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Altered proteomic pattern in platelets of rats with sepsis
Blood Cells, Molecules, and Diseases 48 (2012) 30–35
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Altered proteomic pattern in platelets of rats with sepsis Jin-yu Hu, Chang-Lin Li, Ying-Wei Wang ⁎ Department of Anesthesiology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
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Article history: Submitted 14 August 2011 Revised 26 September 2011 Available online 20 October 2011 (Communicated by M. Lichtman, M.D., 26 September 2011) Keywords: Platelet dysfunction Platelet proteome Two-dimensional electrophoresis Mass spectrometry Immunoblotting
a b s t r a c t Platelet dysfunction and thrombocytopenia are common responses to sepsis, but how sepsis changes platelet function is not completely understood. This is due, in part, to our lack of understanding of how sepsis alters platelet protein patterns. The aim of the present study, accordingly, was to investigate the response of the platelet proteome to sepsis. We applied proteomic technology to analyze platelet samples of rats with sepsis. Rats were divided into two groups: 1) sham surgery and 2) sepsis induced by cecal ligation and puncture (CLP) surgery. Platelet samples were collected from surviving rats 12 and 24 h after surgery, and platelet proteins were separated by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry (MS). In the CLP group, there were 20 spots that were statistically significantly different at 12 h. Of these spots, 16 spots were increased and four spots were decreased. At 24 h, there were six spots increased in the CLP group. Of the 26 spots, 12 proteins associated with platelet activation, acute phase proteins, cytoskeleton structure, and energy production were identified. Of interest, alpha-1-antitrypsin precursor (AAT) and ATP synthase beta subunit (ATPB) were both increased at 12 and 24 h of sepsis by 2-DE and immunoblotting. By providing the platelet profiles, our results demonstrate that this proteomic approach brings us closer to understanding how platelet dysfunction develops after sepsis. © 2011 Elsevier Inc. All rights reserved.
Introduction Sepsis is the systemic inflammatory response syndrome due to infection and is associated with multi-organ failure and a high mortality rate of approximately 20% to 30% [1]. The pathophysiology and molecular mechanisms of sepsis are complicated and remain unclear. Coagulation abnormalities and thrombocytopenia are common in sepsis [2]. Of interest, the severity of hemostatic disorders positively correlates with sepsis severity. In particular, a low platelet count predicts a poor prognosis [3]. Platelets play a central role in hemostasis and thrombosis, providing the first line of defense following injury. Platelets form thrombi as an essential mechanism of hemostasis [4]. In addition, there is increasing evidence that platelets actively participate in the pathogenesis of inflammation and infection [5]. Since platelets are anucleated cells and cannot be evaluated by examining mRNA levels, proteomics offers a powerful approach to provide information on how the platelet catalog changes during sepsis [6]. Proteomics allows the simultaneous monitoring of large sets of proteins and can identify patterns of protein alterations in the platelet protein data set [7]. In recent years, different proteomic approaches have been applied to investigate various aspects concerning platelet biology [8–10]. Specifically, the separation of proteins using 2-DE
⁎ Corresponding author. E-mail address: [email protected] (Y.-W. Wang). 1079-9796/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2011.09.010
with identification by MS has proven to be an effective way to analyze the platelet proteome [11,12]. The sepsis model of CLP in the rat exhibits many features of clinical sepsis [13,14]. By using the CLP model of sepsis, the present report contributes to a more complete understanding of platelet biology in sepsis, helping to build the foundation for future identification of new drug targets and therapeutic strategies. Materials and methods Animals and CLP model Male Sprague–Dawley (SD) rats (6–8 weeks old, 220–250 g) were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). The rats were acclimatized in our facility for 1 week before surgery. All procedures involving animals were conducted according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal protocol was approved by the Bioethics Committee of Shanghai Xinhua Hospital (Shanghai, China). On the day of surgery, the rats were randomly divided into the control sham surgery group (sham; n = 15); a CLP 12 h group (n= 15), and a CLP 24 h group (n = 15). To induce sepsis, the widely accepted CLP model described by Wichterman et al. in 1980 was used [13]. Briefly, after weighing the animals, rats were anesthetized by intraperitoneal injection of 0.5 mL/100 g of 7% chloraldurat. Under aseptic conditions, rats of the sepsis group underwent laparotomy, a
J. Hu et al. / Blood Cells, Molecules, and Diseases 48 (2012) 30–35
1 cm midline abdominal incision was made and the cecum was exposed. The cecum was ligated at two-thirds of the distance from the distal tip and was punctured twice with an 18-gauge needle. Thereafter, the incision was closed and 4 mL of normal saline was administered subcutaneously for fluid resuscitation. The sham operation consisted of laparotomy and cecal exposure without any more manipulation as a control for the surgery. After the surgery, the animals had free access to food and water. Of the 45 rats that underwent surgery, 36 rats survived: 15 rats in the sham group (100%), 12 rats in the 12 h CLP group (80%), and 9 rats in the 24 h CLP group (60%). Platelet isolation
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In-gel digestion and MALDI-TOF MS/MS analysis Twenty six protein spots were excised from the 2-DE gels and washed with water before digestion. Digestion and peptide extraction were performed according to reference methods [18]. 2.5 mL 0.5% TFA (v/v) was added to dissolve the peptides. The proteins were analyzed with a Bruker Autoflex III® MALDI-TOF/TOF 200 mass spectrometer (Bruker, Germany). Laser shots of 200 per spectrum were used to acquire spectra with a mass range of 800–3000 Da. Spectra were calibrated using trypsin autodigested ion peaks (m/z=842.510 and 2211.1046) as internal standards. The PMF data were used to search for candidate proteins using MASCOT (http://www.matrixscience.com) software. Individual ions scores greater than 32 were defined as significant (pb 0.05).
Platelets were isolated as reported previously [15]. Briefly, 6 mL of blood was collected from the ventral aorta of rats at 12 h and 24 h after surgery and placed into vacutainer tubes containing 0.129 mol/L trisodium citrate. Each blood sample was processed individually, and samples were not pooled during any part of the experiment. In order to exclude erythrocytes and leukocytes, the citrated whole blood was centrifuged at 50 ×g for 20 min at room temperature. The resulting supernatant was the platelet-rich plasma. A plasma-free platelet suspension was prepared by passing the platelet-rich plasma fraction through a size-exclusion chromatography (SEC) column. One mL of plasma was applied to an 11 mL Sepharose 2B (Sigma, Steinheim, Germany) packed column (BioRad, Hercules,CA, USA; 15 mm diameter) equilibrated in calcium-free Dulbecco's phosphate buffered saline. Platelet fractions (1.5 mL) of each individual were collected after an elution volume of 2.5 mL, and platelet concentration was counted on a MicroDiff 18 Blood Analyzer (Coulter Electronics, Miami, FL, USA). The platelets were then lysed in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 0.8% ampholytes) and submitted to ultrasonication on ice for 10 min. The lysed platelets were then centrifuged and the supernatant containing the solubilized proteins was used for 2-DE analysis and for immunoblotting.
