Article ID: WMC002444 ISSN 2046-1690

A Novel Approach to Treating FVIII Inhibitors: InVitro Results for Combination Therapy with HumanProthrombin Complex Concentrate andPlasma-Derived FVIII/VWF ComplexCorresponding Author:Dr. Juergen R Roemisch,Senior Vice President Plasma R&D, Research & Development Octapharma PPGmbH, Oberlaaer Strasse 235,1100 - Austria

Submitting Author:Dr. Juergen R Roemisch,Senior Vice President Plasma R&D, Research & Development Octapharma PPGmbH, Oberlaaer Strae 235,1100 - Austria

Article ID: WMC002444

Article Type: Research articles

Submitted on:18-Nov-2011, 10:39:19 AM GMT Published on: 18-Nov-2011, 04:01:42 PM GMT

Article URL: http://www.webmedcentral.com/article_view/2444

Subject Categories:HAEMATOLOGY

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

How to cite the article:Roemisch J R, Pock K , Laher-kheirallah I , Janisch S , Walter O , Knaub S , Berntorp E .A Novel Approach to Treating FVIII Inhibitors: In Vitro Results for Combination Therapy with Human ProthrombinComplex Concentrate and Plasma-Derived FVIII/VWF Complex . WebmedCentral HAEMATOLOGY2011;2(11):WMC002444

Source(s) of Funding:

None

Competing Interests:

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

Additional Files:

FVIII submission

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A Novel Approach to Treating FVIII Inhibitors: InVitro Results for Combination Therapy with HumanProthrombin Complex Concentrate andPlasma-Derived FVIII/VWF ComplexAuthor(s): Roemisch J R, Pock K , Laher-kheirallah I , Janisch S , Walter O , Knaub S , Berntorp E

Abstract

Coagulation factor VIII (FVIII) inhibitors are the mostsevere complications of haemophilia A treatment. Westudied the effect of a non-activated prothrombinconcentrate (PCC) combined with FVIII in vitro. FVIIIantibodies minimised the in vitro thrombin generationof standard human plasma and prolonged the lagphase at a residual FVIII activity of ≤ 0.01 IU/ml. Timeto clotting of FVIII-inhibited fresh whole blood was notmeasurable until 55 minutes (observation time).Combining a PCC and a plasma-derived FVIII/VWFconcentrate normalised TG and ROTEM® parameters.Our findings support an approach to moderately shiftthe haemostatic balance to a more procoagulantpotential.

Introduction

Haemophilia A (HA) is caused by a deficiency in theamount or activity of coagulation factor VIII (FVIII) inplasma and is largely managed through theadministration of plasma-derived or recombinanthuman FVIII concentrates. Inhibitory antibodies toexogenously administered FVIII can develop, therebyneutralising its therapeutic effect and resulting insevere bleeding complications. Inhibitory antibodiesrepresent the most significant clinical complication inthe modern day treatment of HA [1]. In patients whodevelop inhibitory antibodies, prophylaxis with FVIIIcannot be performed until complete and sustainedelimination of the inhibitor has been achieved. As agreat deal of clinical progress has been made inrecent years in the management of HA characterisedby persistent FVIII inhibitors, several treatment optionsare now available in the armamentarium [2-4].Immediate and persistent initiation of immunetolerance induction (ITI) therapy, involving repeatedFVIII dosing over months or years, is the treatment ofchoice and the only proven strategy for achievingsustained inhibitor eradication [5]. ITI has generallybeen more successful when FVIII concentrates

containing von Willebrand factor (VWF) were used[5-7]. Besides its direct haemostatic role, VWF has akey function in the protection, transport andpresentation of FVIII with respect to the immunesystem, and this is an important area of currentresearch. For the patients who do not respond to ITItreatment alone, bypassing agents represent the nexttherapeutic step. These bypassing agents includenon-activated prothrombin complex concentrates(PCCs), activated PCCs (aPCCs) and recombinantfactor VIIa (rFVIIa) [3,4]. Several case reports and invitro studies indicate that combining different types ofbypassing agents, either sequentially or ascombination therapy, can be an efficacious way totreat monotherapy-resistant inhibitors [8-15]. Inparticular Klintman and colleagues showed in vitro thatcombining any two of aPCC, rFVIIa and FVIIIconcentrates resulted in an additive effect on thrombinproduction [15]. While aPCCs and rFVIIa are wellestablished for patients displaying FVIII inhibitors,some individuals are refractory to these treatments,and although thrombotic side effects are rare, apotential risk remains with the application of highconcentrations of activated coagulation factors [16-18].Moreover, the high cost of these products limits theiravailability in some patient populations and countries.In clinical practice, the successful use of non-activatedPCCs has been demonstrated in many casesthroughout the last decades to counteractinhibitor-associated bleeding complications [19,20].Human non-activated PCC (Octaplex® [OctapharmaPPGmbH, Vienna, Austria]), which contains abalanced ratio of the coagulation factors II, VII, IX andX and the anticoagulant proteins C and S, is currentlylicensed for use in perioperative prophylaxis andtreatment of bleeding following treatment or overdosewith vitamin K antagonists and for congenitaldeficiencies of factors II and X when specific factorproducts are not available [21-23]. Followingencouraging results with this PCC in reducing bleedingcomplications of HA patients displaying high-titreinhibitors, we were interested to establish whetherplasma-derived (pd)FVIII/VWF concentrates mayconfer an additional clinical advantage when used as a

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supplementary add-on therapy to PCC in acutebleeding situations [24]. Using a thrombin generation(TG) assay [25-27], thrombelastometry (ROTEM®)[28,29] and quantification of FVIII activity (FVIII:C), weinvestigated the effect of spiking samples with both thePCC and pdFVIII/VWF concentrates (Octanate® orWilate®), while also experimentally adding FVIIIantibodies to standard human plasma (SHP) orpatient-derived plasma samples.

Material and Methods

FVIII: C quantification for inhibitor determinationInhibitory activity was determined using the FVIIIchromogenic method and an inhibitor reagent kit(Technoclone GmbH, Vienna, Austria).Residual FVIII: C activities after incubationFVIII activity was determined in the low range,0.088-0.006 IU FVIII:C/ml using the chromogenicassay (Coamatic FVIII from Chromogenix [IL Spa,Milano, Italy]) in a microplate format as follows: anadapted standard curve was prepared between 0.006and 0.088 IU FVIII:C/ml from dilutions of standardhuman plasma (SHP) (Siemens HealthcareDiagnostics [SHD] GmbH, Marburg, Germany)calibrated against fresh normal plasma and the bufferblank. The samples were diluted, if necessary, into therange of the standard curve. Control SHP calibratedagainst fresh normal plasma served as control. Theincubation steps and the final dilution for samples,standards and controls were performed according tothe manufacturers’ instructions. The absorbance wasread at 405 nm.Thrombin generation assayThrombin generation was measured using theTechnothrombin TGA kit and RC high reagent(Technoclone GmbH, Vienna, Austria). The readoutparameters were lag time (phase) and peak thrombinconcentration. To mimic plasma with a FVIII inhibitor,monoclonal antibodies (mAb, ESH-4 and ESH-8,American Diagnostica Inc., Stamford, USA) and/orpolyclonal (pAb) sheep FVIII antibodies (EnzymeResearch Laboratories Ltd., South Bend, USA) wereadded to SHP in order to achieve final inhibitoryactivities of 10 and 31 Bethesda Units (BU)/ml.Accordingly, 100 µl SHP or whole blood were eachspiked with 2 µl ESH-4 and ESH-8, resulting in finalconcentrations of 20 µg/ml. The pAb was added at 3 µlto achieve a final plasma concentration of 30 µg/ml. To80 µl of the spiked SHP, 20 µl of a preparationcontaining the human PCC (Octaplex® [OctapharmaPPGmbH, Vienna, Austria]) and/or a pdFVIII/VWFconcentrate (Octanate® or Wilate® [Octapharma

PPGmbH, Vienna, Austria]) or aPCC (FEIBA®, BaxterAG, Vienna, Austria), rFVIIa (NovoSeven® [NovoNordisk A/S, Glostrup, Denmark]) or sodium chloride(NaCl) control solution, were added to obtain a finalsample volume of 100 µl. In general, SHP with orwithout spiked antibodies were incubated for 1 hour at37°C. These conditions were found to result in themaximum inhibitory capacity.For experiments performed in the presence ofplatelets, controls without platelets were run in parallel.Lyophilised platelets (MöLab GmbH, Langenfeld,Germany) were reconstituted in 5 ml diluent andadded 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. Inhibitionof FVIII was performed in the presence and absenceof platelets (1 hour, 37°C) so as to exclude anyFVIII-related activity displayed by the reconstitutedplatelets themselves.The FVIII/VWF concentrate was reconstitutedaccording to the manufacturer’s instructions to obtaina solution of 100 IU FVIII:C/ml.The PCC was reconstituted with water for injection toa FIX concentration of 25 IU/ml. Because the PCCused contains an average of 0.45 IU heparin per 1 IUFIX (with low variation; 0.2-0.5 IU heparin/IU FIXaccording to specification), pre-experiments wereperformed including the heparin-free in-process bulksolution to exclude any significant impact on testparameters [30]. In general, the PCC concentrationsindicated refer to the final FIX concentrations added. Aconcentration of 0.25 IU FIX/ml (corresponding to <0.12 IU heparin/ml) in the assay neither prolonged thelag phase nor reduced the peak TG (data not shown).No impact was observed on the ROTEM® results. Asexpected, the PCC at 0.5 IU FIX/ml showed someimpact and, therefore, the heparin-free in-processsolution was used for studies ? 0.5 IU FIX/ml.The PCC and pdFVIII/VWF concentrate were notmixed before spiking in the experiments illustratedhere, but were added to samples separately.Lag phase (minutes) and peak thrombin concentration(nM) were assessed for the PCC and pdFVIII/VWFconcentrate separately and in combination in SHPcontaining inhibitors. TG assays were performed forthe 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 wasinvestigated separately at a concentration of 0.25IU/ml, and combined with 0.5 IU/ml pdFVIII/VWF(Octanate® or Wilate®). In the absence ofhaemostatic treatments, TG parameters wereinvestigated for SHP with or without pAb/mAbinhibitors, and as controls, in SHP containing plateletswith or without pAb/mAb inhibitors.

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In addition, in vitro/ex vivo effects of aPCC usingFEIBA® (2 U/ml) or rFVIIa (2µg/ml) in inhibitorplasmas were studied, without or in combination with0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF in thepresence of platelets.Kinetics of FVIII inhibition after addition of PCCand pdFVIII/VWFAfter 1 hour incubation of antibody-spiked SHP, lagphases and peak thrombin concentrations wereassessed at baseline and after addition of 0.25 IU/mlPCC and 0.5 IU/ml pdFVIII/VWF. FVIII:C wasdetermined in parallel. These parameters wereassessed for the following incubation times of thiscombination of concentrates: 0, 15, 30, 45, and 60minutes. SHP with and without inhibitors were run ascontrols.Rotation thrombelastometry (ROTEM®) of wholebloodROTEM® (TEM Innovations GmbH, Munich, Germany)was performed at 37°C using the NATEM® systemaccording to the manufacturer’s description. pAb wasadded to freshly drawn whole blood samples after aresting time of 30 minutes at room temperature, andincubated for 1 hour at 37°C in order to achieve a finalinhibitory activity of 31 BU/ml. Samples forROTEM®/NATEM® analysis were treated with thefollowing haemostatic interventions: 0.5 IU/ml PCCand 1.0 IU/ml pdFVIII/VWF or 0.25 IU/ml PCC and 0.5IU/ml pdFVIII/VWF. Samples were analysed at thefollowing time points after the addition of the twoconcentrates: 0, 5, 15, 30, 45 and 60 minutes. Wholeblood samples with and without antibodies were run ascontrols.Ex vivo studiesPlasma samples obtained from HA patients wereobtained and stored as described previously [31]. Theinhibitory potencies of the four individuals ranged from30 to 159 BU/ml. TG parameters with or withoutaddition of 0.5 IU/ml PCC and 0.5 IU/ml or 1.0 IU/mlpdFVIII/VWF, FEIBA® or rFVIIa were investigated.

Results

Impact of pdFVIII/VWF and PCC/pdFVIII/VWF onTG parameters in plasma with inhibitorsAs characteristic of these studies, the addition of pAbor pAb/mAb to SHP resulted in the minimisation of TGand a prolongation of the lag phase. In the presence ofplatelets, these effects were still considerable,although thrombin generation was not completelyeradicated (Illustration 1). After incubation of SHP andplatelets with antibodies, the addition of pdFVIII/VWFwithin the concentration range of 0.1-1.5 IU/ml

