10.4081/btvb.2023.89
Emicizumab, a FVIII-mimetic bispecific antibody, is insensitive to degradation by activated protein C (APC) and may thus induce a procoagulant state. We investigated the effect of emicizumab on protein C-mediated inhibition of coagulation under in vitro conditions mimicking physiological and pathological clotting activation. Thrombin generation (TG) in tissue factor-triggered hemophilic plasma containing emicizumab (50 μg/mL) was inhibited by APC or thrombomodulin in a concentration-dependent manner, and to a similar extent as in plasma added with FVIII (Kovaltry, 1 IU/mL). However, when clotting was activated via the intrinsic pathway, emicizumab-plasma displayed resistance to APC, manifested by a barely detectable prolongation of the lag time of TG, and by the lack of inhibition of FXa generation. Moreover, in contact-activated plasma added with APC, the generation of a second wave of thrombin, following prothrombin replenishment, was much greater in emicizumab-plasma than in Kovaltry-plasma, suggesting that the insensitivity of emicizumab to APC may translate in greater thrombin formation. Considering the major role played by the contact system in the thrombotic process, hemophilia A patients under emicizumab treatment might be at increased thrombotic risk.
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Mario Colucci
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Bleeding, Thrombosis and Vascular Biology
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Influence of emicizumab on protein C-mediated clotting regulation
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Original Articles
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Bleed Thromb Vascul Biol
Mario Colucci
2023/10/25
10.4081/btvb.2023.89
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Fredenburgh JC, Weitz JI. News at XI: moving beyond factor Xa inhibitors. J Thromb Haemost 2023;21:1692-702.
Influence of emicizumab on protein C-mediated clotting regulation
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                                                                                                           ORIGINAL ARTICLE

Influence of emicizumab on protein C-mediated clotting regulation
Federica Mancazzo, Antonia Vitulli, Lavinia Dirienzo, Concetta T. Ammollo, Fabrizio Semeraro, Mario Colucci

Department of Precision and Regenerative Medicine and Ionian Area, Aldo Moro University, Bari, Italy



ABSTRACT
     Emicizumab, a FVIII-mimetic bispecific antibody, is insensitive to degradation by activated protein C (APC) and may thus induce a
procoagulant state. We investigated the effect of emicizumab on protein C-mediated inhibition of coagulation under in vitro conditions
mimicking physiological and pathological clotting activation. Thrombin generation (TG) in tissue factor-triggered hemophilic plasma con-
taining emicizumab (50 µg/mL) was inhibited by APC or thrombomodulin in a concentration-dependent manner, and to a similar extent
as in plasma added with FVIII (Kovaltry, 1 IU/mL). However, when clotting was activated via the intrinsic pathway, emicizumab-plasma
                                                                         displayed resistance to APC, manifested by a barely detectable
                                                                         prolongation of the lag time of TG, and by the lack of inhibition
                                                                         of FXa generation. Moreover, in contact-activated plasma added
   Correspondence: Mario Colucci, Piazzale Giulio Cesare 11, 70124
                                                                         with APC, the generation of a second wave of thrombin, follow-
   Bari, Italy.




                                                                                                       ly
   Tel.: +39.0805478471.                                                 ing prothrombin replenishment, was much greater in emicizumab-
   E-mail: mario.colucci@uniba.it                                        plasma than in Kovaltry-plasma, suggesting that the insensitivity




                                                                                               on
                                                                         of emicizumab to APC may translate in greater thrombin forma-
   Citation: Mancazzo F, Vitulli A, Dirienzo L, et al. Influence of emi- tion. Considering the major role played by the contact system in
   cizumab on protein C-mediated clotting regulation. Bleeding,          the thrombotic process, hemophilia A patients under emicizumab




                                                                                         e
   Thrombosis, and Vascular Biology 2023;2:89.                           treatment might be at increased thrombotic risk.
  Key words: emicizumab, protein C resistance, intrinsic pathway,
  thrombotic risk.
                                                                                 us
                                                                            Introduction
                                                                            al
  Acknowledgments: the authors thank Prof. Nicola Semeraro for
  helpful commentary on the manuscript.                                         Emicizumab (Hemlibra) is a FVIII-mimetic bispecific anti-
                                                                  ci

                                                                            body licensed for the prevention of bleeding in hemophilia A
  Contributions: FM, AV, LD, CTA, FS, performed research and con-
                                                                er


