Inhibitors of complement activation and complement breakdown products

324 Plasma Ther Ttattsfus Technol 1987; 8:324-332

Inhibitors of Complement Activation and Complement Breakdown Products P.

The complement system, together with other blood protease systems, such as the coagulation, the kinin, and the fibrinolytic systems, serve to maintain homeostasis. Seen from a biochemical viewpoint, the activation and control of the four plasma protein systems follow the same pattern: Both are achieved by limited proteolysis {Figure I}. The complexity of the plasma protein systems mentioned, especially the complement system, is due to the wide variety of biologic activities expressed by the reaction products of the limited proteolysis. Limited proteolysis of the "native" form of a protein of the four plasma protein systems can either lead to the appearance of an enzymic activity or, in cases of the complement and the coagulation systems, might generate an activity, which enables one of the two cleavage products to (covalently) bind to an appropriate surface. Such a surface may be a macromolecule [e.g., complexed immunoglobulin), a bacterial or cell surface, or an artificial membrane. In cases where generation of an enzyme activity or of a surface-binding activity results in two cleavage products, the peptide without enzymatic or surface-binding activity Individual complement components are termed according to recommendation of the WHO [Editorial, WHO, 1968; Editorial, WHO, 1981) From the Central Laboratory of the Swiss RedCross, Blood Transfus ion Service, Bern, Switzerland. Reprint reque sts to P. J. Spath, Central Laboratory SRC, Blood Tran sfusion Service, CH·3000 Bern·22, Switzerl and.

J.

Spath

usually expresses other very potent biologic activities. The surface-binding activity (CDEFin Figure I) generally serves to achieve a transition from the fluid to the solid phase, to localize the reaction, and to modify the surface in such a way that it can accept and noncovalently bind an enzyme, which itself may be a product of limited proteolysis. The basic principle of assembly of binding unit and enzyme activity is followed in both the activation and inactivation processes . This principle is even conserved for initial activation steps: preformed binding unit-zymogen complexes circulate (Table I). In case of factor XII, the initial limited proteolysis results in a two-chain molecule where one chain expresses surface-binding and the other enzyme activity. Among the four plasma protein systems, the complement system is responsible for humoral immune defense and is involved in the regulation of immune responses. The complement system is also a major mediator of inflammatory reactions. Indeed, activation of the complement system by limited proteolysis leads to cleavage products that mediate such phenomena as modulation of circulating and extravascular immune complexes, phagocytosis, chemotaxis, anaphylaxis, acute shock, numerous inflammatory reactions, acute allergic reactions, and increased vascular permeability. Assembly of the late-acting components of complement induces swelling and lysis of cells and bacteria and disturbance of the

Inhibitors of Complement Activation and Breakdown Products 325

ABQ...s.S-PEF*

GENERATION

~

OFANENZYME

ABCD + EF*

ACTIVITY

limited

ABCDEF proteolysis

BINDING TO

AB + CDEF

CIRCULATING

SURFACE; INTERACTION WITH CELLRECEPTORS

RESTING STATE NO BINDING TO SURFACE; GENERATION OF VARIOUS NON-ENZYMATIC ACTIVITIES; INTERACTION WITHCELL RECEPTORS

Figure 1. Schematic representation of limited proteolysis as it is effectuated in some activation and some control processes of the complement, the coagulation, the kinin, and the fibrinolytic plasma protein systems. For all four plasma protein systems the model protein ABCDEFrepresents the circulating, native, nonactive, proenzyme, zymogen form or the resting state of the proteins. Generation of an enzyme activity by limited proteolysis does not necessarily result in two cleavage . products as schematically demonstrated by ABCs_s DEP, where the peptides ABC and DEF* are linked by S-S-bridge/s).

lipid bilayer of enveloped viruses. Formation of the membrane-attack complex in sublethal quantities on membranes of nucleated cells triggers arachidonic pathway metabolites such as prostaglandins, leukotrienes, or thromboxanes, depending on the cell type involved. Functional or even inactivated surface-bound complement ICDEF and CDEF breakdown products in Figure 1) might have receptors on blood cells. Binding of such complement triggers a series of cellular events. ACTIVATION AND CONTROL OF THE COMPLEMENT SYSTEM

Nineteen complement proteins are well described today. Fourteen of them participate in complement activation and are organized, as in the coagulation system, in two initial activation pathways and one terminal pathway, with the third component of complement, C3, as the "central

molecule" of the system [Figure 21. The proteins of the classical pathway of complement are Cl, C2, and C4. The alternate pathway of complement consists of fac-

S-F.-otein

Figure 2. Simplified schema of the complement system.

326 Plasma Therapy

Vol. 8, No.4

Thble 1. Some Cofactor-Zymogen Complexes of Activation and Control of Plasma Protein Systems Circulating Preformed Binding Unit-Zymogen Complexes Binding Unit Zymogen(s) System Clq HMW-kininogen HMW-kininogen Surface Modulating Unit Activation processes C3b

Clr and CIs XI Prekallikrein

First component of complement Coagulation Kinin

Binding Unit-Enzyme Complexes Assembling on Surfaces Enzyme Function

Bb Bb

C4b

C2a

C4bC3bn

C2a

VIla Va

IXa Xa

C3-convertase of the alternate pathway of complement Cfi-convertase of the alternate pathway of complement C3·convertase of the classical pathway of complement CS-convertase of the classical pathway of complement Activation of factor X Conversion of prothrombin to thrombin

Regulation processes H

I

C4-BP' Protein S'

Protein Ca

Acceleration of decay of alternate pathway C3-convertase and degradation of C3b Acceleration of decay of classical pathway C3 convertase and degradation of C4b Degradation of coagulation factors Va and VIla

• In plasma, protein S exists in a free form and in a bimolecular, noncovalent complex with C4·BP.

C4·Bl'·protein S complex in plasma has no protein Ca cofactor function. Binding of protein S to C4·BP has no direct effect on C4·BP function.

tors B, 0, and P. C3 participates in the activation of the alternate pathway. The components C5, C6, C7, C8, and C9 belong to the lytic pathway of complement and they can assemble to form the "membrane attack complex:' C5b·9.1n addition, the complement system consists of a pathway termed the amplification loop, which is built entirely by the components of the alternate pathway and C3. Five of the complement proteins regulate complement activation. CIesterase-inhibitor or Cl-inhibitor (CI-

INRI, Cd-binding protein IC4-BPI, and factor I control classical pathway activation at several levels. The alternate pathway together with the amplification loop is controlled at a single level by factor H and I. Sprotein, which should not be confused with the vitamin Kdependent control protein of coagulation, protein S, acts as an inhibitor of the insertion capacity of C5b-7 into neighboring bystander cells and functions as an inhibitor of C9 polymerization. S-protein appears to have a further function: It protects thrombin

Inhibitors of Complement Activation and Breakdown Products 327

from rapid inactivation by antithrombin III (ATIII) in the presence of low levels of heparin by formation of a trimolecular complex between S-protein, thrombin, and ATIII during clotting. Activation of the alternate pathway of complement is a continuous event, i.e., a low level of a surface-binding activity is always being generated by limited proteolysis. The molecule involved is C3, and the surface-binding activity is represented by cleavage product C3b. Binding of C3b to a surface is a random event. In the absence of a surface, the C3b molecule interacts with water molecules. Binding of C3b to a surface represents a modification or preparation of the surface such that it is able to accept an enzyme activity generated by limited proteolysis of zymogen factor B. The complex formed by surfaceC3b (cofactor) and Bb (enzyme activity) is termed the alternate pathway (amplification) C3-convertase. The activation of the alternate path- . way and the amplification loop is dependent on the microenvironment provided by the surface. Nonactivating surfaces al'low a rapid and effective but reversible interaction of factor H with the surfaceC3b. The noncovalent binding of factor H to surface-C3b results in decreased formation and the rapid decay of formed surface-C3bBb. Binding of factor H to surface-C3b represents a second modification of the surface. This modification prepares surface-C3b to accept factor I, which cleaves C3b to result in its inactivated form, iC3b. This molecule is no longer able to bind enzyme Bb. The cleavage of surface-C3b is irreversible, it represents a further limited proteolysis, and it shuts down alternate complement pathway activation. An activating surface provides a microenvironment that impairs the action of factor H. The complex of surfaceC3bBb survives for a while and is able to produce further complexes of surfaceC3bBb (on the basis of limited proteolysis); thus the amplification loop of complement is activated. An alternate pathway activating surface does not need

