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Recombinant human factor VIIa and a factor VIIa‐analogue reduces heparin and low molecular weight heparin (LMWH)‐induced bleeding in rats

2008; Elsevier BV; Volume: 6; Issue: 5 Linguagem: Inglês

10.1111/j.1538-7836.2008.02933.x

1538-7933

Brian Lauritzen, Ulla Hedner, Peter Johansen, Mikael Tranholm, Mirella Ezban,

Blood Coagulation and Thrombosis Mechanisms

Journal of Thrombosis and HaemostasisVolume 6, Issue 5 p. 804-811 ORIGINAL ARTICLEFree Access Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats B. LAURITZEN, B. LAURITZEN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorU. HEDNER, U. HEDNER Research and Development, Novo Nordisk A/S, Måløv, Denmark University of Lund, Lund, SwedenSearch for more papers by this authorP. B. JOHANSEN, P. B. JOHANSEN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorM. TRANHOLM, M. TRANHOLM Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorM. EZBAN, M. EZBAN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this author B. LAURITZEN, B. LAURITZEN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorU. HEDNER, U. HEDNER Research and Development, Novo Nordisk A/S, Måløv, Denmark University of Lund, Lund, SwedenSearch for more papers by this authorP. B. JOHANSEN, P. B. JOHANSEN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorM. TRANHOLM, M. TRANHOLM Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this authorM. EZBAN, M. EZBAN Research and Development, Novo Nordisk A/S, Måløv, DenmarkSearch for more papers by this author First published: 26 February 2008 https://doi.org/10.1111/j.1538-7836.2008.02933.xCitations: 22 Brian Lauritzen, Haemostasis Pharmacology, Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, F6.2.30, DK-2760 Måløv, Denmark.Tel.: +45 44420682; fax: +45 44444537.E-mail: blz@novonordisk.com AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Summary. Background: Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives: To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. Methods: Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results: rFVIIa (5, 10 and 20 mg kg−1) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg−1 (n = 8) and 500 IU kg−1 (n = 9), respectively. Similarly, 10 mg kg−1 NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg−1 tinzaparin, 10 mg kg−1 NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg−1 rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions: This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH. Introduction Heparin and low molecular weight heparin (LMWH) are widely used for treatment of venous thromboembolic events, as well as in thromboprophylaxis. Heparin is a heterogeneous mixture of branched glycosaminoglycans, which accelerates anti-thrombin (AT)-derived inactivation of thrombin and to a lesser extent coagulation factor (F) Xa and FIXa [1, 2]. LMWHs are derived from cleavage of heparin into a more homogenous mixture of glycosaminoglycans with a mean molecular mass of ∼5 kDa, and inhibit FXa more effectively than thrombin. Compared with heparin, LMWHs have superior bioavailability, less non-specific binding, and non-dose-dependent half-lives, which facilitate once- or twice-daily subcutaneous dosing based solely on weight and without laboratory monitoring [2]. Consequently, LMWHs have generally replaced heparin for management of thrombotic diseases [3, 4]. Both heparin and LMWHs may cause uncontrollable bleeding. While protamine can reverse the anticoagulant effect of heparin, no effective antidote for LMWHs is currently available [5]. Recombinant human FVIIa (rFVIIa, NovoSeven®, Novo Nordisk A/S, Bagsværd, Denmark) is an effective hemostatic in severe hemophilia patients with inhibitors and in patients with other