Abstract
Platelets have the shortest shelf-life of all major blood components and are the most difficult to store complicating platelet transfusion practices. Transfused fresh radiolabeled autologous platelets differ significantly in recovery and survival among healthy subjects, however the cause of the inter-individual differences remains unclear. We demonstrated that the loss of sialic acid from the surfaces of cold-stored and transfused platelets promotes their clearance by Ashwell Morell receptors. The loss of platelet surface sialic acid correlates with increases in surface sialidase activity during platelet storage. Here we investigated whether fresh platelets from individual donors exhibit differences in surface sialidase expression and glycan exposure and sialic acid content changes with storage.
Methods
Platelets were isolated by standard methods from the venous blood of healthy volunteers or from standard platelet concentrates (PCs) and analyzed by flow cytometry for surface β-galactose using FITC-conjugated E. cristagalli lectin (ECL). Platelet surface sialidase expression was measured by flow cytometry using antibodies to sialidases Neu1 and Neu3. Sialidase activities were assayed using standard methods, Platelet uptake by hepatocytes was measured by using the human hepatoma cell line HepG2. To further elucidate these issues in a structural biology context we performed baseline study of the N- and O-linked glycans and glycosphingolipids (GSLs) in platelets, and any structural changes observed during storage, by employing HPLC, LC-MS/(MS), and sequential mass spectrometry (MSn) approaches.
Results
We found that terminal galactose on freshly-isolated platelet glycoproteins varies considerably among healthy subjects: Seven of ten individuals had low levels of exposed galactose (15.3 ± 4.1, MFI) and three subjects exhibited significantly higher levels of terminal galactose as detected by flow cytometry using lectins. Reduced sialic acid content correlated with increased surface sialidase activity and expression. Platelets with high terminal galactose were ingested with a higher rate by HepG2 cells, i.e via Ashwell Morell receptors. Importantly, individuals with low sialic acid levels correlate with low platelet counts at steady state. Structural analysis revlealed that fresh platelet N-glycan pools include a significant amount of high-mannose (Man5-Man9) and asialo complex glycans, however, are dominated by a diverse range of complex sialylated structures with two to four antennae, up to four NeuAcs, and include antennary fucosylation, and five or more lactosamine extensions. The O-linked fractions are comprised of core-1 and core-2 glycans having zero, one, or two NeuAc residues. A significant decrease in sialylation during conventional platelet storage at room temperature was confirmed at the level of individual O-glycan structures. Quantitative analysis of the more structurally complex N-glycan pools is ongoing.
Conclusion
Our results show that fresh platelets from healthy individuals vary in surface sialidase activity and sialic acid content and exhibit a high complexity in glycan structures. Collectively we propose that individual platelet counts may be dependent on surface sialic acid content and that the surface sialic acid could represent a factor that affects the recovery and survival of transfused platelets.
Disclosures:
No relevant conflicts of interest to declare.
Sialic Acid and Sialidase Activity in Acute Stroke