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

Platelets are small anucleated blood particles derived from megakaryocytes in the bone marrow, and they play a key role in the control of bleeding and hemostasis.1‒10 Platelets need to be activated to perform their functions, a process mediated by many physiological activators. The best known and characterized activators at the level of signaling transduction mechanisms are: thrombin, arachidonic acid and its derivatives (thromboxane A2), collagen, and the adenosine nucleotides (ADP and ATP).2‒8 Activated platelets undergo vast cytoskeleton, organelles, and secretory protein reorganization accompanied by many posttranslational modifications (PTM).5‒14 Due to their anucleate nature, platelets have limited protein synthesis, and therefore it is expected that most of the changes encountered at the level of the proteome under different pathophysiological conditions will be determined mostly by changes in protein expression (translational and post‒translational regulated), at the level of PTM, proteolysis, or secretion during platelets degranulation processes associated with their activation.11‒15 It is well documented that selected platelet messages are translated into proteins after activation, regulating the inflammatory and hemostatic responses of the platelet.2‒8 Platelets can be affected by many physiological conditions during blood circulation that can ultimately lead to vascular complications initiated by thrombus formation.12‒16 During activation, release of platelet microparticles can generate various pathophysiological effects, such as initiation and exacerbation of stroke.16,17 One of the most advanced studies applied to the research of platelets biology under different activation states is the mass spectrometry coupled with profiling of protein changes applied to both resting and activated whole platelet proteomes, or to platelet subproteomes, including the platelet granules (alpha and dense), membrane systems, lipid rafts, and enriched phosphoproteome. Research conducted in the last five years acknowledges the identification of 5000‒5500 expressed proteins in human platelets, a proteome that is highly similar between different healthy individuals.10‒21 Recent emerging analytical technologies coupling the fractionation of the cellular proteome with the multidimensional nano LC/ESI/MS/MS sequencing, together with systems biology approaches empowered by bioinformatics analysis and data mining enabled a revolution in platelet proteomics that led to the development of new, mass spectrometric‒based assays for