by flow cytometry. Platelets were isolated from primary human megakaryocytes and primary mouse megakaryocytes. Primary human platelets were stained with CD41-FITC, and primary mouse platelets were stained with CD61-PE then treated with 10 μg/mL of collagen. Cells were analyzed for collagen-induced CD61/CD41 up-regulation and change in size and shape using flow cytometry.

Cells were fixed and permeabilized with 0.1% Triton-100 for 1 hour at room temperature (RT). Subsequently, cells were blocked with 1% BSA in phosphate buffered saline (PBS)–Tween 20 (0.1%) for 1 hour at RT, then stained with phalloidin-Alexa Fluor 488 (Invitrogen, 1:200) at RT for 2 hours. Cells were mounted and visualized using an Olympus BX51 light microscope (Olympus, Melville, NY).

Cells were fixed for 4 hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. After fixation, cells were placed in 1% osmium tetroxide in phosphate buffer, dehydrated in ethanol, embedded in Epon/Araldite epoxy resin, and analyzed on a Hitachi H-7100 electron microscope.

Coverslips were coated with 100 μg/mL fibrinogen for 1 hour and blocked with 0.5 mg/mL BSA for 1 hour. Platelets were added to the fibrinogen-coated coverslips in Tyrode buffer and fixed with glutaraldehyde, postfixed with 1% osmium tetroxide, and dehydrated. Platelets were then coated with a gold/palladium film. Images were acquired using the Zeiss-Leo 982 FE-SEM (scanning electron microscope).

Meg-01 cells were plated at a density of 1.6 × 10⁷ cells per well in a 6-well plate and transfected, using lipofectamine LTX (Invitrogen), with a PPRE (peroxisome proliferator response element)–luciferase reporter plasmid containing 3 copies of the ACO-PPRE (a gift from Dr B. Seed, Massachusetts General Hospital, Boston, MA). Cells were then treated with 10 μM 15d-PGJ₂, 9,10 dihydro-15d-PGJ₂, or rosiglitazone 24 hours after transfection. Transfection efficiency was determined by transfecting the cells with green fluorescent protein (GFP) plasmid and determining the percentage of cells that were GFP+ by flow cytometry. All cell treatment groups exhibited similar transfection efficiencies. Luciferase activity was assessed 24 hours after ligand treatment using the promega luciferase assay system, and relative light units (RLUs) were determined using a lumicount microplate luminometer (Packard Instrument, Meriden, CT).

A 19-bp target sequence was selected to knock down PPARγ. PPARγ was cloned downstream of the RNA polymerase III U6 promoter and subcloned into a FG12 lentiviral vector, which expresses GFP under the ubiquitin C promoter. HEK 293 cells expressing this siRNA PPARγ had reduced PPARγ protein levels (> 90%; data not shown). To make the lentivirus, HEK 293 cells were cotransfected with 3 plasmids: the envelope vector (CMV-VSVG), the transfer vector (FG12-shRNAPPAR), and the packaging construct (pCMVΔ89.2 [gag/pol proteins]). Viral supernatants were collected every 8 hours for 2 days and concentrated by ultracentrifugation at 50 000g for 2 hours at 4°C using a Beckman SW 28 rotor. Viral titers were determined by infection of HeLa cells with serial dilutions of the viral stocks. Meg-01 cells were infected at a multiplicity of infection (MOI) of 20. After 48 hours, approximately 56% of the PPARγ siRNA cells were transduced as determined by flow cytometry. The GFP+ cells were then sorted by FACS and grown in RPMI with 5% FBS.

Western blot for PPARγ was performed as previously described.^13,14  Briefly, a rabbit antihuman polyclonal primary antibody against PPARγ (Biomol) was added at a 1:1000 dilution in 5% nonfat milk for 1 hour at room temperature. The secondary Ab (Jackson ImmunoResearch Laboratories) was added at a 1:10 000 dilution for 1 hour in 2.5% nonfat milk. Membranes were visualized with enhanced chemiluminescence (ECL; Pierce, Rockford, IL).

