Preparation of washed platelets

Washed human platelets were prepared from blood collected from healthy drug-free volunteers, as previously described (41). Briefly, 9 volumes of blood were collected into 1 volume of 4% (w/v) sodium citrate solution. One volume of ACD solution (1.5% [w/v] citric acid, 2.5% [w/v] sodium citrate and 1% [w/v] glucose) was added to the anti-coagulated blood before being centrifuged at 200g for 20 min at room temperature. Platelet rich plasma (PRP) was collected, to which 2 nM prostacyclin was added, before being centrifuged at 1000g for 10 min. Platelets were washed in 25 mL of modified Tyrode’s-HEPES buffer pH 7.3 (134 nM NaCl, 2.9 mM KCl, 20 mM HEPES, 12 mM NaHCO₃, 1 mM MgCl₂, 5 mM glucose) containing 3 mL of ACD and 1 nM prostacyclin. Platelets were centrifuged at 1000g for 10 min and resuspended at 5 × 10⁸/mL in modified Tyrode’s-HEPES buffer. Platelets were counted with a Coulter Z₂ Particle Count and Size Analyzer (Beckman Coulter Ltd, High Wycombe, UK).

WGA affinity chromatography

Washed platelets (10 mL at 5 × 10⁸/mL) were lysed with an equal volume of 2 × lysis buffer (2% NP-40, 300 mM NaCl, 20 mM Tris, 10 mM ethylenediaminetetraacetic acid pH 7.4), containing protease inhibitors (1 mM AEBSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin and 1 μg/mL pepstatin A). The platelet lysate was pre-cleared with 2 mL of Sepharose 4B beads for 30 min at 4°C and centrifuged at 10,000g for 15 min at 4°C. WGA conjugated to Sepharose 4B (2 mL) was added to the supernatant. The sample was incubated overnight at 4°C with mixing. The WGA resin was transferred to a column and washed three times with 1 × lysis buffer. Bound proteins were eluted from the WGA resin with 3 mL of 0.3 M N-acetyl-D-glucosamine and concentrated to 200 μL using an Amicon Centriprep YM-10 and Ultrafree 0.5 centrifugal filter devices. A fifth of the volume of 5 × SDS-PAGE sample buffer was added to samples and heated to 100°C for 5 min. Samples were prepared in this way in three separate experiments.

Biotinylation of surface proteins and isolation by NeutrAvidin affinity chromatography

Platelet surface proteins were biotinylated according to the manufacturer’s instructions, with a few minor modifications. Platelets (10 mL at 5 × 10⁸/mL) were washed twice with 25 mL PBS pH 7.4 containing 1 μM prostacyclin. They were then resuspended in 10 mL of 412 μM EZ-link sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) in PBS pH 7.4 for 30 min at room temperature. Unreacted biotinylation reagent was quenched by adding Tris pH 8.0 to a final concentration of 50 mM; platelets were pelleted at 1000g for 10 min at room temperature; washed twice in 10 mL 0.025 M Tris, 0.15 M NaCl (TBS) pH 7.4 containing 1 μM prostacyclin; and lysed in 500 μL lysis buffer (proprietary) by sonicating on low power at 10 min intervals for 30 min on ice. Lysates were centrifuged at 10,000g for 2 min at 4°C to remove cell debris. Clarified supernatants were incubated with 250 μL NeutraAvidin (NA)-beads for 1 hr at room temperature then centrifuged for 1 min at 1,000g. The gel was washed 3 × 500 μL wash buffer (proprietary). Proteins were eluted in 2 × sample buffer containing 50 mM DTT and heated to 100°C for 5 min. Samples were prepared in this way in three separate experiments.

Preparation of platelet plasma membranes (PM) and intracellular membranes (IM) by free flow electrophoresis

Platelet PM and IM were prepared as described in detail previously (3). Briefly, platelets were separated from freshly obtained platelet concentrates (National Blood Service, Tooting, London, UK) and treated with neuraminidase (type X, 0.05 U/mL) for 20 min at 37°C. After 2 washings, platelets were disrupted by sonication and the platelet homogenate layered on a linear (1-3.5 M) sorbitol density gradient followed by centrifugation at 42,000g for 90 min, to obtain a mixed membrane fraction (free of granular contamination). This membrane fraction was separated into PM and IM by free-flow electrophoresis using an Octopus electrophoresis apparatus (Dr. Weber Gmbh, Germany) running at 750 V, 100 mA. Two discrete peaks comprising PM and IM (more electronegative) were obtained. Tops of peaks were pooled, centrifuged (100,000g for 60 min) and resuspended in 0.4 M sorbitol, 5% glycerol and 10 mM triethanolamine pH 7.2 and kept at −80°C until further analysis. The purity of fractions was checked by analyzing by SDS-PAGE and western blotting for the absence of actin in IM and of SERCA2 Ca²⁺ATPase in PM fractions, as previously described (3). Samples were prepared in this way on two separate occasions.

