Research articles
 

By Dr. Juergen R Roemisch , Dr. Katharina Pock , Ms. Isabella Laher-kheirallah , Mrs. Sandra Janisch , Dr. Olaf Walter , Dr. Sigurd Knaub , Dr. Erik Berntorp
Corresponding Author Dr. Juergen R Roemisch
Research & Development Octapharma PPGmbH, Oberlaaer Strasse 235 - Austria 1100
Submitting Author Dr. Juergen R Roemisch
Other Authors Dr. Katharina Pock
Octapharma PPGmbH, Research & Development, Oberlaaer Strasse 235 - Austria 1100

Ms. Isabella Laher-kheirallah
Octapharama PPGmbH, Research & Development, Oberlaaer Strasse 235 - Austria 1100

Mrs. Sandra Janisch
Octapharma PPGmbH, Research & Development, Oberlaaer Strasse 235 - Austria 1100

Dr. Olaf Walter
Octapharma AG, International Business Units, Seidenstrasse 2 - Switzerland 8853

Dr. Sigurd Knaub
Octapharma AG, Clinical R&D Haematology, Seiden strasse 2 - Switzerland 8853

Dr. Erik Berntorp
Skane University Hospital, Centre for Thrombosis and Haemostasis, Malmö - Sweden 20502

HAEMATOLOGY

FVIII/VWF concentrate, Non-activated prothrombin complex concentrate, Factor VIII inhibitors, Haemophilia A, Thrombin generation, Octaplex, Octanate, Wilate

Roemisch JR, Pock K, Laher-kheirallah I, Janisch S, Walter O, Knaub S, et al. A Novel Approach to Treating FVIII Inhibitors: In Vitro Results for Combination Therapy with Human Prothrombin Complex Concentrate and Plasma-Derived FVIII/VWF Complex. WebmedCentral HAEMATOLOGY 2011;2(11):WMC002444
doi: 10.9754/journal.wmc.2011.002444
No
Submitted on: 18 Nov 2011 10:39:19 AM GMT
Published on: 18 Nov 2011 04:01:42 PM GMT

Abstract


Coagulation factor VIII (FVIII) inhibitors are the most severe complications of haemophilia A treatment. We studied the effect of a non-activated prothrombin concentrate (PCC) combined with FVIII in vitro. FVIII antibodies minimised the in vitro thrombin generation of standard human plasma and prolonged the lag phase at a residual FVIII activity of ≤ 0.01 IU/ml. Time to clotting of FVIII-inhibited fresh whole blood was not measurable until 55 minutes (observation time). Combining a PCC and a plasma-derived FVIII/VWF concentrate normalised TG and ROTEM® parameters. Our findings support an approach to moderately shift the haemostatic balance to a more procoagulant potential.

Introduction


Haemophilia A (HA) is caused by a deficiency in the amount or activity of coagulation factor VIII (FVIII) in plasma and is largely managed through the administration of plasma-derived or recombinant human FVIII concentrates. Inhibitory antibodies to exogenously administered FVIII can develop, thereby neutralising its therapeutic effect and resulting in severe bleeding complications. Inhibitory antibodies represent the most significant clinical complication in the modern day treatment of HA [1]. In patients who develop inhibitory antibodies, prophylaxis with FVIII cannot be performed until complete and sustained elimination of the inhibitor has been achieved. As a great deal of clinical progress has been made in recent years in the management of HA characterised by persistent FVIII inhibitors, several treatment options are now available in the armamentarium [2-4]. Immediate and persistent initiation of immune tolerance induction (ITI) therapy, involving repeated FVIII dosing over months or years, is the treatment of choice and the only proven strategy for achieving sustained inhibitor eradication [5]. ITI has generally been more successful when FVIII concentrates containing von Willebrand factor (VWF) were used [5-7]. Besides its direct haemostatic role, VWF has a key function in the protection, transport and presentation of FVIII with respect to the immune system, and this is an important area of current research. For the patients who do not respond to ITI treatment alone, bypassing agents represent the next therapeutic step. These bypassing agents include non-activated prothrombin complex concentrates (PCCs), activated PCCs (aPCCs) and recombinant factor VIIa (rFVIIa) [3,4]. Several case reports and in vitro studies indicate that combining different types of bypassing agents, either sequentially or as combination therapy, can be an efficacious way to treat monotherapy-resistant inhibitors [8-15]. In particular Klintman and colleagues showed in vitro that combining any two of aPCC, rFVIIa and FVIII concentrates resulted in an additive effect on thrombin production [15]. While aPCCs and rFVIIa are well established for patients displaying FVIII inhibitors, some individuals are refractory to these treatments, and although thrombotic side effects are rare, a potential risk remains with the application of high concentrations of activated coagulation factors [16-18]. Moreover, the high cost of these products limits their availability in some patient populations and countries. In clinical practice, the successful use of non-activated PCCs has been demonstrated in many cases throughout the last decades to counteract inhibitor-associated bleeding complications [19,20]. Human non-activated PCC (Octaplex® [Octapharma PPGmbH, Vienna, Austria]), which contains a balanced ratio of the coagulation factors II, VII, IX and X and the anticoagulant proteins C and S, is currently licensed for use in perioperative prophylaxis and treatment of bleeding following treatment or overdose with vitamin K antagonists and for congenital deficiencies of factors II and X when specific factor products are not available [21-23]. Following encouraging results with this PCC in reducing bleeding complications of HA patients displaying high-titre inhibitors, we were interested to establish whether plasma-derived (pd)FVIII/VWF concentrates may confer an additional clinical advantage when used as a supplementary add-on therapy to PCC in acute bleeding situations [24]. Using a thrombin generation (TG) assay [25-27], thrombelastometry (ROTEM®) [28,29] and quantification of FVIII activity (FVIII:C), we investigated the effect of spiking samples with both the PCC and pdFVIII/VWF concentrates (Octanate® or Wilate®), while also experimentally adding FVIII antibodies to standard human plasma (SHP) or patient-derived plasma samples.

Material and Methods


FVIII: C quantification for inhibitor determination
Inhibitory activity was determined using the FVIII chromogenic method and an inhibitor reagent kit (Technoclone GmbH, Vienna, Austria).
Residual FVIII: C activities after incubation
FVIII activity was determined in the low range, 0.088-0.006 IU FVIII:C/ml using the chromogenic assay (Coamatic FVIII from Chromogenix [IL Spa, Milano, Italy]) in a microplate format as follows: an adapted standard curve was prepared between 0.006 and 0.088 IU FVIII:C/ml from dilutions of standard human plasma (SHP) (Siemens Healthcare Diagnostics [SHD] GmbH, Marburg, Germany) calibrated against fresh normal plasma and the buffer blank. The samples were diluted, if necessary, into the range of the standard curve. Control SHP calibrated against fresh normal plasma served as control. The incubation steps and the final dilution for samples, standards and controls were performed according to the manufacturers’ instructions. The absorbance was read at 405 nm.
Thrombin generation assay
Thrombin generation was measured using the Technothrombin TGA kit and RC high reagent (Technoclone GmbH, Vienna, Austria). The readout parameters were lag time (phase) and peak thrombin concentration. To mimic plasma with a FVIII inhibitor, monoclonal antibodies (mAb, ESH-4 and ESH-8, American Diagnostica Inc., Stamford, USA) and/or polyclonal (pAb) sheep FVIII antibodies (Enzyme Research Laboratories Ltd., South Bend, USA) were added to SHP in order to achieve final inhibitory activities of 10 and 31 Bethesda Units (BU)/ml. Accordingly, 100 µl SHP or whole blood were each spiked with 2 µl ESH-4 and ESH-8, resulting in final concentrations of 20 µg/ml. The pAb was added at 3 µl to achieve a final plasma concentration of 30 µg/ml. To 80 µl of the spiked SHP, 20 µl of a preparation containing the human PCC (Octaplex® [Octapharma PPGmbH, Vienna, Austria]) and/or a pdFVIII/VWF concentrate (Octanate® or Wilate® [Octapharma PPGmbH, Vienna, Austria]) or aPCC (FEIBA®, Baxter AG, Vienna, Austria), rFVIIa (NovoSeven® [Novo Nordisk A/S, Glostrup, Denmark]) or sodium chloride (NaCl) control solution, were added to obtain a final sample volume of 100 µl. In general, SHP with or without spiked antibodies were incubated for 1 hour at 37°C. These conditions were found to result in the maximum inhibitory capacity.
For experiments performed in the presence of platelets, controls without platelets were run in parallel. Lyophilised platelets (MöLab GmbH, Langenfeld, Germany) were reconstituted in 5 ml diluent and added to plasma (1 part plasma and 1 part platelets) to achieve a platelet count of 200,000/µl (± 10%), resulting in a corresponding plasma dilution. Inhibition of FVIII was performed in the presence and absence of platelets (1 hour, 37°C) so as to exclude any FVIII-related activity displayed by the reconstituted platelets themselves.
The FVIII/VWF concentrate was reconstituted according to the manufacturer’s instructions to obtain a solution of 100 IU FVIII:C/ml.