Ten micromoles carboxy-H₂DCFDA was added to cells for 20 minutes at RT. The cells were washed and immediately analyzed by flow cytometry. Five micromoles MitoSOX Red dye in Hanks balanced salt solution (HBSS) containing Mg ²⁺ and Ca ²⁺ was added to the cells for 15 minutes at 37°C. The cells were washed in HBSS and analyzed by flow cytometry.

Cells were fixed in 95% ethanol, treated with RNAse (Sigma-Aldrich) followed by 20 μg/mL propidium iodide (Sigma-Aldrich) to stain DNA, and analyzed by flow cytometry.

Primary mouse bone marrow cells were suspended in IMDM at a concentration of 8 × 10⁶ cells/mL. Two hundred thousand cells were plated in IMDM supplemented with 20% BIT9500, 0.2% 2-mercaptoethanol, 2% glutamax, and 20% cellgro-H₂O (Mediatech, Herndon, VA), and cultured with 50 ng/mL rhTPO, 10 ng/mL IL-3, 20 ng/mL IL-6, 50 ng/mL IL-11 (all Preprotech, Rocky Hill, NJ), and collagen at 37° for 7 days.²⁴  Collagen gels were dehydrated, fixed, and labeled with GPIbβ and GPV, modified by ABC (Vector Labs, Burlingame, CA), and developed in Vector Red (Vector Labs). Meg-CFCs were defined by their ability to generate colonies containing at least 3 megakaryocytes.

Seven- or eight-week-old male C57BL/6 mice (Jackson Laboratories) were either exposed to 5 Gy cesium (Cs¹³⁷) total body irradiation (TBI) using a Model 8114 6000 Curie Shepherd Cs¹³⁷ irradiator (approximately 3200 Curie sealed Cs¹³⁷ source) with a dose rate of 177.9 Gy/min or left unexposed. Mice (n = 4) received intravenous injections of vehicle (8% DMSO) or 15d-PGJ₂ (1 mg/kg) for 4 consecutive days from day 1 (days 1, 2, 3, and 4 after irradiaton). Blood was obtained from the aorta or the orbital sinus. Platelets were counted before radiation exposure and on days 10, 22, and 31 after radiation exposure using the Heska CBC-Diff veterinary analyzer (Fort Collins, CO). Data are expressed as average platelet counts from 2 separate experiments. For the unirradiated mice, platelets were counted on days 4, 10, and 15.

Results are expressed as the mean plus or minus standard deviation (SD). Statistical analysis was performed using a paired, 2-tailed Student t test with P values less than .05 deemed statistically significant. All experiments were repeated at least 3 times unless otherwise stated.

Our initial studies demonstrated that 3 well-described megakaryocytic cell lines (Meg-01, M07e, Dami) all increased platelet production after exposure for 24 hours to 15d-PGJ₂ (Figure 1A). Further studies were completed on Meg-01 cells, as this cell produces platelets similar in structure and function to freshly isolated human platelets.²⁵  Treatment of Meg-01 cells with increasing concentrations of 15d-PGJ₂ caused a dose-dependent increase in platelet production (Figure 1B). However, not every type of prostaglandin enhanced platelet production. For example, PGE₂, PGI_2, and PGF_2α, which are not PPARγ ligands, failed to enhance platelet production (Figure 1B). Platelets derived from Meg-01 cells express the platelet marker CD61 (glycoprotein IIIa, Figure 1C).

We next determined whether the platelets produced by 15d-PGJ₂ treatment were responsive to known activators. In normal human platelets, collagen promotes shape-change and phosphatidylserine becomes highly expressed on the surface of platelets.^26-28  Therefore, annexin V staining was used to distinguish platelets from apoptotic bodies and as a quantitative measure of platelet activation. Meg-01 cells were untreated or treated with 15d-PGJ₂ and the Meg-01–derived platelets were isolated and treated with collagen. After collagen activation, Meg-01–derived platelets changed size and shape, as indicated by a decrease in forward scatter (size) and an increase in side scatter (granularity; Figure 1D). In addition, collagen increased the binding of annexin V in both Meg-01–derived platelets and normal human platelets (Figure 1E). These data indicate that Meg-01–derived platelets and normal human platelets have similar responses to collagen stimulation.