To confirm that the proteins identified by mass spectrometry contributed to the differential spot intensities, we selected four identified proteins for further confirmation by immunoblotting. In brief, total protein (10 μg) was separated on 12% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore) for 2 h at 60 V. Subsequently, the PVDF membranes were blocked with Tris-buffered saline with 3% bovine serum albumin for 1 h at room temperature. The membranes were then incubated with a rabbit polyclonal anti-rat antibody (1:500) at room temperature for overnight. The primary antibodies used were: ATPB (US Biological, Swampscott, MA, USA), AAT (Cell Sciences, Canton, MA, USA), tubulin alpha 6 (US Biological, Swampscott, MA, USA) and glucose-6-phosphate dehydrogenase (Rockland, Gilbertsville, PA, USA). The secondary antibody used was against rabbit IgG coupled with horseradish peroxidase (1:500). Peroxidase activity was detected with an ECL kit (Amersham Pharamacia Biotech, Uppsala, Sweden).
2-DE analysis
All data were expressed as mean ± SD. Differences between groups were analyzed for statistical significance by a two-way ANOVA test. A p value b 0.05 was accepted as statistically significant.
We conducted 2-DE gel analysis on the rat platelet to separate proteins. IPG strips (non-linear gradient, 13 cm, pH 3–10; Amersham) were rehydrated with total protein (100 μg) at 30 V for 12 h, and then isoelectric focusing was conducted at 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10 h to reach a total of approximately 60–80 kV h. After the IEF run was complete, strips were incubated in equilibration buffer I (1% DTT, 50 mM pH 6.8 Tris–HCl, 6 M Urea, 30% Glycerol, 2% SDS, bromophenol blue) with gentle shaking for 10 min each and equilibration buffer II (DTT was replaced with 2.5% IAA). The IPG strips added to 12.5% acrylamide gels for separation in the second dimension with 30 mA for 3 h, until the bromophenol blue front reached the bottom of the gel. The separated proteins were visualized by a silver diamine-staining as described by Yu et al. that is compatible with mass spectrometry analysis [16,17]. For preparative 2-DE, 100 μg of total protein was separated as described above. Image acquisition and data analysis The silver-stained 2-DE gels were scanned by ImageScanner (Amersham Biosciences, Uppsala, Sweden) in the transmission mode, and the image analysis was conducted with ImageMaster 2-DE Platinum (Amersham Biosciences, Uppsala, Sweden). To ensure comparable data for quantitative analysis, several key parameters in the image analysis were fixed as constants. The relative volume of each spot was used as an index to eliminate the density differences caused by individual experimental errors. The threshold defined as the significant change in spot volume was ≤2-fold change, upon comparison of the average gels between the CLP and the sham samples and between 12 and 24 h samples.
Confirmation by immunoblotting
Statistical analysis
Results Indicators of sepsis Rats recovered rapidly after CLP surgery, but soon became ill due to sepsis. After surgery, the CLP rats showed several significant physiological changes, including feeble movement, increased rate of respiration, and shapeless feces. Effects were most marked after 24 h, in accordance with findings of Wichterman et al. [13]. Of the total 45 animals used for surgery, 15 sham rats, 12 rats in the 12 h CLP group, and 9 rats in the 24 h CLP group survived after surgery. As shown in Table 1, laboratory parameters reflecting hepatic, renal and respiratory function exhibited significant abnormality 12 h and 24 h later when compared with sham rats, which was consistent with histological examination of the lung and kidney specimens (data not shown). In addition, septic rats also showed thrombocytopenia compared to control animals. No abnormalities were noted in the sham group rats. Analysis of 2-DE gels to catalog the rat platelet proteomes All 36 samples of rat platelet protein extracts were run on 2-DE gels, and each sample was run in a minimum of triplicate experiments to ensure reproducibility. Protein spots that showed b10% coefficient of variation among the replicate samples were selected for quantification by image analysis. In Figs. 1 and 2, two representative 2-DE gels are shown for the comparisons of the platelet proteome between the sham and the CLP samples. For the sham group, an average of 890 ± 87 spots were
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Table 1 Physiological variables in the three groups: sham rats vs. rats at 12 h and 24 h after CLP surgery (n = 15, 12, 9 respectively). Groups
PaO2 (mm Hg)
PaCO2 (mm Hg)
ALT (U/L)
AST (U/L)
Creatinine (μmol/L)
Urea (mmol/L)
Thrombocytes (109/L)
Sham CLP 12 h CLP 24 h
94.8 ± 4.1 82.0 ± 5.1⁎ 77.8 ± 4.9⁎
41.4 ± 3.4 28.7 ± 6.0⁎ 26.1 ± 5.8⁎
54.1 ± 8.4 112.9 ± 36.0⁎ 147.3 ± 39.1⁎
85.5 ± 21.0 316.8 ± 38.1⁎ 357.2 ± 39.0⁎
19.2 ± 4.4 43.4 ± 3.9⁎ 57.0 ± 4.6⁎
4.44 ± 0.61 8.17 ± 1.35⁎ 9.85 ± 1.77⁎
792.5 ± 108.1 460.7 ± 51.3⁎ 412.0 ± 59.1⁎
Data are presented as mean ± SD. ⁎p b 0.05 vs sham group. Abbreviations: PaO2, partial pressure of oxygen in artery; PaCO2, partial pressure of carbon dioxide in artery; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
resolved, while 928 ± 90 and 921 ± 87 spots were resolved on the 12 h CLP and 24 h CLP gels (p b 0.05 for both CLP groups vs sham controls). This indicates that some 2-DE spots indeed responded to the development of sepsis. In the 12 h CLP group, there were 20 differential spots detected, with 16 spots showing up-regulation and 4 spots showing down-regulation compared to the sham controls. In the 24 h CLP group, there were six differential spots detected, all of which were increased compared to sham controls. The expression changes ranged from 0.47 (a down-regulation of 2.12-fold) to 9.07 (an up-regulation of 9.07-fold) in comparison to the control group. Identification of the differential 2-DE spots by mass spectrometry Identification of the 26 different 2-DE spots was carried out by MALDI-TOF-MS/MS. Platelet proteins were successfully identified in 12 differential spots through PMF analyses. Spots 774, 797, 870, 912, 1061, 1359, and 1403 could not be identified because PMF did not match with mouse protein database. Fibrinogen gamma chain was identified in two spots, possibly reflecting two isoforms of the protein or two differentially post-translationally modified forms. Identities, degrees of changes, and other related information of all the identified differential proteins were summarized in Table 2. Among all of the differentially expressed proteins detected by 2-DE analysis, only four proteins were decreased. The decreased proteins were protein disulfide isomerase associated 3, tubulin alpha, glucose-6-phosphate dehydrogenase, and myosin regulatory light polypeptide 9. The proteins with altered expression were grouped into four functional categories, as shown in Table 2. Confirmation of differential protein by immunoblotting To further confirm the reliability of our two-dimensional data, a subset of proteins that showed 2–3 fold change from sham levels and had commercially available antibodies were selected for examination by immunoblotting. The proteins that were selected were
ATP synthase beta subunit, alpha-1-antitrypsin precursor (AAT), tubulin alpha-6, and glucose-6-phosphate dehydrogenase proteins. Figs. 3 and 4 show the immunoblotting results of AAT and ATPB, respectively. For these two proteins, the changes detected by 2-DE gels and by immunoblotting were in the same direction and of comparable magnitude.
