Complement system is an important part of immune system that helps in the defense of body against invading microorganisms. It is present in the blood and tissue fluid in inactive form. It contains more than 30 plasma proteins which are activated during infection or immune reaction.

These proteins are mostly present as inactive proenzymes. When a suitable stimulus comes, they are activated by a proteolytic cascade. In this process, one protein cleaves and activates another protein, and the reaction continues in a sequential manner. This gives rapid amplification of immune response.

The activation of complement system occurs mainly by three pathways. These are classical pathway, alternative pathway, and lectin pathway. The classical pathway is generally started by antigen-antibody complex. The alternative pathway is started directly on microbial surface. The lectin pathway is started when lectin binds with carbohydrate present on the pathogen surface.

All the three pathways finally meet at the cleavage of C3 protein. C3 is the central complement component. It is cleaved into C3a and C3b. C3b helps in opsonization of pathogen, while C3a helps in inflammatory response.

The activated complement system performs three major functions. It causes opsonization, where microbes are coated for easy phagocytosis. It also produces inflammation by attracting immune cells at the site of infection. It can also cause direct lysis of pathogen by formation of membrane attack complex (MAC).

Membrane attack complex (MAC) forms pores on the surface membrane of pathogen. Due to this pore formation, water and ions enter into the pathogen cell. Finally the pathogen cell swell, rupture and die. Thus, complement system acts as a bridge between innate immunity and adaptive immunity.

Complement Activation Pathway

Complement activation pathway is the process by which complement proteins are activated in a sequential manner. It is used to detect microbes, immune complexes and foreign surface in the body. The pathway finally produces immune reactions that help in removal of pathogens.

The complement system is activated by three main pathways. These are classical pathway, lectin pathway, and alternative pathway. These pathways are started by different substances, but finally all of them meet at the formation of C3 convertase.

Classical pathway is mainly started when C1q binds with antigen-antibody complex. The antibody may be IgG or IgM. Sometimes C1q can also bind directly with pathogen surface. This pathway connects adaptive immunity with innate immunity.

Lectin pathway is independent of antibody. It is started when mannose-binding lectin (MBL) or ficolins bind with carbohydrate molecules present on microbial surface. These carbohydrate molecules are commonly present on bacteria, fungi and some viruses. This binding activates the next complement proteins in a cascade form.

Alternative pathway works as a continuous surveillance system in blood. It starts by spontaneous hydrolysis of C3 protein. If the activated C3 fragment binds to microbial surface and host protective proteins are absent, then the pathway becomes active. It also makes an amplification loop, so more C3 is cleaved.

All the three pathways finally form C3 convertase. This enzyme complex cleaves C3 into C3a and C3b. C3b attaches to the pathogen surface and helps in opsonization. C3a helps in inflammation and recruitment of immune cells.

After this, C5 convertase is formed and it cleaves C5 into C5a and C5b. C5a is a strong inflammatory molecule. C5b starts the formation of membrane attack complex (MAC) by joining with other complement proteins.

Membrane attack complex (MAC) makes pores in the membrane of invading cells. Due to pore formation, water and ions enter into the cell. Finally the microbial cell swell, burst and die. Thus, complement activation pathway helps in opsonization, inflammation and direct cytolysis of pathogens.

What is Classical Pathway?

Classical pathway of complement activation is a pathway of complement system which is started by binding of C1q with antigen-antibody complex. The antibodies mainly involved are IgG and IgM. It finally forms C3 convertase and helps in opsonization, inflammation and lysis of pathogen.

Classical pathway of complement activation is one of the important pathway of complement system. It mainly starts after the formation of antigen-antibody complex on the surface of pathogen. This pathway helps to connect adaptive immune response with innate immune response.

The classical pathway is started by C1 complex. This complex is made up of C1q, C1r, and C1s. The C1q part binds with antibody attached to antigen on microbial surface. The antibodies which mainly take part in this pathway are IgG and IgM.

This pathway may also be started without antibody in some cases. It can be activated by C-reactive protein (CRP), viral proteins and apoptotic cells. These substances act as danger signal and are recognized by the complement proteins.

After binding of C1q, a structural change occurs in the C1 complex. Due to this change, C1r becomes activated by autoactivation. Then activated C1r cleaves and activates C1s. The activated C1s acts as an enzyme for the next complement proteins.

In this step, C1s cleaves C4 into C4a and C4b. The C4b fragment attaches to the pathogen surface. Then C2 is cleaved into C2a and C2b. The larger fragment C2a joins with C4b and forms C4b2a, which is called classical C3 convertase.

The C3 convertase cleaves large number of C3 protein into C3a and C3b. C3b binds on the microbial surface and causes opsonization. This helps the phagocytic cells to engulf the pathogen more easily. C3a helps in inflammatory reaction at the site of infection.

After this, C3b joins with C3 convertase and forms C5 convertase. It cleaves C5 into C5a and C5b. C5a is important for inflammation and chemotaxis. C5b starts the formation of membrane attack complex (MAC).

Membrane attack complex (MAC) forms pore on the surface membrane of target cell. Due to pore formation, the cell loses its normal membrane balance. Water and ions enter into the cell and finally the target cell swell, rupture and die. Thus classical pathway helps in opsonization, inflammation and direct lysis of pathogen.

Complement was first discovered as a heat-labile substance present in normal plasma.

It was observed that this substance helps the antibodies in killing of bacteria.

It also increased the opsonization of bacteria by antibodies, so bacteria can be easily removed by phagocytic cells.

This activity was called complementing the antibacterial activity of antibodies.

From this complementing activity, the name complement system was given.

The classical pathway was the first activation pathway of complement system which was completely defined.

Later another pathway was discovered, which was called alternative pathway.

It was named alternative pathway because it was found after the classical pathway was already known.

Importance of the Classical Pathway

• Bridging immune systems – Classical pathway is an important pathway which connect innate immunity with adaptive humoral immune response.
• Pathogen opsonization and neutralization – It coats bacteria, viruses and fungi with complement proteins so that these pathogens are easily recognized and engulfed by phagocytic cells.
• Direct cell lysis – It forms membrane attack complex (MAC) on the pathogen membrane. The MAC makes pores and causes rupture of the cell.
• Inflammatory response – It produces inflammatory reaction at the site of infection or injury. It attracts immune cells and increases blood vessel permeability.
• Clearance of immune complexes and debris – It removes antigen-antibody complexes, apoptotic cells and cell debris from the tissue. This prevents harmful accumulation and tissue damage.
• Tissue remodeling and homeostasis – It also has role in normal body processes like wound healing, angiogenesis and pruning of inactive synapses in central nervous system.

Components of the Classical Pathway

• C1 complex – C1 complex is the first component of classical pathway. It starts the pathway after binding with antigen-antibody complex or other danger surface. It is made up of three subcomponents.
  • C1q – It is the recognition part. It binds with IgG or IgM present on antigen surface. It can also bind with pathogen surface and danger signals.
  • C1r – It is a serine protease. After binding of C1q, C1r becomes activated by itself. This is called autoactivation.
  • C1s – It is also a serine protease. It is activated by C1r and then cleaves C4 and C2.
• Complement component 4 (C4) – C4 is cleaved by activated C1s into C4a and C4b. C4a is a small inflammatory peptide. C4b is the larger fragment which attach on the target surface.
• Complement component 2 (C2) – C2 is cleaved by activated C1s and forms active enzymatic fragment. It is traditionally called C2a, but in modern name it may be called C2b.
• Classical C3 convertase – It is formed by joining of surface bound C4b with active C2 fragment. It is written as C4b2a or C4b2b. It cleaves C3 protein.
• Complement component 3 (C3) – C3 is the central and most abundant complement protein. It is cleaved by C3 convertase into C3a and C3b. C3a causes inflammatory response and C3b works as opsonin.
• Classical C5 convertase – It is formed when C3b binds with the already formed C3 convertase. It is written as C4b2a3b or C4b2b3b. It cleaves C5.
• Complement component 5 (C5) – C5 is cleaved by C5 convertase into C5a and C5b. C5a is a strong chemoattractant. C5b starts the terminal cell lysis pathway.
• Terminal components – These are late acting complement proteins which form membrane attack complex (MAC).
  • C6, C7, C8 and C9 – These proteins bind one after another with C5b. They form MAC, which makes pore in pathogen membrane and causes rupture of cell.
• Classical pathway regulators – These proteins control the pathway and protect normal host tissue from damage.
  • C1 inhibitor (C1-INH) – It inactivates C1r and C1s.
  • C4b-binding protein (C4BP) – It interferes with the formation of C3 convertase.

Step by Step Detail Process of Classical Pathway

Step 1- Initiation and binding

In the first step, C1q binds with the target surface. C1q is the recognition molecule of C1 complex.

The target is mostly antigen-antibody complex present on pathogen surface. The antibodies are mainly IgG and IgM. Sometimes C1q can also bind directly with non-antibody danger signals present on microbial surface.

Step 2- Activation of C1 complex

After binding of C1q, a conformational change occurs in the C1 complex. This change activates the enzyme part of the complex.

First C1r becomes activated by autoactivation. In some condition, nearby C1 complexes also activate each other by cross-cleavage. Then activated C1r cleaves and activates C1s.

Step 3- Cleavage of C4

The activated C1s now acts on C4 protein. It cleaves C4 into two parts.

The smaller part is C4a and the larger part is C4b. C4a is released and acts as inflammatory peptide. C4b exposes a reactive thioester bond.

Due to this thioester bond, C4b attaches covalently with pathogen surface. So C4b remains fixed on the target surface.

Step 4- Cleavage of C2 and formation of C3 convertase

After C4b attaches on the surface, C2 binds with C4b. This brings C2 close to the activated C1s enzyme.

Then C1s cleaves bound C2 into two fragments. The smaller fragment is released and the larger active fragment remains attached with C4b.

The active fragment is called C2b in modern name, but old name is C2a. The complex C4b2b or C4b2a is formed. This is called classical C3 convertase.

Step 5- Cleavage of C3

The classical C3 convertase now cleaves C3 protein. C3 is present in large amount in plasma.

It is cleaved into C3a and C3b. C3a acts as inflammatory mediator. C3b attaches on pathogen surface and acts as opsonin.

Due to C3b coating, the pathogen becomes easy for recognition by phagocytic cells. This step also produces amplification of complement reaction.

Step 6- Formation of C5 convertase

Some newly formed C3b binds with already formed C3 convertase. This changes the activity of enzyme complex.

Now the complex becomes C5 convertase. It is written as C4b2b3b or old name C4b2a3b. This enzyme acts on C5 protein.

Step 7- Cleavage of C5

The C5 convertase cleaves C5 into two fragments. These are C5a and C5b.

C5a is a strong chemoattractant and inflammatory molecule. It attracts immune cells at the site of infection. C5b starts the terminal cell lysis pathway.

Step 8- Formation of membrane attack complex (MAC)

The newly formed C5b binds one by one with C6, C7 and C8. This complex enters into the membrane of target cell.

After this, many C9 molecules come and polymerize around the complex. These C9 molecules form a tube like pore. This complete structure is called membrane attack complex (MAC).

Step 9- Target cell lysis

The MAC makes pore in the lipid bilayer of pathogen membrane. Due to this pore, the membrane integrity is lost.

Water and solutes enter into the target cell. The cell becomes swollen and finally rupture. Thus the pathogen cell die by complement mediated lysis.

Initiation of the Classical Pathway

Step 1- Target recognition and binding

In the first step, C1q recognizes and binds with the target surface. C1q is the recognition part of C1 complex. It mostly binds with the Fc region of antibody present in antigen-antibody complex.

The antibodies involved are mainly IgG and IgM. This is called antibody-dependent activation. C1q may also bind directly with C-reactive protein (CRP), apoptotic cells, viral proteins and bacterial surface. This is called antibody-independent activation.

For stable binding, at least two globular heads of C1q must attach with the target surface. This makes the binding strong and proper activation can start.

Step 2- Conformational change

After binding of C1q with the target, a structural change occurs in the C1q molecule. This change is called conformational change.

This change is important because it passes the activation signal to the other parts of C1 complex. So the inactive enzyme parts become ready for activation.

Step 3- Activation of C1r

In this step, the conformational change is transferred to C1r present in the C1 complex. C1r is present as inactive zymogen. After the change, its active site becomes exposed.

Then C1r becomes activated. In some condition, many C1 complexes present close to each other can activate one another. The C1r of one complex may cleave the C1r of another neighbouring complex. This is referred to as intermolecular cross-cleavage.

Step 4- Activation of C1s

After activation of C1r, it cleaves the nearby C1s molecule. C1s is also present in inactive zymogen form.

After cleavage, C1s becomes active serine protease. Now the C1 complex is enzymatically active and can act on the next complement components.

Step 5- Cascade propagation

In this step, active C1s cleaves the downstream complement proteins. These are C4 and C2.

The cleavage products of C4 and C2 later join on the target surface and form classical C3 convertase. After this, the complement cascade continues and more complement proteins are activated. Thus initiation phase of classical pathway is completed.

Activation of C1 Complex and Sequential Cleavage of C4 and C2

Step 1- Target binding

In the first step, C1q binds with the target surface. C1q is the recognition part of C1 complex. The target may be antigen-antibody complex present on pathogen surface.

It can also bind directly with pathogen surface in some condition. After this binding, the classical pathway starts.

Step 2- Conformational change

After attachment of globular heads of C1q with the target, a structural change occurs in C1q molecule. This is called conformational change.

This change is necessary because it sends activation signal to the enzyme part of C1 complex.

Step 3- Activation of C1r

The structural change is transferred to C1r proteases. C1r is present in inactive zymogen form.

After receiving the signal, C1r undergo auto-cleavage and become activated. In some condition, closely placed C1 complexes can activate each other by intermolecular cross-cleavage.

Step 4- Activation of C1s

Activated C1r cleaves C1s zymogen. After cleavage, C1s becomes a fully active serine protease.

At this stage, the C1 complex becomes active. Now it can cleave the next complement components.

Step 5- Cleavage of C4

The active C1s first acts on C4 protein present in plasma. It cleaves C4 into two fragments.

The smaller fragment is C4a and the larger fragment is C4b. C4a is released and it has inflammatory role.

Step 6- Surface anchoring of C4b

After cleavage, C4b gets a reactive thioester bond. Due to this, C4b can quickly attach with the target surface.

It forms covalent bond with pathogen surface. So C4b remains fixed on the surface and helps in next step.

Step 7- Recruitment of C2

The surface bound C4b now works as a scaffold. It binds with C2 protein.

This binding brings C2 close to active C1s. So C2 can be cleaved in proper position.

Step 8- Cleavage of C2 and convertase assembly

The active C1s cleaves the bound C2 into two fragments. One smaller fragment is released away.

The larger active fragment remains attached with C4b. This fragment is historically called C2a, but in modern name it may be called C2b.

Finally C4b and active C2 fragment form classical C3 convertase. It is written as C4b2a or C4b2b. This enzyme then cleaves C3 and continues the complement cascade.

Formation of C3 Convertase and C5 Convertase

Formation of C3 Convertase in Classical and Lectin Pathway

Step 1- Cleavage of C4

In the first step, activated serine protease cleaves C4 protein. In classical pathway, this enzyme is C1s. In lectin pathway, this enzyme is MASP-2.

C4 is cleaved into two fragments. These are C4a and C4b. C4a is smaller fragment and C4b is the larger fragment.

Step 2- Surface attachment of C4b

After cleavage, C4b exposes a reactive thioester bond. Due to this bond, C4b can attach with microbial surface.

It forms covalent bond with the pathogen surface. So C4b remains fixed on the surface and acts as base for next component.

Step 3- Binding and cleavage of C2

The surface attached C4b binds with C2 protein. After binding, C2 comes near to the active enzyme.

Then C1s or MASP-2 cleaves C2 into two fragments. These fragments are C2a and C2b.

Step 4- Formation of C3 convertase

The larger active fragment of C2 remains attached with C4b. This fragment is now called C2b, but in old naming it is called C2a.

Together C4b and active C2 fragment forms C3 convertase. It is written as C4b2b or old name C4b2a. This enzyme cleaves C3 protein.

Formation of C3 Convertase in Alternative Pathway

Step 1- Generation of C3b

In alternative pathway, C3 undergoes spontaneous hydrolysis in blood. It may also be cleaved by other enzymes.

This produces C3b fragment. The C3b attaches on microbial surface by covalent bond.

Step 2- Binding of Factor B

After attachment of C3b, Factor B binds with it. Factor B is a plasma protein.

This binding forms a complex on the microbial surface. Now it becomes ready for cleavage by Factor D.

Step 3- Cleavage of Factor B

Factor D is a plasma serine protease. It acts on the bound Factor B.

It cleaves Factor B into Ba and Bb. Ba is smaller fragment and it is released. Bb is larger active fragment and remains attached with C3b.

Step 4- Formation of alternative C3 convertase

The attached Bb joins with C3b and forms C3bBb complex. This is called alternative pathway C3 convertase.

This complex is not very stable alone. Properdin helps to stabilize this complex on the microbial surface.

Formation of C5 Convertase in All Pathways

Step 1- Cleavage of C3

After formation, C3 convertase acts on C3 protein. In classical and lectin pathway, the C3 convertase is C4b2b or C4b2a.

In alternative pathway, the C3 convertase is C3bBb. These enzymes cleave C3 into C3a and C3b. C3a is released and C3b attaches on the surface.

Step 2- Binding of C3b with C3 convertase

For the next step, newly formed C3b binds with the already present C3 convertase. This binding changes the nature of the enzyme complex.

Now the enzyme does not mainly act on C3. It becomes able to cleave C5 protein.

Step 3- Formation of C5 convertase

In classical and lectin pathway, C3b joins with C4b2b and forms C4b2b3b. In old naming, it may be written as C4b2a3b. This is the classical and lectin pathway C5 convertase.

In alternative pathway, C3b joins with C3bBb and forms C3bBbC3b. This is the alternative pathway C5 convertase.

The C5 convertase cleaves C5 into C5a and C5b. C5a helps in inflammation and chemotaxis. C5b starts the terminal pathway and formation of membrane attack complex (MAC).

Formation of Membrane Attack Complex (MAC)

Step 1- Cleavage of C5

In the first step, C5 convertase cleaves C5 protein. The C5 convertase may come from classical pathway, lectin pathway or alternative pathway.

C5 is cleaved into two fragments. These are C5a and C5b. C5a acts as strong inflammatory mediator and C5b starts the terminal pathway.

Step 2- Formation of C5b67 complex

The newly formed C5b first binds with C6. After this, C7 also binds with it.

Together these proteins form C5b67 complex. This complex is important for attachment with the membrane of target cell.

Step 3- Membrane insertion

After binding of C7, a conformational change occurs in the complex. Due to this change, hydrophobic site of C7 becomes exposed.

This exposed hydrophobic site allows C5b67 complex to insert into the lipid bilayer of pathogen membrane. Now the complex becomes fixed in the target membrane.

Step 4- Binding and insertion of C8

The membrane attached C5b67 works as a receptor for C8 protein. C8 has C8β and C8α-γ subunits.

The C8β part binds with C5b. Then hydrophobic region of C8α-γ enters into the lipid bilayer. This makes the membrane damage more strong.

Step 5- Polymerization of C9

After insertion of C8, the C8α-γ part helps in binding of C9 molecules. Around 10 to 16 molecules of C9 come and join together.

These C9 molecules polymerize and form a tubular channel in the membrane. This step completes the pore forming structure.

