Abstract
Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (pā<ā0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.
Similar content being viewed by others
Introduction
Unconjugated hyperbilirubinemia is an incidental verdict on routine laboratory tests in most liver diseases including chronic liver disease, haemolytic jaundice, Gilbertās syndrome and Crigler-Najjar syndrome1,2,3. Liver disorders quite often present haematological aberrations particularly thrombocytopenia (low platelet count). As high as 58% of liver disease patients are observed to have thrombocytopenia correlating increased concentration of bilirubin4. Consequently, the extent of thrombocytopenia is found to serve as an effective prognostic marker for liver diseases4,5. Thrombocytopenia not only leads to increased bleeding and haemorrhage, but also is an impediment to invasive procedures including diagnosis and therapeutic regimen. One of the reasons behind thrombocytopenia might be rapid destruction or clearance of platelets in the peripheral circulation. This is supported by various reports lately including our own, that demonstrate the intensely sensitive and fragile nature of platelets, as well as their tendency to undergo apoptosis in response to a wide array of intrinsic and extrinsic stimuli6. The anuclear platelets possess the necessary cellular machinery to undergo apoptosis like any other nucleated cell, with the exception of nuclear apoptotic events. The intrinsic apoptotic pathway is more commonly demonstrated in platelets, which transpires via mitochondrial dysfunction in response to stress and is presented with augmented levels of reactive oxygen species (ROS), mitochondrial membrane potential (ĪĪØm) depolarization, intracellular calcium ions (Ca2+), cytosolic cytochrome c (cyt. c), caspases ā9 and ā3 activities and phosphatidylserine (PS) scrambling7,8.
Although the link between thrombocytopenia and liver diseases is quite apparent, at present the scientific community does not acknowledge liver disease as a probable cause of thrombocytopenia. Therefore, the present study sought to delve into the mechanism behind this phenomenon. One common hallmark in the liver diseases, including haemolytic jaundice, Gilbertās syndrome and Crigler-Najjar syndrome, is elevated level of serum unconjugated bilirubin i.e. hyperbilirubinemia. Unconjugated bilirubin (UCB) is the reduced product of biliverdin and is an end-product of heme (Fe-protoporphyrin IX) catabolism. It is made in the Kupffer (hepatic) cells and in monocytic macrophages of bone marrow and spleen, which is then released into plasma9. UCB behaves as a physiological antioxidant in human extracellular fluids10,11. However, it is also demonstrated that bilirubin in the presence of the transition metal ion Cu (II) induces DNA strand cleavage via generation of ROS, particularly hydroxyl radical12. Hence, UCB acts as an antioxidant at normal physiological concentration and as a pro-oxidant at higher concentration, where it may reach up to 300āĪ¼M11. High concentration of UCB is linked to cell cytotoxicity and neurological dysfunctions13. On the other hand, there are studies that have reported the platelets undergo apoptosis in response to oxidative stress14,15,16. Accordingly, it is hypothesized that elevated levels of UCB during a disease condition might exert oxidative stress on platelets and thus trigger the process of apoptosis. Therefore, the present study demonstrates the pro-apoptotic potential of UCB on human platelets as an attempt to correlate hyperbilirubinemia and thrombocytopenia and also explores the underlying molecular signalling pathway. We also report the observed incidences of thrombocytopenia in hyperbilirubinemia patients, as well as activated apoptotic markers in their platelets. Taken together, we demonstrate the clinical significance of UCB in depleting platelet count during hyperbilirubinemic conditions, which demands a collective treatment strategy considering the risks associated with thrombocytopenia during liver diseases.
Results
UCB induces oxidative stress and apoptosis in platelets
To investigate whether UCB affects platelet function and life span, we examined the oxidative stress and apoptotic markers of platelets in presence or absence of UCB. UCB-treated platelets were assessed for oxidative stress parameters like endogenous ROS generation, intracellular calcium content and ĪĪØm depolarization. FACS analysis of UCB-treated platelets showed concentration-dependent increase in ROS generation measured using DCF fluorescence (Fig. 1A). To rule out the interference of UCB fluorescence at the similar excitation and emission range of DCF, UCB -treated platelets were monitored for fluorescence without adding DCF dye. We found no observable fluorescence in UCB-treated platelets suggesting that UCB do not interfere with DCF fluorescence (Supplementary Figure 1). It is established that impaired functions of ETC complex is the primary event leading to mitochondrial ROS production17,18. Therefore, effect of UCB on ETC complex was evaluated. UCB significantly inhibited complex II and complex IV activities in a concentration-dependent manner (Fig. 1B). Additionally, ROS generation induced by disturbances in ETC was confirmed using Mito-TEMPO, a mitochondria targeted anti-oxidant. It significantly abolished UCB-induced endogenous ROS generation, indicating that mitochondria are the primary source of ROS in unconjugated hyperbilirubinemia (Fig. 1C). Fluorometric analysis of UCB-treated platelets showed concentration-dependent increase in intracellular calcium levels (Fig. 1D) together with decreased ĪĪØm (Fig. 1E). As discussed earlier, to exclude the interference of UCB with JC1 fluorescence, (used to detect ĪĪØm) unstained UCB-treated platelets were monitored for fluorescence and we observed no change suggesting UCB do not interfere with JC1 fluorescence (Supplementary Figure 2). Altered mitochondrial dysfunction and calcium homeostasis are marked with peroxidation of cardiolipin, release of cyt. c into cytosol via mPTP, downstream caspase activation and PS externalization. Accordingly, UCB triggered cardiolipin peroxidation (Fig. 2A) and mPTP formation as determined by calcein-CoCl2 quenching assay (Fig. 2B). Further, downstream apoptotic events like cyt. c release, activation of caspase-9 and caspase-3 were evaluated using immunoblotting (Fig. 2C). It is demonstrated that during apoptosis cellular proteins can be phosphorylated through the activation of protein kinases and then exposed to caspase cleavage19. Consistent with this notion, we observed increased phosphorylated protein content in UCB-treated platelets using immunoblot (Fig. 2D) and flow cytometric analysis endorsed that there was concentration-dependent PS externalization in UCB-treated platelets (Fig. 2E). In all the assays, vehicle control (DMSO) was found to be non-toxic to platelets and did not induce either apoptosis or oxidative stress to platelets.
In order to assess the toxicity of UCB towards platelets, UCB-treated platelets were assessed for their viability. There was a significant decrease in G6PDH activity (Fig. 3A), increase in GGT activity of platelets (Fig. 3B) suggesting that platelets were in oxidative stress due to UCB treatment. The increased levels of lysosomal alkalinization, LDH release and MTT assay in platelets confirm the cytotoxic effect of UCB on platelets and a significant reduction in the viability of UCB-treated platelets (Fig. 3CāE). Further, we evaluated the macrophage-mediated clearance of UCB treated platelets by FACS analysis. We observed a significant increase in calcein fluorescence in macrophages incubated with UCB-treated platelets (Fig. 3F) suggesting that platelets undergo apoptosis upon UCB treatment.
Our next question was to determine whether UCB treatment activates platelets. To verify the activation of platelets, platelet aggregation and platelet adhesion assays were performed. UCB did not induce platelet aggregation by itself; however, it inhibited collagen-induced platelet aggregation in washed platelets at a lower dose (Supplementary Figure 3AāC). In addition, UCB altered the adhesive property of washed platelets towards collagen (Supplementary Figure 3D). Together, these findings attest a prior knowledge that UCB affects platelet function and primes them into apoptosis and not their activation.
