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Keywords:

  • recombinant factor VIIa;
  • analogue;
  • severe haemophilia;
  • thrombelastography

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

This study evaluated and compared the haemostatic potential of a recombinant factor VIIa (rFVIIa) analogue (V158D/E296V/M298Q-FVIIa, NN1731, Novo Nordisk, Denmark) with rFVIIa (NovoSeven®, Novo Nordisk). In vitro studies were performed using freshly drawn whole blood (WB) from 14 patients with severe haemophilia A and two patients with inhibitory antibodies to FVIII, comparing NN1731 and rFVIIa against a buffer control. Fourteen healthy males served as controls. Dynamic WB coagulation profiles were recorded, quantitatively illustrating the initiation [clotting time = CT (s)], propagation [maximum velocity = MaxVel (mm*100/s)] and termination [maximum clot firmness = MCF (mm*100)] as determined by thromboelastography with minute amounts of tissue factor (TF, Innovin®– final dilution 1:50 000, c. 0·12 pM) serving as activator. WB clot stability was assessed using a separate set-up including TF plus tissue plasminogen activator (final concentration 2 nmol/l), evaluating the MCF as well as the area under the elasticity curve (AUEC) after 60 min (mm*100*s). NN1731 shortened the CT more markedly than rFVIIa. At the dose tested, NN1731 even shortened CT in haemophilia below the value of healthy males. NN1731 accelerated MaxVel giving a value indistinguishable from that in healthy males. Furthermore, NN1731 increased clot stability more markedly than rFVIIa. Altogether, these in vitro studies on WB revealed a favourable haemostatic potential of NN1731 compared with rFVIIa in severe haemophilia A, both in the absence and presence of enhanced fibrinolytic activity.

Today, the development of inhibitory antibodies against FVIII during substitution therapy constitutes the most serious complication amongst patients with severe haemophilia A and occurs in previously untreated patients with incidences ranging from 20 to 33% (Ehrenforth et al, 1992; Ljung et al, 1992; Kreuz et al, 1999; Scharrer et al, 1999; Wight & Paisley, 2003). The presence of high titre inhibitors (>5 Bethesda Units (BU)/ml) often require the use of bypassing agents, such as plasma-derived activated prothrombin complex concentrate (aPCC) or recombinant FVIIa (rFVIIa) to obtain haemostasis (Ingerslev et al, 1991; Sørensen & Ingerslev, 2004a). Currently, rFVIIa is successfully used for the treatment of bleeds in patients with congenital severe haemophilia with inhibitors (Abshire & Kenet, 2004). However, in a few haemophilia patients with inhibitors, the haemostatic response to rFVIIa, as well as to aPCC, appears rather low (Martinowitz et al, 2005). Moreover, considerable inter-individual response variations indicate a possible need for individualised dose titration (Sørensen & Ingerslev, 2004a). Hence, in a subgroup of difficult-to-treat haemophilia patients with inhibitors, a novel form or formulation of rFVIIa with optimised efficacy and a more predictable haemostatic response profile would appear attractive.

During recent years innovations have resulted in the development of novel, potent rFVIIa analogues that exhibit an increased intrinsic activity, as defined by an enhanced ability of tissue factor (TF)-independent activation of factor X (FX) on the phospholipid surface of activated platelets (Persson et al, 2001a,b; Persson & Olsen, 2002; Persson et al, 2004). Some of these analogues were reported to possess an enhanced potential to facilitate haemostasis, as evaluated by studies in FVIII-deficient plasma (Lisman et al, 2003) and haemophilia A mice models (Tranholm et al, 2003). Although the mechanisms of action of rFVIIa are subject to debate, accumulating data stress the importance of TF (Butenas et al, 2003a) and phospholipids as well as the existence of a TF-independent, platelet-dependent activation of FX (Hoffman & Monroe, 2001). The importance of the latter is illustrated by data on the rFVIIa analogues, which have a significantly higher intrinsic activity when not bound to TF (Persson et al, 2001b). Appreciating the important interactions of platelets and white blood cells in the overall regulation of haemostasis (Polgar et al, 2005), whole blood (WB) seems the optimal medium for in vitro evaluation of haemostatic capacity. Moreover, previous studies performed with rFVIIa in animal models have documented a need for very high doses to achieve a haemostatic effect in certain species, such as rodents. Thus, WB coagulation analyses on blood from patients represent the most appropriate means of exploring the haemostatic potential of rFVIIa and analogues of rFVIIa under various clinical conditions. Our laboratory has developed a thrombelastographic model of continuous WB coagulation, employing activation with minute amounts of TF, and novel data processing of the raw coagulation data signal to provide dynamic parameters of clot formation (Sørensen et al, 2003a). So far, a comprehensive evaluation of the model has been performed in various conditions of abnormal haemostasis, demonstrating the modified thrombelastographic method as an attractive and applicable approach for the characterisation of haemostatic deterioration and prediction of the effect of rFVIIa, aPCC, and other types of haemostatic intervention (Ingerslev et al, 2003; Sørensen & Ingerslev, 2003; Sørensen et al, 2003a; Sørensen et al, 2003b; Sørensen & Ingerslev, 2004a,b; Fenger-Eriksen et al, 2005; Sørensen et al, 2005a). Hence, in addition to detailed and important measurements of thrombin generation (Rand et al, 1996; Cawthern et al, 1998; Butenas et al, 2003a,b), analyses of thrombelastographic WB clot formation and WB clot stability in blood from patients with severe haemophilia A may provide desirable information about the haemostatic potential of rFVIIa and rFVIIa analogues. The present study hypothesised that a rFVIIa analogue (Mutations: V158D/E296V/M298Q-FVIIa, designated: NN1731) would provide a significantly accelerated WB clot formation when compared with rFVIIa, and that NN1731 would significantly improve WB clot stability compared to rFVIIa. Utilising the above-mentioned thrombelastographic model (Sørensen et al, 2003a; Sørensen & Ingerslev, 2004a), the present study aimed to evaluate the haemostatic potential of NN1731 (Novo Nordisk, Bagsvaerd, Denmark) in comparison with wild-type rFVIIa (NovoSeven®, Novo Nordisk) on clot formation characteristics and clot resistance to fibrinolysis in WB from patients with severe haemophilia A.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

Patients

A total of 14 patients with severe haemophilia A that was well characterised both biochemically and clinically, with an average age of 43 years (range 21–56 years) were enroled. None of the patients had signs of inhibitors (Bethesda titre <0·6 IU/ml) nor had they received FVIII substitution for at least 5 d prior to blood sampling. Mean values (range) of standard laboratory parameters were: activated partial thromboplastin time (APTT), 99 s (84–115 s); FVIII coagulant activity (FVIII:C) <0·01 IU/ml; platelet count, 259 × 109/l (158–354 × 109/l); prothrombin time (PT), 8·3 s (8–9 s); fibrinogen, 3·2 g/l (2·5–4·3 g/l); and haematocrit, 0·44 (0·39–0·48). One additional patient, aged 36 years, who suffered from severe haemophilia A (APTT = 101 s, FVIII:C<0·01) and inhibitors (Inhibitor titre at 9 BU/ml) donated blood samples for an in vitro titration experiment with rFVIIa at 1, 2, 5, 10, and 20 μg/ml versus NN1731 at 0·1, 0·2, 0·5, 1, and 2 μg/ml. In addition, we studied a female person, aged 74 years, who had acquired haemophilia A (APTT = 51 s, FVIII:C = 0·08 IU/ml, Bethesda titre = 3·6 BU/ml) and donated blood samples for in vitro dose titration with rFVIIa at 1, 2, 4, and 8 μg/ml versus NN1731 at 2 μg/ml.

Healthy controls

Healthy males [n = 14] with an average age of 43 years (range 20–66 years) and normal APTT, PT, platelet count, EVF (erythrocyte volume fraction), and fibrinogen level constituted the reference group.

Blood sampling

Using minimum stasis and a 21 gauge butterfly needle, blood samples for coagulation analyses were drawn into citrated tubes mixing one part of 3·8% trisodium citrate and nine parts of blood. In addition, EDTA blood was collected for haematological parameters.

Reagents

Recombinant human coagulation FVIIa (rFVIIa, NovoSeven®, Novo Nordisk) and an analogue of recombinant FVIIa (Mutations: V158D/E296V/M298Q-FVIIa, designated: NN1731, Novo Nordisk) were obtained from the manufacturer.

Study design

The protocol was approved by the local Ethics Committee and all participants provided spoken and written informed consent prior to enrollment for blood sampling. Analyses were performed following in vitro addition of buffer versus rFVIIa or NN1731 to citrated blood from each participating haemophilia patient. Reaction mixtures were prepared by taking 280 μl of citrated WB and spiking it with 20 μl buffer, 20 μl rFVIIa, or 20 μl NN1731 (both giving a final concentration of 2 μg/ml). The concentration of 2 μg/ml was based on the most commonly used therapeutic dose of rFVIIa at c. 100 μg/kg (Abshire & Kenet, 2004) as well as experiences gained from previous laboratory investigations with rFVIIa in severe haemophilia A (Sørensen et al, 2003a; Sørensen & Ingerslev, 2004a,b). WB clot formation was evaluated using TF as activator, whereas clot stability was assessed in a new experimental setup containing TF and tissue plasminogen activator (t-PA) for simultaneous induction of clot formation and fibrinolysis.

Coagulation analyses

Standard coagulation analyses

Prothrombin time, APTT, and the level of functional fibrinogen were analyzed employing recombinant human TF (Innovin®, Dade Behring®, Marburg, Germany), APTT test reagent (Platelin® LS, bioMérieux, Geneva, Switzerland) and fibrinogen test reagents (Multifibren® U, Dade Behring®), respectively. The BCT® Analyzer instrument was from Dade Behring®. Determination of FVIII:C was performed by a one-stage method utilising Platelin LS as activator and a FVIII deficiency plasma as test base (prepared in house using plasma from a severe FVIII-deficient patient). Measurements of FVIII:C inhibitor titres were performed by the Bethesda assay in accordance with the Nijmegen modification.

Dynamic thrombelastographic whole blood coagulation

Profiles of continuous WB clot formation were recorded by adopting our modified thrombelastographic model (Sørensen et al, 2003a). Dynamic WB coagulation profiles describing initiation [clotting time = CT (s)], propagation [maximum velocity = MaxVel (mm*100/s)] and termination [maximum clot firmness = MCF (mm*100)] were recorded. Analyses were performed using the ROTEM® thromboelastometry (Pentapharm, Munich, Germany) and ROTEM® plastic cups and pins. Citrated WB was rested for 30 min at ambient temperature. Volumes of 280 μl were transferred to pre-warmed ROTEM® plastic cups. Twenty μl buffer (HEPES 20 mmol/l, NaCl 150 mmol/l, pH = 7·4) with or without rFVIIa or NN1731 (32 μg/ml) were added and coagulation was started by the addition of 20 μl TF diluted in buffer with calcium (HEPES 20 mmol/l, NaCl 150 mmol/l, CaCl2 200 mmol/l, pH = 7·4), providing a final dilution of Innovin at 1:50 000 (final concentration of c. 0·12 pM). Raw coagulation data were processed in DyCoDerivAnTM GOLD (AvordusoL, Risskov, Denmark) to obtain dynamic parameters of clot formation, such as MaxVel and t, MaxVel (see Fig 1). WB clot stability was evaluated using a reaction mixture containing TF (final dilution 1:50 000) and t-PA (final concentration 2 nmol/l), followed by evaluation of MCF and the area under the elasticity curve [AUEC (mm*100*s)].

image

Figure 1.  Whole blood clot formation in severe hemophilia A following in vitro addition of rFVIIa or NN1731. Illustrates representative velocity profiles of clot formation initiated with minute amounts of tissue factor (final dilution of Innovin® 1:50 000) in whole blood from patients with severe hemophilia A (solid line) and following in vitro addition of rFVIIa 2 μg/ml (dotted line) or NN1731 2 μg/ml (broken line). In addition, a characteristic whole blood clotting profile from a healthy male is depicted (dotted/broken line).

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Statistical considerations

All statistical analyses were performed using Analyse-itTM version 1.62 (Analyse-it Software, Ltd., Leeds, UK), a statistical add-in program for Excel® (Microsoft®, Redmond, WA, USA). Descriptive statistics of parameters of WB clot formation (CT, MaxVel, t, MaxVel and MCF) showed that the data did not follow a normal distribution, hence, non-parametric Wilcoxon signed rank test were performed to assess differences. Data are presented as mean, 95% confidence intervals (CI), and Wilcoxon statistical results. The variability amongst the different groups was assessed using a two-sample variance comparison calculator in Intercooled Stata 8·2 (StataCorp LP, College Station, TX, USA). P-values < 0·05 were considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

Whole blood clot formation

Figure 1 depicts characteristic velocity profiles of clot formation in WB from a healthy male and a patient with severe haemophilia A before and after in vitro addition of 2 μg/ml rFVIIa or NN1731. Patients with severe haemophilia A had a WB coagulation profile characterised by a prolonged initiation phase as indicated by late-occurring clotting (Table I). Furthermore, WB from patients with severe haemophilia A also displayed a severely compromised propagation phase as illustrated by a reduced maximum rate of clot formation and a prolonged time to reach the peak (Table I). The rFVIIa analogue NN1731 at 2 μg/ml normalised the compromised propagation phase and induced clotting times significantly shorter than those observed in WB from healthy males (Table I). In contrast, addition of the same concentration of rFVIIa significantly reduced the clotting time and accelerated the propagation phase, but failed to normalise the haemostatic profile (Table I). WB clot firmness was significantly lower in patients with severe haemophilia A but could be reverted to normal values by both rFVIIa and NN1731, each at 2 μg/ml (Table I).

