Peter J Lenting
email: peter.lenting@inserm.fr

Laboratory monitoring of Hemophilia A treatments: new challenges

Key points

-Laboratory monitoring of non-factor therapies in hemophilia raises new challenges -Dependent on the goal of the monitoring exercise, different types of assays should be applied

INTRODUCTION

During the previous four to five decades, hemophilia A treatment mostly involved replacement therapy using concentrates enriched in factor VIII (FVIII). Early management options consisted of cryo-precipitate concentrates, which over time evolved into high-purity, plasma-derived FVIII concentrates and recombinant FVIII concentrates. [START_REF] Muczynski | Emerging therapeutic Strategies in the treatment of hemophilia A[END_REF][START_REF] Sankar | The evolution of recombinant factor replacement for hemophilia[END_REF] Laboratory monitoring of FVIII-replacement therapy is performed via FVIII-specific assays, in most cases by using aPTT-based one-stage clotting assays or via two-stage chromogenic activity assays that use purified proteins. [START_REF] Potgieter | One-stage vs. chromogenic assays in haemophilia A[END_REF] In the aPTT-based coagulation assay, the patient's plasma is mixed with FVIII-deficient plasma, and via the addition of an activating reagent and Ca 2+ions, the coagulation cascade is initiated. The activating agent usually consists of a surface activator (micronized silica, ellagic acid or kaolin), which starts the contact activationpathway. [START_REF] Peyvandi | A critical appraisal of one-stage and chromogenic assays of factor VIII activity[END_REF] In the chromogenic assay, the patient's plasma is diluted and mixed with purified factor X (FX), factor IXa (FIXa), phospholipids, thrombin and Ca 2+ -ions. [START_REF] Rosen | Clinical application of a chromogenic substrate method for determination of factor VIII activity[END_REF] This leads to the generation of FXa, the quantity of which is analyzed by using a small synthetic substrate that is hydrolyzed by FXa.

When analyzed in the plasma of patients, the cofactor activity of plasma-derived and fulllength recombinant FVIII correlates well between one-stage and chromogenic assay systems. In contrast, differences have been detected when assessing high-purity FVIII concentrates towards plasma standards. [START_REF] Barrowcliffe | Factor VIII and factor IX Sub-Committee. Recommendations for the assay of high-purity factor VIII concentrates[END_REF] To avoid these differences, two distinct WHOapproved standards are now available: one plasma standard (NIBSC-code 07/316) has been developed to assign FVIII activity values in the plasma of patients, while a separate standard (NIBSC-code 07/350) has been made available for the assignment of FVIII activity levels in concentrates. [START_REF] Raut | Calibration of human coagulation factor VIII concentrate Ph. Eur. BRP Batch 4 for use in potency assays[END_REF][START_REF] Hubbard | Value assignment of the WHO 6th International Standard for blood coagulation factor VIII and von Willebrand factor in plasma (07/316)[END_REF][START_REF] Raut | Value assignment of the WHO 8th International Standard for factor VIII, concentrate (07/350)[END_REF] Importantly, clinical and laboratory experiences over the last several decades have taught us (within limits, of course) to what extent levels of FVIII measured in the activity assays correlate with the clinical phenotype of the patients.

The first issues on FVIII activity measurements arose upon the introduction of recombinant B-domainless FVIII. These concentrates were associated with an assay discrepancy, in which levels measured using a one-stage clotting assay were 20-50% lower compared to the values obtained using a chromogenic assay. [START_REF] Mikaelsson | Influence of phospholipids on the assessment of factor VIII activity[END_REF][START_REF] Hubbard | Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies[END_REF][START_REF] Morfini | A multicenter pharmacokinetic study of the Bdomain deleted recombinant factor VIII concentrate using different assays and standards[END_REF] To alleviate this discrepancy, a product specific standard was developed. [START_REF] Ingerslev | Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays[END_REF][START_REF] Pouplard | Discrepancies between one stage assay and chromogenic substrate assay in patients treated with recombinant or plasma-derived FVIII and usefulness of a specific standard in ReFacto AF((R)) -treated patients[END_REF] The issue of assay discrepancy between one-stage and chromogenic assays has regained attention with the advent of modified FVIII molecules having an extended half-life. These modifications (fusion to the Fc-portion of IgG, the attachment of polyethylene glycol (PEG), or a combination of different types of modifications) alter the physical properties of the FVIII molecule, and may therefore affect its behavior in the different assay systems. That such modifications indeed affect FVIII activity assays, has elegantly been reviewed by Kitchen and coworkers. [START_REF] Kitchen | Factor activity assays for monitoring extended half-Life Factor VIII and Factor IX replacement therapies[END_REF] We are currently experiencing revolutionary changes in the clinical management of haemophilia A, where apart from replacement-therapy using FVIII molecules or FVIII gene therapy, also so-called non-factor therapies have become available or are in advanced clinical development. These include the bispecific antibody emicizumab, a small interfering RNA (siRNA)-based approach that reduces expression of antithrombin (fitusiran) and antibodies blocking the activity of tissue factor pathway inhibitor (TFPI). [START_REF] Weyand | New therapies for hemophilia[END_REF] These non-factor therapies force us into a re-assessment on how and when to monitor these patients. It is relevant therefore not only to get insight into the mechanism of action of these new therapeutic agents, but also to understand how these agents perform in the biochemical assay systems that are used to monitor hemophilia A patients.

