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The blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormal interactions with complement. The activity of the alternative pathway C3 convertase on the platelets of 9 out of 19 patients with PNH was elevated. 10 patients had C3 convertase activity within the normal range even though 80-95% of their platelets lacked the complement regulatory protein decay accelerating factor (DAF) that is absent from the affected blood cells in PNH. PNH and normal platelets released factor H when C3 was bound to their surfaces. This may account for the apparent regulation of C3 convertase activity on platelets that lack DAF. The abnormal uptake of the membrane attack complex of complement by PNH III erythrocytes was not seen in PNH platelets. 111Indium-labeled platelet survival times were normal in five of eight patients, which suggests that the lack of the membrane attack complex defect results in normal platelet survival in PNH.

Monocyte-mediated lysis in vitro of human red cells coated with measured amounts of immunoglobulin G (IgG) or complement were studied. 1,000-1,500 molecules of IgG anti-D are necessary to effect measurable lysis, and lysis increases linearly with increasing levels of antibody sensitization. 100 microgram/ml of IgG1 abolished lysis even at maximal levels of anti-D sensitization (15,000 molecules/cell). Two isoimmune IgG anti-A or anti-B antisera were 5 to 10-fold less efficient in promoting phagocytosis or lysis per molecule of IgG bound; however, because of the greater antigen density of A or B, more than 100,000 molecules IgG/cell could be bound, producing equivalent lysis to anti-D-coated cells. Although inhibition by IgG1 was similar at equivalent levels of sensitization with anti-A, anti-B, or anti-D at high levels of coating with anti-A or anti-B (not attainable with anti-D), lysis was not inhibited by IgG1. Cells coated with human complement components alone were not lysed by monocytes; however, complement coating augmented IgG-mediated lysis and reduced the quantity of anti-D necessary to produce lysis to less than 1,000 molecules/cell. After thorough degradation of C3b by serum to C3d, complement augmentation persisted.

To investigate the greater fixation of C3 to the erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) upon activation of complement, we have examined the formation and the reaction of the C3 nephritic factor-stabilized alternative pathway convertase made with purified components on normal and PNH erythrocytes. Each convertase complex converts four to five times more fluid-phase C3 to C3b when affixed to a PNH cell than when affixed to a normal cell. The greater activity of the convertase on PNH cells is not due to differences in the intrinsic or extrinsic stability of the convertase complex. The excessive binding of C3 to PNH cell si due to this increased conversion of fluid-phase C3, because the efficiency of binding of nascent C3b was identical for the two cell types. This is the first instance in which the enzyme activity of a complement complex has been shown to be increased by being affixed to an abnormal surface.

The affected erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH II and PNH III cells) are abnormally sensitive to complement-mediated lysis. Normal human erythrocytes chemically modified by treatment with 2-amino-ethylisothiouronium bromide (AET) have been used as models for PNH cells inasmuch as they also exhibit an enhanced susceptibility to complement. To investigate the bases for the greater sensitivity of these abnormal cells to complement-mediated lysis, we compared binding of C3 and constituents of the membrane attack complex to normal, PNH II, PNH III, and AET-treated cells after classical pathway activation by antibody and fluid-phase activation by cobra venom factor complexes. When whole serum complement was activated by antibody, there was increased binding of C3 and C9 to PNH II, PNH III, and AET-treated cells, although the binding of these complement components to PNH II and PNH III cells was considerably greater than their binding to the AET-treated cells. In addition, all of the abnormal cell types showed a greater degree of lysis per C9 bound than did the normal erythrocytes. PNH III and AET-treated cells were readily lysed by fluid-phase activation of complement, whereas normal and PNH II erythrocytes were not susceptible to bystander lysis. The greater hemolysis of PNH III and AET-treated cells in this reactive lysis system was due to a quantitative increase in binding of constituents of the membrane attack complex. This more efficient binding of the terminal components after fluid-phase activation of whole serum complement was not mediated by cell-bound C3 fragments. These investigations demonstrate that the molecular events that characterize the enhanced susceptibility of PNH II, PNH III, and AET-treated erythrocytes to complement-mediated lysis are heterogeneous.

A crude, urine-derived, erythropoietin-containing preparation was subjected to inactivation by high-energy electrons and low-energy x rays. The molecular weight was calculated to be about 27,000 by use of target theory computations. Inactivation characteristics to low-energy x rays suggest that the molecule is asymmetric and is about 10 times as long as it is wide. The result of irradiation of erythropoiesis-stimulating material from renal and cerebellar hemangioblastoma cysts from patients with polycythemia suggests that the biologically active molecule from these sources has the same or nearly the same size as that derived from urine of an anemic patient.

