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,

We have recently shown that human monocytes and U937 cells possess two molecular classes of Fcy receptor. One, a 72,000-mol-wt sialoglycoprotein, has high affinity for certain subclasses of human and murine monomeric IgG. The other is a 40,000-mol-wt protein (p40) with low affinity for monomeric IgG but with the capacity to bind IgG aggregates or IgG-coated particles.In the present study, a 40,000-mol-wt single chain protein, apparently identical to p40 from U937 cells, was isolated from surface-radioiodinated human platelets by affinity purification using a murine IgG2b monoclonal antibody to p40. This 40,000-mol-wt protein was not seen when control IgG2b or unrelated murine monoclonal antibodies were employed in place of antip40. The same 40,000-mol-wt protein was also recovered from an IgG-Sepharose affinity adsorbent, but not from ovalbuminor myoglobin-Sepharose. The 72,000-mol-wt Fcvy receptor of monocytes was not identified on platelets.Monoclonal anti-p4O and Fab fragments derived from this antibody blocked platelet aggregation by heat-aggregated human IgG, whereas a control murine IgG2b protein had little or no inhibitory effect at 500-1,000-fold higher concentrations. A murine IgG1 monoclonal antibody, reactive with an unrelated platelet-specific membrane antigen, did not inhibit platelet responses to aggregated IgG. Anti-p4O did not affect platelet aggregation by thrombin, collagen, or fibrinogen plus ADP. Although antip40 did not directly aggregate platelets in the concentrations employed, cross-linking with F(ab'`) goat anti-murine Ig induced apyrase-sensitive aggregation of anti-p40-treated platelets. This indicates that p40 possesses transmembrane linkage for platelet activation and secretion.These observations strongly suggest that this newly recognized 40,000-mol-wt platelet membrane protein serves as an Fcy receptor.

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Immunoglobulin G (IgG) autoantibodies of 20 patients with autoimmune hemolytic anemia (AHA) were used in immunoaffinity assays with surface-radioiodinated human red was not seen in the absence of p34, and both proteins are likely to be members of the Rh family. Indeed, a 34-kD polypeptide band and 37-55-kD polydisperse "smear," isolated concurrently from the same labeled RBCs by IgG allo-anti-e, were indistinguishable from their autoantibody-isolated counterparts and may well be the same protein identified at different epitopes by the auto-and allo-antibodies. Individual AHA patients' autoantibodies isolated p34 and gp37-55, alone or in combination with band 3 (nine cases); strong band 3 alone (five cases); and combinations of band 3 with GPA (six cases). The autoantibodies of three additional patients whose AHA had been induced by a-methyldopa also isolated p34 and gp37-55. (J. Clin. Invest. 1993. 91:1672-1680

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A B S T R A C T The first recognized human kindred with hereditary deficiency of the fifth component of complement (C5) is described. The proband, a 20-year-old black female with systemic lupus erythematosus since age 11, lacked serum hemolytic complement activity, even during remission. C5 was undetectable in her serum by both immunodiffusion and hemolytic assays. Other complement components were normal during remission of lupus, but C1, C4, C2, and C3 levels fell during exacerbations.A younger half-sister, who had no underlying disease, was also found to lack immunochemically detectable C5. By hemolytic assay, she exhibited 1-2% of the normal serum C5 level and normal concentrations of other complement components. C5 levels of other family members were either normal or approximately half-normal, consistent with autosomal codominant inheritance of the gene determining C5 deficiency.Normal hemolytic titers were restored to both homozygous C5-deficient (CSD) sera by addition of highly purified human C5. In specific C5 titrations, however, it was noted that when limited amounts of C5 were assayed in the presence of low dilutions of either C5D serum, curving rather than linear dose-response plots were consistently obtained, suggesting some inhibitory effect. Further studies suggested that low dilutions of C5D serum contain a factor (or factors) interfering at some step in the hemolytic assay of C5, rather than a true C5 inhibitor or inactivator.

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A B S T R A C T The fourth component of human complement (C4) in 102 individual plasma samples has been examined by the technique of antigen-antibody crossed electrophoresis (AACE). Electrophoretic heterogeneity of C4 was manifested by the repeated occurrence of seven different precipitin patterns. These patterns were formed by varying combinations of three subtypes of C4, differing in electrophoretic mobility. The subtypes were designated C, A, and A,, in order of increasing electrophoretic mobility toward the anode. The evidence that the observed electrophoretic heterogeneity of the C4 molecule represents structural polymorphism rests on five points: the pattern obtained from the plasma of a given individual was reproducible in different runs and with different bleedings; all seven patterns could be demonstrated on the same electrophoretic run; C4 of a given subtype retained its characteristic mobility after purification, when run alone or mixed with plasma containing C4 of other subtypes; the subtypes A, and C comprising pattern 6 could be separated chromatographically as well as electrophoretically; and the characteristic relative mobilities of different C4 subtypes, in plasma or after purification, were retained even after the rather large shift in mobility associated with conversion to C4i. The ratio of C4 hemolytic activity to protein concentration varied according to the subtype composition of individual samples, with highest ratios occurring with patterns composed of subtype C alone, intermediate values with patterns consisting of A and C, and lower values occurring with patterns containing subtype A alone. Although the mechanism of inheritance of this polymorphism is not yet clear, the data suggest that subtypes A and A, are inherited as autosomal codominant characteristics, independent of the inheritance of subtype C.Dr.