UCB-induces platelet apoptosis through p38-mediated mitochondrial dysfunction
Apoptosis in UCB-treated platelets was so profound that nearly half of the treated population had undergone death as conferred by PS externalization and MTT assay. Therefore, we found it interesting to delineate the molecular mechanism of UCB-induced platelet apoptosis; hence phosphorylation of proteins involved in apoptosis signalling cascade was evaluated using immunoblotting. It is well established in platelets that MAP kinases play a significant role during mitochondrial dysfunction-mediated apoptosis20. Interestingly, there was increased phosphorylation of p38 (Thy180/Tyr182) as well as phosphorylation of p53 (Ser15) in platelets treated with UCB (Fig. 4A). Alongside, there was significant up-regulation of pro-apoptotic Bcl-2 family protein Bax, together with down-regulation of pro-survival proteins Bcl-2, phospho Bad, demonstrating that mitochondrial dysfunction was mediated by p53 (Fig. 4B). Further, there was an increased localization of Bax and phospho p53 in mitochondrial fraction and cyt. c in cytosolic fraction of platelets treated with UCB suggesting that p53 has a definite role in mitochondrial dysfunction (Fig. 4C).
In order to confirm the involvement of MAPK kinases in UCB-triggered platelet apoptosis, specific inhibitors of MAPK pathway were used. Under physiological conditions, albumin binds to UCB in circulation and hence inhibits its action towards the cells that are in close proximity. As expected, the immunoblotting data showed the decreased phosphorylation of p38 and p53 in UCB-BSA conjugate-treated platelets (Fig. 5A). Additionally, Mito-TEMPO (mitochondrial ROS scavenger) also inhibited p38 and p53 phosphorylation, indicating that their activation was due to mitochondrial ROS production (Fig. 5A). To explore whether p53 was being activated by p38, SB203580 a specific p38 inhibitor was used. Phosphorylation of p53 was inhibited by SB203580 suggesting that the phosphorylation was mediated by p38 activation (Fig. 5B). Further to confirm that UCB mediates apoptosis in platelets through p38 MAPK, the downstream apoptotic markers like Bcl-2, Bax and caspase-3 expression were evaluated both in presence and absence of SB203580 (Fig. 5B). Interestingly, SB203580 restored the altered levels of Bcl-2, Bax and caspase-3 in UCB-treated platelets as compared to untreated platelets. Furthermore, Pifithrin-Ī¼, a specific blocker of p53- Bcl-xL/Bcl-2 interaction, inhibited cyt. c release and caspase-3 activation and increased PS levels in UCB-treated platelets as compared to untreated platelets (Fig. 5C), indicating that UCB triggers apoptosis in platelets via p53-mediated activation of Bcl-2 family proteins and caspases. The specific caspase inhibitor z-DEVD-fmk, also restored the elevated PS levels establishing further that UCB- induced platelet apoptosis is caspase dependent (Fig. 5D,E).
UCB alters platelet apoptosis markers in rat model
Since, it was clear from the previous set of results that UCB induces platelet apoptosis, it was ideal to ascertain the effects of UCB in vivo. Therefore, phenylhydrazine (PHZ)-induced hyperbilirubinemic animals were initially evaluated for platelet count. PHZ administration effectively induces haemolytic hyperbilirubinemia in the rats, which is close enough to human haemolytic jaundice with elevated UCB levels21. We observed a significant decrease in the population of circulating platelets (Fig. 6A) with elevated serum UCB levels (Fig. 6B). The platelet apoptotic markers like endogenously generated ROS, intracellular calcium, ĪĪØm, cardiolipin peroxidation and PS externalization were also altered in PHZ-induced hyperbilirubinemic rats in a similar pattern as the in vitro results (Fig. 6C). In the previous set of experiments, activation of p38 and p53 were shown to be critical in UCB-mediated mitochondrial damage, hence platelets from the hyperbilirubinemic rats were evaluated for the same. Increased phosphorylation of p38 and p53 along with decreased Bcl-2 levels and increased levels of Bax, cyt. c and caspase-3 were clearly evident from respective immunoblots suggesting that the decreased platelet count was due to hyperbilirubinemia (Fig. 6D). To exclude the fact that PHZ itself interferes with altered platelet properties, platelets were treated with PHZ in vitro. As the results demonstrate, the alterations in ROS content, intracellular calcium levels, ĪĪØm, cardiolipin peroxidation, mPTP formation, PS externalization and LDH activity were found to be negligible (Supplementary Figure 4). In addition, altered serum cytokine levels were also observed in PHZ-induced hyperbilirubinemic rats compared to control (Fig. 7A). A significant difference in macroscopic and microscopic structure of both liver (Fig. 7B) and spleen (Fig. 7C) was observed in PHZ-induced hyperbilirubinemic rats compared to saline control.
UCB-triggers platelet apoptosis in hyperbilirubinemic human subjects
Further, to validate the link between thrombocytopenia and hyperbilirubinemia, hyperbilirubinemic (HB) patients were recruited and the correlation between indirect bilirubin level and platelet count was determined. As hypothesized, there was a significant decrease in the number of circulating platelets (Fig. 8A) along with elevated UCB levels (Fig. 8B) in HB subjects compared to HS. Therefore, platelet apoptotic markers and intracellular signalling events were evaluated in HS and HB subjects. There was significant alteration in platelet apoptotic markers like endogenous generation of ROS, intracellular calcium, ĪĪØm, cardiolipin peroxidation, mPTP formation and PS externalization (Fig. 8C). The obtained results were further backed up by demonstrating the decreased lysosomal acidity (Fig. 8D) and increased GGT activity (Fig. 8E). Moreover, the phosphorylated forms of p38 and p53 were also elevated significantly together with apoptotic markers like decreased Bcl-2 and increased levels of Bax, cyt. c and caspase-3 (Fig. 8F). The obtained results manifestly demonstrate the subliminal path of UCB priming circulating platelets into their deaths via an intricate cellular mechanism (Fig. 8G).
