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Coagulation the basics and recombinant Factor VIIa Mechanism of Action

Normal Hemostasis. First step in hemostasis is formation of a platelet aggregateAt the molecular level interaction of coagulation factors takes place on the surface of activated plateletsThe Tissue Factor

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Coagulation the basics and recombinant Factor VIIa Mechanism of Action

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    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

    3. Hemostasis Hemostasis refers to the prevention of blood loss, and is accomplished by vasoconstriction and coagulation by cellular and coagulation factors. Undue bleeding is controlled and the fluidity of the blood is maintained by counterbalances within the coagulation and fibrinolytic systems. Blood vessel injury or disruption, platelet defects, abnormalities of the normally circulating anticoagulants and fibrinolytic mechanisms may upset the balance between fibrinolysis and coagulation. Blood normally circulates through endothelium-lined vessels without coagulation or platelet activation occurring and without appreciable hemorrhage. Injury to the endothelial cells triggers the hemostatic process, which typically begins with the attachment of platelets (“Adhesion”) to the damaged endothelium or exposed subendothelial proteins such as collagen and von Willebrand factor (vWf). The platelets then change form (“Activate”) and release factors that stimulate the clotting process. They also bind together (“Aggregate”). At the same time, plasma proteins may react with elements in the subendothelium, activating the “contact” phase of coagulation. Exposed fibroblasts and macrophages present tissue factor, a membrane protein, to the blood at the injured site, thereby triggering the “Extrinsic “phase of blood coagulation. Under normal conditions, hemostasis protects the individual from massive bleeding secondary to trauma. In abnormal states, life-threatening bleeding can occur or thrombosis can occlude the vascular tree. Hemostasis is influenced by a number of different factors including: (a) vascular extracellular matrix and alterations in endothelial reactivity, (b) platelets, (c) coagulation proteins, (d) inhibitors of coagulation, and (e) fibrinolysis. Cotran RS, Kumar V, Robbins SL, eds. Robbins pathologic basis of disease, 5th ed. Philadelphia: W.B. Saunders, 1994 pp 99-106. Goodnight S. Physiology of coagulation and the role of vitamin K. In: Ansell JE, Oertel LB, Wittkowsky AK, eds. Managing oral anticoagulation therapy, Gaithersburg: Aspen Publishers, 1997 pp 1B-1:1-5.Hemostasis refers to the prevention of blood loss, and is accomplished by vasoconstriction and coagulation by cellular and coagulation factors. Undue bleeding is controlled and the fluidity of the blood is maintained by counterbalances within the coagulation and fibrinolytic systems. Blood vessel injury or disruption, platelet defects, abnormalities of the normally circulating anticoagulants and fibrinolytic mechanisms may upset the balance between fibrinolysis and coagulation. Blood normally circulates through endothelium-lined vessels without coagulation or platelet activation occurring and without appreciable hemorrhage. Injury to the endothelial cells triggers the hemostatic process, which typically begins with the attachment of platelets (“Adhesion”) to the damaged endothelium or exposed subendothelial proteins such as collagen and von Willebrand factor (vWf). The platelets then change form (“Activate”) and release factors that stimulate the clotting process. They also bind together (“Aggregate”). At the same time, plasma proteins may react with elements in the subendothelium, activating the “contact” phase of coagulation. Exposed fibroblasts and macrophages present tissue factor, a membrane protein, to the blood at the injured site, thereby triggering the “Extrinsic “phase of blood coagulation. Under normal conditions, hemostasis protects the individual from massive bleeding secondary to trauma. In abnormal states, life-threatening bleeding can occur or thrombosis can occlude the vascular tree. Hemostasis is influenced by a number of different factors including: (a) vascular extracellular matrix and alterations in endothelial reactivity, (b) platelets, (c) coagulation proteins, (d) inhibitors of coagulation, and (e) fibrinolysis. Cotran RS, Kumar V, Robbins SL, eds. Robbins pathologic basis of disease, 5th ed. Philadelphia: W.B. Saunders, 1994 pp 99-106. Goodnight S. Physiology of coagulation and the role of vitamin K. In: Ansell JE, Oertel LB, Wittkowsky AK, eds. Managing oral anticoagulation therapy, Gaithersburg: Aspen Publishers, 1997 pp 1B-1:1-5.