to express an intrinsic biologic activity; such a surface acquires this activity. Artificial organs, which come in close contact with blood or plasma, and which are able to disturb regulatory function expressed by complement proteins H and I, are potential activators of the alternate pathway of complement and may trigger rapid production of vasoactive and other substances. The accelerated cleavage of C3 and factor B while the alternate pathway and the amplification loop are activated has several consequences: binding of newly generated C3b in close proximity or to the surface-C3bEb to result in surfaceC3b nBb complexes; a shift of specificity of the enzyme complex from C3 to C5 allows consumption of C5; enzyme Bb may convert plasminogen to plasmin; and as a consequence of the limited proteolysis, biologically highly active peptides C3a, C5a, and Ba are generated. 'In conclusion, the activation and the control of the alternate pathway and the amplification loop is based on continuous, low-level, limited proteolysis; on preparation of a surface to accept an enzyme activity; and on impairment of the control function exerted by factors H and I. As mentioned above, control of the classical pathway of complement is achieved on several levels. The control of activation of C3 by the classical pathway C3-convertase, C4b2a, is achieved by C4-binding protein (C4-BPI and factor I. The principle is the same as the control of the (amplification) C3-convertase of the alternate pathway (Figure 3). Instead of factor H it is the C4-BP that competes for binding and prevents the survival not of enzyme Bb but of enzyme C2a. Binding of C4-BP again alters surface-Cab so as to accept the enzyme factor I, which cleaves C4b and renders it unable to bind C2a and shuts down activation of C3 by the classical pathway of complement. C4-EP and factor I do not control the initial activation of the classical pathway; . activation of the first component of cornplement, CI , is controlled by CI -INH. CI

328 Plasma Th erapy Vol. 8, No.4

recognitioil of antigen by antibody

01-Ni

01-1+1··01 ~tofthe

control by C1-Ni of autoactlvaUon "'"""'...IlL........ of C1r

Icla88lcal1 INITIATION

C3CONVERTASE

Iafternative I

contnwa low C1ad9 activation

C3a

sa

Figure 3. Comparison of control of classical and alternate pathway (amplification) C3-convertase by regulatory proteins factor H, factor I, and C4-binding protein. Squares represent surface binding/modulating units and circles functionally active enzymes of activation processes. Triangles and elongated circle depict binding/modulating cofactor unit and enzyme activity, respectively, of regulation processes.

is a pentamolecular complex composed of one molecule of Clq and two molecules of Clr and CIs. Clq represents a surfacebinding or surface-modifying factor, Clr and CIs are zymogens. Thus, CI represents something like a preformed convertase. According to the most recent hypothesis, CI in circulation forms a weak and labile complex with CI-INH. The role of CI-INH in such a complex would be to prevent the autoactivation of the zymogen Clr. As soon as Clr escapes control by CI-INH, the autoactivated form con. verts by limited proteolysis zymogen CIs to its enzyme form, the Cl-esterase. Autoactivation of Clr in absence of CI-INH

is apparently a slow process that is thought to be accelerated by possible conformational changes of Clq upon binding to surface. Thus, all macromolecules that are able to displace Cl-INH from CI and are able to bind Clq, are potential activators of Cl. However, activation of Cl does not necessarily result in the activation of C2 and C4 and the formation of the classical pathway C3-convertase, because CI-INH exerts its regulatory function on a second level. It is able to interact with the active enzymes Clr and CIs. The nature of the control of enzymatically active Clr and CIs by CI-INH involves the for-

Inhibitors of Complement Activation and Breakdown Products 329

pathway. The initial step of activation can again be considered as an impairment of regulation. In contrast to the alternate pathway, it is not a permanent activation that escapes control, but the autoactivatability of Clr.

mation of covalent CI-INH-enzyme complexes involving the active site of these enzymes and the dissociation of the active CI complex into a surfacebound Clq and two complexes of CIINH-Clr-Cls-CI-INH. Activation of C2 and C4 by active CI apparently depends on the macromolecule or surface that has displaced CI-INH from CI and may have bound CI via its binding unit, Clq. Such macromolecules or surfaces may be found in dialyzer membranes. Indeed, saponified cellulose membranes contain a "Limulus amebocyte lysate reactive material:' which is definitely not endotoxin, is released from membranes in the first 30 minutes of hemodialysis, and mediates activation of C4 as assessed by appearance of C4a. Although the Limulus amebocyte lysate reactive material can be shown in plasma of patients on hemodialysis in vivo, no adverse reactions could be associated with cellulose-based material. In conclusion} the classical pathway of complement is controlled on several levels. Control at the level of classical pathway C3-convertase is biochemically similar to the control of the alternate

INHIBITORS COMMON TO PLASMA PROTEIN SYSTEMS Besides the control of the initial steps of activation of the classical pathway of complement, CI-INH further controls initiation of the coagulation} the kinin, and the fibrolytic systems (Figure 41. The key molecule of initial activation of the coagulation and the kinin systems is factor XIIa} a product of limited proteolysis of factor XII} the Hageman factor. It is thought that factor XIIa might be generated continuously on an extreme low level. Activity of XIIa is controlled exclusively by Cl·INH. If continuous generation of low levels of XIIa occur, formation of larger quantities of XIIa and activation of the initial steps of the coagulation and the kinin system can again be considered as an impairment of control. Control of the initial proteases of the

COMPLEMENT SYSTEM C11tt C1

1 XII

COAGULATION SYSTEM

PLASMINOGEN

•

~ C1-INH

~

Xlla"

~flSM'N

..,.

