CHARACTERIZATION OF PORCINE PLATELET GLYCOPROTEINS RECOGNIZED BY HUMAN NATURAL ANTI-GAL ANTIBODIES

Human natural ''anti-Gal'' antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on nonprimate mammal a nd new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA ). IgG and IgM were isolated from human plasma, and anti-Gal antibodie s were purified by affinity chromatography on a Synsorb-14 column (Che mbiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG clas ses represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal huma n sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kD pig platelet proteins, wherea s purified anti-Gal antibodies of both IgM and IgG classes mainly boun d to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xeno antibodies are not solely directed to galactosyl epitopes. These antib odies revealed bands of 115, 125, and 240 kD. alpha-Galactosidase trea tment of porcine platelet glycoproteins (gps) enriched by affinity chr omatography abrogated the reactivity of 135- and 210-kD proteins. N- a nd O-glycosidase treatments demonstrated that Lu-galactosyl residues a re located on the O-glycans of the 135-kD component. Finally, glycopro teins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and Ilb, respectively, wh ereas the 240-kD component was identified as the porcine fibrinogen, u sing a new murine monoclonal antibody (NaM147-7B6; IgG1) specific for its beta-chain. (C) 1996 by The American Society of Hematology.