Non-factor therapies

The molecules that qualify as non-factor therapies for hemophilia A (emicizumab, fitusiran and monoclonal anti-TFPI antibodies) have extensively been reviewed elsewhere [START_REF] Weyand | New therapies for hemophilia[END_REF][START_REF] Lenting | Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII?[END_REF][START_REF] Spadarella | Paradigm shift for the treatment of hereditary haemophilia: Towards precision medicine[END_REF][START_REF] Nogami | New therapies using nonfactor products for patients with hemophilia and inhibitors[END_REF][START_REF] Butterfield | A Molecular Revolution in the Treatment of Hemophilia[END_REF] , and only a brief summary will be given here: First, emicizumab is a recombinant humanized bispecific antibody that consists of two different antigen-binding domains. One domain recognizes FIX/FIXa and a second domain has its epitope on FX/FXa. The mode of action of this molecule resides in bridging FIXa and FX, and by bringing enzyme and substrate in close proximity, FIXa-mediated activation of FX is increased. [START_REF] Lenting | Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII?[END_REF][START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF][START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF] The availability of more FXa molecules will support more thrombin generation, ultimately allowing a stronger hemostatic response. Clinical trials investigating emicizumab indeed demonstrated a strong reduction in bleeding events in hemophilia A patients with and without inhibitors. [START_REF] Oldenburg | Emicizumab prophylaxis in hemophilia A with inhibitors[END_REF][START_REF] Mahlangu | Emicizumab prophylaxis in patients who have hemophilia A without inhibitors[END_REF][START_REF] Pipe | Efficacy, safety, and pharmacokinetics of emicizumab prophylaxis given every 4 weeks in people with haemophilia A (HAVEN 4): a multicentre, open-label, non-randomised phase 3 study[END_REF][START_REF] Young | A multicenter, open-label, phase 3 study of emicizumab prophylaxis in children with hemophilia A with inhibitors[END_REF] A second approach, named fitusiran, is based on the reduction of antithrombin expression in the hepatocytes. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF] Transcriptional silencing in eukaryotic cells was first reported in 1988, when antisense cDNAs were used to silence expression of the chalcone synthase gene in tobacco and petunia plants, resulting in modified flower color patterns. [START_REF] Van Der Krol | An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation[END_REF][START_REF] Van Der Krol | Antisense genes in plants: an overview[END_REF] This approach has evolved into therapeutically applied oligonucleotides that use the natural RNA-interference pathway to inactivate the corresponding mRNA. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF][START_REF] Setten | The current state and future directions of RNAi-based therapeutics[END_REF] In the case of fitusiran, a GalNAc-tagged oligonucleotide is used for the post-transcriptional silencing of the antithrombin gene in hepatocytes. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF][START_REF] Setten | The current state and future directions of RNAi-based therapeutics[END_REF] Consequently, protein levels of antithrombin in plasma are reduced, eliminating part of the anticoagulant pathway that targets several coagulation enzymes, including thrombin, FXa and FIXa. Reduced antithrombin levels will thus increase the overall hemostatic potential in the absence of FVIII. Indeed, clinical data that have been reported so far, indicate a strong reduction in bleeding episodes in patients receiving fitusiran. [START_REF] Pasi | Targeting of antithrombin in hemophilia A or B with RNAi therapy[END_REF] In a third alternative approach, another member of the anticoagulant pathway is targeted: ie. tissue factor pathway inhibitor (TFPI). [START_REF] Chowdary | Anti-tissue factor pathway inhibitor (TFPI) therapy: a novel approach to the treatment of haemophilia[END_REF][START_REF] Peterson | Targeting TFPI for hemophilia treatment[END_REF] To my knowledge, four monoclonal antibody-based therapeutical agents have been subject to clinical evaluation: concizumab (by NovoNordisk), marstacimab (by Pfizer), BAY-1093884 (by Bayer) and MG1113 (by Green Cross Corporation).