Discussion Sepsis refers to a systemic infection that is accompanied by a severe inflammatory response and can lead to multi-organ failure. Sepsis affects over 750,000 Americans each year [19]. The pathophysiology of sepsis is complex and multifactorial, with platelets playing a major role in disease pathogenesis. Additional research on the role of platelet activation in the development of septic complications is needed in order to provide mechanistic insight to develop novel treatment strategies and to evaluate treatment efficacy. In this study, we provide a comprehensive proteomic analysis of platelets isolated from rats with sepsis. We investigated alterations in platelet protein expression over an observational window of 12 to 24 h after sepsis, in order to catalog the response of the platelet proteome to sepsis. The time-points of 12 and 24 h after CLP-surgery were chosen, because the sepsis model shows two septic phases over the time-course of response [20]. After 12 h, animals typically display a hyperdynamic sepsis (“early sepsis”) response that turns into a hypodynamic phase (“late sepsis”) after 16–24 h [13,20]. As disturbances in hemostasis during sepsis are of particular clinical interest, it is necessary to establish a platelet protein catalog to identify those proteins that change upon cell activation in response to sepsis [21]. Studies characterizing the human platelet proteome have been carried in past years, which have provided important insights into platelet biology [9,11,22–24]. We were unable, however, to find any proteomic data relating to rat platelets in the literature, and this report is the first to elucidate platelet protein changes in response to sepsis.
Fig. 1. Proteome map of platelet proteome during sepsis in sham and CLP rats (n = 15, 12, and 9, respectively). Selected gel images were representative gels derived from gel scans of individual groups.
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Fig. 3. Immunoblotting for AAT expression in platelet after sham and CLP surgery (n = 15, 12, and 9, respectively). AAT levels were increased in the CLP groups, compared with the sham controls.
those protein spots with a ≥2-fold change in expression compared with the non-septic controls, and additionally, those spots with statistical significance of p ≤0.05 by ANOVA. Each sample was run three times to minimize inter-gel variability. In our analysis, some spots yielded identical proteins, which likely represent different variations in post-translational modifications [26]. This demonstrates the ability of a proteome approach to separate the same proteins with different posttranslational modifications. In our study, we found eight proteins with increased expression and four proteins with decreased expression in platelets compared with control. These total 12 proteins were grouped by biological system occurrence into following four categories to facilitate the discussion of their physiological significance: (1) platelet activation; (2) acute phase protein; (3) cytoskeleton structure; and (4) energy production. Fig. 2. Typical 2-DE protein profile of platelet. Spots marked with circles and numbers show the location of the proteins that were ≥2.0-fold differentially expressed between sham and CLP rats (n = 15, 12, and 9 respectively).
Confirmation of sepsis In our study, three and six animals died in the sepsis groups, after 12 h and 24 h respectively, and these mortality rates are in accordance with previous reports using the same animal model [25]. The appearance of typical indicators (e.g., changes in behavioral, vital signs, and laboratory parameters) confirmed that sepsis occurred in all of the rats in these two treated groups. These typical changes in vital parameters and laboratory parameters shown in our study have been shown to influence mortality. Proteome analysis In the present study, the differentially expressed proteins in platelets of rats with sepsis were analyzed using a proteomic approach, which had been shown to be very useful in the study of platelet function. To ensure biological significance, we regarded as differentially expressed only
Platelet activation Several proteins that were increased, including growth factor receptor-bound protein 2 (Grb2), thrombospondin 1 and fibrinogen gamma chain, are directly involved in platelet activation. In platelets, Grb2 was shown to play an important role in the inside-out signaling after activation by thrombin[27]. Inhibition of Grb2 interactions with signal transduction proteins would down-regulate thrombin-induced platelet activation and potentiate Fc receptor-mediated platelet activation. TSP-1 is abundantly stored in platelet alpha-granules. Endogenous TSP-1 is necessary for platelet aggregation in vitro in the presence of physiological levels of nitric oxide [28,29]. Fibrinogen is directly involved in platelet activation. Fibrinogen is a component of the storage organelles of platelets, the α-granules. In our study, fibrinogen levels were significantly higher after stimulation with sepsis. Alt et al. found a similar result in human platelets [30]. Importantly, the fibrinogen gamma chain is closely involved in the downstream response to platelet activation. Our study highlights the need to further study the role of these proteins in sepsis.
Table 2 Platelet proteins that were identified in significantly differentially expressed spots on 2-DE gels of cell extracts. Functional categories
Spot
Target protein
NCBI accession number
Fold change Fold change Protein (12 h) (24 h) score
Theoretical Mr Sequence coverage (kDa)/pI (%)
Platelet activation
P908 P910 P1304 P1702 P772 P885 P1337 P1851 P1615 P916 P1035 P1294
Fibrinogen gamma chain Fibrinogen gamma chain Growth factor receptor-bound protein 2 Thrombospondin 1 Protein disulfide-isomerase associated 3 Alpha-1-antitrypsin precursor Thioredoxin Myosin regulatory light polypeptide 9 Tubulin alpha 6 ATP synthase beta subunit Glucose-6-phosphate dehydrogenase Succinate dehydrogenase complex subunit B
gi|61098186 gi|61098186 gi|914957 gi|61556835 gi|149023097 gi|20367 gi|16758644 gi|198278553 gi|58865558 gi|1374715 gi|058702 gi|149024456
2.73 2.01 2.18 9.07 − 2.12 2.07 3.26 − 2.04 − 2.09 2.04 − 2.51 9.07
50.2/5.85 50.2/5.85 23.5/6.31 129.6/4.74 54.1/7.10 45.9/5.70 11.7/4.80 19.8/4.80 49.9/4.96 51.1/4.92 128.3/4.60 34.1/9.05
Acute phase protein Cytoskeleton structure Energy production
1.26 3.02 2.96 1.68 − 1.09 2.15 1.26 − 1.71 1.62 3.01 1.27 1.82
130 200 96 99 249 202 79 119 107 109 115 66
39 67 49 57 53 41 61 52 59 72 68 31
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reorganization of cytoskeleton structure is necessary and important for activation of platelets [42]. Thus, the regulation of cytoskeleton-related protein levels might also influence platelet activation.
Energy production Fig. 4. Western blotting for ATPB expression in platelet after sham and CLP surgery (n = 15, 12, and 9 respectively). ATPB levels were increased in the CLP groups, compared with sham controls.
Acute phase proteins The differentially expressed proteins include several acute phase proteins: protein disulfide isomerase associated 3 (PDI), alpha-1antitrypsin precursor (AAT), and thioredoxin. A number of reports have shown that the acute phase response results in a series of physiological changes in response to sepsis. Upregulation of acute phase proteins, including heat shock proteins (HSP), may be one mechanism through which cells sense and respond to environmental oxidative stress [31]. Therefore, these proteins may play a role in cellular stress pathways [32]. In general, acute phase proteins represent a group of intracellular proteins that may protect cells against exposure to heat, toxins, infection, or other cellular stresses [33]. Nevertheless, a global study for the acute phase proteins of platelet corresponding with septic development has not been reported to date. In fact, such information is important to gain insight into the molecular mechanisms of sepsis, since these proteins are thought to reduce the proteolysis of oxidized proteins [34]. Our data showed that the expression of PDI in platelets was decreased, while AAT and thioredoxin were up-regulated. Protein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an important role in cell survival under various stress conditions. PDIs have been identified recently as crucial mediators of wound healing by activating the extrinsic coagulation pathway and rapidly delivering NO-related signals from donors of all NO redox derivatives [35–37]. Zhou et al. found that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production [38]. Thus, PDI may be an intracellular anti-inflammatory molecule [39]. PDI has also recently been shown to be an important protein involved with protein folding in cells. PDI was down-regulated in platelets during sepsis, which suggests that folding of some proteins may be incomplete. The incomplete folding of proteins could alter platelet function. The main function of AAT is to maintain the proper shape of proteins. An increase in AAT could attenuate injury induced by platelets during sepsis. Likewise, thioredoxin could participate in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol–disulfide exchange reactions.
Several of the differentially expressed proteins, including ATP synthase beta subunit, succinate dehydrogenase complex subunit B (SDH), and glucose-6-phosphate dehydrogenase, are directly involved in energy production pathways. ATP-synthase is responsible for the production of the majority of cellular ATP that eukaryotic organisms need to adapt to the changing energy needs. SDH is also an important enzyme in the energy synthesis process, and SDH serves a pivotal role in the tricarboxylic acid cycle. As our present data showed, the concentration of the β-chain of the ATPsynthase protein and the SDH protein were both considerably increased in the septic rats, suggesting an increase in ATP synthase activity, possibly as a means to compensate for the low level of ATP. A similar finding has been reported by Estesoa et al. in porcine platelets activated by thrombin. The increase in ATP synthase could be explained by a response to thrombin [15]. Glucose-6-phosphate dehydrogenase is the first and essential step of the hexose monophosphate shunt, a main source of cellular NADPH that is consumed by reactive oxygen detoxifying pathways [43]. Downregulation of glucose-6-phosphate dehydrogenase after 12 h of sepsis therefore, may indicate decreased energy production in platelet and may explain the damage of cell function. This view is further strengthened by the fact that oxidative stress was found to be higher in glucose-6-phosphate dehydrogenase deficient animals [43]. Spolarics et al. have showed that glucose-6-phosphate dehydrogenase deficient mice show aggravated responses to sepsis [43]. The down-regulation of metabolic enzymes may result in less energy production in the cell, which in turn could hinder protein translation, an ATP-requiring process.
Comparison with the human platelet proteome We selected the rat as the model species for our study. Compared with the mouse, the rat is a pathologically more relevant model for investigating human diseases [44,45]. In order to place our results in the context of the clinical scenario, we compared previously published proteome analyses of rat and human platelets, and found significant similarities in the hydrophobicity, pI distribution, subcellular localization, and functional distribution patterns between the rat and human platelet protein profiles[46–49]. This fact highlights the utility of the rat model to study platelet-related thrombosis diseases. Based on this comparison, the proteins altered in the rat during sepsis are proteins most likely present in the human platelet.
Cytoskeleton structure
Study limitations
Proteins related to cytoskeleton structure include myosin regulatory light chain 9 and tubulin alpha 6. Myosin regulatory light chain 9 is a regulatory cytoskeleton protein and belongs to the myosin light chain family. This family regulates diverse cell activities, including shape change, secretion, contractile function, and cytokinesis. In patients with impaired platelet aggregation associated with a mutation in transcription factor CBFA2, the expression of myosin regulatory light chain 9 was selectively decreased approximately 77-fold [40,41]. In this study, we also found myosin regulatory light chain 9 was down-regulated. Tubulin alpha-6 belongs to the tubulin family, which is a major constituent of microtubules. Tubulin alpha-6 expression in our study was also down-regulated. It is well accepted that the
To ensure biological significance, only proteins that showed a change in expression ≥2-fold vs. the controls and had p values ≤ 0.05 were regarded as differentially regulated. This common selection standard was used to strictly exclude proteins with random or slight up- or down-regulation [50]. Using this selection, only significantly changed proteins were selected and insignificantly changed proteins were eliminated from analysis. Based on this strict selection, however, we cannot exclude the possibility that additional proteins may be differentially expressed or biologically relevant to the septic response. In addition, our approach is limited in the total number of proteins that can be resolved. Finally, because only surviving animals were evaluated, there is the potential that a survival bias affected results.
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Conclusion In conclusion, we found that sepsis induces a complex regulation of platelet protein changes, which suggests that platelets occupy one of the fundamental crossroads in the complicated interaction between inflammation and coagulation. To date, this study is the first to use proteomic techniques to search globally for the possible sepsis-related proteins in rat platelets. In addition to identifying previously known players, we also identified novel proteins that have not previously been associated with the platelet response to sepsis. These modulated proteins are part of the platelet content or surface, it means most probably that these modulations occurred during thrombocytopoiesis. Exploring the roles of those sepsis-related proteins may lead to the identification of new therapeutic targets to improve treatment outcome in sepsis. These results may help to understand the development of septic coagulation abnormalities, and further research is needed to identify more detailed functional implications and to investigate the resulting clinical implications. References [1] J.M. O'Brien Jr., N.A. Ali, S.K. Aberegg, Sepsis, Am. J. Med. 120 (2007) 1012–1022. [2] A.C. Mavrommatis, T. Theodoridis, V. Orfanidou A:Christopoulou-Kokkinou, S. Zakynthinos, Coagulation system and platelets are fully activated in uncomplicated sepsis, Crit. Care Med. 28 (2000) 451–457. [3] R. Strauss, M. Wehler, K. Mehler, Thrombocytopenia in patients in the medical intensive care unit: bleeding prevalence, transfusion requirements, and outcome, Crit. Care Med. 30 (2002) 1765–1771. [4] J. Gibbins, Platelets adhesion signalling and the regulation of thrombus formation, J. Cell Sci. 117 (2004) 3415–3425. [5] P. von Hundelshausen, C. Weber, Platelets as immune cells: bridging inflammation and cardiovascular disease, Circ. Res. 100 (2007) 27–40. [6] S. Lindemann, M. Gawaz, The active platelet: translation and protein synthesis in an anucleate cell, Semin. Thromb. Hemost. 