ALBERT

Description: Microparticles (MPs) are small vesicles shed from stimulated cells that permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin and differs when produced by stimulation versus apoptosis. Whether or not MP properties vary according to stimulus is not currently known. Monocyte-derived MPs are of particular interest as they are known to promote inflammation and are highly procoagulant. We studied the characteristics of MPs produced from monocytic THP-1 cells upon four different types of stimulation: lipopolysaccharide, a soluble P-selectin chimera (P-sel-Ig), an IgG control, and PBS (to study spontaneously-generated MPs). We developed a novel criterion for defining an MP using calcein-AM staining which only fluoresces when inside an intact cell or vesicle. Using proteomics, flow cytometry, western blotting, and electron microscopy, we were able to compare the properties of the four MP populations. Through our proteomic analysis, we identified 331 proteins in the MPs produced by P-sel-Ig stimulation, 830 in the MPs produced by LPS stimulation, 199 in the MPs produced by IgG stimulation, and 457 in the MPs produced spontaneously. We found that MP populations were similar with respect to size distribution and expression of certain antigens such as the β2 and αL integrins. Not only did all the MPs express cytoskeletal proteins, verified by both proteomics and western blotting, but electron microscopy revealed that these MPs contain an internal three-dimensional protein scaffolding that is similar in structure to the highly branched cytoskeleton of cells. The MPs also shared the same level of tissue factor expression and procoagulant activity. Additionally, we found that MPs have distinct characteristics depending on stimuli. The MPs differed in phosphatidylserine expression with less phosphatidylserine-positive MPs produced by P-sel-Ig stimulation. There were 52 proteins identified by proteomics to be in only the MPs produced upon P-sel-Ig stimulation, and 408 proteins found only in the MPs produced upon LPS stimulation. The MPs also differed in the expression of proteins from specific subcellular locations. For example, the MPs produced via LPS stimulation contained many more mitochondrial and nuclear proteins; whereas, the MPs produced by P-sel-Ig stimulation contained more plasma membrane proteins involved in cell adhesion and signaling. Specifically, these MPs produced by P-sel-Ig stimulation expressed the inhibitory receptor leukocyte-associated immunoglobin-like-receptor-1 (LAIR-1) which binds to collagen. The expression of LAIR-1 suggests MPs generated from P-sel-Ig stimulation could accumulate at sites of vascular injury where collagen is exposed, allowing the procoagulant MPs to promote hemostasis. Our finding that the properties of MPs depend on the stimulus that produced them supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Description: Activated platelets release exosomal vesicles which contain a wide range of proteins and nucleic acids including microRNAs (miRNAs) and crucially these can be delivered to other cells1. The expression of approximately 50% of genes are regulated by miRNAs, therefore, they serve as powerful modulators of signalling networks within cells. In this study, we assess the miRNAs which platelets have the potential to deliver into target cells via secreted exosomes and we particularly focus on how this influences signalling through the WNT pathway in endothelial cells. Exosomes were isolated by differential ultracentrifugation from thrombin-activated platelet releasate and were assessed for microparticle vesicle contamination using western blotting, transmission electron microscopy and nanoparticle tracking analysis. miRNAs (372) were quantified using low density arrays using real-time PCR (Qiagen miScript miRNA PCR Arrays) from both whole platelets and isolated platelet exosomes. Identification of putative miRNA target genes was performed using multiple prediction algorithms (TargetScan, miRDB, DIANA-MicroT). Pathway analysis of the target genes was performed using ClueGO/Clupedia within Cytoscape (http://www.cytoscape.org/; version 3.01). Luciferase reporter assay and miRNA transfections in EA.hy926 endothelial cells were performed using Attractene (Qiagen). Exosome uptake by target cells was monitored using a lipophilic fluorescent stain (Dil) followed by fluorescent imaging. We successfully quantified 370 miRNAs in platelets and 277 miRNAs specifically in platelet-derived exosomes and identified a 32 miRNA signature which was preferentially enriched (〉2 fold, p

