Natascha C.J. Mathijssen, UMC St Radboud Nijmegen, Central Haematology Laboratory (CHL 539), PO box 9101, 6500 MB Nijmegen, The Netherlands. E-mail: N.Mathijssen@chl.umcn.nl
Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder associated with a bleeding tendency. We describe three patients with congenital FVII deficiency who have been treated with activated recombinant factor VII (rVIIa). Two patients had novel mutations and were treated prophylactically with 1·2 mg rVIIa two to three times a week. Patients 1 and 2 had a severe bleeding tendency. The frequency and severity of bleeding decreased by treatment with rVIIa compared with similar treatment with plasma-derived FVII. The third patient with a moderate bleeding phenotype was treated on demand and showed no change in the frequency of bleeding upon treatment with rVIIa or plasma products. The beneficial effect of rVIIa cannot be explained by the rVIIa half-lives. Pharmacokinetical analysis showed rVIIa activity half-lives of 35, 50 and 54 min for patients 1, 2 and 3, respectively. In conclusion, prophylactic treatment of FVII deficient patients with rVIIa appears to be applicable, safe and successful, although the mechanism of action remains to be elucidated.
Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder, with an estimated incidence of one in 500 000. Patients with severe homozygous FVII deficiency are at risk of umbilical and intracranial bleeding. In addition, joint bleeds in FVII deficient patients have been reported, as well as epistaxis and gum bleeding, menorrhagia and mucous membrane type bleeding (Perry, 2002). The cause of the bleeding complication is the absence of functional FVII, because of either the absence of total FVII or the presence of ineffective FVII. FVII is involved in the initiation of coagulation via binding of activated FVII (FVIIa) to tissue factor. In normal plasma, some FVII circulates in its active form (c. 10–100 pmol/L, 2·5–25 IU/dl FVIIa compared with 10 nmol/L FVII) (Wildgoose et al, 1992).
Treatment of congenital FVII deficiency consists of replacement therapy with fresh frozen plasma, prothrombin complex concentrates, FVII concentrates and/or recombinant FVIIa (rVIIa) (NovoSeven®, NovoNordisk, Bagsvaerd, Denmark) (Perry, 2002). As fresh frozen plasma, prothrombin complex concentrates and FVII concentrates are plasma-derived products, the use of these products is associated with a risk of transmission of pathogens. In addition, replacement therapy with prothrombin complex concentrates is associated with both arterial and venous thrombosis (Wilde, 2002). Material of human origin is not used in the production of rVIIa, thereby eliminating the transmission of pathogenic agents of human origin (Lund-Hansen, 1996).
Fresh frozen plasma and prothrombin complex concentrates are mainly administered in case of a patient-bleed or before, during and after surgery (Perry, 2002). FVII concentrates are also administered as prophylaxis, one to three times a week 10–50 U/kg (Perry, 2002).
Relatively small amounts of rVIIa are required for replacement therapy in FVII deficient patients. rVIIa for FVII deficiency is usually administered upon bleeding or around surgery. A FVII clotting activity level of 15–25% of normal is considered to be sufficient for effective haemostasis in this patient group. Upon bleeding or prior to surgery, a dose of 25 μg/kg (≅1250 IU/kg), administered every 4–6 h, appears to be sufficient treatment for patients with FVII deficiency during a bleed or surgical procedures (Mariani et al, 1999). Because of its relatively short half-life of c. 3 h both in FVII deficient patients (Berrettini et al, 2001) and in other cases (Lindley et al, 1994), rVIIa has until now not been regarded as a prophylactic treatment option for FVII deficiency, although prophylaxis with rVIIa has been described as an effective therapy in a case report of a boy with haemophilia A and inhibitory antibodies (Saxon et al, 2001).
We report treatment of three FVII deficient patients with rVIIa. Two of them received rVIIa as prophylaxis, while the third patient only used rVIIa on demand. The bleeding tendency of the patients before and after diagnosis of FVII deficiency and upon rVIIa treatment are discussed as well as the half-lives of rVIIa for these patients and their genotypic abnormalities.
Materials and methods
Informed consent and questionnaire
Informed consent was obtained from each of the patients. FVII deficient patients received a questionnaire concerning their disease before diagnosis, when treated with plasma products and when treated with rVIIa. The questions concerned the frequency, severity and nature of the bleed and treatment. The results from the questionnaire in combination with clinical data from patient files were used to determine the nature, frequency and severity of each bleed as well as treatment regimens.
Three patients with FVII deficiency participated in this study. Patients 1 and 2 used rVIIa as prophylaxis. Patient 3 received rVIIa on demand. Patient 1, a 28-year-old female, weighing 68 kg, received tranexamic acid and oral contraceptives in addition to rVIIa. Patient 2 was a 17-year-old female, weighing 63 kg, who used oral contraceptives. Patient 3 was a 35-year-old male, weighing 105 kg. He did not receive additional therapy.
The patients received several different products before rVIIa treatment was started. Among these were FVII concentrate (Immuno, Vienna, Austria) and prothrombin complex concentrates (PPSB; Cofact; CLB, Amsterdam, The Netherlands).
Pharmacokinetics of rVIIa
Patients received 2·4 mg of rVIIa. Venous blood was collected, before administration and 5, 10, 15, 30 min and 1, 2, 3, 4, 6, 24, and 48 h after administration. Blood drawn into EDTA was used for DNA isolation, citrated plasma was used for FVII antigen and activity determination. Before analysis, citrated blood was centrifuged at 4200 g at 4°C for 10 min to obtain platelet poor plasma. In these samples, FVII clotting activity and FVIIa activity were measured as described later. The individual plasma FVIIa concentration versus time data were analysed according to a one-compartment model with an intravenous bolus infusion and a first order elimination rate, by using the nonlinear least square regression program software, WinNonlin version 1.1 (Scientific Consulting, Inc. Apex, NC, USA). Pharmacokinetic parameters determined from each curve were the half-life (t1/2), total body clearance (Cl), mean residence time (MRT), area under the curve (AUC), and volume of distribution at steady-state (VSS). The goodness-of-fit was evaluated as the deviation between the observed and model predicted values as R2 = 1 − (Dev)2/(Obs)2, where (Obs)2 is the observed sum of squared observations and (Dev)2 the sum of squared deviations.
FVII antigen and activity levels
Factor VII antigen levels were determined in citrated plasma, using an enzyme-linked immunosorbent assay (ELISA) kit for FVII (Asserachrom® VII:Ag; Diagnostica Stago, Asnières, France). Normal pooled plasma was the positive control, FVII deficient plasma served as a negative control.
To determine FVIIa activity levels, a modified one-stage clotting assay was used. A stock solution consisting of 2·25 μg/ml rVIIa in FVII deficient plasma was made. This stock solution was serially diluted with FVII deficient plasma to obtain a calibration curve ranging from 225 to 5·6 ng/ml (correlation coefficient: 0·995).
A normal one-stage clotting assay was performed to determine FVII clotting activity levels. Here, normal pooled plasma was serially diluted with FVII deficient, resulting in a calibration curve ranging from 100 to 2·5% (correlation coefficient: 0·996).
Factor VII inhibitor assay
Factor VII inhibitors were determined by an assay similar to the Nijmegen-Bethesda assay for FVIII inhibitors (Verbruggen et al, 1995). In the FVII inhibitor assay, 0·1 mol/l imidazole-buffered normal pooled plasma was mixed (1:1) with patient plasma. After incubation for 2 h at 37°C, FVII clotting activity is measured by one-stage clotting assay (STA; Diagnostica Stago). These results were divided by the FVII activity levels of FVII deficient plasma mixed with normal pooled plasma (residual activity = patient FVII activity/FVII deficient FVII activity × 100%). Inhibitor concentration = [2 − log (residual activity)]/0·30103.
DNA isolation and mutation analysis
The DNA was isolated from EDTA blood using a DNA isolation kit (Puregene Genomic DNA Isolation Kit; Gentra, Minneapolis, MN, USA). Mutations in the F7 gene of these patients were determined by sequencing of the F7 exons, intron–exon junctions, promoter and polyadenylation site.
Factor V Leiden and prothrombin G20210A polymorphisms
Patients were screened for the presence of the factor V Leiden and prothrombin G20210A polymorphisms using the Light Cycler (Roche, Basel, Switzerland). The manufacturers instructions were followed (Light Cycler Factor V Leiden mutation detection kit and Light Cycler Prothrombin G20210A mutation detection kit; Roche, Basel, Switzerland).
After birth, patient 1 experienced severe bleeding episodes including an intracranial bleed. Following these bleeding episodes she was diagnosed with FVII deficiency. Both FVII activity and antigen levels were <1%. She was homozygous for an insertion of a G at position Leu13/Gln14 in exon 2 (GGA/GGGA), which causes a stop codon in exon 3 at position 47 (Table I).
