Lysine acetyltransfer supports platelet function

Abstract

Summary 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. 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. 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.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach

“…Reversible lysine acetylation as catalyzed by lysine acetyltransferases such as p300 and deacetylase enzymes such as histone deacetylase 6 (HDAC6) and sirtuin (SIRT) proteins have also been shown to modify RhoGDIs to regulate their function (38). Indeed, our recent characterization of the platelet lysine acetylome identified Ly-GDI as a lysine-acetylated protein in platelets with a putative role in regulating actin-mediated platelet processes (6,12). Future work is needed to better understand how the phosphorylation and lysine acetylation of Ly-GDI as well as Fig.…”

Data detailing the platelet acetyl-lysine proteome

“…Purified platelets were isolated from 100 ml human venous blood drawn from healthy volunteers by venipuncture into sodium citrate in accordance with an IRB-approved protocol at Oregon Health & Science University as previously described [1] , [4] . Briefly, citrated blood was centrifuged at 200 g (20 min) to obtain platelet rich plasma (PRP).…”

“…Tandem mass spectrometry data was collected using a Thermo Orbitrap Fusion Tribrid Mass Spectrometer configured with an EasySpray NanoSource (Thermo Scientific). Survey scans were performed in the Orbitrap mass analyzer, and data-dependent MS2 scans in the linear ion trap following HCD fragmentation in the instrument׳s ion routing multipole, as detailed [1] . Peptides were identified using Protein Discoverer, version 1.4 (Thermo Scientific), using Sequest HT, a human Swiss-Prot database downloaded in April 2013 containing 20,221 protein entries, static modification of +57.021 for C residues, variable modifications of +15.995 and +42.011 for M and K residues, respectively, parent ion tolerance of +/−10 ppm, fragment ion tolerance of 0.6 Da, monoisotopic masses, and trypsin cleavage (max 2 missed cleavages).…”

“… Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al, 2015 [1] ). An affinity purification – mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets.…”

Identification, Quantification, and System Analysis of Protein N‐ε Lysine Methylation in Anucleate Blood Platelets

“…We recently detailed acetyl‐lysine modifications of cytosolic proteins through a proteomic analysis of human blood platelets . Platelets are well known as small (≈2 μm diameter), anucleate cells that mediate clot formation and vascular repair .…”