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Blood Bank

Pathway Mechanisms

The complement system is a fascinating and powerful part of our immune system – think of it as a cascade of protein dominos that, once tipped over, leads to some major downstream effects like inflammation, marking targets for destruction, and even directly blowing holes in cells! It’s part of the innate immune system but forms a crucial bridge to the adaptive system (especially via antibodies)

In blood banking, complement activation is super important because it’s a major way that incompatible red blood cells get destroyed, either inside the blood vessels (intravascular hemolysis) or by being flagged for removal by macrophages (extravascular hemolysis). Understanding how it gets activated helps us understand transfusion reactions and interpret our tests (like the DAT!)

There are three main pathways to activate complement, but we’ll focus on the two most relevant for us: the Classical and Alternative pathways. They start differently but eventually merge to create the same powerful effector molecules

The Classical Pathway: Antibody-Driven Activation

  • The Trigger: This pathway is typically initiated when antibodies bind to antigens, forming immune complexes. In blood banking, this usually means:
    • IgM: Just ONE IgM molecule bound to a red cell antigen is enough to kick things off! IgM is a pentamer, and its structure is perfect for binding the first complement protein, C1q. This is why ABO mismatches (involving IgM anti-A/anti-B) can cause such rapid and severe complement activation
    • IgG: It’s less efficient. You generally need two IgG molecules bound close together on the cell surface to effectively bind C1q. Not all IgG subclasses are equal: IgG3 is the best activator, followed by IgG1. IgG2 is very poor, and IgG4 does not activate the classical pathway. This is relevant for antibodies like anti-K (often IgG1) or anti-Jka (can be IgG3)
    • (Less common triggers include C-reactive protein binding to microbes)
  • The Mechanism (The Dominoes Falling)
    1. Initiation (C1 Complex) The process starts with the C1 complex, which consists of C1q (the recognition unit) and two molecules each of C1r and C1s (serine proteases - enzymes that cut other proteins)
      • C1q binds: to the Fc region (the “stem” of the Y) of the bound IgM or adjacent IgG antibodies
      • This binding causes a shape change in C1q, which activates C1r
      • Activated C1r then cleaves and activates C1s. Now C1s is an active enzyme
    2. Activating C4 and C2
      • Activated C1s finds and cleaves C4 into two pieces: C4a (a small fragment that floats away, acting as a weak anaphylatoxin - promoting inflammation) and C4b (a larger fragment)
      • C4b: is chemically unstable and quickly tries to form a covalent bond with the nearby cell surface (e.g., the red blood cell membrane). If it doesn’t bind quickly, it gets inactivated
      • Activated C1s also cleaves C2 into C2a and C2b. (Note: convention has changed; C2a is now considered the larger enzymatic fragment)
      • C2a: binds to the surface-bound C4b
    3. Formation of the C3 Convertase
      • The complex formed by C4b2a is the Classical Pathway C3 Convertase. Its job is to chop up lots of C3 molecules. This is a major amplification point!
    4. Activating C3 (The Central Step)
      • The C4b2a enzyme cleaves C3 into C3a (another anaphylatoxin, more potent than C4a, causing inflammation and attracting phagocytes) and C3b (the larger fragment)
      • C3b: is crucial! Like C4b, it tries to bind covalently to nearby surfaces. It acts as a powerful opsonin, meaning it coats the cell and flags it for destruction by phagocytes (like macrophages in the spleen/liver) that have C3b receptors. This leads to extravascular hemolysis
      • C3b also participates in forming the next enzyme complex
    5. Formation of the C5 Convertase
      • Some of the newly generated C3b binds to the existing C4b2a complex, forming C4b2a3b. This is the Classical Pathway C5 Convertase
    6. Activating C5
      • The C4b2a3b enzyme cleaves C5 into C5a (a very potent anaphylatoxin and chemotactic factor, strongly recruiting inflammatory cells) and C5b (the larger fragment)
      • C5b: is the initiating factor for the final stage – the Membrane Attack Complex (MAC)

