Blood First Edition Paper, prepublished online September 16, 2014; DOI /blood

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1 Blood First Edition Paper, prepublished online September 16, 2014; DOI /blood In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia Cecilia Augustsson and Egon Persson* Haemophilia Biology, Novo Nordisk A/S, Måløv, Denmark Running title: Therapeutic mechanism of rfviia assessed in vitro Word count text: 1303 Word count abstract: 147 Figures: 2 References: 17 Scientific category: Thrombosis and Hemostasis Presented in abstract form at the ISTH congress, The Netherlands, 2013 Corresponding Author* Egon Persson Novo Nordisk A/S Novo Nordisk Park DK-2760 Måløv Denmark Tel.: egpe@novonordisk.com 1 of 7 Copyright 2014 American Society of Hematology

2 Abstract Key points The negative impact on thrombin generation of zymogen FVII competing with rfviia for TF is counteracted by FVII (auto)activation. Correction of hemophilia A occurs in a rfviia concentration range where detectable effects of FVII competition are minimal or absent. Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rfviia) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)fviia:tf complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but virtually absent at pharmacological amounts of rfviia. Moreover, corrections of the propagation phase in hemophilia A required rfviia concentrations above the range where a physiological level of FVII was capable to down-regulate thrombin generation. These data strongly suggest that rfviia acts TF-independently in hemophilia therapy and that FVII displacement by rfviia is a negligible mechanistic component. 2 of 7

3 Introduction Recombinant factor VIIa (rfviia, NovoSeven ) is approved as a bypassing agent to treat hemophilia patients with inhibitory antibodies against factor VIII (FVIII) or IX 1. It is debated whether the pharmacological effect of rfviia, taking place after (r)fviia has functioned in the initiation phase which serves to prime the coagulation process with a non-hemostatic amount of thrombin, primarily results from its occupation of tissue factor (TF) by displacing zymogen factor VII (FVII) or from TF-independent factor X activation upon its localization to activated platelets 2. In vivo, FVII is present in large excess over FVIIa. Even though blood clotting upon physiological TF exposure is initiated by as little as picomolar concentrations of FVIIa, along the TF-dependent avenue it has been suggested that a high dose of rfviia (typically 90 µg/kg, plasma concentration ~25 nm) would be needed in order to efficiently compete with 10 nm endogenous FVII for TF and ensure hemostasis 3, 4. As an alternative, the TF-independent mechanism postulates that a similar dose would provide an adequate amount of rfviia bound to activated platelets in order to mediate a sufficient rate of factor X activation 5, 6. The most recent mechanistic studies also arrive at conflicting conclusions, one suggesting that a TFdependent component dominates 7 and the other demonstrating that rfviia acts in a TFindependent manner 8. We used thrombin generation measurements in hemophilia A plasma to demonstrate zymogen FVII competition with FVIIa for TF and a counteractive effect of FVII (auto)activation, and assessed the net outcome of these two events at physiological and pharmacological levels of rfviia. We then monitored the dose-dependent effect of rfviia and could assign the major contribution to a TF-independent mode of action. Methods Materials FVIII- and FVII-deficient plasmas were from George King Bio-Medical (Overland Park, KS) and normal plasma from Precision BioLogic Inc. (Dartmouth, Canada). Double-deficient plasma was FVII-deficient plasma treated with 0.1 mg/ml polyclonal sheep anti-human FVIII antibody (Haematologic Technologies Inc., Essex Junction, VT). rfviia (NovoSeven ), FVII and FVIIR152A were produced in-house 9. The FVIIa content in the FVII preparation was 0.3% as measured by the amidolytic activity of 300 nm FVII in the presence of 1 µm soluble TF using a FVIIa standard curve. Hepes/Tyrodes buffer consisted of 10 mm Hepes, 137 mm NaCl, 2.7 mm KCl, 1.7 mm MgCl2, 5 mm D-Glucose, and 0.4 mm NaH2PO4, ph 6,5 or 7,4. Prostaglandin E1 was from Sigma (St. Louis, MO), relipidated TF (Dade-Innovin ) from Siemens Healthcare (Erlangen, Germany), SFLLRN agonist peptide from Bachem (Bubendorf, Switzerland), convulxin from Santa Cruz Biotechnology (Dallas, TX) and reagents for thrombin generation assay from Thrombinoscope (Maastricht, Netherlands). Isolation of platelets Human blood was obtained from healthy donors, members of the Danish National Corps of Voluntary Blood Donors, as approved by the Danish National Committee on Biomedical Research Ethics (H-D ). Platelets were isolated from citrate-stabilized plasma by centrifugation 10. Briefly, the platelet pellet was washed in Hepes/Tyrodes buffer, ph 6.5, containing prostaglandin E1 (5 µg/ml), and resuspended in Hepes/Tyrodes buffer, ph 7.4. Thrombin generation assay Thrombin generation was performed according to the calibrated automated thrombogram method 11. Citrate-stabilized, platelet-rich human plasma (80 µl, 150,000 platelets/µl) was placed at 37 C for 5 min, followed by the addition of 10 µl FVIIa ( nm) and buffer with or without added FVII or FVIIR152A (10 µl, 10 or 100 nm). Thrombin generation was initiated with 20 µl FluCa reagent supplemented with either TF (1 pm) or convulxin (100 ng/ml) plus SFLLRN (30 µm). Final concentrations are noted in parentheses. 3 of 7

