MEMBRANE PROTEIN DEFICIENCY OF PNH ERYTHROCYTES

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The decay accelerating factor (DAF) is an integral membrane protein expressed on the surface of normal human erythrocytes. DAF inhibits the classical and alternative complement pathway C3 convertases by accelerating the subunit dissociation of these bimolecular enzymes. An assay based on this activity permitted chromatographic isolation of DAF. Antibody raised against isolated DAF was used to assess the quantity of DAF antigen on the surface of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes. Type II PNH-erythrocytes are relatively deficient and type III PNH erythrocytes are totally deficient in DAF antigen. The abnormal sensitivity to complement-mediate lysis expressed by the DAF deficient PNH erythrocytes is strong evidence that the C3 convertase regulation provided by DAF has an important role in protecting normal erythrocytes and probably other cells, from damage by autologous complement. The objectives of the proposed studies are to: 1) Produce monoclonal anti-DAF antibodies to facilitate the purification and subsequent studies of DAF. 2) Characterize DAF's subunit structure, composition, active site(s), capacity to bind the relevant complement ligands, topographic position in the membrane, and relation to other membrane moieties as defined by "nearest neighbor" cross-linking studies. 3) Asses the capacity of DAF to inhibit the formation, as well as the decay of C3 convertases, and to act as a cofactor for the C4b/C3b inactivator's (I) cleavage of C4b and C3b. 4) Extend the studies of the DAF-deficiency in PNH cells by characterizing the subunits of DAF in type II PNH erythrocytes and in leukocytes and platelets from PNH donors. Abnormal subunit(s) or the absence of 1 or 2 subunits would suggest a structural gene mutation, whereas a reduced number of normal appearing subunits would suggest a mutation in regulatory gene. Cytofluorography of PNH cells reacted with anti-DAF antibody will allow for the first time the quantitation of the proportion of abnormal cells of each cell type in PNH donors' blood. The proposed studies will give (A) a more complete understanding of how DAF performs its important complement regulatory function in the membrane of normal cells, and (B) a better definition of the molecular basis of the DAF deficiency in PNH cells. This knowledge will provide the necessary background for future studies of the nature of the somatic mutation responsible for PNH. The reagents and methodologies developed for the proposed studies may provide the basis for future therapeutic intervention in PNH.