resulted in a general trend towards reduced lag timewith increasing pdFVIII/VWF concentrations, but thisdid not reach the control level (SHP, Illustration 1,Panel A and platelets). Peak thrombin concentrationsincreased with higher pdFVIII/VWF level, but reacheda plateau at 0.5 IU/ml FVIII corresponding toapproximately half of that observed for the control(Illustration 1, Panel B). The addition of 0.25 IU/mlPCC to SHP with platelets containing pAb/mAbnormalised the lag time, whereas peak thrombinconcentration was significantly improved but notnormalised. The addition of 0.25 IU/ml PCC and 0.5IU/ml pdFVIII/VWF from Octanate® resulted innormalisation of both lag time and peak thrombinconcentration. Also, the addition of the otherpdFVIII/VWF product (Wilate®) to the PCC revealedcomparable effects at the same FVIII:C concentrations(data not shown).Correspondingly, ROTEM® analysis performed withfreshly drawn blood showed prevention of clotformation upon addition of FVIII antibodies.Normalisation of ROTEM® parameters was achievedafter addition of 0.25 IU/ml PCC and 0.5 IU/mlpdFVIII/VWF (Illustration 2; Panels A, B and C).Kinetics of FVIII inhibition after addition ofPCC/pdFVIII/VWF: Impact on TG and clot formation(ROTEM®)SHP incubation in the presence of platelets and afteraddition of pAb showed a minimum residual FVIII:C of0.01 IU/ml after 60 minutes at 37°C, whichreproducibly did not further decrease upon longerincubation times (up to 180 minutes, data not shown).Accordingly, this procedure was defined as thestandard pre-incubation time.After standard pre-incubation of SHP with pAb in thepresence of added platelets, 0.25 IU/ml PCC and 0.5IU/ml pdFVIII/VWF was added and aliquots weredrawn for immediate measurements of FVIII:C activityand TG at time point 0 and up to 60 minutes afteraddition. According to the FVIII:C added (0.5 IU/ml)and the residual activity after inhibitor incubation, acalculated FVIII:C of 0.51 IU/ml resulted after additionof PCC/pdFVIII/VWF. Immediate measurementrepeatedly resulted in residual FVIII:C of 0.26 to 0.29IU/ml, which decreased to 0.08-0.10 IU/ml and 0.05IU/ml after 15 and 30 minutes, respectively, reaching astable baseline activity of 0.03 IU/ml at 60 minutes(Illustration 3; Panel A).TG assays revealed that lag time in SHP containingpAb was severely prolonged (55 minutes until theinitiation of clotting) (Illustration 3; Panel A). Incubationof SHP containing pAb inhibitors with 0.25 IU/ml PCCand 0.5 IU/ml pdFVIII/VWF for 0 and 15 minutesresulted in normalisation of the lag phase (effect was

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greatest at 0 minutes). Although longer incubationtimes with this combination of concentrates wereassociated with a lengthening of the lag phase, it wasstill in the range of 20 to 25 minutes. Incubation withpAb eliminated thrombin generation in SHP(Illustration 3; Panel B). Subsequent addition of 0.25IU/ml PCC and 0.5 IU/ml pdFVIII/VWF resulted in apeak thrombin concentration close to SHP at timepoint 0 minutes, declining to about 30% of SHP after10 minutes and to 10% after 60 minutes.ROTEM® of whole blood of healthy donors revealedno onset of clot formation after incubation with pAb forup to 55 minutes of measurement. Addition of 0.25IU/ml PCC and 0.5 IU/ml pdFVIII/VWF completelyrestored the clotting time (CT) and maximum clotstrength (MCF) as compared to the whole bloodcontrol at time point 0 minutes (Illustration 2; Panel A).A higher concentration of this combination ofconcentrates (0.5 IU/ml PCC and 1.0 IU/mlpdFVIII/VWF) essentially did not affect ROTEM®variables relative to the lower concentration (data notshown). After 15 minutes incubation and subsequentROTEM® performance, the CT was prolonged by2-2.5 fold compared with immediate measurement orwhole blood control, respectively (Illustration 2; PanelD). The MCFs remained in the normal range. After 60minutes, the CT was prolonged 3-fold, correspondingly(Illustration 2; Panel E).Impact on TG parameters of PCC/pdFVIII/VWFcompared with aPCC and rFVIIaTG assays were performed in order to assess thehaemostatic effects of 0.25 IU/ml PCC and 0.5 IU/mlpdFVIII/VWF, aPCC and rFVIIa after addition toSHP/platelets containing pAb/mAb. rFVIIa shortenedthe lag time somewhat below the SHP/platelet control,but did not normalise peak thrombin concentration(Illustration 4; Panels A, B). In contrast, thecombination of PCC and pdFVIII/VWF normalised bothparameters. aPCC revealed a significant shortening ofthe lag phase at the concentration used, while peakthrombin concentration was in the normal range.Combining PCC/pdFVIII/VWF with rFVIIa or aPCCshowed further moderate shortening of lag times andincreases in peak thrombin concentrations comparedwith experiments where these components wereevaluated 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 thefollowing haemostatic interventions: 0.5 IU/ml PCCand 0.5 IU/ml pdFVIII/VWF; 0.5 IU/ml PCC and 1.0IU/ml FVIII; rFVIIa (2µg/ml); and aPCC (2 U/ml). In theabsence of haemostatic treatments, TG was severelyimpaired relative to SHP containing platelets, as