Protein preparation for MS/MS

Proteins were resolved on 4-20% Tris-glycine SDS-PAGE gels and stained with Colloidal Coomassie G-250 stain. Twelve to 32 gel slices each with a width of 1-2 mm were manually excised with a razor for subsequent in-gel trypsinization and LC-MS/MS analysis. Bands were excised from three separate WGA affinity purification experiments; three biotin/NA affinity purification experiments and two FFE experiments. Proteins were trypsinized within gel slices and peptides extracted using the method described by Shevchenko (42).

LC-MS/MS and data anlysis

Tryptic peptides were analyzed by LC-MS/MS, using a ThermoFinnigan LCQ Deca XP Plus ion-trap (Thermo Electron Corporation, Hemel Hempstead, UK) coupled to a Dionex/LC Packings nanobore HPLC system (Dionex/LC Packings, Sunnyvale, CA, USA), configured with a 300 μm id/1 mm C18 Pepmap pre-column (LC Packings, San Francisco, CA, USA) and a 75 μm id/15 cm C18 PepMap analytical column (LC Packings). Tryptic peptides were eluted into the ion-trap mass spectrometer using a 45 min 5-95% acetonitrile gradient containing 0.1% formic acid at a flow rate of 200 nL/min. Spectra were acquired in an automatic data dependent fashion using a full MS scan (400-2000 m/z) to determine the 5 most abundant ions which were sequentially subjected to MS/MS analysis. Each precursor ion was analyzed twice before it was placed on an exclusion list for 1 min. MS/MS spectra were converted into dta-format files by Bioworks Browser (3.1) and searched against the NCBInr database (released April 2004) using the TurboSequest (3.1) search algorithm (ThermoFinnigan). Both the precursor mass tolerance and the fragment mass tolerance were set at 1.4 Da. Two missed tryptic cleavages and carbamidomethylation of cysteine residues as a fixed modification were allowed. Positive peptide hits using TurboSequest had a minimum cross-correlation factor of 2.5, a minimum delta correlation value of 0.25 and a preliminary ranking of one. The same dta-format files generated with the LC-MS/MS ion-trap and Bioworks Browser set up were also searched against the NCBInr database using the Mascot 1.8 search algorithm (Matrix Science, London, UK). Mascot searches were restricted to the human taxonomy allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as a potential variable modification. Both precursor mass tolerance and MS/MS tolerance of 1.4 Da, allowing for up to two missed cleavages. Positive identification was only accepted when the data satisfied the following criteria: (i) MS/MS data were obtained for at least 80% y-ions series of a peptide comprising at least eight amino acids and no missed tryptic cleavage sites; (ii) MS/MS data with more than 50% y-ions were obtained for two or more different peptides comprising at least eight amino acids long and no more than two missed tryptic cleavage sites. Swiss-Prot/TrEMBL accession numbers were obtained for all proteins identified.

MS/MS analysis of tryptic fragments was also carried out with a Q-TOF 1 mass spectrometer (Micromass, Manchester, UK) as a means of verifying proteins identified with the ion-trap mass spectrometer, and of improving both protein and proteome coverage by using complementary instruments for the MS/MS analysis (13). The Q-TOF 1 mass spectrometer was coupled to a CapLC HPLC system (Waters, Milford, MA, USA) configured with a 300 μm id/5 mm C18 pre-column (LC Packings) and a 75 μm id/25 cm C18 PepMap analytical column (LC Packings). Tryptic peptides were eluted to the mass spectrometer using a 45 min 5-95% acetonitrile gradient containing 0.1% formic acid at a flow rate of 200 nL/min. Spectra were acquired in an automatic data dependent fashion with a 1 sec survey scan followed by three 1 sec MS/MS scans of the most intense ions. The selected precursor ions were excluded from further analysis for 2 min. MS/MS spectra were converted into pkl-format files using Mass Lynx 3.4 and searched against the NCBInr database with the Mascot search algorithm, as described above.

All proteins identified by both Sequest and Mascot were checked for predicted transmembrane domains (TMDs) with TMHMM v. 2.0 (25).

Construction of decoy database and estimation of the false positive rate of protein identification by LC-MS/MS

A randomized version of the NCBInr database used in this study was generated by a Perl programme downloaded from Matrix Science Ltd. (London, UK), decoy.pl. This programme was run using the random and append command line switches that appended a random set of sequences, with the same average amino acid composition as those in the original dataset, onto the database. The decoy.pl programme was modified to work correctly with the long header format of the NCBInr database. Database searches with all of the dta-format files generated by LC-MS/MS ion-trap and Sequest were searched against the decoy database using the same search parameters described above for the original searches. The percent false positive rate of protein identification was calculated by dividing the number of “random” proteins identified by the sum of “random” and “real” proteins identified and multiplying by 100. The false positive rate was calculated for random proteins identified by two or more peptide hits and for those identified by 1 peptide hit.

