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Use of the Thrombin Generation Test to Evaluate Response to Treatment With Recombinant Activated Factor VII
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Use of the Thrombin Generation Test to Evaluate Response to Treatment With Recombinant Activated Factor VII Yesim Dargaud,a Jean-Claude Bordet,b Anne Lienhart,a and Claude Negriera
R
ecombinant activated factor VII (rFVIIa; NovoSeven®, Novo Nordisk, Bagsværd, Denmark) represents an effective treatment for hemophilia patients with inhibitors. However, there is no consensus on the optimal dosing regimen. Using the recommended dose of 90 g/kg, hemostatic efficacy is obtained in 85% of cases. The efficacy and safety of a single high dose of rFVIIa (270 g/kg) also have been demonstrated.1–3 As the final product generated by rFVIIa is thrombin, the thrombin generation test (TGT) could theoretically be used to monitor individual responses to rFVIIa by each hemophilia patient with an inhibitor. In this study, we assessed the role of TGT in monitoring the response to rFVIIa in three patients with severe hemophilia A with an inhibitor. Thrombin generation (TG) curves were obtained in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) samples spiked in vitro with rFVIIa at doses of 35, 90, 120, 160, 200, 240, and 270 g/kg. The ex vivo TG capacity was measured before and 15 minutes, 1 hour, and 2 hours after the infusion of a therapeutic dose of rFVIIa. The in vitro results showed that the addition of rFVIIa dose-dependently increased TG capacity. The comparison of in vitro and ex vivo results suggested that in vitro TG spiking experiments could be helpful in predicting the individual minimal effective dose of rFVIIa. The ex vivo results showed an important increase in TG capacity following an injection of rFVIIa with a variable rate of correction in the three patients studied. These data are in agreement with clinical knowledge showing an individual response to rFVIIa. In addition, our results clearly demonstrate that the monitoring of rFVIIa by TGT using PPP underestimated the hemostatic ef-
ficacy by approximately 30% (Figure 1). These data emphasize the need to use PRP for the monitoring of rFVIIa by TGT. This result is in accordance with the platelet-dependent action of rFVIIa, previously reported by Monroe et al.4 We also measured thrombin generation in hemophilic PRP supplemented with 45 to 90 g/kg and 270 g/kg rFVIIa. The fibrin network generated was studied using scanning electron microscopy and clots obtained from the TGT (Figure 2). In this particular patient, we observed that only rFVIIa 270 g/kg generated a normal fibrin network with thinner fibrin fibers (139 ⫾ 85 nm; median ⫾ SD), tightly packed fibrin strands, and the highest thrombin generating capacity (endogenous thrombin potential [ETP] ⫽ 969 nmol/L per minute) compared with lower rFVIIa doses. Using rFVIIa 90 g/kg, the TG capacity was partially corrected (ETP ⫽ 898 nmol/L per minute), while the fibrin thickness was not significantly different from the PRP with hemophilia A (218 ⫾ 91 nm v 216 ⫾ 71
aClinical
Hemostasis Unit, Hôpital Edouard Herriot, Lyon, France. Laboratory, Hôpital Edouard Herriot, Lyon, France. STATEMENT OF CONFLICT OF INTEREST: None of the authors declares any conflict of interest. Address correspondence to Yesim Dargaud, MD, PhD, Assistant Hospitalier, Universitaire en Hémostase Angiologue, Unité d’Hémostase Clinique, Hopital Edouard Herriot, Pavillon E - 5, place d’Arsonval, 69003 Lyon, France. E-mail: [email protected] bHemostasis
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0037-1963/08/$-see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2008.03.008
Figure 1 Ex vivo endogenous thrombin potential (ETP) in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) following infusion of a therapeutic dose of recombinant activated factor VII (rFVIIa) in three patients with severe hemophilia A with an inhibitor (mean ⫾ SD).