The PCC was reconstituted with water for injection to a FIX concentration of 25 IU/ml. Because the PCC used contains an average of 0.45 IU heparin per 1 IU FIX (with low variation; 0.2-0.5 IU heparin/IU FIX according to specification), pre-experiments were performed including the heparin-free in-process bulk solution to exclude any significant impact on test parameters [30]. In general, the PCC concentrations indicated refer to the final FIX concentrations added. A concentration of 0.25 IU FIX/ml (corresponding to < 0.12 IU heparin/ml) in the assay neither prolonged the lag phase nor reduced the peak TG (data not shown). No impact was observed on the ROTEM® results. As expected, the PCC at 0.5 IU FIX/ml showed some impact and, therefore, the heparin-free in-process solution was used for studies ? 0.5 IU FIX/ml.
The PCC and pdFVIII/VWF concentrate were not mixed before spiking in the experiments illustrated here, but were added to samples separately.
Lag phase (minutes) and peak thrombin concentration (nM) were assessed for the PCC and pdFVIII/VWF concentrate separately and in combination in SHP containing inhibitors. TG assays were performed for the pdFVIII/VWF at FVIII concentrations of 0.1, 0.3, 0.5, 0.75, 1.0 or 1.5 IU/ml. In addition, the PCC was investigated separately at a concentration of 0.25 IU/ml, and combined with 0.5 IU/ml pdFVIII/VWF (Octanate® or Wilate®). In the absence of haemostatic treatments, TG parameters were investigated for SHP with or without pAb/mAb inhibitors, and as controls, in SHP containing platelets with or without pAb/mAb inhibitors.
In addition, in vitro/ex vivo effects of aPCC using FEIBA® (2 U/ml) or rFVIIa (2µg/ml) in inhibitor plasmas were studied, without or in combination with 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF in the presence of platelets.
Kinetics of FVIII inhibition after addition of PCC and pdFVIII/VWF
After 1 hour incubation of antibody-spiked SHP, lag phases and peak thrombin concentrations were assessed at baseline and after addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF. FVIII:C was determined in parallel. These parameters were assessed for the following incubation times of this combination of concentrates: 0, 15, 30, 45, and 60 minutes. SHP with and without inhibitors were run as controls.
Rotation thrombelastometry (ROTEM®) of whole blood
ROTEM® (TEM Innovations GmbH, Munich, Germany) was performed at 37°C using the NATEM® system according to the manufacturer’s description. pAb was added to freshly drawn whole blood samples after a resting time of 30 minutes at room temperature, and incubated for 1 hour at 37°C in order to achieve a final inhibitory activity of 31 BU/ml. Samples for ROTEM®/NATEM® analysis were treated with the following haemostatic interventions: 0.5 IU/ml PCC and 1.0 IU/ml pdFVIII/VWF or 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF. Samples were analysed at the following time points after the addition of the two concentrates: 0, 5, 15, 30, 45 and 60 minutes. Whole blood samples with and without antibodies were run as controls.
Ex vivo studies
Plasma samples obtained from HA patients were obtained and stored as described previously [31]. The inhibitory potencies of the four individuals ranged from 30 to 159 BU/ml. TG parameters with or without addition of 0.5 IU/ml PCC and 0.5 IU/ml or 1.0 IU/ml pdFVIII/VWF, FEIBA® or rFVIIa were investigated.

