1. Biomaterials ResearchBiomaterials Research - Manfred Maitz The Complement System and Kinin System Manfred Maitz www.manfred.maitz-online.de

2. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz The Membrane Attack Complex C6 C7 C8 C9 C5 C5b C5a 70-100 Å

6. Biomaterials ResearchBiomaterials Research - Manfred Maitz The Complement Cascade C5 C5a Ba Properidin D C3P C3a B The Alternative Pathway C3bC3bBC3bBbC3bBbPC3bBb3b B C3C3iC3iB Ba C3iBb C3 C3a The Tick-over Activation C5b C4 C4a C2 C2b C3 C3a The Classical Pathway Antibody + C1qrs C4bC4b2aC4b2a3b

7. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz Complement Activation General –Hydrophobic surfaces –Oxides –Strong binding of C3(b) to nucleophilic groups (-NH2, -OH) –Higher absorption of C3 to crystalline TiO 2 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz Complement Receptors ReceptorLigandCellular distribution CR1 (CD35)C3b>iC3b C4b B-Cells Phagocytes RBC follicular dendritic cells CR2 (CD21)iC3b C3dg B cells epithelial cells CR3 (CD18/11b)iC3b Zymosan ICAM-1 Phagocytes NK Cells follicular dendritic cells CR4 (CD18/11c)iC3bPhagocytes

12. Biomaterials ResearchBiomaterials Research - Manfred Maitz Anaphylatoxins Fragments C5a, C3a, C4a Degranulation of Phagocytes –Reactive oxygen species –Prostaglandins –Monocytes  IL-1, IL-6 –Mast cells  Histamine Chemotaxis –Only C5a

13. Biomaterials ResearchBiomaterials Research - Manfred Maitz Consequences in vitro Lysis of “innocent” neighbour cells –Red blood cells Activation of phagocytic cells –Release of reactive oxygen species –Release of mediators

14. Biomaterials ResearchBiomaterials Research - Manfred Maitz Consequences in vivo Factors of complement activation at revised hip implants One single study (Tang L. et al. J Biomed Mater Res 41: 333-340 (1998)) Au-Mercaptoglycerol induces strong inflammatory response in control animals. No reaction in Complement-depleted animals.

15. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz The Complement Cascade C5 C5a Ba Properidin D C3P C3a B The Alternative Pathway C3bC3bBC3bBbC3bBbPC3bBb3b B C3C3iC3iB Ba C3iBb C3 C3a The Tick-over Activation C5b C4 C4a C2 C2b C3 C3a The Classical Pathway Antibody + C1qrs C4bC4b2aC4b2a3b

17. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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 ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz The Clotting Cascade Fibrinogen Fibrin monomers Prothrombin I Thrombin Factor F XaFactor F X F VIIF VIIa F IXaF IX F XIaF XI Surface Contact Collagen FXII activator F XIIaF XII F III (Tissue Thromboplastin) Tissue/Cell Defect Intrinsic Pathway Extrinsic Pathway Platelet Factor 3 Ca 2+ Fibrin polymers Crosslinked Fibrin Meshwork F XIIIaF XIII F VF Va F VIIIaF VIII Ca 2+

22. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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 Pre-Kallikrein Kallikrein Kininogen HMWK, LMWK Kinin e.g. Bradykinin F XIIaF XII SurfaceFibrinolytic Pathway Plasminogen Plasmin Complement Activation Clotting Cascade (intrinsic pathway)

23. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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

25. Biomaterials ResearchBiomaterials Research - Manfred Maitz

26. Biomaterials ResearchBiomaterials Research - Manfred Maitz 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