Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially transferred to newly released extracellular vesicles

Abstract

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC‐EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC‐EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC‐EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC‐EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine‐1‐phosphate (−53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC‐EVs were enriched in lysophosphatidic acid (60‐fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A‐I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC‐EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.

Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets

“…Lipid extraction from PL‐EVs (corresponding to 100 μg of total protein), lipid processing, and quantification by lipid mass spectrometry of cholesterol, glycerophospholipids, and major sphingolipids were carried out as earlier (http://onlinelibrary.wiley.com/doi/10.1111/trf.12874/suppinfo). Minor sphingolipids and glycerophospholipids including cardiolipin (CL) and bis(monoacylglycero)phosphate were analyzed by hydrophilic interaction according to Scherer and colleagues .…”

Platelet Lipidomics

“…In contrast to this, human plasma contains S1P and also dihydrosphingosine, but little ceramide 25 . Separately, stored platelets were found to transfer phospholipids and sphingolipids to newly released extracellular vesicles resulting in increased ceramide and decreased S1P in the cells 28 .…”

“…Using traditional methods, individual lipid pools first had to be isolated using TLC, then hydrolyzed to release FAs for determination as free acid species, a considerably slower approach that requires more material 1-4 . Recently, the composition of stored platelets and extracellular vesicles were characterized, showing that vesicles became enriched with LPA, cholesterol, and other lipids 28, 29 .…”

Sources, metabolism, and regulation of circulating sphingosine-1-phosphate

“…The concentration of S1P in human platelets decreases upon storage, which is associated with elevation in ceramide level ( 31 ). It was shown that thrombocytes are able to dephosphorylate S1P (most likely with the help of LPP) and incorporate released sphingosine into ceramide and then sphingomyelin via the action of ceramide synthase and sphingomyelin synthase ( 25,28 ).…”