33 (2007) 144–150. [7] F. Tao, A. Lazarev, Clinical proteomics: opportunities for diagnostics, pharmaceuticals and the clinical laboratory, Expert Rev. Proteomics 4 (2007) 9–11. [8] Y. Yao, W.Y. Wu, S.H. Guan, Proteomic analysis of differential protein expression in rat platelets treated with notoginsengnosides, Phytomedicine 15 (2008) 800–807. [9] W. Winkler, M. Zellner, M. Diestinger, Biological variation of the platelet proteome in the elderly population and its implication for biomarker research, Mol. Cell. Proteomics 7 (2008) 193–203. [10] B. Walkowiak, M. Kaminska, W. Okrój, The blood platelet proteome is changed in UREMIC patients, Platelets 18 (2007) 386–388. [11] E. O'Neil, C. Brock, A. von Kriegsheim, Towards complete analysis of platelets proteome, Proteomics 2 (2002) 288–305. [12] A. García, Y. Senis, R. Antrobus, A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter, Proteomics 6 (2006) 5332–5343. [13] K.A. Wichterman, A.E. Baue, I.H. Chaudry, Sepsis and septic shock: a review of laboratory models and a proposal, J. Surg. Res. 29 (1980) 189–201. [14] Ping-bo Xu, Zhong-ying Lin, Hai-bing Meng, A metabonomic approach to early prognostic evaluation of experimental sepsis, J. Infect. 56 (2008) 474–481. [15] G. Esteso, M.I. Mora, J.J. Garrido, Proteomic analysis of the porcine platelet proteome and alterations induced by thrombin activation, J. Proteomics 71 (2008) 547–560. [16] L.R. Yu, R. Zeng, X.X. Shao, Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry, Electrophoresis 21 (2000) 3058–3068. [17] J.X. Yan, R. Wait, T. Berkelman, A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry, Electrophoresis 21 (2000) 3666–3672. [18] J.Y. Zhu, H.Q. Huang, X.D. Bao, Q.M. Lin, Z.W. Cai, Acute toxicity profile of cadmium revealed by proteomics in brain tissue of Paralichthys olivaceus: potential role of transferrin in cadmium toxicity, Aquat. Toxicol. 78 (2006) 127–135. [19] G.S. Martin, D.M. Mannino, M. Moss, The effect of age on the development and outcome of adult sepsis, Crit. Care Med. 34 (2006) 15–21. [20] S. Yang, W.G. Cioffi, K.I. Bland, Differential alterations in systemic and regional oxygen delivery and consumption during the early and late stages of sepsis, J. Trauma 47 (1999) 706–712. [21] S. Watson, W. Bahou, D. Fitzgerald, Mapping the platelets proteome: a report of the ISTH platelets Physiology Subcommittee, J. Thromb. Haemost. 3 (2003) 2098–3101.
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Altered proteomic pattern in platelets of rats with sepsis Jin-yu Hu, Chang-Lin Li, Ying-Wei Wang ⁎ Department of Anesthesiology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
a r t i c l e
i n f o
Article history: Submitted 14 August 2011 Revised 26 September 2011 Available online 20 October 2011 (Communicated by M. Lichtman, M.D., 26 September 2011) Keywords: Platelet dysfunction Platelet proteome Two-dimensional electrophoresis Mass spectrometry Immunoblotting
a b s t r a c t Platelet dysfunction and thrombocytopenia are common responses to sepsis, but how sepsis changes platelet function is not completely understood. This is due, in part, to our lack of understanding of how sepsis alters platelet protein patterns. The aim of the present study, accordingly, was to investigate the response of the platelet proteome to sepsis. We applied proteomic technology to analyze platelet samples of rats with sepsis. Rats were divided into two groups: 1) sham surgery and 2) sepsis induced by cecal ligation and puncture (CLP) surgery. Platelet samples were collected from surviving rats 12 and 24 h after surgery, and platelet proteins were separated by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry (MS). In the CLP group, there were 20 spots that were statistically significantly different at 12 h. Of these spots, 16 spots were increased and four spots were decreased. At 24 h, there were six spots increased in the CLP group. Of the 26 spots, 12 proteins associated with platelet activation, acute phase proteins, cytoskeleton structure, and energy production were identified. Of interest, alpha-1-antitrypsin precursor (AAT) and ATP synthase beta subunit (ATPB) were both increased at 12 and 24 h of sepsis by 2-DE and immunoblotting. By providing the platelet profiles, our results demonstrate that this proteomic approach brings us closer to understanding how platelet dysfunction develops after sepsis. © 2011 Elsevier Inc. All rights reserved.
Introduction Sepsis is the systemic inflammatory response syndrome due to infection and is associated with multi-organ failure and a high mortality rate of approximately 20% to 30% [1]. The pathophysiology and molecular mechanisms of sepsis are complicated and remain unclear. Coagulation abnormalities and thrombocytopenia are common in sepsis [2]. Of interest, the severity of hemostatic disorders positively correlates with sepsis severity. In particular, a low platelet count predicts a poor prognosis [3]. Platelets play a central role in hemostasis and thrombosis, providing the first line of defense following injury. Platelets form thrombi as an essential mechanism of hemostasis [4]. In addition, there is increasing evidence that platelets actively participate in the pathogenesis of inflammation and infection [5]. Since platelets are anucleated cells and cannot be evaluated by examining mRNA levels, proteomics offers a powerful approach to provide information on how the platelet catalog changes during sepsis [6]. Proteomics allows the simultaneous monitoring of large sets of proteins and can identify patterns of protein alterations in the platelet protein data set [7]. In recent years, different proteomic approaches have been applied to investigate various aspects concerning platelet biology [8–10]. Specifically, the separation of proteins using 2-DE
⁎ Corresponding author. E-mail address: [email protected] (Y.-W. Wang). 1079-9796/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2011.09.010
with identification by MS has proven to be an effective way to analyze the platelet proteome [11,12]. The sepsis model of CLP in the rat exhibits many features of clinical sepsis [13,14]. By using the CLP model of sepsis, the present report contributes to a more complete understanding of platelet biology in sepsis, helping to build the foundation for future identification of new drug targets and therapeutic strategies. Materials and methods Animals and CLP model Male Sprague–Dawley (SD) rats (6–8 weeks old, 220–250 g) were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). The rats were acclimatized in our facility for 1 week before surgery. All procedures involving animals were conducted according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal protocol was approved by the Bioethics Committee of Shanghai Xinhua Hospital (Shanghai, China). On the day of surgery, the rats were randomly divided into the control sham surgery group (sham; n = 15); a CLP 12 h group (n= 15), and a CLP 24 h group (n = 15). To induce sepsis, the widely accepted CLP model described by Wichterman et al. in 1980 was used [13]. Briefly, after weighing the animals, rats were anesthetized by intraperitoneal injection of 0.5 mL/100 g of 7% chloraldurat. Under aseptic conditions, rats of the sepsis group underwent laparotomy, a
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1 cm midline abdominal incision was made and the cecum was exposed. The cecum was ligated at two-thirds of the distance from the distal tip and was punctured twice with an 18-gauge needle. Thereafter, the incision was closed and 4 mL of normal saline was administered subcutaneously for fluid resuscitation. The sham operation consisted of laparotomy and cecal exposure without any more manipulation as a control for the surgery. After the surgery, the animals had free access to food and water. Of the 45 rats that underwent surgery, 36 rats survived: 15 rats in the sham group (100%), 12 rats in the 12 h CLP group (80%), and 9 rats in the 24 h CLP group (60%). Platelet isolation
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In-gel digestion and MALDI-TOF MS/MS analysis Twenty six protein spots were excised from the 2-DE gels and washed with water before digestion. Digestion and peptide extraction were performed according to reference methods [18]. 2.5 mL 0.5% TFA (v/v) was added to dissolve the peptides. The proteins were analyzed with a Bruker Autoflex III® MALDI-TOF/TOF 200 mass spectrometer (Bruker, Germany). Laser shots of 200 per spectrum were used to acquire spectra with a mass range of 800–3000 Da. Spectra were calibrated using trypsin autodigested ion peaks (m/z=842.510 and 2211.1046) as internal standards. The PMF data were used to search for candidate proteins using MASCOT (http://www.matrixscience.com) software. Individual ions scores greater than 32 were defined as significant (pb 0.05).