The Alternative Pathway: Spontaneous Activation & Amplification

  • The Trigger: This pathway doesn’t rely on antibodies for initiation. It’s triggered by:
    • Spontaneous “Tick-over”: C3 molecules in the plasma are inherently slightly unstable and can spontaneously hydrolyze (react with water) at a low rate, forming C3(H2O). This happens continuously
    • Foreign Surfaces: Certain microbial surfaces (like bacterial LPS or yeast cell walls) or other activating surfaces lack the regulatory proteins that normally protect our own cells
    • (IgA immune complexes can sometimes activate this pathway too)
  • The Mechanism
    1. Initiation (Tick-over)
      • C3(H2O): forms in the plasma
      • This altered C3 can bind a plasma protein called Factor B
      • Another plasma protease, Factor D, which is always active but at low concentration, cleaves the bound Factor B into Ba (released) and Bb
      • This forms the complex C3(H2O)Bb, which is a fluid-phase C3 convertase. It’s not very stable, but it can cleave a few C3 molecules into C3a and C3b
    2. Deposition and Amplification on Surfaces
      • The C3b generated by the tick-over (or by the classical pathway!) can covalently attach to nearby surfaces – including host cells and foreign particles (like bacteria or potentially even red cells under certain conditions)
      • Crucial Difference: Our own cells have regulatory proteins (like Factor H) that quickly bind deposited C3b and allow Factor I to cleave and inactivate it (preventing damage to self). Foreign surfaces often lack these regulators
      • On an activating surface (lacking regulators), the bound C3b can bind Factor B
      • Factor D: again cleaves Factor B into Ba and Bb
      • This forms C3bBb, the surface-bound Alternative Pathway C3 Convertase
    3. Stabilization and Amplification
      • The C3bBb convertase is inherently unstable, but it can be stabilized by another protein called Properdin (Factor P). Properdin binding extends the half-life of the convertase, allowing it to cleave many more C3 molecules
      • This creates a powerful amplification loop each C3bBb complex generates more C3b, which can then form more C3bBb complexes on the activating surface
    4. Formation of the C5 Convertase
      • When an additional C3b molecule binds to the C3bBb complex, it forms C3bBb3b. This is the Alternative Pathway C5 Convertase
    5. Activating C5
      • Just like the classical pathway C5 convertase, C3bBb3b cleaves C5 into C5a (inflammation/chemotaxis) and C5b (initiates MAC)

Convergence and The Terminal Pathway: Membrane Attack Complex (MAC)

  • Convergence: Both the Classical (C4b2a3b) and Alternative (C3bBb3b) pathways generate a C5 convertase. From this point on, the steps are the same
  • Mechanism
    1. Initiation C5b is generated and binds transiently to the target cell surface near the C5 convertase
    2. Assembly
      • C5b sequentially binds C6, then C7. The C5b-7 complex undergoes a conformational change and inserts itself into the lipid bilayer of the target cell membrane
      • C8: then binds to the C5b-7 complex and also inserts into the membrane, creating a small pore
      • C9: molecules (10-16 of them!) are recruited to the site. They bind to the C5b-8 complex and polymerize, inserting through the membrane to form a large, ring-like channel
    3. Lysis This complete structure, C5b-9, is the Membrane Attack Complex (MAC). It forms a stable pore through the cell membrane, disrupting the osmotic balance. Water and ions rush in, causing the cell to swell and burst (lysis)
  • Blood Bank Relevance: MAC formation on red blood cells causes intravascular hemolysis, the rapid destruction of RBCs within the blood vessels. This is characteristic of severe, acute hemolytic transfusion reactions (like ABO incompatibility)

Regulation is Key!

It’s crucial that complement activation is tightly controlled to prevent it from damaging our own healthy tissues. Numerous regulatory proteins exist in plasma and on our cell surfaces (e.g., C1 Inhibitor, Factor H, Factor I, DAF/CD55, Protectin/CD59) that limit the cascade at various steps. Lack of these regulators on foreign surfaces allows complement to target them effectively

Blood Bank Summary Points

  • Classical Pathway: Triggered mainly by IgM and IgG (subclasses matter!) binding to RBC antigens. Key in HTRs, HDFN, AIHA
  • Alternative Pathway: Activated by surfaces lacking regulators; acts as a major amplification loop for C3b deposited by either pathway
  • C3b Deposition: Leads to opsonization and extravascular hemolysis (removal by macrophages)
  • C3d: A stable breakdown product of C3b that remains on the RBC surface. Detected by anti-C3d in AHG reagent (DAT/IAT)
  • MAC Formation (C5b-9): Leads to intravascular hemolysis. Visible hemolysis in test tubes is a sign of complement activation to completion
  • Anaphylatoxins (C3a, C4a, C5a): Contribute to inflammatory symptoms sometimes seen in transfusion reactions

Key Terms

  • Complement System: A group of plasma proteins that act in a cascade to opsonize pathogens, recruit inflammatory cells, and lyse cells
  • Classical Pathway: Complement activation pathway typically initiated by IgM or IgG immune complexes binding to C1q
  • Alternative Pathway: Complement activation pathway initiated by spontaneous C3 hydrolysis (“tick-over”) and amplified on activating surfaces lacking regulators; involves Factors B, D, and Properdin
  • C3 Convertase: Enzyme complex (C4b2a in classical; C3bBb in alternative) that cleaves C3 into C3a and C3b. Central amplification step
  • C5 Convertase: Enzyme complex (C4b2a3b in classical; C3bBb3b in alternative) that cleaves C5 into C5a and C5b, initiating the MAC
  • Membrane Attack Complex (MAC): The terminal complex of complement (C5b-9) that forms a pore in target cell membranes, causing lysis
  • Opsonization: The coating of a particle (e.g., RBC) with molecules (like C3b or IgG) that enhance its phagocytosis
  • Anaphylatoxin: Complement fragments (C3a, C4a, C5a) that cause histamine release from mast cells and other inflammatory effects
  • C3b: Major opsonin and component of C5 convertases; covalently binds to surfaces
  • C3d: Stable breakdown product of C3b that remains bound to cell surfaces; detected by anti-C3d in AHG reagent
  • Factor B, Factor D, Properdin (Factor P): Key protein components unique to the Alternative Pathway activation and regulation
  • Factor H, Factor I, C1 Inhibitor (C1-INH): Key regulatory proteins that control complement activation to prevent damage to host tissues