~+

Surface - - - - - - - '

PREKAl.L1KREIN HMW-KJninogen (Surface)

FIBRINOLYTIC SYSTEM

... KALLIKREIN HMW-KJninogen (Surface)

KININ SYSTEM Figure 4. Control of initial activation steps by CI-INH of various plasma protein systems.

330

Plasma Tllerapy Vol. 8, No.4

complement, the coagulation, and the kinin systems is achieved by formation of stable C1-INH-enzyme complexes (Table 21. Indeed, C1-INH can form irreversible complexes in a molar ratio of 1:1 with the coagulation factors Xlla, xnr, Xla, with plasma kallikrein, and activated complement subcomponents C1r and CIs. Thus, the escape from control of these initial activation cascades leads to a consumption of a functionally active C1-INH, which further decreases inhibitory capacity of total CI-INH in the circulation. If CI-INH concentration falls below the critical level needed to control the previously mentioned proteases, a cascade of reactions may follow. Some of these possible reactions are depicted in Figure 5. These reactions may potentiate each other, and many of them generate highly active biologic peptides, including anaphylatoxins. The question might arise as to how far such a complex reaction pattern can go in vivo. It is noteworthy that kallikrein, factor Xllf, and factor XIa arc controlled by other plasma protease inhibitors besides C1·INH (Table 21. Nevertheless, excessive activation of the initial steps of these protease systems and accumulation of vasoactive substances may take place in the absence of functional CI-INH. This can be demonstrated in patients suffering from hereditary angiolneurotic] edema (HA(N)EI, which is biochemically characterized by drastically diminished levels of functionally active CI-INH in circulation. Clinically it is characterized by recurrent, cold, non itching swelling episodes of the subcutaneous and submucous tissues.

Vasoactive reaction products of uncontrolled activation of the complement and/or the kinin system are thought to be responsible. Indeed, in the blood of these patients a clear consumption of complement, factor XIII and prekallikrein can be demonstrated. Functional C1-INH is consumed while reacting with proteases of the initial sequence of the complement, coagulation, and kinin systems. What is the critical level of C1-INH that maintains homeostasis? To control autoactivation of zymogen Clr and to inhibit active CI , a concentration of C1-INH of 35% to 40% of normal is apparently sufficient; it maintains C1-INH function within the normal range. This estimate is supported by the assessment of CI-INH concentration immediately prior to or after onset of swelling attacks in patients suffering from the common form of HA{NIE. Indeed, no edematous attacks have been observed with C1-INH concentrations at or above 40% of normal. Thus, treatment regimens that markedly reduce the molar ratio of CI-INH to enzymes to be controlled or that influence functional activity of CI-INH may disturb homeostasis. In large-scale plasma exchange CI-INH concentration is drastically diminished. However, other components of serum are diminished as well; thus the impairment of CI-INH function is without drastic effect. However, todays situation of replacement fluids is not satisfactory. The use of fresh frozen plasma is dangerous because infective agents can be transferred. Protein solutions such as albumin or im-

Table 2. Plasma Enzymes Able to Form Complexes With CI·INH Complement System Clr (CI-INH only) CIs (Cl-INH only)

Kinin System Kallikrein ICI-INH and others)

Coagulation System XIIa ICI-INH only) XIIf (mainly CI-INHj XIa I CI-INH and others]

Fibrinolytic System Plasmin (mainly others)

Inhibitors of Complement Activation and Breakdown Products 331

C1--t> C1

+t+

XII

~

1

XI [ HMW-K

Surface

'-- X l a -

)

(Surface)

COAGULATlON

PLASMIN

I

V

FIBRINOLYSIS I I

PREKALlIKREIN

Surface

V V

I

I

HMW-K

C-;.

I

KALLIKREIN (fluid phase)

t

KALUKRE'N.~1? ~

e:: 11

INACTIVATION +-OF COAGULANT . ACTNITY

HMW·K (Surface)

CASCADE

1

\

HMW-K

•

PLASMINOGEN

\

1- xu.lx,"-

Surface

COMPLEMENT

...