Platelets were isolated as reported previously [15]. Briefly, 6 mL of blood was collected from the ventral aorta of rats at 12 h and 24 h after surgery and placed into vacutainer tubes containing 0.129 mol/L trisodium citrate. Each blood sample was processed individually, and samples were not pooled during any part of the experiment. In order to exclude erythrocytes and leukocytes, the citrated whole blood was centrifuged at 50 ×g for 20 min at room temperature. The resulting supernatant was the platelet-rich plasma. A plasma-free platelet suspension was prepared by passing the platelet-rich plasma fraction through a size-exclusion chromatography (SEC) column. One mL of plasma was applied to an 11 mL Sepharose 2B (Sigma, Steinheim, Germany) packed column (BioRad, Hercules,CA, USA; 15 mm diameter) equilibrated in calcium-free Dulbecco's phosphate buffered saline. Platelet fractions (1.5 mL) of each individual were collected after an elution volume of 2.5 mL, and platelet concentration was counted on a MicroDiff 18 Blood Analyzer (Coulter Electronics, Miami, FL, USA). The platelets were then lysed in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 0.8% ampholytes) and submitted to ultrasonication on ice for 10 min. The lysed platelets were then centrifuged and the supernatant containing the solubilized proteins was used for 2-DE analysis and for immunoblotting.
To confirm that the proteins identified by mass spectrometry contributed to the differential spot intensities, we selected four identified proteins for further confirmation by immunoblotting. In brief, total protein (10 μg) was separated on 12% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore) for 2 h at 60 V. Subsequently, the PVDF membranes were blocked with Tris-buffered saline with 3% bovine serum albumin for 1 h at room temperature. The membranes were then incubated with a rabbit polyclonal anti-rat antibody (1:500) at room temperature for overnight. The primary antibodies used were: ATPB (US Biological, Swampscott, MA, USA), AAT (Cell Sciences, Canton, MA, USA), tubulin alpha 6 (US Biological, Swampscott, MA, USA) and glucose-6-phosphate dehydrogenase (Rockland, Gilbertsville, PA, USA). The secondary antibody used was against rabbit IgG coupled with horseradish peroxidase (1:500). Peroxidase activity was detected with an ECL kit (Amersham Pharamacia Biotech, Uppsala, Sweden).
2-DE analysis
All data were expressed as mean ± SD. Differences between groups were analyzed for statistical significance by a two-way ANOVA test. A p value b 0.05 was accepted as statistically significant.
We conducted 2-DE gel analysis on the rat platelet to separate proteins. IPG strips (non-linear gradient, 13 cm, pH 3–10; Amersham) were rehydrated with total protein (100 μg) at 30 V for 12 h, and then isoelectric focusing was conducted at 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10 h to reach a total of approximately 60–80 kV h. After the IEF run was complete, strips were incubated in equilibration buffer I (1% DTT, 50 mM pH 6.8 Tris–HCl, 6 M Urea, 30% Glycerol, 2% SDS, bromophenol blue) with gentle shaking for 10 min each and equilibration buffer II (DTT was replaced with 2.5% IAA). The IPG strips added to 12.5% acrylamide gels for separation in the second dimension with 30 mA for 3 h, until the bromophenol blue front reached the bottom of the gel. The separated proteins were visualized by a silver diamine-staining as described by Yu et al. that is compatible with mass spectrometry analysis [16,17]. For preparative 2-DE, 100 μg of total protein was separated as described above. Image acquisition and data analysis The silver-stained 2-DE gels were scanned by ImageScanner (Amersham Biosciences, Uppsala, Sweden) in the transmission mode, and the image analysis was conducted with ImageMaster 2-DE Platinum (Amersham Biosciences, Uppsala, Sweden). To ensure comparable data for quantitative analysis, several key parameters in the image analysis were fixed as constants. The relative volume of each spot was used as an index to eliminate the density differences caused by individual experimental errors. The threshold defined as the significant change in spot volume was ≤2-fold change, upon comparison of the average gels between the CLP and the sham samples and between 12 and 24 h samples.
Confirmation by immunoblotting
Statistical analysis
Results Indicators of sepsis Rats recovered rapidly after CLP surgery, but soon became ill due to sepsis. After surgery, the CLP rats showed several significant physiological changes, including feeble movement, increased rate of respiration, and shapeless feces. Effects were most marked after 24 h, in accordance with findings of Wichterman et al. [13]. Of the total 45 animals used for surgery, 15 sham rats, 12 rats in the 12 h CLP group, and 9 rats in the 24 h CLP group survived after surgery. As shown in Table 1, laboratory parameters reflecting hepatic, renal and respiratory function exhibited significant abnormality 12 h and 24 h later when compared with sham rats, which was consistent with histological examination of the lung and kidney specimens (data not shown). In addition, septic rats also showed thrombocytopenia compared to control animals. No abnormalities were noted in the sham group rats. Analysis of 2-DE gels to catalog the rat platelet proteomes All 36 samples of rat platelet protein extracts were run on 2-DE gels, and each sample was run in a minimum of triplicate experiments to ensure reproducibility. Protein spots that showed b10% coefficient of variation among the replicate samples were selected for quantification by image analysis. In Figs. 1 and 2, two representative 2-DE gels are shown for the comparisons of the platelet proteome between the sham and the CLP samples. For the sham group, an average of 890 ± 87 spots were
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Table 1 Physiological variables in the three groups: sham rats vs. rats at 12 h and 24 h after CLP surgery (n = 15, 12, 9 respectively). Groups
PaO2 (mm Hg)
PaCO2 (mm Hg)
ALT (U/L)
AST (U/L)
Creatinine (μmol/L)
Urea (mmol/L)
Thrombocytes (109/L)
Sham CLP 12 h CLP 24 h
94.8 ± 4.1 82.0 ± 5.1⁎ 77.8 ± 4.9⁎
41.4 ± 3.4 28.7 ± 6.0⁎ 26.1 ± 5.8⁎
54.1 ± 8.4 112.9 ± 36.0⁎ 147.3 ± 39.1⁎
85.5 ± 21.0 316.8 ± 38.1⁎ 357.2 ± 39.0⁎
19.2 ± 4.4 43.4 ± 3.9⁎ 57.0 ± 4.6⁎
4.44 ± 0.61 8.17 ± 1.35⁎ 9.85 ± 1.77⁎
792.5 ± 108.1 460.7 ± 51.3⁎ 412.0 ± 59.1⁎
Data are presented as mean ± SD. ⁎p b 0.05 vs sham group. Abbreviations: PaO2, partial pressure of oxygen in artery; PaCO2, partial pressure of carbon dioxide in artery; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
