Demonstration of the heterogeneity of epitopes of the platelet‐specific alloantigen, Bak^a

Abstract

It is well known that the platelet-specific alloantigen, Baka is carried on glycoprotein (GP) IIb, but little is known about the biochemical characteristics of its epitopes. To clarify the characteristics of the epitopes, we examined the interaction of four anti-Baka sera (Yam, Lin, Kl and MO) with their epitopes, either with or without modifications by sodium dodecyl sulphate (SDS) and/or neuraminidase. By immunoprecipitation, all four antisera bound to the intact GP IIb/IIIa complex from a Baka-positive subject. In contrast, immunoblotting demonstrated that Yam, Lin and Kl bound to SDS-denatured GP IIb, while MO did not. When blotted GP IIb was treated with neuraminidase, Yam and Lin did not bind to desialylated GP IIb, while Kl still did. When the purified GP IIb/IIIa complex or washed platelets were treated first with neuraminidase followed by immunoblotting, the molecular weight of GP IIb decreased from 145 kD to 138 kD; Yam did not bind to desialylated GP IIb, but Kl did. Furthermore, to eliminate the effect of SDS, we examined the interaction of Yam and Lin with neuraminidase-treated platelets using flow cytometry. The results were the same as those obtained using immunoblotting. Our results thus demonstrate that the expression of the Baka epitopes is not uniform and that sialic acid contributes to the expression of some actual allogenic epitopes.

ABO Blood Group as a Model for Platelet Glycan Modification in Arterial Thrombosis

“…Take et al tested the hypothesis that post-translational sialylation of GP IIb affected the expression of Bak epitopes. The authors demonstrated that the binding of four anti-Bak sera to Bak changed depending on whether neuraminidase was administered to desiaylate GP IIb (64). A subsequent study by Goldberger et al, demonstrated changes in electrophoretic mobility and reactivity to antisera when Bak a and Bak b platelets were exposed to neuraminidase, thereby desiaylating GP IIb.…”

Neonatal Alloimmune Thrombocytopenia in Taiwan Due to an Antibody against a Labile Component of HPA-3a (Bak^a)

“…Although HPA-3a is found on GPIIb, it ap pears likely that GPIIb (or Ilb/lIIa) has a labile component (s) which is denatured after fixation, strong detergent treat ment, or prolonged storage and even reduced after extensive washing in saline (under the latter two conditions, dissocia tion of the labile component is possible). The antigenicity of HPA-3a has been found to be strongly influenced by glyco sylation, and HPA-3a antibodies from different individuals have been found to vary in their reactions with GPIIb [8,15]. However, we found that there was no obvious reduction in the reactivity after neuraminidase treatment in our case; however, we found that the reactivity was weakened after extensive washing in saline (after 6 washes), suggesting that the labile component(s) of HPA-3a may be located in the carbohydrate-rich 'fuzzy' coat (glycocalyx) which extends 15-20 nm beyond the outer surface of the lipid bilayer as observed by electron microscopic studies on the platelet membrane [16].…”

“…The frequency of HPA-3a in this report of 84% using SPRCA is close to, but slightly higher than, our previous report of 77% using MPHA [1], The difference may be due to the fact that the HPA-3a anti gen of some HPA-3a-positive platelets may only be detected when the platelets are fresh. Alternatively, population bias, heterogeneity of HPA-3a epitopes or differences in the spec ificity of different anti-HPA-3a antisera [8] could also be factors involved. Further study is needed in this respect.…”

“…The wells were then fixed with 50 pi 7% formaldehyde (pH 6.0) for 20 min and again washed 5 times with saline. [8] Washed platelets (2.5x107ml) were suspended in 0.05 M PBS (pH 6.2) and treated with neuraminidase from Vibrio cholerae (Hoechst-Behring. Germany) at a concentration of 0.5 p/ml for 3 h at 37°C.…”

Heterogeneity of Platelet Alloantigens and Alloantibodies: New Insights into Structure and Function

“…This difference in reactivity may be related to differences in the nature of these diseases. In contrast to HPA-1 epitopes [40], several studies have shown that carbohydrate residues contributed critically to the integrity of HPA-3a epitopes [49][50][51]. Djaffar et al [52] demonstrated that presence of O-linked rather than N-linked carbohydrate moieties preserves HPA-3a alloantigenic determinants.…”