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The Role of Reactive Oxygen Species and Ferroptosis in Heme-Mediated Activation of Human Platelets Somanathapura K NaveenKumar, Bidare N Sharathbabu, Mahadevappa Hemshekhar, Kempaiah Kemparaju, Kesturu S. Girish, and Govindasamy Mugesh ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00458 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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ACS Chemical Biology

Proposed mechanism for the heme-induced platelet activation and ferroptosis. 92x56mm (300 x 300 DPI)

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The Role of Reactive Oxygen Species and Ferroptosis in HemeMediated Activation of Human Platelets Somanathapura K. NaveenKumar,$,§ Bidare N. Sharathbabu,§ Mahadevappa Hemshekhar,# Kempaiah Kemparaju,*,$ Kesturu S. Girish,*,$,ǁ Govindasamy Mugesh*,§

$

DOS in Biochemistry, University of Mysore, Manasagangotri, Mysuru 570 006, India

§

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore

560012, India #

Department of Internal Medicine, Manitoba Centre for Proteomics and Systems

Biology, University of Manitoba, Winnipeg- R3E3P4, Canada ǁ

Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru

572 103, India *Corresponding authors Email addresses: [email protected] (Govindasamy Mugesh); [email protected] (Kesturu S. Girish); [email protected] (Kempaiah Kemparaju)

Abstract. Hemolysis, a process by which the destruction of red blood cells leads to the release of hemoglobin, is a critical event observed during hemolytic disorders. Under oxidative stress conditions, hemoglobin can release its heme prosthetic group, which is highly cytotoxic and can catalyze the generation of reactive oxygen species (ROS), leading to several undesired redox reactions in the cells. Herein, we demonstrate for the first time that heme can mediate the activation and death of human platelets through ferroptosis, which is an iron-dependent form of nonapoptotic cell death. This study also suggests that the heme-mediated lipid peroxidation and ferroptosis in platelets may play an important role in hemolytic disorders.

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Heme proteins such as hemoglobin, myoglobin, cytochromes, oxygenases, catalases and peroxidases play key roles in various biological functions, including oxygen transport, electron transfer reactions and hormone synthesis.1-3 For example, hemoglobin (Hb), present in the red blood cells (RBCs), is responsible for the oxygen transport in vertebrates and the heme prosthetic group in this protein is essential for the oxygen binding.4 Hemolysis, often known as destruction of RBCs, inevitably results in the release of Hb and free heme into the circulation (Supporting Information Figure S1), leading to the generation of reactive oxygen species (ROS) via iron-mediated Fentontype reactions and to oxidative stress.5,6 Hb released from the RBCs during hemolytic conditions is the major source of circulatory free heme (up to 20 µM in plasma).7 The toxic heme is generally scavenged by the plasma protein hemopexin, and subsequently catabolized into carbon monoxide (CO), biliverdin and ferric iron (Fe3+) by heme oxygenase-1 (HO-1).7 However, the increased release of heme from Hb and decreased levels of hemopexin under hemolytic conditions contribute to the elevated level of circulatory heme. The free heme is highly toxic and can cause cell damage and tissue injury.8 Heme is known to induce the activation of endothelial cells via TLR4 signalling, leading to increased expression of P-selectin, E-selectin, NF-ƙB, von Willebrand Factor (vWF), and vaso-occlusion in transgenic SCD mice.8 An elevated level of heme in circulation also contributes to the activation of coagulation cascade in the presence of tissue factors and the depletion of plasma hemopexin level induces oxidative stress in endothelial or renal tubular cells.9 Similarly, heme induces apoptosis and necroptosis in RBCs and cerebral microvascular cells by increasing the intracellular calcium level and depleting the cellular glutathione (GSH).9 In this paper, we demonstrate for the first time