Table I. Genotypic analysis and factor VII (FVII) antigen and activity levels. The FVII antigen and activity are expressed as percentages of controls.
Ex2 Insertion of G at position Leu13/Gln14 (GGA/GGGA) homozygous
Following diagnosis of FVII deficiency she received prophylaxis initially consisting of PPSB (CLB; dosage unknown), later consisting of FVII concentrates (Immuno) at a dose of 600 IU every other day, and after 1996, rVIIa at a dose of 1·2 mg every other day. For bleeding episodes she received additional rVIIa.
The patient experienced bleeding of soft tissues, joints and muscle (Table II). During rVIIa prophylaxis, the frequency of bleeding episodes decreased from over 160 bleeding episodes to five episodes per year (Table II). Subclassification of bleed types revealed the complete absence of soft tissue, gum and nosebleeds after initiation of rVIIa treatment. The duration of bleeding episodes varied from several weeks before treatment and under FVII treatment with plasma products, to 2 days under treatment with rVIIa.
Table II. Bleeding characteristics with plasma products and NovoSeven. Patient characteristics are described in the upper section of this table. The lower section reports the number and nature of the bleeding episodes and possible complications of therapy.
As a complication of treatment with plasma products, the patient was infected with hepatitis C. The patient showed sustained response to α-interferon monotherapy. No complications of rVIIa prophylaxis have been reported. She did not develop inhibitors to FVII. The patient was negative for facto V Leiden and the prothrombin G20210A polymorphism (Table I).
This patient was diagnosed with FVII deficiency at the age of 4 years. She had experienced many bruises and joint bleeds. She had a FVII activity level of <1% and a FVII antigen level of 8%. The patient was a compound heterozygote with a Arg290His transition, caused by a CGT/CAT nucleotide substitution in exon 8, and a Gln100Arg transition, caused by a CAG/CGG nucleotide substitution in exon 5 (Table I).
Until 6 years of age, patient 2 did not receive treatment for FVII deficiency. Thereafter she received prophylactic treatment with plasma products (prothrombin complex concentrates, FVII concentrates). As prophylaxis the patient received 500 U plasma FVII concentrate three times a week. Upon bleeding she received 1000 U followed by 500 U/d for 3 d. As prophylaxis for abdominal surgery she received 1000 U, followed by 2 weeks of 1000 U three times a day.
In 2001, rVIIa prophylactic treatment with 1·2 mg twice weekly was started at 15 years of age. Additional rVIIa was administered when bleeding episodes occurred.
Under rVIIa treatment the patient only experienced bleeding episodes in muscles and sometimes in her ankle, including one episode that occurred during discontinuance of prophylaxis (Table II). She experienced serious menorrhagia under rVIIa treatment when she stopped taking oral contraceptives. This was treated with additional rVIIa and with tranexamic acid (1000 mg three times a day) for 3 d. The patient reported a modest decrease in the number of bleeding episodes during rVIIa prophylaxis from 20 to 12 bleeding episodes per year (Table II). Use of rVIIa almost eliminated haemarthroses and she is now able to take part in sports and lead a normal life. The patient reported that rVIIa not only had an effect on her bleeding tendency, but also on her general condition.
No complications occurred during treatment with either rVIIa or plasma-derived products. She did not develop inhibitors to FVII. The patient was negative for Factor V Leiden and the prothrombin G20210A polymorphisms (Table I).
Patient 3 was diagnosed shortly after birth when several bleeding episodes occurred. He experienced bleeding of the umbilical cord and a large haematoma arose after a fall on the head. FVII activity was <1% and FVII antigen was 12%. The patient was homozygous for the Gln100Arg transition (Table I) and received ‘on demand’ therapy. Bleeding episodes lasted 1–2 weeks both when treated with prothrombin complex concentrates and with rVIIa.
The patient received several products. Upon bleeding he received prothrombin complex concentrate containing 350 U FVII, followed by several weeks of intensive treatment; the first week 300 U three times a day, the second week 300 U twice a day, and the third 300 U once a day. For synovectomy 600 U FVII concentrate were administered followed by 400 U after 8 and 16 h. On the second and third day, 400 U (×2) were administered. Approximately 10 bleeding episodes per year occurred during treatment with prothrombin complex and the bleeding frequency was 5–10 episodes per year while he received rVIIa. As a complication of treatment with plasma products, the patient developed hepatitis C. He showed sustained response to α-interferon monotherapy.
