An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies

Abstract

To date, there has been no ideal method for blood platelet isolation which allows one to obtain a preparation devoid of contaminations, reflecting the activation status and morphological features of circulating platelets. To address these requirements, we have developed a method which combines the continuous density gradient centrifugation with washing from PGI-supplemented platelet-rich plasma (PRP). We have assessed the degree of erythrocyte and leukocyte contamination, recovery of platelets, morphological features, activation status, and reactivity of isolated platelets. Using our protocol, we were able to get a preparation free from contaminations, representing well the platelet population prior to the isolation in terms of size and activity. Besides this, we have obtained approximately 2 times more platelets from the same volume of blood compared to the most widely used method. From 10 ml of whole citrated blood we were able to get on average 2.7 mg of platelet-derived protein. The method of platelet isolation presented in this paper can be successfully applied to tests requiring very pure platelets, reflecting the circulating platelet state, from a small volume of blood.

Pyrethroid Insecticide Effect on Platelet Biomembranes of Rats

“…Platelet membrane was isolated based on the method adopted by Aneta et al [27]. An equal volume of platelet suspension and Triton X-100 lysis buffer was taken in microfuge tubes and mixed by inversion.…”

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.…”

Galectina-1: detección, caracterización, actividades biológicas e interacciones en plaquetas humanas

“…Por lo tanto, para eludir resultados artificiales, existe la necesidad urgente de estandarizar los protocolos de preparación y activación de plaquetas, así como también del aislamiento de proteínas (Wrzyszcz et al, 2017).…”