expected. Both PCC/pdFVIII/VWF combinationsnormal ised lag t imes and peak thrombinconcentrations. Compared to SHP, the area under theconcentration-time curve (AUC) values were increasedby this combination of factors. This was even morepronounced with aPCC and was accompanied by ashorter lag phase in the example presented. rFVIIa (2mg/ml) elicited a poor TG response in the HApatient-derived inhibitor plasma. In contrast, additionof 2 U/ml aPCC (plasma level reached upon standardtreatment with aPCC) resulted in a very pronouncedthrombin generation. The AUC profile for aPCC wasnotably greater than that of the control condition(consisting of SHP not containing FVIII inhibitors).All HA inhibitor plasmas investigated so far respondedto PCC (applied separately) with regard to TGparameters and showed more pronounced effects toPCC/pdFVIII/VWF spiking, although different individualresponses were observed, e.g. in the extent ofthrombin generation induced by aPCC (data notshown).Pla te le ts cont r ibute to the e f fec t o fPCC/pdFVIII/VWF on TG in SHP in the presence ofpAb/mAbIn order to evaluate the contribution of platelets in thedescribed setting, TG assays were performed usingSHP containing pAb/mAb. In contrast to theexperiments performed with pAb alone (data notshown) , p la te le ts were requ i red for thePCC/pdFVIII/VWF combination to bring peak thrombinconcentrations into the normal range. In contrast,conditions without platelets were associated with onlya low response (Illustration 6). There was nosignificant difference between thrombin generation ofSHP and SHP/platelets.

Discussions and Conclusion

In HA patients with inhibitors, recent data suggest thatthe PCC used in this study may be a useful alternativeto aPCCs or rFVIIa as a bypassing agent [24]. Thedata presented in this in vitro/ex vivo studydemonstrate that PCC, given in combination with pdFVIII/VWF concentrates (Octanate® or Wilate®),results in normalisation of plasma/platelet TG. Thisheld true for experimentally (FVIII) inhibited normalplasma as well as for the investigated HA inhibitorplasmas.Both assay systems, TG and ROTEM®, have beenextensively investigated for monitoring of aPCC andrFVIIa effects in the treatment of HA patients withspecific factor inhibitors and rare coagulation disorders[16,32-34]. In the present study, ROTEM® analyses

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supplemented the data gained by the TG assay,showing that clotting activity was eliminated in wholeblood containing pAb, and application of a combinationof PCC and pdFVIII/VWF restored clot formationparameters in vitro.In general, rFVIIa alone confirmed the poor in vitroresponse reported by other working groups [35-37],even in the presence of platelets, though rFVIIa hasbeen shown to be clinically effective [38]. It thus poorlyfacilitates a prediction of in vivo effects. In contrast,aPCC application resulted in the expected pronouncedshortening of lag time and increased peak thrombinconcentration [39]. The in vitro/ex vivo effectsobserved upon combination of PCC/pdFVIII/VWF andrFVIIa or aPCC appeared not to be synergistic, butthis will have to be carefully observed clinically withregard to thromboembolic adverse events if used totreat emergency bleeding episodes.Based on the fact that non-activated PCCs show aclinical usefulness for treating bleeding events in HAinhibitor patients, cases responding poorly tomonotherapy of either PCC, aPCC or rFVIIa maybecome of particular interest for a potentialPCC/pdFVIII/VWF combination treatment. In vitroresults indicated that even a moderate increase innon-activated clotting factors can shift the haemostaticbalance to a more procoagulant state, which can bebrought about via intervention with a PCC. It isplausible that application of exogenous FVIII may drivecoagulation if exceeding the total inhibitor capacity or ifescaping the immediate neutralisation by theantibodies. The significant haemostasis enhancingeffect of even very low FVIII:C is known, and thus thekinetics of FVIII inhibition are decisive for the extentand duration of cofactor effect and treatment efficacy.Therefore, we used the model to mimic moderate tohigh FVIII inhibitory capacities in plasma by spikingsamples with pAb or a mixture of pAb/mAb. Epitopespecificities and neutralising capabilities of the latterwere reported by another working group [40].After inhibition of FVIII by a 1–hour incubation withpAb 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. Activitiesdecreased with time, but even after 30 and 45 minutes,residual activities of 5% and 3%, respectively, werequantified. Peak thrombin concentrations remained inthe 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 theseparameters remained clearly measurable abovebaseline. Although ROTEM® results were obtained