Most bleeds occurred in joints with both treatments (Table II). The patient showed a mild to moderate bleeding tendency and rarely had spontaneous bleeding episodes. He only used rVIIa on demand. For small bleeds, e.g. nosebleed, no treatment was required.
Patient 3 developed synovitis in his left ankle. He reported fewer small bleeding episodes and more importantly, had undergone several surgical procedures under protection with rVIIa and tranexamic acid, such as the extraction of his wisdom teeth and surgery of the vocal cords, without excessive bleeding. Patient 3 did not develop inhibitory antibodies to FVII. He was negative for factor V Leiden and the prothrombin G20210A polymorphism (Table I).
In order to determine rVIIa half-lives of these patients, the pharmacokinetics of rVIIa were determined. All three patients showed half-lives of FVIIa activity of less than 1 h; 35 min (0·58 h), 50 min (0·84 h) and 55 min (0·91 h) for patients 1, 2 and 3, respectively (Table III). Factor VIIa concentrations reached levels of 190, 260 and 185 IU/dl upon administration of 2·4 mg of rVIIa (Fig 1). Half-lives based on FVII clotting activity were similar to half-lives based on FVIIa concentrations (data not shown). Half-lives of FVII antigen were considerably longer than those of FVII activity. FVII antigen half-lives were 82 min (1·36 h), 102 min (1·70 h) and 103 min (1·71 h) for patients 1, 2 and 3, respectively. Again, the half-life was shortest for patient 1, while patients 2 and 3 showed similar half-lives (Table IV). Antigen levels reached maxima of 138%, 189% and 113% for patients 1, 2 and 3, respectively.
Table III. Factor VIIa pharmacokinetics. Factor VIIa was determined before the administration of 2·4 mg NovoSeven and 5, 10, 15, 30, 60 min, and 2, 3, 4, 6, 24 h afterwards. The results were used to calculate factor VIIa pharmacokinetics. The standard deviations were obtained by comparison of the found and expected results.
AUC, area under the curve; Vss, volume at steady-state; Cl, clearance; t1/2, half-life.
1080 ± 90 ml/kg
890 ± 130 ml/kg
677 ± 53 ml/kg
97 ± 9 μg·h/l
50 ± 10 μg·h/l
43 ± 4 μg·h/l
1080 ± 90 ml/h/kg
720 ± 140 ml/h/kg
520 ± 50 ml/h/kg
0·83 ± 0·09 h
1·2 ± 0·3 h
1·3 ± 0·2 h
0·58 ± 0·06 h
0·84 ± 0·20 h
0·91 ± 0·11 h
Table IV. Factor VII antigen pharmacokinetics. Factor VII antigen levels were determined before administration of 2·4 mg NovoSeven and 5, 10, 15, 30, 60 min, and 2, 3, 4, 6, 24 h after administration. The results were used to calculate factor VII antigen pharmacokinetics. Basal factor VII antigen levels were deducted. The standard deviations were obtained by comparison of the found and expected results.
AUC, area under the curve; Vss, volume at steady-state; Cl, clearance; t1/2: half-life.
270 ± 20 ml/kg
260 ± 20 ml/kg
250 ± 20 ml/kg
254 ± 28%·h
378 ± 56%·h
251 ± 19%·h
138 ± 15 ml/h/kg
97 ± 12 ml/h/kg
90 ± 8 ml/h/kg
2·0 ± 0·3 h
2·46 ± 0·43 h
2·5 ± 0·2 h
1·36 ± 0·20 h
1·70 ± 0·30 h
1·71 ± 0·15 h
We report the successful rVIIa prophylaxis of two FVII deficient patients (patients 1 and 2) with a severe bleeding tendency and on-demand treatment of a third patient (patient 3) with moderate bleeding tendency. The frequency of bleeding episodes in the patients receiving rVIIa prophylaxis decreased considerably compared with plasma-derived FVII, whereas the frequency of bleeding in the patient treated with rVIIa on-demand was similar to that with plasma-derived FVII. No adverse events occurred during rVIIa therapy of these three patients; inhibitors were not detected and thrombotic complications did not occur.