Discussion
The current study for the first time, probed the cause of thrombocytopenia in unconjugated hyperbilirubinemia. Unconjugated hyperbilirubinemia is a common characteristic feature in liver diseases such as haemolytic jaundice, Gilbertās syndrome and Crigler-Najjar syndrome1,2,3. Thereby, it is conjectured that there might be a link between hyperbilirubinemia and thrombocytopenia. Of note, thrombocytopenia is frequently observed in 58% of patients with liver diseases, which correlates with increased concentration of bilirubin4. The consequences of thrombocytopenia include increased risk of internal and external bleeding, delay in wound healing and coagulation defects. Moreover, it can become a clinically significant predicament in liver disease patients who have to undergo invasive diagnosis/therapy, interferon treatment, liver transplantation, cancer chemotherapy and surgery22. Though UCB is a metabolic waste product, it is also shown to be an endogenous antioxidant in mammalian tissues and makes up a greater part of the antioxidant activity in human serum10,11. Bilirubin and biliverdin are potent modulators of cell signalling as well23,24. Nevertheless, unconjugated hyperbilirubinemia is associated with pathological conditions such as, increased haemolysis or haemolytic jaundice, Gilbertās syndrome and Crigler-Najjar syndrome3. UCB is also shown to exert cytotoxic and pro-apoptotic effects on neural cells and tumour cells25. Therefore, the present study investigated whether UCB affects platelet life span to trigger thrombocytopenia in liver diseases associated with elevated UCB levels. The results of the present study indicate that UCB induces platelet apoptosis in vitro at the concentration range 0ā200āĪ¼M, due to the presence of UCB-binding proteins like albumin and other plasma proteins we deliberate our data with high concentration of UCB (0ā200āĪ¼M). The mitochondrial/intrinsic pathway of apoptosis was mainly probed because of its susceptibility to physiological and pathological oxidative stress-inducing factors. Bilirubin is reported to be as efficient as anti-oxidants like glutathione and vitamin E in foraging hydroxyl radicals. However, elevated serum levels of UCB often exhibit pro-oxidative effect, which might be responsible for its anti-microbial properties. In the present study, UCB was found to considerably trigger the endogenous generation of ROS in platelets. Previously, few anti-oxidants such as resveratrol, sesamol and melatonin were also reported to trigger platelet apoptosis via ROS generation6. High level of UCB can lead to kernicterus (brain dysfunction) and studies suggest that oxidative stress is an important factor in bilirubin-induced neurotoxicity26. ROS plays a pivotal role in setting off the apoptotic events in platelets via the mitochondrial pathway by ĪĪØm dissipation. Mitochondria perform the function of biological switches that decide the fate of the respective cells by supplying the required energy for cell survival and when there are lethal stimuli they trigger cell death27. The current results show that UCB significantly stimulates cardiolipin oxidation, ĪĪØm depolarization, which underscores the noxious effect of UCB on mitochondria. Bilirubin was previously reported to be toxic to astrocytes and neurons by causing damage to mitochondria and thus, leading to impaired energy metabolism and apoptosis28. Furthermore, oxidative stress can cause perturbations in intracellular Ca2+ homeostasis. At high concentrations (>1āĪ¼M), Ca2+ could inhibit respiration, cause cardiolipin oxidation, mitochondria to undergo permeability transition and release pro-apoptotic proteins leading to cell death29,30. Cardiolipin oxidation is a key event in mitochondrial dysfunction as well as in the early stages of the mitochondrial apoptotic pathway as it is the primary target of ROS31. Externalization of oxidized cardiolipin from the inner mitochondrial membrane to the outer, allows mPTP formation resulting in cyt. c leakage into the cytosol32. In the present study UCB was found to bring forth a remarkable increase in intracellular Ca2+ concentration and cardiolipin oxidation. Endoplasmic reticulum stress-induced perturbation in Ca2+ homeostasis and ROS generation leading to hepatocyte apoptosis has been observed as a pathological event in several liver diseases. The transcription factor C/EBP homologous protein, the mitogen activated protein kinase c-jun N-terminal kinase (JNK), Bcl-2 family proteins and caspase activation has been linked to ER-stress induced apoptosis33,34. UCB was found to decrease the activity of complex II and IV of ETC, increase mPTP formation and cytosolic cyt. c. Further, it was also observed to up-regulate pro-apoptotic Bcl-2 family protein Bax and tBid, down-regulate Bcl-2, activate caspases-9 and -3 and increase protein phosphorylation in a dose-dependent manner in platelets. Activated caspases can cleave a wide range of cellular substrates to expose new sites on proteins for phosphorylation19 and hence, increased levels of tyrosine-phosphorylated proteins are observed in UCB-treated platelets. Furthermore, UCB was capable of provoking PS externalization, an indispensable biochemical feature of an apoptotic cell that signals the phagocytic cells, finally leading to cell death. To rule out the possibility of exteriorized PS as a consequence of platelet activation, platelet aggregation studies were carried out wherein UCB was found to have no influence on platelet aggregation. UCB can easily enter the cells by simple passive diffusion and trigger toxicity35. Hence, overall toxic effects of UCB towards platelets were further confirmed by the positive results for MTT and LDH assays, decreased G6PDH activity and lysosomal stability and increased GGT activity. G6PDH is involved in the generation of NADPH responsible for the recycling of glutathione and thus combating oxidative stress. Decreased platelet G6PDH activity in oxidative stress-induced pathologic conditions and in neonatal indirect hyperbilirubinemia has been reported36,37. Similarly, GGT is shown to cleave GSH into glutamic acid and cysteinylglycine, resulting in GSH depletion; hence, elevated levels of GGT indicate ongoing oxidative stress38. If the platelets are undergoing apoptosis, it should be cleared by the reticuloendothelial system through phagocytosis. Several studies have demonstrated the phagocytosis of apoptotic platelets using macrophage39,40,41. In the present study, phagocytosis of apoptotic platelets by macrophages was observed and confirms the UCB induced platelet apoptosis. Altogether, the findings suggest the platelet pro-apoptotic nature of UCB in vitro. The study was further extended to in vivo PHZ-induced hyperbilirubinemia rat model as well as hyperbilirubinemic human subjects. Interestingly, in both the cases it was found that there was not only a marked decrease in platelet count, but also all the above-mentioned events of mitochondrial apoptotic pathway were observed. These findings provide a substantial evidence for the pro-apoptotic effect of UCB in vivo.
The next phase of the study aimed to probe the molecular mechanism of UCB-induced platelet apoptosis. Various stress stimuli including ROS can activate JNK and p38 MAPK signalling. Platelets have also been shown to comprise of key signalling proteins (Bcl family proteins), which regulate mitochondria-mediated apoptosis34. Tang et al.42 have also reported the phosphorylation of p38 in human platelets by elevated glucose levels, which in turn phosphorylate p53. Both expression of p53 and its phosphorylation are shown both in mitochondrial and cytosolic platelet fractions43. In addition, it is demonstrated that activated p53 induces mitochondrial damage by interacting with Bcl-xL. It was studied using pifithrin-Ī¼, a p53-specific inhibitor that inhibits p53 binding affinity towards mitochondrial Bcl-xL/ Bcl-2 as well as inhibits p53-mediated mitochondrial dysfunction and apoptosis44. On the other hand, it was previously reported that UCB induces oligodendrocyte precursor cell death via JNK activation, mitochondrial dysfunction and ROS generation45. In our study, UCB was found to trigger the phosphorylation of p38 and p53 in a dose-dependent manner. From the in vitro findings of the study, the molecular pathway was worked out as: UCB induces mitochondrial ROS generation, which in turn activates p38 and then causes the downstream activation of p53, eventually resulting in up-regulation of pro-apoptotic protein Bax and down-regulation of pro-survival proteins Bcl-2 and phospho Bad. Further, specific inhibitors of p38 and p53 evidently demonstrate that UCB induces apoptosis in platelets via p38 and p53 activation. Hence, we clearly demonstrate that activation of p53 is the ultimate key downstream event in UCB-driven apoptosis in platelets. In addition, we also reveal that mitochondria are the chief resources for ROS production in platelets and are accountable for UCB-induced p38 and p53 activation and platelet toxicity by using Mito-TEMPO. The findings were further confirmed through in vivo rat model as well as hyperbilirubinemic human subjects.
