When platelets are chilled for short times (<4 hours), βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered GPIbαmolecules are recognized by the lectin domain of αMβ2 receptors on hepatic macrophages, and are removed from circulation. Clearance can be prevented by galactosylation of the exposed βGlcNAc residues. Galactosylation, however, does not improve the circulation of platelets refrigerated for longer times (>48 h-refrigerated) (Wandall et al. Blood, 2008). Investigating the clearance mechanism of 48 h-refrigerated platelets, we now demonstrate macrophage-independent hepatic removal, that is mediated by the Ashwell-Morell asialoglycoprotein receptor (AMR). This conclusion is based on the following evidence:
macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that dramatically improves the circulation of 4 h chilled platelets, does not prevent the clearance of 48 h chilled platelets;
streptavidin-POD staining reveals abundant long-term refrigerated and biotinylated platelets in hepatocytes;
48 h-refrigeration increases by ~1.7-fold the binding of the βgalactose-recognizing lectin RCA, but not of the βGlcNAc recognizing lectin sWGA lectin to platelets;
KO mice lacking Asgr-1 or Asgr-2 subunits of the AMR support 48 h-refrigerated platelet circulation times comparable to room temperature stored platelets;
Co-injection of asialofetuin, a competitive inhibitor of the asialoglycoprotein-receptors, restore the survival of 48 h-refrigerated platelets in WT mice; and
hepatocyte HepG2 cells ingest fluorescently labeled long-term refrigerated platelets in culture, and asialofetuin prevents this ingestion.
Regarding the platelet target of the Asgr-1/Asgr-2 receptors, circulation of 48 h-refrigerated platelets is markedly improved by removal of GPIbα’s N-T domain using O-sialopeptidase. We conclude that longterm refrigeration exposes galactose residues on GPIbα subunits of platelets to generate ligands recognized by hepatocyte Ashwell-Morell receptors.
Disclosures: Wandall:Zymequest, Inc.: Consultancy. Hoffmeister:Zymequest, Inc.: Consultancy.