Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system

Abstract

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.

Evidence for the involvement of complement proteins in platelet aggregation by Streptococcus sanguis NCTC 7863

“…Just how complement-coated particles, such as zymosan or S. sanguis, induce platelet aggregation is not yet known. Human platelets do not possess the C3 receptor, CR1, but a number of other C3 binding sites have been identified on platelets (Yu et al, 1986;Nicholson-Weller et al, 1985;Vik & Fearon, 1987;Seya et al, 1988;Chen et al, 1994;Shenoy-scaria et al, 1992), whose roles, if any, in platelet activation remain undefined. There is evidence, however, for stimulation of platelets or modulation of platelet activation by the terminal complement complex, C5b-9.…”

“…Oral streptococci, like many other bacteria, would be expected to activate complement in plasma, and there is some evidence that platelets can be activated and secretion induced by elements of the complement activation pathways (Breckenridge et al, 1977;Sims & Wiedmer, 1991). Various complement regulatory molecules and some complement receptors have been found on the platelet surface, although their roles have not been clearly defined (Yu et al, 1986;Nicholson-Weller et al, 1985;Vik & Fearon, 1987;Seya et al, 1988;Chen et al, 1994). In this paper we report a requirement for complement activation in the aggregation of platelets by S. sanguis NCTC 7863 and show that the major route of complement activation by these micro-organisms is via the alternative pathway.…”

Blockage of complement regulators in the conjunctiva and within the eye leads to massive inflammation and iritis

“…3±8,24,25 Important among this evidence are studies of acquired haemolytic anaemia and paroxysmal nocturnal hemoglobinuria (PNH) (reviewed in ref. 1), in which de®ciency of two of the regulators, DAF 4,13,26,27 and CD59, 6,28 leads to increased uptake of autologous C3b 13,29 and C9 30 on affected erythrocytes, eventuating in their lysis in vivo. Surprisingly, it has been found that expression levels of DAF are many-fold 9,10 and of MCP 12 and CD59 11,12 more than twofold higher on corneal, conjunctival and uveal cells than on most blood cells.…”

“…Intrinsic membrane regulators of complement are species-speci®c proteins that protect self-cells from activation of autologous complement on their surfaces (reviewed in refs 1 and 2). In humans, these proteins consist of the decay-accelerating factor (DAF or CD55), 3,4 the membrane cofactor protein (MCP or CD46) 4 and CD59 (homologous restriction factor 20 [HRF20] or the membrane inhibitor of reactive lysis [MIRL]). 5,6 DAF and MCP act early in the activation sequence to disable the classical and alternative pathway C3 convertases, 3,4,7 the central ampli®cation enzymes of the cascade.…”

“…In humans, these proteins consist of the decay-accelerating factor (DAF or CD55), 3,4 the membrane cofactor protein (MCP or CD46) 4 and CD59 (homologous restriction factor 20 [HRF20] or the membrane inhibitor of reactive lysis [MIRL]). 5,6 DAF and MCP act early in the activation sequence to disable the classical and alternative pathway C3 convertases, 3,4,7 the central ampli®cation enzymes of the cascade. CD59 functions later in the cascade to prevent binding of C9 to C5b-8 5,6 and consequent formation of membranolytic poly C9 channels that bring about cell lysis.…”