The study also demonstrates that UCB induces apoptosis in platelets, but did not activate them as evident from platelet aggregation assay. UCB did not affect platelet aggregating properties, however it inhibited collagen-induced platelet aggregation in washed platelets at lower doses. Previous studies have reported that UCB at lower doses had no effect on platelet aggregation of platelet rich plasma, but it did inhibit at higher doses. This justifies that the albumin and some plasma proteins would bind UCB through non-covalent interaction, thus nullifying its effect on platelets. Further, the effect of UCB on platelet aggregation with collagen was confirmed with the platelet adhesion assay. In which, a significant inhibition UCB pre-treated collagen-coated wells and UCB pre-treated WP directly added to collagen-coated wells are observed. This might be due to the existence of an interaction between UCB and collagen. The results of the present study are contradictory to those presented in earlier studies that showed platelets undergoing activation upon exposure to increased levels of UCB in washed platelets46,47. This disparity in the results could be attributed to the difference in the protocols and solubility of UCB as discussed by Kundur et al.48.
Apoptotic platelets release negatively charged PS-positive plasma membrane vesicles, which are known as microparticles (MPs). MPs not only provide surface area for thrombus formation but also stimulate coagulation and influence vascular functions due to their pro-inflammatory nature. These events eventually lead to development of thrombotic disorders, CVDs and arthritis. Therefore, the future perspective of the study is to probe the link between hyperbilirubinemia and other pathophysiological conditions, especially inflammatory diseases. Overall the current study gives a better understanding of a novel approach to deal with thrombocytopenia associated with liver diseases with elevated UCB. Unconjugated hyperbilirubinemia should be taken more seriously and not treated as just one of the symptoms of liver diseases. Since UCB decreases platelet count by triggering platelet apoptosis via mitochondrial ROS-induced activation of p38 and p53, platelet protective antioxidants could be used to deal with thrombocytopenia in liver diseases. Moreover, the existing medications for liver diseases might themselves stimulate platelet apoptosis. Therefore, it is necessary to deal with hyperbilirubinemia and thrombocytopenia in human pathologies associated with the conditions of elevated unconjugated bilirubin.
Materials and Methods
Chemicals
All fluorogenic, non-fluorogenic dyes, Histopaque-1077 and standard platelet agonists were from Sigma Chemicals, USA. Monoclonal anti-cytochrome c antibody was from Epitomics, USA. z-DEVD-fmk, SB203580, ABT-737, Pifithrin-Ī¼, antibodies against caspase-3, caspase-9, Bax, Bcl-2, BAD, phospho-BAD (ser136), Bid and tBid were from Santa Cruz Biotechnology, Inc., USA. Phospho-p38 (Thr180/Tyr182), p38, phospho-p53 (ser15), p53, COX-IV and Ī²-tubulin antibodies were from Cell Signalling and Technology, USA. TNF-Ī±, IL-1Ī² and IL-6 antibodies were from Komabiotech, Korea. IL-10 and IL-23 antibodies were from Thermo Fisher Scientific, USA. LysoSensor green DND-189 was from Molecular probes, USA. Phenylhydrazine hydrochloride (PHZ) was from Fisher Scientific, USA. Commercial bilirubin and LDH estimation kits were purchased from Agappe Diagnostics Limited, India.
Preparation of UCB solution
Unconjugated bilirubin (UCB, purity- 99%) used for the study was purchased from Sisco Research Laboratories, India. Purity of UCB was again confirmed by RP-HPLC which showed 99% pure (Data not shown). UCB was dissolved in dimethyl sulfoxide (DMSO)48, as UCB is partially soluble and forms precipitate in 0.02% cetyltrimethylammonium bromide (CTAB) and 0.1 N NaOH. Further, UCB solution was centrifuged at 4000āĆāg to pellet the precipitate and the concentration was determined by spectrophotometric method and using bilirubin estimation kit according to the manufacturerās protocol. We found that there was no change in UCB concentration solubilised in DMSO, but there was decreased UCB concentration in CTAB and NaOH solubilised UCB. (Supplementary Figure 5). Therefore for the further assays, UCB was dissolved in DMSO to obtain a 10āmM stock solution from which the working solutions of 5āmM and 1āmM were prepared in DMSO. The final concentration of DMSO was limited to less than 4% in the reaction mixture. The UCB stock/working solutions were prepared in amber tubes and reactions were performed in dark/dim light to avoid photo oxidation of UCB. UCB solution was prepared freshly every time within 10āmin, before treatment.
Preparation of platelet-rich plasma and washed platelets
Venous blood was drawn from healthy volunteers (drug-free/non-smokers) with informed consent and was approved as per the guidelines of Institutional Human Ethical Committee (IHEC-UOM No. 40Res/2013-14), University of Mysore, Mysuru. It was immediately mixed with acid-citrate dextrose (ACD) anti-coagulant (2.5% tri-sodium citrate, 2% D-glucose, 1.5% citric acid) in the ratio of 7:1 (blood : ACD, v/v). The anti-coagulated blood was then centrifuged at 90āĆāg at 37āĀ°C for 15āmin and the supernatant thus obtained was the platelet-rich plasma (PRP). The PRP was centrifuged at 700āĆāg for 15āmin at 37āĀ°C. The platelet pellet thus obtained was suspended in CGS buffer (123āmM NaCl, 33āmM D-glucose, 13āmM tri-sodium citrate, pH 6.5) and washed thereafter at 700āĆāg for 15āmin at 37āĀ°C. The previous washing step was repeated one more time. Finally, the washed platelets (WP) were suspended in Tyrodeās buffer (2.5āmM HEPES, 150āmM NaCl, 2.5āmM KCl, 12āmM NaHCO3, 1āmM CaCl2, 1āmM MgCl2, 5.5āmM D-glucose, pH 7.4)49. The cell count was determined in both PRP and WP suspension using a Neubauer chamber and adjusted to 5āĆā108 cells/mL in the final suspension using platelet poor plasma/Tyrodeās buffer.
Recruitment of hyperbilirubinemia (HB) subjects
HB subjects (nā=ā35) from Government Ayurvedic Medical College (GAMC), Mysuru were recruited for the study (Supplementary Table 1). HB subjects were recruited purely on basis of serum bilirubin level (reference range: 1.5ā15āmg of total bilirubin/dL of blood). Cirrhotic, viral and obstructive jaundice patients were excluded from the study. All the experiments were in accordance and approved by the Institutional Human Ethical Committee (IHEC-UOM No. 47Res/2014-15), University of Mysore, Mysuru. Venous blood was drawn from HB subjects and non-smoking/drug-free healthy subjects (HS, nā=ā21) by trained professionals from GAMC, Mysuru. Platelets and serum samples were separated for further analysis. About 1/3 fraction of the drawn blood was used to separate serum and the remaining 2/3 fraction was immediately mixed with ACD in the ratio of 7:1 (blood: ACD, v/v). About 100āĪ¼L aliquots of anti-coagulated blood was used to determine cell count in an automated hemo-analyser (Sysmex KX-21, Japan). Further, the remaining anti-coagulated blood was then centrifuged to obtain PRP and WP. The cell count was determined in both PRP and WP suspension using a Neubauer chamber and adjusted to 5āĆā108 cells/mL in the final suspension using platelet poor plasma/Tyrodeās buffer. Serum was stored at ā80āĀ°C until further use.