The anti-TFPI antibodies are designed to neutralize the inhibitory activity of TFPI towards FXa and as such also the factor VIIa/tissue factor (FVIIa/TF) complex. A prolonged functional availability of the FVIIa/TF complex will result in the production of larger quantities of FIXa and FXa, thereby stimulating thrombin generation. The clinical data that have been presented are indeed compatible with an enforced hemostatic system in patients with hemophilia A, manifested by a reduced bleeding tendency in these patients when under anti-TFPI therapy. [START_REF] Shapiro | Subcutaneous concizumab prophylaxis in hemophilia A and hemophilia A/B with inhibitors: phase 2 trial results[END_REF] Of note, as of this date (April 3, 2020), clinical trials with concizumab have been paused, whereas those with BAY-1093884 have been terminated [START_REF] Ferrante | Anti-TFPI antibody BAY 1093884: early termination of phase II dose escalation study due to thrombosis[END_REF] , both because of thrombotic events. Thrombotic events have also been observed in patients treated with emicizumab and fitusiran. [START_REF] Oldenburg | Emicizumab prophylaxis in hemophilia A with inhibitors[END_REF][START_REF] Machin | An investigational RNAi therapeutic targeting antithrombin for the treatment of hemophilia A and B[END_REF] Apart from their mode of action, these non-factor therapies also modify the management of hemophilia in a more general sense (Figure 1). Classic replacement therapy using FVIII concentrates requires regular intravenous infusions. The frequency of these infusions varies between patients, and may be as often as three times per week. These intravenous infusions are associated with peak levels of FVIII after infusion, which decrease gradually until the next infusion. Such peak and trough levels are absent in the non-factor therapies, which are characterized by subcutaneous administrations and stable levels of the therapeutic agent.

These differences in mode of action, route of administration and absence of peak and troughs will have a major impact on how patients receiving these non-factor therapies are being monitored. Indeed, a series of publications have recently discussed laboratory testing in perspective of non-factor therapies, particularly with regard to emicizumab. [START_REF] Adamkewicz | Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Function, on Coagulation Assays[END_REF][START_REF] Tripodi | Advances in the treatment of hemophilia: implications for laboratory testing[END_REF][START_REF] Jenkins | Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline[END_REF][START_REF] Aleman | Assay challenges (and opportunities) with non-factor VIII therapies for Hemophilia A[END_REF] In this review, focus will be on the biochemical aspects when monitoring non-factor therapies.

Laboratory monitoring of non-factor therapies

When considering monitoring of patients receiving non-factor therapy, the first and most important question that comes to mind is: what is it exactly that needs to be monitored? Several answers to this question are possible, and include for instance:

1) monitoring levels of the therapeutic agent.

2) monitoring the effect of non-factor therapies on the hemostatic potential of patients.

3) monitoring the FVIII-equivalence of non-factor therapies.

As each of these options requires a different approach and a different assay to generate the desired response, let's address them separately.

Monitoring levels of the therapeutic agent

Prophylactic home treatment has played an important role in the emancipation of hemophilia A therapy, providing numerous advantages to the patients. In contrast to prophylactic diabetes treatment (where patients can monitor themselves by analyzing blood sugar levels), monitoring FVIII activity levels is currently not yet accessible to the patients at home. Consequently, when patients return for a clinical visit, this occasion is frequently used to test for FVIII activity and/or FVIII inhibitors. With the non-factor therapies, such monitoring will also be difficult to perform at home, at least for now. If and when levels of the therapeutic agent should be monitored will of course be decided at the discretion of the clinician (and I assume guidance will be provided by national or international expert committees). But in case this will be done, each non-factor variant will require a different test.