Table I.   Parameters of whole blood clot formation (n,= 14). Thrombelastographic parameters obtained following activation with tissue factor (Innovin®, final dilution 1:50 000, c. 0·12 pM).
 Severe haemophilia AHealthy males
BufferrFVIIa [2 μg/ml]NN1731 [2 μg/ml]Buffer
  1. CT, clotting time; MaxVel, maximum velocity; MCF, maximum clot firmness; 95% CI, 95% Confidence interval; SD, standard deviation.

  2. *rFVIIa/NN1731 versus buffer in severe haemophilia A, P < 0·05.

  3. †Severe haemophilia A versus healthy males, P < 0·05.

  4. ‡NN1731 versus healthy males, P < 0·05.

  5. §NN1731 versus rFVIIa in severe haemophilia A, P < 0·05.

Clot initiation
 CT [s]1424†881*119*§‡294
 95% CI981–1866502–125986–152271–317
 SD697†65558*§49
Clot propagation
 MaxVel [mm*100/s]3·8†9·2*14·5*§14·1
 95% CI2·2–5·46·6–11·812·2–16·712·5–15·6
 SD2·84·53·93·3
 t, MaxVel [s]2158†1395*323*§‡486
 95% CI1541–2776808–1982294–351459–513
 SD1022†101749*§54
Clot termination
 MCF [mm]44†59*59*60
 95% CI32–5754–6556–6156–64
 SD22†10*4*§‡9

Whole blood clot resistance to t-PA-induced fibrinolysis

The reaction mixture containing TF and t-PA initially stimulated clot formation, while the developing clot was subject to continuous fibrinolysis. As illustrated in Fig 2, a gain in absolute elasticity during clot formation was soon followed by a reduction during clot lysis. When employing the reaction mixture of TF at a final dilution of 1:50 000 together with t-PA at 2 nmol/l in WB from patients with severe haemophilia A, virtually no coagulation signal was seen (Fig 2). In vitro addition of rFVIIa and NN1731, each at a final concentration of 2 μg/ml, significantly improved clot formation as illustrated by a shortened time to clot initiation as well as a distinctly elevated clot propagation. As seen in the assay without t-PA, NN1731 significantly reduced the CT when compared with those recorded in the age-matched healthy reference group. Focusing on the parameters used to evaluate WB clot stability, such as MCF and AUEC, NN1731 at 2 μg/ml significantly increased the WB MCF when compared with rFVIIa at 2 μg/ml (Table II). Furthermore, the overall clot (AUEC) was increased 15-fold by NN1731 compared with the buffer control (eightfold by rFVIIa (P < 0·05)).

image

Figure 2.  Whole blood clot stability in severe hemophilia A following in vitro addition of rFVIIa or NN1731. Illustrates representative whole blood clotting profiles following initiation with tissue factor (final dilution of Innovin® 1:50 000) and t-PA (final concentration 2 nmol/l) in blood from patients with severe hemophilia A (solid line) and following in vitro addition of rFVIIa 2 μg/ml (dotted line) or NN1731 2 μg/ml (broken line). In addition, a characteristic whole blood clot stability pattern of a healthy male is depicted (dotted/broken line).

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Table II.   Parameters of whole blood clot stability (n = 14). Thrombelastographic parameters obtained following simultaneous activation with tissue factor (Innovin®, final dilution 1:50 000, c. 0·12 pM) and tissue plasminogen activator (t-PA, final concentration 2 nmol/l).
 Severe haemophilia AHealthy males
BufferrFVIIa [2 μg/ml]NN1731 [2 μg/ml]Buffer
  1. CT, clotting time; MaxVel, maximum velocity; MCF, maximum clot firmness; AUEC, area under the elasticity curve; 95% CI, 95% Confidence interval; SD, standard deviation.

  2. *rFVIIa/NN1731 versus buffer in severe haemophilia A, P < 0·05.

  3. †Severe haemophilia A versus healthy males, P < 0·05.

  4. ‡NN1731 versus healthy males, P < 0·05.

  5. §NN1731 versus rFVIIa in severe haemophilia A, P < 0·05.

  6. ¶Average of n = 4, in the remaining patients the clotting time could not be defined.

Clot initiation
 CT [s]2140†¶989*165*§‡436
 95% CI551–1426140–190379–492
 SD72343§‡79
Clot propagation
 MaxVel [mm*100/s]0·9†5·2*12·2*§11·6
 95% CI0·2–1·52·3–8·29·7–14·69·3–14·0
 SD1·1†4·9*4·1*3·3
 t, MaxVel [s]2069†1240*329*§‡643
 95% CI1443–2694748–1733298–360573–713
 SD748†81552*§‡98
Clot termination
 MCF [mm]3†16*29*§‡46
 95% CI0·6–5·58·6–23·720·6–36·740·7–50·4
 SD4·213·1*14·0*‡6·7
Overall clot area
 AUEC [mm*s]/1002·9†22·5*43·1*§‡87·1
 95% CI0·5–5·37·3–37·720·2–65·969·5–104·7
 SD4·0†25·2*37·8*24·6

In conclusion, as judged from all parameters, NN1731 at 2 μg/ml appeared to have a significantly stronger haemostatic effect according to the thrombelastographic analyses than rFVIIa at 2 μg/ml (Table II).

Variability of the clotting profiles before and after ex vivo addition of rFVIIa and NN1731

In an attempt to obtain a surrogate measure of the predictability of NN1731 compared with rFVIIa, we evaluated the variability of the WB clot formation and WB clot stability as illustrated by the standard deviation in each group. Following addition of NN1731, the variation in CT and the t, MaxVel of clot formation assay were significantly lower than upon addition of rFVIIa, and NN1731 produced variability of the same order as observed in healthy individuals (Table I). No differences in the variability of the MaxVel of clot formation was observed between the tested groups (Table I). The variability of the MCF following addition of rFVIIa was significantly less than in the buffer control, and NN1731 induced a distinctly lower variability than rFVIIa and that observed amongst the healthy individuals. Analysis of WB clot stability showed that the variability of clot initiation following addition of NN1731 was lower than in the rFVIIa group as well as amongst healthy controls. Similar results were found with the t, MaxVel. Regarding the MaxVel, the variability after NN1731 or rFVIIa spiking was the same as in the healthy controls. With respect to the MCF, the variability was not different comparing patients at baseline with healthy individuals, whereas addition of rFVIIa or NN1731 increased the variability. Finally, the addition of rFVIIa or NN1731 to haemophilic WB revealed variations in the overall clot area not statistically different from that observed in the healthy controls.

Ex vivo titration with rFVIIa vs NN1731

In blood from one patient with severe haemophilia A and inhibitors, titration experiments demonstrated that NN1731 was superior to rFVIIa in terms of its ability to shorten the initiation phase as well as to accelerate the propagation phase (Fig 3, panels A and B). In blood from the patient with acquired haemophilia A, titration experiments with rFVIIa showed only modest improvements in the WB clotting profile, whereas in vitro addition of NN1731 induced complete normalisation (Fig 4).

image

Figure 3. In vitro titration experiment in blood from a patient with severe hemophilia A and inhibitors – rFVIIa vs NN1731. Tissue factor activated thromboelastographic coagulation parameters. Depicts changes in the clot initiation (clotting time) and clot propagation (maximum velocity – MaxVel) following in vitro addition of various concentrations of rFVIIa versus NN1731.

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image

Figure 4. In vitro titration experiment in blood from a patient with acquired hemophilia A – rFVIIa vs NN1731. Tissue factor activated thromboelastographic dynamic coagulation profiles. Illustrates the base-line profile as well as the changes induced by in vitro addition of rFVIIa at 1 μg/ml, 2 μg/ml, 4 μg/ml and 8 μg/ml versusin vitro addition of NN1741 at 2 μg/ml.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