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that hemin can induce platelet activation and cell death through an iron-mediated ferroptosis pathway involving lipid peroxidation, which is distinct from the other forms of regulated cell death such as apoptosis and necroptosis.10 To understand whether extracellular hemin (Figure 1A) can exert cytotoxic effect in human platelets, the metabolic activity (cell viability, MTT assay) and release of lactate dehydrogenase (LDH) were evaluated by treating the platelets with various concentrations of hemin. It has been observed that hemin significantly reduced the cell viability (Figure 1B) and increased the release of LDH within 30 min of treatment, confirming the cytotoxic effect of hemin in platelets. To further understand the mechanism by which hemin decreases the cell viability, the hemin-treated platelets were analyzed for the well-known forms of cell death namely apoptosis and necroptosis.11 As the apoptotic cell death occurs by the activation of the cysteinecontaining enzymes caspases,12 the platelet viability, the protein expression level and enzyme activity were determined in the presence and absence of hemin. When platelets were treated with hemin and z-VAD-FMK, a cell-permeable pan caspase inhibitor, no improvement in the platelet viability was observed. Furthermore, the caspase-3 specific inhibitor, z-DEVD-FMK, also did not have any effect on the platelet viability (Figure 1C). The immunoblots of cytochrome c (cyt c), pro-caspase-3 and active caspase-3 obtained after treatment with different concentrations of hemin indicate that hemin does not mediate the release of cyt c from mitochondria, which is a key initial step in the apoptotic process (Figure 1D, Supporting Information Figure S3). The enzyme activity assay also confirms that hemin does not induce the activation of caspase-3 in platelets (Supporting Information Figure S4). ABT-737, a small-molecule inhibitor that specifically


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inhibits anti-apoptotic proteins of the Bcl-2 family, was used as control. The lack of cyt c release by hemin is surprising as it has been shown that Fe(II)/ascorbate can strongly induce the release of cyt c from mitochondria.11 These observations clearly indicate that hemin does not induce apoptosis in platelets.

Figure 1. (A) Chemical structure of hemin. (B) Dose-dependent effect of hemin on platelet viability by MTT and LDH assays. ABT-737 (inducer of apoptosis) and A23187 (calcium ionophore) are used as positive controls. (C) Platelet viability in the presence of apoptosis inhibitors z-VAD-FMK (a pan caspase inhibitor) and z-DEVD-FMK (caspase-3 specific inhibitor). (D) Immunoblots of cytochrome C and pro- and active-caspase-3 obtained after treatment with different concentrations of hemin. (E) Chemical structure of necrostatin-1 (NS-1), an inhibitor of necroptosis. (F) Dose-dependent effect of hemin on platelet viability in the presence and absence of NS-1. 4 ACS Paragon Plus Environment


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As platelets do not undergo apoptosis by hemin, it was thought worthwhile to understand whether the cytotoxicity observed in the presence of hemin is due to another form of cell death, necroptosis. It is known that necroptosis do not involve caspase activation and the cell death results in leakage of cell contents into the extracellular space.11 Therefore, platelet viability was studied in the presence of necroptosis-specific inhibitor necrostatin-1 (NS-1, Figure 1E). The anti-necroptosis activity of NS-1 is due to its ability to selectively inhibit the death domain receptorinteracting serine/threonine-protein kinase 1 (RIP1).11 Interestingly, it was observed that NS-1 at concentrations up to 25 µM neither improved the metabolic activity (MTT assay) nor inhibited the LDH release (Figure 1F), indicating that the heme-mediated platelet death is independent of necroptosis. The scanning electron microscopic (SEM) analyses indicate that hemin certainly has altered the morphological structure of the platelets in a dose-dependent manner (Figure 2A). Hemin at a dose of 5 to 10 µM induces the formation of filopodia-like structures, and at higher dose (25 µM), the filopodia gets destroyed, leading to membrane damage and blebbing. The activation of platelets by reactive oxygen species (ROS) is physiologically relevant and several studies have reported that ROS (hydrogen peroxide, superoxide, and hydroxyl radicals) play a pivotal role in inducing platelet activation, apoptosis and thrombosis.13 Therefore, we studied the effect of hemin on the level of cytosolic ROS using the standard DCFDA assay. It was found that hemin induced a dose-dependent increase in the level of ROS in human platelets (Figure 2B, Supporting Information Figure S5). Almost a 6-fold increase in the ROS level was observed at 25 µM concentration of hemin. Further, to understand the nature of ROS