with whole blood, we utilised this methodologyfollowing inhibition of FVIII by the addition ofantibodies (as in the TG kinetic studies) todemonstrate that small amounts of thrombin that aregenerated are sufficient to drive clot formation, asindicated in the plasma/platelet system. Taking intoaccount that the plasma was diluted by 50% byplatelet addition, extrapolated peak thrombinconcentrations in whole plasma of equal or less than60 nM should be sufficient to initiate clot formation, asobserved 60 minutes after addition of PCC andpdFVIII/VWF. This is in agreement with recentpublications suggesting that initial thrombinconcentrations as low as 20-30 nM are sufficient forinitiation of clotting, while the subsequent thrombinboost may be required for stability of the clot [41,42].Nevertheless, at present there is no proven generalthreshold of peak thrombin generation sufficient tostop bleeding in HA inhibitor patients. Individualclinical conditions have to be considered.The corresponding lag times increased withdecreasing FVIII:C and peak thrombin concentrationsover time, as expected. Notably, they did not reach thelevel of the inhibited plasma/platelet control, reflectingthe basic effect of PCC in decreasing FVIII:C, which isin line with the ROTEM® results.The experiments performed in this study underline thepivotal role of platelets in PCC/pdFVIII/VWF-mediatedclotting activity and thrombin generation. Followingtreatment with the combination of PCC andpdFVIII/VWF, substantial clotting activity wasobserved in SHP containing lyophilised platelets withpAb/mAb inhibitors. However, this was not the case forconditions with no platelets, which warrants furtherinvestigation. Experiments in this study wereperformed using lyophilised platelets, which could beviewed as a consequential limitation of the results.While the lyophilised platelets have many of themolecular functions of fresh platelets, they do notperfectly reflect the multi-facetted functionality ofviable fresh platelets, and they were not particular tothe specific patients’ samples [43-45]. Nevertheless,the lyophilised platelets across all experiments herecame from the same standardised batch. Thisstandardisation of platelets thereby removedinter-patient or inter-batch platelet variability otherwiseunderlying differences in the results from theexperimental samples.Thrombin generation and clot formation werenormalised for as long as 15 minutes after applicationof PCC/pdFVIII/VWF, remaining measurable until 60minutes (limit of observation period). Other inhibitorprofiles will also certainly have an influence on FVIIIinhibition kinetics. However, coagulation factors

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present in the PCC should remain present over alonger period of time relative to the decreasing FVIII:Cfollowing application of PCC and pdFVIII/VWF. Thislonger-lasting presence should hypotheticallysafeguard against recurrent bleeds [14,46,47].The HA inhibitor patient samples spiked with plateletsand PCC/pdFVIII/VWF revealed an excellent ex vivoresponse in all cases investigated. The addition of0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWFconcentrate (Octanate® or Wilate®), administered asseparate preparations (i.e. not in immediatecombination), would appear to be a reasonablestarting point for clinical trials.In conclusion, the in vitro/ex vivo results reported hereare encouraging and support the investigation of thecombination of PCC and pdFVIII/VWF in aproof-of-principle clinical trial, in order to furtherestablish the viability of this approach as an effectiveand safe strategy for the treatment of bleeding eventsin HA inhibitor patients.

Acknowledgements

The authors would like to thank Margoth Gunnarssonand Anne-Marie Thämlitz (Wallenberg Laboratory,Malmö University Hospital). Medical writing serviceswere provided by nspm ltd, Meggen, Switzerland, withfinancial support from Octapharma AG, Lachen,Switzerland.