                                                                            patients with and without inhibitors.1,2 It mimics the function of
  tributed to data analysis; MC designed and supervised the study
  and wrote the manuscript. All authors read and approved the final         FVIII by simultaneously binding FIXa and factor X, thereby
                                                               m



  version to be published.                                                  promoting the co-localization of the enzyme-substrate complex
                                                                            on the phospholipid surface.3 Compared to FVIII, emicizumab
                                                    om




  Conflict of interest: the authors declare no potential conflict of in-    presents several differences from biochemical and mechanistic
  terest.                                                                   perspectives.4 FVIII is highly regulated as it needs to be acti-
                                                                            vated by thrombin to express its full cofactor activity and, once
  Funding: the work was supported by the Aldo Moro University of
                                             -c




                                                                            activated (FVIIIa), it is degraded by activated protein C (APC),
  Bari.
                                                                            which represents one of the major physiological control mech-
                                    on




  Availability of data and material: data and materials are available       anisms of coagulation.5 Moreover, FVIIIa activity decays spon-
  by the authors.                                                           taneously as a result of A2 subunit dissociation.4 In contrast,
                                                                            emicizumab is constitutively active, is stable, and cannot be in-
                              N




  Informed consent: the manuscript does not contain any individual          activated by APC,4 and therefore it might tilt the coagulation
  person’s data in any form.                                                balance towards a procoagulant state.
                                                                                APC is a serine protease, derived from the activation of pro-
  Received: 20 July 2023.                                                   tein C (PC) by the thrombin-thrombomodulin complex, which
  Accepted: 21 September 2023.
                                                                            downregulates thrombin generation by inactivating the cofactors
  Publisher’s note: all claims expressed in this article are solely those   FVa and FVIIIa through limited proteolysis at specific arginine
  of the authors and do not necessarily represent those of their affili-    residues.5 The best-known condition associated with refractori-
  ated organizations, or those of the publisher, the editors and the re-    ness to APC is the FV Leiden mutation,6 which, in rare homozy-
  viewers. Any product that may be evaluated in this article or claim       gous carriers, increases the thrombotic risk by approximately
  that may be made by its manufacturer is not guaranteed or endorsed        80-fold.7 In the physiological clotting cascade, FVIIIa serves as
  by the publisher.                                                         cofactor of FIXa in the intrinsic tenase complex whereas FVa is
                                                                            part of the prothrombinase complex, which leads to the genera-
  ©
   Copyright: the Author(s), 2023                                           tion of the final clotting enzyme thrombin.8 Moreover, FV is
  Licensee PAGEPress, Italy                                                 able to function as cofactor of APC in the inactivation of
  Bleeding, Thrombosis and Vascular Biology 2023; 2:89
                                                                            FVIIIa.9 In view of the different roles of the two cofactors, a key
  doi:10.4081/btvb.2023.89
                                                                            question is whether APC-resistant FVIII has the same effect on
  This work is licensed under a Creative Commons Attribution-               thrombin generation and thrombotic risk as APC-resistant FV.
  NonCommercial 4.0 International License (CC BY-NC 4.0).                   Wilhelm et al. reported that a FVIII variant (FVIII-
                                                                            R336Q/R562Q) insensitive to degradation by APC made FVIII-


                                              Bleeding, Thrombosis and Vascular Biology 2023; 2:89
                                    Influence of emicizumab on protein C-mediated clotting regulation                                  49