Phenylhydrazine (PHZ)-induced hyperbilirubinemia
Wistar rats of 4ā6 week old were used for the experiments. The experimental animals were grouped as: Group I - Saline control; Group II - PHZ-induced and each group consisted of 6 rats. Unconjugated hyperbilirubinemia was induced by injecting a single dose of PHZ (75āmg/kg) intraperitoneally and housed for two days50. Thereafter, animals were euthanized and blood was collected immediately through cardiac puncture. About 1/3 fraction of the collected blood was used to separate serum and the remaining 2/3 fraction was immediately mixed with ACD in the ratio of 5:1 (blood: ACD, v/v). About 100āĪ¼L aliquots of anti-coagulated blood was used to determine cell count in an automated hemo-analyser. Further, the remaining anti-coagulated blood was then centrifuged to obtain PRP and WP. The cell count was determined in both PRP and WP suspension using a Neubauer chamber and adjusted to 5āĆā108 cells/mL in the final suspension using platelet poor plasma/Tyrodeās buffer. Liver and spleen tissues from experimental animals were harvested and rinsed in ice cold saline, serum was separated and stored at ā80āĀ°C for further analysis.
Flow cytometry
WP (1āĆā106 cells/mL) were independently treated with UCB (0ā200āĪ¼M) and ABT-737 (1āĪ¼M) as positive control and incubated for 30āmin at 37āĀ°C. After incubation, cells were stained using CM-H2DCFDA, JC-1, NAO (10-nonyl acridine orange) and Annexin V-FITC, washed and analysed using FACSVerseTM flow cytometer (BD Biosciences, USA).
Determination of reactive oxygen species (ROS)
Endogenous ROS generation in platelets was determined according to the method of Thushara et al.51. WP (5āĆā106 cells/mL) were taken separately in 96-well plates and treated with A23187 (1āĪ¼M) as positive control or UCB (0ā200āĪ¼M) as test and the final volume was made up to 200āĪ¼L with Tyrodeās buffer and incubated for 30āmin at 37āĀ°C. For inhibition studies, pre-loaded platelets with UCB (200āĪ¼M), were incubated with Mito-TEMPO (10āĪ¼M) and incubated for 30āmin at 37āĀ°C. The untreated and treated platelets were then incubated with CM-H2DCFDA (10āĪ¼M) for 30āmin at 37āĀ°C and the fluorescence was recorded using a Varioskan multimode plate reader (Thermo Scientific, USA) by exciting the samples at 488ānm and measuring the resulting fluorescence at 530ānm. The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Estimation of intracellular calcium
Intracellular Ca2+ concentration was measured in WP according to the method of Thushara et al.51. WP (5āĆā106 cells/mL) were treated with A23187 (1āĪ¼M) or UCB (0ā200āĪ¼M) and the final volume was made up to 200āĪ¼L with Tyrodeās buffer containing 1āmM Cacl2 and incubated for 30āmin at 37āĀ°C followed by incubation with fura-2/AM (2āĪ¼M). The fura-2/AM fluorescence was determined by exciting the samples at 340 and 380ānm and the resulting fluorescence was measured at 500ānm. Data were presented as absorption ratios (340/380ānm). The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Determination of changes in mitochondrial membrane potential (ĪĪØm)
The cationic dye JC-1 was used to detect changes in the ĪĪØm according to the method of Thushara et al.51. WP (5āĆā106 cells/mL) were treated with UCB (0ā200āĪ¼M) and the final volume was made up to 200āĪ¼L with Tyrodeās buffer and incubated for 30āmin at 37āĀ°C. The samples were loaded with JC-1 (2āĪ¼M) at 37āĀ°C for 10āmin. The cells were then excited at 488ānm and emission was detected at 585ānm for JC-1 aggregates and 516ānm for JC-1 monomers using multimode plate reader. Data were presented as emission ratios (585/516). The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Assessment of cardiolipin peroxidation
10-Nonyl acridine orange (NAO), a fluorescent probe was used to detect peroxidation of cardiolipin. NAO loses its affinity for peroxidised cardiolipin resulting in decreased fluorescence. WP (5āĆā106 cells/mL) were processed as discussed above with either A23187 (1āĪ¼M) or UCB (0ā200āĪ¼M) and then loaded with NAO (1āĪ¼M) and incubated for 30āmin at 37āĀ°C. After incubation the fluorescence was recorded by exciting the samples at 499ānm and emission was recorded at 530ānm52. The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Assessment of mPTP formation
Formation of mPTP in platelets was assessed using calcein AM53. WP (5āĆā106 cells/mL) were treated with A23187 (1āĪ¼M), UCB (0ā200āĪ¼M) and incubated for 30āmin at 37āĀ°C. Following incubation calcein AM (1āĪ¼M) along with CoCl2 (1āmM) for quenching cytosolic calcein fluorescence was added. Fluorescence was recorded by exciting the samples at 488ānm and emission was detected at 585ānm. The same procedure was followed for untreated WP obtained from HS and HB.
Determination of PS externalization
PS externalization was determined according to the method of Thushara et al.51. WP (5āĆā106 cells/mL) were treated with UCB (0ā200āĪ¼M) and for inhibition studies, pre-loaded platelets with UCB (200āĪ¼M) were incubated with Pifithrin-Ī¼ (5āĪ¼M)/z-DEVD-fmk (5āĪ¼M) and the final volume was made up to 200āĪ¼L with Tyrodeās buffer and incubated for 30āmin at 37āĀ°C. The untreated and treated platelets were stained with Annexin V-FITC (0.6āĪ¼g/mL) and fluorescence was measured in a multimode plate reader by exciting the samples at 496ānm and emission was recorded at 516ānm. The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Subcellular fractionation of platelets
UCB (0ā200āĪ¼M) treated WP (1āĆā108 cells/mL) were suspended in isolation buffer (225āmM mannitol, 75āmM sucrose, 0.1āmM EGTA, 1āmg/mL fatty acid-free BSA, 10āmM HEPES-KOH, proteinase inhibitor mixture, phosphatase inhibitor mixture, pH 7.4) and were frozen in liquid nitrogen for 1āmin and then thawed at 37āĀ°C for 3āmin. This freeze-and-thaw sequence was repeated for two more cycles and then the samples were centrifuged at 700āĆāg for 10āmin at 4āĀ°C. The supernatant was further centrifuged at 15,000āĆāg for 10āmin at 4āĀ°C. The pellet was regarded as the mitochondria-rich fraction and the supernatant was considered as the cytosolic fraction54.
Immunobloting
To determine the expression level of various proteins, WP (1āĆā108 cells/mL) were treated with UCB (0ā200āĪ¼M) and for inhibition studies, UCB (200āĪ¼M) treated platelets were pre-incubated with Mito-TEMPO (2āĪ¼M)/SB203580 (2āĪ¼M)/Pifithrin-Ī¼ (5āĪ¼M)/z-DEVD-fmk (5āĪ¼M)/ BSA (5āmg/mL) and incubated at 37āĀ°C for 30āmin. Further, platelet suspensions were lysed by adding 10āĪ¼L lysis buffer (20āmM Tris-HCl, pH 8, containing 0.5% Triton-X 100, 150āmM NaCl along with cocktail of protease inhibitors). Following centrifugation, the supernatants were separated on SDS-PAGE (4ā14%) and proteins were transferred onto PVDF membrane. After blocking, the blots were probed with antibodies against phospho-p38 (Thy180/Tyr182), p38, phospho-p53 (ser15), p53, cyt. c, caspase-9, caspase-3, Bax, Bcl-2, BAD, phospho BAD, tBid, COX-IV, GAPDH and Ī²-tubulin at 4āĀ°C overnight. Blots were then incubated with horseradish-peroxidase (HRP)-conjugated secondary antibody and developed by enhanced chemiluminescence method and the bands were visualized using chemiluminescence imaging system (Alliance 2.7, Uvitec, UK). For re-probing, blots were incubated in stripping buffer (200āmM glycine, pH-2.2, 1% Tween-20 and 0.1% SDS) for 2āmin and washed with TBST. Blots were again treated with stripping buffer for 5āmin and washed thrice with TBST, 2āmin each wash. The stripped membrane was blocked in TBST containing 5% non-fat milk powder over night at 4āĀ°C and probed with desired antibodies54. The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.