4 Results and discussion Competition between FVII and FVIIa for TF Zymogen FVII and FVIIa bind TF with equal affinity 12, and no or slow (auto)activation would lead to inhibition of TF-dependent rfviia activity by FVII (reduced fraction of rfviia:tf complexes). FVII can potentially be activated by FVIIa (TF-dependent autoactivation) or by the other products FVIIa generates, FXa or FIXa (feedback activation), and the term (auto)activation as used herein is intended to cover FVII activation mediated by any of these activators because they are not distinguishable in our plasma system. Using FVII-deficient platelet-rich human plasma where FVIII had been neutralized to mimic hemophilia A, we investigated whether the presence of FVII influenced FVIIa-mediated, TF-induced thrombin generation. FVII competition with rfviia for TF became evident at high FVII/rFVIIa ratios. One-hundred nm FVII did not affect thrombin generation at 25 nm rfviia, but the peak was reduced by 40% at 6 nm rfviia and by more than 50% when rfviia dropped below 1 nm (Figure 1A). As expected, the reduction was more modest at 10 nm FVII, reaching 25% at physiological FVIIa level. However, the degree of inhibition did not reflect the molar FVII/FVIIa ratio. By using 100 nm FVIIR152A (zymogen incapable of conversion to FVIIa), and comparing the thrombin generation to that obtained in the presence of the same amount of FVII, we could show that the actual competition was more pronounced (Figure 1B and C). This strongly suggested that (auto)activation of FVII to a large extent neutralized the inhibitory effect of competing with FVIIa for TF. The lowering of the effective TF concentration by 100 nm FVIIR152A in the presence of 25 nm rfviia is equivalent to reducing TF to one fifth in the presence of rfviia alone. While the former condition reduced the functional TF:FVIIa density on the TF-containing vesicles, the latter reduced the number of vesicles (with unaltered TF density), but both gave similar effective rfviia:tf concentrations and parallel reductions of the thrombin peak (Figure 1B; also at 6 nm FVIIa (Figure 1C)). Thus FVII (auto)activation has a remarkable ability to counteract the competitive effect of FVII. Even though competition between FVII and rfviia is an inevitable phenomenon, detectable under certain conditions, it has limited or no effect at physiological (0.1 nm) or clinically relevant concentrations (above 5 nm) of FVIIa and a physiological concentration of FVII (10 nm; Figures 1A and D). Mode of action of rfviia in FVIII deficiency In order to assess the contributions of TF-dependent and -independent events, rfviia titrations were made in platelet-rich hemophilia plasma and coagulation was initiated with either TF or direct platelet activators (convulxin and SFLLRN). When triggered with TF, addition of rfviia up to 6 nm had hardly any effect on the thrombin generation profile, followed by a successively increasing thrombin peak at higher rfviia concentrations (Figure 2, black bars). The effect of rfviia on thrombin generation in plasma triggered with direct platelet activators was qualitatively very similar, with an increase in thrombin peak becoming evident and measurable at 6 nm rfviia (Figure 2, grey bars). This indicated that the TF-independent (platelet surface) factor Xa generation of rfviia is manifested at 6 nm, where the zymogen competition effect is no longer observed (Figure 1B). The fact that endogenous FVII competition with rfviia for TF ceases to have an effect on the thrombin generation at the concentration of rfviia (around 6 nm) needed for detectable restoration of thrombin generation in hemophilia suggests that the dose-effect relationships of rfviia reflect a TF-independent mode of action (Figure 2). In other words, competition between FVII and rfviia for TF cannot explain the increased hemostatic potential, according to the thrombin generation measurements, of 6 nm and higher concentrations ( 25 nm) of rfviia. Nevertheless, TF doubtlessly plays a crucial role, not for the hemostatic effect of rfviia but by being instrumental in generating the initial thrombin needed especially for platelet activation. It is important to keep in mind that the initiation of the blood coagulation cascade, presumably also facilitated by FVII (auto)activation, is effectively mediated by sub-nanomolar concentration of FVIIa, which makes it unlikely that pharmacological amounts of rfviia dramatically improve hemostasis through a TF-dependent mechanism. Our overall conclusion 4 of 7