rare congenital bleeding disorders, like FVII-deficiency and Glanzmann's thrombasthenia [6, 7]. Recently, rFVIIa has also demonstrated efficacy in other bleeding conditions [8, 9]. The hemostatic effect of rFVIIa is mediated by enhanced thrombin-generation on the surface of activated platelets and may, thus, be effective in neutralizing the effect of heparins/LMWHs. The literature is, however, limited and conflicting in this regard, as clinical case-reports indicate that rFVIIa may be able to control bleeding associated with LMWH treatment [10-13], while a study in rabbits failed to show effect of rFVIIa against tinzaparin-induced (a LMWH) bleeding [14]. The purpose of the present study was to study the effect of rFVIIa and NN1731, an analogue of rFVIIa with increased FX-activating activity [15], on bleeding caused by heparin or tinzaparin in a rat tail bleeding model. We also examined the effect of rFVIIa and NN1731 on bleeding in severely anticoagulated rats, using the same dosage of tinzaparin as Chan et al. [14]. Materials and methods This study consists of three separate sub-studies testing the effects of rFVIIa and NN1731 on (i) heparin-induced bleeding, (ii) tinzaparin-induced bleeding and (iii) bleeding induced by severe anticoagulation following a high dose of tinzaparin imitating the anticoagulation used by Chan et al. [14]. Animals Female Sprague-Dawley rats (Taconic, Ry, Denmark) weighing 247 ± 27 (mean ± SD), were housed in a Scantainer type IV box with aspen bedding. The rats had access to nesting material, a biting stick and a hide and received 'Altromin 1324' (Brogaarden/Altromin GMBH, Gentofte, Denmark) and water ad libitum. The rats were acclimatized for 1 week before the experiment. Room temperature was kept at 18–22 °C, humidity at 30–70% and the animals were subjected to a 12 h light:12 h dark cycle. Experiments were performed according to guidelines from The Danish Animal Experiments Council, Danish Ministry of Justice. Study protocol Dose-titration studies Initially, the dose of heparin and tinzaparin to be used for the main experiments was determined by dose-titration. Thus, tail bleeding experiments were performed in rats (n∼4) dosed with increasing doses of heparin (0, 50, 100, 150 and 200 IU kg−1) or tinzaparin (0, 200, 400, 500 and 600 IU kg−1). The doses for the main experiments were chosen as the lowest possible dose, which caused a continuous bleeding for the full observation period (1800 s) in more than 50% of the animals and at least a 5-fold increase in blood loss. This procedure ensured a window for improvement and avoided too severe anticoagulation, which might render improvement impossible. Heparin study On the day of experiment the rats were weighed, and anaesthetized with an intraperitoneal injection of 50 mg kg−1 pentobarbital sodium. Under local anaesthesia with lidocain (Xylocain, AstraZeneca, Albertslund, Denmark) a PE90 catheter (Intramedic, Lyngby, Denmark) was inserted in the right carotic artery for blood sampling. The catheter was kept open by infusion of isotonic saline (3.8 mL h−1) by an infusion pump (VI Secura FT, B. Braun, Frederiksberg, Denmark). Similarly, a PE50 (Intramedic) was inserted in the jugular vein for administration of heparin and test compounds. The body temperature of the rats was monitored by a rectal thermometer and kept at approximately 38 °C throughout the study using a heating blanket. After an initial blood sampling (Fig. 1), the tail of the rat was placed in a plastic tube containing 50 mL isotonic saline kept at 37 °C in a water bath (TYP V3/8; Julabo, Seelbach, Germany). After 5 min, 200 IU kg−1 heparin (2 mL kg−1; Leo Pharma, Ballerup, Denmark) or isotonic saline (control group) was administered intravenously. After another 2 min, blood