The average haemostatic effect of rFVIIa in haemophilia patients with inhibitors is favourable, with reported efficacy rates around 85% in trivial bleeds and an excellent safety profile (Abshire & Kenet, 2004). However, the haemostatic response to a specified dose of rFVIIa may display considerable inter-individual variation, reducing the overall predictability of the haemostatic efficacy. In addition, in a minority of patients, even repeated high doses of rFVIIa may fail to control haemorrhage (Schneiderman et al, 2004; Martinowitz et al, 2005). For the subgroup of haemophilia patients with inhibitors who showed a reduced haemostatic effect of rFVIIa or aPCC, an analogue of rFVIIa with enhanced haemostatic potency, providing an improved therapeutic predictability and a higher efficacy rate may represent an attractive addition to the existing haemostatic armamentarium available to such patients. Experimental studies employing human haemophilic plasma (Lisman et al, 2003) and haemophilic animals (Tranholm et al, 2003) have suggested that a rFVIIa analogue, such as NN1731, may be able to close this therapeutic gap and offer a more potent haemostatic agent in treatment of haemophilia patients with inhibitors. However, studying the haemostatic effect of rFVIIa or NN1731 in plasma carries the risk of misinterpretation, leaving out the essential contribution of viable platelets and other blood cells in the overall regulation of haemostasis. Animal models also have drawbacks, since interactions between human FVIIa and animal FX (and other substrates) most probably differ from the human situation in various species. Moreover, rFVIIa (and NN1731) inhibition kinetics may be different. Studies with rFVIIa and rFVIIa analogues in certain animals required >100-fold elevated doses to observe a significant haemostatic effect (Tranholm et al, 2003) when compared with studies in human blood (Sørensen & Ingerslev, 2004a). In order to avoid many of these pitfalls and take into account the participation of human platelets in the pharmacological mechanisms of rFVIIa in haemophilia, freshly drawn WB from patients with severe haemophilia appears to be the most favourable test model. In addition, several investigations have provided a wide range of arguments for the use of low-level TF-activated thromboelastography in visualising and evaluating the haemostatic capacity of rFVIIa in various situations of compromised haemostasis(Ingerslev et al, 2003; Sørensen & Ingerslev, 2003; Sørensen et al, 2003a; Sørensen et al, 2003b; Sørensen & Ingerslev, 2004a; Sørensen & Ingerslev, 2004b; Fenger-Eriksen et al, 2005; Sørensen et al, 2005a). In contrast to previous studies in animals (Tranholm et al, 2003) and in plasma from patients (Lisman et al, 2003), the present investigation represents the first report on the haemostatic potential of the novel rFVIIa analogue NN1731 in freshly drawn WB from patients with severe haemophilia A through recording of WB clot formation and WB clot stability using thromboelastography.

In the present study, NN1731 demonstrated the capacity to induce a complete normalisation of WB clot formation in blood from several patients with well-characterised severe haemophilia A as well as in two patients with inhibitor haemophilia and acquired haemophilia A, respectively. We observed a normalised MaxVel, as well as a CT and t, MaxVel that were even slightly shorter than those seen in blood from healthy individuals. A recent report concluded that the thromboelastographic WB coagulation profile represented a useful surrogate measure of WB thrombin generation (Rivard et al, 2005; Sørensen & Ingerslev, 2005), and thus our findings indirectly point to the likelihood that the NN1731 analogue may produce a complete normalisation of thrombin generation in vivo in patients with severe haemophilia A. In addition, NN1731 increased WB clot stability in the presence of t-PA with MCF and AUEC values that were twofold higher than those obtained with rFVIIa, although complete normalisation of the resistance to t-PA-induced fibrinolysis was not reached. Eventually, NN1731 in vitro diminished the pronounced inter-person variability of the parameters associated with WB clot initiation, and it may be hypothesised that this finding mirrors an ability of NN1731 to produce a more predictable and effective haemostatic response compared with rFVIIa. A recent publication demonstrated that a shortened APTT may predict the risk of venous thrombosis (Tripodi et al, 2004), and it may be questioned whether the quite dramatic shortening of the CT of blood from severe haemophilia A patients in the presence of N1731 might reflect a potential state of hypercoagulation at the concentration level tested. Against this possible side effect is the observation that the velocity of fibrin formation was no greater than in healthy persons. However, other recent data suggest that it is not merely the shortened clotting time but also the concomitant acceleration of the clot propagation that characterise a hypercoagulable clotting profile (Sørensen et al, 2005b; Hvitfeldt et al, 2006). Dose titration studies showed saturation effects of NN1731 and rFVIIa on the time to and maximum rate of clot formation. Much lower concentrations of NN1731 were required to obtain maximal effects, albeit the differences in the maximal effects between NN1731 and rFVIIa were modest. Finally, we reported a case with acquired haemophilia A that had a very limited haemostatic response pattern to in vitro addition of rFVIIa, even at concentrations corresponding to infusion of approximately 400 μg/kg. In this case, in vitro addition of NN1731 also induced a complete normalisation the WB clotting profile.

In summary, these in vitro studies revealed an increased haemostatic potential of NN1731, a novel analogue of recombinant FVIIa, when compared with wild-type rFVIIa when added to WB from patients with severe haemophilia A as well as patients with inhibitors. In vitro addition of 2 μg/ml of NN1731 caused the CT and t, MaxVel to shorten significantly compared to non-substituted healthy male blood, whereas the MaxVel and MCF were normalised. NN1731 further normalised WB clot formation and significantly improved WB clot stability in our series of patients with severe haemophilia A in a manner that was superior to that seen with an equivalent amount of rFVIIa. Moreover, the effects of NN1731 showed less variation amongst blood from different individuals. It may be hypothesised that NN1731 might be a useful haemostatic tool, particularly when currently available drugs fail. Further investigations are warranted to assess the clinical implications of the present in vitro laboratory data.

