High Precision Platelet Releasate Definition by Quantitative Reversed Protein Profiling—Brief Report

Abstract

However, these direct methods experience a high false-positive rate because of contamination by uncontrolled platelet lysis. Here, we take a reverse approach, monitoring quantitatively the concentration changes of all proteins in the platelets after platelet stimulation, whereby we assume that the released protein content should be detectable in the reduction of its level from the whole platelet proteome. For accuracy, our strategy uses stable isotope-labeling ( Figure 1A) to discriminate the released proteins from uncontrolled lysis products. In other words, most platelet proteins do not change except the ones that are significantly released on activation. Materials and MethodsMaterials and Methods are available in the online-only Supplement. 10-13 ResultsPerforming the reversed releasate quantitative proteomics experiments on 3 individuals resulted in the identification of 4375 unique proteins, of which 2970 (68%) could be quantified in ≥2 of 3 individuals (Table I in the online-only Data Supplement). Identity of the Platelet ReleasateFirst, we set out to identify the releasate on full platelet activation. The releasate was evaluated in each individual by the determination of the dimethyl intensity ratio between the light (resting) and intermediate (activated) labeled peptides ( Figure 1A). As expected, the vast majority of © 2013 American Heart Association, Inc. Objective-Platelet activation and subsequent protein release play an important role in healthy hemostasis and inflammatory responses, yet the identity and quantity of proteins in the platelet releasate are still debated. Here, we present a reversed releasate proteomics approach to determine unambiguously and quantitatively proteins released from activated platelets. Approach and Results-Isolated platelets were mock and fully stimulated after which the released proteins in the supernatant were removed. Using high-end proteomics technology (2D chromatography, stable isotope labeling, electron transfer dissociation, and high collision dissociation fragmentation) allowed us to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of ≈4500 platelet proteins, we observed that after stimulation via thrombin and collagen, only 124 (<3%) proteins were significantly released (P<0.05). The released proteins span a concentration range of ≥5 orders, as confirmed by ELISA. The released proteins were highly enriched in secretion tags and contained all known factors at high concentrations (>100 ng/mL, eg, thrombospondin, von Willebrand factor, and platelet factor 4). Interestingly, in the lower concentration range of the releasate many novel factors were identified. proteins (>95%) had a resting/activated ratio close to 1.0. These ratios were highly reproducible between replicates with a median RSD between ratios of 9.6% (Table I and Figure I in the online-only Data Supplement). Using the statistical criteria described in the Methods section in the online-only Data Supplement, 124 proteins were observed to be ...

Immunothrombosis in Acute Respiratory Distress Syndrome: Cross Talks between Inflammation and Coagulation

“…During endothelial damage, the exposure to subendothelial collagen leads to platelet activation, consisting of a change of their shape and subsequent release of cell membrane proteins and granular contents, including chemokines, cytokines, coagulation proteases, adhesive molecules, growth factors, and mediators of angiogenesis [60,61] . Platelet-derived mediators lead to further platelet aggregation and activation, and promote hemostatic mechanisms and clot formation.…”

What Can Proteomics Tell Us About Platelets?

“…115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches. COFRADIC indicates combined fractional diagonal chromatography; and MudPIT, multidimensional protein identification technology.…”

Caracterização proteômica comparativa da agregação plaquetária induzida pela trombina e pela PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca

“…Avanços importantes podem ser vistos em diversos trabalhos sobre a composição das proteínas plaquetárias humanas, analisando plaquetas em repouso e ativadas (Marcus et al, 2000;O'Neill et al, 2002;García et al, 2004a;Martens et al, 2005;Qureshi et al, 2009;Májek et al, 2010;Burkhart et al, 2012), proteínas pertencentes à membrana plasmática (Moebius et al, 2005;Maguire et al, 2005;Senis et al, 2007;Tucker et al, 2009), proteínas secretadas após ativação com diferentes agonistas (McRemond et al, 2004;Coppinger et al, 2004Coppinger et al, e 2007Piersma et al, 2009;Wijten et al, 2013;van Holten et al, 2014;Vélez et al, 2015), proteínas presentes em micropartículas plaquetárias (García et al, 2005;Capriotti et al, 2013;Aatonen et al, 2014;Milioli et al, 2015) e nos grânulos alfa e densos Embora exista uma extensa literatura sobre função plaquetária, ainda pouco se sabe sobre os mecanismos que regem a secreção, tanto de proteínas como de pequenas moléculas, e também, de vesículas extracelulares (micropartículas e exossomos) durante processos fisiológicos e patológicos. O conhecimento mais acurado sobre o proteoma e o secretoma de plaquetas, em diferentes condições de ativação, poderá contribuir para o entendimento dos mecanismos que regem este sistema e também, no desenvolvimento de novos alvos para terapias antiplaquetárias.…”

“…A maioria dessas proteínas já foi descrita por outros autores que analisaram o secretoma de plaquetas Wijten et al, 2013;van Holten et al, 2014;Vélez et al, 2015), e também serão descritas adiante em nossa análise do sobrenadante de plaquetas ativadas.…”

“…Entre as proteínas identificadas no sobrenadante de plaquetas ativadas por PA-BJ ou trombina, a trombospondina-1, o fator XIII da coagulação, e o fibrinogênio, estão envolvidas na formação do trombo plaquetário, e já foram extensivamente descritas como presentes no secretoma de plaquetas ativadas, em outros estudos Maynard et al, 2007;Piersma et al,2009;Wijten et al, 2013;van Holten et al, 2014).…”