Prophylaxis with rVIIa for FVII deficiency has not been reported before, although prophylaxis with rVIIa has been described for a boy with haemophilia A and inhibitory antibodies (Saxon et al, 2001). As the presence of rVIIa is thought to be required when haemostasis is challenged, prophylaxis with rVIIa would seem unlikely to succeed because of its short half-life in blood. On-demand therapy with rVIIa for FVII deficiency has been described before in small studies and case reports (Bauer, 1996; Ingerslev et al, 1997; Mariani et al, 1999; Hunault & Bauer, 2000; Berrettini et al, 2001; Charpiat et al, 2002; Eskandari et al, 2002). Furthermore, rVIIa has been administered for many indications, such as haemophilia A and B with inhibitors, for which the product was originally developed, surgical procedures or trauma in otherwise healthy patients (Hedner, 2003). For these indications, including FVII deficiency, rVIIa has been reported to be effective and safe, in that very few adverse events were reported (Roberts, 2001).
Half-lives for rVIIa of 2·49–3·11 h have been reported in patients with severe FVII deficiency (Berrettini et al, 2001). We report FVIIa activity half-lives of less than 1 h in our patients (35 min; 50 min; 55 min). Surprisingly, the antigen half-lives were longer (82 min; 102 min; 103 min), although they were still shorter than reported half-lives. The cause of this difference remains to be elucidated. All three patients showed clearances which were higher than reported both in FVII deficiency (Berrettini et al, 2001) and in other cases (Lindley et al, 1994). These are the result of relatively low volumes of distribution. Higher clearances and low volumes of distribution, possibly the result of sustained use of coagulation factors, would result in shorter half-lives. This is consistent with the fact that the highest clearance was seen in the patient with the shortest half-life and the most severe bleeding tendency. Although the half-lives found in our patients were very short, this did not seem to impair the prophylactic or on-demand therapy of these patients. This suggests that an additional mechanism with t1/2 much longer than rVIIa plays a role in prophylaxis.
The maximal FVIIa concentrations (190, 260 and 185 IU/dl) reached after administration of 2·4 μg rVIIa are higher than the concentration of FVIIa in the plasma of normal individuals (2·5–25 IU/dl). They are similar to the FVII antigen levels of normal individuals.
Of these three patients, patient 1 displayed the most severe bleeding tendency. The underlying genotypic abnormality of this patient was a homozygous insertion of a guanine at position Leu13/Gln14 in exon 2. This insertion causes a frame shift starting in the Gla domain and resulting in a stop codon in exon 3 at position 47. The position of this insertion renders the detection of FVII antigen in plasma not possible. Patient 2 was compound heterozygous for a Gln100Arg in exon 5 and an Arg290His transition in exon 8. The Gln100Arg transition is located in the EGF2 domain. This domain is involved in tissue factor/FVII(a) interaction (Banner et al, 1996). The Arg290His transition is located in the catalytic domain of FVII. Patient 3 was homozygous for the Gln100Arg transition in the EGF2 domain. Patients 2 and 3 do have detectable FVII antigen levels, as they are carriers of missense mutations. The Gln100Arg transition in exon 5 has been reported in the FVII mutation database (http://europium.csc.mrc.ac.uk). It is associated with a highly variable phenotype leading to both severe and asymptomatic FVII deficiency. The Arg290His transition in exon 8 and the guanine insertion in exon 2 have not been reported in the FVII database. Whether this transition results in impaired FVII levels remains to be elucidated.
Patients 2 and 3 had similar antigen and activity levels. However, patient 2 needs prophylactic therapy while on-demand therapy is sufficient for patient 3. The Arg290His transition in the catalytic domain of one of the FVII alleles of patient 2 might be the cause of this difference. While the Gln100Arg transition impairs tissue factor binding and thus tissue factor-dependent effects of FVII(a), the Arg290His transition is very likely to impair both tissue factor-dependent and independent activity of FVII(a). Genetic heterogeneity has been reported to influence the severity of disease independently of FVII clotting activity levels (Giansily-Blaizot et al, 2002).
The severity of bleeding was determined based on information from the patients themselves, therefore a prospective study comparing different treatments would be very useful to determine effects on not only the frequency but also the severity of bleeding episodes.
In conclusion, prophylactic treatment of FVII deficient patients with recombinant FVIIa was applicable, safe and successful in our patient group although the mechanism of action remains to be determined.
This study was supported by a research grant of the ‘Stichting De Erven Leeuwenhart’. Furthermore, the study was supported by an unrestricted educational grant of NovoNordisk AS (Bagsvaerd, Denmark). In addition, Selene Schoormans and Gwendolyn van Beurden-Lourrensen are highly acknowledged.