Morphologic changes, characteristic of megakaryopoiesis, in Meg-01 cells were evident within 1 to 2 hours of 15d-PGJ₂ treatment, as revealed by staining for filamentous (f)-actin (Figure 1F-G). Arrows in Figure 1G show actin bundles protruding from the cell membrane. The formation of these protrusions are associated with megakaryopoiesis, as actin is found in proplatelet extensions and is important for the bending and bifurcation of the branches.^8,29  These membrane protrusions were absent in untreated Meg-01 cells (Figure 1F). Meg-01 cells were also analyzed by transmission electron microscopy (TEM). Within 24 hours of 15d-PGJ₂ treatment, Meg-01 cells exhibited structural characteristics consistent with differentiating cells, such as granule formation (g), a horseshoe-shaped nucleus (n), and the elongation of cytoplasmic extensions (c) (Figure 1I). Figure 1I confirms the presence of the cytoplasmic extensions seen with phalloidin staining. These phenotypic changes are absent in untreated cells (Figure 1H).

To evaluate whether 15d-PGJ₂ affects platelet production from normal mouse megakaryocytes, primary mouse bone marrow cells were cultured for 5 days with rhTPO to promote megakaryocyte enrichment and maturation. After culture, bone marrow cells were treated with 15d-PGJ₂ for 24 hours. 15d-PGJ₂ increased the percentage of CD61 + platelets in culture by approximately 14% (Figure 2A). These mouse bone marrow–derived platelets exhibited normal responses to collagen, as they changed shape (decreased in size and increased in granularity) and increased their surface expression of CD41 (GPIIb, Figure 2B,C). Geometric mean fluorescence intensity increased from 9 to 20 after collagen treatment. Mouse bone marrow culture–derived platelets show functionality, as they spread in response to fibrinogen (Figure 2D). 15d-PGJ₂ also induced the appearance of ruffled edges on the megakaryocytes, commonly observed during membrane blebbing of platelets, whereas vehicle-treated megakaryocytes exhibited a smooth surface (Figure 2E).

Cord blood–derived CD34+ cells (99% pure) were cultured for 14 days with rhTPO, at which time more than 90% of the cells expressed CD61 (data not shown). The cultures consisted of a mixed population of both immature and mature megakaryocytes as determined by ploidy observed by Diff-Quik staining (data not shown). These CD61+ cells were then treated with 15d-PGJ₂ (10 μM) for 24 hours and platelet production was assessed by flow cytometry. Treatment with 15d-PGJ₂ doubled the number of platelets generated from these human megakaryocytes as determined by presence of the platelet marker CD61 and the absence of DNA (Figure 3A). Flow cytometric data showed that human culture–derived platelets exhibited normal responses to collagen, as they changed shape (decreased in size and increased in granularity) and increased their surface expression of CD61 (Figure 3B,C). Geometric mean fluorescence intensity increased from 5 to 20 after collagen treatment. In addition, these culture-derived platelets showed functionality, as they spread in response to fibrinogen (Figure 3D). 15d-PGJ₂ also induced proplatelet formation (Figure 3E).

We next investigated whether 15d-PGJ₂ enhanced platelet production was PPARγ-dependent or -independent. All PPARγ ligands tested (15d-PGJ₂, 9,10 dihydro-15d-PGJ₂, and rosiglitazone) activated PPARγ (Figure 4A). Despite this, only the electrophilic prostaglandins (PGJ₂ and 15d-PGJ₂) enhanced Meg-01 platelet production (Figure 4B). Nonelectrophilic thiazolidinedione-type drugs such as rosiglitazone and the nonelectrophilic prostaglandins,9,10 dihydro-15d-PGJ₂ and 15d-PGD₂, failed to enhance platelet production. (Figure 4B). This suggests that the effects of these agents are independent of PPARγ. The involvement of PPARγ in the platelet-generating effects of 15d-PGJ₂ was further analyzed by infecting Meg-01 cells with a PPARγ siRNA lentivirus (Figure 4C). As shown in Figure 4D, knocking down PPARγ protein failed to attenuate the platelet-enhancing effects of 15d-PGJ₂. In addition, the ability of 15d-PGJ₂ to enhance platelet production was not blocked by the small molecule, irreversible PPARγ antagonist, GW9662.³⁰  This further supports that the effects of 15d-PGJ₂ are PPARγ-independent.