To determine emicizumab levels, various options are possible. During clinical trials, an ELISA-based assay using anti-idiotype antibodies was used for the analysis of emicizumab concentrations. [START_REF] Uchida | A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects[END_REF] This assay is not commercially available, but other assays can be employed instead. Since emicizumab mimics FVIII cofactor function (at least to some extent), it is possible to use FVIII activity assays to monitor emicizumab levels. Unfortunately, the classic one-stage APTT-based assay is not useful in this regard, as the APTT is normalized already at sub-therapeutical emicizumab concentrations (3-5 µg/ml of emicizumab normalizes the APTT, while therapeutic levels are around 55 µg/ml). [START_REF] Adamkewicz | Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Function, on Coagulation Assays[END_REF][START_REF] Nogami | Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti-idiotype monoclonal antibodies[END_REF] To overcome this limitation, two other activity assays could be used. A modified APTT-assay has been described, in which plasma samples are further diluted, allowing a linear relationship between emicizumab concentrations and clotting times. [START_REF] Shinohara | Evaluation of emicizumab calibrator and controls with a modified one-stage FVIII assay on an Automated Coagulation Analyzer[END_REF][START_REF] Wilmot | Laboratory measurement of emicizumab requires a product specific calibrator[END_REF] A second option is to use chromogenic FVIII activity assays that contain human FIXa and FX. Also in this assay, a near linear relationship between emicizumab concentrations and FXa generation can be obtained. [START_REF] Adamkewicz | Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Function, on Coagulation Assays[END_REF] For both assays, specific emicizumab calibrators are commercially available (r 2 Diagnostics, South Bend, IN, USA). Of note, these calibrators are not (yet) approved for clinical use in the USA. Importantly, the activity obtained using these tests provides surrogate FVIII-like activities. These values do not relate to a true FVIII equivalence. [START_REF] Lenting | Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII?[END_REF][START_REF] Leksa | Intrinsic differences between FVIIIa mimetic bispecific antibodies and FVIII prevent assignment of FVIII-equivalence[END_REF] Indeed, emicizumab activity relies on the amount of FIXa that is available. These amounts are not only variable between tests from different manufacturers, but may also differ in a lot-to-lot manner in chromogenic assay kits that use the same reagents. Consequently generate different FVIII-like activity values. Likewise, different activating compounds in the APTT will generate different quantities of FIXa, thereby influencing emicizumab activity. It is further important to note that the outcome of these assays can be affected by the presence of other procoagulant molecules (e.g. FVIII or activated prothrombin complex concentrates (APCC)) present in emicizumab-containing plasma samples. [START_REF] Adamkewicz | Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Function, on Coagulation Assays[END_REF][START_REF] Jenkins | Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline[END_REF] As for fitusiran, it will obviously be unpractical to measure residual oligonucleotide levels within the hepatocytes of the patients. However, it would be possible to measure residual antithrombin levels, either via antigen assays or via thrombin-or FXa-based activity assays. [START_REF] Cooper | The phenotypic and genetic assessment of antithrombin deficiency[END_REF] Again, the presence of other therapeutics, like APCC, in such samples could modify the results of the activity assays.

To measure anti-TFPI antibody levels, no specific assays for the antibodies are yet available, meaning that TFPI levels should be analyzed instead. Antigen levels of TFPI can be measured in established ELISA systems. However, it is unclear whether all of the therapeutic anti-TFPI antibodies actually reduce plasma TFPI levels or whether these monoclonal antibodies interfere in ELISA-assays. Alternatively, specific activity assays are available to measure residual TFPI activity, including a diluted prothrombin time-based assay and tissue factor-dependent chromogenic assays. [START_REF] Dahm | A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI)[END_REF][START_REF] Berrettini | A simple chromogenic substrate assay of tissue factor pathway inhibitor activity in plasma and serum[END_REF] Note should be taken that plasma contains but 10-30% of all TFPI [START_REF] Maroney | New insights into the biology of tissue factor pathway inhibitor[END_REF] , and these assays will therefore be unable to give a full comprehension of how much TFPI is inhibited at the vascular lining.

In conclusion, direct measurement of emicizumab, fitusiran or anti-TFPI antibodies is complicated, but alternative, indirect activity assays could be used to monitor whether these agents are still present and/or active.

Monitoring the effect of non-factor therapies on the hemostatic potential of patients

When analyzing the hemostatic potential in normal individuals using global coagulation tests, a large range between individuals can be observed. [START_REF] Calzavarini | Thrombin generation measurement using the ST Genesia Thrombin Generation System in a cohort of healthy adults: Normal values and variability[END_REF] To analyze the effect of the non-factor therapies on the hemostatic potential of a patient, it is thus important to perform such global coagulation test before and after onset of treatment in order to obtain a proper impression of the changes in hemostatic potential. A diverse arsenal of global test is available, including clot-waveform analysis, thromboelastography and thrombin generation assays. [START_REF] Lance | A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis[END_REF] The focus in this section will be on the thrombin generation assay (TGA). As illustrated in Figure 2, the addition of emicizumab, nanobodies against antithrombin [START_REF] Barbon | Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors[END_REF] , or polyclonal antibodies against TFPI [START_REF] Marlu | Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex[END_REF] has a strong stimulating effect on the thrombin generation profile in FVIII-deficient plasma. Thus, the presence of the procoagulant antibody emicizumab or the functional absence of antithrombin or TFPI results in increased thrombin peaks and the endogenous thrombin potential compared to FVIII-deficient plasma alone. This is in line with an ameliorated hemostatic potential induced by these agents. Indeed, thrombin generation profiles appear to be ameliorated in the vast majority of patients that receive emicizumab, fitusiran or anti-TFPI antibodies. [START_REF] Pasi | Targeting of antithrombin in hemophilia A or B with RNAi therapy[END_REF][START_REF] Kizilocak | Management of perioperative hemostasis in a severe hemophilia A patient with inhibitors on emicizumab using global hemostasis assays[END_REF][START_REF] Chowdary | Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial[END_REF][START_REF] Patel-Hett | Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas[END_REF] . Thus, these types of assays are excellent tools to verify that whether hemostatic profile of the patient has been changed since the onset of treatment. Nevertheless, care should be taken with the interpretation of these data. First, it is yet unclear how well the individual thrombin generation potential in vitro correlates with the actual hemostatic response in a patient, ie. whether these assays accurately predict clinical outcome. [START_REF] Chelle | Prediction of individual factor VIII or IX level for the correction of thrombin generation in haemophilic patients[END_REF][START_REF] Olsson | Bleeding phenotype in carriers of haemophilia A does not correlate with thrombin generation[END_REF][START_REF] Dargaud | Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B[END_REF][START_REF] Santagostino | Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile[END_REF][START_REF] Young | Thrombin generation and whole blood viscoelastic assays in the management of hemophilia: current state of art and future perspectives[END_REF] Second, these global assays will not give information about the true FVIII-equivalence of the agent. This is easily illustrated by using emicizumab as an example. The functioning of emicizumab is fully dependent on the amount of FIXa available during the test.