was collected and after a total of 15 min, tail bleeding was induced by cutting off the outermost 2 mm of the tail by the use of a nail scissor, whereafter the tail was re-positioned in the plastic tube containing saline. The amputated tail tip was weighed after the experiment. Figure 1Open in figure viewerPowerPoint Timeline of the experiments. After 5 min of bleeding, the rats were randomized into six groups, each of eight animals, receiving 5, 10 or 20 mg kg−1 rFVIIa (NovoSeven; Novo Nordisk A/S), 10 mg kg−1 NN1731 (Novo Nordisk A/S) or isotonic saline (9.1 mL kg−1). Likewise, a non-heparinized control group received isotonic saline. After repositioning of the tail in a new plastic tube containing saline, the bleeding was observed for 30 min, whereafter a last blood sample was collected and the animals were euthanized by an overdose of pentobarbital. Total bleeding time was defined as the cumulated bleeding time over the 30-min observation period, including rebleedings. A bleeding episode was recorded as long as a visible 'thread' was observed in the water. This 'thread' may be constituted of erythrocytes and/or plasma proteins. Thus, bleeding time may be observed with a limited loss of hemoglobin. Blood loss was determined spectrophotometrically (SpectraMAX; Molecular Devices Corp., Sunnyvale, CA, USA) after conversion of hemoglobin into cyanmethemoglobin by a hemoglobin reagent (JT Baker, 3073; Bie & Berntsen A/S, Rødovre, Denmark), and using human hemoglobin standards (JT Baker, 3074; Bie & Berntsen A/S). All persons involved in the practical work were blinded regarding the treatment. Tinzaparin study The protocol for the tinzaparin study was identical to the protocol for the heparin study, except for the following differences. Rats were dosed with 500 IU kg−1 tinzaparin (innohep, Leo Pharma, Ballerup, Denmark) or saline in a volume of 1 mL kg−1 10 min before initiation of tail bleeding. After 5 min of bleeding, the rats were randomized into five groups of nine animals receiving 9.1 mL kg−1 of 5, 10 or 20 mg kg−1 rFVIIa, 10 mg kg−1 NN1731 or histidine buffer (10 mm histidine, 100 mm NaCl, 10 mm CaCl2, pH 6.0). Furthermore, a control group received isotonic saline rather than tinzaparin. High-dose tinzaparin study The experimental procedures were generally as described above. Three groups of 10 rats were dosed with 1800 IU kg−1 tinzaparin in a volume of 1 mL kg−1. After 10 min, the tail was cut, and after another 5 min the animals were treated with 20 mg kg−1 rFVIIa, 10 mg kg−1 NN1731 or histidine buffer (9.1 mL kg−1). Blood samples Blood from the carotid catheter was sampled prior to, 2 min after administration of anticoagulant and at the end of the study. At each time-point, EDTA-stabilized blood was used for hematological analysis, and citrate-stabilized blood was centrifuged (4000 g, 5 min, 20 °C) and processed for plasma. At the last sampling point, a plasma sample was diluted 1:10 in 1% SPA-buffer (0.01 m sodium phosphate buffer; 0.145 m NaCl, 0.05% Tween 20, 1% BSA, pH 7.6) for rFVIIa/NN1731 ELISA. Plasma was stored at −80 °C until analysis. Analyses Hematological analysis was performed on EDTA-stabilized whole blood using a Medonic CA 620 (Boule Nordic, Kastrup, Denmark). PT and aPTT were determined on an ACL 9000 Autoanalyzer (ILS Laboratories, Allerød, Denmark) using reagents from the same company (IL Test PT-Fibrinogen Recombinant and HaemosIL SynthAfax, respectively). Test range for the aPTT-assay was 6–247.5 s. In case the aPTT was above 247.5 s after heparin/tinzaparin-treatment and before treatment with rFVIIa/NN1731, the aPTT was set at 247.5 s. Thrombin/anti-thrombin (TAT) complexes were measured using an enzyme immunoassay (Enzygnost TAT micro, Dade Behring, Marburg, Germany). rFVIIa/NN1731 antigen concentration in the final blood sample was determined by ELISA [16]. Statistics The effect of heparin/tinzaparin vs. vehicle control was tested using the Mann–Whitney U-test (bleeding time) and a two-tailed t-test (blood loss). The differences in bleeding time were tested by the Kruskal–Wallis test, followed by Dunn's test. Blood loss data were analyzed using one-way anova followed by Bonferroni′s test. Blood loss data were log(x + 1)-transformed before analysis in order to achieve variance homogeneity. TAT was tested using paired t-test (change over time) and a one-way anova (difference between groups). PT and aPTT were tested using Wilcoxon's matched pairs test. Dose-response relationships were determined by Spearman's rank test. Data are mean ± SD unless otherwise indicated. Significance level was 5%. Results Effect of rFVIIa/NN1731 on heparin-induced bleeding Dose-titration An initial heparin dose-titration study was performed in order to establish the appropriate dose of heparin. In untreated animals, the total bleeding time was 428 s (median; range 190–960 s; n = 4) and the blood loss was 583 ± 275 nmol hemoglobin (n = 4). Heparin dose-dependently prolonged total bleeding time, reaching 1800 s (1470–1800 s; n = 4) and increased blood loss to 7346 ± 8381 nmol hemoglobin (n = 4), respectively, with the highest tested dose (200 IU kg−1). Effect study Pretreatment with heparin (200 IU kg−1) prolonged total bleeding time (P < 0.001; Fig. 2A). rFVIIa caused a dose-dependent reduction in total bleeding time, reaching significance at a dose of 20 mg kg−1 (P < 0.01). Similarly, 10 mg kg−1 NN1731 reduced the total bleeding time (P < 0.01) to the level observed in control animals. Figure 2Open in figure viewerPowerPoint Total tail bleeding time (A; single observations and medians) and blood loss (B; mean + SD) in rats treated with vehicle, rFVIIa (5, 10 or 20 mg kg−1; n = 8) or NN1731 (10 mg kg−1; n = 8) following pretreatment with 200 IU kg−1 heparin. Observation period was 1800 s. Control animals received saline rather than heparin. Accordingly, pretreatment with heparin caused a significant increase in blood loss following tail transection (P < 0.01; Fig. 2B). Blood loss was reduced to the control level by rFVIIa (P < 0.01 and P < 0.05 for 10 and 20 mg kg−1, respectively) as well as by 10 mg kg−1 NN1731 (P < 0.01). Effect of rFVIIa/NN1731 on tinzaparin-induced bleeding Dose-titration In the initial tinzaparin dose-titration study, untreated animals had a total bleeding time of 530 s (median; range 250–1165 s; n = 4) and a blood loss of 605 ± 430 nmol hemoglobin (n = 4). Tinzaparin dose-dependently prolonged total bleeding time, reaching 1800 s (420–1800 s; n = 7) and increased blood loss to 3895 ± 4247nmol hemoglobin (n = 7) with the chosen dose (500 IU kg−1). Effect study Total tail bleeding time in rats was significantly prolonged by pretreatment with tinzaparin (P < 0.001; Fig. 3A). rFVIIa caused a dose-dependent shortening in total bleeding time, reaching significance at 20 mg kg−1 (P < 0.05). The effect of 10 mg kg−1 NN1731 was even more pronounced, with a shortening of the total bleeding (P < 0.001) to the level in normal rats. Figure 3Open in figure viewerPowerPoint Total tail bleeding time (single observations and medians) in rats receiving 500 IU kg−1 (A; n = 9) or 1800 IU kg−1 (B; n = 10) tinzaparin followed by treatment with vehicle, rFVIIa (5, 10 or 20 mg kg−1) or NN1731 (10 mg kg−1). Observation period was 1800 s. Control animals received saline rather than tinzaparin. Pretreatment with 500 IU kg−1 tinzaparin caused a significant increase in blood loss (P < 0.001; Fig. 4A). Blood loss was significantly reduced by rFVIIa (P < 0.001 and P < 0.05 for 10 and 20 mg kg−1, respectively) and