Organellar Proteomics of Human Platelet Dense Granules Reveals

Organellar Proteomics of Human Platelet Dense Granules Reveals That 14-3-3ζ Is a Granule Protein Related to Atherosclerosis Laura Herna´ ndez-Ruiz,† Federico Valverde,‡ Maria D. Jimenez-Nun ˜ ez,† Esther Ocan ˜ a,† § | ⊥ Ana Sa´ ez-Benito, Javier Rodrı´guez-Martorell, Juan-Carlos Boho´ rquez, Aurelio Serrano,‡ and Felix A. Ruiz*,† Unidad de Investigacion, Hospital Universitario Puerta del Mar, Facultad de Medicina, Universidad de Cadiz, Cadiz, Spain, Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla, CSIC, Seville, Spain, and Servicios de Ana´lisis Clıńicos, Hematologıá, and Cirugıá Vascular, Hospital Universitario Puerta del Mar, Cadiz, Spain Received June 18, 2007

Dense granules, a type of platelet secretory organelle, are known to accumulate high concentrations of small molecules such as calcium, adenine nucleotides, serotonin, pyrophosphate, and polyphosphate. Protein composition of these granules has been obscure, however. In this paper, we use proteomics techniques to describe, for the first time, the soluble protein composition of platelet dense granules. We have isolated highly enriched human platelet dense granule fractions that have been analyzed using two proteomics methods. Using this approach, we have identified 40 proteins, and most of them, such as actin-associated proteins, glycolytic enzymes, and regulatory proteins, have not previously been related to the organelle. We have focused our efforts on studying 14-3-3ζ, a member of a conserved family of proteins that interact with hundreds of different proteins. We have demonstrated that 14-3-3ζ is localized mostly on dense granules and that it is secreted after platelet activation. As some proteins secreted from activated platelets could promote the development of atherosclerosis and thrombosis, we have studied the expression of 14-3-3ζ in sections of human abdominal aorta of patients with aneurysm, identifying it at the atherosclerotic plaques. Together, our results reveal new details of the composition of the platelet dense granule and suggest an extracellular function for 14-3-3ζ associated with atherosclerosis. Keywords: platelets • dense granules • proteomics • 14-3-3ζ

Introduction Platelet adhesion and secretion of its stored substances have been related to the development of atherosclerosis, a systemic inflammatory disease of the arterial wall.1 Platelets adhere to each other and to the injured arterial endothelium, by the interaction of platelet glycoprotein (Gp) Ib/V/IX with its ligand von Willebrand factor (VWF).2 It has recently been shown that GpIb/V/IX associations with VWF are regulated inside the cell by the adapter protein 14-3-3ζ.3-6 143-3ζ is a member of a conserved family of proteins that interact with numerous cytoplasmic and nuclear molecules, mainly phosphoproteins.7 14-3-3 proteins were initially found as activators of tyrosine and tryptophan hydroxyhydrolases,8 and recent research is still revealing new regulatory roles for them.9 Platelets secrete stored pro-aggregatory and/or inflammatory moieties from their secretory granules after platelet activation.10 * To whom correspondence should be addressed. Tel: +(34) 956003156. Fax: +(34) 956002347. E-mail: [email protected]. † Unidad de Investigacion, Hospital Universitario Puerta del Mar. ‡ Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla, CSIC. § Servicio de Ana´lisis Clıńicos, Hospital Universitario Puerta del Mar. | Servicio de Hematologıá, Hospital Universitario Puerta del Mar. ⊥ Servicio de Cirugıá Vascular, Hospital Universitario Puerta del Mar. 10.1021/pr070380o CCC: $37.00

 2007 American Chemical Society

Platelets contain three types of these secretory organelles: lysosomes, alpha granules, and dense granules. Lysosomes hold various hydrolytic proteins, and alpha granules store several adhesion and repair proteins.11 Dense granules are the only platelet secretory organelles that accumulate principally small nonprotein components, such as calcium, adenine nucleotides, serotonin, and pyrophosphate, which are believed to exist as insoluble complexes inside the granule.11 Recently, we have reported that platelet dense granules also accumulate large amounts of inorganic polyphosphate (polyP),12 a ubiquitous phosphate polymer with ATP-like bonds associated with the modulation of coagulation and fibrinolysis.13 The presence of several membrane proteins has been described in platelet dense granules. Dense granules contain a vacuolar proton pump, a serotonin transporter, GTP-binding proteins, lysosome-associated membrane protein (LAMP) 1, LAMP2, granulophysin (CD63), P-selectin (CD62), GpIb, and GpIIbIIIa.11 A specific localization of a nucleotide transporter (called MRP4) in dense granules has been recently reported.14 In addition, the presence of an orphan nucleotide sugar transporter (Slc35d3) in dense granules has been proposed.15 Nevertheless, the soluble protein composition of platelet dense granules remains obscure. These organelles are difficult to study Journal of Proteome Research 2007, 6, 4449-4457

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Published on Web 10/06/2007

research articles because obtaining sufficient quantities of purified organelles is an intricate process and because of the extremely high content of nonprotein components. Therefore, the present study analyzes the soluble protein contents of fractions enriched in platelet dense granules using a proteomics approach. We have identified 40 proteins, most of which have not previously been associated with this organelle. We focus on studying one of these newly identified granular proteins, 14-3-3ζ, and demonstrate its presence in human atherosclerotic lesions. Our results suggest a new extracellular task for this protein, associated with atherosclerosis.