    4. Initiation of coagulation

    5. Coagulation Pathways Coagulation may be initiated by vascular injury, however, multiple coagulation pathways are involved in the actual formation of clot. Vasoconstriction occurs immediately following vascular injury and is followed by platelet adhesion to collagen in the vessel wall exposed by injury. Subsequently platelet aggregation results in a platelet plug which is later strengthened by fibrin. Fibrin production may begin with the conversion of factor X to factor Xa. Factor X can be activated by means of two reaction sequences. One requires tissue factor (TF) which is exposed to the blood as a result of vascular injury. Because TF is not in the blood, it is an extrinsic element in coagulation, hence the name "extrinsic" pathway for this sequence. The catalytic action of TF is the central precipitating event in the clotting cascade. TF acts in concert with factor VIla and phospholipid (PL) to convert factor IX to IXa and factor X to Xa. The "intrinsic" pathway is initiated by the "contact" activation of factor XI by the XIIa/activated high molecular weight kininogen (HKa) complex. Factor XIa also converts factor IX to IXa and factor IXa in turn converts factor X to Xa, in concert with factors VIIIa and phospholipid (the “tenase complex”). However factor Xa is formed, it is the active catalytic ingredient of the "Prothrombinase” complex, which includes factor Va and PL and converts prothrombin to thrombin. Thrombin cleaves fibrinopeptides (FPA, FPB) from fibrinogen, allowing the resultant fibrin monomers to polymerize, and converts factor XIII to XIIIa which crosslinks the fibrin clot. Thrombin accelerates the clotting cascade by its potential to activate factors V and VIII, but continued proteolytic action also activates protein C which degrades Va and VIIIa. Adapted from: Colman RW, Hirsh J, Marder VJ, Salzman EW. Overview of hemostasis.Overview of the thrombotic process and its therapy. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and thrombosis, 3rd ed. Philadelphia: J.B. Lippincott, 1994 p 9.1154-1155. Colman RW, Hirsh J, Marder VJ, Salzman EW. Overview of the thrombotic process and its therapy. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and thrombosis, 3rd ed. Philadelphia: J.B. Lippincott, 1994 pp 1154-1155. Goodnight S. Physiology of coagulation and the role of vitamin K. In: Ansell JE, Oertel LB, Wittkowsky AK, eds. Managing oral anticoagulation therapy, Gaithersburg: Aspen Publishers, 1997 pp 1-7.Coagulation may be initiated by vascular injury, however, multiple coagulation pathways are involved in the actual formation of clot. Vasoconstriction occurs immediately following vascular injury and is followed by platelet adhesion to collagen in the vessel wall exposed by injury. Subsequently platelet aggregation results in a platelet plug which is later strengthened by fibrin. Fibrin production may begin with the conversion of factor X to factor Xa. Factor X can be activated by means of two reaction sequences. One requires tissue factor (TF) which is exposed to the blood as a result of vascular injury. Because TF is not in the blood, it is an extrinsic element in coagulation, hence the name "extrinsic" pathway for this sequence. The catalytic action of TF is the central precipitating event in the clotting cascade. TF acts in concert with factor VIla and phospholipid (PL) to convert factor IX to IXa and factor X to Xa. The "intrinsic" pathway is initiated by the "contact" activation of factor XI by the XIIa/activated high molecular weight kininogen (HKa) complex. Factor XIa also converts factor IX to IXa and factor IXa in turn converts factor X to Xa, in concert with factors VIIIa and phospholipid (the “tenase complex”). However factor Xa is formed, it is the active catalytic ingredient of the "Prothrombinase” complex, which includes factor Va and PL and converts prothrombin to thrombin. Thrombin cleaves fibrinopeptides (FPA, FPB) from fibrinogen, allowing the resultant fibrin monomers to polymerize, and converts factor XIII to XIIIa which crosslinks the fibrin clot. Thrombin accelerates the clotting cascade by its potential to activate factors V and VIII, but continued proteolytic action also activates protein C which degrades Va and VIIIa. Adapted from: Colman RW, Hirsh J, Marder VJ, Salzman EW. Overview of hemostasis.Overview of the thrombotic process and its therapy. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and thrombosis, 3rd ed. Philadelphia: J.B. Lippincott, 1994 p 9.1154-1155. Colman RW, Hirsh J, Marder VJ, Salzman EW. Overview of the thrombotic process and its therapy. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and thrombosis, 3rd ed. Philadelphia: J.B. Lippincott, 1994 pp 1154-1155. Goodnight S. Physiology of coagulation and the role of vitamin K. In: Ansell JE, Oertel LB, Wittkowsky AK, eds. Managing oral anticoagulation therapy, Gaithersburg: Aspen Publishers, 1997 pp 1-7.