Dependent on the concentrations of TF or FXIa used to initiate the reaction, the amounts of FIXa generated will be different. This may lead to more or less thrombin generated in the presence of emicizumab, and as such a different equivalence to FVIII. Another example concerns the functional absence of antithrombin in FVIII-deficient plasma. The initial part of a normal thrombin generation curve is dependent on FVIIa/TF generated FIXa and FXa, as well as the temporary presence of the labile FVIIIa molecule (the activity of which disappears within a few minutes). In contrast, the second part of the curve depends for an important part on the inhibition of FIXa, FXa and thrombin by antithrombin. Reduced antithrombin activity in FVIII-deficient plasma will thus extend the lifespan of these enzymes in the assay, which translates into an increased thrombin potential, visualized by a delayed decline of the thrombin peak (Fig. 2; see also Sehgal et al. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF] ). It is possible therefore that despite a lower thrombin peak, the total amount of thrombin generated under these conditions approaches or even exceeds that of normal plasma. However, we should not forget that a2-macroglobulinmediated inhibition of thrombin is not taken into account in this assay (a2-macroglobulinbound thrombin hydrolyzes the small fluorogenic substrate that is used in this assay) and only a small portion of the TFPI population is present (about 10% of total TFPI in vivo).

Moreover, the thrombomodulin-dependent activated Protein C pathway is present to a minor extent, whereas the platelet thrombin inhibitor Protease Nexin-1 is completely absent. The TGA will thus exaggerate the amount of thrombin generated under these conditions, because portions of the anticoagulant pathway are not present to compensate the absence of antithrombin.

In summary, global assays are an excellent tool to monitor the hemostatic potential of a patient before and after the onset of non-factor therapies. They may even be used to analyze the effect of additive treatments, like FVIII or APCC, before they will be administered to the patient. [START_REF] Kizilocak | Management of perioperative hemostasis in a severe hemophilia A patient with inhibitors on emicizumab using global hemostasis assays[END_REF][START_REF] Dargaud | Use of thrombin generation assay to personalize treatment of breakthrough bleeds in a patient with hemophilia and inhibitors receiving prophylaxis with emicizumab[END_REF] In contrast, the global assays cannot be used to assign a FVIII-equivalence to the therapeutic agent itself.

Monitoring the FVIII-equivalence of non-factor therapies

An issue that often comes up in the discussions around non-factor therapies is: how do they Again, the mode of action for each of the non-factor approaches is fundamentally different from that of FVIII. For instance, in the FVIII-dependent FXa generation, FVIIIa is the limiting factor, and an excess of FIXa is present in the reaction. In the emicizumab-based reaction, it is FIXa that represent the limiting factor. It is thus not possible in an in vitro biochemical assay to establish a true FVIII-equivalence for emicizumab, as it will depend on how much FIXa is used in the assay. Perhaps a more unbiased response may be obtained by using animal models. When inflicting an injury in an animal model, there will be a natural hemostatic response, with all elements of the pro-and-anticoagulant pathways present.

A first example of an animal model to obtain a FVIII-equivalence for emicizumab was reported by Kitazawa and colleagues, who used a primate model for acquired hemophilia A (Fig. 3). [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF] Primates were given an anti-FVIII antibody that specifically inhibits primate FVIII.

The animals then received either emicizumab to a concentration of 6 µg/ml or porcine FVIII (which is not recognized by the anti-FVIII antibody) to a concentration of 0.01 U/ml (1%).

Both treatments resulted in a similar prevention of blood loss (measured by hemoblobin levels). In contrast, porcine FVIII proved somewhat more efficient than emicizumab in reducing the bruised area in these animals. [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF] The authors conclude that emicizumab activity at these concentrations corresponds to about 1% FVIII activity.

