Defining the Molecular Mechanisms Responsible for the ABO High Expresser Phenotype.

Abstract 2119

Poster Board II-96

ABO(H) blood group antigen expression on platelets varies widely among normal blood donors. An ABO ‚High Expresser' phenotype (HXP) that exhibits significantly increased A and/or B antigen expression on platelets has been identified in ∼7% of normal donors. HXP has been implicated in both platelet-refractoriness and neonatal alloimmune thrombocytopenic purpura, however, the underlying molecular and genetic elements that mediate this phenomenon are not well-defined.

To investigate the mechanisms underlying HXP, blood samples were collected from 231 group A (180 A₁and 51 A₂) and 310 group O individual apheresis platelet donors. Quantitative expression of platelet A and H antigen were then assessed by flow cytometry of platelet-rich plasma. In total, 10 A₁ donors (5.6%) exhibited HXP. Analysis of the platelet A antigen expression in these individuals identified 8 HXP donors who exhibited ‚type I' HXP (normal bimodal population of platelets, but with predominant A antigen expression) whereas 2 individuals exhibited ‚type II' HXP (a single uniform population of platelets, strongly positive for blood group A expression). Both types of HXP were found to be a stable donor characteristic.

ABO(H) determinants have also been identified on the N-linked glycans of the plasma von Willebrand factor (VWF), and influence plasma VWF levels and susceptibility to proteolysis by ADAMTS13. To determine whether HXP was platelet-specific, blood group A antigen expression on plasma VWF from group A donors was determined. Interestingly, blood group A antigen expression on plasma VWF was concordantly increased in donors with type I and type II HXP, indicative of increased glycosyltransferase expression in HXP individuals.

To ascertain whether increased glycosyltransferase expression contributes to HXP, ABO genotype was determined for all 231 group A donors by PCR-RFLP analysis. Genotype at the ABO locus on 9q34 exerts a dosage effect on glycosyltransferase expression. 80% HXP (all type I) donors were genotyped A₁A_1. suggesting increased A transferase activity contributes to type I HXP. Despite this, the majority of A₁A₁individuals (67%) did not exhibit HXP, and 2 HXP donors were found to possess the A₂ allele, which expresses limited A transferase enzymatic activity. Collectively, this data clearly demonstrates the contribution of additional factors to ABO genotype that contribute to HXP.

To identify additional HXP modifiers, potential enhancer repeat elements upstream of the ABO gene were examined in group A donors, including those with HXP. Typically, A₁alleles contain a single 43-base pair repeat within a minisatellite positive regulatory region upstream of the ABO gene. In contrast, A₂and O₁alleles contain four 43bp repeats, which are associated with a 100-fold enhancement of transcriptional activity. Analysis of this enhancer region demonstrated two HXP donors with A₁alleles containing four copies of the 43-base pair repeat. Consequently, this allele would be predicted to modulate A transferase expression via enhanced ABO gene transcription.

In conclusion, we have demonstrated the multi-factorial nature of the regulatory elements mediating platelet type I and type II HXP. A₁alleles containing novel upstream enhancer repeats identified in donor individuals may represent a novel genetic mediator of HXP, and contribute to the pathophysiology associated with this phenomenon independently of ABO genotype.