A B S T R A C T The intracellular content of Ki in thalassemia minor red blood cells is markedly reduced after incubation in autologous serum for 24 hr at 370C. There is no compensatory increase in intracellular Na' concentration of the cell thus reduced. This change is due to an acquired increase in selective permeability of the membrane to K+. This phenomenon follows the depletion of energy sources in the thalassemia minor cells but does not follow comparable depletion in normal cells. The loss of osmotically active intracellular contents probably accounts for the increased resistance of incubated thalassemia minor red blood cells to osmotic lysis.

The concentration of cell-bound and serum antibody was determined in a series of patients with warm antibody immune hemolytic anemia by determining the amount of C[unk]1 fixed to the cells by anti-IgG. This was compared to the rate of hemolysis as determined by hemoglobin concentration and reticulocyte count, or the endogenous production of carbon monoxide. The rate of hemolysis was, in general, proportional to the concentration of cell-bound antibody. In splenectomized patients, the rate of hemolysis was very much less than in unsplenectomized patients for a given concentration of cell-bound antibody. When prednisone was given, three effects were noted: (a) at high doses of drug, the concentration of cell-bound antibody decreased rapidly and the concentration of serum antibody increased, suggesting that the affinity of antibody for antigen had been altered; (b) in patients achieving remission, the concentration of serum antibody fell to low levels but rose again if the dose of prednisone was insufficient; (c) in one patient, prednisone appeared to inhibit sequestration of highly sensitized cells.

The fixation of the first component of complement (C′1a) by human antibodies and human cells has been studied by the use of the C′1a fixation and transfer test (C′1a FT test) of Borsos and Rapp. Cold agglutinin antibodies appear to require no more than one antibody molecule to fix one molecule of C′1a. Most warm agglutinin antibodies are IgG in immunoglobulin type and require at least two molecules of antibody to fix a molecule of C′1a. Donath-Landsteiner antibody has the same requirements for C′1a fixation. A single example of a warm agglutinin antibody which appears to require one molecule of antibody for the fixation of C′1a was found. Antibodies of the Rh system do not fix significant amounts of C′1a in the absence of anti-antibody when antiserum of a single Rh specificity was used. However, when three antisera at different specificity are present, C′1a may be fixed. Under these conditions cells from a patient with paroxysmal nocturnal hemoglobinuria may be lysed when fresh serum is added to provide the other components of complement. The presence of IgG antibodies could be detected by the use of anti-IgGHu antiserum and a one-to-one relationship between the concentration of antiserum in the reaction and the amount of C′1a fixed could be established. The effect of temperature, ionic strength, papainization of the red cells, and repeated washing of the red cell-antibody aggregates on the amount of C′1a fixed was investigated. Conditions of maximal C′1a fixation were established for each class of antibodies. Globulins present in normal isologous or autologous serum are absorbed in small amounts to normal red cells in a manner analogous to warm agglutinin antibody. Their presence is detectable by the C′1a fixation and transfer test only with antiglobulin antiserum. Within certain limits, the C′1a fixation and transfer test provides a quantitative measure of the reaction of human red cells and antibodies to antigens on their surface.

The effect of five different reactions which activate complement (antibody activation, reduction in ionic strength, acidification, cobra venom factor (CoF) activation, and inulin activation) upon normal and PNH cells was investigated, using normal serum and serum devoid of the fourth component of complement (C4) activity from patients with hereditary angioneurotic edema (HANE) as a source of complement. Both normal and HANE serum lysed paroxysmal nocturnal hemoglobinuria (PNH) cells when complement was activated by acidification, CoF and inulin, indicating that the terminal steps of complement were activated in the absence of C4, presumably by the alternate or properdin pathway. Normal but not HANE serum lysed cells coated with anti-I, indicating that complement was activated by the C1-dependent classic pathway. HANE serum partially supported lysis by serum at reduced ionic strength, indicating that the activation of terminal complement components had occurred through both of the pathways of activation. The amount of the third component of human complement (C3) which was bound to the membrane of lysed and unlysed cells by these procedures was determined by anti-C3 absorption and was found to differ for each method of complement activation. In general, more C3 was bound to lysed cells than to unlysed cells. For given conditions, more was bound to PNH cells than to normal cells. However, very much less bound C3 was required for lysis of the PNH cells than for lysis of normal cells. These two phenomena, especially the latter, account for the marked lysis of PNH cells when complement is activated. Normal cells treated with AET (aminoethylisothiouronium bromide) did not bind more C3 than untreated cells and the lysed cells had less bound C3 than lysed PNH cells.