    6. Normal Hemostasis: Pivotal role of TF/VIIa This slide illustrates the pivotal role of FVIIa/tissue factor activation in producing hemostasis. This slide represents a schematic model of normal hemostasis that requires activation of both FX and FIX. FVIIa/tissue factor (TF)-activated FXa and FIXa play distinct roles in coagulation. FXa cannot move to the platelet surface because of the presence of normal plasma inhibitors, but instead remains on the TF-bearing cell and activates a small amount of thrombin. This thrombin is not sufficient for fibrinogen cleavage but is critical for hemostasis since it can activate platelets, activate and release FVIII from von Willebrand factor (vWF), activate platelet and plasma FV, and activate FXI. FIXa moves to the platelet surface, where it forms a complex with FVIIIa and activates FX on the platelet surface. This platelet surface FXa is relatively protected from normal plasma inhibitors and can complex with platelet surface FVa, where it activates thrombin in quantities sufficient to provide for fibrinogen cleavage. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65.This slide illustrates the pivotal role of FVIIa/tissue factor activation in producing hemostasis. This slide represents a schematic model of normal hemostasis that requires activation of both FX and FIX. FVIIa/tissue factor (TF)-activated FXa and FIXa play distinct roles in coagulation. FXa cannot move to the platelet surface because of the presence of normal plasma inhibitors, but instead remains on the TF-bearing cell and activates a small amount of thrombin. This thrombin is not sufficient for fibrinogen cleavage but is critical for hemostasis since it can activate platelets, activate and release FVIII from von Willebrand factor (vWF), activate platelet and plasma FV, and activate FXI. FIXa moves to the platelet surface, where it forms a complex with FVIIIa and activates FX on the platelet surface. This platelet surface FXa is relatively protected from normal plasma inhibitors and can complex with platelet surface FVa, where it activates thrombin in quantities sufficient to provide for fibrinogen cleavage. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65.

    7. Platelet Activation Pathways Multiple pathways are responsible for platelet activation. Platelets adhere to damaged blood vessels via cell surface adhesion molecules and their membrane receptors such as glycoprotein Ib/IX (GP Ib/IX), the ligand for von Willebrand factor (VWF), which in turn can activated platelets and cause conformational changes. Further, other activators including thrombin, adrenaline, ADP, and collagen can also activate platelets. When activation occurs, the glycoprotein IIb/IIIa membrane receptor (GP IIb/IIIa) is exposed. This receptor forms bridges using fibrinogen resulting in aggregation. Platelet activation also exposes a phospholipid surface (meeting place) upon which coagulation proteins carry out their reactions. The sequential activation of these coagulation factors ultimately leads to the formation of fibrin, which is a critical component in stabilizing the hemostatic plug. Thrombin when generated, plays a pivotal role in hemostasis, via both fibrin conversion and platelet activation. Multiple pathways are responsible for platelet activation. Platelets adhere to damaged blood vessels via cell surface adhesion molecules and their membrane receptors such as glycoprotein Ib/IX (GP Ib/IX), the ligand for von Willebrand factor (VWF), which in turn can activated platelets and cause conformational changes. Further, other activators including thrombin, adrenaline, ADP, and collagen can also activate platelets. When activation occurs, the glycoprotein IIb/IIIa membrane receptor (GP IIb/IIIa) is exposed. This receptor forms bridges using fibrinogen resulting in aggregation. Platelet activation also exposes a phospholipid surface (meeting place) upon which coagulation proteins carry out their reactions. The sequential activation of these coagulation factors ultimately leads to the formation of fibrin, which is a critical component in stabilizing the hemostatic plug. Thrombin when generated, plays a pivotal role in hemostasis, via both fibrin conversion and platelet activation.

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

    13. HEMOSTASIS: ROLE OF FACTOR VII and TISSUE FACTOR

    14. Normal Hemostasis Current data suggest that high-dose FVIIa can enhance thrombin generation when normal levels of all of the coagulation factors are present. FVIIa on the platelet surface generates additional FX (and probably FIXa), so that thrombin generation is significantly increased. This observation may account for the efficacy of FVIIa in patients with thrombocytopenia. With high-dose FVIIa, each platelet can produce more thrombin than it would normally. So even if there are fewer platelets at the site of an injury, each platelet that does localize is more efficient at generating thrombin. The following slides illustrate the interactive steps involved in hemostatic activation associated with TF-FVIIa activation. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65. Current data suggest that high-dose FVIIa can enhance thrombin generation when normal levels of all of the coagulation factors are present. FVIIa on the platelet surface generates additional FX (and probably FIXa), so that thrombin generation is significantly increased. This observation may account for the efficacy of FVIIa in patients with thrombocytopenia. With high-dose FVIIa, each platelet can produce more thrombin than it would normally. So even if there are fewer platelets at the site of an injury, each platelet that does localize is more efficient at generating thrombin. The following slides illustrate the interactive steps involved in hemostatic activation associated with TF-FVIIa activation. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65.

    15. Normal Hemostasis

    16. Normal Hemostasis

    17. Normal Hemostasis

    18. Normal Hemostasis

    19. Normal Hemostasis

    20. Normal Hemostasis Normal Hemostasis Current data suggest that high-dose FVIIa can enhance thrombin generation when normal levels of all of the coagulation factors are present. FVIIa on the platelet surface generates additional FX (and probably FIXa), so that thrombin generation is significantly increased. This observation may account for the efficacy of FVIIa in patients with thrombocytopenia. With high-dose FVIIa, each platelet can produce more thrombin than it would normally. So even if there are fewer platelets at the site of an injury, each platelet that does localize is more efficient at generating thrombin. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65.Normal Hemostasis Current data suggest that high-dose FVIIa can enhance thrombin generation when normal levels of all of the coagulation factors are present. FVIIa on the platelet surface generates additional FX (and probably FIXa), so that thrombin generation is significantly increased. This observation may account for the efficacy of FVIIa in patients with thrombocytopenia. With high-dose FVIIa, each platelet can produce more thrombin than it would normally. So even if there are fewer platelets at the site of an injury, each platelet that does localize is more efficient at generating thrombin. Hoffman M et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61–S65.

    21. References: Andersen H. Greenberg DL. Fujikawa K. Xu W. Chung DW. Davie EW. Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proceedings of the National Academy of Sciences of the United States of America. 96(20):11189-93, 1999. Camerer E. Huang W. Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proceedings of the National Academy of Sciences of the United States of America. 97(10):5255-60, 2000. Friederich PW. Levi M. Bauer KA. Vlasuk GP. Rote WE. Breederveld D. Keller T. Spataro M. Barzegar S. Buller HR. Ability of recombinant factor VIIa to generate thrombin during inhibition of tissue factor in human subjects. Circulation. 103(21):2555-9, 2001. .Hedner U. NovoSeven as a universal haemostatic agent. Blood Coagulation & Fibrinolysis. 11 Suppl 1:S107-11, 2000. .Pike AC. Brzozowski AM. Roberts SM. Olsen OH. Persson E. Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proceedings of the National Academy of Sciences of the United States of America. 96(16):8925-30, 1999. .Siegbahn A. Cellular consequences upon factor VIIa binding to tissue factor. Haemostasis. 30 Suppl 2:41-7, 2000. .Wiiger MT. Pringle S. Pettersen KS. Narahara N. Prydz H. Effects of binding of ligand (FVIIa) to induced tissue factor in human endothelial cells. Thrombosis Research. 98(4):311-21, 2000. References: Andersen H. Greenberg DL. Fujikawa K. Xu W. Chung DW. Davie EW. Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proceedings of the National Academy of Sciences of the United States of America. 96(20):11189-93, 1999. Camerer E. Huang W. Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proceedings of the National Academy of Sciences of the United States of America. 97(10):5255-60, 2000. Friederich PW. Levi M. Bauer KA. Vlasuk GP. Rote WE. Breederveld D. Keller T. Spataro M. Barzegar S. Buller HR. Ability of recombinant factor VIIa to generate thrombin during inhibition of tissue factor in human subjects. Circulation. 103(21):2555-9, 2001. .Hedner U. NovoSeven as a universal haemostatic agent. Blood Coagulation & Fibrinolysis. 11 Suppl 1:S107-11, 2000. .Pike AC. Brzozowski AM. Roberts SM. Olsen OH. Persson E. Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proceedings of the National Academy of Sciences of the United States of America. 96(16):8925-30, 1999. .Siegbahn A. Cellular consequences upon factor VIIa binding to tissue factor. Haemostasis. 30 Suppl 2:41-7, 2000. .Wiiger MT. Pringle S. Pettersen KS. Narahara N. Prydz H. Effects of binding of ligand (FVIIa) to induced tissue factor in human endothelial cells. Thrombosis Research. 98(4):311-21, 2000.

    22. Recombinant Factor VIIa (7) Mechanism of Action

    23. Recombinant Factor VIIa (7) Mechanism of Action

    24. Recombinant Factor VIIa (7) Mechanism of Action

    25. Recombinant Factor VIIa (7) Mechanism of Action

    26. Recombinant Factor VIIa (7) Mechanism of Action

    27. Recombinant Factor VIIa (7) Mechanism of Action

    28. FVIIa Binding to Platelets FVIIa Binding to Platelets To assess the interaction of FVIIa with platelets, the binding of FVIIa to platelets was examined using indirect immunofluorescence and flow cytometry. The results are shown on this slide. The units of measure in flow cytometry are relative fluorescence. The fluorescence levels from several different experiments were normalized so that binding from several people could be compared. FVIIa did not bind to the unactivated platelets (shown in red), nor did it bind to activated platelets in the absence of calcium (shown in orange). However, in the presence of calcium, FVIIa bound weakly to activated platelets with a half-maximal binding at 100 nM (in contrast to binding to tissue factor [TF], which is half maximal at less than 0.01 nM). Monroe DM et al. Br J Haematol 1997;99:542–547.FVIIa Binding to Platelets To assess the interaction of FVIIa with platelets, the binding of FVIIa to platelets was examined using indirect immunofluorescence and flow cytometry. The results are shown on this slide. The units of measure in flow cytometry are relative fluorescence. The fluorescence levels from several different experiments were normalized so that binding from several people could be compared. FVIIa did not bind to the unactivated platelets (shown in red), nor did it bind to activated platelets in the absence of calcium (shown in orange). However, in the presence of calcium, FVIIa bound weakly to activated platelets with a half-maximal binding at 100 nM (in contrast to binding to tissue factor [TF], which is half maximal at less than 0.01 nM). Monroe DM et al. Br J Haematol 1997;99:542–547.

    29. FVIIa Activates FX on Platelets in the Absence of TF FVIIa Activates FX on Platelets in the Absence of TF The ability of FVIIa to activate FX was examined using platelets activated with a thrombin receptor agonist peptide (SFLLRN), plasma concentrations of FX, calcium, and varied concentrations of FVIIa. Increasing concentrations of FVIIa resulted in increasing activation of FX. Addition of an anti-TF antibody did not alter FX activation. The investigators concluded that FVIIa was acting on activated platelets to generate FXa, even though platelets do not contain TF on their cell surface. Monroe DM et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S15–S20.FVIIa Activates FX on Platelets in the Absence of TF The ability of FVIIa to activate FX was examined using platelets activated with a thrombin receptor agonist peptide (SFLLRN), plasma concentrations of FX, calcium, and varied concentrations of FVIIa. Increasing concentrations of FVIIa resulted in increasing activation of FX. Addition of an anti-TF antibody did not alter FX activation. The investigators concluded that FVIIa was acting on activated platelets to generate FXa, even though platelets do not contain TF on their cell surface. Monroe DM et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S15–S20.

    30. Recombinant Factor VIIa Mechanism of action: Conclusion

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