5 that rfviia in hemophilia treatment works primarily through a TF-independent mechanism, most likely (primarily) on platelets, is also in agreement with the hemostatic equipotency of murine FVIIa and a variant thereof unable to bind TF in hemophilia B mice 8. Acknowledgments We would like to thank Dr. Mirella Ezban, Novo Nordisk A/S, for her contributions to the study initiation and for valuable discussions. Authorship C.A. designed and performed experiments, analyzed data and wrote the manuscript; E.P. designed experiments and wrote the manuscript. Disclosure of Conflicts of Interest The authors are employees of Novo Nordisk A/S. 5 of 7

6 References 1. Persson E, Bolt G, Steenstrup TD, Ezban M. Recombinant coagulation factor VIIa--from molecular to clinical aspects of a versatile haemostatic agent. Thromb Res. 2010;125(6): doi: /j.thromres ; /j.thromres Roberts HR, Monroe DM, White GC. The use of recombinant factor VIIa in the treatment of bleeding disorders. Blood. 2004;104(13): doi: /blood van 't Veer C, Golden NJ, Mann KG. Inhibition of thrombin generation by the zymogen factor VII: Implications for the treatment of hemophilia A by factor VIIa. Blood. 2000;95(4): Butenas S, Brummel KE, Branda RF, Paradis SG, Mann KG. Mechanism of factor VIIadependent coagulation in hemophilia blood. Blood. 2002;99(3): Hoffman M, Monroe DM, Roberts HR. Platelet-dependent action of high-dose factor VIIa. Blood. 2002;100(1):364-5; author reply Monroe DM, Hoffman M, Oliver JA, Roberts HR. Platelet activity of high-dose factor VIIa is independent of tissue factor. Br J Haematol. 1997;99(3): Shibeko AM, Woodle SA, Lee TK, Ovanesov MV. Unifying the mechanism of recombinant FVIIa action: Dose dependence is regulated differently by tissue factor and phospholipids. Blood. 2012;120(4): doi: /blood ; /blood Feng D, Whinna H, Monroe D, Stafford DW. FVIIa as used pharmacologically is not TF dependent in hemophilia B mice. Blood. 2014;123(11): doi: /blood [doi]. 9. Persson E, Olsen OH. Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa. FEBS J. 2009;276(11): doi: /j x; /j x. 10. McNicol A. Platelet preparation and estimation of functional responses. In: Watson SP, Authi KS, eds. Platelets: A Practical Approach. Oxford: IRL Press at Oxford University Press; 1996: Hemker HC, Giesen P, Al Dieri R, et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb. 2003;33(1):4-15. doi: Kelley RF, Yang J, Eigenbrot C, et al. Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity. Biochemistry. 2004;43(5): doi: /bi035738i. 13. Radcliffe R, Nemerson Y. Activation and control of factor VII by activated factor X and thrombin. isolation and characterization of a single chain form of factor VII. J Biol Chem. 1975;250(2): Rao LV, Rapaport SI, Bajaj SP. Activation of human factor VII in the initiation of tissue factor-dependent coagulation. Blood. 1986;68(3): Seligsohn U, Osterud B, Brown SF, Griffin JH, Rapaport SI. Activation of human factor VII in plasma and in purified systems: Roles of activated factor IX, kallikrein, and activated factor XII. J Clin Invest. 1979;64(4): doi: /JCI Yamamoto M, Nakagaki T, Kisiel W. Tissue factor-dependent autoactivation of human blood coagulation factor VII. J Biol Chem. 1992;267(27): Neuenschwander PF, Fiore MM, Morrissey JH. Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. implications of a two-dimensional enzyme kinetic mechanism. J Biol Chem. 1993;268(29): of 7

