Evaluation of platelet surface antigens: localization of the Pl A1 alloantigen

S ummary . An approach is described for localizing antigenic determinants to specific platelet proteins. Platelets are solubilized, electrophoresed and transferred to nitrocellulose paper where they are immobilized. After incubation with radiolabelled specific antibody, the antigenic determinants are localized by radioautography. Purified proteins ranging in size from 50 000 to 540 000 could be studied in this manner and still retain their antigenicity. Solubilized immobilized platelet proteins were reacted with serum IgG from two patients with post‐transfusion purpura known to have anti‐Pl A1 antibodies. The radioactivity was associated with a major protein band with an apparent molecular weight of 100 000 daltons. However, when purified anti‐Pl A1 antibody eluted from Pl A1 (+) platelets was used, at least six additional minor bands were noted with molecular weights ranging from about 86 000 to 175 000. No radioactive bands were noted using Pl A1 negative platelets or reduced Pl A1 (+) platelet proteins. Reaction of the antibody from one patient with proteins from thrombasthenic platelets resulted in a marked reduction in the intensity of all radioactive bands. In addition, a new high molecular weight band was noted which was not present in reactions containing Pl A1 (+) platelets. These data suggest that the Pl A1 antigen is primarily but not entirely localized to glycoprotein IIIa. This approach is relatively simple, requires only minimal amounts of antibody and should be generally useful in the study of cellular antigens and other complex mixtures of proteins.