Platelet A and H antigen expression

Platelet–rich plasma was prepared by centrifugation of whole blood. Platelet count was then measured using a Cell-Dyn 3200 analyser (Abbott laboratories, IS). To assess A antigen expression levels, 10⁶ platelets were incubated with Phycoerythrin (PE)-labeled anti-human CD41a (BD Biosciences Pharmingen; San Diego, CA) and Fluorescein (FITC)-labelled Helix pomatia (HPA, Anti-A) (Sigma-Aldrich; St. Louis, MO) for 30 min as previously described²⁸. Samples were then analyzed using a BD FacsCanto II (BD Biosciences, San Jose, CA). Platelets staining dual-positive for both CD41a and anti-A were quantified, together with mean fluorescence intensity for CD41a and HPA expression. Data were quantified using FACSDiva software (BD Biosciences, San Jose, CA). In keeping with previous studies, HXP was defined as greater than 75% platelet A antigen positivity, and LXP defined as less than 15% platelet positivity^23,24. Similarly, FITC-labeled Ulex europaeus (UEA, anti-H) (Sigma-Aldrich; St. Louis, MO) was used to investigate H antigen expression on group O platelets. Finally, platelet α2-6 linked sialic acid expression was analyzed using FITC-labeled Sambucus nigra agglutinin (SNA) (Vector Laboratories Inc: Burlingame, CA). For each glycan analysis, a parallel sample was analyzed using isotype control (FITC-labelled mouse IgG1κ isotype; BD Biosciences Pharmingen, San Jose, CA).