Thrombin generation test to evaluate rFVII response
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Figure 2 Representative scanning electron microscopy images showing normal clot formation (first image) and fibrin network structures following thrombin generation tests on hemophilic platelet-rich plasma (second image) supplemented with 90 and 270 g/kg recombinant activated factor VII.
nm, respectively). These results are in agreement with data reported by Lisman et al,5 who showed that activated platelets in combination with rFVIIa can support thrombin and fibrin generation. Taken together, these ex vivo and in vitro results strongly suggest that evaluation of TGT using PRP could be a useful tool for monitoring the pharmacokinetics of rFVIIa and optimizing the dose to be infused. These results also emphasize the potential interest of high-dose rFVIIa, which may allow a better correction of the fibrin clot than standard dosing.
References 1. Kavakli K, Makris M, Zulfikar B, Erhardtsen E, Abrams ZS, Kenet G, et al: Home treatment of haemarthroses using a single dose regimen of recombinant activated factor VII in patients with haemophilia and inhibitors. A
2.
3.
4.
5.
multi-centre, randomised, double-blind, cross-over trial. Thromb Haemost 95:600-605, 2006 Santagostino E, Mancuso ME, Rocino A, Mancuso G, Scaraggi F, Mannucci PM: A prospective randomized trial of high and standard dosages of recombinant factor VIIa for treatment of haemarthroses in hemophiliacs with inhibitors. J Thromb Haemost 4:367-371, 2006 Young G, Shafer FE, Rojas P, Seremetis S: Single 270 g/kg-dose rFVIIa versus standard 90 g/kg-dose rFVIIa and APCC for home treatment of joint bleeds in haemophilia patients with inhibitors: a randomized comparison. Haemophilia 14:287-294, 2008 Monroe DM, Hoffman M, Oliver JA, Roberts HR: Platelet activity of high-dose factor VIIa is independent of tissue factor. Br J Haematol 99:542-547, 1997 Lisman T, Adelmeijer J, Heijnen HF, de Groot PG: Recombinant factor VIIa restores aggregation of alphaIIbbeta3-deficient platelets via tissue factor-independent fibrin generation. Blood 103:17201727, 2004
Use of the Thrombin Generation Test to Evaluate Response to Treatment With Recombinant Activated Factor VII Yesim Dargaud,a Jean-Claude Bordet,b Anne Lienhart,a and Claude Negriera
R
ecombinant activated factor VII (rFVIIa; NovoSeven®, Novo Nordisk, Bagsværd, Denmark) represents an effective treatment for hemophilia patients with inhibitors. However, there is no consensus on the optimal dosing regimen. Using the recommended dose of 90 g/kg, hemostatic efficacy is obtained in 85% of cases. The efficacy and safety of a single high dose of rFVIIa (270 g/kg) also have been demonstrated.1–3 As the final product generated by rFVIIa is thrombin, the thrombin generation test (TGT) could theoretically be used to monitor individual responses to rFVIIa by each hemophilia patient with an inhibitor. In this study, we assessed the role of TGT in monitoring the response to rFVIIa in three patients with severe hemophilia A with an inhibitor. Thrombin generation (TG) curves were obtained in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) samples spiked in vitro with rFVIIa at doses of 35, 90, 120, 160, 200, 240, and 270 g/kg. The ex vivo TG capacity was measured before and 15 minutes, 1 hour, and 2 hours after the infusion of a therapeutic dose of rFVIIa. The in vitro results showed that the addition of rFVIIa dose-dependently increased TG capacity. The comparison of in vitro and ex vivo results suggested that in vitro TG spiking experiments could be helpful in predicting the individual minimal effective dose of rFVIIa. The ex vivo results showed an important increase in TG capacity following an injection of rFVIIa with a variable rate of correction in the three patients studied. These data are in agreement with clinical knowledge showing an individual response to rFVIIa. In addition, our results clearly demonstrate that the monitoring of rFVIIa by TGT using PPP underestimated the hemostatic ef-