Conflict of Interest Statement

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

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Chavanne H, Chamouard V, Marin S, Negrier C. Majorsurgery in a severe haemophilia A patient with hightitre inhibitor: use of the thrombin generation test in thetherapeutic decision. Haemophilia 2005; 11:552-558.15. Klintman J, Astermark J, Berntorp E. Combinationof FVIII and by-passing agent potentiates in vitrothrombin production in haemophilia A inhibitor plasma.Br J Haematol 2010; 151:381-386.16. Hayashi T, Tanaka I, Shima M, Yoshida K, FukudaK, Sakurai Y, Matsumoto T, Giddings JC, Yoshioka A.Unresponsiveness to factor VIII inhibitor bypassingagents dur ing haemostat ic t reatment forlife-threatening massive bleeding in a patient withhaemophilia A and a high responding inhibitor.Haemophilia 2004; 10:397-400.17. Astermark J, Morado M, Rocino A, van den BergHM, Von DM, Gringeri A, Mantovani L, Garrido RP,Schiavoni M, Villar A, Windyga J. Current Europeanpractice in immune tolerance induction therapy inpatients with haemophilia and inhibitors. Haemophilia2006; 12:363-371.18. Sarode R, Matevosyan K. Prothrombin complexconcentrate and fatal thrombosis: poor evidence toimplicate a relatively safe drug. Ann Emerg Med 2009;54:481-482.19. Abildgaard CF, Britton M, Harrison J. Prothrombincomplex concentrate (Konyne) in the treatment ofhemophilic patients with factor VIII inhibitors. J Pediatr1976; 88:200-205.20. Lusher JM, Shapiro SS, Palascak JE, Rao AV,Levine PH, Blatt PM. Efficacy of prothrombin-complexconcentrates in hemophiliacs with antibodies to factorVIII: a multicenter therapeutic trial. New Engl J Med1980; 303:421-425.21. Franken T, Rees W, Hubner N, et al. Octaplex inroutine clinical use for prophylaxis and therapy ofbleeding in patients with prothrombin complex factordeficiency [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 aprothrombin complex concentrate (Octaplex) for rapidreversal of oral anticoagulation. Thromb Res 2004;113:371-378.23. Riess HB, Meier-Hellmann A, Motsch J, Elias M,Kursten FW, Dempfle CE. Prothrombin complexconcentrate (Octaplex) in patients requiring immediatereversal 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. Aretrospective study of Octaplex in the treatment ofbleeding in patients with haemophilia A complicated byinhibitors. Blood Coagul Fibrinolysis 2010; 21:577-583.25. Berntorp E, Salvagno GL. Standardization and

clinical utility of thrombin-generation assays. SeminThromb Hemost 2008; 34:670-682.26. Dargaud Y, Beguin S, Lienhart A, Al DR, TrzeciakC, Bordet JC, Hemker HC, Negrier C. Evaluation ofthrombin generating capacity in plasma from patientswith haemophilia A and B. Thromb Haemost 2005;93:475-480.27. Hemker HC, Al DR, Beguin S. Thrombingeneration assays: accruing clinical relevance. CurrOpin Hematol 2004; 11:170-175.2 8 . L u d d i n g t o n R J .Thrombelastography/thromboelastometry. Clin LabHaematol 2005; 27:81-90.29. Reikvam H, Steien E, Hauge B, Liseth K, HagenKG, 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 compositionregulates differential effects of prothrombin complexconcentrates on thrombin generation. J ThrombHaemost 2009; 7:2154-2156.31. Salvagno GL, Astermark J, Lippi G, Ekman M,Franchini M, Guidi GC, Berntorp E. Thrombingeneration assay: a useful routine check-up tool in themanagement of patients with haemophil ia?Haemophilia 2009; 15:290-296.32. Sorensen B, Johansen P, Christiansen K, WoelkeM, Ingerslev J. Whole blood coagulat ionthrombelastographic profiles employing minimal tissuefactor activation. J Thromb Haemost 2003; 1:551-558.33. Sorensen B, Ingerslev J. Whole blood clotformation phenotypes in hemophilia A and rarecoagulation disorders. Patterns of response torecombinant factor VIIa. J Thromb Haemost 2004;2:102-110.34. Yoshioka A, Nishio K, Shima M. Thrombelastgramas a hemostatic monitor during recombinant factorVIIa treatment in hemophilia A patients with inhibitor tofactor VIII. Haemostasis 1996; 26 Suppl 1:139-142.35. Allen GA, Hoffman M, Roberts HR, Monroe DM.Manipulation of prothrombin concentration improvesresponse to high-dose factor VIIa in a cell-basedmodel of haemophilia. Br J Haematol 2006;134:314-319.36. Butenas S, Brummel KE, Branda RF, Paradis SG,Mann KG. Mechanism of factor VIIa-dependentcoagulation in hemophilia blood. Blood 2002;99:923-930.37. Hedner U. Factor VIIa and its potential therapeuticuse in bleeding-associated pathologies. ThrombHaemost 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

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factor VIII inhibitors after infusion of recombinant factorVIIa. Eur J Clin Invest 2009; 39:707-713.39. Turecek PL, Varadi K, Gritsch H, Schwarz HP.FEIBA: mode of action. Haemophilia 2004; 10 Suppl2:3-9.40. Egler C, Albert T, Brokemper O, Zabe-Kuhn M,Mayer G, Oldenburg J, Schwaab R. Kineticparameters of monoclonal antibodies ESH2, ESH4,ESH5, and ESH8 on coagulation factor VIII and theirinfluence 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 bloodcoagulation. Blood 2002; 100:148-152.42. Mann KG, Brummel K, Butenas S. What is all thatthrombin 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, ReadMS. Thrombus formation with rehydrated, lyophilizedplatelets. Hematology 2002; 7:359-369.44. Read MS, Reddick RL, Bode AP, Bellinger DA,Nichols TC, Taylor K, Smith SV, McMahon DK, GriggsTR, Brinkhous KM. Preservation of hemostatic andstructural properties of rehydrated lyophilized platelets:potential for long-term storage of dried platelets fortransfusion. Proc Natl Acad Sci U S A 1995;92:397-401.45. Bode AP, Read MS, Reddick RL. Activation andadherence of lyophilized human platelets on caninevessel strips in the Baumgartner perfusion chamber. JLab 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. Nouveauxanticoagulants : tout n’est pas permis pour l’instant.Annales Fr Anesth Reanim 2009; 28:836-837.

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TG parameters are shown for 0.25 IU/ml of PCC and for a combination of 0.25 IU/ml of PCC with 0.5 IU/ml pdFVIII/VWF in SHP spiked with

platelets and pAb/mAb (31 BU/ml). TG parameters are also shown for SHP with or without pAb/mAb, and SHP containing platelets with or

without inhibitors (served as controls). SHP with platelets (Plt) and pAb/mAb are abbreviated as A in the legend.

Panel A

Illustrations

Illustration 1

Impact of PCC/pdFVIII/VWF on in vitro TG parameters in SHP containing FVIII antibodies

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Panel B

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Representative thrombelastograms are shown for freshly drawn whole blood (Panel A) and blood incubated with pAb (10 BU/ml) at 37°C for one

hour (Panel B). Subsequently, 0.25 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF was added and ROTEM®/NATEM® was started immediately. Time

points are shown for time 0 (Panel C), after 15 minutes (Panel D) and after 60 minutes of incubation (Panel E).

Panel A

Illustration 2

ROTEM analysis of PCC/pdFVIII/VWF in whole blood containing neutralising FVIII antibodies

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Panel B

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Panel C

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Panel D

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Panel E

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Panel A: FVIII:C (line) and lag time (bars) were assessed in SHP incubated with 10 BU/ml of pAb. The impact of 0.25 IU/ml PCC/0.5 IU/ml

pdFVIII/VWF were investigated at different time points. A theoretical value of 0.51 IU/ml of FVIII:C was calculated (first left point of line).

Panel B: peak thrombin concentrations (bars) and FVIII:C activities (line) according to Panel A. A: SHP with pAb.

Panel A

Illustration 3

FVIII inhibition kinetics and impact on TG parameters upon application of PCC/pdFVIII/VWF.

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Panel B

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TG parameters (Panel A: lag phase; Panel B: peak thrombin concentration) are shown using SHP withplatelets (Plt) and pAb/mAb (31 BU/ml). The impact of rFVIIa (2 μg/ml), aPCC (2 IU/ml), andrFVIIa/aPCC in combination with 0.25 IU/ml PCC/0.5 IU/ml pdFVIII/VWF are also shown. TGparameters are shown for SHP with platelets not containing pAb/mAb served as a control. A: SHP withplatelets and pAb/mAb.

Panel A

Illustration 4

Impact of PCC/pdFVIII/VWF, rFVIIa and aPCC on TG parameters in SHP containing FVIII antibodies.

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Panel B

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The plasma aliquots containing inhibitors (34 BU/ml) were spiked with platelets and TG curves were monitored for the following

conditions: no treatment (squares), 0.5 IU/ml PCC and 0.5 IU/ml pdFVIII/VWF (crosses), 0.5 IU/ml PCC and 1.0 IU/ml

pdFVIII/VWF (triangles), 2 µg/ml rFVIIa (circles), 2 IU/ml aPCC (dotted line) and SHP (diamonds, dashed line).

Illustration 5

Impact on TG parameters after ex vivo addition of PCC/pdFVIII/VWF, rFVIIa or aPCC to a plasma sample of an HA patientdisplaying inhibitors.

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Peak thrombin concentrations were measured using SHP, SHP spiked with pAb/mAb, and SHP spikedwith pAb/mAb and PCC/pdFVIII/VWF, each in the presence (grey bars) or absence of (added) platelets(black bars). Prior to addition of 0.25 IU/ml PCC/0.5 IU/ml pdFVIII/VWF, SHP with and withoutplatelets had been incubated for 60 minutes at 37°C. SHP with pAb/mAb is abbreviated as A in thelegend.

Illustration 6

The impact of platelets on the effect of PCC/pdFVIII/VWF on TG in SHP containing FVIII antibodies.

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