Step 6- Pore formation and cell lysis

The polymerized C9 forms complete membrane attack complex (MAC). It makes a pore of about 10 nm or 100 Å in the target cell membrane.

Due to this pore, water and solutes move freely through the membrane. The lipid bilayer and proton gradient are disturbed. Finally the pathogen cell swells, rupture and die by osmotic lysis.

Role of Antigen-Antibody Complex in Classical Pathway Activation

• Primary initiators – Antigen-antibody complex is the main substance which starts the classical complement pathway. It is formed when antibody binds with antigen present on pathogen surface. The antibodies mainly involved are IgM and all subclasses of IgG, except IgG4.
• Exposure of binding sites – When IgM or IgG bind with antigen, the antibody molecule undergo some conformational change. Due to this change, specific binding sites become exposed on the Fc region of the antibody.
• Attachment of C1q – C1q is the recognition part of C1 complex. It uses its globular heads to bind with the exposed Fc region. In IgG, C1q binds with CH2 domain. In pentameric IgM, it binds with CH4 domain.
• Requirement for multiple connections – For stable activation, C1q must bind with at least two antibody molecules. IgM is pentameric in structure, so it gives many binding sites at one time. For this reason IgM is a more powerful activator than IgG.
• Conformational shift – After attachment of C1q with antigen-antibody complex, a structural change occurs in the C1q molecule itself. This change is important for activating the other parts of C1 complex.
• Triggering the cascade – The structural change is passed to C1r proteases. The active sites of C1r become exposed and C1r activate itself. Then activated C1r cleaves and activates C1s. After this C1s starts the cleavage of C4 and C2, and the classical complement cascade begins.

Biological Functions of the Classical Pathway

• Opsonization – Classical pathway helps in coating of bacteria, viruses and fungi by complement proteins. This makes the pathogen easy for recognition by phagocytic cells and then they are engulfed and destroyed.
• Neutralization of pathogens – It binds with pathogen and make them inactive. Due to this, pathogen cannot attach properly with host cell and spreading is reduced.
• Cytolysis – It forms membrane attack complex (MAC) on the target membrane. The MAC makes pore in the membrane and finally the cell rupture and die.
• Inflammatory response – During this pathway, inflammatory molecules are produced. C5a is one important anaphylatoxin. It attracts immune cells at infection site and also causes vasodilation and more vascular permeability.
• Clearance of immune complexes and apoptotic cells – It removes antigen-antibody complexes and apoptotic cells from tissue. This removal is important because their accumulation may cause tissue damage and autoimmune reaction.
• Synaptic pruning – In the central nervous system, this pathway tags inactive and extra neuronal synapses. These synapses are removed by microglia during development.
• Tissue remodeling and angiogenesis – It also takes part in normal tissue remodeling and wound healing. It helps in endothelial cell migration and formation of new capillary vessels.
• Macrophage tolerance induction – C1q interact with macrophages and reduce the release of pro-inflammatory cytokines. This helps to make anti-inflammatory and tolerogenic condition.

Regulation of the Classical Pathway

• C1 inhibitor (C1-INH) – C1-INH is a plasma protein which controls the first step of classical pathway. It binds with C1r and C1s and make them inactive. Due to this, C1r and C1s are removed from C1q and initiation of pathway is stopped.
• C4b-binding protein (C4BP) – C4BP is an important fluid phase regulator. It prevents binding of C2 with C4b and so formation of C3 convertase is reduced. It also helps in decay of convertase and works as cofactor for Factor I.
• Complement factor I (CFI) – CFI is a serine protease which cleaves C4b and C3b into inactive fragments. It forms inactive products like C4d and iC3b. Thus these fragments cannot form new convertase. But CFI needs cofactor proteins for its activity.
• Decay-accelerating factor (DAF or CD55) – DAF is present on host cell membrane. It protects host cell by increasing breakdown of C3 convertase and C5 convertase. It removes active catalytic subunits from the convertase complex.
• Membrane cofactor protein (MCP or CD46) – MCP is present on all nucleated host cells. It acts as local cofactor for Factor I. It helps Factor I to cleave C4b and C3b which are accidentally deposited on normal host tissue.
• Complement receptor 1 (CR1 or CD35) – CR1 is present on immune cells and red blood cells. It increases decay of convertase and also acts as cofactor for Factor I. It also helps in safe removal of immune complexes from blood.
• CD59 (Protectin) – CD59 is present on most host cell membrane. It controls the terminal stage of complement pathway. It binds with C8 and C9 and prevents their polymerization. Due to this membrane attack complex (MAC) is not formed on healthy host cells.

