Label Free Quantitative Mass Spectrometry Identifies Processes Linked to Platelet Degranulation As Early Events during Platelet Storage
Authors: Rijkers Maaike , van Alphen Floris P. , van der Meer Pieter F. , de Korte Dirk , Leebeek Frank W.G. , Meijer Alexander B. , Voorberg Jan , Jansen Gerard AJG 
Published: October 2019

Abstract
Background
Platelet concentrates (PCs) are stored at room temperature to preserve their biological activity. To minimize the risk of bacterial outgrowth, storage time is limited to 7 days. It is well-established that prolonged storage of PCs results in modifications that result in a decreased hemostatic efficacy. This loss of platelet functionality during storage is commonly referred to as the platelet storage lesion (PSL). Typical events linked to development of the PSL are shape changes, platelet activation and loss of receptors crucial for platelet functionality. Two-dimensional (2D) differential gel electrophoresis (DIGE), isotope tagging and isotope-coded affinity tagging (ICAT) have been previously used to monitor changes in protein composition during storage. These studies have provided valuable insights into the changes associated with the PSL, however, these studies generally focused on a limited set of proteins.
Aim
We aimed to generate an overview of changes in the platelet proteome during storage using label free quantitative mass spectrometry. Furthermore, we employed Gene ontology (GO) enrichment analysis to identify pathways and biological processes that were linked to development of the PSL.
Methods
Three independently pooled PCs were stored in plasma under standard blood bank conditions for 16 days. Tryptic peptides were separated by nanoscale C18 reverse phase chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer. The RAW mass spectrometry files were processed with the MaxQuant computational platform. The global changes in protein level during platelet storage were assessed employing the analysis-of-variance functions of PERSEUS. Gene ontology enrichment analysis of biological processes, molecular functions and cellular compartments of the significantly different proteins was performed using the Cytoscape plug-in BiNGO.
Results
A total number of 2501 proteins was detected in all 3 biological replicates in at least one of the time points analyzed. The analysis showed that 18 proteins were down-regulated over time, whereas the level of 3 proteins was found to increase. CytoScape BinGo analysis of these significantly downregulated proteins revealed that the majority of this set was linked to GO-terms platelet degranulation, secretion and regulated exocytosis. This set of proteins included von Willebrand factor (VWF), serglycin (SRGN), SPARC, amyloid beta A4 protein (APP), multimerin-1 (MMRN1) and platelet factor 4 (PF4). A significant decline in these protein levels was observed at day 5 of storage, suggesting that release of α-granules is a relatively early event during platelet storage. At day 5 also a marked decline in S100A9 was observed. S100A9 has been implicated in degranulation in neutrophils, and may therefore also be linked to platelet granule release. Levels of membrane surface platelet glycoproteins such as glycoprotein Ibα did not significantly change at day 5. Only one single protein, histone H2A, was found to be consistently decreased already at two days of storage, but the significance of this finding is not clear. Upon prolonged storage (13 and 16 days) an increase in the level of α-2-macroglobulin (A2M), immunoglobulin M (IGM) and glycogenin-1 (GYG1) was observed suggesting that platelets acquire an (increased) potential to bind and/or internalize proteins from their environment. Consistent with this notion we also detected significant levels of several serine protease inhibitors, although levels of these proteins did not change upon storage.
Conclusions
Overall, our findings highlight dynamic changes in protein composition of platelets during storage. Our data provide evidence for sustained release of α-granules over time which becomes significant at day 5. Our data also suggest that during storage, platelets can bind or ingest proteins from their environment which may have impact on the hemostatic properties of stored platelets.

Disclosures
Leebeek: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dutch Hemphilia Foundation: Research Funding.