Taking into account that the actual therapeutic dose that is now used in patients is ten-fold higher (about 55 µg/ml), it seems conceivable that the FVIII-equivalence of emicizumab at these therapeutic levels will be around 10%. Indeed, in a more extensive analysis using this primate model for acquired hemophilia, the same group were able to estimate the FVIIIequivalence to 10-20% FVIII. [START_REF] Muto | Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation[END_REF] Primate studies are costly, and not accessible to all laboratories, which limits more in-depth studies to the in vivo behavior of emicizumab. Unfortunately, emicizumab is very specific to human and primate factors IX and X, complicating its assessment in more accessible animal models for hemophilia A, like mice, rats and dogs. To overcome this limitation, we have developed an adapted mouse model (Fig. 3), that combines FVIII-deficiency with the presence of human factors IX and X (manuscript under revision). Without going into the specific details, as they will be outlined in a separate manuscript, this model allowed us to directly compare the activity of emicizumab to that of FVIII in a tail clip-bleeding model.

Basically, mice were given different doses of FVIII in order to establish a calibrator curve, in which a specific FVIII concentration correlated with a specific amount of blood loss. We then measured blood loss in mice that were given emicizumab. The calibration curve with FVIII was subsequently used to estimate the FVIII-equivalence for emicizumab in this particular model. By doing so, we observe that the reduction in blood loss observed in the presence of therapeutic levels of emicizumab corresponds to a FVIII concentration of 9%, which is very close to the 10% estimated in the primate model.

Of course, each model has its limitations and a semi-humanized mouse model is no exception. The FVIII-equivalence obtained is true for this particular type of injury in this particular tissue (amputation of the terminal 3 mm of the tail tip). It is possible that in another bleeding model (e.g. a tail vein transection or saphenous vein injury), the activity of emicizumab will be different. Its activity will be fully dependent on the local production of FIXa, which in turn is dependent on how much TF or FXIa will be present following the injury.

It seems conceivable that this will also be true in the human setting: the severity of the injury and its location will determine how much FIXa will be generated and will be available to support emicizumab activity. Continuous clinical observation of patients receiving emicizumab will help us to further understand how efficient emicizumab is compared to FVIII in different conditions of injury.

As for fitusiran, a saphenous vein bleeding model has been used to investigate its in vivo efficacy in comparison to FVIII. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF] FVIII-deficient mice were treated with fitusiran to obtain a reduction of 70% antithrombin antigen or with FVIII at a dose of 25 U/kg (which results in FVIII levels of about 0.5 U/ml or 50%). In this model, the reduction in antithrombin proved as efficient as FVIII. However, it is unclear if lower levels of FVIII would have been sufficient to stop the bleeding in this model. Based on recent data using nanobodies that block antithrombin activity in a tail clip model using FVIII-deficient mice [START_REF] Barbon | Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors[END_REF] , it would be fair to assume that a reduction of antithrombin levels to 30% of normal corresponds to a FVIII-equivalence of at least 20% FVIII.

With regard to anti-TFPI antibodies, preclinical data on the analysis of Concizumab (presented under its original name mAb2021) in a cuticle bleeding model in FVIII-deficient rabbits have been published. [START_REF] Hilden | Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model[END_REF] When given intravenously at a dosing range of 0.5 to 8 mg/kg 35 minutes before the induction of bleeding, a dose of 0.5 mg/kg was already sufficient to markedly reduce the bleeding tendency in this model. Moreover, all doses of ≥1 mg/kg reduced blood loss to levels observed in non-hemophilic rabbits. Unfortunately, it is not reported what were the levels of the antibody in plasma. Assuming a 90% availability and a plasma volume of 40 ml/kg in rabbits, the antibody concentration following a 1 mg/kg intravenous injection would be about 22 µg/ml. Noteworthy, a FVIII calibrator curve could not be used in this acquired hemophilia model, complicating the estimation of a true FVIIIequivalence. However, given the strong reduction in blood loss it seems fair to assume that the FVIII-equivalence is al least 20% FVIII.

Taken together, animal models provide an alternative approach to establish a FVIIIequivalence for non-factor therapies, as they will generate a natural, unbiased response to an injury. By doing so, we should keep in mind that animal models are inherently hampered by species differences, which could influence the reactivity of these agents. Nevertheless, when considering the animal models currently available, it seems that emicizumab provides a FVIII-equivalence of about 10-20%. Unfortunately, FVIII-calibrators were lacking in the reports assessing fitusiran and concizumab. Based on their capacity to fully correct the bleeding in these models, it seems fair to assume that their FVIII-equivalence is ≥20% FVIII.