Author statement

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

All authors have contributed in the design and performance of the research, data analyses, result interpretation, as well as the writing of the manuscript.

Financial support

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

This study was supported by an unrestricted research grant (#22-03-0386) from the Danish Research Agency. The pharmaceutical company Novo Nordisk delivered rFVIIa and rFVIIa analogue free of charge.

Conflicts of interest

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References

EP is an employee of the Novo Nordisk A/S. JI and BS have received consultancy and speakers fees from pharmaceutical companies including Novo Nordisk, Bayer, Baxter, and Wyeth.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Author statement
  7. Financial support
  8. Conflicts of interest
  9. References
  • Abshire, T. & Kenet, G. (2004) Recombinant factor VIIa: review of efficacy, dosing regimens and safety in patients with congenital and acquired factor VIII or IX inhibitors. Journal of Thrombosis and Haemostasis, 2, 899909.
  • Butenas, S., Brummel, K.E., Paradis, S.G. & Mann, K.G. (2003a) Influence of factor VIIa and phospholipids on coagulation in ‘‘acquired’’ hemophilia. Arteriosclerosis, Thrombosis, and Vascular Biology, 23, 123129.
  • Butenas, S., Brummel, K.E., Bouchard, B.A. & Mann, K.G. (2003b) How factor VIIa works in hemophilia. Journal of Thrombosis and Haemostasis, 1, 11581160.
  • Cawthern, K.M., Van't Veer, C.V., Lock, J.B., DiLorenzo, M.E., Branda, R.F. & Mann, K.G. (1998) Blood coagulation in hemophilia A and hemophilia C. Blood, 91, 45814592.
  • Ehrenforth, S., Kreuz, W., Scharrer, I., Linde, R., Funk, M., Gungor, T., Krackhardt, B. & Kornhuber, B. (1992) Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. Lancet, 339, 594598.
  • Fenger-Eriksen, C., Anker-Moller, E., Heslop, J., Ingerslev, J. & Sørensen, B. (2005) Thrombelastographic whole blood clot formation after ex vivo addition of plasma substitutes: improvements of the induced coagulopathy with fibrinogen concentrate. British Journal of Anaesthesia, 94, 324329.
  • Hoffman, M. & Monroe, III, D.M. (2001) The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis. Seminars in Hematology, 38, 69.
  • Hvitfeldt, P.L., Christiansen, K., Sorensen, B. & Ingerslev, J. (2006) Whole blood thrombelastographic coagulation profiles using minimal tissue factor activation can display hypercoagulation in thrombosis-prone patients. Scandinavian Journal of Clinical and Laboratory Investigation, 66, 329336.
  • Ingerslev, J., Feldstedt, M. & Sindet-Pedersen, S. (1991) Control of haemostasis with recombinant factor VIIa in patient with inhibitor to factor VIII. Lancet, 338, 831832.
  • Ingerslev, J., Poulsen, L.H. & Sørensen, B. (2003) Potential role of the dynamic properties of whole blood coagulation in assessment of dosage requirements in haemophilia. Haemophilia, 9, 348352.
  • Kreuz, W., Escuriola-Ettingshausen, C., Martinez-Saguer, I., Kaiml, M. & Kornhuber, B. (1999) Epidemiology of inhibitor development in haemophilia A patients treated with virus-inactivated plasma-derived clotting factor concentrates. Vox Sanguinis, 77(Suppl. 1), 38.
  • Lisman, T., De Groot, P.G., Lambert, T., Rojkjaer, R. & Persson, E. (2003) Enhanced in vitro procoagulant and antifibrinolytic potential of superactive variants of recombinant factor VIIa in severe hemophilia A. Journal of Thrombosis and Haemostasis, 1, 21752178.
  • Ljung, R., Petrini, P., Lindgren, A.C., Tengborn, L. & Nilsson, I.M. (1992) Factor VIII and factor IX inhibitors in haemophiliacs. Lancet, 339, 1550.
  • Martinowitz, U., Livnat, T., Zivelin, A., Luboshitz, J., Seligsohn, U. & Kenet, G. (2005) Concomitant infusion of low doses of rFVIIa and FEIBA in severe haemophilia A patients with an inhibitor to factor VIII who are refractory to rFVIIa or FEIBA has an excellent haemostatic effect and synergistically enhances thrombin generation. Journal of Thrombosis and Haemostasis, 3, P0637 (Abstract).
