PB0388 - Platelet glycoprotein Ibα carries ABO blood group antigens

Background: ABO blood group is associated with risk of thrombosis and bleeding. Research into this association has focused largely on the impact of ABO antigens on von Willebrand factor (VWF). Platelet ABO antigens may also play an important role in hemostasis and thrombosis. Few studies have investigated the expression of ABO antigens on the platelet binding partner of VWF, GPIbα.

Aims: To confirm that platelet GPIbα carries ABO antigens, to determine if these antigens are present on N- vs O-glycans, and to estimate the number of ABO antigens carried by a single molecule of GPIbα.

Methods: The ectodomain of GPIbα was purified from human platelets of known blood type. The purified protein was treated with recombinant α-N-acetylgalactosaminidase (cleaves terminal α-N-acetylgalactose found in the A antigen, Fig 1A), α-galactosidase (cleaves terminal α-galactose found in the B antigen, Fig 1B), or Peptide N-glycosidase F (PNGaseF, cleaves N-glycans) and subjected to immunoblotting using monoclonal anti-A, anti-B, or Ulex europeaus (UEA) lectin (binds H antigen). The monosaccharide composition of the purified glycoprotein was determined by high-performance anion-exchange chromatography.

Results: GPIbα purified from donors of blood type A and B showed reactivity with antibodies against blood group A and B antigens, respectively (Fig 1C). GPIbα purified from donors of blood type O, A, and B showed reactivity with UEA lectin (Fig 1D). Treatment of type A GPIbα with α-N-acetylgalactosaminidase but not PNGase F abolished anti-A reactivity (Fig 1E). Treatment of type B GPIbα with α-galactosidase eliminated anti-B antigen reactivity (Fig 1C). Monosaccharide analysis showed a mean molar fucose-to-GPIbα ratio of 5.83 (Table 1).

Conclusion(s): Human platelet GPIbα carries ABO blood group antigens, most likely on O-glycans. On average, a single molecule of GPIbα carries less than 6 ABO antigens. These data confirm and extend prior research that has suggested a role for GPIbα ABO antigen expression in hemostasis and thrombosis.