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Classical Pathway of Complement Activation

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Classical Pathway of Complement Activation

  • The complement components react in a cascade in a specified order, culminating in cell lysis, and this process is known as the classical pathway.
  • Antibody-antigen complexes, but not native or unbound antibodies, trigger this response.
  • IgG and IgM antibodies are the primary initiators of the classical complement pathway.
  • In order to cleave the C3 protein, a series of activation-induced protein recruitment events must occur, at which point C3 convertase (C4b2b, formerly known as C4b2a) is produced.
  • C3 convertase (C4b2b) attaches to the C3b subunit of cleaved C3, generating C5 convertase (C4b2b3b), which cleaves the C5 protein.
  • The cleavage products signal for the arrival of phagocytes and mark infected cells for phagocytic clearance.
  • The membrane attack complex is assembled near the end of the complement system’s life cycle, which is triggered by the C5 convertase (MAC).
  • By forming a breach in the membrane of the victim cell, the membrane assault complex triggers the cell’s internal organelles to lyse and kill it.
  • In addition to apoptotic cells, necrotic cells, and acute phase proteins can activate the classical complement pathway.

Activators of the classical pathway

Among the activators of classical pathways are:

  • Immunoglobulins IgM and IgG. IgG3 immunoglobulins are the most effective at activating the complement, followed by IgG1 and IgG2 immunoglobulins. Not activated by IgG4 immunoglobulins is the traditional route.
    • IgG and IgM that are native and free do not activate the complement system. A single molecule of natural IgG will not bind to and activate the complement system. However, if IgG antibodies are aggregated as a result of antigen binding, complement fixation and activation will occur. The development of an antigen–antibody complex produces conformational changes in the Fc region of the IgM molecule that expose a C1-binding site.
  • Staphylococcal protein A.
  • C-reactive protein.
  • DNA 

Steps of activation of classical pathway

Typically, the conventional process of complement activation begins with the production of soluble antigen–antibody complexes (immune complexes) or with the binding of antibody to antigen on an appropriate target, such as a bacterial cell. The sequential steps in the activation of the classical route are as follows:

Steps of activation of classical pathway
Steps of activation of classical pathway

1. Activation of C1

  • The activation of the classical route begins with the activation of C1.
  • It turns out that C1 is a complex made up of three distinct molecules: C1q, C1r, and C1s.
  • The Fc domain of the attached antibody (IgM or IgG) is the first site where C1q binds.
  • This causes C4, C2, and C3 to become active in that order. Activation of C1 requires binding to at least two contiguous Fc regions.
  • That’s why it’s important to have a high concentration of IgG antibodies and for the antigenic determinants recognised by those antibodies to be in close proximity to one another.
  • Pentameric IgM assumes a so-called stable conformation when bound to antigen on a target surface, in which at least three C1q binding sites are accessible.
  • With only one IgM molecule required to initiate the complement pathway, the lower valency of IgG molecules necessitates roughly a thousand of them.

2. Activation of C1r and C1s / C3 convertase formation

  • The binding of C1q in the presence of calcium ions activates C1r and C1s.
  • Activated C1s is an esterase that divides C4 into two fragments: a tiny, soluble fragment (C4a) and a bigger fragment (C4b) (C4b).
  • C4a is an anaphylatoxin, whereas C4b attaches to the cell membrane alongside C1.
  • In the presence of Mg2, C4b breaks C2 into C2a and C2b. C2b diffuses away, but C2a remains bound to C4b.
  • C4b2a complex contains enzymatic activity and is referred to as C3 convertase, which transforms C3 into an active form.

3. Activation of C3 molecules

  • The C3 convertase activates and divides hundreds of C3 molecules into C3a and C3b.
  • A single molecule of C3 convertase can produce over 200 molecules of C3b, resulting in an enormous amplification at this phase in the sequence.
  • Activated C3b and C4b are biologically significant because they can bind to C3b/C4b receptors (now identified as CR1 receptors) present on virtually all host cells, most notably phagocytes.
  • Immune adherence is the increased affinity of phagocytic cells for C3b (or iC3b)/C4b-coated particles.
  • The latter is responsible for a considerable increase in phagocytosis, one of the body’s primary defence systems.

4. Formation of C5 convertase

  • Some C3b binds to C4b2a to create the C4b2a3b complex, also known as C5 convertase.
  • C5 convertase breaks C5 down into C5a and C5b.
  • C5a diffuses away, whereas C5b binds to C6 and initiates the creation of the C5b–9 complex, also known as the membrane assault complex (MAC).
  • C5b67 complexes that have been released are capable of inserting into the membrane of surrounding cells and triggering “innocent-bystander” lysis.
  • Normally, regulator proteins in human sera prevent this from happening, but in some disorders, this process of innocent-bystander lysis can cause cell and tissue damage.
  • The membrane-bound C5b–6–7 complex functions as a C8 and C9 receptor. The binding of C8 to the complex stabilises the complex’s adhesion to the foreign cell membrane.
  • The C5b–8 complex functions as a catalyst for C9, a single-chain glycoprotein that tends to spontaneously polymerize.
Steps of activation of classical pathway
Steps of activation of classical pathway

5. Formation of MAC

  • The C5b–8 complex undergoes polymerization upon attachment to C9 molecules, resulting in the creation of the C5b–9 complex, also known as MAC.
  • The MAC forms a 100 transmembrane channel within the cell. This transmembrane channel enables the exchange of ions between the cell and its environment.
  • The osmotic pressure inside the cell increases rapidly due to the rapid input of ions and their interaction with cytoplasmic proteins.
  • This results in an influx of water, enlargement of the cell, and, for certain cell types, the rupture and lysis of the cell membrane.
Membrane attack complex
Membrane attack complex Formation

What is a Membrane attack complex?

All three distinct processes, including the traditional pathway, culminate in the development of the MAC. At the step involving the creation of the MAC, all three paths converge. The complement subcomponents C5b, C6, C7, C8, and C9 contribute to the development of the MAC. In the soluble phase, C5b, C6, and C7 form a complex, which then adheres to the cell membrane via the hydrophobic amino acid groups of C7. C7 becomes exposed as a result of its association with the C5b–C6 complex.

Action of membrane attack complex
Action of membrane attack complex

Deficiencies in the Classical Pathway: C1q, C1r, C1s, C4, C2, C1-Inh

  • Normal CPs are capable of efficiently removing immune complexes, dead cells, and debris from injured tissue. It is believed that a primary shortage of C1q, C1r, C1s, or C4 contributes to the development of systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), due in part to the complement system’s failure to eliminate immune complexes and dying cells.
  • Small complexes are eliminated from circulation when they attach to complement receptors on spleen and liver macrophages.
  • Without a complement, the complexity may become unmanageable. The resultant aggregates can trigger the alternative pathway, allowing C3 to be deposited into the matrix, along with re-solubilized complexes that can be eliminated via liver and spleen clearance.
  • In the absence of this, these massive complexes become insoluble, deposit in the tissues, and become sites of inflammation.
  • If dying cells are not eliminated by non-inflammatory CP activity, they may serve as sources of altered self-antigens capable of generating autoantibodies.
  • C2 deficiency is the most prevalent complement deficiency, with prevalence estimates ranging from 1 in 10,000 to 1 in 20,000 for homozygous C2-deficient individuals.
  • SLE patients had a slightly greater prevalence of C2 deficiency than healthy controls.
  • C2 deficiency is observed in young children with primary immunodeficiency and recurrent infections, mainly upper respiratory infections with Streptococcus pneumoniae or related pathogens.
  • These youngsters frequently suffer from ear infections and colds. Hereditary angioedema (HAE) is a condition caused by a C1-Inh deficiency. Generally, symptoms begin around puberty, but they might begin sooner.
  • These individuals experience recurring swelling of the limbs, face, lips, throat, or gastrointestinal tract. Patients report a feeling of fullness but no discomfort or itching in the affected location, with the exception of individuals with abdominal swellings, who frequently experience severe abdominal pain.
  • The latter two manifestations are the most worrisome because asphyxia can occur if the airways are constricted, and acute abdominal swelling causes severe discomfort that frequently necessitates exploratory surgery.
  • Not the complement enzymes but the kinin-generating pathway is responsible for the creation of the edoema.
  • Changes in tissue permeability are brought about by the synthesis of Bradykinin via this route.
  • Acute treatments include the replacement therapy C1 inhibitor (both plasma-derived and recombinant medicines are available), the kallikrein inhibitor ecallantide, and the bradykinin-2 receptor antagonist icatibant. Included in preventative therapy are attenuated androgens and a C1 inhibitor.


  • Textbook of Microbiology & Immunology by Subhash Chandra Parija 
  • Holsbach Beltrame, Marcia & Catarino, Sandra & Goeldner, Isabela & Boldt, Angelica & Reason, Iara. (2014). The Lectin Pathway of Complement and Rheumatic Heart Disease. Frontiers in pediatrics. 2. 148. 10.3389/fped.2014.00148. 
  • Brown, J. S., Hussell, T., Gilliland, S. M., Holden, D. W., Paton, J. C., Ehrenstein, M. R., … Botto, M. (2002). The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice. Proceedings of the National Academy of Sciences, 99(26), 16969–16974. doi:10.1073/pnas.012669199 
  • https://www.creative-diagnostics.com/complement-system.htm
  • https://www.svarlifescience.com/knowledge/focus-areas/complement-system-overview/classical-pathway
  • https://www.creative-diagnostics.com/complement-system.htm
  • https://www.creative-biolabs.com/complement-therapeutics/classical-pathway.htm
  • https://aklectures.com/lecture/immunology/classical-pathway-of-complement-system
  • https://en.wikipedia.org/wiki/Classical_complement_pathway
  • https://primaryimmune.org/about-primary-immunodeficiencies/specific-disease-types/complement-deficiencies


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