ficacy by approximately 30% (Figure 1). These data emphasize the need to use PRP for the monitoring of rFVIIa by TGT. This result is in accordance with the platelet-dependent action of rFVIIa, previously reported by Monroe et al.4 We also measured thrombin generation in hemophilic PRP supplemented with 45 to 90 g/kg and 270 g/kg rFVIIa. The fibrin network generated was studied using scanning electron microscopy and clots obtained from the TGT (Figure 2). In this particular patient, we observed that only rFVIIa 270 g/kg generated a normal fibrin network with thinner fibrin fibers (139 ⫾ 85 nm; median ⫾ SD), tightly packed fibrin strands, and the highest thrombin generating capacity (endogenous thrombin potential [ETP] ⫽ 969 nmol/L per minute) compared with lower rFVIIa doses. Using rFVIIa 90 g/kg, the TG capacity was partially corrected (ETP ⫽ 898 nmol/L per minute), while the fibrin thickness was not significantly different from the PRP with hemophilia A (218 ⫾ 91 nm v 216 ⫾ 71
aClinical
Hemostasis Unit, Hôpital Edouard Herriot, Lyon, France. Laboratory, Hôpital Edouard Herriot, Lyon, France. STATEMENT OF CONFLICT OF INTEREST: None of the authors declares any conflict of interest. Address correspondence to Yesim Dargaud, MD, PhD, Assistant Hospitalier, Universitaire en Hémostase Angiologue, Unité d’Hémostase Clinique, Hopital Edouard Herriot, Pavillon E - 5, place d’Arsonval, 69003 Lyon, France. E-mail: [email protected] bHemostasis
S72
0037-1963/08/$-see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2008.03.008
Figure 1 Ex vivo endogenous thrombin potential (ETP) in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) following infusion of a therapeutic dose of recombinant activated factor VII (rFVIIa) in three patients with severe hemophilia A with an inhibitor (mean ⫾ SD).
Thrombin generation test to evaluate rFVII response
S73
Figure 2 Representative scanning electron microscopy images showing normal clot formation (first image) and fibrin network structures following thrombin generation tests on hemophilic platelet-rich plasma (second image) supplemented with 90 and 270 g/kg recombinant activated factor VII.
nm, respectively). These results are in agreement with data reported by Lisman et al,5 who showed that activated platelets in combination with rFVIIa can support thrombin and fibrin generation. Taken together, these ex vivo and in vitro results strongly suggest that evaluation of TGT using PRP could be a useful tool for monitoring the pharmacokinetics of rFVIIa and optimizing the dose to be infused. These results also emphasize the potential interest of high-dose rFVIIa, which may allow a better correction of the fibrin clot than standard dosing.
References 1. Kavakli K, Makris M, Zulfikar B, Erhardtsen E, Abrams ZS, Kenet G, et al: Home treatment of haemarthroses using a single dose regimen of recombinant activated factor VII in patients with haemophilia and inhibitors. A
2.
3.
4.
5.
multi-centre, randomised, double-blind, cross-over trial. Thromb Haemost 95:600-605, 2006 Santagostino E, Mancuso ME, Rocino A, Mancuso G, Scaraggi F, Mannucci PM: A prospective randomized trial of high and standard dosages of recombinant factor VIIa for treatment of haemarthroses in hemophiliacs with inhibitors. J Thromb Haemost 4:367-371, 2006 Young G, Shafer FE, Rojas P, Seremetis S: Single 270 g/kg-dose rFVIIa versus standard 90 g/kg-dose rFVIIa and APCC for home treatment of joint bleeds in haemophilia patients with inhibitors: a randomized comparison. Haemophilia 14:287-294, 2008 Monroe DM, Hoffman M, Oliver JA, Roberts HR: Platelet activity of high-dose factor VIIa is independent of tissue factor. Br J Haematol 99:542-547, 1997 Lisman T, Adelmeijer J, Heijnen HF, de Groot PG: Recombinant factor VIIa restores aggregation of alphaIIbbeta3-deficient platelets via tissue factor-independent fibrin generation. Blood 103:17201727, 2004