C3 PROACTIVATOR CONVERTASE AND ITS MODE OF ACTION

The activity in human serum which is responsible for conversion of C3 proactivator (C3PA) to C3 activator was shown to reside in a 3S α-globulin. The factor, called C3PA convertase (C3PAse), was obtained in partially purified form. For conversion of C3PA, C3PAse required participation of metal ions and of a C3 fragment, which in physicochemical and antigenic properties resembled C3b. Isolated, native C3 failed to substitute for the fragment, but did restore the impaired C3 activator system in hydrazine-treated serum. Unlike native C3, the C3 fragment initiated conversion of C3PA in whole serum. A hypothetical concept which envisions the C3 fragment as effector of C3PAse has been proposed

LYSIS OF ERYTHROCYTES BY COMPLEMENT IN THE ABSENCE OF ANTIBODY

A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement

ENHANCEMENT OF THE HEMOLYTIC ACTIVITY OF THE SECOND COMPONENT OF HUMAN COMPLEMENT BY OXIDATION

A method has been described for the chemical modification of human C'2 which results in a pronounced enhancement of its hemolytic activity and a marked increase in the stability of the intermediate complex EAC'1a,4,2a prepared with the modified C'2. Both effects are fully explained by the observed increase in activity and stability of the C'3 converting enzyme, C'4,2a, following its generation with modified C'2. Evidence has been presented in support of the hypothesis that the modification resulting from treatment of C'2 with a critical concentration of iodine consists of oxidation of one or more sulfhydryl group within the molecule

THE SECOND COMPONENT OF HUMAN COMPLEMENT: ITS ISOLATION, FRAGMENTATION BY C'1 ESTERASE, AND INCORPORATION INTO C'3 CONVERTASE

A method has been described for the purification and isolation of the second component of complement (C'2) from human serum. The protein is a β1-globulin with an approximate molecular weight of 117,000. Immunochemical analysis using a variety of specific antisera, including a monospecific antiserum to the isolated protein, indicate that the C'2 protein represents a heretofore unrecognized human serum constituent. Isolated C'2 contained 2 x 109 "effective molecules" per microgram and 1000 hemolytically active C'2 molecules were required to produce a single hemolytically effective C'2 site on erythrocytes undergoing immune cytolysis. C'1 esterase treatment of C'2 resulted in reduction of both its electrophoretic mobility and its molecular size, the latter observation indicating fragmentation of the molecule. Direct evidence was presented for the physical presence of C'2 as an integral part of the enzyme C'3 convertase

THE MEMBRANE ATTACK MECHANISM OF COMPLEMENT : VERIFICATION OF A STABLE C5-9 COMPLEX IN FREE SOLUTION

The membrane attack mechanism of complement, C5 to C9, has previously been postulated to associate on the target cell surface to a stable decamolecular complex with a calculated mol wt of 995,000. A soluble and stable complex consisting of C5, C6, C7, C8, and C9 has now been demonstrated to arise as a consequence of complement activation by the classical or alternate pathway. It has a sedimentation coefficient of 22.5S and a mol wt of 1 million daltons, and it migrates on electrophoresis at pH 8.6 as an α-globulin. The stable and soluble C5b-9 complex cannot bind to erythrocytes and has no demonstrable cytolytic activity. However, due to partially unsaturated binding sites for C9, it can bind additional C9 and thus function as an inhibitor of lysis of EAC1-8 by C9. These results support the concept according to which the membrane-bound attack system of complement represents a stable, decamolecular assembly of C5b-9. Unlike its analogue in free solution, the membrane-bound complex is cytolytically active

ISOLATION OF β1F-GLOBULIN FROM HUMAN SERUM AND ITS CHARACTERIZATION AS THE FIFTH COMPONENT OF COMPLEMENT

At least 3 complement factors were found necessary for the conversion of the thermolabile intermediate complex EAC'1a,4,2a to a thermostable state. One of these factors is the earlier described β1C-globulin. The second, a heretofore unrecorded serum protein, β1F-globulin. The third factor has not yet been defined as a discrete serum protein entity. Kinetic experiments indicated that β1C reacted prior to β1F, which in turn seemed to precede the third factor in the reaction sequence. Therefore, the 3 components were tentatively designated the third (C'3), the fifth (C'5), and the sixth (C'6) components of complement, respectively. A procedure was developed allowing the isolation of highly purified β1C-(C'3) and β1F-globulin (C'5) and of partially purified C'6. With respect to its function in immune hemolysis, β1F-globulin or C'5 was found to be closely dependent on the simultaneous presence of C'6. The hypothesis that C'5 and C'6 form a functional unit was supported by the finding that both components interact with each other in solution resulting in the formation of a complex. A similar complex was also found in fresh human serum