Discussion

Laboratory monitoring of hemophilia A goes back a long time, and the presence of different sorts of FVIII (plasma-derived versus recombinant, plasma samples versus concentrates, Bdomainless versus full-length, standard half-life versus extended half-life) has had an important impact on FVIII analysis. It is because of these complications that correct monitoring of FVIII activities are performed by specialized clinical laboratories. The advent of non-factor therapies will add another layer of difficulty in this regard. For each patient, its treatment should be well indicated in order for the laboratory to perform the correct assay. In turn, clinical laboratories should be equipped with the necessary assays to be performed, whether it is needed to monitor levels of the therapeutic agent or to investigate how the patient reacts to the treatment.

As to the assays themselves, it is important to have a thorough biochemical knowledge on the properties of the novel therapies, as this is pertinent for the interpretation of the obtained results. It is also relevant to have an understanding on how the novel therapies affect other clinical assays or how other types of medications affect the analysis of the non-factor therapies, issues that have recently been discussed for emicizumab. [START_REF] Adamkewicz | Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Function, on Coagulation Assays[END_REF][START_REF] Tripodi | Advances in the treatment of hemophilia: implications for laboratory testing[END_REF][START_REF] Jenkins | Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline[END_REF] Hemophilia treatment is entering into a new era, and its laboratory monitoring should follow in parallel. Different types of animal models have been used to estimate FVIII-equivalence for non-factor therapies. For emicizumab, a primate acquired hemophilia A model has been used [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF][START_REF] Muto | Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation[END_REF] and a semi-humanized mouse model for FVIII-deficiency (P.J. Lenting, revised manuscript submitted). In both models, the use of a FVIII-calibrator allowed to estimate the FVIIIequivalence to be 10-20%. For fitusiran, FVIII-deficient mice displayed a strong reduction of their bleeding tendency. [START_REF] Sehgal | An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia[END_REF] However, no FVIII calibrator curve was used to determine a FVIII equivalence. In an alternative approach using anti-antithrombin nanobodies, inhibition of antithrombin activity appeared to correspond to a FVIII-equivalence of ≥20%. [START_REF] Barbon | Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors[END_REF] Regarding concizumab, a rabbit model for acquired hemophilia has been described. [START_REF] Hilden | Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model[END_REF] Also in this model, a FVIII-calibrator was unavailable. Based on the observed efficiency in the cuticle bleeding model, the FVIII-equivalence seems to be ≥20% FVIII.

LEGENDS

  compare to FVIII in terms of efficiency? The long clinical experience with FVIII-replacement therapy has established what levels of FVIII are needed for certain types of activities, such as sports or surgical interventions. It would be convenient to know therefore what would be the FVIII-equivalence of these non-factor therapies, in order to understand what level of protection will be present during this type of activities.

Figure 1 :

 1 Figure 1: Comparison of traditional and novel Hemophilia therapiesSimilarities and differences of hemophilia A treatment between FVIII-replacement therapy, emicizumab, fitusiran and anti-TFPI antibodies with regard to patient target group, mode of administration and therapeutic levels over time.

Figure 2 :

 2 Figure 2: Thrombin generation in FVIII-deficient plasmaThrombin generation in FVIII-deficient plasma in the presence of emicizumab (panel A), antiantithrombin nanobodies (panel B) or anti-TFPI antibodies (panel C). In all cases, the addition of the antibodies increases thrombin generation compared to FVIII-deficient plasma alone. Panel A represents factor XIa-induced thrombin generation in the absence or presence of Emicizumab (0-50 µg/ml; P.J. Lenting, unpublished data). Panel B represents tissue factor-induced thrombin generation in the absence or presence of anti-antithrombin nanobodies.[START_REF] Barbon | Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors[END_REF] Panel C represents tissue factor-induced thrombin generation in the absence or presence of polyclonal anti-TFPI antibodies.[START_REF] Marlu | Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex[END_REF]

Figure 3 :

 3 Figure 3: Animal models to estimate FVIII-equivalence of non-factor therapies

Conflict of interest:

PJL received speaker fees from Biotest, Chugai, NovoNordisk, Roche, Sanofi, Sobi and Takeda.