Difference Between Classical, Lectin, and Alternative Pathways

Inhibitors of Classical Pathway

• Natural host regulatory proteins – These inhibitors are present in the body and protect normal host tissue from complement damage.
  • C1 inhibitor (C1-INH) – C1-INH is a plasma serine proteinase inhibitor. It binds with C1r and C1s and makes them inactive. Due to this, C1r and C1s are separated from C1q and pathway is stopped at first step.
  • C4b-binding protein (C4BP) – C4BP is a fluid phase regulator. It binds with C4b and blocks the binding of C2. It also increases decay of classical C3 convertase and works as cofactor for Factor I.
  • Complement factor I (CFI) – CFI is a plasma serine protease. It cleaves C4b with the help of cofactor protein. It forms inactive fragments like C4c and C4d, so C4b cannot form convertase.
  • Decay-accelerating factor (DAF or CD55) – DAF is a membrane bound protein. It protects host cell by breaking the C3 convertase and C5 convertase complex. It removes catalytic subunit from the convertase.
  • Membrane cofactor protein (MCP or CD46) – MCP is present on all nucleated host cells. It works as cofactor for Factor I. It helps in cleavage of C4b which are accidentally attached on normal host tissue.
  • Complement receptor 1 (CR1 or CD35) – CR1 is found on immune cells and red blood cells. It has decay accelerating activity and cofactor activity for Factor I. It also helps in control of convertase formation.
• Therapeutic inhibitors – These inhibitors are prepared for treatment of diseases where classical pathway is over activated.
  • Sutimlimab (Enjaymo) – Sutimlimab is a humanized monoclonal antibody. It binds with C1s serine protease and inhibits it. It is used in adults with cold agglutinin disease (CAD) to stop C1 activated hemolysis.
  • TNT003 and TNT009 (BIVV009) – These are monoclonal antibodies against C1s. They inhibit complement activation and reduce microvascular inflammation and C3d deposition. They may be used in autoimmune condition and transplant rejection.
  • Anti-C1q monoclonal antibodies – These antibodies are developed against C1q. They block complement dependent cytotoxicity. They may be useful in neuroinflammatory and autoimmune diseases like neuromyelitis optica.
  • Macrocyclic peptide cL3 – cL3 is an engineered peptide. It binds with globular domains of C1q. It acts as competitive inhibitor and prevents C1q binding with antibodies.
• Pathogen-derived evasion inhibitors – These molecules are produced by pathogens. They block classical pathway and helps pathogen to survive inside the host.
  • BBK32 – BBK32 is produced by Borrelia burgdorferi. It binds directly with C1r and blocks its proteolytic active site. Due to this enzymatic amplification is stopped.
  • TcCRT – TcCRT is produced by Trypanosoma cruzi. It competes with C1q for binding to C1r/C1s tetramer. It also disturbs the enzymatic activity of C1s.
  • CIP and Eap – CIP is secreted by Streptococcus agalactiae and Eap is secreted by Staphylococcus aureus. These proteins bind with C4b and inhibit the formation of classical proconvertase.
  • CNA-like MSCRAMMs and HAstV-1 coat protein – CNA-like MSCRAMMs are found in S. aureus and HAstV-1 coat protein is found in human astrovirus. They bind with C1q and remove C1r/C1s tetramer from C1q.

