El SMIC (Sistema de matrices de impactos cruzados)

The proteins of the alternative pathway of complement are factor D, factor B, C3 and C5 (Figure 1.1). Components C5-C9 are common to both pathways and will be discussed in the terminal components section. C3 has a central role in both pathways. The regulatory proteins factor H, factor I and properdin have major roles in controlling activation of the alternative pathway. Activation of the alternative pathway can be both antibody dependent and antibody independent. Antibody dependent activation can occur via IgG, IgA and (rarely) IgE immune complexes. Antibody independent activation can be effected by a whole spectrum of substances located on the surfaces of bacteria, fungi, viruses, multicellular parasites and tumour cells.

1.4.1 Activation

The mechanism by which the targets are recognised by the alternative pathway is less well understood than for the classical pathway. In the alternative pathway, several proteins are involved simultaneously in recognition. Perturbations in the interaction between C3b deposited on the activating surface and regulatory molecules is important for determining whether complement is activated or not.

Table 1.2 Membrane bound regulators of complement.

Regulator Action Characteristics

DAF Di s s o c i a t e s C3 70 kDa glycoprotein present on the convertases. membranes of peripheral blood cells,

vascular endothelial cells, placenta and epithelial cells. Extracellular portion contains 4 SCR domains. Soluble forms occur.

MCP Binds C3b and acts as 50-60 kDa integral membrane

(CD 46) a cofactor to factor I glycoprotein present on all circulating m e d i a t i n g C 3 cells except erythrocytes and most other cleavage. Cellular

receptor for measles virus.

cell types. Contains 4 SCR domains.

Protectin Binds C5d-8 complex 20 kDa glycoprotein present on the (CD 59) and prevents formation membranes of all circulating blood cells.

(pl8) of the polymeric C9 endothelial cells, epithelial cells and

(MACIF) (HRF20) (MIRL)

complex. spermatozoa.

Homologous HRF/C8bp prevents Poorly characterised to date. restriction factor the binding of C9 to

(HRF) C8ay and its insertion

(C8 binding protein) (C8bp)

into membranes.

Abbreviations used: DAF, decay accelerating factor; MCP, membrane cofactor protein; MIRL, membrane inhibitor of reactive lysis; HRF, homologous restriction factor; C8bp, C8 binding protein; SCR, short consensus repeat

Native C3, like C4, contains an internal thiolester in the a-chain which can be hydrolysed to form C3i (also known as C3(H20)) and is capable of attaching to membranes (Pangbum and Müller-Eberhard, 1980; Pangbum et al, 1981). Although C3i is structurally different from C3 b in that it has an intact a-chain, both C3 i and C3b are functionally similar. C3 is continually activated at a slow rate in the fluid phase by three mechanisms: (i) C3 may be cleaved to C3b by serum proteases; (ii) small nucleophiles, or water, may gain access to C3 and react with the thiolester; (iii) C3 may be subj ect to non-specific perturbation leading to exposure and hydrolysis of the thiolester. Thus the alternative pathway is in a permanently activated state. If activated C3 is deposited on an activating surface of the alternative pathway, it can serve as a seed for the positive amplification loop which operates explosively. Activated C3 is able to form a C3 convertase with factor B in the presence of factor D. Removal of C3a by proteolytic cleavage by the C3 convertase induces a conformational change in C3b which leads to the exposure of the internal thiolester, which is normally buried in native C3. The exposed thiolester is extremely reactive with nucleophiles, including water and molecules bearing hydroxyl or amino groups. C3b, activated at the surface of a foreign cell, is largely restricted to binding to the surface of the same cell or to being inactivated by water. This puts a limit on the spatial range of the activated C3b. The deposition of C3b is minimised on host cells because of their inability to activate the host’s own complement pathways. C3 activation is kept to a low level by the control proteins, factor H and factor I in the blood. The C3 and C5 convertases decay quite rapidly by the dissociation of the enzymatic components in the absence of control proteins. The survival of the first C3b molecule deposited on the surface of a substance determines whether it will be an activator or non-activator of the alternative pathway

Activation of the alternative pathway does not depend upon antibodies recognising specific molecules on the target cell surface: rather it relies on molecular structures on the target cell to upset the delicate balance of proteins involved to focus their activation and deposition on its surface. Activators include polysaccharides, fungi, bacteria, viruses, parasites and certain mammalian cells. It is not known what structure all the activators have in common but it is possible that C3b deposited on activators is protected from proteolytic degradation,

enabling the formation of the C3bBb complex (Fearon, 1978; Pangbum etal, 1980; Fearon and Austen, 1975). C4b bound to a surface can also activate the alternative pathway by binding to C3bBbP, which may be important in cases of C2 deficiency (Parries et al, 1990).

The most obvious similarity between the classical and alternative pathway is the C3 convertase enzyme. The C3 convertase of the classical pathway is C4b2a which is very similar to the alternative pathway C3 convertase, C3bBb. C4b is a homologue of C3b and C2 a homologue of factor B. Factor D is a serine protease that has a role similar to the classical pathway Cls. The alternative pathway does not have proteins similar to Cl q or Cl r. A paradoxical situation arises in which C3b is required in order to assemble the enzymes responsible for C3 cleavage. In vivo the classical pathway is also present and the amplification of the C3b deposition via the alternative pathway occurs if the classical pathway C3 convertase (C4b2a) is the source of the initial C3b molecule. Multiple C3b molecules are deposited in clusters on the complement activator. This cluster formation is important in mediating multiple interactions with C3 receptors on phagocytic cells.

1.4.2 Control proteins

Control of the alternative pathway is comparable to that of the classical pathway in that homologous proteins fulfill similar roles. Both positive and negative control mechanisms exist. The C3bBb convertase is stabilized by the binding of the control protein properdin (P) to form a C3bBbP complex (Fearon and Austen, 1975; Medicus eta l, 1976; Farries etal,

1987), which facilitates the binding of factor B and prevents cleavage of the convertase by factor I (Farries et al, 1988a). The versatility of complement to recognise all types of foreign material is desirable, but its binding to host cells must be minimised. Negative control of activation is governed by factor H which acts in a similar way to C4bp in the classical pathway by acting as a cofactor for factor I-mediated cleavage of C3b and C3i to form the fi*agments iC3b + C3f and iC3i + C3f respectively (Pangbum and Müller-Eberhard, 1983). The factor H molecule can interact with C3b via three binding sites and so prevent the formation of the C3bBb convertase and it can dissociate the complex (decay accelerating activity). Factor H is also able to discriminate between activator and non-activator bound C3 molecules. Non­

activators, which include host cells, carry polyanionic substances like sialic acid or glycosaminoglycans on the cell surface. The high affinity of factor H for non-activator bound C3b seems to be a joint recognition of both C3b and surface structures. Various microbes have capsules rich in sialic acid (group B streptococcus, E. coli capsule type K1 and group B meningococcus) which allows them to escape alternative pathway activation, opsonisation and phagocytosis.

1.4.3 Protection mechanisms

The majority of C3b and C4b molecules (70 to 90%) formed as a result of complement activation do not become covalently linked to surfaces but remain in the fluid phase (Hourcade et al., 1989). Of those that do bind, some will do so to host cells. These are protected against complement-mediated cytolysis by the membrane proteins DAF and MCP (Atkinson and Farries, 1987). DAF prevents assembly of C3 convertase on membranes and will dissociate enzymes already bound. MCP has cofactor activity for factor I mediated cleavage (Liszewski et al., 1991; Fearon, 1979).

1.5 Terminal pathway