Recombinant activated factor VII (rFVIIa; Novoseven, Novo Nordisk, Bagsvaerd, Denmark) was first developed for the treatment of spontaneous or traumatic bleeding in haemophiliac patients.1 The tissue factor (TF) activated factor VII (FVIIa) complex initiates coagulation by the massive activation of factors X and IX, thereby promoting thrombin generation.2,3 High FVIIa concentrations reportedly activate factor X in a TF-independent and platelet-dependent manner.3 Thus, rFVIIa is generated at sites of TF exposure and/or platelet accumulation such as vascular injuries; rFVIIa induces a thrombin burst on the surface of activated platelets.
The withdrawal of aprotinin has generated an immediate need for pharmacological therapies to control postoperative haemorrhage.4,5 For the past decade, rFVIIa has increasingly been used as an adjunctive therapy for uncontrolled and life-threatening bleeding in various situations (trauma, surgery, gynaecology). Despite numerous case reports and case series describing its successful use, our understanding of published randomized controlled trials leads us to conclude that the effectiveness of rFVIIa as a haemostatic drug remains unclear.6,7 Large-powered randomized studies are difficult to perform for uncommon therapeutics.8–10 Intractable bleeding cases are rare and heterogeneous and, frequently, must be addressed as an emergency, making systematic observation and analysis difficult such as in retrospective war wounded studies.11 These findings highlight the need for the proof of concept of rFVIIa efficacy in reproducibly injured animals.
We hypothesized that rFVIIa could be used in the management of a major arterial lesion, encountered during cardiovascular surgery or as a result of trauma, that cannot be controlled by the haemostatic system, even though it is completely normal. This therapy would not replace surgical treatment of the leak, but could be useful in some circumstances such as when surgery is not rapidly available (trauma) or when surgery may be harmful for the patient (damage control).11 We assumed that the boost of coagulation initiated by rFVIIa might allow a plug to be formed quickly, ensuring haemostasis at the site of vascular injury where a normal clot would not form.
We designed a major arterial bleeding wound model and compared the effects of two doses of rFVIIa with those of the vehicle in a randomized and blinded study. We chose rabbit, as this animal is widely used to test new haemostatic drugs12,13 and the rabbit carotid is easily exposed. In line with European guidelines, the animals were noncoagulopathic, haemostatically competent and were treated under normothermic conditions;14,15 no resuscitation or blood dilution manoeuvres were performed after treatment injection to avoid confounding factors.
The primary objective of our study was to bring the proof of concept that systemic administration of a haemostatic drug, rFVIIa, can attenuate and even stop an arterial bleeding due to a major lesion in a haemostatically competent animal.
The secondary objectives were
- to compare two doses, the high dose (200 μg kg−1) matches the dose investigated in trauma studies and the low dose (80 μg kg−1) is within the range of the most frequently used dose in clinical practice,
- to look at circulating microthrombus formation by examination of the microvasculature of the liver and kidney and
- to look at major intraluminal extension of the haemostatic plug formed at the site of the arterial lesion.
Animals were treated according to the ethics rules of the ‘Institut National de la Santé et de la Recherche Médicale’ in France and European directive no. 86/609/CEE. Sixty New Zealand male rabbits (Cegav, St Mars d'Egrenne, France) were housed one per cage and provided with tap water and food ad libidum. Animals were used in experiments after an acclimation period of at least 1 week. Animals were sacrificed at the end of the experiments with an intravenous (i.v.) injection of pentobarbital (60 mg kg−1; Penthotal, Abbot, Illinois, USA).
Rabbits (∼2.5 kg) were anaesthetized with a mixture of ketamine (15 mg kg−1, ketamine 1000; VIRBAC Santé Animale, Carros, France) and xylazine (1 mg kg−1, Rompun; Bayer, Leverkusen, Germany) given through a 22-G catheter (Introcan Certo; B Braun, Melsungen, Germany) placed in the left ear marginal vein. Anaesthesia was maintained during the course of the experiment with a continuous i.v. injection of ketamine (40 mg kg−1 h−1, sufficient to maintain a slight corneal reflex) in saline (9% NaCl, Baxter, Abbot).
After a local anaesthetic (1 ml s.c. lidocaine; Aguettant, Lyon, France) was provided, a median neck incision was made and tracheotomy was performed to ensure mechanical ventilation (respiratory rate, 45 cycles min−1; tidal volume, 5 ml kg−1). Ventilation parameters were previously established to maintain physiological pH and oxygen saturation levels.16 Body temperature was maintained in the range of 38–38.5°C using an electric blanket (medium blanket system; Harvard Apparatus, Massachusetts, USA). A 22-G catheter was surgically placed in the left femoral artery for continuous haemodynamic monitoring and blood sample collection. Data were collected and treated with a monitoring system (Acqknowledge 3.7; Biopac, California, USA) and a PC (Presario; Compaq, California, USA).
The carotid lesion, the bleed control and the schematic of the protocol design are shown in Fig. 1. The total length of the right common carotid artery was gently isolated and cleared of the surrounding fascia. An arterial lesion was produced by a 1 cm catheterization of the carotid with an 18-G needle catheter close to the thyroidal branch, marking the beginning of the observation period (T0). The catheter diameter was chosen to be close to the carotid lumen to make a reproducible and large break of the artery. The size of the catheter was chosen after prior validation of its long-lasting bleeding characteristics.
The catheter was removed and a gauze ball (to measure blood loss) and metal weight (to control bleeding) were immediately placed on the lesion. Based on prior validation with a Doppler control, the chosen weight was heavy enough to provide appropriate compression to avoid rapid and lethal haemorrhage, but light enough to maintain blood flow through the vessel. Bleeding was visually controlled each minute for 1 h by quickly and gently removing and placing the gauze and weight. The gauze balls were weighed before the experiment (tare weight), after treatment administration [preinjection blood mass loss (BML)] and at the end of the experiment (postinjection BML) to collect and compare the lost blood mass before and after treatment injection.
After a 5 min observation period of ongoing pulsate bleeding, animals (n = 60) were randomly assigned to receive either a 1 ml i.v. bolus of vehicle (solvent for lyophilized drug) or 80 or 200 μg kg−1 rFVIIa in the left marginal ear vein. The treatment aliquots were prepared and frozen before the beginning of the study. The investigator was blinded to the treatment group.
When the carotid stopped bleeding, the time was noted and the vessel was left uncovered until the end of the experiment. In the case of bleeding recurrence, which occurred in six rabbits, the gauze and weight were replaced and the time of bleeding was recorded and added. At the end of the experiment when bleeding had stopped, the permeability of the vessel was assessed by visual examination (pulsatile vessel, no gross thrombosis) and by a needle puncture close to the haemostatic plug to assess pulsate bleeding. When bleeding was still ongoing after 60 min of observation, the length of bleeding noted was 60 min. No animal died.
To assess systemic thrombus formation in the animals, we examined the liver and kidney microvasculature. The abundant microvasculature of these organs makes them highly sensitive targets for intravascular emboli. Slices (0.5–1.0 cm thick) of the left liver lobe and right kidney were harvested from each animal through a median laparotomy at the end of each experiment. These were stored in 4% (v/v) buffered formaldehyde (pH 7.4) for 48 h. The slices were then routinely processed and embedded in paraffin. Paraffin blocks were used to generate 5 μm thick haematoxylin and eosin-stained sections that were analysed by an experienced pathologist blinded to the experimental conditions.17
Arterial femoral blood was collected into tubes containing citrate (Coagulation 9NC/1.4 ml, citrate 0.106 mol l−1; Sarstedt Monovette, Numbrecht, Germany) before carotid injury and at 10 and 55 min after the start of intravenous vehicle or rFVIIa. The haematocrit, haemoglobin level and platelet count were measured in duplicate with an automated device (Micros 60; ABX, Montpellier, France). Platelet poor plasma (PPP) was obtained by centrifugation at 2500 × g for 10 min at 20°C and was frozen at −80°C. The prothrombin time (PT)16 and rFVIIa activity were measured after investigators were unblinded. The PT was determined using an automated device (STA-R; Diagnostica Stago, Asnières-sur-Seine, France) with 100 μl of frozen thawed PPP and 200 μl of Neoplastine CI Plus (Diagnostica Stago) at 37°C. The rFVIIa clotting activity, measured only for blood samples 2 and 3 and for rabbits treated with rFVIIa, was measured using frozen thawed PPP with the same automated device and FVII-deficient plasma (STA-Deficient VII; Diagnostica Stago).
The primary endpoint for bleeding (efficacy) was bleeding duration. The secondary endpoint was the BML. The main endpoint for intravascular microthrombosis (safety) was the presence of microthrombi in the liver and kidney microvasculature. The other endpoint was the carotid patency (both macroscopic and after distal puncture).
Data are expressed as the mean ± SD or median (range). The level of significance was set at P less than 0.05. Comparisons between dosage groups were performed using Wilcoxon's test and intragroup differences were tested using the Wilcoxon's signed rank test. Results are shown in box-and-whiskers plots showing the mean, median and 5th, 25th, 75th and 95th percentiles.
Haemodynamic and blood sample results
The haemoglobin level, haematocrit, platelet count, mean arterial pressure, heart rate, arterial pH and temperature remained within physiological ranges throughout the experiments in the three groups, with no statistical difference except for platelet count (Table 1). The mean PTs and FVII activities for the three groups are shown in Tables 2 and 3.
Bleeding duration and blood mass lost
The bleeding duration was statistically reduced by rFVIIa injection in both treatment groups. For the vehicle, 80 and 200 μg kg−1 rFVIIa groups, the median bleeding durations were 56 min (range 7–60 min), 15 min (range 5–60 min) and 10 min (range 5–60 min), respectively, but no statistically significant difference was found between the 80 and 200 μg kg−1 doses (Fig. 2a). Thirteen rabbits were still bleeding at the end of the experiment, including nine in the vehicle group and two in each rFVIIa group. Although the preinjection BML did not differ among the three groups (data not shown), the median postinjection BML of the vehicle group was significantly higher than that of the 80 or 200 μg kg−1 rFVIIa groups (Fig. 2b). No significant difference was seen between the postinjection BMLs of the 80 and 200 μg kg−1 rFVIIa group animals.
Pathology and carotid patency
Examination of the liver and kidney fragments revealed no evidence of treatment-related pathological changes, with no macroscopic or microscopic thrombosis in any animal. When carotid bleeding stopped before the end of the experiment (47/60 cases), no macroscopic thrombosis was noted, as indicated by the pulsating vessels. Furthermore, no carotid thrombosis was found, as needle puncture always revealed pulsate bleeding.
In this randomized, placebo-controlled rabbit study, we showed that rFVIIa might be successfully used to control an intractable bleed caused by a surgical wound or trauma by reducing both bleeding duration and BML. No thrombus was found in our macroscopic or microscopic analysis. The 80 μg kg−1 dose seemed large enough to facilitate haemostasis, as no statistical difference was noted between the 80 and 200 μg kg−1 doses.
To our knowledge, this is the first time that rFVIIa has been shown to be effective in treating severe arterial bleeds in a healthy rabbit model without resuscitation or blood dilution. This result was obtained with the lowest dose we studied, which is similar to that currently used in clinical practice, based on the PT decrease and FVII activity measurements.18 Interactions between animal TFs and human FVIIa vary depending on the animal species. As most standard clinical in-vitro coagulation assays are performed with rabbit brain thromboplastin, the choice of a rabbit model seems to be more relevant than another species model.
In the only published prospective trial of human penetrating trauma using rFVIIa, Boffard et al.19 found that repeated doses of 100 μg kg−1 rFVIIa reduced blood loss only in the case of blunt trauma and not in penetrating trauma cases. In 2009, Gill et al.20 published the only known randomized controlled trial for rFVIIa in cardiac surgery. Although the results were encouraging, their conclusion was very cautious. Their dose escalation study was stopped at 80 μg kg−1 for safety reasons; increased adverse events, including stroke, were seen in patients treated with rFVIIa, although this increase was not significant. The bleeding rate inclusion criterion was not spectacular.
Two recent studies in swine have demonstrated the efficacy of rFVIIa in major aortic wounds.21,22 Using resuscitated animals, Sapsford et al.21 found that rFVIIa prolonged the survival time by a few hours. McMullin et al.22 observed that swine prophylactically treated with 90 μg kg−1 rFVIIa prior to aortotomy and subsequent resuscitation displayed reduced bleeding volumes compared with controls.21,22 Although suitable conditions for rFVIIa use include appropriate blood product compensation in terms of haemoglobin level, PT, platelet count, fibrinogen and temperature,14 animal models used to study rFVIIa frequently employ blood dilution, thrombopathic animals or hypothermic conditions.12 In contrast with these previous studies, we ensured appropriate conditions for rFVIIa use by using normothermic, nonacidotic and noncoagulopathic rabbits, bleeding was quantified before rFVIIa administration to ensure group homogeneity and wound reproducibility, and we avoided the use of intravascular fluids, as intravascular fluids can potentially affect haemostasis. The rFVIIa efficacy would be affected by the degree of haemodilution and the diluent used23 and potential thrombotic side-effects could be masked.
We established an original model of long-lasting (1 h) arterial wound bleeding in rabbits. This model was implemented in our laboratory and validated by preliminary studies regarding the reproducibility and stability of the lesion and the carotid compression (unpublished data). We found no difference among the 60 rabbits in their preinjection BML (∼13 g), indicating that our animals were similar with respect to the arterial lesion.
These results must be viewed within the context of the limitations of the study. The study was conducted in rabbits. This arterial wound model management does not mimic any common clinical setting, as no resuscitation had to be performed. Pathological examination was restricted to a selected area of the liver and kidney, so we cannot rule out a deleterious prothrombotic effect. Although the baseline platelet counts were different among the three groups, all counts were within the normal range for rabbits. It is unlikely that such a small difference with counts remaining within the normal range would have biased the results. The baseline PT was slightly different between the vehicle and 80 μg kg−1 rFVIIa group, although both values were also within the normal range. We assume that this was a random variation that should not have biased the results.
With this animal model, we sought to reproduce a severe arterial wound such as may occur during human cardiovascular surgery or trauma. We suggest that rFVIIa at a low dose can attenuate life-threatening bleeding when time or safety issues or technical difficulties preclude surgical treatment. The treatment is not administered to compensate for a coagulation factor deficiency, but as an adjunctive therapy to boost a natural process in an emergency. Our results support that, in the case of intractable bleeding, coagulation has to be improved with blood product transfusion before rFVIIa administration. We conclude that this animal study provides the proof of concept that systemic administration of rFVIIa, known to promote microvascular bleeding cessation when haemostasis is deficient, can also attenuate a major arterial bleed at least when haemostasis is not deficient.
The authors are grateful to Novonordisk for drug supply without participating in the study design, analysis or writing of the paper. The authors declare neither financial support nor conflict of interest.
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