Results


Impact of pdFVIII/VWF and PCC/pdFVIII/VWF on TG parameters in plasma with inhibitors
As characteristic of these studies, the addition of pAb or pAb/mAb to SHP resulted in the minimisation of TG and a prolongation of the lag phase. In the presence of platelets, these effects were still considerable, although thrombin generation was not completely eradicated (Illustration 1). After incubation of SHP and platelets with antibodies, the addition of pdFVIII/VWF within the concentration range of 0.1-1.5 IU/ml resulted in a general trend towards reduced lag time with increasing pdFVIII/VWF concentrations, but this did not reach the control level (SHP, Illustration 1, Panel A and platelets). Peak thrombin concentrations increased with higher pdFVIII/VWF level, but reached a plateau at 0.5 IU/ml FVIII corresponding to approximately half of that observed for the control (Illustration 1, Panel B). The addition of 0.25 IU/ml PCC to SHP with platelets containing pAb/mAb normalised the lag time, whereas peak thrombin concentration was significantly improved but not normalised. The addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF from Octanate® resulted in normalisation of both lag time and peak thrombin concentration. Also, the addition of the other pdFVIII/VWF product (Wilate®) to the PCC revealed comparable effects at the same FVIII:C concentrations (data not shown).
Correspondingly, ROTEM® analysis performed with freshly drawn blood showed prevention of clot formation upon addition of FVIII antibodies. Normalisation of ROTEM® parameters was achieved after addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF (Illustration 2; Panels A, B and C).
Kinetics of FVIII inhibition after addition of PCC/pdFVIII/VWF: Impact on TG and clot formation (ROTEM®)
SHP incubation in the presence of platelets and after addition of pAb showed a minimum residual FVIII:C of 0.01 IU/ml after 60 minutes at 37°C, which reproducibly did not further decrease upon longer incubation times (up to 180 minutes, data not shown). Accordingly, this procedure was defined as the standard pre-incubation time.
After standard pre-incubation of SHP with pAb in the presence of added platelets, 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF was added and aliquots were drawn for immediate measurements of FVIII:C activity and TG at time point 0 and up to 60 minutes after addition. According to the FVIII:C added (0.5 IU/ml) and the residual activity after inhibitor incubation, a calculated FVIII:C of 0.51 IU/ml resulted after addition of PCC/pdFVIII/VWF. Immediate measurement repeatedly resulted in residual FVIII:C of 0.26 to 0.29 IU/ml, which decreased to 0.08-0.10 IU/ml and 0.05 IU/ml after 15 and 30 minutes, respectively, reaching a stable baseline activity of 0.03 IU/ml at 60 minutes (Illustration 3; Panel A).
TG assays revealed that lag time in SHP containing pAb was severely prolonged (55 minutes until the initiation of clotting) (Illustration 3; Panel A). Incubation of SHP containing pAb inhibitors with 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF for 0 and 15 minutes resulted in normalisation of the lag phase (effect was greatest at 0 minutes). Although longer incubation times with this combination of concentrates were associated with a lengthening of the lag phase, it was still in the range of 20 to 25 minutes. Incubation with pAb eliminated thrombin generation in SHP (Illustration 3; Panel B). Subsequent addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF resulted in a peak thrombin concentration close to SHP at time point 0 minutes, declining to about 30% of SHP after 10 minutes and to 10% after 60 minutes.
ROTEM® of whole blood of healthy donors revealed no onset of clot formation after incubation with pAb for up to 55 minutes of measurement. Addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF completely restored the clotting time (CT) and maximum clot strength (MCF) as compared to the whole blood control at time point 0 minutes (Illustration 2; Panel A). A higher concentration of this combination of concentrates (0.5 IU/ml PCC and 1.0 IU/ml pdFVIII/VWF) essentially did not affect ROTEM® variables relative to the lower concentration (data not shown). After 15 minutes incubation and subsequent ROTEM® performance, the CT was prolonged by 2-2.5 fold compared with immediate measurement or whole blood control, respectively (Illustration 2; Panel D). The MCFs remained in the normal range. After 60 minutes, the CT was prolonged 3-fold, correspondingly (Illustration 2; Panel E).
Impact on TG parameters of PCC/pdFVIII/VWF compared with aPCC and rFVIIa
TG assays were performed in order to assess the haemostatic effects of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF, aPCC and rFVIIa after addition to SHP/platelets containing pAb/mAb. rFVIIa shortened the lag time somewhat below the SHP/platelet control, but did not normalise peak thrombin concentration (Illustration 4; Panels A, B). In contrast, the combination of PCC and pdFVIII/VWF normalised both parameters. aPCC revealed a significant shortening of the lag phase at the concentration used, while peak thrombin concentration was in the normal range. Combining PCC/pdFVIII/VWF with rFVIIa or aPCC showed further moderate shortening of lag times and increases in peak thrombin concentrations compared with experiments where these components were evaluated separately.
Plasma obtained from HA patients with FVIII inhibitors (an example is presented in Illustration 5; 34 BU/ml) was used in TG assays to assess the effects of the following haemostatic interventions: 0.5 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF; 0.5 IU/ml PCC and 1.0 IU/ml FVIII; rFVIIa (2µg/ml); and aPCC (2 U/ml). In the absence of haemostatic treatments, TG was severely impaired relative to SHP containing platelets, as expected. Both PCC/pdFVIII/VWF combinations normalised lag times and peak thrombin concentrations. Compared to SHP, the area under the concentration-time curve (AUC) values were increased by this combination of factors. This was even more pronounced with aPCC and was accompanied by a shorter lag phase in the example presented. rFVIIa (2 mg/ml) elicited a poor TG response in the HA patient-derived inhibitor plasma. In contrast, addition of 2 U/ml aPCC (plasma level reached upon standard treatment with aPCC) resulted in a very pronounced thrombin generation. The AUC profile for aPCC was notably greater than that of the control condition (consisting of SHP not containing FVIII inhibitors).
All HA inhibitor plasmas investigated so far responded to PCC (applied separately) with regard to TG parameters and showed more pronounced effects to PCC/pdFVIII/VWF spiking, although different individual responses were observed, e.g. in the extent of thrombin generation induced by aPCC (data not shown).
Platelets contribute to the effect of PCC/pdFVIII/VWF on TG  in SHP in the presence of pAb/mAb
In order to evaluate the contribution of platelets in the described setting, TG assays were performed using SHP containing pAb/mAb. In contrast to the experiments performed with pAb alone (data not shown), platelets were required for the PCC/pdFVIII/VWF combination to bring peak thrombin concentrations into the normal range. In contrast, conditions without platelets were associated with only a low response (Illustration 6). There was no significant difference between thrombin generation of SHP and SHP/platelets.