by 10 mg kg−1 NN1731 (P < 0.001) to the level of normal rats. Figure 4Open in figure viewerPowerPoint Blood loss (mean + SD) in rats receiving 500 IU kg−1 (A; n = 9) or 1800 IU kg−1 (B; n = 10) tinzaparin followed by treatment with vehicle, rFVIIa (5, 10 or 20 mg kg−1) or NN1731 (10 mg kg−1). Observation period was 1800 s. Control animals received saline rather than tinzaparin. #Below detection limit. High-dose tinzaparin study Using a more severe anticoagulation with 1800 IU kg−1 of tinzaparin, no significant effect of 20 mg kg−1 rFVIIa on bleeding time (Fig. 3B) or blood loss (Fig. 4B) was observed, although both variables decreased numerically. In contrast, a highly significant effect of 10 mg kg−1 NN1731 was observed on bleeding time (P < 0.001) as well as blood loss (P < 0.001), reaching the normal level for both variables. Effects on TAT, aPTT and PT Plasma TAT decreased significantly following treatment with 1800 IU kg−1 tinzaparin (P < 0.001; Fig. 5). Treatment with 20 mg kg−1 rFVIIa and 10 mg kg−1 NN1731 increased TAT significantly (P < 0.05 and P < 0.001, respectively), with a statistical significant difference between the two treatments in favour of NN1731 (P < 0.01), even though the dose was 2-fold lower. Figure 5Open in figure viewerPowerPoint Plasma thrombin/anti-thrombin complex (TAT)-concentrations in rats, at baseline, 2 min after injection of 1800 IU kg−1 tinzaparin and 30 min after administration of vehicle, 20 mg kg−1 rFVIIa or 10 mg kg−1 NN1731 (n = 10). A: indicates statistical significant difference (P < 0.01) compared with 20 mg kg−1 rFVIIa. Administration of 200 IU kg−1 heparin to the rats caused a significantly prolonged aPTT (Table 1). rFVIIa dose-dependently shortened the aPTT (P < 0.001 for dose-dependency) 30 min after treatment, reaching the normal level or even below. Similarly, 10 mg kg−1 NN1731 caused a significantly shortened and normalized aPTT. Table 1. Prothrombin time (PT; left) and activated partial thromboplastin time (aPTT; right) in rats undergoing tail transection following pretreatment with 200 IU kg−1 heparin (above; n = 8) or 500 IU kg−1 tinzaparin (below; n = 9), and being treated with vehicle, rFVIIa (5, 10 or 20 mg kg−1) or NN1731 (10 mg kg−1) 5 min after induction of bleeding Group PT aPTT Baseline After anticoagulation 30 min after treatment Baseline After anticoagulation 30 min after treatmment Heparin (n = 8) Control 12.2 ± 1.2 12.5 ± 1.8 12.3 ± 1.5 28.6 ± 5.3 34.4 ± 5.6 29.5 ± 2.9 Heparin 200 IU kg−1 11.0 ± 1.3 16.0 ± 4.4a 13.1 ± 0.9 34.6 ± 7.4 228 ± 42a 217 ± 28 Hep. + 5 mg kg−1 rFVIIa 12.3 ± 1.1 16.7 ± 3.1a 10.1 ± 1.0d 32.7 ± 7.5 237 ± 31b 24.5 ± 4.0d Hep. + 10 mg kg−1 rFVIIa 11.7 ± 1.5 16.0 ± 1.5a 9.9 ± 0.5c 38.9 ± 14.3 242 ± 15b 17.8 ± 1.8d Hep. + 20 mg kg−1 rFVIIa 12.3 ± 0.5 15.9 ± 3.3a 8.2 ± 0.9 36.9 ± 7.1 248 ± 0.0b 11.3 ± 0.9d Hep. + 10 mg kg−1 NN1731 12.4 ± 0.9 16.5 ± 2.6a 14.5 ± 1.5 34.9 ± 5.7 248 ± 0.0b 16.2 ± 4.8d Tinzaparin (n = 9) Control 12.5 ± 2.9 12.1 ± 1.0 12.4 ± 0.8 26.9 ± 6.0 27.9 ± 4.0 30.6 ± 6.2 Tinzaparin 500 IU kg−1 11.3 ± 1.1 19.5 ± 4.8b 16.7 ± 5.7c 26.7 ± 3.6 212 ± 75b 242 ± 11 Tinz. + 5 mg kg−1 rFVIIa 11.4 ± 1.2 18.8 ± 3.0b 12.0 ± 1.8d 32.2 ± 15.1 224 ± 71b 26.4 ± 4.7d Tinz. + 10 mg kg−1 rFVIIa 11.7 ± 0.7 17.7 ± 2.2b 9.9 ± 0.8d 30.6 ± 8.4 248 ± 0.0b 16.6 ± 2.8d Tinz. + 20 mg kg−1 rFVIIa 11.7 ± 1.0 17.6 ± 3.2b 8.1 ± 2.5c 28.6 ± 5.4 232 ± 46b 12.3 ± 3.0c Tinz. + 10 mg kg−1 NN1731 11.7 ± 1.3 17.2 ± 2.8b 12.4 ± 2.3c 26.2 ± 3.5 228 ± 37b 14.2 ± 3.1d Control animals received saline rather than heparin/tinzaparin. Data are mean + SD. Superscripts indicate: aP < 0.05 vs. baseline; bP < 0.01 vs. baseline; cP < 0.05 vs. after anticoagulation; dP < 0.01 vs. after anticoagulation. The PT was prolonged by heparin treatment (Table 1). The