15d-PGJ₂ can act independently of PPARγ by modulating cellular redox status.^17,31,32  Subsequent experiments were performed to address whether 15d-PGJ₂ and other prostaglandins alter intracellular ROS levels in megakaryocytes. We used carboxy-H₂DCFDA to detect a broad range of oxidants, including superoxide, peroxynitrate, hydrogen peroxide, and nitric oxide (NO). Flow cytometric analysis (Figure 5A) demonstrates an increase in the percentage of Meg-01 cells that express ROS from 52% to 91% after 1 hour and 97% after 6 hours of 15d-PGJ₂ treatment. The nonelectrophilic compounds, 9,10 dihydro-15d-PGJ₂ and 15d-PGD₂, failed to increase the percentage of cells generating ROS. However, another electrophilic prostaglandin, PGJ₂, also increased the percentage of cells expressing ROS (65% at 1 hour and 76% at 6 hours).

In addition to total intracellular ROS, we next investigated the ability of different prostaglandins to increase mitochondrial superoxide levels using MitoSOX Red. MitoSOX Red is a live-cell permeant indicator that is rapidly targeted to the mitochondria, where it reacts with superoxides and binds to nucleic acids, resulting in fluorescence.³³  Flow cytometric analysis (Figure 5B) demonstrates an increase in the percentage of Meg-01 cells that produce mitochondrial superoxide at 1 hour (43%) and 6 hours (31%) after 15d-PGJ₂ treatment (10 μM). Similar to carboxy-H₂DCFDA analysis, the nonelectrophilic 9,10 dihydro-15d-PGJ₂ and 15d-PGD₂ failed to increase the percentage of cells generating mitochondrial superoxide. However, the electrophilic PGJ₂ also increased the percentage of cells expressing superoxide at 1 hour (33%) and 6 hours (25%). Thus, electrophilic prostaglandins induce ROS in Meg-01 cells.

We extended our Meg-01 studies by examining intracellular ROS production in primary human megakaryocytes in response to 15d-PGJ₂ and 9,10 dihydro-15d-PGJ₂. Flow cytometric analysis (Figure 5C) demonstrates an increase in the per-centage of ROS-positive cells in the 15d-PGJ₂ cultures after carboxy-H₂DCFDA staining and Mitosox Red staining at 1 hour and 6 hours.

The effects of 15d-PGJ₂ on platelet production are partially reversed with pretreatment using thiol antioxidants such as GSH-EE and NAC. Meg-01 cells and primary human megakaryocytes were pretreated with 5 mM of GSH-EE or 1 mM of NAC for 2 hours and extensively washed before 15d-PGJ₂ addition. After 24 hours, cells were stained for CD61 expression and platelet number was evaluated. Flow cytometric data (Figure 5D) reveal that both GSH-EE and NAC attenuate 15d-PGJ₂–induced platelet production by reducing the number of platelets produced to approximately control levels. Collectively, these data suggest that the production of ROS is important for platelet production from megakaryocytes.

To determine the mechanism of increased platelet production, we examined the effect of 15d-PGJ₂ on megakaryocyte colony formation and maturation. Meg-01 cells were treated with 15d-PGJ₂ for 24, 48, or 72 hours. Although DNA content was unchanged at 24 and 48 hours (data not shown), histograms show that 15d-PGJ₂ addition increased DNA content in Meg-01 cells by 72 hours (Figure 6A). In addition, DNA histograms show that 15d-PGJ₂ increased DNA content in primary mouse megakaryocytes by 24 hours (Figure 6B). To determine the effect of 15d-PGJ₂ on early-stage thrombopoiesis, primary mouse bone marrow cells were harvested from mice that were injected with 1 mg/kg of 15d-PGJ₂ or 1 mg/kg of vehicle for 2 consecutive days. After 7 days of culture, Meg-CFCs were identified by labeling with GPV and GPIbβ. 15d-PGJ₂ did not influence megakaryocyte colony number or colony size (Figure 6C), however, 15d-PGJ₂ significantly augmented the percentage of megakaryocytes producing proplatelets (Figure 6D,E).