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generated in the hemin-treated platelets, we determined the level of H2O2 and superoxide (O2−) using Amplex Red and dihydroethidium (DHE), respectively. Interestingly, the H2O2 level was only marginally increased by hemin, and almost no change in the superoxide level was observed (Figure 2B). These observations indicate that hydroxyl radical (•OH) is probably the major ROS generated in the presence of hemin. Therefore, we analyzed the lipid peroxidation induced by •OH using a BODIPYbased dye as the probe. It was found that hemin drastically increased the lipid peroxidation and at 25 µM concentration of hemin, a 6-fold increase in the fluorescence was observed (Figure 2C). To further confirm the lipid peroxidation, we estimated the malondialdehyde (MDA, Supporting Information Figure S6) level in platelets treated with hemin and found that hemin dose-dependently increased the MDA level (Figure 2D). These results indicate that hemin-induced lipid peroxidation is the major contributor of platelet death. The increased lipid peroxidation in platelets suggests that hemin probably releases free Fe2+ ions, which can mediate Fenton-type reactions to produce hydroxyl radicals. It is known that heme oxygenase-1 (HO-1) metabolizes heme to generate carbon monoxide (CO), biliverdin and iron(II).7 Thus, HO-1-mediated iron release is the major intracellular source of labile iron.7 To understand the metabolism of hemin by HO-1, we treated the platelets with various concentrations of hemin and analyzed the expression levels of HO-1 using a specific antibody and quantified the cytosolic free iron spectrophotometrically using the iron chelator, ferrozine.14 These experiments reveal that hemin not only increases the expression of HO-1, but also significantly elevates the cytosolic iron level in platelets (Figures 2E,F). Furthermore, a significant decrease in the



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Figure 2. (A) SEM images of platelets treated with hemin (0-25 µM). (B) The levels of total ROS, hydrogen peroxide, superoxide measured by using appropriate fluorescent probes. H2O2/Rotenone mixture was used as positive control. (C) Flow cytometric analysis of lipid peroxidation using a BODIPY-based probe with cumene hydroperoxide (CHP) as positive control. (D) Estimation of malondialdehyde (MDA, nmol/mg protein) using thiobarbituric acid. (E) The expression level of heme oxygenase-1 (HO-1) determined by immunoblotting using HO-1specific antibody. (F) Quantification of cytosolic free iron levels (nmol/mg protein) by using ferrozine. (G) The effect of hemin on GSH/GSSG ratio and γ-glutamyltransferase (GGT) activity (nmol min-1mg-1 protein). 7 ACS Paragon Plus Environment

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GSH/GSSG ratio and an increase in the γ-glutamyltransferase (GGT) activity were observed in the platelets treated with hemin (Figure 2G). These results suggest that the cellular GSH level is significantly altered and the HO-1-mediated release of iron induces oxidative stress in platelets. Recently, Stockwell and co-workers have reported that the oncogenic RAS-selective small molecule erastin can trigger a nonapoptotic cell death, which they termed ferroptosis. They showed that ferroptosis is dependent upon intracellular iron and is morphologically, biochemically, and genetically distinct from apoptosis and necrosis.10 It has been shown that ferroptosis does not involve the activation of caspases, ATP depletion and mitochondrial ROS generation (mediators of necroptosis).10 Instead, ferroptosis is driven by accumulation of lipid peroxides, depletion of cellular GSH and inhibition of cysteine-glutamate antiporter.15 Conrad and co-workers reported an interesting study on the importance of selenium in the mammalian enzyme glutathione peroxidase 4 (GPx4) in suppressing peroxide-induced ferroptosis.16 Interestingly, Hb and free-iron are shown to induce ferroptosis in neuronal cells via generation/ accumulation of lipid radicals.15 Also, it has been shown that hemin/HO-1 accelerates erastin-induced ferroptotic cell death in cancer cells.17 Taken together, our results suggest that the lipid peroxidation and ferroptosis may play key roles in the hememediated platelet death. In human platelets, hemin treatment increases the cellular labile iron by increasing the expression of HO-1 and decreases the GSH level, leading to lipid peroxidation and ferroptosis. Therefore, we studied the effect of tin protoporphyrin IX dichloride (SnPP), a specific inhibitor of HO-1, deferoxamine (DFO), an iron chelator, and ferrostatin-1 (FS8 ACS Paragon Plus Environment