resolved, while 928 ± 90 and 921 ± 87 spots were resolved on the 12 h CLP and 24 h CLP gels (p b 0.05 for both CLP groups vs sham controls). This indicates that some 2-DE spots indeed responded to the development of sepsis. In the 12 h CLP group, there were 20 differential spots detected, with 16 spots showing up-regulation and 4 spots showing down-regulation compared to the sham controls. In the 24 h CLP group, there were six differential spots detected, all of which were increased compared to sham controls. The expression changes ranged from 0.47 (a down-regulation of 2.12-fold) to 9.07 (an up-regulation of 9.07-fold) in comparison to the control group. Identification of the differential 2-DE spots by mass spectrometry Identification of the 26 different 2-DE spots was carried out by MALDI-TOF-MS/MS. Platelet proteins were successfully identified in 12 differential spots through PMF analyses. Spots 774, 797, 870, 912, 1061, 1359, and 1403 could not be identified because PMF did not match with mouse protein database. Fibrinogen gamma chain was identified in two spots, possibly reflecting two isoforms of the protein or two differentially post-translationally modified forms. Identities, degrees of changes, and other related information of all the identified differential proteins were summarized in Table 2. Among all of the differentially expressed proteins detected by 2-DE analysis, only four proteins were decreased. The decreased proteins were protein disulfide isomerase associated 3, tubulin alpha, glucose-6-phosphate dehydrogenase, and myosin regulatory light polypeptide 9. The proteins with altered expression were grouped into four functional categories, as shown in Table 2. Confirmation of differential protein by immunoblotting To further confirm the reliability of our two-dimensional data, a subset of proteins that showed 2–3 fold change from sham levels and had commercially available antibodies were selected for examination by immunoblotting. The proteins that were selected were
ATP synthase beta subunit, alpha-1-antitrypsin precursor (AAT), tubulin alpha-6, and glucose-6-phosphate dehydrogenase proteins. Figs. 3 and 4 show the immunoblotting results of AAT and ATPB, respectively. For these two proteins, the changes detected by 2-DE gels and by immunoblotting were in the same direction and of comparable magnitude.
Discussion Sepsis refers to a systemic infection that is accompanied by a severe inflammatory response and can lead to multi-organ failure. Sepsis affects over 750,000 Americans each year [19]. The pathophysiology of sepsis is complex and multifactorial, with platelets playing a major role in disease pathogenesis. Additional research on the role of platelet activation in the development of septic complications is needed in order to provide mechanistic insight to develop novel treatment strategies and to evaluate treatment efficacy. In this study, we provide a comprehensive proteomic analysis of platelets isolated from rats with sepsis. We investigated alterations in platelet protein expression over an observational window of 12 to 24 h after sepsis, in order to catalog the response of the platelet proteome to sepsis. The time-points of 12 and 24 h after CLP-surgery were chosen, because the sepsis model shows two septic phases over the time-course of response [20]. After 12 h, animals typically display a hyperdynamic sepsis (“early sepsis”) response that turns into a hypodynamic phase (“late sepsis”) after 16–24 h [13,20]. As disturbances in hemostasis during sepsis are of particular clinical interest, it is necessary to establish a platelet protein catalog to identify those proteins that change upon cell activation in response to sepsis [21]. Studies characterizing the human platelet proteome have been carried in past years, which have provided important insights into platelet biology [9,11,22–24]. We were unable, however, to find any proteomic data relating to rat platelets in the literature, and this report is the first to elucidate platelet protein changes in response to sepsis.
Fig. 1. Proteome map of platelet proteome during sepsis in sham and CLP rats (n = 15, 12, and 9, respectively). Selected gel images were representative gels derived from gel scans of individual groups.
J. Hu et al. / Blood Cells, Molecules, and Diseases 48 (2012) 30–35
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Fig. 3. Immunoblotting for AAT expression in platelet after sham and CLP surgery (n = 15, 12, and 9, respectively). AAT levels were increased in the CLP groups, compared with the sham controls.
those protein spots with a ≥2-fold change in expression compared with the non-septic controls, and additionally, those spots with statistical significance of p ≤0.05 by ANOVA. Each sample was run three times to minimize inter-gel variability. In our analysis, some spots yielded identical proteins, which likely represent different variations in post-translational modifications [26]. This demonstrates the ability of a proteome approach to separate the same proteins with different posttranslational modifications. In our study, we found eight proteins with increased expression and four proteins with decreased expression in platelets compared with control. These total 12 proteins were grouped by biological system occurrence into following four categories to facilitate the discussion of their physiological significance: (1) platelet activation; (2) acute phase protein; (3) cytoskeleton structure; and (4) energy production. Fig. 2. Typical 2-DE protein profile of platelet. Spots marked with circles and numbers show the location of the proteins that were ≥2.0-fold differentially expressed between sham and CLP rats (n = 15, 12, and 9 respectively).
Confirmation of sepsis In our study, three and six animals died in the sepsis groups, after 12 h and 24 h respectively, and these mortality rates are in accordance with previous reports using the same animal model [25]. The appearance of typical indicators (e.g., changes in behavioral, vital signs, and laboratory parameters) confirmed that sepsis occurred in all of the rats in these two treated groups. These typical changes in vital parameters and laboratory parameters shown in our study have been shown to influence mortality. Proteome analysis In the present study, the differentially expressed proteins in platelets of rats with sepsis were analyzed using a proteomic approach, which had been shown to be very useful in the study of platelet function. To ensure biological significance, we regarded as differentially expressed only
Platelet activation Several proteins that were increased, including growth factor receptor-bound protein 2 (Grb2), thrombospondin 1 and fibrinogen gamma chain, are directly involved in platelet activation. In platelets, Grb2 was shown to play an important role in the inside-out signaling after activation by thrombin[27]. Inhibition of Grb2 interactions with signal transduction proteins would down-regulate thrombin-induced platelet activation and potentiate Fc receptor-mediated platelet activation. TSP-1 is abundantly stored in platelet alpha-granules. Endogenous TSP-1 is necessary for platelet aggregation in vitro in the presence of physiological levels of nitric oxide [28,29]. Fibrinogen is directly involved in platelet activation. Fibrinogen is a component of the storage organelles of platelets, the α-granules. In our study, fibrinogen levels were significantly higher after stimulation with sepsis. Alt et al. found a similar result in human platelets [30]. Importantly, the fibrinogen gamma chain is closely involved in the downstream response to platelet activation. Our study highlights the need to further study the role of these proteins in sepsis.