Electron transport chain (ETC) assays
Assays for ETC complexes I, II, III and IV were performed as described previously55. Briefly, platelet mitochondria were isolated as described above and were treated with UCB (0ā200āĪ¼M) along with vehicle control (DMSO). Complex I activity (NADH: ubiquinone oxidoreductase) was measured by the oxidation of NADH and the activity was expressed as mM NADH oxidised/min/mg protein. Complex II activity (succinate: ubiquinone oxidoreductase) was determined by the reduction of dichlorophenolindophenol (DCIP) and the activity was expressed as mM DCIP reduced/min/mg protein. Complex III activity (coenzyme Q: cytochrome c-oxidoreductase) was measured by cyt. c reduction and the activity was expressed as mM cyt. c reduced/min/mg protein. Complex IV (cyt. c oxidase) activity was measured by the oxidation of cyt. c and the activity was expressed as first-order rate constant (k) of mM cyt. c oxidized/min/mg protein.
Evaluation of lysosomal acidity
For monitoring the changes in pH of lysosomes, pH sensitive dye LysoSensor Green DND-189 (LSG) was used56. LSG accumulates in intact lysosomes and its fluorescence decreases upon lysosome alkalinisation. WP (5āĆā106 cells/mL) were independently treated with UCB (0ā200āĪ¼M) or A23187 (1āĪ¼M) and incubated for 30āmin at 37āĀ°C. Further untreated and treated samples were loaded with LSG (1āĪ¼M) and incubated for 30āmin at 37āĀ°C. Fluorescence was recorded by exciting the samples at 488ānm and emission at 510ānm. The same procedure was followed for untreated WP obtained from HS and HB.
Estimation of glucose-6-phosphate dehydrogenase (G6PDH) enzyme activity
G6PDH activity was determined in WP treated with either A23187 (1āĪ¼M) or UCB (0ā200āĪ¼M) and incubated for 30āmin at 37āĀ°C. After incubation, platelets were pelleted, suspended in distilled water and lysed by sonication. Activity was monitored by adding platelet lysate (50āĪ¼g) to 1āmL reaction volume (50āmM Tris-HCl, pH 7.5 containing 3.8āmM NADP, 3.3āmM glucose-6-phosphate and 6.3āmM MgCl2). Increase in absorbance was recorded at 340ānm for 3āmin due to NADP+-dependent glucose 6-phosphate transformation and the activity was expressed as mM NADPH formed/min/mg protein57.
Measurement of Ī³-Glutamyltransferase (GGT) activity
To determine the GGT activity, WP (5āĆā106 cells/mL) were treated with either A23187 (1āĪ¼M) or UCB (0ā200āĪ¼M) and incubated for 30āmin at 37āĀ°C. After incubation, platelets were pelleted, suspended in distilled water and lysed by sonication. The resulting lysate was used to determine GGT activity according to the method of Sener et al.16. Activity was monitored in 1āmL reaction volume consisting of Ī³-glutamyl-p-nitroanilide (4āmM), glycylglycine (40āmM) in Tris-HCl buffer (185āmM, pH 8.2). The results were calculated using molar extinction coefficient for p-nitroanilide (9,900āMā1cmā1) at 405ānm expressed as mM p-nitroanilide formed/min/mg protein. The same procedure was followed for untreated WP obtained from HS and HB.
Measurement of LDH leakage
WP (5āĆā106 cells/mL) were treated either with UCB (0ā200āĪ¼M) or with A23187 (1Ī¼M) for 30āmin at 37āĀ°C and platelets were pelleted by centrifugation at 3,700āĆāg for 10āmin. Supernatants were used to detect LDH release by using LDH kit, according to the manufacturerās protocol. The assay was performed in a time course of decrease in NADH absorbance at 340ānm for 3āmin.
Evaluation of platelets viability by MTT assay
MTT colorimetric assay was performed to assess the cell viability58. WP (1āĆā106 cells/mL) was taken separately in 96-well microtiter plates and treated with either A23187 (1āĪ¼M) or with UCB (0ā200āĪ¼M) and the final volume was made up to 200āĪ¼L with Tyrodeās buffer. After 30āmin of incubation, 250āĪ¼M of MTT was added and incubated for additional 3āh. Thereafter, MTT was removed and remaining formazan crystals were completely dissolved in DMSO and the absorbance was recorded at 570ānm.
Platelet aggregation assay
Platelet aggregation was determined by turbidimetric method with a dual channel Chrono-log model 700-2 aggregometer (Havertown, USA). Briefly, 250āĪ¼L of WP (1.5āĆā108 cells/mL) was taken in siliconized glass cuvette and pre-incubated for 5āmin at 37āĀ°C with UCB (0ā200āĪ¼M) and the aggregation was initiated by the addition of collagen (2āĪ¼g/mL). The aggregation was then followed with constant stirring at 1200ārpm for 6āmin at 37āĀ°C59.
Platelet adhesion assay
Platelet adhesion assay was performed according to the method by Kumar et al.60. Briefly, 20āĪ¼g of collagen type I in 200āĪ¼L PBS was added independently to 96-well polystyrene microtiter plates and kept for 16āh at 4āĀ°C. The coated wells were then blocked by adding 200āĪ¼L of 1% BSA in PBS for 1āh at 37āĀ°C and washed with PBS. In the first set of experiment, UCB (0ā200āĪ¼M) was directly added to the collagen type I coated wells, incubated for 10āmin followed by washing with PBS and then WP (1.5āĆā108 cells/mL) was added. In the second set of experiment, WP pre-treated with UCB (0ā200āĪ¼M) for 10āmin at 37āĀ°C was added to the pre-coated collagen type I wells. The total reaction volume was made up to 200āĪ¼L with PBS. The reaction mixture was incubated at 37āĀ°C for 90āmin and then washed with PBS. The adherent platelets were then lysed by adding 150āĪ¼L lysis buffer (100āmM citrate buffer pH 5.4 containing 5āmM p-nitrophenyl phosphate and 0.1% Triton X-100) at 37āĀ°C for 90āmin. The reaction was terminated by inactivating the membrane bound acid phosphatase with the addition of 100āĪ¼L stopping reagent (2 N NaOH). The colour developed was measured at 405ānm. Platelet adhesion was expressed as percent adhesion, considering PBS-treated platelet suspension as 100%.
