PB0393 - Storage associated changes in apheresis platelet components using glycomics.

Background: Platelet transfusions are lifesaving treatments for bleeding and thrombocytopaenia. Currently, platelets are stored at 22°C for 7 days, but they deteriorate gradually. While there have been many metabolomic, proteomic and lipidomic studies of stored platelets, the glycome of stored platelets has not been well-studied (Lee-Sundlov, RPTH 2022;6(S1).

Aims: The aim was to characterise the platelet glycome, storage-associated changes and their impact on functionality.

Methods: The platelet N-glycome was investigated in lysates prepared from washed apheresis platelets on day 1, 5 and 7 post-collection (n=6). N-glycans were released enzymatically using PNGase F and the N-glycome determined using porous graphitised carbon (PGC)-nanoscale liquid chromatography (nanoLC)-Electrospray (ESI) tandem mass spectrometry (MS/MS), and quantified using Skyline 22.2.0.255 software. All major glycoproteins and surface glycans (lectin binding) were analysed by flow cytometry, as well as activation markers (P-selectin, PAC-1 binding, phosphatidylserine-exposure), and following activation with 20 µM TRAP-6.

Results: 118 different N-glycan structures were observed: complex type, sialylated N-glycans were most abundant. While most fucosylated N-glycans carried core fucose, some contained Lewis-X type fucose residues. Despite considerable donor variability, abundance of five complex and hybrid sialylated and fucosylated N-glycans increased during storage (Figure 1), while three hybrid fucose/complex sialylated and oligomannose-Man8 N-glycans decreased. Independent of storage, core-fucosylated N-glycans preferentially carried 2-3-linked sialic acid. Sialic acid was largely 2-6-linked if core fucose was absent.
On day 7 of storage, basal RCA-1 (galactose; r=0.64), sWGA-binding (βGlcNAc) correlated with αIIbβ3-integrin (r=0.75). ECL (galactose) and SNA (sialic acid) binding were inversely correlated with GPIbα abundance decrease over storage (r=-0.72 and r=-0.84). Basal sWGA and MAL binding correlated with P-selectin post-TRAP (r=0.78 and r=0.77).

Conclusion(s): Following platelet storage, specific N-glycans change and become sialylated, suggesting activity of various sialyltransferases for different N-glycan structures. Platelet surface glycans appear to be associated with GPIbα abundance and P-selectin post-TRAP.