Materials and Methods Materials. 1,4-Diazabicyclo[2.2.2]octane (DABCO), phosphatase-conjugated anti-rabbit and anti-goat IgG, anti-human von Willebrand factor IgG, protease inhibitor cocktail for mammalian cells, 4′,6-diamidino-2-phenylindole (DAPI), cytochrome c oxidase assay kit, and PolyPs were purchased from Sigma Chemicals Co. (St. Louis, MO). 5-[2-14C]-Hydroxytryptamine binoxalate was obtained from GE Healthcare (Uppsala, Sweden). Mouse monoclonal antibodies (mAb) against LAMP1 were provided by Becton Dickinson (San Jose, CA). Rabbit polyclonal antibodies (pAb) against 14-3-3ζ, mAb against pan cadherin, mAb against cytochrome c oxidase, and goat pAb against MRP4 were provided by Abcam (Cambridge, UK). Alexa488-labeled anti-rabbit, Alexa488-labeled anti-mouse, Alexa647-labeled anti-rabbit, and Alexa647-labeled anti-mouse IgGs were from Molecular Probes (Eugene, OR). Development reagents and molecular weight protein standards were provided by Bio-Rad (Hercules, CA). The Escherichia coli strain CA38 pTrcPPX1 was kindly provided by Prof. Arthur Kornberg, Stanford University School of Medicine (Stanford, CA). All other reagents were of analytical grade. Blood Platelets. Subcellular fractionation was performed using expired platelet-rich plasma units, from the hematology service at the “Puerta del Mar” Hospital (Cadiz, Spain). Plateletrich plasma units were routinely stored for 5 days and processed on their sixth day to obtain isolated dense granules. All other experiments were carried out using fresh human platelets from healthy volunteers and from three patients with delta storage pool disease (δSPD). δSPD diagnosis was confirmed after analysis of mepacrine incorporation by flow cytometry and ADP secretion by luminescence. Approval was obtained from the institutional review board (Ethics Committee). Informed consent was provided according to the Declaration of Helsinki. Platelet Dense Granule Isolation. Dense granules were isolated as previously described12 with some modifications. Two units of expired platelet-rich plasma were centrifuged twice at 200g for 10 min to eliminate contaminant red blood cells and leukocytes. Aspirin (100 µM) was added to the supernatant followed by centrifugation at 1000g for 15 min. The resulting pellet was resuspended at 2 × 109 platelet/mL in lysis buffer (25 mM Hepes pH 6.5, 0.25 M sucrose, 12 mM sodium citrate, 1 mM ethylenediaminetetraacetic acid (EDTA), and a 1/500 dilution of Sigma protease inhibitor cocktail for mammalian cell extracts). Platelets were sonicated five times on ice (5 s on and 15 s off at 15% intensity in a Branson sonifier, model 150), and unbroken cells were separated by centrifugation (1000g for 15 min at 4 °C). Sonication and centrifugation were repeated twice with the pellets resuspended in a similar volume of lysis buffer. The three supernatants were combined and centrifuged for 20 min at 19000g at 4 °C. The pellet was resuspended in 3 4450

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Herna´ ndez-Ruiz et al.

mL of 20% Histodenz (w/v) in lysis buffer. Suspension was applied at the top of a discontinuous gradient of 3 mL steps of 35, 38, and 40% Histodenz (w/v) in lysis buffer. The gradient was centrifuged at 100000g using a Sorvall Superspin 630 rotor for 60 min. The dense granule-rich fraction was pelleted at the bottom of the tube and resuspended in lysis buffer. Serotonin incorporation, short chain polyP content, and acid phosphatase activity (lysosome marker) of gradient fractions was measured as previously described.12 Fractions were assayed as well for cytochrome c oxidase activity (mitochondria marker) using a colorimetric assay kit (Sigma). Dense granule samples were concentrated in Nanosep 10K filters (PALL, Ann Arbor, MI), resuspended in the same volume of nanopure water, precipitated with cold acetone, and dried for subsequent proteomic analyses. Two-Dimensional Gel Electrophoresis and MALDI-TOF Mass Spectrometry. For two-dimensional gel electrophoresis (2DGE), dense granule samples were resuspended in 200 µL of sample rehydration buffer (7 M urea, 2 M thiourea, 1% (v/ v) NONIDET P-40, and traces of bromophenol blue) with the fresh addition of 30 mM dithiothreitol (DTT) and 0.5% (v/v) ampholytes pH 3-10 or pH 5-8 (Fluka). After brief centrifugation at 4 °C in a microfuge (10000g, 5 min) the supernatant was loaded on 11 cm Bio-Rad NL IPG strips (pH 3-10 or pH 5-8), and the first dimension electrophoresis was performed in standard conditions in a Bio-Rad Protean IEF cell. Strips were then suspended on trays containing 1.5 mL of re-equilibration buffer (50 mM Tris HCl pH 8.8, 6 M urea, 30% glycerol (v/v), 2% sodium dodecyl sulfate (SDS) (w/v), and traces of bromphenol blue) in two 15 min steps on a shaker. The first reequilibration step contained 15 mg of fresh DTT and the second 75 mg of freshly added iodoacetamide. Strips were then soaked for 15 min in SDS electrophoresis buffer, loaded on SDSpolyacrylamide denaturing gels, and run in a SE 600 Hoefer vertical midi-cell. Invitrogen benchmark markers were used as molecular mass standards. Gels were finally stained with colloidal Coomassie stain (EZ Blue, Sigma) following manufacturer’s instructions. Stained proteins were extracted and digested with trypsin following standard protocols, and peptides were subjected to matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) on an Ultraflex apparatus (Bruker Daltonics, Bremen, Germany). Mass spectra were acquired with the Flex control v. 2.0 and data processed with the Flex analysis v. 2.0 and Biotools v. 2.2 software (Bruker Daltonics, Bremen, Germany). Proteins were eventually identified with the MASCOT v 2.2 peptide mass fingerprint search engine (Matrix Science, London, UK) against the 20063108 release of MSDB database. Searching parameters were as follows: Type of search: Peptide mass fingerprint; Taxonomy: All entries; Enzyme: Trypsin, Fixed modifications: Carbamidomethylation of cysteines; Variable modifications: Oxidation of methionine; Mass value: Monoisotopic; Protein mass: Unrestricted; Peptide mass tolerance: (100 ppm; Peptide charge state: 1+; Maximum missed cleavages: 1. Liquid Chromatography Electrospray Ionization and MS/ MS Analysis. The liquid chromatography electrospray ionization (LC-ESI) MS/MS analysis of dense granule fractions was performed at the Proteomics Service of the Spanish National Center for Biotechnology (CNB, Madrid), as follows. Samples were resuspended in 180 µL of digestion buffer (urea 8 M, ammonium bicarbonate 25 mM, DTT 10 mM) and centrifuged, and supernatant aliquots of 50 µg (5 µL) were incubated at 37 °C for 1 h. Iodoacetamide at a final concentration of 50 mM