Author contributions:

PJL wrote the manuscript.


est destinée au dépôt

  Hydrogenation of the ternary silicides RENiSi (RE = Ce, Nd) crystallizing in the tetragonal LaPtSi-type structure Mathieu Pasturel, François Weill, Françoise Bourée, Jean-Louis Bobet, Bernard Chevalier To cite this version: Mathieu Pasturel, François Weill, Françoise Bourée, Jean-Louis Bobet, Bernard Chevalier. Hydrogenation of the ternary silicides RENiSi (RE = Ce, Nd) crystallizing in the tetragonal LaPtSi-type structure. Journal of Alloys and Compounds, 2005, 397 (1-2), pp.17-22. 10.1016/j.jallcom.2005.01.015


Dennis Bodewits
email: dennis@auburn.edu

Damian Christian

Michael S P Kelley

Jian Yang

Silvia Protopapa

Tony Farnham

Kip D Kuntz

Dimitra Koutroumpa

Jennifer Carter

Davide Farnocchia

Tim Lister

Matthew M Knight

John Noonan

Walt Harris

Joel Parker

Kathleen Mandt

Charles Bell

Characterizing the physical and chemical behaviour of comet 46P/Wirtanen

Introduction

Comet nuclei are remnants from the planet formation era but cannot be observed directly from Earth. When they approach the Sun a cloud of gas dust obscures their nuclei, and their properties must be inferred from gas and dust surrounding them. Understanding the connection between the coma and nucleus is therefore critical. Comet 46P/Wirtanen passed Earth with an historic closest approach distance of 0.077 au (12 million km) on Dec. 16, 2018. Its apparition was widely anticipated by planetary scientists and was the target of a world-wide observing campaign. Studies of comet Wirtanen around the time of its close approach to Earth provided spatial resolutions high enough to permit valuable comparisons to the Rosetta observations of 67P/Churyumov-Gerasimenko and the Deep Impact/EPOXI fly-by results of 103P/Hartley 2. In this contribution, we will summarize the preliminary results from our global campaign that combined telescopes in and around Earth to characterize the physical and chemical behavior of comet 46P/Wirtanen.

Chandra and Hubble Space Telescope

Chandra X-ray observations were obtained on Dec. 3, 2018 and Dec. 13, 2018 to use the comet's X-ray emission to create a tomographic image of the bulk distribution of gas in the coma, and to compare the emission from solar wind charge exchange reactions between water molecules in the comet's atmosphere with reactions between H and He in the heliosphere. X-ray background subtraction is a common problem when observing extended sources, and we acquired long exposures of the part of the sky that the comet moved through. Despite this careful approach, only a marginal detection of 46P was made with ACIS-S likely owing to the detector's degradation, and analysis is on-going.

To connect the X-ray observations with the nucleus and the gas and dust surrounding it, parallel observations were scheduled with HST/WFC3. Tracking issues resulted in a loss of the observations acquired on Dec. 3, 2018, and to make up for these observations HST/WFC3 observed the comet again on Dec. 25, 2018.

Neil Gehrels Swift Observatory

Swift observed 46P four times between Nov. 28, 2018 and Jan. 12, 2019. The main goal of the observations was to acquire high cadence UVOT broad band imaging in the U, V, and UVW1 filters to measure variations in the gas and dust production (cf. [1]). Swift cannot track non-sidereal objects and significant trailing of the comet was avoided by using a large number of images with short exposures. In addition, part of the images was acquired in 'event mode', which allows time tagging of each incoming photon (at significant telemetry expense).

Las Cumbres Observatory

The robotic 0.4m and 1.0m telescopes of the global Las Cumbres Observatory were used between Sep. 9, 2018 and May 5, 2019 to monitor the long-term baseline of the activity of 46P and to use extended high-cadence monitoring to investigate the evolution of the comet's variability around its perihelion. Two filters (g' and r') were used to search for color changes and as a proxy of the gas and dust content of the coma.

HST COS/STIS campaign

HST operations requires the use of guide stars when the sensitive COS NUV-FUV and STIS/MAMA detectors are used, but the apparent motion of the comet was too high during closest approach for this. HST spectroscopy observations were possible after Jan. 9, 2019 and were obtained between Jan 9, 2019 and Jan. 20, 2019. The observations aimed to use the unique close proximity of 46P to study the inner part of the coma (within 100 km of the nucleus), a region rarely accessible to remote observations. Using multiple FUV spectroscopy settings we will search for the emission of short-lived species (such as S2, O2), of molecules not accessible from the ground (CO2), and we investigated the transitions between regions where either electrons or photons drive most of the chemistry in the coma. The results of the HST spectroscopy campaign are also discussed in another contribution [START_REF] Noonan | Hubble Space Telescope Cosmic Origins Spectrograph Observations of 46P/Wirtanen During Close Approach[END_REF].

Acknowledgements

We are grateful to observers around the world who reported their observations of 46P to the MPC, which allowed us to successfully observe the comet with HST. These include Nick Moskovitz, Marco Micheli, Bill Ryan, Dave Tholen, Paolo Bacci, Brian Skiff, Emmanuel Jehin, Chunua Qi, and Martin Cordiner. These observations were made possible by the efforts of the scheduling teams on CXO, HST, and Swift. We acknowledge support from Chandra GI grant 19100360; HST GO-15372 and GO-15625; Swift GI 1417065; and LCO observing time through NOAO programs 2018B-0221 and 2019A-0118.