PT was significantly and dose-dependently shortened 30 min after treatment with rFVIIa (P < 0.001 for dose-dependency) to the normal level or below. In contrast, treatment with NN1731 did not shorten the PT significantly at this time-point. In the tinzaparin-experiment, the changes in PT and aPTT were generally similar to the changes in the heparin experiment. Thus, tinzaparin caused a significant prolonged PT and aPTT for all groups (Table 1), and rFVIIa dose-dependently shortened and normalized the PT and aPTT at 30 min after treatment (P < 0.001 for dose-dependency). Similarly, 10 mg kg−1 NN1731 significantly shortened and normalized the aPTT. In contrast to what was seen in the heparin study, treatment with NN1731 shortened the PT significantly (P < 0.05), although to a minor degree compared with rFVIIa. Discussion Numerous animal bleeding models are available, all having their advantages and disadvantages. In the present study, a rat tail bleeding model was chosen. Difficulties in standardization of the tail clip, for example due to variable tail-anatomy between animals and lack of knowledge of the direct clinical relevance of a tail bleeding, are examples of the weaknesses of tail bleeding models. However, they are rapid and easy to perform, and have in our hands proven to be reproducible. In the present study, an increased bleeding was induced by administration of heparin or LMWH to rats. In order to ensure a window for effect of the tested procoagulants, achievement of an appropriate degree of anticoagulation of the animals is important, as too low a dose of heparin/LMWH may not give the desired anticoagulant effect, whereas too severe anticoagulation may render it difficult for the procoagulant to reverse the anticoagulation. The used doses of heparin and tinzaparin were chosen by pilot dose-titration studies, and are close to the clinical doses recommended by the manufacturers. In the study by Chan et al. [14], no effect of rFVIIa was found on rabbit ear-bleeding, induced by a very high dose of tinzaparin (1800 IU kg−1). The lack of effect of rFVIIa in that study may have been due to the severe anticoagulation. Consistent with these findings, rFVIIa did not reduce the bleeding following anticoagulation with 1800 IU kg−1 tinzaparin significantly in the present study, whereas rFVIIa reduced bleeding following a lower (and more clinically relevant) degree of anticoagulation. Furthermore, the dose of rFVIIa (0.4 mg kg−1) used in the rabbit study [14] may have been too low [17]. Likewise, too low rFVIIa dose may explain the lack of effect of 1 mg kg−1 rFVIIa against heparin-induced bleeding in rats reported by Ghrib et al. [18]. The effective dose of rFVIIa used in the present rat study was 100-fold higher than the recommended clinical dose for hemophilia patients with inhibitors. Thus, the present study is primarily a proof of concept study, and the observed effect of rFVIIa/NN1731 may not be directly extrapolated to a clinical effect in humans. This difference in dose requirement is not currently fully understood. However, human rFVIIa may have a reduced reactivity with rat coagulation factors and inhibitors, similar to what has been described between human rFVIIa and murine TF [19]. Furthermore, species differences in platelet glycoproteins [20] may also contribute to the reduced effect of human rFVIIa in rodents, as it recently has been shown that platelet glycoproteins may contribute to the TF-independent generation by rFVIIa on the activated platelets [21]. It should, therefore, be emphasized that dose requirements of rFVIIa are species specific, and cannot be extrapolated between animal species. The observed hemostatic effect of rFVIIa and NN1731, in the presence of heparin and LMWH, suggests that