Discussions and Conclusion


In HA patients with inhibitors, recent data suggest that the PCC used in this study may be a useful alternative to aPCCs or rFVIIa as a bypassing agent [24]. The data presented in this in vitro/ex vivo study demonstrate that PCC, given in combination with  pdFVIII/VWF concentrates (Octanate® or Wilate®), results in normalisation of plasma/platelet TG. This held true for experimentally (FVIII) inhibited normal plasma as well as for the investigated HA inhibitor plasmas.
Both assay systems, TG and ROTEM®, have been extensively investigated for monitoring of aPCC and rFVIIa effects in the treatment of HA patients with specific factor inhibitors and rare coagulation disorders [16,32-34]. In the present study, ROTEM® analyses supplemented the data gained by the TG assay, showing that clotting activity was eliminated in whole blood containing pAb, and application of a combination of PCC and pdFVIII/VWF restored clot formation parameters in vitro.
In general, rFVIIa alone confirmed the poor in vitro response reported by other working groups [35-37], even in the presence of platelets, though rFVIIa has been shown to be clinically effective [38]. It thus poorly facilitates a prediction of in vivo effects. In contrast, aPCC application resulted in the expected pronounced shortening of lag time and increased peak thrombin concentration [39]. The in vitro/ex vivo effects observed upon combination of PCC/pdFVIII/VWF and rFVIIa or aPCC appeared not to be synergistic, but this will have to be carefully observed clinically with regard to thromboembolic adverse events if used to treat emergency bleeding episodes.
Based on the fact that non-activated PCCs show a clinical usefulness for treating bleeding events in HA inhibitor patients, cases responding poorly to monotherapy of either PCC, aPCC or rFVIIa may become of particular interest for a potential PCC/pdFVIII/VWF combination treatment. In vitro results indicated that even a moderate increase in non-activated clotting factors can shift the haemostatic balance to a more procoagulant state, which can be brought about via  intervention with a PCC. It is plausible that application of exogenous FVIII may drive coagulation if exceeding the total inhibitor capacity or if escaping the immediate neutralisation by the antibodies. The significant haemostasis enhancing effect of even very low FVIII:C is known, and thus the kinetics of FVIII inhibition are decisive for the extent and duration of cofactor effect and treatment efficacy. Therefore, we used the model to mimic moderate to high FVIII inhibitory capacities in plasma by spiking samples with pAb or a mixture of pAb/mAb. Epitope specificities and neutralising capabilities of the latter were reported by another working group [40].
After inhibition of FVIII by a 1–hour incubation with pAb only, about 1% of residual FVIII:C was measured. Addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF, immediate mixing and dilution for measurement, provoked a 40-50% increase in FVIII:C. Activities decreased with time, but even after 30 and 45 minutes, residual activities of 5% and 3%, respectively, were quantified. Peak thrombin concentrations remained in the normal to lower–limit–of–normal range, respectively, until 15 minutes after application. Minimum values were approached by 60 minutes; however, in the presence of platelets these parameters remained clearly measurable above baseline. Although ROTEM® results were obtained with whole blood, we utilised this methodology following inhibition of FVIII by the addition of antibodies (as in the TG kinetic studies) to demonstrate that small amounts of thrombin that are generated are sufficient to drive clot formation, as indicated in the plasma/platelet system. Taking into account that the plasma was diluted by 50% by platelet addition, extrapolated peak thrombin concentrations in whole plasma of equal or less than 60 nM should be sufficient to initiate clot formation, as observed 60 minutes after addition of PCC and pdFVIII/VWF. This is in agreement with recent publications suggesting that initial thrombin concentrations as low as 20-30 nM are sufficient for initiation of clotting, while the subsequent thrombin boost may be required for stability of the clot [41,42]. Nevertheless, at present there is no proven general threshold of peak thrombin generation sufficient to stop bleeding in HA inhibitor patients. Individual clinical conditions have to be considered.
The corresponding lag times increased with decreasing FVIII:C and peak thrombin concentrations over time, as expected. Notably, they did not reach the level of the inhibited plasma/platelet control, reflecting the basic effect of PCC in decreasing FVIII:C, which is in line with the ROTEM® results.
The experiments performed in this study underline the pivotal role of platelets in PCC/pdFVIII/VWF-mediated clotting activity and thrombin generation. Following treatment with the combination of PCC and pdFVIII/VWF, substantial clotting activity was observed in SHP containing lyophilised platelets with pAb/mAb inhibitors. However, this was not the case for conditions with no platelets, which warrants further investigation. Experiments in this study were performed using lyophilised platelets, which could be viewed as a consequential limitation of the results. While the lyophilised platelets have many of the molecular functions of fresh platelets, they do not perfectly reflect the multi-facetted functionality of viable fresh platelets, and they were not particular to the specific patients’ samples [43-45]. Nevertheless, the lyophilised platelets across all experiments here came from the same standardised batch. This standardisation of platelets thereby removed inter-patient or inter-batch platelet variability otherwise underlying differences in the results from the experimental samples.
Thrombin generation and clot formation were normalised for as long as 15 minutes after application of PCC/pdFVIII/VWF, remaining measurable until 60 minutes (limit of observation period). Other inhibitor profiles will also certainly have an influence on FVIII inhibition kinetics. However, coagulation factors present in the PCC should remain present over a longer period of time relative to the decreasing FVIII:C following application of PCC and pdFVIII/VWF. This longer-lasting presence should hypothetically safeguard against recurrent bleeds [14,46,47].
The HA inhibitor patient samples spiked with platelets and PCC/pdFVIII/VWF revealed an excellent ex vivo response in all cases investigated. The addition of 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF concentrate (Octanate® or Wilate®), administered as separate preparations (i.e. not in immediate combination), would appear to be a reasonable starting point for clinical trials.
In conclusion, the in vitro/ex vivo results reported here are encouraging and support the investigation of the combination of PCC and pdFVIII/VWF in a proof-of-principle clinical trial, in order to further establish the viability of this approach as an effective and safe strategy for the treatment of bleeding events in HA inhibitor patients.

