PB0749 - Human Platelet Glycoprotein IIb is Glycosylated at the Polymorphic Serine Residue that Defines Human Platelet Antigen 3b

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening condition that is driven by anti-platelet antibodies targeting human platelet antigens (HPAs). The HPA-3 antigen system is located on platelet glycoprotein IIb (GPIIb). There are two isoforms of GPIIb which correspond to two different HPA-3 epitopes: HPA-3a has Ile at amino acid 874, and HPA-3b has Ser874. Prior work suggests that anti-HPA-3 antibodies may bind to O-glycans (carbohydrate polymers that can modify Ser) located on or near amino acid 874. The O-glycosylation profile of GPIIb has previously been largely uncharacterized, hampering efforts to understand the nature of the epitopes that are relevant for anti-HPA-3 FNAIT.

Aims: The aim of this study was to analyze GPIIb O-glycosylation in a site-specific manner.

Methods: Purified human platelet GPIIb/IIIa was analyzed by mass spectrometry glycomics and glycoproteomics to determine which amino acids are O-glycosylated (the glycosites) and the structure of the attached O-glycans. To determine the nature of the linkage of O-glycan neuraminic acid, GPIIb/IIIa was treated with the following enzymes: PNGaseF to remove N-glycans, Neuraminidase A to remove all neuraminic acid, and Neuraminidase S to remove ⍺2-3-linked neuraminic acid. Following enzymatic incubations, GPIIb/IIIa was analyzed by blotting with Maackia amurensis lectin II (MAL II), which preferentially binds to ⍺2-3-linked neuraminic acid.

Results: GPIIb is glycosylated at Ser 874, 876, and 878 (Figure 1). All three glycosites can be modified with a neuraminic acid-containing core 1 O-glycan. GPIIb O-glycans carry predominantly ⍺2-3-linked neuraminic acid (Figure 2).

Conclusion(s): This is the first study to report an in-depth analysis of human platelet GPIIb O-glycosylation. This work shows that the polymorphic Ser874 which defines HPA-3b can be glycosylated with an ⍺2-3-linked neuraminic acid core 1 O-glycan. This glycan may be important for binding of pathogenic FNAIT anti-HPA-3b antibodies to platelets.