7 Figure Legends Figure 1. Impact of competition between FVII and FVIIa for TF. Thrombin generation was measured in FVII-deficient, FVIII-neutralized plasma supplemented with rfviia in the absence or presence of FVII or FVIIR152A and initiated with 1 pm TF (Innovin) unless otherwise stated. A) The effect of the presence of 10 or 100 nm FVII with nm rfviia. The degree of inhibition of thrombin generation was calculated at each [rfviia] as the decrease in thrombin peak level compared with a sample without added FVII. The FVIIa present in FVII (0.3%) was taken into account when 100 nm FVII was used at a total FVIIa concentration below 6 nm. Data are mean of repeated experiments (n=2 or 3). B) The effect of FVII, FVIIR152A and reduced TF level on the thrombin generation profile at 25 nm rfviia. The curves represent the thrombin generation obtained in the presence of rfviia alone initiated with either 1 pm TF (thick solid line) or 1/5 pm TF (dotted line) or initiated with 1 pm TF in the presence of either 100 nm FVII (thin solid line) or 100 nm FVIIR152A (dashed line). C) The effect of FVII, FVIIR152A and reduced TF level on the thrombin generation profile at 6 nm FVIIa. Same set-up as in B but initiation was with 1/15 pm TF in the low-tf sample (dotted line). D) The effect of endogenous level (10 nm) of FVII on the thrombin generation profile at therapeutic concentrations of rfviia. Thin solid and dotted lines depict the results with 6 nm rfviia, and thick solid and dashed lines the corresponding results with 25 nm rfviia, in the absence and presence of 10 nm FVII, respectively. The presence of 100 nm FVIIR152A together with 6 or 25 nm FVIIa reduced the functional TF density as measured by a significant decrease in the thrombin generation, but still no inhibitory effect of 10 nm zymogen FVII could be revealed (data not shown). In panels B-D, the thrombin generation profiles were normalized for easier comparison, and a peak level of 1 was assigned to the samples initiated with 1 pm TF without added zymogen FVII. Data are mean of repeated experiments (n=3). Figure 2. Assessment of rfviia mode of action under FVIII-deficient conditions. rfviia was added to FVIII-deficient plasma and thrombin generation was initiated with 1 pm TF (Innovin, black bars) or direct platelet activators (100 ng/ml convulxin and 30 µm SFLLRN, grey bars). The thrombin peak level is plotted against increasing rfviia concentration (0-300 nm). The presence of polyclonal goat-anti-human TF antibodies did not significantly influence the thrombin peak after initiation with the platelet activators (data not shown). Thrombin peak levels are expressed in percentage relative to the thrombin peak in normal human plasma. Data are mean with SD of repeated experiments (n=3). 7 of 7

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10 Prepublished online September 16, 2014; doi: /blood In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia Cecilia Augustsson and Egon Persson Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include digital object identifier (DOIs) and date of initial publication. Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.