Acknowledgements


The authors would like to thank Margoth Gunnarsson and Anne-Marie Thämlitz (Wallenberg Laboratory, Malmö University Hospital). Medical writing services were provided by nspm ltd, Meggen, Switzerland, with financial support from Octapharma AG, Lachen, Switzerland.

Conflict of Interest Statement


Erik Berntorp has received speaker fees from Octapharma. Juergen Roemisch, Katharina  Pock, Isabella Laher-Kheirallah, Sandra Janisch, Olaf Walter and Sigurd Knaub are employees of Octapharma.

References


1. Gouw SC, van der Bom JG, van den Berg M. Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study. Blood 2007; 109:4648-4654.
2. Astermark J, Donfield SM, DiMichele DM, Gringeri A, Gilbert SA, Waters J, Berntorp E. A randomized comparison of bypassing agents in hemophilia complicated by an inhibitor: the FEIBA NovoSeven Comparative (FENOC) Study. Blood 2007; 109:546-551.
3. Ingerslev J. Hemophilia. Strategies for the treatment of inhibitor patients. Haematologica 2000; 85:15-20.
4. Lusher JM. Inhibitor antibodies to factor VIII and factor IX: management. Semin Thromb Hemost 2000; 26:179-188.
5. Escuriola-Ettingshausen C, Kreuz W. Role of von Willebrand factor in immune tolerance induction. Blood Coagul Fibrinolysis 2005; 16 Suppl 1:S27-S31.
6. Goudemand J, Rothschild C, Demiguel V, Vinciguerrat C, Lambert T, Chambost H, Borel-Derlon A, Claeyssens S, Laurian Y, Calvez T. Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A. Blood 2006; 107:46-51.
7. Kreuz W, Escuriola-Ettinghausen C, Auerswald G, Heidemann P, Kemkes-Matthes B, Schneppenheim R, Behnisch W, Kobelt R, Martinez Saguer I, Mentzer D, Gnekow A, Klingebiel T. Immune tolerance induction (ITI) in haemophilia A - patients with inhibitors- the choice of concentrate affecting success. Haematologica 2001; 86:16-22.
8. Key NS, Christie B, Henderson N, Nelsestuen GL. Possible synergy between recombinant factor VIIa and prothrombin complex concentrate in hemophilia therapy. Thromb Haemost 2002; 88:60-65.
9. Schneiderman J, Rubin E, Nugent DJ, Young G. Sequential therapy with activated prothrombin complex concentrates and recombinant FVIIa in patients with severe haemophilia and inhibitors: update of our previous experience. Haemophilia 2007; 13:244-248.
10. Livnat T, Martinowitz U, Zivelin A, Seligsohn U. Effects of factor VIII inhibitor bypassing activity (FEIBA), recombinant factor VIIa or both on thrombin generation in normal and haemophilia A plasma. Haemophilia 2008; 14:782-786.
11. van Veen JJ, Gatt A, Bowyer AE, Cooper PC, Kitchen S, Makris M. The effect of tissue factor concentration on calibrated automated thrombography in the presence of inhibitor bypass agents. Int J Lab Hematol 2009; 31:189-198.
12. Tomokiyo K, Nakatomi Y, Araki T, Teshima K, Nakano H, Nakagaki T, Miyamoto S, Funatsu A, Iwanaga S. A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone. Vox Sang 2003; 85:290-299.
13. Habermann B, Hochmuth K, Hovy L, Scharrer I, Kurth AH. Management of haemophilic patients with inhibitors in major orthopaedic surgery by immunadsorption, substitution of factor VIII and recombinant factor VIIa (NovoSeven): a single centre experience. Haemophilia 2004; 10:705-712.
14. Dargaud Y, Lienhart A, Meunier S, Hequet O, Chavanne H, Chamouard V, Marin S, Negrier C. Major surgery in a severe haemophilia A patient with high titre inhibitor: use of the thrombin generation test in the therapeutic decision. Haemophilia 2005; 11:552-558.
15. Klintman J, Astermark J, Berntorp E. Combination of FVIII and by-passing agent potentiates in vitro thrombin production in haemophilia A inhibitor plasma. Br J Haematol 2010; 151:381-386.
16. Hayashi T, Tanaka I, Shima M, Yoshida K, Fukuda K, Sakurai Y, Matsumoto T, Giddings JC, Yoshioka A. Unresponsiveness to factor VIII inhibitor bypassing agents during haemostatic treatment for life-threatening massive bleeding in a patient with haemophilia A and a high responding inhibitor. Haemophilia 2004; 10:397-400.
17. Astermark J, Morado M, Rocino A, van den Berg HM, Von DM, Gringeri A, Mantovani L, Garrido RP, Schiavoni M, Villar A, Windyga J. Current European practice in immune tolerance induction therapy in patients with haemophilia and inhibitors. Haemophilia 2006; 12:363-371.
18. Sarode R, Matevosyan K. Prothrombin complex concentrate and fatal thrombosis: poor evidence to implicate a relatively safe drug. Ann Emerg Med 2009; 54:481-482.
19. Abildgaard CF, Britton M, Harrison J. Prothrombin complex concentrate (Konyne) in the treatment of hemophilic patients with factor VIII inhibitors. J Pediatr 1976; 88:200-205.
20. Lusher JM, Shapiro SS, Palascak JE, Rao AV, Levine PH, Blatt PM. Efficacy of prothrombin-complex concentrates in hemophiliacs with antibodies to factor VIII: a multicenter therapeutic trial. New Engl J Med 1980; 303:421-425.
