2D‐DIGE‐based proteomic analysis of intracoronary versus peripheral arterial blood platelets from acute myocardial infarction patients: Upregulation of platelet activation biomarkers at the culprit site

Abstract

Purpose: Platelets play a fundamental role in the atherothrombotic events that lead to an acute myocardial infarction. In the present study we compared the proteome of intracoronary and peripheral arterial platelets from ST-elevation myocardial infarction (STEMI) patients in the search for potential platelet biomarkers/drug targets related to what is happening at the culprit site. Experimental design: Ten STEMI patients were recruited and blood collected from the occluded coronary artery, at the culprit site, in the moment of reperfusion. Systemic blood obtained from the radial artery of the same patients was used as control. Proteome analysis was based on high-resolution 2D-DIGE and mass spectrometry. Validations were by western blotting in a group of 11 patients. Results: Sixteen differentially regulated protein features were identified, corresponding to 15 ORFs, mostly related to cytoskeletal and signaling proteins. We demonstrate the up-regulation of integrin ␣IIb (ITA2B), the adapter Src kinase-associated phosphoprotein-2 (SKAP2), and thrombospondin-1 isoforms in intracoronary platelets. Conclusion and clinical relevance:This study constitutes the first analyzing in detail the proteome of arterial intracoronary platelets from STEMI patients. We show variations in the platelet proteome when comparing intracoronary and peripheral platelets. Observed differences might be related to platelet activation events at the culprit site. Keywords:Acute myocardial infarction / Intracoronary / Integrin ␣IIb / Platelet proteome / SKAP-2 / Thrombospondin-1 Additional supporting information may be found in the online version of this article at the publisher's web-site

Alteration of platelet GPVI signaling in ST-elevation myocardial infarction patients demonstrated by a combination of proteomic, biochemical, and functional approaches

“…There are precedents of intracoronary studies, using a similar approach, that allowed the detection of platelet activation biomarkers in the intracoronary blood. Indeed, we recently showed an up-regulation of signaling proteins in platelets from intracoronary blood at the culprit site - in comparison with peripheral platelets - in STEMI patients25, which highlights the relevance of studying platelets from occluded blood in direct contact with the thrombus as activation biomarkers are more prompted to be elevated in these platelets. As another example, Gresele and colleagues recently showed that platelets release matrix metealloproteinase-2 in the coronary circulation of patients with ACS as a consequence of platelet activation26.…”

“…Intracoronary samples from STEMI patients were taken during normal hemodynamics procedure using a Pronto V3 catheter (Vascular Solutions), which occludes antegrade flow allowing collecting culprit site blood from the distal artery252728. Peripheral arterial samples were obtained from the radial artery through which the sheath was inserted.…”

Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

“…showing that these proteins had more than two-fold increase in their expression profiles in the "thrombinactivated" vs "resting" platelets. We recovered a high overlap of proteins members with the kinome family reported already by other proteomic analyses, [20][21][22][23][24][25][26][27][28][29][30] that are also indexed in many other research studies related to the proteomic profiling of agonists activated platelets, such as the "Adhesome" and "Platelet Web" databases, as described later in the results section. Other major pathways predicted by IPA to be up-regulated in the proteome of "thrombin-activated platelets" were mapped to the cytoskeleton, actin and RhoA mediated cellular signaling ( Figure 18).…”

“…The "in solution" digestion with three enzymes (trypsin/LysC/Glu-C), enabled the extraction of peptides from the total platelet proteome, including the membrane-derived and microvesicles contained in the releasates, as reported previously by other studies on platelets proteomics. [20][21][22][23][24][25][26][27][28][29][30] The extracted peptides were subjected to nano LC/MS/MS sequencing on a Q Exactive quadrupole orbitrap mass spectrometer, as summarized in the protocol shown in Figure 1. The label-free proteomics analysis employed the label free quantification (LFQ) module provided by PEAKS 8.5 (Bioinformatics Solutions Inc.), In addition, the changes in the protein expression profiles were complemented by an additional analysis involving the Scaffold and per SPECtives software (Proteome Software Inc.).…”

“…[13][14][15][16] Using highly sensitive and highly specific mass spectrometers (providing mass accuracy and resolution), and proteome fractionations assays involving denaturant, and reducing one-dimensional or two-dimensional protein electrophoretic systems (1-DE and 2-DE, respectively) followed by protein digestions with multiple enzymes (trypsin, LysC, Glu-C and Asp-N, among others), and further fractionations of peptides by means of orthogonal chromatographic systems, thousands of proteoforms can be characterized from highly pure fractions consisting of only 2x10 8 platelets, purified from small volumes of blood, usually in the range of few milliliters. [20][21][22][23][24][25][26][27][28] Consequently, the future advances in the mass spectrometric profiling of protein expression and their corresponding highly-regulated PTM are expected to allow platelet proteomics to be implemented in many research and clinical laboratories as a reliable tool for characterizing the molecular and cellular pathways that affect platelet homeostasis in healthy and disease states. Many original research articles and recent reviews highlighted the wide area of research dedicated to label or label-free, targeted or untargeted proteomics profiling of changes in the platelets purified from patients experiencing cardiovascular, inflammatory, and bleeding disorders.…”