research articles

Proteomics of Platelet Dense Granules and 14-3-3

was then added, and samples were incubated at room temperature for 45 min in darkness. Samples were diluted 5× with ammonium bicarbonate 25 mM, 1 µg of recombinant trypsin (Roche, Mannheim, Germany) was added, and samples were incubated overnight at 37 °C. Resulting tryptic peptides were dried and dissolved in ESI Buffer A (0.5% acetic acid in water). Fractions were loaded in a 100 mm × 100 µM i.d. column (New Objective, Woburn, MA) and fractionated in a Famos-SwitchosUltimate chromatographic system (LCPackings, The Netherlands) with a linear gradient of 5-25% ESI Buffer B (90% acetonitrile, 0.5% acetic acid in water) at 500 nL/min for 180 min. Peptides eluting from the column were directly analyzed on a Esquire 3000Plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). Data-dependent MS/MS spectra were acquired by automatic switching between MS and MS/ MS mode using dynamic exclusion. Data processing was carried out using Bruker Daltonics’ software (DataAnalysis version 3.2). Searches using home-licensed MASCOT software (version 2.1.0) (Matrix Science, London) were performed in the latest version of NCBI database (NCBInr 20051213). Searching parameters were as follows: Type of search: MS/MS search; Taxonomy: Homo sapiens (108559 sequences); Enzyme: Trypsin; Fixed modifications: Carbamidomethylation of cysteines; Variable modifications: Oxidation of methionine; Peptide mass tolerance: (2 Da; Fragment mass tolerance: (0.8 Da; Maximum number of missed clevages allowed: 1. Minimum scoring value accepted was 40. Peptide fragmentation spectra of proteins identified through one or two individual peptides were manually validated. Estimated false discovery rate was calculated according to a statistical method previously described16 and was on the order of 0.002. Briefly, false discovery rate (FDR) was defined as the proportion of false positives among the population of spectra passing a given p-threshold, and it was calculated by the equation: FDR = (T - Op/Op)(p/1 - p), where T is the total number of spectra and Op is the observed number of spectra to score with probability lower than or equal to p.16 One-Dimensional Gel Electrophoresis and Western Blot Analyses. Washed platelets, isolated granules, or aliquots of platelet-secreted material were resuspended in 4× electrophoresis loading buffer (Sigma) and applied to 10% SDSpolyacrylamide gels. Electrophoresced proteins were transferred to Immobilon-P membranes (Millipore). Blotting was performed using standard techniques, as previously described.17 A 1/500 dilution was used for anti-pan cadherin and anticytochrome c oxidase Abs, and a 1/1000 dilution was used for other primary-specific Abs. Phosphatase-conjugated secondary Abs was used at a 1/5000 dilution. Electron Microscopy. Whole mount and conventional electron microscopy of the dense granule fractions were performed as described previously.12 Analysis of Platelet-Released Material. Aliquots of secreted material from thrombin-activated platelets were obtained as described previously.12 Briefly, washed platelets were resuspended in New Tyrode’s buffer at 108 cell/mL and placed in a cuvette with agitation. Thrombin was added at a final concentration of 1.2 units/mL, and aliquots were taken at different times. Samples were directly filtered trough a 0.2 µm filter to separate the secreted material from the suspension. Short-chain polyP in the samples was measured as described before,12 and contents of 14-3-3ζ were analyzed by western blot as described above. Abdominal Aorta Wall Samples. Abdominal aorta wall samples were obtained as described previously.18 Briefly, fresh

aneurysmal wall samples from three male patients with infrarenal abdominal aortic aneurysms were obtained during an elective surgical repair. Diagnosis was made by physical examination and computed tomographic scan. All samples from patients with aortic aneurysms presented atherosclerotic plaques at the intima layer. Control samples of abdominal aorta wall were obtained from cadaver donors for solid organs that did not present evidence of atherosclerosis. The tissues were maintained frozen in liquid nitrogen until processing; samples were fixed in 10% formaldehyde (v/v), embedded in paraffin, and sliced in sections. The study was carried out following ethical committee approval and informed consent from patients. Immunostaining and Fluorescence Confocal Microscopy. For isolated platelets, protein localization was determined by immunostaining as described,19 using 1/50 dilutions of primaryspecific Abs and 1/100 dilutions of labeled-secondary Abs (Alexa488-labeled anti-rabbit, Alexa488-labeled anti-mouse, Alexa647-labeled anti-rabbit, or Alexa647-labeled anti-mouse). For aorta tissues, sections were fixed with 4% paraformaldehyde (v/v) in phosphate buffer saline (PBS) for 20 min at 4 °C, washed, and permeabilized with PBS containing 0.1% Triton X-100 (w/v) and 1% bovine serum albumin (BSA) (w/v). Sections were blocked for 15 min in PBS plus 1% BSA (w/v) and 5% fetal calf serum (v/v), followed by an overnight incubation at 4 °C with 14-3-3ζ antibodies (1/50 dilution). Samples were reblocked as above and incubated 1 h at room temperature with Alexa647-conjugated anti-rabbit IgG (1/100 dilution). Samples were mounted with DABCO. Fluorescence localization in the samples was visualized and recorded using a laser TCS-SL confocal imaging system (Leyca Microsystems). Negative controls of all preparations were done in parallel with preimmune serum or without the primary antibodies. Haematoxylin/Eosin Staining. A Mayer’s hematoxylin staining was done over sections of aorta tissue samples using standard procedures.18 Low-magnification images (40×) were obtained with an Olympus BX-40 microscope employing an Olympus DP71 digital image system.

Results Isolation of Human Platelet Dense Granules. In order to isolate the relatively large quantities of human platelet dense granules required for the proteomic analysis, we utilized a method previously described in our group12 adapted to the use of expired platelet plasma units. It has been described that platelet proteome remains stable on the first 5 to 9 days of storage;20 therefore we used 6-day stored platelets that were broadly available to us. In the procedure, total platelet membranes were separated using a density gradient in which dense granules accumulated at the densest fraction of the gradient (designated below as “fraction 13”). This fraction was examined by electron microscopy (Figure 1A). The observation of fraction 13 samples air-dried, without fixation and staining, showed only electron-dense vacuoles (Figure 1A, left panel) similar to the descriptions of platelet dense granules reported by several authors.11 An examination of fraction 13 using conventional transmission electron microscopy with fixation, dehydration, and staining procedures showed membrane-bound organelles of similar size that appeared to be empty (Figure 1A, right panel). Immunoblot analysis of protein markers (Figure 1B) in whole platelet lysates (P) and fraction 13 samples (dense granule, DG) showed that the enrichment of LAMP1, a protein generally considered a dense granule and lysosomal marker,11,21 had been obtained by the isolation. In addition, MRP4, a Journal of Proteome Research • Vol. 6, No. 11, 2007 4451

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Figure 1. Isolation of dense granules from human platelets. (A) Electron microscopy of dense granule fractions. Before observation, preparations were unfixed and unstained (left) or fixed and sectionated (right). Bars: 0.2 µm (B) Immunoblot analysis of protein markers. Five to 25 µg of protein from whole platelets (P) or dense granule fractions (DG) was tested against the following antibodies: LAMP1 (lysosome and dense granule marker), MRP4 (dense granule marker), VWF (alpha granule marker), cytochrome c oxidase (COX, mitochondria marker), cadherin (CAD, plasma membrane marker).