Mice that received intravenous injections of 15d-PGJ₂ for 4 consecutive days had higher platelet numbers compared with mice that received vehicle (Figure 7A). To evaluate the effect of 15d-PGJ₂ on platelet counts in a model of thrombocytopenia, we exposed mice to Cs¹³⁷ (5 Gy). Mice exhibited a platelet nadir at approximately 10 days after irradiation (approximately 250 000/μL). The mice that received intravenous injections of 15d-PGJ₂ for 4 consecutive days exhibited accelerated platelet recovery on days 22 and 31 compared with the vehicle mice (Figure 7B). Platelet numbers completely recovered (approximately 800 000 μL) by day 31 in the 15d-PGJ₂–treated mice. There were no differences in weight between vehicle-treated and 15d-PGJ₂–treated mice (data not shown).

Thrombocytopenia causes significant morbidity and mortality, and few therapies are useful except for transfusion. Understanding the molecular mechanisms that underlie both megakaryocyte maturation and platelet release will provide insight into new ways to enhance platelet production from their precursor cells, the megakaryocytes. The results presented herein demonstrate that 15d-PGJ₂ enhances platelet production from both mouse and human megakaryocytes. We show that ROS generation is critical for platelet production and that small electrophilic prostaglandins, such as 15d-PGJ₂, which enhance ROS, may provide therapeutic benefit in the treatment of thrombocytopenia.

Meg-01 cells undergo differentiation reflective of megakaryocyte maturation and platelet release.²⁵  Our data indicate that 15d-PGJ₂ has a strong platelet production–enhancing effect in this cell line. When Meg-01 cells were treated with 15d-PGJ₂, they lost their smooth rounded appearance, exhibited significant cytoplasmic protrusions, and increased their granule content (Figure 1I). Meg-01 cells also up-regulated active-caspase protein in the presence of 15d-PGJ₂ (data not shown). All these features are consistent with a megakaryocyte that is actively making platelets.^34-36  These platelets appeared to be functional, as they expressed platelet surface markers and changed shape and elevated surface levels of phosphatidylserine in response to collagen (Figure 1D,E). Phosphatidylserine is up-regulated on the surface of platelets with collagen activation and is important for blood coagulation.^26,27  We expanded our findings based on Meg-01 cells by also evaluating differentiating megakaryocytes from mouse bone marrow and from human cord blood–derived CD34+ cells. In both mouse and human cultures, more platelets were generated from megakaryocytes after 15d-PGJ₂ treatment. Culture-derived platelets showed similar morphologic and functional features to normal human platelets. They expressed platelet-specific receptors and were activated in response to either collagen or fibrinogen. Culture-derived platelets up-regulated CD61 and CD41, changed shape in response to collagen stimulation, and spread on fibrinogen-coated slides. Platelet spreading is an irreversible process necessary for platelet-surface contact during hemostasis and is, therefore, a good measure of platelet function.³⁷ 

