1. Coagulation (the basics) and recombinant Factor VIIa Mechanism of Action Jerrold H. Levy, MD Emory University School of Medicine and Emory Healthcare Atlanta, Georgia

2. Normal Hemostasis First step in hemostasis is formation of a platelet aggregateFirst step in hemostasis is formation of a platelet aggregate At the molecular level interaction of coagulation factors takes place on the surface of activated plateletsAt the molecular level interaction of coagulation factors takes place on the surface of activated platelets The Tissue Factor–FVIIa complex is the physiological activator of normal hemostasisThe Tissue Factor–FVIIa complex is the physiological activator of normal hemostasis

3. Hemostasis Subendothelial matrix Nitric oxide Endothelial cell

4. Initiation of coagulation

5. Contact Tissue Factor + VII XIII a XIII Thrombin Fibrin(strong) Fibrinogen Fibrin(weak) IX XI XI a IX a XaXaXaXa VaVaVaVa XII a Prothrombin TF-VII a (Prothrombinase) PL PL (Tenase) VIII a PL X Intrinsic Pathway HK a Extrinsic Pathway Common Pathway TF Pathway Coagulation Pathways Protein C, Protein S, Antithrombin III

6. Hoffman et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61. TF-Bearing Cell Activated Platelet Platelet TF VIIIa Va VIIIa Va Va VIIa TF VIIa Xa X II IIa IX VVa II VIII/vWF VIIIa II IXa X IX X IXa IXa VIIa Xa IIa IIa Xa Normal Hemostasis: Pivotal role of TF/VIIa

7. Adhesion GpIIb/IIIa Platelet Activation Pathways GpIIb/IIIa Aggregation ADP Adrenaline Platelet GpIb Exposed Collagen Endothelium vWF COLLAGEN GpIIb/IIIa Aggregation GpIIb/IIIa Aggregation Adhesion ADP Adrenaline THROMBIN

8. COAGULATION and recombinant Factor VIIa (NovoSeven ) Mechanism of Action

9. TissueFactor Factor VIIa The first step in all coagulation: The Tissue Factor-The first step in all coagulation: The Tissue Factor- Factor VIIa complex formation Bleeding through a cut in a vessel wall

10. TissueFactor- Factor VIIa Complex The first step in all coagulation: The Tissue Factor-The first step in all coagulation: The Tissue Factor- Factor VIIa complex formation Factor VIIa complex formation This catalysis the coagulation cascade in normalThis catalysis the coagulation cascade in normal persons and in patients with bleeding disorders Bleeding through a cut in a vessel wall

11. Platelets rFactorVIIa Recombinant Factor VIIa (rFVIIa) in high concentration Recombinant Factor VIIa (rFVIIa) in high concentration binds to platelets; this complex catalysis further coagulation. The local coagulation activation is greatly enhanced The local coagulation activation is greatly enhanced TissueFactor- Factor VIIa Complex Recombinant Factor VIIa Platelet Binding

12. rFVIIa Platelets High peak levels of recombinant Factor VIIa (rFVIIa) induces formation of a strong fibrin network. This network cross-binds and forms a solid hemostatic plug TissueFactor- rFVIIa Complex Further formation of a hemostatic plug

13. HEMOSTASIS: ROLE OF FACTOR VII and TISSUE FACTOR

14. TF-Bearing Cell Va TF VIIa Xa X II IIa (Thrombin) Normal Hemostasis

15. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa Normal Hemostasis

16. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa VVa Platelet Normal Hemostasis

17. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa VVa Platelet Activated Platelet Normal Hemostasis

18. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa VVa Platelet TF VIIa IX IXa Activated Platelet Normal Hemostasis

19. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa VVa Platelet TF VIIa IX IXa Activated Platelet VIIIa Va IXa Xa IIa II Normal Hemostasis X

20. Hoffman et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61. Activated Platelet Platelet TF VIIIa Va VIIa X II TF-Bearing Cell Va TF VIIa Xa IIa IX VVa II VIII/vWF VIIIa IXa X IXa IIa Xa Normal Hemostasis

21. FACTOR VIIa Mechanism of Action Increases the tissue factor (TF) occupancyIncreases the tissue factor (TF) occupancy In pharmacological doses binds to activated plateletsIn pharmacological doses binds to activated platelets Activates Factor X independent of tissue factorActivates Factor X independent of tissue factor Proceedings of the National Academy of Sciences 97(10):5255-60, 2000. Circulation. 103(21):2555-9, 2001. Blood Coagulation & Fibrinolysis. 11 Suppl 1:S107-11, 2000. Proceedings of the National Academy of Sciences. 96(16):8925-30, 1999. Haemostasis. 30 Suppl 2:41-7, 2000. Thrombosis Research. 98(4):311-21, 2000.

22. TF-Bearing Cell Va TF Xa X II IIa Recombinant Factor VIIa ( 7 ) Mechanism of Action

23. TF-Bearing Cell Va TF Xa X II IIa VIII/vWF VIIIa VVa Platelet Activated Platelet Recombinant Factor VIIa ( 7 ) Mechanism of Action

24. TF-Bearing Cell Va TF Xa X II IIa VIII/vWF VIIIa VVa Platelet TF IX IXa Activated Platelet Recombinant Factor VIIa ( 7 ) Mechanism of Action

25. TF-Bearing Cell Va TF VIIa Xa X II IIa VIII/vWF VIIIa VVa Platelet TF IX IXa Activated Platelet Recombinant Factor VIIa ( 7 ) Mechanism of Action X Xa VIIIa IXa

26. Activated Platelet X Xa Recombinant Factor VIIa binding to activated platelets binding to activated platelets

27. Recombinant Factor VIIa ( 7 ) Mechanism of Action Activated Platelet X Xa IIa II Recombinant Factor VIIa binding to activated platelets binding to activated platelets

28. [FVIIa] (nM) RelativeFluorescence Monroe et al. Br J Haematol 1997;99:542. 2.0 1.5 1.0 0.0 0.5 0150300450600 Activated Unactivated Activated, without Ca +2 FVIIa Binding to Platelets

29. Monroe et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S15. Time (min) [FXa] (nM) 50 nM VIIa 20 nM VIIa 10 nM VIIa 5 nM VIIa 5 nM VIIa 2 nM VIIa 2 nM VIIa None None 065 4 3 2 1 0 10203040 FVIIa Activates FX on Platelets in the Absence of TF

30. Recombinant Factor VIIa Mechanism of action: Conclusion Recombinant Factor VIIa increases the Tissue Factor occupancyRecombinant Factor VIIa increases the Tissue Factor occupancy Recombinant Factor VIIa in pharmacological doses binds to activated plateletsRecombinant Factor VIIa in pharmacological doses binds to activated platelets Recombinant Factor VIIa provides Factor X activation independent of Tissue FactorRecombinant Factor VIIa provides Factor X activation independent of Tissue Factor