Figure 2. Two-dimensional gel electrophoresis of platelet dense granules. Fractions enriched in dense granules were subjected to 2DGE, and gels were stained with colloidal Coomassie Blue dye. The indicated spots were excised and digested with trypsin, and the resultant peptides were analyzed by MALDI-TOF MS. Protein markers of molecular weight and gradients of pI are indicated. Gels were repeated at least three times; a representative experiment is shown.

protein recently described as a dense granule marker,14 was increased in the dense granule fraction (Figure 1B). Crosscontamination with alpha granules, mitochondria, and plasma membrane was discounted by detecting specific markers for these organelles (Figure 1B). In addition, lysosomal and mitochondrial enzymatic activities were reduced at the dense granules fraction in relation with the other fractions of the density gradients (Figure 3B). Protein Identification of Platelet Dense Granule Fractions by 2DGE MALDITOF MS and LC-ESI MS/MS. Isolated platelet 4452



dense granule fractions were separated by 2DGE, and 20 protein spots were excised from gels, digested with trypsin, and analyzed by MALDI-TOF MS (Figure 2). The proteins identified using Mascot software are presented in Table 1. Eighteen of the proteins were identified on the basis of their highest ranking hit, MOWSE scores higher than 75, and sequence coverage over 20% (Table 1). Then LC-ESI MS/MS was used to further characterize soluble proteins from platelet dense granule fractions. Peptides, from trypsin-digested dense granule fractions, were separated, in optimized elution conditions, through a nanocapillary column. In this case, the proteins were considered as identified when they had MOWSE scores higher than 40 and were reproduced at least in two independent samples of isolated granules (Table 1). Using this method, 35 proteins were identified (Table 1). With these two experimental methods, we identified a total of 40 different proteins. Among these, 11 identifications were identical independently of the method used (Table 1). All of the identified proteins have previously been described as platelet proteins, but this is the first report describing the localization of most of them in the dense granule. To our knowledge, from the list presented in Table 1, only the platelet glycoprotein IIb has been reported as being localized in the dense granule.22 Information on functions of the identified proteins was obtained from the SwissProt Protein Knowledgebase (http://www.expasy.org/ sprot/) and from published literature (Table 1). Of the 40 proteins identified, 15 were from the cytoskeleton, 11 were directly related to platelet function, 5 were involved with the glycolytic pathway, 5 were molecular chaperones, and 4 were related to general cell signaling. Western Blot Analysis Confirms the Presence of 14-3-3ζ in Platelet Dense Granules. From the list of proteins identified above in the platelet dense granules, we focused on the study 14-3-3ζ, a protein associated with intracellular signaling and regulation in platelets in conjunction with GpIb/V/IX (recently reviewed in ref 23). We found there was high expression of 143-3ζ in lysates of whole platelets (P), and in dense granule fractions (DG), by western blot (Figure 3A). In addition, analysis of platelet membranes separated by density gradients showed that distribution of 14-3-3ζ and the dense granule markers, serotonin and polyP, increased toward the densest fractions of the gradient and they were enriched 8 to 16 times at fraction 13 (Figure 3B). In contrast, protein markers of other organelles, such as lysosome (acidic phosphatase) and mitochondria (cytochrome c oxidase), presented a different distribution and lower enrichments at fraction 13 (Figure 3B). Furthermore, we analyzed the levels of 14-3-3ζ in whole platelet lysates of three controls and three patients of δSPD by immunoblot. Similar levels of 14-3-3ζ were found in controls (Ct) and patients (Pt), as shown at Figure 3C for representative samples. Immunocytochemistry of 14-3-3ζ in Human Platelets and Its Co-localization with Dense Granule Markers. Distribution of 14-3-3ζ was analyzed by immunofluorescence and confocal microscopy (Figure 4). We observed a strong signal of 14-3-3ζ localized in cytoplasmic structures and a faint signal around the cytoplasm and/or plasma membrane (Figure 4A,B). Colocalization images (Figure 4A) show that the distribution of 14-3-3 ζ was similar to that of MRP4 protein (dense granule marker14). We also found a partial co-localization of 14-3-3ζ with LAMP1 (dense granule and lysosomal marker11,21) (Figure 4B). As a control, we also show the partial co-localization of LAMP1 with MRP4 (Figure 4C).


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Figure 3. Immunoblot analysis of 14-3-3ζ in human platelets and dense granule fractions. (A) Presence of 14-3-3ζ in isolated dense granules. Four micrograms of protein from whole platelet lysates (P) or dense granule fractions (DG) was tested against 14-3-3ζ antibodies. (B) Distribution of 14-3-3ζ and organelle markers on platelet membrane fractions separated by density gradients. Total levels of 14-3-3ζ, serotonin (dense granules), inorganic polyphosphate (PolyP) (dense granules), acidic phosphatase (lysosome), cytochrome c oxidase (mitochondria), and total protein are shown. The numbers given in parentheses indicate the enrichment of each protein marker on dense granule fraction (fraction 13). 14-3-3ζ amounts were determined by western blot and densitometry. (C) Levels of total 14-3-3ζ in delta storage pool disease. Ten micrograms of protein from whole platelets from control healthy volunteers (Ct) and from patients with delta storage pool disease (Pt) was tested against 14-3-3ζ antibodies. Results of one representative patient are shown.

Figure 4. Confocal immunofluorescence analysis in platelets of dense granule markers and 14-3-3ζ. The figure shows the colocalization of (A) 14-3-3ζ and MRP4 (dense granule maker); (B) 14-3-3ζ and LAMP1 (lysosome and dense granule marker); and (C) LAMP1 and MRP4. Bars: 2 µm.

Secretion Kinetics of 14-3-3 ζ from Thrombin-Activated Platelets. Previous proteomic studies of platelet-released material have demonstrated that 14-3-3ζ is the only isoform of the 14-3-3 family that is secreted by thrombin-activated platelets.24,25 To confirm the granular origin of this protein, we studied the time course of secretion of 14-3-3ζ from platelets after thrombin addition (Figure 5). We used polyP as a marker of secretion from platelet dense granules because we had demonstrated before that this polymer is secreted by thrombin-

Figure 5. Time course of thrombin-induced polyP release compared with 14-3-3ζ protein. Release of polyP (squares) and 14-3-3ζ (triangles) in platelets after the addition of thrombin as described under Methods. Results are expressed as percentage of the material released at 300 s.

activated platelets with similar kinetics to platelet dense granule components: serotonin, PPi, and ADP.12 Figure 5 shows that the time courses of polyP and 14-3-3ζ secretion are similar. Presence of 14-3-3ζ in Aorta Walls of Patients with Abdominal Aortic Aneurysm. We studied the distribution of 143-3ζ in sections of aorta wall from three patients with abdominal aortic aneurysm, using immunofluorescence (Figure 6). To localize the aorta wall components, low-magnification haematoxylin/eosin staining from representative controls (Figure 6A) and patients (Figure 6D) is shown. Atheromatous plaques are observed in samples of patients with aneurysm (Figure 6D). Labeling of 14-3-3ζ appears only in the aneurysm patients, concentrated in patches on the extreme luminal side of Journal of Proteome Research • Vol. 6, No. 11, 2007 4453

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Journal of Proteome Research • Vol. 6, No. 11, 2007 23031 8483 27899 47202 18229 72402 71082 83584 57480 40536 41321 103563 51926 18719 18719 283301 86043 86043 76475 19914 17091 227646 40456 15014 15216 272762 22563 28619 29243 29109 124161 117220 39851 39706 47350 47244 36202 36900 58339 58411 14171 83728 49708 56577 55545 50092 36596 7992 114446 14171 67690 71316 133321

gi/10863927 gi/7229462 gi/5729877 gi/306891 gi/2144545 gi/54036678 gi/14250401 gi/4501891 gi/5453595 gi/105664 gi/5031635 gi/57284201 gi/71649 gi/4504165 gi/41281905 gi/188586 gi/188590 gi/12667788 gi/4505879 gi/130979 gi/4826898 gi/16753233 gi/12803567 gi/107978 gi/88935 gi/9508585 gi/21903479 gi/4507877 gi/113606 gi/28614 gi/119339 gi/16878083 gi/31645 gi/54696396 gi/20178296 gi/35505 gi/129874 gi/119720 gi/223918 gi/279672 gi/182430 gi/71827 gi/121039 gi/34810164 gi/7447665 gi/4505981 gi/113576 gi/28592 gi/135717

MW

gi/539679 gi/16974825 gi/80477445 gi/16741280

NCBI acc. no.

