1. Terry Kotrla, MS, MT(ASCP)BB
Complement
Terry Kotrla, MS, MT(ASCP)BB

2. Introduction
Complement refers to a complex set of 14 distinct serum proteins (nine components) that are involved in two separate pathways of activation.
Components numbered in order of discovery.
Sequence of activation is not in numerical order.
Components circulate in inactive precursor form, develop activity upon activation.
Complement proteins designated by “C” followed by numbers and letters.

3. Two major functions: Promote the inflammatory response.
Alter biological membranes to cause direct cell lysis or enhanced susceptibility to phagocytosis.

4. Complement

5. General Properties of Complement
Primary role is cell lysis.
Activity of complement destroyed by heating sera to 56 C for 30 minutes.
IgM and IgG are the only immunoglobulin capable of activating complement (classical pathway).
Complement activation can be initiated by complex polysaccharides or enzymes (alternative or properdin pathway).
Portions of the complement system contribute to chemotaxis, opsonization, immune adherence, anaphylatoxin formation, virus neutralization, and other physiologic functions

6. Sequential interaction of complement components
Cleavage of components generates a small fragment which is released, and a larger molecule which attaches to cell surface and continues in reaction sequence.
Sequence of activation referred to as a cascade reaction.

7. Three Pathways

8. Classical Pathway – C1: The Recognition Unit
C1 consists of 3 subunits: C1q, C1r, and C1s.
C1q molecule consists of a collagenous region with six globular head groups globe end serves as recognition unit
When antibody binds to antigen, binding sites for the globular head groups of C1q are exposed on the Fc region of the antibody.

9. Classical Pathway – C1: The Recognition Unit
For C1q to initiate the cascade it must attach to at least 2 Fc fragments, requires at least 2 molecules of IgG or one molecule of IgM.
C1q binding causes C1r to enzymatically activate C1s.

10. Classical Pathway – C1: The Recognition Unit

11. The Activation Unit (C4b2a3b)
C1s cleaves C4 into C4a and C4b
C1s cleaves C2 into C2a and C2b
C4b2a (C4b2b in some texts) is enzymatically active and can cleave many molecules of C3 into C3a and C3b.

12. Membrane Attack Unit
In the presence of C5b, molecules of C6, C7, C8 and a variable number of C9 molecules assemble themselves into aggregates.
This molecular complex causes a change in membrane permeability.
Exact cause of lysis unknown, one theory is change in lipid membrane causes exchange of ions and water molecules across membrane.
Cells can lyse without C9 but it’s slower.

13. Membrane Attack Unit

14. Order of Activation in Classical Pathway
C1, C4, C2, C3, C5, C6, C7, C8, C9

16. Alternative Pathway (Properdin Pathway)
Cleavage of C3 and activation of the remainder of the complement cascade occurs independently of antibody.
Triggers for the alternative pathway include
bacterial cell walls
bacterial lipopolysaccharide
fungal cell walls
some virus infected cells
and rabbit erythrocytes

17. Alternative Pathway (Properdin Pathway)
Molecules of C3 undergo cleavage at continuous low level in normal plasma.
At least 4 serum proteins (factor B, factor D, properdin (P), and initiating factor (IF) function in this pathway.
C3b attaches to appropriate site (activating surface) which is actually a protective surface

18. Alternative Pathway (Properdin Pathway)
Action by the 4 serum proteins on C3b proceeds to the C3 activator stage without participation of C1, C4 or C2.
Activation sequence: C3, C5, C6, C7, C8, C9.

19. Lectin Pathway
Activation of the lectin pathway begins when mannan-binding protein (MBP) binds to the mannose groups of microbial carbohydrates.
Two more lectin pathway proteins called MASP1 and MASP2 (equivalent to C1r and C1s of the classical pathway) bind to the MBP
This forms an enzyme similar to C1 of the classical complement pathway that is able to cleave C4 and C2 to form C44bC2a, the C3 convertase capable of enzymatically splitting hundres of molecules of C3 into C3a and C3b

20. Lectin Pathway
The beneficial results are the same as in the classical complement pathway:
Trigger inflammation (C5a>C3a>C4a);
Chemotactically attract phagocytes to the infection site (C5a);
Promoate the attachment of antigens to phagocytes via enhanced attachment or opsonization (C3b>C4b;
Serves as a second signal for the activation of naive B-lymphocytes (C3d);
Cause lysis of gram-negative bacteria and human cells displaying foreign epitopes (MAC).
And remove harmful immune complexes from the body (C3b>C4b).

21. Lectin Pathway

22. Lectin Pathway

23. Regulation of the Complement Cascade
Modulating mechanisms are necessary to regulate complement activation and control production of biologically active split products

24. Regulation of the Complement Cascade
First means of control is extreme lability of activated complement
If activated complement does not combine within milliseconds the activity is lost or decreased.
Active fragments rapidly cleared from the body.

25. Regulation of the Complement Cascade
Second type of control involves specific control proteins
C1 inhibitor blocks activity of C1r and C1s.
Factor I in activator in the presence of certain cofactors inactivates C3b and C4b.
A number of proteins act to control membrane attack unit

26. References http://pathmicro.med.sc.edu/ghaffar/complement.htm