Deficiencies and Disorders Associated with the Classical Pathway

• Systemic lupus erythematosus (SLE) – Deficiency of early classical pathway proteins like C1q, C1r, C1s, C4 and C2 are strongly related with early onset SLE. In this condition apoptotic cells and immune complexes are not cleared properly. These materials accumulate in the body and starts autoimmune response.
• Periodontal Ehlers-Danlos syndrome (pEDS) – pEDS is a rare genetic disorder. It shows early onset periodontitis, joint hyperlaxity, dermal hyperelasticity and tissue fragility. It is caused by mutation in C1R and C1S genes, which disturb normal control of C1 complex and causes continuous activation.
• Hereditary and acquired angioedema – This disorder occurs due to deficiency or dysfunction of C1 inhibitor (C1-INH). Due to lack of proper control, C1r and C1s remain active. This causes excess formation of vasoactive peptides like bradykinin and C2 kinin, producing repeated swelling of tissues. Swelling of trachea may be life threatening.
• Cold agglutinin disease (CAD) – CAD is a chronic autoimmune hemolytic anemia. In this disease, IgM autoantibodies bind with red blood cells at low temperature. Since IgM is strong activator of classical pathway, it causes continuous complement mediated hemolysis. This leads to severe anemia, fatigue and risk of thromboembolic events.
• Increased susceptibility to infections – Deficiency of classical pathway components like C1s, C4 or C2 reduces opsonization and formation of C3 convertase. Due to this, pathogens are not removed properly. The patient becomes more susceptible to repeated bacterial and viral infections, specially by Neisseria species.
• Rheumatoid arthritis (RA) – Abnormal activation of classical pathway is related with joint inflammation in RA. Activated C1s may be present in degraded cartilage matrix. This shows that classical pathway can take part in local destruction of cartilage and bone.
• Neuromyelitis optica spectrum disorder (NMOSD) – NMOSD is a severe neuroinflammatory disease. It causes inflammation and demyelination in central nervous system. Autoantibodies against AQP4 bind with astrocytes and activate classical complement pathway. This causes complement dependent cytotoxicity and tissue damage.
• Cancer progression – In some cancers, local production of classical pathway proteins like C1q and C1s may help tumor progression. In clear cell renal cell carcinoma and gliomas, C1q interaction and C1s activation helps tumor cell adhesion, migration, angiogenesis and immunosuppressive environment. This is related with poor clinical prognosis.

Clinical Significance of Classical Pathway

• Diagnostic biomarker in transplantation – C4d is a stable cleavage product of C4. Its deposition shows active classical pathway activation. It is used as important clinical marker for diagnosis of antibody-mediated renal and cardiac graft rejection.
• Target for autoimmune and inflammatory therapeutics – Classical pathway components are used as target for treatment of autoimmune and inflammatory diseases. Sutimlimab (Enjaymo) is a monoclonal antibody which targets C1s and stops classical complement mediated hemolysis in cold agglutinin disease (CAD). Other antibodies are also developed against C1s in bullous pemphigoid and against C1q in neuromyelitis optica (NMO).
• Role in cancer prognosis and treatment – In some cancers, local production of C1q and C1s may help tumor progression. In clear cell renal cell carcinoma, activation of C1s is related with poor clinical prognosis. But in cancer treatment, this pathway can also be used to destroy tumor cells by recruiting C1q and activating complement cascade.
• Development of infectious immunotherapies – Classical pathway is used for development of targeted infectious immunotherapy. Some synthetic peptides can produce specific IgG response and then activate classical complement pathway to detect and kill HIV infected cells. Some IgM variants also activate this pathway and help in destruction of methicillin-resistant Staphylococcus aureus (MRSA).
• Indicator of immune deficiency and disease risk – Deficiency of C1q, C2 or C1 inhibitor has important clinical effect. These deficiencies reduce clearance of immune complexes and apoptotic cells. This may cause systemic lupus erythematosus (SLE), recurrent Neisseria infection and angioedema which may become fatal.
• Link with obesity and metabolic disorders – Classical pathway also takes part in immune inflammation inside adipose tissue. In obesity, production of C1 complex becomes high. This causes chronic tissue inflammation and systemic insulin resistance.

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