Table 2 Platelet proteins that were identified in significantly differentially expressed spots on 2-DE gels of cell extracts. Functional categories
Spot
Target protein
NCBI accession number
Fold change Fold change Protein (12 h) (24 h) score
Theoretical Mr Sequence coverage (kDa)/pI (%)
Platelet activation
P908 P910 P1304 P1702 P772 P885 P1337 P1851 P1615 P916 P1035 P1294
Fibrinogen gamma chain Fibrinogen gamma chain Growth factor receptor-bound protein 2 Thrombospondin 1 Protein disulfide-isomerase associated 3 Alpha-1-antitrypsin precursor Thioredoxin Myosin regulatory light polypeptide 9 Tubulin alpha 6 ATP synthase beta subunit Glucose-6-phosphate dehydrogenase Succinate dehydrogenase complex subunit B
gi|61098186 gi|61098186 gi|914957 gi|61556835 gi|149023097 gi|20367 gi|16758644 gi|198278553 gi|58865558 gi|1374715 gi|058702 gi|149024456
2.73 2.01 2.18 9.07 − 2.12 2.07 3.26 − 2.04 − 2.09 2.04 − 2.51 9.07
50.2/5.85 50.2/5.85 23.5/6.31 129.6/4.74 54.1/7.10 45.9/5.70 11.7/4.80 19.8/4.80 49.9/4.96 51.1/4.92 128.3/4.60 34.1/9.05
Acute phase protein Cytoskeleton structure Energy production
1.26 3.02 2.96 1.68 − 1.09 2.15 1.26 − 1.71 1.62 3.01 1.27 1.82
130 200 96 99 249 202 79 119 107 109 115 66
39 67 49 57 53 41 61 52 59 72 68 31
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reorganization of cytoskeleton structure is necessary and important for activation of platelets [42]. Thus, the regulation of cytoskeleton-related protein levels might also influence platelet activation.
Energy production Fig. 4. Western blotting for ATPB expression in platelet after sham and CLP surgery (n = 15, 12, and 9 respectively). ATPB levels were increased in the CLP groups, compared with sham controls.
Acute phase proteins The differentially expressed proteins include several acute phase proteins: protein disulfide isomerase associated 3 (PDI), alpha-1antitrypsin precursor (AAT), and thioredoxin. A number of reports have shown that the acute phase response results in a series of physiological changes in response to sepsis. Upregulation of acute phase proteins, including heat shock proteins (HSP), may be one mechanism through which cells sense and respond to environmental oxidative stress [31]. Therefore, these proteins may play a role in cellular stress pathways [32]. In general, acute phase proteins represent a group of intracellular proteins that may protect cells against exposure to heat, toxins, infection, or other cellular stresses [33]. Nevertheless, a global study for the acute phase proteins of platelet corresponding with septic development has not been reported to date. In fact, such information is important to gain insight into the molecular mechanisms of sepsis, since these proteins are thought to reduce the proteolysis of oxidized proteins [34]. Our data showed that the expression of PDI in platelets was decreased, while AAT and thioredoxin were up-regulated. Protein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an important role in cell survival under various stress conditions. PDIs have been identified recently as crucial mediators of wound healing by activating the extrinsic coagulation pathway and rapidly delivering NO-related signals from donors of all NO redox derivatives [35–37]. Zhou et al. found that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production [38]. Thus, PDI may be an intracellular anti-inflammatory molecule [39]. PDI has also recently been shown to be an important protein involved with protein folding in cells. PDI was down-regulated in platelets during sepsis, which suggests that folding of some proteins may be incomplete. The incomplete folding of proteins could alter platelet function. The main function of AAT is to maintain the proper shape of proteins. An increase in AAT could attenuate injury induced by platelets during sepsis. Likewise, thioredoxin could participate in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol–disulfide exchange reactions.
Several of the differentially expressed proteins, including ATP synthase beta subunit, succinate dehydrogenase complex subunit B (SDH), and glucose-6-phosphate dehydrogenase, are directly involved in energy production pathways. ATP-synthase is responsible for the production of the majority of cellular ATP that eukaryotic organisms need to adapt to the changing energy needs. SDH is also an important enzyme in the energy synthesis process, and SDH serves a pivotal role in the tricarboxylic acid cycle. As our present data showed, the concentration of the β-chain of the ATPsynthase protein and the SDH protein were both considerably increased in the septic rats, suggesting an increase in ATP synthase activity, possibly as a means to compensate for the low level of ATP. A similar finding has been reported by Estesoa et al. in porcine platelets activated by thrombin. The increase in ATP synthase could be explained by a response to thrombin [15]. Glucose-6-phosphate dehydrogenase is the first and essential step of the hexose monophosphate shunt, a main source of cellular NADPH that is consumed by reactive oxygen detoxifying pathways [43]. Downregulation of glucose-6-phosphate dehydrogenase after 12 h of sepsis therefore, may indicate decreased energy production in platelet and may explain the damage of cell function. This view is further strengthened by the fact that oxidative stress was found to be higher in glucose-6-phosphate dehydrogenase deficient animals [43]. Spolarics et al. have showed that glucose-6-phosphate dehydrogenase deficient mice show aggravated responses to sepsis [43]. The down-regulation of metabolic enzymes may result in less energy production in the cell, which in turn could hinder protein translation, an ATP-requiring process.
Comparison with the human platelet proteome We selected the rat as the model species for our study. Compared with the mouse, the rat is a pathologically more relevant model for investigating human diseases [44,45]. In order to place our results in the context of the clinical scenario, we compared previously published proteome analyses of rat and human platelets, and found significant similarities in the hydrophobicity, pI distribution, subcellular localization, and functional distribution patterns between the rat and human platelet protein profiles[46–49]. This fact highlights the utility of the rat model to study platelet-related thrombosis diseases. Based on this comparison, the proteins altered in the rat during sepsis are proteins most likely present in the human platelet.
Cytoskeleton structure
Study limitations
Proteins related to cytoskeleton structure include myosin regulatory light chain 9 and tubulin alpha 6. Myosin regulatory light chain 9 is a regulatory cytoskeleton protein and belongs to the myosin light chain family. This family regulates diverse cell activities, including shape change, secretion, contractile function, and cytokinesis. In patients with impaired platelet aggregation associated with a mutation in transcription factor CBFA2, the expression of myosin regulatory light chain 9 was selectively decreased approximately 77-fold [40,41]. In this study, we also found myosin regulatory light chain 9 was down-regulated. Tubulin alpha-6 belongs to the tubulin family, which is a major constituent of microtubules. Tubulin alpha-6 expression in our study was also down-regulated. It is well accepted that the
To ensure biological significance, only proteins that showed a change in expression ≥2-fold vs. the controls and had p values ≤ 0.05 were regarded as differentially regulated. This common selection standard was used to strictly exclude proteins with random or slight up- or down-regulation [50]. Using this selection, only significantly changed proteins were selected and insignificantly changed proteins were eliminated from analysis. Based on this strict selection, however, we cannot exclude the possibility that additional proteins may be differentially expressed or biologically relevant to the septic response. In addition, our approach is limited in the total number of proteins that can be resolved. Finally, because only surviving animals were evaluated, there is the potential that a survival bias affected results.
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