rFVIIa may be of benefit in treatment of heparin- and LMWH-induced bleeding. Interestingly, NN1731, which is characterized by an enhanced thrombin generation capacity on the surface of activated platelets, but with similar TF-dependent activity [15], normalized bleeding even after severe anticoagulation by 1800 IU kg−1 tinzaparin. In contrast, double the dose of rFVIIa did not significantly reduce this more severe bleeding. This may indicate a higher potency or efficacy of NN1731 in the treatment of tinzaparin-induced bleeding as compared with rFVIIa, but full dose-response studies are needed in order to prove this hypothesis. Increased potency of NN1731 compared with rFVIIa was previously demonstrated in hemophilic mice [22] and in an in vitro cell-based coagulation model [23]. In accordance with an increased thrombin-generation capacity of the analogue, increased TAT-concentrations were found after NN1731-treatment compared with after rFVIIa-treatment in the present study. Under hemophilia-like conditions, rFVIIa in pharmacological concentrations binds with a low affinity to thrombin-activated platelets and generates thrombin on the platelet surface in the absence of FVIII/FIX. [24]. Thus, it is likely that the hemostatic effect of rFVIIa is mediated by the accelerated thrombin generation on the surface of activated platelets. Anticoagulation with heparin or LMWH severely impairs thrombin formation due to increased AT activity. The results of the present study indicate that rFVIIa and NN1731, even under these conditions, are capable of generating sufficient thrombin locally to induce hemostasis, as indicated by the increased TAT concentration and hemostatic effect of the two compounds. In the present study, rats received a single dose of anticoagulant. However, the experiment was performed within one plasma half-life of heparin and tinzaparin in rats, being 40–70 min [25] and 90 min [26], respectively. Future studies should study the effects of rFVIIa/NN1731 in animals with a constant level of anticoagulant in the blood (e.g. during infusion). A limited number of animal studies indicate some effect of rFVIIa on bleeding following treatment with warfarin [27], phenprocoumon [28] and melagatran [29, 30], and a general use of rFVIIa against anticoagulant-induced bleeding has been proposed [31]. Sixteen hours after phenprocoumon treatment of rats, 0.1 mg kg−1 rFVIIa normalized tail bleeding [28], presumably due to replacement of plasma FVII. In contrast, rFVIIa was not effective after sustained phenprocoumon-treatment, in which case not only FVII, but also the other vitamin K-dependent coagulation factors, were depleted from plasma. It is possible that a higher dose of rFVIIa would have been effective even under these conditions. Elg et al. [29, 30] found a numerical but non-significant hemostatic effect of 10 mg kg−1 rFVIIa against melagatran-induced bleeding in rats, which was similar to what was observed in the present study. Thus, it is likely that an increased dose might have improved the outcome. PT and aPTT were significantly prolonged by both heparin and tinzaparin treatment, as previously observed in dogs [32]. Even the lowest dose of rFVIIa normalized PT as well as aPTT 30 min after treatment. Similarly, NN1731 normalized the aPTT, while the effect on PT was more limited at this time-point. This may theoretically be explained by a shorter plasma half-life of NN1731 as compared with rFVIIa. No adverse effects of NN1731 or rFVIIa were observed in the present study, but the study was not designed for this purpose. The safety of NN1731 has recently been addressed in a dose-escalation study in humans where no adverse effects of NN1731 were found [33]. 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