21. Franken T, Rees W, Hubner N, et al. Octaplex in routine clinical use for prophylaxis and therapy of bleeding in patients with prothrombin complex factor deficiency [abstract]. Crit Care 2007;11(Suppl 2):376.
22. Lubetsky A, Hoffman R, Zimlichman R, Eldor A, Zvi J, Kostenko V, Brenner B. Efficacy and safety of a prothrombin complex concentrate (Octaplex) for rapid reversal of oral anticoagulation. Thromb Res 2004; 113:371-378.
23. Riess HB, Meier-Hellmann A, Motsch J, Elias M, Kursten FW, Dempfle CE. Prothrombin complex concentrate (Octaplex) in patients requiring immediate reversal of oral anticoagulation. Thromb Res 2007; 121:9-16.
24. Berntorp E, Figueiredo S, Futema L, Pock K, Knaub S, Walter O, Trawnicek L, Romisch J. A retrospective study of Octaplex in the treatment of bleeding in patients with haemophilia A complicated by inhibitors. Blood Coagul Fibrinolysis 2010; 21:577-583.
25. Berntorp E, Salvagno GL. Standardization and clinical utility of thrombin-generation assays. Semin Thromb Hemost 2008; 34:670-682.
26. Dargaud Y, Beguin S, Lienhart A, Al DR, Trzeciak C, Bordet JC, Hemker HC, Negrier C. Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B. Thromb Haemost 2005; 93:475-480.
27. Hemker HC, Al DR, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hematol 2004; 11:170-175.
28. Luddington RJ. Thrombelastography/thromboelastometry. Clin Lab Haematol 2005; 27:81-90.
29. Reikvam H, Steien E, Hauge B, Liseth K, Hagen KG, Storkson R, Hervig T. Thrombelastography. Transfus Apher Sci 2009; 40:119-123.
30. Gilmore R, Harmon S, Keane G, Gannon C, O'Donnell JS. Variation in anticoagulant composition regulates differential effects of prothrombin complex concentrates on thrombin generation. J Thromb Haemost 2009; 7:2154-2156.
31. Salvagno GL, Astermark J, Lippi G, Ekman M, Franchini M, Guidi GC, Berntorp E. Thrombin generation assay: a useful routine check-up tool in the management of patients with haemophilia? Haemophilia 2009; 15:290-296.
32. Sorensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost 2003; 1:551-558.
33. Sorensen B, Ingerslev J. Whole blood clot formation phenotypes in hemophilia A and rare coagulation disorders. Patterns of response to recombinant factor VIIa. J Thromb Haemost 2004; 2:102-110.
34. Yoshioka A, Nishio K, Shima M. Thrombelastgram as a hemostatic monitor during recombinant factor VIIa treatment in hemophilia A patients with inhibitor to factor VIII. Haemostasis 1996; 26 Suppl 1:139-142.
35. Allen GA, Hoffman M, Roberts HR, Monroe DM. Manipulation of prothrombin concentration improves response to high-dose factor VIIa in a cell-based model of haemophilia. Br J Haematol 2006; 134:314-319.
36. Butenas S, Brummel KE, Branda RF, Paradis SG, Mann KG. Mechanism of factor VIIa-dependent coagulation in hemophilia blood. Blood 2002; 99:923-930.
37. Hedner U. Factor VIIa and its potential therapeutic use in bleeding-associated pathologies. Thromb Haemost 2008; 100:557-562.
38. Eichinger S, Lubsczyk B, Kollars M, Traby L, Zwiauer K, Gleiss A, Quehenberger P, Kyrle PA. Thrombin generation in haemophilia A patients with factor VIII inhibitors after infusion of recombinant factor VIIa. Eur J Clin Invest 2009; 39:707-713.
39. Turecek PL, Varadi K, Gritsch H, Schwarz HP. FEIBA: mode of action. Haemophilia 2004; 10 Suppl 2:3-9.
40. Egler C, Albert T, Brokemper O, Zabe-Kuhn M, Mayer G, Oldenburg J, Schwaab R. Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity. J Mol Recognit 2009; 22:301-306.
41. Brummel KE, Paradis SG, Butenas S, Mann KG. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100:148-152.
42. Mann KG, Brummel K, Butenas S. What is all that thrombin for? J Thromb Haemost 2003; 1:1504-1514.
43. Fischer TH, Merricks EP, Bode AP, Bellinger DA, Russell K, Reddick R, Sanders WE, Nichols TC, Read MS. Thrombus formation with rehydrated, lyophilized platelets. Hematology 2002; 7:359-369.
44. Read MS, Reddick RL, Bode AP, Bellinger DA, Nichols TC, Taylor K, Smith SV, McMahon DK, Griggs TR, Brinkhous KM. Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion. Proc Natl Acad Sci U S A 1995; 92:397-401.
45. Bode AP, Read MS, Reddick RL. Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber. J Lab Clin Med 1999; 133:200-211.
46. Samama CM. Prothrombin complex concentrates: a brief review. Eur J Anaesthesiol 2008; 25:784-789.
47. Samama CM, Rosencher N. Nouveaux anticoagulants : tout n’est pas permis pour l’instant. Annales Fr Anesth Reanim 2009; 28:836-837.

Source(s) of Funding


None

Competing Interests


Erik Berntorp has received speaker fees from Octapharma. Juergen Roemisch, Katharina  Pock, Isabella Laher-Kheirallah, Sandra Janisch, Olaf Walter and Sigurd Knaub are employees of Octapharma.

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