9.04 5,75 5,48 8.54 8,31 5.70 8,46 8,8 5.21 9,04 5.63 6.05 4,71

8.30 8,34 6.99 7.59 8,26 5.71 7.95 7,58

5.55 5.56 5.25 8.07 8.22 8.22 5.7 5.90 5.9 6.52 5.09 4.51 5.5 8.32 8.46 8.44 5.8 8.41 4.67 4.75 4,79 5.51 5,83

7.68 5.07 5,37 4,97 4.76

5.10 3,9 4,73 5,14

pI

6/40% 3/4% 1/3% 29/48% 1/3% 25/59% 1/4% 2/35% 42/39% 4/25% 39/58% 2/3% 6/6%

24/72% 1/3% 14/21% 1/3% 3/13% 2/8% 28/49% 6/15%

11/43% 6/20% 4/6% 2/6% 12/63% 2/18% 24/14% 27/32% 2/3% 2/3% 1/6% 1/8% 9/6% 1/3% 14/72% 4/40% 21/13% 2/13% 27/71% 34/69% 3/14% 55/40% 11/12%

1/6% 42/55% 1/1% 1/1% 17/25%

16/72% 1/17% 3/14% 1/3%

peptides sec./coverage (%)

61 48 60 218 50 172 45 101 256 125 343 101 175

259 155 137 50 156 76 152 174

141 357 126 75 130 91 714 260 132 105 54 56 297 44 94 187 739 100 243 292 227 388 333

44 364 43 50 147

157 54 198 53

protein score

involved in cell mobility, ubiquitously expressed involved in cell mobility, ubiquitously expressed anchor actin to intracellular structures effectively recycling of cofilin and actin controls actin polymerization controls actin polymerization anchors transmembrane proteins to the actin “end-blocking” of actin (promotes assembly) “end-blocking” of actin (promotes assembly) links actin cytoskeleton to ECM regulatory light chain of myosin (binds Ca+2) regulatory light chain of myosin (does not bind Ca+2) involved in cell shape and secretion (binds to F-actin) PKC substrate -cytoskeletal reorganization prevents or enhances actin polymerization prevents or enhances actin polymerization cytoskeletal protein (links vinculin to integrins) cytoskeletal regulation-actin binding stabilizes cytoskeleton actin filaments stabilizes cytoskeleton actin filaments stabilizes cytoskeleton actin filaments involved in anchor F-actin to the membrane involved in anchor F-actin to the membrane converts F1,6DP into GAP and DHAP converts F1,6DP into GAP and DHAP converts pyruvate into GAP converts pyruvate into GAP converts GAP into 1,3 BPG converts pyruvate into lactate converts pyruvate into phosphoenolpyruvate converts pyruvate into phosphoenolpyruvate heparin neutralization (associated with R granules) cross-linking of fibrin chains polymerizes into fibrin; acts as cofactor in aggreg. polymerizes into fibrin; acts as cofactor in aggreg. polymerizes into fibrin; acts as cofactor in aggreg. polymerizes into fibrin; acts as cofactor in aggreg. antigen binding (associated with R granules) heparin neutralization (associated with R granules) receptor for fibrinogen and fibronectin platelet-derived growth factor, chemokine main protein of plasma (associated with R granules) main protein of plasma (associated with R granules) mediates cell-to-cell and cell-to-matrix interactions

PMF (1)a MS/MSb MS/MSb MS/MSb PMF (9)a MS/MSb MS/MSb PMF (19)a MS/MSb MS/MSb MS/MSb MS/MSb MS/MSb MS/MSb PMF (10)a MS/MSb MS/MSb MS/MSb PMF (2)a PMF (3)a MS/MSb PMF (20)a MS/MSb PMF (13)a MS/MSb PMF (8)a MS/MSb MS/MSb MS/MSb PMF (6)a MS/MSb PMF (11)a MS/MSb MS/MSb PMF (7)a MS/MSb PMF (18)a MS/MSb MS/MSb PMF (14)a MS/MSb PMF (4)a MS/MSb MS/MSb

Protein identifications

peptidylprolyl isomerase A (molecular chaperone) facilitates the assembly of multimeric proteins molecular chaperone (Ca+2 and ATP binding) molecular chaperone catalyzes protein -S-S- bonds

MS/MSb PMF (5)a MS/MSb MS/MSb PMF (15)a

b

regulates Rho proteins calcium binding and signaling actives Tyr 3-hydroxylase phosphatidylserine-binding protein

function

PMF (17)a MS/MSb MS/MSb MS/MSb

identification method

a Protein identifications performed by 2DGE MALDITOF MS and PMF (peptide mass fingerprint). The numbers given in parentheses indicate the spot positions on the 2D gels (Figure 2) performed by LC-ESI MS/MS. c Proteins that have been reported to be secreted by activated platelets (refs 22 and 23).

Cell signaling (4 proteins) 1-Rho-GDP-dissociation inhibitor Ly-GDI/c 2-calcium-calmodulin N-terminal domain/c 3-14-3-3 protein ζ/c 4-serum deprivation response protein/ Molecular chaperones (5 proteins) 5-cyclophilin A/c 6-glucose-regulated protein 78 kDa (grp78)/c 7-heat shock 70kDa protein 8 isoform 1/ 8-heat shock 90 kDa protein/ 9-protein disulfide-isomerase/ Cytoskeletal (15 proteins) 10-actin beta/c 10-actin beta/c 11-actinin alpha 1/c 12-adenylyl cyclase-associated protein/ 13-cofilin 1/c 13-cofilin-1 non muscle/c 14-Filamin A/c 15-gelsolin precursor/c 15-gelsolin isoform a or b/c 16-kindin1/ 17-myosin light chain 2/ 17-myosin light chain 3/ 18-myosin, heavy polypep. 9/ 19-pleckstrin/c 20-profilin, 1 chain A/c 20-profilin I/c 21-talin 1/c 22-transgelin 2/c 23-tropomyosin beta chain/ 23-tropomyosin/ 23-tropomyosin isoform/ 24-vinculin (Metavinculin)/c 24-vinculin isoform VCL/c Glycolysis (5 proteins) 25-aldolase A/c 25-aldolase A/c 25-alpha enolase 1/ 26-enolase 3/ 27-GAPDH/c 28-lactate dehydrogenase B/ 29-pyruvate kinase/c 29-pyruvate kinase/c Directly related to the platelet function (11 proteins) 30-beta-thromboglobulin precursor/ 31-coagulation factor XIII A chain precursor/c 32-fibrinogen alpha/c 33-fibrinogen beta chain precursor/c 33-fibrinogen beta precursor/c 34-fibrinogen gamma-A chain precursor/c 35-Ig gamma-1 chain C region/c 36-platelet factor 4/c 37-platelet membrane glycoprotein IIb/ 38-pro-platelet basic protein precursor/c 39-serum albumin/c 39-serum albumin/c 40-thrombospondin-1 precursor/c

protein no. and name

Table 1. Proteins Identified in Platelet Dense Granules.

research articles Herna´ ndez-Ruiz et al.


Figure 6. Distribution of 14-3-3ζ in normal and aneurysmal aorta wall. Sections of abdominal aorta wall from controls (A, B, C) and patients with abdominal aortic aneurysm (D, E, F, H, I, J) were analyzed. Results of one representative patient are shown. Samples were stained with haematoxylin/eosin (A, D) or with anti-14-3-3ζ by immunofluorescence (B, C, E, F, I, J). Squares at low-magnification haematoxylin/eosin staining (A, D) are provided to indicate the localization of the intima represented in the immunofluorescence experiments (B, C and E, F, respectively). 14-3-3ζ fluorescence labeling appears only in the atherosclerotic plaque (F) and in the inflammatory infiltrates (J). B, E, and I are mergers of bright fields and 14-3-3ζ labeling. Arrows show the inflammatory infiltrates. Bars: 75 µm.

atherosclerotic plaques (Figure 6F) and in the inflammatory infiltrates (Figure 6J). Control immunostaining, using rabbit preimmune sera instead of 14-3-3ζ antibodies, does not reveal any signal in the sections of aneurysm patients (result not shown), indicating the specificity of the signal.

Discussion Subcellular fractionation has become an effective partner to proteomics in the understanding of the intracellular organelles and in the detection of new protein functions.26,27 In this paper, we combine these two techniques to perform the analysis of human platelet dense granules. We have used an adaptation of a dense granule isolation procedure that is free from contamination by lysosomes and mitochondria12 to generate a highly enriched fraction of dense granule proteins, as demonstrated by electron microscopy and western blot (Figure 1). To date, the presence of mainly membrane proteins has been described in platelet dense granules.11,14,15 In order to identify principally luminal and structural proteins of platelet dense granules, we concentrated on the analysis of the soluble subfractions after processing the samples without any ionic detergents. Using this approach, we identified 40 proteins by combining 2DGE MALDITOF MS and LC-ESI MS/MS (Table 1). We compared our protein identifications with previous proteomics studies that analyzed the platelet releasate after thrombin activation.24,25 At least 80% of the proteins identified in this work had previously been reported to belong to the

research articles platelet secretome (Table 1), suggesting their granular localization. In addition, several platelet dense granule proteins reported here (14-3-3 isoforms, enolase, myosin, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and heat shock proteins, among others) have also been identified in recent proteomics studies of isolated neutrophil secretory granules,28 natural killer cell lytic granules,29 and melanosomes.30 All of these secretory compartments have been considered as a group with functional similarities, denominated “secretory lysosomes” or “lysosomal-related organelles”;31,32 therefore it is not surprising that they share a significant proportion of identical proteins. Most of the proteins identified in our study have not been associated previously with the dense granule, such as actinassociated proteins, glycolytic enzymes, and regulatory proteins (Table 1). Cytoskeletal and actin-associated proteins (such as cofilin, gelsolin, profilin, talin, and vinculin) were identified in dense granules (Table 1). Actin-associated proteins found in dense granules could be related to the characteristic regulatory mechanism of secretion of the organelle contents.21 We have previously reported that platelet dense granules accumulate massive quantities of polyP.12 Certain cytoskeletal proteins in dense granules could be connected with polyP content since it has been reported that a polyP synthetic enzyme is also an actin-related protein.33 In this context, we have found that platelets treated with actin-disrupting agents present lower contents of polyP (Hernandez-Ruiz and Ruiz; unpublished data). We also identified five proteins involved in glycolysis: aldolase, enolase, pyruvate kinase, GAPDH, and lactate dehydrogenase (Table 1). Glycolytic enzymes have been found also in the platelet releasate,24,25 and within the lumen of other lysosomal-related organelles28-30 isolated by different methods; therefore we excluded a possible contamination with other membrane fractions. We found also some proteins related directly to the platelet function (Table 1). Most of these proteins have been previously associated with R granules such as platelet factor 4, betathromboglobulin, fibrinogen, Ig gamma-1, and serum albumin. As possible contamination with R granules was ruled out by western blot analysis with VWF antibodies (Figure 1B), our results suggest that these proteins are located in dense granules as well. Alpha and dense granules are both formed from the multivesicular bodies in megakaryocytes,34 and their membranes share some similar proteins such as IIb3, GpIb, and P-selectin.22,35 From the list of identified proteins, we focused on studying 14-3-3ζ, a member of a conserved family of proteins that interact with hundreds of different proteins.7 Human platelets express ζ, β, γ, , and η 14-3-3 isoforms,36 but only 14-3-3ζ is released by activated platelets.24,25 14-3-3ζ is related to platelet signaling and regulation by its association with GpIb/V/IX (reviewed in ref 23). In addition, a recent proteomic study has identified 14-3-3ζ as a platelet signaling protein that is tyrosinephosphorylated after the activation of the collagen receptor GpVI.37 We confirmed the presence of high levels of 14-3-3ζ in platelet dense granules by western blot, immunofluorescence, and functional analysis of platelet secretion (Figures 3-5). Our results indicate that 14-3-3ζ is secreted by an activated platelet from its dense granules, suggesting new extracellular roles for this protein. The family of 14-3-3 proteins has been considered by many to be relevant in intracellular functions, Journal of Proteome Research • Vol. 6, No. 11, 2007 4455