15d-PGJ₂ is a potent PPARγ ligand.¹⁵  Although PPARγ is important in adipogenesis and inflammation,¹¹  recent studies suggest that PPARγ ligands influence the hematopoietic system.³⁸  Nagasawa et al published that certain PPARγ ligands impaired erythrocyte maturation,³⁹  and Kasono et al demonstrated that some PPARγ ligands elevated platelet numbers in a mouse model of thrombocytopenia by reducing the phagocytic activity of macrophages.⁴⁰  Our laboratory previously reported that megakaryocytes and platelets express PPARγ.¹³  Therefore, herein, we examined the effects of PPARγ ligands on megakaryocytes. Our findings show that prostaglandins such as PGE₂, PGI₂, and PGF₂α, which are poor activators of PPARγ, failed to enhance platelet production in Meg-01 cells. In contrast, the natural PPARγ ligand, 15d-PGJ₂, potently enhanced platelet production, a novel finding. Interestingly, 15d-PGJ₂ induced platelet production in Meg-01 cells that was independent of PPARγ. We demonstrated this by first showing that 2 highly selective synthetic PPARγ ligands, rosiglitazone and 9,10 dihydro-15d-PGJ₂, do not have the same platelet enhancing effects as 15d-PGJ₂. Next, we demonstrated PPARγ independence by knocking down PPARγ protein or by inhibiting PPARγ activation with the irreversible PPARγ antagonist. Under these conditions, no changes were observed in the ability of 15d-PGJ₂ to induce platelet production. The PPARγ-independent effects of 15d-PGJ₂ may be due to its electrophilic carbon in the cyclopentanone ring.^10,16,41  9,10 dihydro-15d-PGJ₂, a structural analog of 15d-PGJ₂, which binds to PPARγ with the same affinity as 15d-PGJ₂ but lacks the electrophilic carbon in the cyclopentanone ring, failed to enhance platelet production. In contrast, the electrophilic PGJ₂ also enhanced platelet production similarly to 15d-PGJ₂. We conclude that the electrophilic properties of 15d-PGJ₂ are important for its platelet-enhancing effects.

The electrophilic nature of 15d-PGJ₂ promotes mechanisms that accompany apoptotic related events such as cytoskeletal rearrangement and ROS generation.^10,18,42  Our data are consistent with these findings because both cytoskeletal changes (Figure 1G,I, Figure 2E, Figure 3E) and ROS generation (Figure 5A-C) occurred after 15d-PGJ₂ exposure, supporting the observation that platelet production is, in part, a specialized form of apoptosis.^35,43  One recent study showed that the overexpression of scinderin, an f-actin–severing protein, in Meg-01 cells led to megakaryoblast differentiation and platelet production.⁴⁴  In addition, 15d-PGJ₂ can interact with the cytoskeleton and oxidize susceptible cysteine residues leading to f-actin depolymerization.^18,42,45  Actin is a main scavenger of electrophilic lipids because of its high abundance and nucleophilic cysteine residues.⁴⁶  Thus, 15d-PGJ₂ may promote platelet production by a mechanism involving interaction with cytoskeletal proteins and f-actin depolymerization.

Several studies have implicated the cytoskeleton as an important regulator of the redox state of the cell, and conversely, the redox state of the cell may also influence the cytoskeleton.^47,48  Our data show that 15d-PGJ₂ promotes the generation of ROS within 1 hour. While it is well-known that redox status regulates cell proliferation, differentiation, and survival, the role of ROS in platelet production has been unclear until now. We demonstrate that ROS, and more specifically, mitochondrial superoxide, play a role in the dynamic process of megakaryocyte maturation and platelet release. Only the electrophilic molecules that generated ROS enhanced platelet production (Figure 5A-C). Pretreating Meg-01 cells or primary human megakaryocytes with either GSH-EE or NAC before 15d-PGJ₂ treatment attenuated both ROS induction (data not shown) and platelet formation (Figure 5D). Interestingly, many disorders associated with oxidative stress have platelet abnormalities, such as those seen in type 2 diabetes and atherosclerosis.⁴⁹  Further studies will be useful to determine whether oxidative stress in the bone marrow and, more specifically, in the megakaryocytes affects megakaryocyte maturation and platelet function.

