The reaction mechanisms of C3 are central to both the classical and alternative pathways of complement activation and have in consequence been extensively studied (see 1 and 2 for reviews). The activation of C3 by cleavage to C3b is brought about by the C3-converting enzymes of the classical and alternative pathways, as well as by a number ofnoncomplement enzymes. C3b is subsequently inactivated and prevented from further participation in the C3b feedback cycle and in the hemolytic reaction by cleavage to C3bi by factor I, which splits C3b when the latter is combined with factor H. C3bi is the C3 conversion product that appears in serum in vitro after complement activation by either pathway, and it is fairly clear that the C3 product, which was known as "beta 1A" in much of the earlier complement literature, is C3bi and not C3c, the fragment with which beta 1A has usually been identified (3). C3bi does, however, undergo further breakdown both in serum and when cell bound. It was originally shown by Lachmann and Mi.iller-Eberhard (4) that the conglutinable form of C3 bound on cells (cell-bound C3bi) could be cleaved by 1/~g/ml of trypsin to lose its conglutinability. This was accompanied by the elution of C3c from the cell, C3d remaining bound. In serum, too, C3 gave rise, on aging, to two fragments, one of which was described as beta 1A and the other, a fast-migrating fragment, as alpha 2D (5). Alpha 2D was generally equated with C3d (3). As will be pointed out in this paper, this identification is not wholly correct.Antigenic analysis with polyclonal antisera to C3 had shown the existence of distinct antigenic specificities in C3a, C3c, and C3d, as well as antigenic determinants found only in native C3 (5, 6). Studies with monoclonal anti-C3 antibodies (7) showed one that reacted with C3c, one with C3d, and one that showed an unusual pattern of reactivity that has now been found to react with the fragment here called C3g. By the use of the monoclonal antibodies, the breakdown of C3bi both on cells and in plasma has been reinvestigated, and the results of this investigation are presented herein. Materials and MethodsMonoclonal Anti-C3 Antibodies. The three monoclonal antibodies are described as clone 3, clone 4, and clone 9 and have been fully described (7). Unless stated to the contrary, ascites from tumor-bearing rats was used as the source of monoclonal antibodies without further purification.

Increasing evidence suggests that complement activation might represent an important mechanism in early atherogenesis. Thus, complement components, in particular the membrane attack complex (MAC) C5b-9(m), have been isolated from human atherosclerotic lesions. Furthermore, complement activation is known to occur in atherosclerotic lesions induced in experimental animals, and the severity of cholesterol-induced plaques is markedly reduced in complement-deficient animals. During atherogenesis monocytes are recruited into the arterial wall, and a potent chemoattractant for monocytes, monocyte chemotactic protein-1 (MCP-1), is expressed by vascular smooth muscle cells (SMCs). We hypothesized that generation of MACs on SMCs during the activation of complement might lead to the release of MCP-1 and hence to monocyte recruitment. In this study, MACs were generated on human SMCs in vitro by sequential addition of the purified complement components C5b6, C7, C8, and C9. This supernatant of the culture was chemotactic for freshly isolated peripheral blood monocytes in a modified Boyden chamber. The chemotactic activity of the supernatant was abolished by anti-MCP-1 blocking antibodies but not by an isotype-matched antibody against an irrelevant antigen. The release of chemotactic activity was dependent on the dose of MAC formed on SMCs and was demonstrated within 10 minutes of exposure of the cells. The data support the hypothesis that complement-mediated release of MCP-1 from SMCs might be important in the recruitment of monocytes into the developing atherosclerotic lesion and could be an important initiating event in atherogenesis.

Paroxysmal nocturnal hemoglobinuria (PNH) results from the expansion of a hematopoietic clone that is deficient in glycosylphosphatidylinositol-anchored molecules. PNH is characterized by chronic hemolysis with acute exacerbations due to the uncontrolled activity of complement on PNH cells, which lack the inhibitor of homologous complement, CD59. Symptoms include severe fatigue, hemoglobinuria, esophageal spasm, erectile dysfunction, and thrombosis. We report the use of a novel synthetically modified recombinant human CD59, rhCD59-P, a soluble protein that attaches to cell membranes. In vitro treatment of PNH erythrocytes with rhCD59-P resulted in levels of CD59 equivalent to normal erythrocytes and effectively protected erythrocytes from complement-mediated hemolysis. The administration of rhCD59-P to CD1 mice resulted in levels of CD59 on erythrocytes, which protected them from complement-mediated lysis. Thus, rhCD59-P corrects the CD59 deficiency in vitro and can bind to erythrocytes in an in vivo murine model, protecting the cells from the activity of human complement, and represents a potential therapeutic strategy in PNH. IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, venous thrombosis, and an association with aplastic anemia. 1 PNH arises through a somatic mutation of the phosphatidylinositol glycan complementation class A (PIGA) gene in a hematopoietic stem cell and the expansion of the abnormal hematopoietic clone. PIGA encodes a protein involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. 2 GPI structures anchor a wide variety of proteins to the cell surface via their lipid moiety. Deficiency of GPI-linked proteins from PNH erythrocytes can be complete (PNH type III cells) or partial (PNH type II cells). Type I cells express normal levels of GPI-linked proteins. The terminology derives from different complement lysis sensitivities of the 3 types of PNH erythrocytes, type III being 15 to 25 times more sensitive to complement than normal cells. 3 This variability in the severity of the deficiency, as well as in the proportion of the affected cell population, defines the clinical manifestations of the disease. 4,5 The increased sensitivity to complement results in chronic hemolysis with acute intravascular hemolytic exacerbations causing symptoms of severe fatigue, dark urine, esophageal spasm, and erectile dysfunction in men. Thrombosis is a major cause of morbidity, and even mortality, in this disease.Among the many proteins missing from the surface of PNH cells are the complement regulatory proteins CD55 (decay accelerating factor) and CD59 (also known as protectin and membrane inhibitor of reactive lysis). [6][7][8] The increased complement sensitivity of PNH cells leads to hemolysis and thrombosis. 9 CD55 inhibits C3 and C5 convertases, whereas CD59 is the sole membrane regulator of membrane attack complex (MAC) assembly. As there are additional inhibitors of C3 convertases in human plasma, bystander activation of compleme...