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1), a ferroptosis inhibitor, in hemin-treated platelets. When we analysed the effect of hemin on platelet viability and lipid peroxidation after pre-treating the platelets with SnPP and co-treatment with DFO and FS-1, we found that both DFO and FS-1 rescued the platelets significantly from the hemin-mediated cytotoxicity (Figure 3B, Supporting Information Figure S7). Crucially, all three compounds, SnPP, DFO and FS-1, almost completely inhibited the lipid peroxidation (Figure 3C, Supporting Information Figure S8) and controlled the ROS level (Figure 3D). It is known that the peroxidation of n-6polyunsaturated fatty acids such as arachidonic and linoleic acids produces 4hydroxynonenal (4-HNE) (Supporting Information Figure S6), which is one of the most abundant and active lipid peroxides in the cells. 4-HNE can react with amino acid residues, such as cysteine, lysine or histidine, to form stable adducts with proteins. Therefore, the levels of 4-HNE are used as a marker of oxidative stress. When the 4HNE-modified proteins were studied by immunoblotting, 5 or 10 µM of hemin significantly increased the amount of protein-HNE adducts and the treatment with SnPP, DFO and FS-1, reduced the amount of such protein adducts (Figure 3E). We also studied the total glutathione (GSH) level in platelets after treatment with hemin by incubating the cell lysates with the non-fluorescent monochlorobimane (MCB) that reacts with GSH to produce a fluorescent GSH-bimane (GSB) conjugate. Interestingly, a significant increase in the GSH level was observed in the presence of SnPP, DFO and FS-1, although the increase in the GSH level was found to be relatively lower in platelets treated with FS-1 (Figure 3F, Supporting Information Figure S9). The substantial depletion in the GSH level indicates that the heme-mediated ferroptosis may occur through inhibition of system Xc− that transports extracellular cystine into the cell


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Figure 3. (A) Chemical structure of SnPP (tin protoporphyrin IX, HO-1 inhibitor), DFO (deferoxamine, iron chelator) and Ferrostatin-1 (FS-1, ferroptosis inhibitor). (B) Platelet viability (MTT assay) in the presence of hemin, SnPP, DFO and FS-1. (C) Quantification of lipid peroxidation and ROS level (DCFDA assay). (D) Measurement of lipid peroxidation by flow cytometry using a BODIPY-based fluorescent probe. (E) Immunoblot of 4-HNE-modified proteins in platelets in the presence of SnPP, DFO and FS-1. (F) Confocal images of hemininduced GSH depletion and the effect of SnPP, DFO and FS-1 on the GSH level. Monochlorobimane (MCB) was used as a fluorescent tag.



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for the biosynthesis of GSH.18 As β‐mercaptoethanol (β‐ME) can enhance cysteine uptake through other pathways,18 we treated the platelets with β-ME in the presence and absence of cysteine (Supporting Information Figure S10). Interestingly, the treatment of platelets with β‐ME significantly improved the cell viability, suggesting the involvement of system XC− in the heme-mediated ferroptosis in platelets. The above observations clearly indicate that HO-1-catalysed release of iron from hemin induces lipid peroxidation and ferroptosis in platelets. It is well-known that platelets are exposed to adhesive proteins and soluble agonists upon blood vessel injury, and such exposure initiates platelet activation.19 However, platelet activation under pathological conditions can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke19 To understand whether hemin induces platelet activation, the expression levels of Pselectin, a biomarker for platelet activation, was studied. In unactivated platelets, Pselectin is stored in α-granules, but during platelet activation, it is translocated to the plasma membrane and acts as a cell adhesion molecule (CAM) on the surface of activated platelets19 Interestingly, the FACS analysis showed that P-selectin level was significantly increased in the hemin-treated platelets in a dose-dependent manner (Figure 4A). This observation was further supported by fluorescence microscopic experiments, where we analyzed the actin dynamics and P-selectin translocation in adherent platelets by using Alexa Fluor-546 conjugated Phalloidin and FITC-conjugated anti-P-selectin antibody, respectively. In agreement with the SEM analysis (Figure 2A), these actin dynamics experiments indicate that platelets develop filopodia-like structures at lower doses of hemin (5-10 µM), whereas at a higher concentration of