BLOOD COAGULATION INITIATION BY A COMPLEMENT-MEDIATED PATHWAY

A variety of complement-activating substances, including inulin, immunoglobulin aggregates, bacterial endotoxins, and staphylococcal protein A, were found to initiate blood coagulation through a complement-mediated pathway. These substances markedly accelerated blood coagulation in normal rabbit blood. That this clot-promoting activity requires an intact complement system was demonstrated by an almost total lack of effect on blood from rabbits with an inherited deficiency of the sixth component of complement (C6). Small amounts of isolated C6 conferred to C6-deficient blood the ability to respond with accelerated coagulation upon activation of the complement system. In addition, it was determined that activation of complement through the previously described C3 activator system resulted in the initiation of blood coagulation. The participation of C1, C2, and C4 was not necessary

C'1 ESTERASE EFFECT ON ACTIVITY AND PHYSICOCHEMICAL PROPERTIES OF THE FOURTH COMPONENT OF COMPLEMENT

Highly purified C'1 esterase of human serum is capable of inactivating isolated fourth component of human complement (β1E-globulin). Inactivation is accompanied by changes in electrophoretic and ultracentrifugal properties of β1E-globulin. If non-sensitized sheep erythrocytes are present during the action of C'1 esterase on β1E-globulin, a complex is formed consisting of cells and cytolytically active fourth component (EC'4). Thus, inactivation of β1E-globulin by C'1 esterase appears to be preceded by a state of activation enabling β1E-molecules to combine with cell membrane receptors. Acceptor groups appear to be present also in 7S γ-globulin and in β1E-globulin itself, since C'1 esterase can induce the formation of β-β and of β1E-7S γ-globulin complexes

FOURTH COMPONENT OF HUMAN COMPLEMENT: DESCRIPTION OF A THREE POLYPEPTIDE CHAIN STRUCTURE

The fourth component of human complement (C4) was shown to be composed of three distinct polypeptide chains linked by disulfide bonds and noncovalent forces. The sum of the molecular weights of the chains equalled that of the intact molecule. The mol wt of the α-, β-, and γ-chains were respectively, 93,000, 78,000, and 33,000 daltons. Action of C1s on C4 affected only the α-chain, reducing its mol wt to 87,000 daltons. The size of the activation peptide. C4a, is therefore estimated to be 6,000 and that of the major fragment C4b, 198,000 daltons. Periodic acid-Schiff-stained SDS polyacrylamide gels of reduced C4 revealed carbohydrate to be associated with all three chains. A modification of the original method of isolation of C4 is presented

FORMATION OF C3a AND C5a ANAPHYLATOXINS IN WHOLE HUMAN SERUM AFTER INHIBITION OF THE ANAPHYLATOXIN INACTIVATOR

Two biologically and chemically distinct anaphylatoxins (ATs) could be generated in whole human serum after removal of the AT inactivator (AI) by immune-absorption or after inhibition of AI with 1 M epsilon-aminocaproic acid (EACA). Both human ATs could be generated by treatment of serum with antigen-antibody complexes, which activate the classical complement pathway, and with inulin or yeast, both of which trigger the alternate pathway. The ATs were isolated from serum in active form and characterized as C3a and C5a. Although human C3a had been characterized previously, C5a had not. The molecular weight of human C5a AT was 17,500; its electrophoretic mobility at pH 8.5 was –1.7 x 10–5 cm2 V–1 s–1. The minimal effective concentration in vitro was 7.5 x 10–10 M. The minimal effective doses of human C5a in producing a wheal and erythema in the human skin was 1 x 10–15 mol. The results strongly suggest a biological function for both ATs and indicate that the expression of their activity is controlled by the AI of normal blood plasma