Hepatocytes Ingest Longterm Refrigerated Platelets: A Novel Platelet Clearance Mechanism.

We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (>8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.