15d-PGJ₂ not only stimulated platelet production, but also stimulated megakaryocyte maturation. While 15d-PGJ₂ failed to increase megakaryocyte number (data not shown), megakaryocyte polyploidization was increased. 15d-PGJ₂ did not significantly enhance Meg-01 ploidy until 72 hours after treatment, suggesting that this is a longer-term effect and did not directly promote the platelet release that was demonstrated by 24 hours. In contrast, 15d-PGJ₂ enhanced primary mouse megakaryocyte ploidy by 24 hours, suggesting that megakaryocyte maturation may play a direct role in promoting the platelet release. In addition, while 15d-PGJ₂ failed to promote the proliferation of megakaryocyte progenitors, the number of megakaryocytes with proplatelet extensions was significantly higher. Collectively, these results suggest that 15d-PGJ₂ increases platelet numbers by stimulating megakaryocyte maturation and/or subsequent proplatelet formation.

The platelet-enhancing activity of 15d-PGJ₂ in vitro raised the possibility that it may exhibit a similar effect in vivo. As shown, 15d-PGJ₂ had a significant effect on enhancing platelet production (Figure 7A) and accelerating platelet recovery in our model of radiation-induced thrombocytopenia (Figure 7B). We observed a significant increase in platelet number at days 22 and 31 after radiation exposure. These data suggest that 15d-PGJ₂ may be having an indirect effect on platelet number by up-regulating a cytokine, initiating a protein cascade, or influencing another cell type that regulates megakaryocyte maturation and platelet release; these effects may take additional time to manifest a phenotype. The elevation in platelet number has potential clinical significance through promoting platelet formation in immune-mediated thrombocytopenias where megakaryocyte numbers in the marrow are normal or increased and through reducing the risk for hemorrhage during platelet recovery after myelosuppression. The latter platelet-enhancing effect may be particularly important after chemotherapy or radiation exposure, where platelet depletion is accompanied by endothelial cell damage. Another complication associated with radiation exposure is scarring of vital organs such as lung and bone marrow. Importantly, 15d-PGJ₂ has demonstrated significant anti-scarring activities in models of lung scarring.⁵⁰ 

Our findings suggest that 15d-PGJ₂ is a promising therapeutic target for treating thrombocytopenia and may have advantages over other thrombopoietic agents that are being developed. 15d-PGJ₂ is small, cheap to make, and can readily penetrate tissues. In addition, 15d-PGJ₂ is highly conserved between species and is endogenously produced, raising the possibility that high endogenous levels of 15d-PGJ₂ in the bone marrow could potentially be associated with conditions characterized by high platelet counts.

Bone marrow suppression is the most common severe adverse effect after cytotoxic chemotherapy or radiation exposure and results in anemia, leucopenia, and thrombocytopenia. These can be ameliorated by transfusion support, but this has unwanted side effects including allergy, disease transmission, alloimmunization, limited availability, and expense. Pharmacologic treatment with myeloid growth factors and with erythropoietin has improved our ability to accelerate myeloid and erythroid recovery, respectively. However, no comparable cytokine therapy is currently available to accelerate thrombopoiesis. Our discovery that the electrophilic prostaglandin 15d-PGJ₂ exerts a potent thrombopoietic effect provides insight into the molecular mechanisms regulating both megakaryopoiesis and thrombopoiesis. This may lead to identification of new therapeutic agents to accelerate platelet recovery after marrow injury.

The authors thank the technical support of the Electron Microscope Research Core, specifically Karen L. de Mesy Bentley, Ian M. Spinelli, and Gayle Schneider. The authors also thank Dr Craig T. Jordan and Randall M. Rossi for use of the Heska Veterinary Analyzer.

This work was supported by National Institutes of Health (Bethesda, MD) grants T32ES07026, DE011390, ES01247, HL078603, HL086367, EY017123, and DK09361, and by the PhRMA Foundation (Washington, DC).

National Institutes of Health

Contribution: J.J.O'B. designed and performed research, analyzed data, and wrote the paper. S.L.S. contributed vital new reagents, analyzed data, and edited the manuscript. J.T. and K.E.S. performed research. N.B., C.W.F., and M.B.T. analyzed data and edited the manuscript. J.M.G. contributed vital new reagents. J.P. edited the manuscript. R.P.P. designed research, analyzed data, and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Richard P. Phipps, Department of Environmental Medicine, Box 850, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642; e-mail: richard_phipps@urmc.rochester.edu.