Purification and characterization of papain-solubilized HLA antigens from human platelets.

Human platelet membranes were isolated by the hypotonic glycerol lysis technique from donors who had their HLA antigens identified on lymphocytes by lymphocytotoxicity. The HLA antigens of the platelets were solubilized by papain treatment of the isolated membranes. The solubilized material retained antigenic activity and specificity. This crude antigenic material was further purified by column chromatographic techniques. On Sephadex chromatography the antigenic material had an estimated molecular weight of 40.000 ± 3500 d (1 SD). It contained material that reacted immunologically with an antibody specific for 82-microglobulin. Further purification of this material by DEAE-cellulose chromatography followed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) showed it to be composed of two pieces. One piece had a molecular weight of 12.000 d. and the larger piece had a molecular weight of 26,000 d. The purification procedure resulted in a 540-fold increase in HLA-A2 specific activity, with a yield of 0.26 mg per 6 X 1011 platelets. $2-microglobulin cochromatographed with the HIA antigen activity on exclusion and ion-exchange chromatography and migrated with HLA antigen activity on non-SDS-PAGE gels. The 1 2.000-d band on SDS-PAGE gels was believed to represent /92-microglobulin. These results suggest that HLA antigens of platelets and lymphocytes are similar in structure when solubilized by papain and that platelets can be used as a source of HIA antigens for further structural analysis.