Description: Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Description: Upon activation platelets release a myriad of soluble proteins as well as two distinct membrane vesicle populations- exosomes and microparticles (MPs) into the external milieu [1]. Although there have been many studies characterising platelet MPs and their increased circulating numbers in disease [2, 3], platelet exosomal vesicles have been characterised to date only by transmission electron microscopy (TEM) [4]. Here, we characterize the proteome of human platelet exosomes and reveal that a population of these vesicles carry active WNT glycoproteins on their surface that can modulate WNT signalling activity in both endothelial and monocytic cells. To establish the exosomal proteome of human platelets we compared platelet exosomes isolated from three healthy volunteers by (i) differential ultracentrifugation (DC) and (ii) ExoQuickTM (ExoQ) precipitation. Assessment of both exosomal populations was performed using nanoparticle tracking analysis (NTA), TEM, western blot and proteomics analysis (in triplicate) using a high performance Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer coupled with quantitative computational analysis using MaxQuant software. We concluded that DC was the superior isolation method for platelet exosomes, for example, our NTA revealed a 2-fold greater enrichment (6x108 /ml) of platelet exosomes following isolation by DC in comparison to ExoQ. Proteomic analysis of platelet exosomes identified 704 proteins across 18 MS runs, 119 of which were present in all 18 runs and represented the core human platelet exosomal proteome. Using western blotting and immunogold TEM, we confirmed the presence of numerous well-established exosomal membrane markers (CD63, CD9, and HSP70) in our proteome, as well as several platelet-specific proteins including GPIb, as previously described [4], and GPV. We also detected several of the 19 human WNT glycoproteins to be specifically secreted in the exosomal fraction upon platelet activation and verified their surface expression on platelet-derived exosomes using immunogold TEM. As we have previously demonstrated, platelets themselves can be modulated directly by WNT glycoproteins [5], hence here, we assessed the paracrine impact of these WNT-positive platelet exosomes on intracellular signalling events in monocytes and endothelial cells, both cell types closely linked to the pathogenesis of atherosclerosis. Using fluorescence microscopy, we found that these platelet exosomes were readily endocytosed by both endothelial (EAHY296) and monocytic (THP-1) cell lines where they specifically modulated beta-catenin nuclear expression and canonical WNT signalling. In conclusion our proteomic analysis has revealed that platelet exosomes are new players in the regulation of WNT signalling in both endothelial and monocytic cells. 1. Coppinger, J.A., et al., Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood, 2004. 103(6): p. 2096-104. 2. Knijff-Dutmer, E.A., et al., Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum, 2002. 46(6): p. 1498-503. 3. van der Zee, P.M., et al., P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem, 2006. 52(4): p. 657-64. 4. Heijnen, H.F., et al., Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood, 1999. 94(11): p. 3791-9. 5. Steele, B.M., et al., Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A, 2009. 106(47): p. 19836-41. Disclosures No relevant conflicts of interest to declare.

Description: Modulation of the proteins released during activation is one mechanism whereby aspirin may influence platelet-mediated human disease. We investigated the effect of aspirin on the platelet releasate using mass spectrometry and found that different agonists evoked different releasate profiles, with aspirin having a general moderating effect on the amount of protein released regardless of the agonist. These observations were confirmed for several cytokines using an antibody array approach.

Description: Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6−/−), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

Description: Lipid rafts are specialised membrane regions, rich in dynamic multi-protein complexes that are implicated in the regulation of platelet activation processes, particularly those mediated by GPVI and GPIb/IX/V. To elucidate the role of rafts in platelet function, we carried out a comprehensive characterization of the detergent-insoluble proteins associated with rafts from control and VWF-activated platelets using liquid chromatography coupled to tandem mass spectrometry. Over 160 proteins were identified including 72 proteins unique to platelet rafts and several novel platelet signaling and vesicle transport proteins e.g., rab 5, rab 8 and syntaxin 11. Indeed, the high level of signaling and trafficking proteins identified implicates rafts as concentrating platforms for the critical platelet functions of activation and secretion. Moreover, activation through GPIb-IX-V resulted in the translocation of many proteins into or out of rafts. Proteins recruited into rafts upon VWF-activation included GPIbαand regulator of G-protein signalling-19 (RGS-19). We confirmed that the novel platelet protein, RGS-19, upon VWF activation resides exclusively within the raft fraction and using confocal microscopy, RGS-19 co-localised with GPIbα and the raft-marker flottilin, to both the plasma and vesicle membranes of VWF-activated platelets. The phosphorylation-dependent interaction of RGS proteins with 14-3-3 leads to the inhibition of their GTPase-activating protein (GAP) activity. Aligning RGS-19 to other members of the RGS family, we identified that it contained a novel 14-3-3 binding motif. Immunoprecipitation with an antibody to 14-3-3ζ demonstrated that RGS-19 and GPIbα co-immunoprecipitated with 14-3-3ζ in VWF-activated rafts. Furthermore, we demonstrated that acid phosphatase treatment could significantly reduce the co-immunoprecipitation of RGS-19 with 14-3-3ζ from activated rafts. Thus, within 15 sec of VWF activation we are suggesting maximal suppression of the GAP activity of RGS-19 in rafts through a phosphorylation-dependent association with 14-3-3ζ. Our results provide the basis for a new hypothesis concerning how RGS proteins might govern signaling within platelet rafts.

Description: Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets.