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J Thromb Haemost. Author manuscript; available in PMC 2016 October 01. Published in final edited form as: J Thromb Haemost. 2015 October ; 13(10): 1908–1917. doi:10.1111/jth.13070.

Lysine Acetyltransfer Supports Platelet Function Joseph E. Aslan1,2,3, Rachel A. Rigg1, Marie S. Nowak1,6, Cassandra P. Loren1, Sandra M. Baker-Groberg1, Jiaqing Pang1, Larry L. David5, and Owen J. T. McCarty1,2,4 1Departments

of Biomedical Engineering, Oregon Health & Science University, Portland, OR

97239, USA 2Cell

and Developmental Biology, Oregon Health & Science University, Portland, OR 97239, USA

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3Knight

Cardiovascular Institute, Oregon Health & Science University, Portland, OR 97239, USA

4Division

of Hematology & Medical Oncology School of Medicine, Oregon Health & Science University, Portland, OR 97239, USA 5Proteomics

Shared Resource, Oregon Health & Science University, Portland, OR 97239, USA

6Department

of Medical Physics, Universite des Sciences et Technologies de Lille, Villeneuve d'Ascq Cedex, France

Summary

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Background and Objectives—The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function.

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Methods and Results—Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments found that platelets express p300, which is activated in a Src kinase-dependent manner upon platelet stimulation with the platelet glycoprotein VI (GPVI) agonist CRP. Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin and cortactin as well as F-actin polymerization, integrin activation and platelet aggregation. Super resolution visualization of platelet actin-rich adhesion structures revealed abundant acetyllysine protein content colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodia formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiological role for lysine acetyltransferase activity in platelet function.

Corresponding author: Joseph E. Aslan, Knight Cardiovascular Institute, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Mail Code UHN-62, Portland, OR 97239, USA, Tel: (503) 418-9350, Fax: (503) 418-9311, [email protected]. Addendum J. E. Aslan, L. L. David and O. J. T. McCarty designed the research. J. E. Aslan, R. A. Rigg, M. S. Nowak, C. P. Loren, S. M. BakerGroberg, J. Pang and L. L. David performed experiments. J. E. Aslan, R. A. Rigg, L. L. David and O. J. T. McCarty analyzed the data. J. E. Aslan and O. J. T. McCarty wrote the paper. The authors state that they have no conflicts of interest.

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Conclusion—Together, our findings uncover lysine acetyltransfer as a potential regulator of platelet actin dynamics and reveal potential roles for lysine acetylation in the molecular coordination of platelet activation and function. Keywords acetyltransferases; actin; metabolism; p300 acetyltransferase; platelets

Introduction

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Platelets serve as the cellular mediators of hemostasis and thrombosis, having critical roles in normal physiology as well as in diverse disease states [1]. After budding from the proplatelet appendages of megakaryocytes, platelets circulate to detect sites of vessel rupture and, in turn, respond to halt bleeding. In addition to maintaining vascular integrity, platelets have roles in angiogenesis, tumor metastasis, inflammation and immunity [2]. Platelets express a diverse set of cell surface receptors that respond to extracellular cues with intracellular signaling events to promote platelet intracellular calcium release and cytoskeletal changes that ultimately lead platelets to aggregate with one another to form hemostatic plugs or thrombi [3]. While intracellular signaling platforms such as kinases [3] and GTPases [4] gate key molecular stages of platelet activation and serve as potential drug targets in the management of disease states, the roles of other signaling systems with emergent cell biological functions remain largely unexplored in platelet biology.

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Similar to the reversible phosphorylation of serine/threonine and tyrosine residues, the modification of protein lysine ε-amino groups by acetylation, catalyzed by lysine acetyltransferases (KATs) and deacetylases, regulates diverse aspects of cellular function [5-7]. Proteomics and functional studies have determined that, like phosphorylation, lysine acetylation is an evolutionarily conserved protein covalent modification program found in diverse organisms, tissues and cell types [5, 6]. While histones and other nuclear proteins are a central focus of KAT studies [7], acetylation of cytosolic proteins has developing roles in cellular function. The acetylation of cytoskeletal proteins, including α-tubulin [8], cortactin [9] and myosin [10] regulates cell contractility, motility and migration processes. Studies of intermediary metabolism suggest that lysine acetylation of metabolic enzymes may connect metabolic state to cellular function [11-13]. Lysine acetyltransfer also has cell biological roles in chaperone function [14], autophagy [15] and apoptosis [5]. Collectively, such studies have developed a model in which acetyllysine modifications target multiple substrates of macromolecular structures to integrate complex cellular programs such as cytoskeletal remodeling, metabolism, protein translation and apoptosis [6, 12, 16].

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We recently demonstrated that in platelets, the cytoskeletal protein α-tubulin is acetylated and specifically deacetylated upon platelet activation, suggesting a role for lysine acetyltransfer in platelet function [17]. Although >4500 proteins are modified by lysine acetylation in other mammalian cell types [6], the presence and regulation of lysineacetylated proteins in platelets beyond α-tubulin remains largely unexamined. In this study, to explore a systems-level role for lysine acetyltransfer in platelet function, we define the platelet lysine acetylome, identifying candidate KAT substrate proteins in platelets with

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roles in cytoskeletal and metabolic functions. We find that lysine acetylation of platelet proteins is, in part, mediated by the KAT p300, which is activated following platelet stimulation with the glycoprotein GPVI agonist collagen-related peptide (CRP). Through platelet functional studies, we validate a role for p300 acetyltransfer in platelet actin regulation and platelet hemostatic function.

Materials and Methods Reagents Reagents are listed in Data S1. In vitro platelet studies

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Purified platelets were isolated from human venous blood drawn from healthy volunteers by venipuncture into sodium citrate in accordance with an IRB-approved protocol at Oregon Health & Science University. Anticoagulated blood was centrifuged (200×g, 20 min) to obtain platelet rich plasma (PRP). PRP was centrifuged (1000×g, 10 min) in the presence of prostacyclin (0.1 μg/ml). PRP-isolated platelets were resuspended in modified HEPES/ Tyrode buffer (129 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1mM MgCl2; pH 7.3) and washed once via centrifugation at 1000×g for 10 min in modified HEPES/Tyrode buffer. Purified platelets (>97.5% purity as determined by flow cytometry) were resuspended in modified HEPES/Tyrode buffer at indicated concentrations. Platelet proteomics, Western blot, immunoprecipitation, flow cytometry and acetyltransferase assay methods are detailed in Data S1. Platelet adhesion, spreading and visualization

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For platelet adhesion assays, video microscopy and immunofluorescence microscopy experiments, #1.5 glass coverslips (Warner Instruments) or #1.5 Glass Bottom Dishes (MatTek) were coated with 50 μg/ml human fibrinogen (FIB3, Enzyme Research Labs) or 100 μg/ml fibrillar collagen (Chronolog) and blocked with fatty-acid free BSA (Sigma) prior to platelet seeding, as described [18]. Platelet static adhesion, immunofluorescence and live video microscopy were performed using Kohler-illuminated Nomarski differential interference contrast (DIC) optics with a Zeiss 63× oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert 200M microscope as previously described [19-21]. The surface areas of individual platelets were measured using Image J software and plotted as previously described [18]. For acK immunofluorescence experiments, fixed platelets were blocked, permeablized and stained in blocking buffer (PBS + 0.1% SDS + 1% BM). Super resolutionstructured illumination microscopy (SR-SIM) experiments were carried out on a Zeiss Elyra SR-SIM microscope as previously described [17, 18]. Statistical analyses For acetyltransferase enzyme activity data, a one-way within-subjects ANOVA was performed, followed with Bonferroni-corrected, unequal variance t-tests for each timepoint versus baseline. For flow cytometry, whole blood flow experiments, and Western blot quantification, a two-way ANOVA was performed (treatment and day as factors), followed by post-hoc analysis with Tukey's test. A square root transformation was applied when data J Thromb Haemost. Author manuscript; available in PMC 2016 October 01.

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were determined to lack equal variances by Bartlett's test for homogeneity of variance. For platelet spreading data, a random sample of 90 platelets per treatment (random seed = 42) was plotted using the density function (geom_density) in ggplot2 in R. In all analyses, p < 0.05 was considered statistically significant. Analyses were performed in R (R Foundation for Statistical Computing, Vienna, Austria).

Results Identification of lysine-acetylated proteins in platelets

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To determine if proteins in addition to α-tubulin are modified by lysine ε-amino acetylation in platelets, we purified platelets from healthy volunteers and analyzed platelet lysates for proteins with N-ε acetyllysine (acK) moieties by Western blot. As seen in Fig. 1A, platelets contain a number of acK positive proteins of various molecular weights, supporting the hypothesis that, in addition to α-tubulin, multiple platelet proteins are regulated by lysine acetylation. Immunofluorescence microscopy of human platelets in fixed in solution as well as platelets adherent to a surface of fibrinogen revealed that acK-modified proteins are widely distributed throughout platelets, suggesting a multifunctional role for acK protein modifications in platelet biology (Fig. 1B and S1).

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Unlike protein phosphorylation, which is routinely analyzed quantitatively by staining with panphosphospecific antisera, Western blotting and immunostaining assays of protein acK modifications are limited, owing to the low affinities of pan acK antibodies for varying acK motifs as well as the high abundance of some acetylated proteins that mask the detection of less prevalent proteins with acK modifications [12, 22]. Thus, to examine the platelet acK proteome, we conducted proteomics studies to identify platelet proteins modified by lysine acetylation. Purified human platelets were lysed by sonication in 8M urea before trypsinization and acK peptide enrichment. Mass spectrometric analysis identified 552 acK modifications mapping on 273 platelet proteins involved in actin cytoskeletal regulation, metabolism, protein translation, ER homeostasis and other cellular functions (Fig. 2, Supporting Table 1). STRING interactome analyses [23] of platelet acK proteins identified functional networks around actin regulation and metabolism while gene ontology (GO) analysis noted network roles in platelet activation (p=3.34×10−22), actin cytoskeletal reorganization (p=2.31×10−11; Fig. 2B) and cellular catabolic processes (p=2.04×10−19; Fig. 2C). In addition to actin, which is modified on 4 lysine residues by acetylation (Supporting Table 1), acK-modified actin regulatory proteins included talin (24 acKs), filamin (19 acKs), vinculin (10 acKs) and actinin (4 acKs). Mitochondrial proteins and metabolic enzymes with roles in glycolysis, TCA cycle, amino acid catabolism and oxidative phosphorylation were also amongst the most modified by lysine acetylation, highlighting a potential role for lysine acetylation in the metabolic regulation of platelet function (Fig. 2C, Supporting Table 1). Other proteins with more specific roles in platelet function and hemostasis, including Factor XIII (3 acKs) and GPIbα, were also modified by lysine acetylation. Histones, which account for ~30% of acK-containing proteins in nucleated cells [6], are not substantially expressed in anucleate platelets [24] and were not detected in our analysis of the platelet acK proteome. Notably, platelet protein acK modification was not directly dependent on protein abundance, supporting the hypothesis that lysine acetylation of platelet proteins is a regulated process;

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acK peptides were identified from highly abundant platelet proteins such as cytosolic actin (ACTB, estimated 750,000 protein copies per platelet [24]) as well as lower abundance proteins such as the Threonine synthase-like 1 protein (THNS1, estimated 620 protein copies per platelet [24]) (Fig. S2). KAT expression and regulation in platelets

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Protein lysine acetylation is catalyzed by lysine acetyltransferase (KAT) enzymes that transfer the acetyl group of acetyl-CoA to the ε-amino group of specific protein lysine residues. To date, 22 KATs, grouped into 3 separate families (p300/CBP, GCN5 and MYST), have been identified in human and mouse cells [5]. While KATs typically localize to the nucleus to regulate histone acetylation and transcriptional activation, KATs such as p300 have increasingly recognized roles in the cytoplasm [25]. To investigate how protein lysine acetylation may be regulated in anucleate platelets, we analyzed identified platelet protein acK peptides for lysine acetyltransferase consensus sequence motifs using Acetylation Set Enrichment-Based (ASEB) analysis [26]. ASEB analysis determined with high confidence (p

Lysine acetyltransfer supports platelet function.

The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in ...
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