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Figure 4. (A) Relative translocation of P-selectin measured by flow cytometry using anti-Pselectin antibody. Thrombin, one of the most potent agonists for platelet activation, is used as positive control. (B) Dose-dependent effect of hemin on the F-actin organization. (C) Confocal images of hemin-induced P-selectin expression and alteration of the F-actin organization in the presence or absence of SnPP, DFO and FS-1.

hemin (25 µM), these structures are destroyed (Figure 4B). Further, hemin induced the formation of platelet microparticles (PMPs) through platelet activation and death (Supporting Information Figure S11). When the PMPs were quantified using anti-CD41 eFluor-450 antibody, it was observed that 25 µM of hemin treatment increased the level of PMPs by almost 4-times, indicating that both the platelet activation and ferroptosis 12 ACS Paragon Plus Environment


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may contribute to the formation of PMPs. It should be noted that elevated levels of PMPs are associated with several diseases, which include thrombocytopenia, arterial thrombosis, sickle cell disease, uremia, malignancy, and rheumatoid arthritis.20 To further verify whether SnPP, DFO and FS-1 can block the P-selectin translocation and rescue the platelets from hemin-induced morphological changes and activation, we performed confocal microscopic studies and FACS analysis. These experiments demonstrated that SnPP, DFO and FS-1 significantly inhibited the P-selectin translocation (Figure 4C, Supporting Information Figure S12). As mentioned earlier, hemin (10 µM) induced the development of filopodia-like structures in platelets. It is believed that actin reorganization is responsible for the shape change.19,20 During platelet activation, actin undergoes dramatic changes in the length and organization of the actin filaments. Therefore, we studied the effect of SnPP, DFO and FS-1 on F-actin organization and found that these compounds significantly inhibited the filopodia formation (Figure 4C). Further, FS-1 remarkably decreased the hemin-induced generation of PMPs. It is interesting to note that FS-1 almost completely blocked all the hemin-induced events, strongly suggesting that the platelet activation and death can be prevented by inhibiting ferroptosis. In this study, we demonstrate for the first time that hemin induces ferroptosis in platelets (Supporting Information Figure S13), which is distinct from the common forms of regulated cell death such as apoptosis and necroptosis. Further, this study provides experimental evidence that the heme oxygenase-1-mediated release of iron from hemin and the subsequent generation of hydroxyl radicals are responsible for the lipid peroxidation. Hemin also induces platelet activation, translocation of P-selectin and 13 ACS Paragon Plus Environment

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reorganization of actin filaments. The ferroptosis inhibitor FS-1 significantly attenuates the hemin-induced toxicity and platelet activation, indicating that the regulation of ferroptosis-like process in platelets can be considered as a potential strategy to treat hemolytic disorders. Methods: Chemicals. Hemin, ferrostatin-1 (FS-1), monochlorobimane (MCB), osmium tetroxide, thrombin from bovine plasma, cumene hydroperoxide (CHP), calcium ionophore (A23187), rotenone and dihydroethidium (DHE) were purchased from Sigma Chemicals, USA. Deferoxamine (DFO) was purchased from Novartis India Ltd. Tin protoporphyrin IX dichloride (SnPP), ABT-737, calcein-AM and caspase-3 were obtained from Santa Cruz Biotechnology, Inc., USA. Antibodies against heme oxygenase-1 (HO-1), 4-HNE and βtubulin were purchased from Cell Signalling and Technology, USA. Antibody against FITC conjugated CD62P (P-selectin) were purchased from BD biosciences, USA. Alexa Fluor -546 Phalloidin, CM-H2DCFDA, Amplex UltraRed Reagent, and BODIPY 581/591 C11 were purchased form Molecular Probes, Thermo Fisher Scientific, USA.

LDH

estimation kits were purchased from Agappe Diagnostics Limited, India. Hydrogen peroxide (H2O2) was from RANKEM, India. All other reagents were of analytical grade and procured from Sisco Research Laboratories, India. Isolation of platelets. Blood was collected from healthy human volunteers with informed consent according to the approved guidelines of Institutional Human Ethical Committee (IHEC-UOM No. 114 Ph.D/2015-16), University of Mysore, Mysuru. Blood was drawn from antecubital vein 14 ACS Paragon Plus Environment


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and was immediately mixed with acid citrate dextrose (ACD) anti-coagulant and platelets were isolated as described previously.21 Briefly, human platelet-rich plasma was prepared by centrifuging anticoagulated blood at 100xg for 15 min and the supernatant was collected and centrifuged at 700xg for 10 min at 37 °C. The platelet pellet was washed twice by suspending them in CGS (13 mM tri-sodium citrate, 33 mM D-glucose, 123 mM NaCl, pH 6.5) buffer and centrifuged at 700xg for 15 min at 37 °C. Finally, the washed platelets (WPs) were suspended in Tyrode’s buffer (2.5 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, pH 7.4). The cell count was determined in WPs suspension using a Neubauer chamber and adjusted to 2 x 107 platelets/mL in the final suspension using Tyrode’s buffer.21 Assessment of Ferroptosis. To study the hemin-iron induced ferroptotic death, we evaluated the cell viability in presence or absence of tin protoporphyrin IX (SnPP); is a synthetic heme analog that selectively inhibits HO-1, deferoxamine (DFO); an intracellular iron chelator and Ferrostatin-1 (FS-1); a potent inhibitor of ferroptosis via inhibiting the accumulation of lipid hydroperoxides. To assess the cell viability and LDH release in presence of ferroptosis inhibitor. MTT colorimetric assay and LDH release assay was performed, platelets (2 x 106 cells/mL) were treated with hemin (10 µM), pre-treatment with SnPP (15 min at 37 °C) and co-treatment with DFO/ FS-1/ vehicle (DMSO), incubation for 30 min at 37 °C. After 30 min of incubation, samples were spined and the supernatant was collected and used to detect LDH release by using LDH kit or samples were plated to 96 well microtiter plate and 250 µM of MTT was added and incubated for additional 3 h.


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Thereafter, MTT was removed and remaining formazan crystals were completely dissolved in DMSO and the absorbance was recorded at 570 nm using a Varioskan multimode plate reader (Thermo Scientific, USA) Further, total ROS and lipid peroxidation was evaluated in presence or absence of ferroptosis inhibitors. Platelet suspension (5 x 106 cells) treated with of hemin (10 µM), pre-treatment with SnPP (15 min at 37 °C) and co-treatment with DFO/ FS-1 followed by incubation for 30 min at 37 °C and were washed with Tyrode’s buffer and incubation with CMDCFDA (5 µM) and BODIPY 581/591 C11 (10 µM) for 20 min at 37 °C in dark. Cells were again washed, and fluorescence was analyzed using flow cytometry (BD FACSCelesta).10,21,22 Acknowledgments. This study was supported by the Science and Engineering Research Board (SERB, EMR/IISc-01/2016), Department of Science and Technology (DST), New Delhi. SKN and BNS thank the UGC-BSR and IISc, respectively, for their fellowship. G. M. thanks the SERB for the award of J. C. Bose National fellowship (SB/S2/JCB-067/2015). Competing financial interests The authors declare no competing financial interests. Supporting Information. Additional experimental details and figures. This material is available free of charge on the ACS Publications website at http://pubs.acs.org.



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