research articles but recent advances have implicated the family in extracellular signals as well. Keratinocytes secrete 14-3-3σ protein, also known as “stratifin”, an anti-fibrogenic factor that regulates the gene expression in dermal fibroblasts.38,39 Several members of the 14-3-3 protein family, including 14-3-3ζ, have been identified in extracellular matrix vesicles of osteoblasts40 and also in exosomes of dendritic cells.41 Secreted 14-3-3 proteins have been involved in the cross-linking of the cell wall glycoproteins in the unicellular green alga Chlamydomonas42 and also in the protection from fungal infection of the pea root tip.43 The δSPDs are a group of disorders characterized by malfunction and/or defective formation of platelet dense granules, as well as other lysosome-related organelles.44 As our results suggested that 14-3-3ζ is localized abundantly in dense granules, we hypothesized that patients with δSPD must show lower levels of this protein than healthy controls. Surprisingly, immunoblot analysis of whole platelet lysates showed similar levels of 14-3-3ζ in all samples (Figure 3C). It has been reported that resting platelets contain a considerable proportion of cytosolic 14-3-3ζ.36 Levels of this soluble protein must be masking the differences between controls and patients with δSPD. Acordingly, a recent study showed relevant differences in the distribution, but not on the total expression, of a dense granule protein on platelets of δSPD patients.14 Likewise, we believe that further studies on the distribution and/or secretion of 14-3-3ζ in platelets from δSPD patients and controls could probably reveal some differences. It has been demonstrated that some proteins secreted from platelets contribute to the progression of atherosclerosis, such as platelet factor 445 and RANTES.46 Thus, we evaluated the localization of 14-3-3ζ in sections of the aorta wall of patients with abdominal aortic aneurysm, which present atherosclerotic plaques. In these samples, 14-3-3ζ was found forming patches on the extreme luminal side of atherosclerotic plaques (Figure 6), suggesting that the protein could originate from activated platelets in the lumen of the artery. As 14-3-3ζ protein has been implicated with adhesion molecules such as integrins47 and GpIb,3-6 our observations could relate this protein to cellular adhesion to the atherosclerotic endothelium, thus resulting in the progression of the disease. We also found an intracellular distribution of 14-3-3ζ in inflammatory infiltrates, presumably from platelets or other mononuclear cells present.48

Conclusions In summary, we have used 2DGE MALDITOF MS and LCESI MS/MS to reveal new features of the soluble protein composition of platelet dense granules. In addition, our results suggest, for the first time, an extracellular role for 14-3-3ζ in the progression of atherosclerosis. Further studies will be needed to elucidate the mechanisms of 14-3-3ζ involvement in atherosclerotic disease, but our observations reveal it as an attractive therapeutic target.

Acknowledgment. We thank Prof. Arthur Kornberg for kindly providing the E. coli CA38 pTrc PPX1, Dr. Alberto Paradela for assistance with the ESI MS/MS analysis, Dr. Carmen Castro for critically reading the manuscript, and Marıá I. Medinilla for technical assistance. This work was supported in part by the Spanish Ministerio de Ciencia y Tecnologıá (Grant BFU2004-06097), the Plan Andaluz de Investigacion, and the Conserjerıá de Salud (Junta de Andalucia). F.A.R. and F.V. are researchers of the “Ramon y Cajal” Programme (Spanish Ministry of Education and Science). F.A.R. was a 2004/2005 4456



“Larramendi” Research Fellow (Fundacion Mapfre-Medicina). L.H-R. is a Predoctoral Fellow of the “Puerta del Mar” Foundation.

Supporting Information Available: Supplementary information 1: Spectra information of protein identifications performed by PMF. Supplementary information 2: Representative spectra used to identify proteins by PMF. Supplementary information 3: Information of protein identifications performed by LC-ESI MS/MS. Supplementary information 4: MS/MS spectra of identifications based on one peptide. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Huo, Y.; Ley, K. F. Role of platelets in the development of atherosclerosis. Trends Cardiovasc. Med. 2004, 14 (1), 18-22. (2) Ware, J. Molecular analyses of the platelet glycoprotein Ib-IX-V receptor. Thromb. Haemost. 1998, 79 (3), 466-78. (3) Du, X.; Harris, S. J.; Tetaz, T. J.; Ginsberg, M. H.; Berndt, M. C. Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex. J. Biol. Chem. 1994, 269 (28), 18287-90. (4) Feng, S.; Christodoulides, N.; Resendiz, J. C.; Berndt, M. C.; Kroll, M. H. Cytoplasmic domains of GpIbalpha and GpIbbeta regulate 14-3-3zeta binding to GpIb/IX/V. Blood 2000, 95 (2), 551-7. (5) Munday, A. D.; Berndt, M. C.; Mitchell, C. A. Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3zeta. Blood 2000, 96 (2), 577-84. (6) Dai, K.; Bodnar, R.; Berndt, M. C.; Du, X. A critical role for 143-3zeta protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications. Blood 2005, 106 (6), 1975-81. (7) Aitken, A.; Jones, D.; Soneji, Y.; Howell, S. 14-3-3 proteins: biological function and domain structure. Biochem. Soc. Trans. 1995, 23 (3), 605-11. (8) Ichimura, T.; Isobe, T.; Okuyama, T.; Yamauchi, T.; Fujisawa, H. Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+, calmodulin-dependent protein kinase II. FEBS Lett. 1987, 219 (1), 79-82. (9) Aitken, A. 14-3-3 proteins: a historic overview. Semin. Cancer Biol. 2006, 16 (3), 162-72. (10) Rendu, F.; Brohard-Bohn, B. The platelet release reaction: granules’ constituents, secretion and functions. Platelets 2001, 12 (5), 261-73. (11) McNicol, A.; Israels, S. J. Platelet dense granules: structure, function and implications for haemostasis. Thromb. Res. 1999, 95 (1), 1-18. (12) Ruiz, F. A.; Lea, C. R.; Oldfield, E.; Docampo, R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J. Biol. Chem. 2004, 279 (43), 44250-7. (13) Smith, S. A.; Mutch, N. J.; Baskar, D.; Rohloff, P.; Docampo, R.; Morrissey, J. H. Polyphosphate modulates blood coagulation and fibrinolysis. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (4), 903-8. (14) Jedlitschky, G.; Tirschmann, K.; Lubenow, L. E.; Nieuwenhuis, H. K.; Akkerman, J. W.; Greinacher, A.; Kroemer, H. K. The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 2004, 104 (12), 3603-10. (15) Chintala, S.; Tan, J.; Gautam, R.; Rusiniak, M. E.; Guo, X.; Li, W.; Gahl, W. A.; Huizing, M.; Spritz, R. A.; Hutton, S.; Novak, E. K.; Swank, R. T. The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet dense granules. Blood 2007, 109 (4), 1533-40. (16) Lopez-Ferrer, D.; Martinez-Bartolome, S.; Villar, M.; Campillos, M.; Martin-Maroto, F.; Vazquez, J. Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST. Anal. Chem. 2004, 76 (23), 6853-60. (17) Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979. Biotechnology 1992, 24, 1459. (18) Ocana, E.; Bohorquez, J. C.; Perez-Requena, J.; Brieva, J. A.; Rodriguez, C. Characterisation of T and B lymphocytes infiltrating abdominal aortic aneurysms. Atherosclerosis 2003, 170 (1), 3948.

research articles

Proteomics of Platelet Dense Granules and 14-3-3 (19) Reales, E.; Mora-Lopez, F.; Rivas, V.; Garcia-Poley, A.; Brieva, J. A.; Campos-Caro, A. Identification of Soluble N-EthylmaleimideSensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for Antibody Secretion. J. Immunol. 2005, 175 (10), 6686-93. (20) Thiele, T.; Steil, L.; Gebhard, S.; Scharf, C.; Hammer, E.; Brigulla, M.; Lubenow, N.; Clemetson, K. J.; Volker, U.; Greinacher, A. Profiling of alterations in platelet proteins during storage of platelet concentrates. Transfusion 2007, 47 (7), 1221-33. (21) Flaumenhaft, R.; Dilks, J. R.; Rozenvayn, N.; Monahan-Earley, R. A.; Feng, D.; Dvorak, A. M. The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion. Blood 2005, 105 (10), 3879-87. (22) Youssefian, T.; Masse, J. M.; Rendu, F.; Guichard, J.; Cramer, E. M. Platelet and megakaryocyte dense granules contain glycoproteins Ib and IIb-IIIa. Blood 1997, 89 (11), 4047-57. (23) Du, X. Signaling and regulation of the platelet glycoprotein IbIX-V complex. Curr. Opin. Hematol. 2007, 14 (3), 262-9. (24) Coppinger, J. A.; Cagney, G.; Toomey, S.; Kislinger, T.; Belton, O.; McRedmond, J. P.; Cahill, D. J.; Emili, A.; Fitzgerald, D. J.; Maguire, P. B. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004, 103 (6), 2096-104. (25) McRedmond, J. P.; Park, S. D.; Reilly, D. F.; Coppinger, J. A.; Maguire, P. B.; Shields, D. C.; Fitzgerald, D. J. Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes. Mol. Cell. Proteomics 2004, 3 (2), 133-44. (26) Huber, L. A.; Pfaller, K.; Vietor, I. Organelle proteomics: implications for subcellular fractionation in proteomics. Circ. Res. 2003, 92 (9), 962-8. (27) Brunet, S.; Thibault, P.; Gagnon, E.; Kearney, P.; Bergeron, J. J.; Desjardins, M. Organelle proteomics: looking at less to see more. Trends Cell. Biol. 2003, 13 (12), 629-38. (28) Lominadze, G.; Powell, D. W.; Luerman, G. C.; Link, A. J.; Ward, R. A.; McLeish, K. R. Proteomic analysis of human neutrophil granules. Mol. Cell. Proteomics 2005, 4 (10), 1503-21. (29) Casey, T. M.; Meade, J. L.; Hewitt, E. W. Organelle Proteomics: Identification of the Exocytic Machinery Associated with the Natural Killer Cell Secretory Lysosome. Mol. Cell. Proteomics 2007, 6 (5), 767-80. (30) Chi, A.; Valencia, J. C.; Hu, Z. Z.; Watabe, H.; Yamaguchi, H.; Mangini, N. J.; Huang, H.; Canfield, V. A.; Cheng, K. C.; Yang, F.; Abe, R.; Yamagishi, S.; Shabanowitz, J.; Hearing, V. J.; Wu, C.; Appella, E.; Hunt, D. F. Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes. J. Proteome Res. 2006, 5 (11), 3135-44. (31) Dell’Angelica, E. C.; Mullins, C.; Caplan, S.; Bonifacino, J. S. Lysosome-related organelles. FASEB J. 2000, 14 (10), 1265-78. (32) Blott, E. J.; Griffiths, G. M. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 2002, 3 (2), 122-31. (33) Gomez-Garcia, M. R.; Kornberg, A. Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (45), 15876-80. (34) Youssefian, T.; Cramer, E. M. Megakaryocyte dense granule components are sorted in multivesicular bodies. Blood 2000, 95 (12), 4004-7.

(35) Israels, S. J.; Gerrard, J. M.; Jacques, Y. V.; McNicol, A.; Cham, B.; Nishibori, M.; Bainton, D. F. Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140). Blood 1992, 80 (1), 143-52. (36) Wheeler-Jones, C. P.; Learmonth, M. P.; Martin, H.; Aitken, A. Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation. Biochem. J. 1996, 315 (Pt 1), 41-7. (37) Garcia, A.; Senis, Y. A.; Antrobus, R.; Hughes, C. E.; Dwek, R. A.; Watson, S. P.; Zitzmann, N. A global proteomics approach identifies novel phosphorylated signaling proteins in GPVIactivated platelets: involvement of G6f, a novel platelet Grb2binding membrane adapter. Proteomics 2006, 6 (19), 5332-43. (38) Ghaffari, A.; Li, Y.; Karami, A.; Ghaffari, M.; Tredget, E. E.; Ghahary, A. Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin. J. Cell. Biochem. 2006, 98 (2), 383-93. (39) Ghahary, A.; Marcoux, Y.; Karimi-Busheri, F.; Li, Y.; Tredget, E. E.; Kilani, R. T.; Lam, E.; Weinfeld, M. Differentiated keratinocytereleasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts. J. Invest. Dermatol. 2005, 124 (1), 170-7. (40) Xiao, Z.; Camalier, C. E.; Nagashima, K.; Chan, K. C.; Lucas, D. A.; de la Cruz, M. J.; Gignac, M.; Lockett, S.; Issaq, H. J.; Veenstra, T. D.; Conrads, T. P.; Beck, G. R., Jr. Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts. J. Cell. Physiol. 2007, 210 (2), 325-35. (41) Thery, C.; Boussac, M.; Veron, P.; Ricciardi-Castagnoli, P.; Raposo, G.; Garin, J.; Amigorena, S. Proteomic analysis of dendritic cellderived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 2001, 166 (12), 7309-18. (42) Voigt, J.; Frank, R. 14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall. Plant Cell 2003, 15 (6), 1399-413. (43) Wen, F.; VanEtten, H. D.; Tsaprailis, G.; Hawes, M. C. Extracellular proteins in pea root tip and border cell exudates. Plant Physiol. 2007, 143 (2), 773-83. (44) Gunay-Aygun, M.; Huizing, M.; Gahl, W. A. Molecular defects that affect platelet dense granules. Semin. Thromb. Hemost. 2004, 30 (5), 537-47. (45) Nassar, T.; Sachais, B. S.; Akkawi, S.; Kowalska, M. A.; Bdeir, K.; Leitersdorf, E.; Hiss, E.; Ziporen, L.; Aviram, M.; Cines, D.; Poncz, M.; Higazi, A. A. Platelet factor 4 enhances the binding of oxidized low-density lipoprotein to vascular wall cells. J. Biol. Chem. 2003, 278 (8), 6187-93. (46) Huo, Y.; Schober, A.; Forlow, S. B.; Smith, D. F.; Hyman, M. C.; Jung, S.; Littman, D. R.; Weber, C.; Ley, K. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med. 2003, 9 (1), 61-7. (47) Bialkowska, K.; Zaffran, Y.; Meyer, S. C.; Fox, J. E. 14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta. J. Biol. Chem. 2003, 278 (35), 33342-50. (48) Pearce, W. H.; Koch, A. E. Cellular components and features of immune response in abdominal aortic aneurysms. Ann. N. Y. Acad. Sci. 1996, 800, 175-85.

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Journal of Proteome Research • Vol. 6, No. 11, 2007 4457

Organellar Proteomics of Human Platelet Dense Granules Reveals

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