Pathways of Complement Activation

The complement activation can be divided into three pathways, Classical, Lectin (mannose binding protein) and Alternative, all of which result in the activation of C5 and lead the formation of the membrane attack complex (MAC).

Stages of Complement Activation

There are three main stages in the activation of complement cascade by any pathway:

  • Formation of C3 Convertase
  • Formation of C5 Convertase
  • Formation of MAC Complex

The three pathways differ in the way C5 is broken down but after that the formation of MAC is essentially the same.


1. Classical Pathway

The classical pathway is triggered primarily by immune complexes (containing antigen and IgG or IgM) in the presence of complement components 1, 4, 2, 3, Ca++ and Mg++ cations. IgG and IgM are most potent activators of this pathway. Since they need Antibody interaction to initiate the pathway its an antibody dependent pathway.

They react in the order C1q, C1r, C1s,C4, C2, C3, C5, C6, C7, C8 and C9. Different IgG subclasses have different potency to activate the pathway. While IgG1, IgG2 and IgG3 (most effective) can activate complement, IgG4 is not able to activate at all.

This pathway begins by the formation and activation of C1qr2s2 (C1 Macro molecular complex) by the antigen-antibody complex and finally ends in the formation of membrane attack complex (MAC).


  • C1 is the first complement component to participate in classical pathway. It act as initiation and recognition unit. It is a macro molecular complex composed of three polypeptide chains – C1q, C1r and C1s. The over all molecule appears like a bunch of six tulips. Each globular head has a binding site for Fc fragment of the antibody. Two molecules each of C1r and C1s are bounded to C1q in space between the heads and stems. The overall molecular structure of C1 complex is C1qr2s2.
  • The C1r and C1s are serine protease enzymes which are activated by C1q. C1r2s2 complex exist in an elongated S form when in free state. But when bound to C1q, it assume a different conformation resembling to figure 8.
  • The activation of classical pathway begins by binding of at least two globular heads of the C1qr2s2 complex with the antigen antibody complex through the Fc fragment of the antibody.
  • C1qrs can also bind to a number of agents including some retroviruses, mycoplasma, poly-inosinic acid and aggregated IgG, and initiate the classical pathway.

Formation of C3 Convertase:

  • Binding of Cqr2s2 globular heads leads to a conformational change in the stems of C1q.This change is sequentially transmitted to C1r2 and then to C1s2.
  • During this change, C1r is activated by auto cleavage. The altered C1r cleaves C1s. At this stage, Cqr2s2 complex is fully activated.
  • This cleaved C1s is capable of cleaving both C4 and C2. Activated C1s enzymatically cleaves C4 into C4a and C4b. C4b binds to the Ag-bearing particle or cell membrane while C4a remains a biologically active peptide at the reaction site.
  • C4b binds C2, which becomes susceptible to C1s and is cleaved into C2a and C2b. C2a remains complexed with C4b whereas C2b is released. C4b2a complex is known as C3 convertase.

Formation of C5 Convertase:

  • C3 convertase, in the presence of Mg++,cleaves C3 into C3a and C3b. C3b binds to the membrane to form C4b2a3b complex whereas C3a remains in the microenvironment. C4b2a3b complex functions as C5 convertase, which cleaves C5 into C5a and C5b. Generation of C5 convertase marks the end of the classical pathway. C5b initiates the formation of membrane attack complex.

Formation of Membrane Attack Complex (MAC):

  • The C3b components of C5 convertase binds C5 and split into C5a and C5b fragments. The smaller fragment diffuses away and the large fragments C5b binds to C5 convertase to form a C4b2a3b5b complex. This act as the catalyst to form the membrane attack complex.
  • The C5b is extremely labile unless stabilized by the binding to C6 and immobilized by binding to cell membrane. it therefore, binds sequentially to C6 and C7 to form the complex C5b67. This binding exposes a hydrophobic region on C7 through which it is able to penetrate the cell membrane.
  • This membrane bound C5b67 act as a receptor for C8 which thus binds to it. C8 also inserts itself into the membrane.
  • This membrane inserted complex, C5b678 has a catalytic property to bind C9 molecule and polymerize it into a 12-15 unit tubular channel of about 10nm diameter . This structure penetrate into the cell membrane and form channels or pores into the cell membrane. It allows the free passage of water into the cell leading to cell swelling and lysis.
  • Since the C5b6789 complex destroys the cell by attacking cell membranes, it is called membrane attack complex (MAC). This form of cytolysis is referred to as complement mediated cytotoxicity.


2.  Alternative Pathway:

  • Alternate pathway is so called because it bypasses the requirement o f antigen-antibody complex, C1, C2 and C4 components and thus it is part of the innate immune system.
  • It begins with the spontaneous activation of C3 in serum and requires three other serum proteins called Factors B, Factor D and properdin, all present in normal serum.
  • C3 is spontaneously broken in serum to C3a and C3b.
  •  The cleaved C3b binds to foreign antigens or to host cells. In the normal cell surface sialic acid residues destroy the bound C3b rapidly and so do not allow the activation of this pathway further. Because of the low concentration of sialic acid levels in foreign antigen surfaces, the bound C3b remains active for a longer period.
  • The bound C3b on the foreign antigen surface binds another protein, factor B, which is cleaved by Factor D to produce C3bBb (now C3 convertase).
  • This has very short life of about 5 minutes unless it is stabilized by properdin which increases the half life to 30 minutes. Hence the alternative pathway is also known as the properdin pathway.
  • Stabilized C3 convertase cleaves more C3 and produces C3bBb3b complex (C5 convertase), which cleaves C5 into C5a and C5b. C5b initiates the formation of membrane attack complex. The sequence of events for the formation of MAC is common in all the pathways.
  • The alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defence against a number of infectious agents.
  • This pathway is initiated without the antibody by various cell surface constituents that are foreign to the host such as those of bacteria, virus, fungi, yeast, parasites and even virally infected or cancerous host cells. Many non pathogenic cells and molecules such as Human antibodies-IgG, IgA and IgE, Cobra venom factor (CVF) and other proteins (e.g. proteases, clotting pathway products) also can activate the alternative pathway.


3. The Lactin Pathway

  • C4 activation can be achieved without antibody and C1 participation via the lectin pathway. Three proteins initiate this pathway namely a mannan-binding lectin/protein (MBL), and two mannan-binding lectin-associated serine proteases (MASP and MASP2), all present in normal serum.
  • MBL binds to certain mannose residues on many bacteria and subsequently interacts with MASP and MASP2. The MBL-MASP-MASP2 complex is similar to Ab-C1qrs complex (of classical pathway) and leads to activation of C4, C2 and C3. The rest follows as in classical pathway.


Complement System

The term “complement” was coined by Paul Ehrlich to describe the activity in serum, which could “complement” the ability of specific antibody to cause lysis of bacteria. Complement historically refers to fresh serum capable of lysing antibody-coated cells.

Complement system is composed of over 20 different proteins produced by hepatocytes,macrophages and intestinal epithelial cells. Fibroblasts and intestinal epithelial cells make C1, while the liver makes C3, C6, and C9. They are present in the circulation as inactive molecules.

Though some components are resistant to heat, heating serum at 56oC for 30 minutes destroys complement’s activity and thus they are not able to kill the microbes or pathogens present in the blood. Thus complement system are heat labile.  Serum complement levels, especially C3, often drop during infection as complement is activated faster than it is produced.

Several complement proteins are zymogens (proenzymes). Which means they are an inactive substance which is converted into an enzyme when activated by another enzyme. They get activated when infected with pathogen. When activated, they become proteases that cut peptide bonds in other complement proteins to activate them by the process called proteolysis. Proteolysis removes an inhibitory fragment exposing an active site. Complement proteins work in a cascade, where the binding of one protein promotes the binding of the next protein in the cascade.

The compliment components are named in various ways e.g. by numerals (C1- C9), by alphabetical symbols (factor D) and by common trivial names (homologus restriction factor). Complement components named by numerals are numbered in the order in which they were discovered. During activation, some complement components are cleaved into two unequal fragments. The larger part of the molecule is designated “b” while the smaller fragment “a”.

Small fragment,known as Anaphylotoxins, may diffuse away into the micro-environment to produce other effects (they act as chemotactic agents) while in most cases it is the “b” fragment that participates in the cascade reaction sequence or in other words binds to the surface of the cell to be lysed (the fragments of C2 are an exception to this rule: C2a binds to the membrane while C2b is freed into serum or tissue spaces).

Inactivated fragments are indicated by a small “i”. Enzymatically active forms are symbolized by a bar over the letter or number.

Activation of complement results in the production of several biologically active molecules such as MAC, which contribute to nonspecific immunity and inflammation. Complement is not antigen-specific and it is activated immediately in the presence of pathogen, so it is considered part of innate immunity. Since antibody also activates some complement proteins, complement activation is also part of humoral immunity. Their activation proceeds via different pathways in a cascade fashion leading to lysis.

A polymeric membrane attack complex (MAC) is formed following the activation of complement system. MACs breakdown the cell membrane leading to “pore” formation. Movement of ions and fluid into cell through these pores leads to cell lysis. Complement products opsonize the antigen which is then easily phagocytosed. Immune complexes also require complement products for their removal from the body. Also byproducts of complements, cascade induce inflammation which again augments the overall cellular response in the affected area. Beside all these, complements play important role in viral neutralization in several ways.

Complement proteins can be quantified directly by ELISA, and complement activity can be measured by the complement fixation test.