Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired-defect of bone marrow stem cells in which the affected clones produce erythrocytes (also granulocytes and platelets) with membranes that are abnormally sensitive to complement-mediated lysis. Abnormal erythrocytes (E) from patients with PNH (PNH-E) are 3-5 times more sensitive (type II PNH-E) or 15-25 times more sensitive (type Im PNH-E) to lysis in vitro by human complement than normal -E from unaffected individuals and the functionally normal E that arise from unaffected clones in PNH patients (type I PNH-E). After complement activation by either the classical or alternative pathway, abnormal amounts of C3b are deposited on the membranes of PNH-E compared with normal E, suggesting that the PNH-E membrane cannot regulate the events responsible for C3b deposition. Two proteins that decrease the stability of the classical and alternative pathway C3 convertases on target cells have been isolated from normal human E stroma: the 70,000 Mr decay accelerating factor of stroma (DAF) and the 250,000 Mr C3b receptor (C3bR pathway (8, 9), by the alternative pathway (10, 11), or by acid treatment of C5 that results in subsequent formation of the lytic complex of complement proteins, C5-9 (12, 13). The relative contribution of each of these pathways to the lysis of PNH-E in vivo is not known.With equivalent amounts of isohemagglutinin sensitization, normal E, type II PNH-E, and type III PNH-E activate the same amount of C1 and bind equivalent amounts of C4b, but 6 times more membrane-bound C3b is deposited on PNH-E than on normal E (14). The classical C3 convertase, C4b,2a, loses its ability to cleave C3 enzymatically and deposit C3b when the C2a subunit "spontaneously" dissociates from the membrane-bound C4b subunit (15, 16). The increased ratio of C3b/ C4b bound to PNH-E suggests that these cells may lack a regulatory mechanism for C3 convertase activity.Two proteins that regulate complement C3 convertases on target cells have been isolated from the membranes of normal E: the 70,000 M, decay accelerating factor of stroma (DAF) (17, 18) and the 250,000 Mr C3b receptor (C3bR) (19,20). When DAF and the C3bR are solubilized and isolated, each can cause the accelerated decay of both membrane-bound classical (C4b,2a) and properdin-stabilized alternative complement (C3b, Bb) C3 convertases; DAF has preferential activity for the C4b,2a convertase and C3bR for the C3b,Bb convertase (18). The availability of an antiserum to each of these moieties allowed the direct determination of their presence or absence on PNH-E. MATERIALS AND METHODSThe following reagents were-obtained as noted: acrylamide, bisacrylamide, temed, 2-mercaptoethanol, NaDodSO4, hydroxyapatite, and nitrocellulose paper from Iodogen (1,3,4,

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol- reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol- reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.