Comparison of proteomics datasets

To compare which proteins were common to both our proteomic dataset reported in this study and that of Moebius et al (31), a non-redundant set of peptide sequences were collected from each study. A total of 295 were obtained from the Moebius study and 136 from the present study. All sequences were subsequently BLAST searched against the Reference Sequence Project peptides. Sixty-two proteins were found to be common to both datasets.

Megakaryocyte culture and purification

Bone marrow cells were flushed from femurs and tibias of 3 to 4 month old C57Bl6 mice, as previously described (12). Mature erythrocytes were lysed with ammonium chloride potassium buffer (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA pH 7.3). CD16/CD32⁺Gr1⁺B220⁺CD11b⁺ cells were depleted using immunomagnetic sheep anti-rat IgG beads and rat-anti mouse antibodies according to the manufacturer’s instructions. The cell depleted population was then cultured in serum-free medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin and 20 ng/mL murine SCF at 37°C and 5% CO₂ for 2 days and 5 more days under the same conditions, in addition to 200 ng/mL recombinant human thrombopoietin. High density mature megakaryocytes were then isolated in a 0-3% BSA gradient (4 mL 3% BSA/PBS in a 15 mL Falcon tube overlaid with 4 mL of 1.5% BSA/PBS and 4 mL of suspension cells in PBS) (11). After standing for 40 min at room temperature, the cells remaining in the lower 2 mL were collected, washed in PBS and subjected to another 0-3% BSA gradient to obtain a pure population. DNA content of cells was determined by staining with 50 μg/mL propidium iodide and analyzing cells with a FACScan analyzer and CellQuest software (Becton Dickinson), as previously described (12).

Serial analysis of gene expression

Primary mouse megakaryocyte RNA was made using the RNeasy Miniprep Kit. The LongSAGE library was generated from 20 μg RNA using the I-SAGE Long Kit and sequenced by Agencourt Bioscience Corporation (Beverly, MA, USA). LongSAGE sequence tags were identified using SAGE2000 4.5 Analysis Software with reference to the SAGEmap_tag_ug-rel database (http://www.ncbi.nlm.nih.gov/SAGE/). To identify megakaryocyte-specific genes, the resulting SAGE library, of 53,046 sequence tags, was compared to 30 other mouse SAGE libraries, from T lymphocyte (14 SAGE libraries), dendritic cells (6), intra-epithelial lymphocytes (2), embryonic stem cells (2), brain (2), B lymphocyte (1), heart (1), 3T3 fibroblast cell line (1) and P19 embryonic carcinoma cell line (1), with a combined total of 1,031,389 tags. The data analysis was performed using custom written software (!SAGEClus) as described in Cobbold et al. (8). Genes with predicted TMDs were identified using TMHMM v. 2.0 (25).

Platelet activation, immunoprecipitations and western blotting

Washed platelets (8 × 10⁸/mL) were stimulated with 10 μg/mL CRP or 5 U/mL thrombin for 90 seconds with constant mixing at 1,200 rpm and 37°C, as previously described (41). Platelets were lysed in 2 × lysis buffer containing 5 mM sodium vanadate in addition to the protease inhibitors described above. Proteins were immunoprecipitated from platelet lysates with 2 μg rabbit anti-SHP-1 antibody and 10 μL rabbit anti-G6b-B serum. Ten microlitres of rabbit pre-immune serum was used as a negative control for immunoprecipitations. Membranes were immunoblotted with 1 μg/mL anti-phosphotyrosine antibody, 0.2 μg/mL anti-SHP-1 antibody and 1/1000 rabbit anti-G6b-B antibody, as previously described (29, 41).

Identification of G6b-B in human platelets: a novel tyrosine phosphorylated ITIM-bearing protein