Macrophage engulfment assay
Engulfment of apoptotic platelets from monocyte derived macrophage was assessed as described by Kumari et al.61. In brief, blood drawn from healthy donors (drug-free/non-smokers) was collected in citrate containing tube and peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque-1077 according to the standard protocol from the manufacturer. The obtained PBMCs were plated onto polystyrene culture flasks for 4āh at 37āĀ°C and washed with PBS to remove non-adherent lymphocytes. Monocytes (200,000 in 500āĪ¼L volume) were then plated on to 6-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum and cultured for 7 days to obtain monocyte-derived macrophages. Control and UCB (200āĪ¼M) - treated platelets labelled with calcein-AM were incubated with adherent monolayer of monocyte-derived macrophages for 45āmin. Following the incubation period, the phagocyte monolayer was washed to remove non-interacting platelets and adherent macrophages were removed by treatment with trypsin at 37āĀ°C for 5āmin, followed by 5āmM EDTA at 4āĀ°C. Monocyte-derived macrophages were recovered by trypsin-EDTA treatment for 15āmin at 37āĀ°C and subjected to flow cytometric analysis.
Histological assessment of liver and spleen tissues
The liver and spleen tissues were dissected out and blotted free of blood, rinsed in ice-cold saline and fixed in 10% buffered formalin for overnight. The tissue samples were subjected to dehydration by processing with different grades of alcohol and chloroform mixture. The processed tissues were embedded in paraffin wax and sections (5āĪ¼m thickness) were prepared, stained with hematoxylin- eosin dye (H&E) and observed under an Axio imager.A2 microscope (Oberkochen, Germany) and photographed.
Protein estimation
The protein estimation was done according to the method of Lowry et al.62 using BSA as standard.
Statistical analysis:
All the results were expressed as mean Ā± SEM of five independent experiments. Statistical significance among groups was determined by t-test and one way analysis of variance (ANOVA) followed by Tukeyās test for comparison of means as appropriate.
Additional Information
How to cite this article: NaveenKumar, S. K. et al. Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation. Sci. Rep. 5, 15045; doi: 10.1038/srep15045 (2015).
References
Sato, H., Adachi, Y. & Koiwai, O. The genetic basis of Gilbertās syndrome. Lancet. 347, 557ā558 (1996).
Aono, S. et al. Identification of defect in the genes for bilirubin UDP-glucuronosyl-transferase in a patient with Crigler-Najjar syndrome type II. Biochem. Biophys. Res. Commun. 197, 1239ā1244 (1993).
Maruo, Y., Nishizawa, K., Sato, H., Doida, Y. & Shimada, M. Association of neonatal hyperbilirubinemia with bilirubin UDP-glucuronosyltransferase polymorphism. Pediatrics. 103, 1224ā1227 (1999).
Hancox, S. H. & Smith, B. C. Liver disease as a cause of thrombocytopenia. Q. J. Med. 106, 425ā431 (2012).
Afdhal, N. et al. Thrombocytopenia associated with chronic liver disease. J. Hepatol. 48, 1000ā1007 (2008).
Thushara, R. M. et al. Therapeutic drug-induced platelet apoptosis: an overlooked issue in pharmacotoxicology. Arch. Toxicol. 88, 185ā198 (2014).
Leytin, V. Apoptosis in the anucleate platelet. Blood. Rev. 26, 51ā63 (2012).
Thushara, R. M. et al. Differential action of phytochemicals on platelet apoptosis: a biological overview. Curr. Med. Chem. 20, 1018ā1027 (2013).
Bosma, P. J. Inherited disorders of bilirubin metabolism. J. Hepatol. 38, 107ā117 (2003).
Erlinger, S., Arias, I. M. & Dhumeaux, D. Inherited disorders of bilirubin transport and conjugation: new insights into molecular mechanisms and consequences. Gastroenterology. 146, 1625ā1638 (2014).
Neuzil, J. & Stocker, R. Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J. Biol. Chem. 269, 16712ā16719 (1994).
Asad, S. F., Singh, S., Ahmad, A. & Hadi S. M. Bilirubin-Cu(II) complex degrades DNA. Biochim. Biophys. Acta. 1428, 201ā208 (1999).
Asad, S. F., Singh, S., Ahmad, A., Khan, N. U. & Hadi, S. M. Prooxidant and antioxidant activities of bilirubin and its metabolic precursor biliverdin: a structure-activity study. Chem. Biol. Interact. 137, 59ā74 (2001).
Stocker, R., Yamamoto, Y., McDonagh, A. F., Glazer, A. N. & Ames, B. N. Bilirubin is an antioxidant of possible physiological importance. Science. 235, 1043ā1046 (1987).
Pietraforte, D. et al. Redox control of platelet functions in physiology and pathophysiology. Antioxid Redox Signal. 21, 177ā193 (2014).
Sener, A., Cevik, O., Yanikkaya-Demirel, G., Apikoglu-Rabus, S. & Ozsavci, D. Influence of platelet Ī³-glutamyltransferase on oxidative stress and apoptosis in the presence of holo-transferrin. Folia. Biol. 58, 193ā202 (2012).
Ide, T. et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ. Res. 88, 529ā535 (2001).
Zhang, Y. et al. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J. Biol. Chem. 265, 16330ā16336 (1990).
Dix, M. M. et al. Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome. Cell. 150, 426ā440 (2012).
Rukoyatkina, N., Mindukshev, I., Walter, U. & Gambaryan. S. Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists. Cell. Death. Dis. 4, e931 (2013).
Zhang, L., Yuan, B., Wang, H. & Gao, Y. Therapeutic effect of Agaricus brasiliensis on phenylhydrazine-induced neonatal jaundice in rats. Biomed Res Int. 2015, 651218 (2015).
Giannini, E. G. & Savarino, V. Thrombocytopenia in liver disease. Curr. Opin. Hematol. 15, 473ā480 (2008).
Otterbein, L. E. & Choi, A. M. Hemeoxygenase: colors of defense against cellular stress. Am. J. Physiol. Lung. Cell. MolPhysiol. 279, 1029ā1037 (2000).
Maines, M. D. Bile pigments: newcomers to the cell signaling arena. Toxicol. Sci. 71, 9ā10 (2003).
Rodrigues, C. M., SolĆ”, S. & Brites, D. Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology. 35, 1186ā1195 (2002).
Brito, M. A. et al. N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity. Mol. Med. 16, 372ā380 (2010).
Chandel, N. S. Mitochondria as signaling organelles. BMC. Biol. 2, 12ā34 (2014).
Ostrow, J. D., Pascolo, L. & Tiribelli, C. Mechanisms of bilirubin neurotoxicity. Hepatology. 35, 1277ā1280 (2002).
Pandya, J. D., Nukala, V. N. & Sullivan, P. G. Concentration dependent effect of calcium on brain mitochondrial bioenergetics and oxidative stress parameters. Front Neuroenergetics. 5, 10 (2013).
Thushara, R. M. et al. Biologicals, platelet apoptosis and human diseases: An outlook. Crit. Rev. Oncol. Hematol. 93, 149ā158 (2015).
Korytowski, W., Basova, L. V., Pilat, A., Kernstock, R. M. & Girotti, A. W. Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis. J. Biol. Chem. 286, 26334ā26343 (2011).
Petrosillo, G., Moro, N., Ruggiero, F. M. & Paradies, G. Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release. Free. Radic. Biol. Med. 47, 969ā974 (2009).
Dara, L., Ji, C. & Kaplowitz, N. The contribution of endoplasmic reticulum stress to liver diseases. Hepatology. 53, 1752ā1763 (2011).
Vanags, D. M., Orrenius, S. & Aguilar-Santelises, M. Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis. Br. J. Haematol. 99, 824ā831 (1997).