•

I

.. C3a(dUNgl C4a

·-.1

CARBOXYPEPTIDASE B

BRADYKININ - . OCTAPEPTIDE

1

C4a(deoAtgl

csa - -....... Csa(de&Algl

MOBILIZATION OF RENIN FROM KIDNEY

Figure 5. Schema of some of the reactions that might occur in the absence of functional CI-INH. Evidence for such reactions was largely obtained by in vitro experiments using purified proteins. Usually a (high) excess of enzymes of one plasma protein system was necessary to obtain limited proteolysis of proteins of another plasma protein system. However, in such experiments the accelerating role of surface and of cofactor was neglected. Evidence of some of the reactions depicted is obtained in vivo in patients suffering from hereditary or acquired deficiency of Cl·INH.

munoglobulin are also not satisfactory. Thus, there is a need for exchange fluids that are more complex than albumin or immunoglobulin solution and that are safe. To achieve safety (with no transmission of infective agents], a chemical or physical treatment is a prerequisite. Such treatments may alter proteins in a way that allows them to impair CI-INH function or to shift the molar ratio of CI-INH to proteases to be controlled. When large volumes of plasma exchange fluids that are more complex than albumin or IgG solution are used, their effect on CI-INH should be evaluated carefully. Besides CI-INH, plasma carboxypeptidase B (pCPB, former serum carboxypeptidase NI is a further inhibitor common

to the coagulation, the kinin, the fibrinolytic, and the complement systems. The survival of the circulating anaphylatoxins is regulated by binding to receptors on lymphocytes and macrophages and by pCPB, which is an exopeptidase that cleaves carboxy terminal arginine or lysine. pCPB modulates the activity pattern or regulates the survival of the physiologically active peptides C3a, C4a, CSa, Ba, bradykinin, fibrinopeptides A and B, kal lidin, hexapeptide enkephalins as well as the plasmin-generated fragments of fibrin. The hereditary deficiency of pCPB clinically manifests as angioedema. A deficiency can also be acquired, as suggested by subnormal activity of pCPB during hemodialysis.

332

1'lasma Therapy Vol. 8, No .4

FOR FURTHER READING 9. 1. Arlaud GJ, Colomb MG, Gagnon J: A functional model of the human Cl complex. Immunol Today 1987; 8:106-111. 2. Dahlback B:Inhibition of protein Ca cofactor function of human and bovine protein S by C4b -binding protein. , BioI Chem 1986; 261:12022-12027. 3. Kaplan ~ Silverberg M: The coagulationkinin pathway of human plasma. Blood 1987; 70:1-15. 4. Kazatchkine MD, Nydegger UE: The human alternative complement pathway: Biology and immunopathology of activation and regulation. Ptog Allergy 1982; 30:193-234. 5. Mathews KP, Curd JG, Hugli TE: Decreased synthesis of serum carboxypeptidase N (SCPN) in familial SCPN deficiency. , Clin Immunol 1986; 6:87-91. 6. Morgan LE, Thoman ML, Hoeprich PO, Hugli TE: Bioactive complement fragm ents in immunoregulation. Immunol Lett 1985; 9:207-213. 7. Nomenclature of complement, editorial. Bull WHO 1968; 39:935 . 8. Nomenclature of the alternative activat-

10.

11.

12.

13.

ing pathway of complement, editorial. Bull WHO 1981; 59:489. Pearson FC, Caruana R, Burkart J, Katz DV, Chenoweth 0, Dubczak J, Weary M: The use of Limulus amebocyte lysate assay to monitor hernodialyzer-associated soluble cellulose material (LAL-reactive material), in Detection of Bacterial Endotoxin with the Limulus Amebocyte Lysate Test. Alan R. Liss Inc, 1987; pp 211-232. Podack ER, Dahlback B, Griffin JH: Interaction of S-protein of complement with thrombin and antithrombin ill during clot· ting . ] BioI Chem 1986; 261:7387-7392. Spath PT, Wuthrich B, Butler R: Quantification of Cl-inhibitor functional activities by immunodiffusion assay in plasma of patients with hereditary angioedema - Evidence of a functionally critical level of Cl-inhibitor concentration. Complement 1984; 1:147-159. Sundsmo JS, Fair OS: Relationships among the complement, kinin, coagulation and fibrinolytic systems. Springer Semin lmmunopathol 1983; 6:231-258. Ziccardi RJ: The first component of corn plement [Cl]: Activation and control. Springer Semin Immunopathol 1983; 6:213-230.