One of the novel platelet PM proteins is the immunoglobulin superfamily member G6b, which is reported to have seven splice variants, G6b-A to G6b-G (10). Two of these splice variants, G6b-A and G6b-B, have transmembrane domains and have been shown to be expressed on the surface of transiently transfected cells (10). The main difference between these two splice variants is in their cytoplasmic tails. The G6b-A isoform lacks any tyrosine residues in this region, whereas the G6b-B isoform contains an ITIM and therefore has the potential to selectively inhibit signalling by the platelet immunoreceptor tyrosine-based activation motif (ITAM)-receptors, GPVI and FcγRIIA. Three unique peptides were identified for different isoforms of G6b by MS/MS. MS/MS spectra for all three peptides are shown in Figure 2. One of the peptides (TVLHVLGDR) could have come from any of the seven splice variants. A second peptide (LPPQPIRPLPR) could only have come from G6b-A, whereas the third peptide (IPGDLDQEPSLLYADLDHLALSR) could have come from either G6b-B, -C or -E. However, neither G6b-C nor G6b-E are predicted to contain transmembrane domains. In order to clarify the ambiguity of the MS/MS result and determine whether G6b-B is expressed in human platelets, we raised a rabbit polyclonal antibody to peptides found in a portion of the cytosolic tail of G6b-B that is absent from G6b-A, and used these to confirm expression of the ITIM-bearing isoform of G6b in platelets by western blotting (Fig. 3A). Whole cell lysate prepared from HEK 293T cells transiently transfected with G6b-B was used as a positive control (Fig. 3A). The specific antibody identified two bands at 32 and 38 kDa on a 4-20% SDS-PAGE gel in platelets, which are most likely to represent differentially glycosylated isoforms of G6b-B, as similar bands were also seen in G6b-B-transfected, but not mock-transfected HEK 293T cells (Fig. 3A). Multiple forms of G6b-B that can be separated by SDS-PAGE have been described in transfection studies in other cell types (10).

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Fig. 2

MS/MS spectra of G6b peptides. Peptides corresponding to each MS/MS spectra are shown in the top right corner of each panel, along with the G6b isoforms from which each peptide may have been derived, the Swiss-Prot/TrEMBL accession number (in parenthesis), and the experiment and band slice identification numbers. The start and end of each peptide are indicated by dots. Adjacent amino acids to the peptide identified are also included (outside dots). Selected b- and y-ions identified are indicated. (A) Peptide TVLHVLGDR is present in all seven isoforms of G6b. (B) Pepetide LPPQPIRPLPR is only present in G6b-A. (C) Peptide IPGDLDQEPSLLYADLDHLALSR is present in G6b-B, -C and -E.

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Fig. 3

Expression of G6b in human platelets. A, (i) whole cell lysates were prepared from human platelets and HEK 293T cells transiently transfected with either plasmid alone (mock) or a G6b-B expression plasmid (G6b-B) were western blotted for G6b-B using a rabbit anti-G6b-B polyclonal antibody raised against two peptides from the cytoplasmic tail of the protein; (ii) as a control, the same samples western blotted in (i) were blotted with pre-immune serum from the same rabbit in which the G6b-B anti-serum was raised. B, G6b-B undergoes an increase in tyrosine phosphorylation in response to CRP and thrombin stimulation, and interacts with SHP-1 in human platelets. G6b-B was immunoprecipitated (IP) from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 μg/mL CRP or 5U/mL thrombin. Samples were western blotted for tyrosine phosphorylated proteins, then stripped and blotted for G6b-B, followed by SHP-1. C, G6b-B is tyrosine phosphorylated in resting and CRP and thrombin activated platelets and interacts with SHP-1. SHP-1 was immunoprecipitated from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 μg/mL CRP or 5 U/mL thrombin. Samples were western blotted for tyrosine phosphorylated proteins, then stripped and blotted for G6b, followed by SHP-1. Results are representative of three experiments.

To investigate a possible functional role for G6b-B in platelets, the protein was immunoprecipitated from resting and stimulated platelets and analysed for tyrosine phosphorylation. Platelets were stimulated with the GPVI-specific peptide, CRP, and the G protein coupled receptor agonist, thrombin. G6b-B was constitutively phosphorylated on tyrosine residues under resting conditions and underwent a small increase in tyrosine phosphorylation upon stimulation by both agonists (Fig. 3B). The tyrosine phosphatase SHP-1, which is regulated by ITIM receptors, was weakly precipitated with G6b-B under basal conditions and more strongly following stimulation by the two agonists. Importantly, G6b-B was also precipitated by an antibody to SHP-1, with the level of G6b-B in the immunoprecipitate increasing upon stimulation with CRP and thrombin (Fig. 3C). Taken together, these results demonstrate that G6b-B associates with SHP-1 in resting and stimulated platelets, consistent with the idea that the immunoglobulin superfamily protein may function as a novel ITIM receptor in platelets.

We would like to thank Miss Donna Holmes and Mr. Neil Shimwell from the Cancer Research-UK Institute, University of Birmingham, for analyzing samples on the ion trap mass spectrometer; Kath Nolan from the Therapeutic Immunology Group, Sir William Dunn School of Pathology, University of Oxford for SAGE advice; and Majd Protty from the Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham for excellent secretarial assistance. AG would like to thank The Oxford Glycobiology Institute Endowment for funding. This research was supported by the BHF, Wellcome Trust and Cancer Research-UK.