Gazzin, S., Strazielle, N., Tiribelli, C. & Ghersi-Egea, J. F. Transport and metabolism at blood-brain interfaces and in neural cells: relevance to bilirubin-induced encephalopathy. Front Pharmacol. 3, 89 (2012).
Harold, W., Thomas, M., Liesel, B. & Lewis, G. Glucose-&Phosphate Dehydrogenase Activity in Platelets. Blood. 17, 314ā318 (1961).
Atay, E., Bozaykut, A. & Ipek, I. O.Glucose-6-phosphate dehydrogenase deficiency in neonatal indirect hyperbilirubinemia. J. Trop. Pediatr. 52, 56ā58 (2006).
FeketeovĆ”, L. et al. Effect of methotrexate on inflammatory cells redistribution in experimental adjuvant arthritis. Rheumatol. Int. 32, 3517ā3523 (2012).
Nayak, M. K., Kulkarni, P. P. & Dash, D. Regulatory role of proteasome in determination of platelet life span. J. Biol. Chem. 288, 6826ā6834 (2013).
Nayak, M. K., Dash, A., Singh, N. & Dash, D. Aspirin delimits platelet life span by proteasomal inhibition. PLoS One. 9, e105049 (2014).
Mason, K. D. et al. Programmed anuclear cell death delimits platelet life span. Cell. 128, 1173ā1186 (2007).
Tang, W. H. et al. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane. J. Clin. Invest. 121, 4462ā4476 (2011).
Tang, W. H. et al. Aldose reductase-mediated phosphorylation of p53 leads to mitochondrial dysfunction and damage in diabetic platelets. Circulation. 129, 1598ā1609 (2014).
Strom, E. et al. Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat. Chem. Biol. 2, 474ā479 (2006).
Malhi, H. & Kaufman, R. J. Endoplasmic reticulum stress in liver disease. J. Hepatol. 54, 795ā799 (2011).
Moiny, G., Thirion, A. & Deby, C. Bilirubin induces platelet aggregation. Thromb. Res. 59, 413ā416 (1990).
Maurer, H. M. & Caul, J. Influence of bilirubin on human platelets. Pediatr. Res. 6, 136ā144 (1972).
Kundur, A. R., Bulmer, A. C. & Singh, I. Unconjugated bilirubin inhibits collagen induced platelet activation. Platelets. 25, 45ā50 (2014).
Zhang W. et al. Cisplatin induces platelet apoptosis through the ERK signaling pathway. Thromb. Res. 130, 81ā91 (2012).
Kamisah, Y., Lim, J. J., Lim, C. L. & Asmadi, A. Y. Inhibitory effects of palm tocotrienol-rich fraction supplementation on bilirubin-metabolizing enzymes in hyperbilirubinemic adult rats. PLoS. One. 9, e89248 (2014).
Thushara, R. M. et al. Sesamol induces apoptosis in human platelets via reactive oxygen species-mediated mitochondrial damage. Biochimie. 95, 2060ā2068 (2013).
Ferlini, C. & Scambia, G. Assay for apoptosis using the mitochondrial probes, Rhodamine 123 and 10-N-nonyl acridine orange. Nat. Protoc. 2, 3111ā3114 (2007).
Petronilli, V. et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys. J. 76, 725ā734 (1999).
Sundaram, M. S. et al. Tamarind seed (Tamarindus indica) extract ameliorates adjuvant-induced arthritis via regulating the mediators of cartilage/bone degeneration, inflammation and oxidative stress. Sci. Rep. 5, 11117 (2015).
Paul, M. et al. Methotrexate promotes platelet apoptosis via JNK-mediated mitochondrial damage: alleviation by N-Acetylcysteine and N-Acetylcysteine amide. PLoS. One. 10, e0127558 (2015).
Zhang, S., Wang, Y. & Li, S. J. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion. Biochem. Biophys. Res. Commun. 448, 424ā429 (2014).
Sundaram, M. S. et al. Tamarind seed extract mitigates the liver oxidative stress in arthritic rats. Food. Funct. 5, 587ā597 (2014).
Girish, K. S. et al. Melatonin elevates apoptosis in human platelets via ROS mediated mitochondrial damage. Biochem. Biophys. Res. Commun. 438, 198ā204 (2013).
Thushara, R. M. et al. Crocin, a dietary additive protects platelets from oxidative stress-induced apoptosis and inhibits platelet aggregation. Mol. Cell. Biochem. 373, 73ā83 (2013).
Kumar, M. S., Girish, K. S., Vishwanath, B. S. & Kemparaju, K. The metalloprotease, NN-PF3 from Naja naja venom inhibits platelet aggregation primarily by affecting Ī±2Ī²1 integrin. Ann. Hematol. 90, 569ā577 (2011).
Kumari, S., Chaurasia, S. N., Nayak, M. K., Mallick, R. L. & Dash, D. Sirtuin Inhibition Induces Apoptosis-like Changes in Platelets and Thrombocytopenia. J. Biol. Chem. 290, 12290ā12299 (2015).
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement using folin-phenol reagent. J. Biol. Chem. 193, 265ā275(1951).
Acknowledgements
NKSK thanks DBT, MSS and MP thanks UGC-BSR for research fellowship. Authors thanks Prof. Sathyanarayana Bhat, Principal and Mr. Dwarkesh, Lab technician, GAMC, Mysuru for helping in recruiting HB subjects for the study, Mr. Katkar GD, Mr. Jagadish S, University of Mysore, Mr. Mahesh Hegade, Mr. Kumar Somyajit, Ms. Sneha Saxena, IISc Bengaluru, Dr. Devaraja S, Tumkur University, Dr. Naveen S and Ms. Anusha MB, DFRL, Mysuru for their kind help. Authors also thanks Central instrumentation facility, Institution of excellence (IOE), University of Mysore, Mysuru and FACS facility, IISc, Bengaluru. Authors acknowledge the recognition of University of Mysore as a University with Potential for Excellence (UPE) from the University Grants Commission (UGC), Govt. of India. Authors also thank UPE-UGC and UGC-SAP for financial assistance.
Author information
Authors and Affiliations
Contributions
K.S.G., K.K., M.H. and K.S.R. designed the experiments; S.K.N., M.S.S. and M.P. performed the experiments; S.K.N., C.T., G.N., S.C.R., K.S.G., H.M. and Basapppa analyzed the data; K.S.G., K.K., R.M.T., H.M. and S.K.N. wrote the paper.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the articleās Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
NaveenKumar, S., Thushara, R., Sundaram, M. et al. Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation. Sci Rep 5, 15045 (2015). https://doi.org/10.1038/srep15045
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep15045
This article is cited by
-
Molecular mechanisms of bilirubin induced G1 cell cycle arrest and apoptosis in human breast cancer cell lines: involvement of the intrinsic pathway
Molecular Biology Reports (2022)
-
Risk factors analysis of hyperbilirubinemia after off-pump coronary artery bypass grafting: a retrospective observational study
Journal of Cardiothoracic Surgery (2021)
-
Endogenous toxic metabolites and implications in cancer therapy
Oncogene (2020)
-
Pamidronate decreases bilirubin-impaired cell death and improves dentinogenic dysfunction of stem cells from human deciduous teeth
Stem Cell Research & Therapy (2018)
-
Relation between unconjugated bilirubin and RDW, neutrophil to lymphocyte ratio, platelet to lymphocyte ratio in Gilbertās syndrome
SpringerPlus (2016)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.