O-linked sialic acid residues on platelet membrane glycoprotein (GP) IIb mask the human HPA-9b alloepitope.

Sialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins. The polymorphisms of Human Platelet Alloantigens (HPA)-3 and HPA-9, locating near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, are adjacent to sialyl-Core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase, or those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2, dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these two O-glycan chains contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached.