Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system. 1985
- 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.
Deficiency of the complement regulatory protein, decay-accelerating factor, on membranes of granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria. 1985
Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria are deficient in decay-accelerating factor, a membrane protein that inhibits the complement C3 convertases. We studied the expression of this protein on leukocytes and platelets from four patients with paroxysmal nocturnal hemoglobinuria, using cytofluorographic analysis and antibody to decay-accelerating factor. The granulocytes and monocytes had a bimodal distribution of fluorescence, indicating antigen-deficient and antigen-positive subpopulations of cells. In contrast, granulocytes and monocytes from normal donors and patients with other diseases had no antigen-deficient cells. Platelets from the four patients with paroxysmal nocturnal hemoglobinuria had less fluorescence than normal platelets. Furthermore, surface-radiolabeled granulocytes and platelets from one of the four patients, which were maximally deficient in decay-accelerating factor, also lacked antigen that was immunoprecipitable by specific antibody to this protein. Thus, paroxysmal nocturnal hemoglobinuria is a clonal disorder characterized by deficient membrane expression of decay-accelerating factor on granulocytes, monocytes, and platelets, as well as on erythrocytes.