  • Persson, E. & Olsen, O.H. (2002) Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes. European Journal of Biochemistry, 269, 59505955.
  • Persson, E., Bak, H. & Olsen, O.H. (2001a) Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity. Journal of Biological Chemistry, 276, 2919529199.
  • Persson, E., Kjalke, M. & Olsen, O.H. (2001b) Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proceedings of the National Academy of Sciences of the United States of America, 98, 1358313588.
  • Persson, E., Bak, H., Ostergaard, A. & Olsen, O.H. (2004) Augmented intrinsic activity of Factor VIIa by replacement of residues 305, 314, 337 and 374: evidence of two unique mutational mechanisms of activity enhancement. Biochemical Journal, 379, 497503.
  • Polgar, J., Matuskova, J. & Wagner, D.D. (2005) The P-selectin, tissue factor, coagulation triad. Journal of Thrombosis and Haemostasis, 3, 15901596.
  • Rand, M.D., Lock, J.B., Van't Veer, C., Gaffney, D.P. & Mann, K.G. (1996) Blood clotting in minimally altered whole blood. Blood, 88, 34323445.
  • Rivard, G.E., Brummel-Ziedins, K.E., Mann, K.G., Fan, L., Hofer, A. & Cohen, E. (2005) Evaluation of the profile of thrombin generation during the process of whole blood clotting as assessed by thrombelastography. Journal of Thrombosis and Haemostasis, 3, 20392043.
  • Scharrer, I., Bray, G.L. & Neutzling, O. (1999) Incidence of inhibitors in haemophilia A patients–a review of recent studies of recombinant and plasma-derived factor VIII concentrates. Haemophilia, 5, 145154.
  • Schneiderman, J., Nugent, D.J. & Young, G. (2004) Sequential therapy with activated prothrombin complex concentrate and recombinant factor VIIa in patients with severe haemophilia and inhibitors. Haemophilia, 10, 347351.
  • Sørensen, B. & Ingerslev, J. (2003) Potential effect of recombinant factor VIIa in ex vivo reversal of pentasaccharide induced anticoagulation. Journal of Thrombosis and Haemostasis, 1, P1108 (Abstract).
  • Sørensen, B. & Ingerslev, J. (2004a) Whole blood clot formation phenotypes in hemophilia A and rare coagulation disorders. Patterns of response to recombinant factor VIIa. Journal of Thrombosis and Haemostasis, 2, 102110.
  • Sørensen, B. & Ingerslev, J. (2004b) Thromboelastography and recombinant factor VIIa-hemophilia and beyond. Seminars in Hematology, 41(Suppl. 1), 140144.
  • Sørensen, B. & Ingerslev, J. (2005) Tailoring haemostatic treatment to patient requirements – an update on monitoring haemostatic response using thrombelastography. Haemophilia, 11(Suppl. 1), 16.
  • Sørensen, B., Johansen, P., Christiansen, K., Wöelke, M. & Ingerslev, J. (2003a) Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. Journal of Thrombosis and Haemostasis, 1, 551558.
  • Sørensen, B., Johansen, P., Nielsen, G.L., Sorensen, J.C. & Ingerslev, J. (2003b) Reversal of the International Normalized Ratio with recombinant activated factor VII in central nervous system bleeding during warfarin thromboprophylaxis: clinical and biochemical aspects. Blood Coagulation and Fibrinolysis, 14, 469477.
  • Sørensen, B., Fenger-Eriksen, C. & Ingerslev, J. (2005a) Recombinant factor VIIa fails to correct coagulopathy induced by haemodilution with colloid. British Journal of Anaesthesia, 94, 862863.
  • Sørensen, B., Johansen, P., Christiansen, K., Norengaard, L., Larsen, O.H., Hvas, A.M. & Ingerslev, J. (2005b) What the standard APTT didn't tell you – dynamic plasma clotting parameters reveal hypercoagulation. Blood, 106, 739740 ( Abstract no. 2634).
  • Tranholm, M., Kristensen, K., Kristensen, A.T., Pyke, C., Rojkjaer, R. & Persson, E. (2003) Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A. Blood, 102, 36153620.
  • Tripodi, A., Chantarangkul, V., Martinelli, I., Bucciarelli, P. & Mannucci, P.M. (2004) A shortened activated partial thromboplastin time is associated with the risk of venous thromboembolism. Blood, 104, 36313634.
  • Wight, J. & Paisley, S. (2003) The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia, 9, 418435.