The Complement System and Kinin System

1. The Complement Systemand Kinin System Manfred Maitz www.manfred.maitz-online.de

2. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

3. Background • Evolutionary old system • Non specific defence system • Constitutive • Very fast • Selectivity and self-tolerance • Activation by antibodies possible • Interaction with the specific immune system • Functions • Lysis of cells/ bacteria and viruses • Opsonization for phagocytes • Immune Clearance • Similarities and crosstalks with the clotting cascade • Cascade of serine proteases, which activate each other • Two (three) pathways of activation • Factor XIIa (Hageman-Factor) activates both the clotting cascade and the complement cascade

4. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

5. C5a 70-100 Å C5b The Membrane Attack Complex C5 C9 C9 C9 C9 C9 C9 C9 C9 C9 C9 C9 C7 C8 C6

6. The Alternative Pathway C3 P C3a Properidin Ba B C3b C3bB C3bBb C3bBbP C3bBb3b D B C3a C3iBb C3iB C3i C3 Ba The Tick-over Activation C3 The Classical Pathway C5b Antibody + C1qrs C4 C4b C4b2a C4b2a3b C2 C3 C2b C3a C4a The Complement Cascade C5a C5

7. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

8. Regulation of the Complement Cascade • Short half-time of • C3b • C3bBb • C5b • C1 inhibitor • Inhibits the C1s activity • Protein S in Serum • Binds to C5b67 • Inhibits Formation of the Membrane Attack Complex • HRF or CD59 • Bind to C8 • Inhibits C9 binding • Factor H • Binds to C3b • Facilitates binding of Factor I • cleaves C3b to inactive iC3b • cleaves C4b to inactive fragments • Decay Accelerating Factor • Increased dissociation of C3 convertase (both pathways)

9. Complement Activation General • Hydrophobic surfaces • Oxides • Strong binding of C3(b) to nucleophilic groups (-NH2, -OH) • Higher absorption of C3 to crystalline TiO2 than to amorphous • Kallikrein directly activates C5 • Plasmin directly activates C5 Classical Pathway • Antibodies IgM, IgG1, IgG2, IgG3 • Lectin via the mannan binding protein (MBP) “Lectin Pathway” • Hageman Factor (F XIIa) • Rough surfaces • C-reactive protein (CRP) • (Zirkonium, transiently) Alternative Pathway • PE debries • Acetylated chitosan

10. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

11. Complement Receptors

12. Anaphylatoxins Fragments C5a, C3a, C4a • Degranulation of Phagocytes • Reactive oxygen species • Prostaglandins • Monocytes  IL-1, IL-6 • Mast cells Histamine • Chemotaxis • Only C5a

13. Consequences in vitro • Lysis of “innocent” neighbour cells • Red blood cells • Activation of phagocytic cells • Release of reactive oxygen species • Release of mediators

14. Consequences in vivo • Factors of complement activation at revised hip implants • One single study(Tang L. et al.J Biomed Mater Res41: 333-340 (1998)) Au-Mercaptoglycerol induces strong inflammatory response in control animals. No reaction in Complement-depleted animals.

15. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

16. The Alternative Pathway C3 P C3a Properidin Ba B C3b C3bB C3bBb C3bBbP C3bBb3b D B C3a C3iBb C3iB C3i C3 Ba The Tick-over Activation C3 The Classical Pathway C5b Antibody + C1qrs C4 C4b C4b2a C4b2a3b C2 C3 C2b C3a C4a The Complement Cascade C5a C5

17. Methods for Investigation General/Common pathway • Lysis of sheep red blood cells • Solid phase methods (ELISA, RIA) • Products: C3a, C5a, sC5b-9 • Consumption of C3 • Ellipsometry Classical Pathway • Measurement of C1qrs • Measurement of C2b or C4b2a Alternative Pathway • Measurement of Ba or C3bBb • Measurement of Properidin

18. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

19. Biomaterials Consequences • Testing for complement activation included in standard test programs • Covalent binding of Factor H to the surface of a blood contacting implant • Reduced C3a, sC5b-9 • Inhibition of FXII activation by Heparin-ATIII

20. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

21. F Va F V The Clotting Cascade Intrinsic Pathway Extrinsic Pathway Surface Contact Collagen FXII activator Tissue/Cell Defect F XII F XIIa F VIIa F VII Ca2+ F XI F XIa Ca2+ F IX F IXa F III (Tissue Thromboplastin) Ca2+ F VIII F VIIIa Platelet Factor 3 Factor F X Factor F Xa Factor F X Ca2+ Ca2+ Prothrombin I Thrombin Ca2+ F XIIIa F XIII Fibrinpolymers Fibrinmonomers Fibrinogen CrosslinkedFibrin Meshwork

22. Surface Fibrinolytic Pathway Clotting Cascade (intrinsic pathway) F XII F XIIa Plasmin Complement Activation Kallikrein Pre-Kallikrein Plasminogen KininogenHMWK, LMWK Kinine.g. Bradykinin The Kinin System • Effects • vascular smooth muscle relaxation • Increased permeability of blood vessels  Oedema • Pain • Interaction with clotting cascade • Interaction with fibrinolytic cascade • Interaction with complement cascade

23. Outline Complement System • Background • Reactions of the complement cascade • Regulation and activation of the complement cascade • Effects of Complement activation • Evaluation methods • Consequences for biomaterials Kinin System • Reactions and interactions with other systems Summary and Conclusions

24. Conclusions • The blood plasma has four cascade systems • Clotting cascade • Fibrinolytic cascade • Complement system • Kinin system • All four systems are proteolytic cascades • Besides the main streams there are many cross connections with minor importance • Common activators and cross activation • Common inhibitors/ regulators • C1 Esterase Inhibitor • Hageman Factor (F XII) • Effective in all systems • Directly activated by surfaces (esp. negatively charged) • 1-3% people have deficiencies without clinical syndromes • Plasmin • Directly effective in three systems • Indirectly also effective on the clotting cascade

26. General Conclusions Biomaterials research is an interdisciplinary field • Physics/Materials science • Modification and check of physical surface properties • Roughness • Surface free energy • Hardness, tribological properties • Surface charges, electrical and electronic properties • Bulk properties • Chemistry • Corrosion • Polymers • Chemical surface modification • Chemical reactions of the biomolecules with the biomaterial • Life science • Knowledge about the normal biochemical situation • Knowledge about changes at certain diseases • Check of influences due to the biomaterial • Support of the physiological situation