deficient plasma resistant to the anticoagulant effect of APC and       amino-4-methylcoumarin (ZGGR-AMC) and thrombin calibra-
displayed a greater prothrombotic activity than wild-type B-do-         tor from Diagnostica Stago (Asnieres, France); human APC (Xi-
mainless FVIII in a ferric chloride thrombosis model in hemo-           gris) from Eli Lilly (Indianapolis, IN, USA); soluble rabbit
philia A mice.10 Moreover, acquired APC resistance is a risk            thrombomodulin (TM) from Sekisui Diagnostics (Darmstadt,
factor for venous thrombosis,11 and one of the mechanisms be-           Germany); hirudin from Abbott GmbH (Ludwigshafen, Ger-
hind this form of APC resistance is the elevation of FVIII lev-         many); purified human FXIa from Life Technologies (Monza,
els,11,12 a finding that provides a likely explanation for the          Italy); purified human prothrombin from Haematologic Tech-
increased thrombosis risk associated with high FVIII levels.13          nologies (Essex Junction, VT, USA); bovine fibrinogen from
Based on these observations, it is conceivable to hypothesize           Sigma (Milan, Italy). Phospholipid vesicles (20% phos-
that the replacement of FVIII with emicizumab will impair the           phatidylserine, 60% phosphatidylcholine, 20% phos-
anticoagulant activity of PC, thereby increasing thrombin gen-          phatidylethanolamine; Avanti Polar Lipids, Alabaster, AL, USA)
eration and, eventually, the thrombotic risk.                           were prepared by sonication.
     To date, only one study has specifically investigated the ef-
fect of emicizumab on APC-dependent control of coagulation.14           Fluorogenic thrombin generation assay
The authors reported that APC addition to hemophilic A plasma
supplemented with emicizumab (referred to as emicizumab-                     All reported concentrations are final concentrations per mL
plasma) resulted in a concentration-dependent reduction of              of plasma (emicizumab, FVIII concentrate, APC, TM) or per ml
thrombin generation, which was entirely due to the inactivation         of the final mixture. Thrombin generation (TG) was evaluated




                                                                                          ly
of FV(a). The study, however, does not allow us to safely con-          according to Hemker et al.,15 with minor modifications as fol-
                                                                        lows. Forty μL plasma, 5 μL emicizumab (50 μg/mL) or Koval-




                                                                                  on
clude that APC-dependent inhibition of coagulation is not im-
paired by emicizumab for the following reasons. First,                  try (1 IU/mL), 2.5 μL phospholipids (5 μg/mL), 5 μL buffer,
emicizumab-plasma was not compared to a proper control con-             APC or TM (at the indicated concentrations), and 2.5 μL clotting
                                                                        trigger were added to round-bottom 96-well plates (Immulon




                                                                            e
sisting of the same FVIII-deficient plasma supplemented with a
“traditional” FVIII preparation (plasma-derived or recombi-             2HB; Dynex Technologies). The reaction was started by the ad-
nant). Therefore, it is not possible to establish to what extent the
inhibition of coagulation by APC in emicizumab-plasma com-
                                                                       us
                                                                        dition of a 10 μL mixture containing CaCl2 and ZGGR-AMC
                                                                        (16 mM and 0.42 mM, respectively). Measurements were taken
pares to that observed in a plasma containing a FVIII sensitive         every 20 seconds in a FluoroScan Ascent fluorometer (Thermo
                                                                  al
to APC degradation. Second, as recognized by the same authors,          Scientific, Dreieich, Germany), and data were analyzed using
                                                                        the Thrombinoscope software (Thrombinoscope BV, Maastricht,
                                                   ci

the concentration of emicizumab added to FVIII-deficient
plasma (30 µg/mL) was lower than that achieved in patients (40-         The Netherlands). The TG parameters evaluated in this study
                                                 er


60 µg/mL) and thus it is unknown if greater concentrations of           were lag time, thrombin peak, and endogenous thrombin poten-
emicizumab would reduce the anticoagulant response to APC.              tial (ETP). All samples and calibrators were run in duplicate.
                                                m



Finally, the thrombin generation experiments were performed                  Unless otherwise specified, coagulation was triggered by
using a single condition in which APC was added to plasma trig-         thromboplastin (1:10000 final dilution, corresponding to approx-
                                      om




gered with a combination of tissue factor (TF) and ellagic acid.        imately 1 pM tissue factor) or human FXIa (10 pM). In some
     Our study was undertaken to further evaluate the influence         experiments, the intrinsic pathway of coagulation was activated
of emicizumab on the inhibition of thrombin generation by the           with 1:50 diluted aPTT reagent or Kovaltry replaced by Beriate
                                -c




PC system, using experimental conditions that mimic both phys-          (1 IU/mL). The effect of APC or TM on TG parameters was cal-
iological and pathological clotting activation. Our data suggest        culated as the ratio between the values recorded in the presence
                        on




that, under specific conditions, the resistance of emicizumab to        and in the absence of APC or TM. Thus, as for lag time, the
APC results in faster and greater thrombin generation that may          greater the ratio the stronger the anticoagulant effect of APC,
                   N




induce a hypercoagulable state.                                         whereas for Peak and ETP, the lower the ratio the stronger the
                                                                        APC effect.

Materials and Methods                                                   Functional thrombin generation assay
Hemophilia A plasma and reagents                                            To evaluate the effect of prothrombin replenishment (after
                                                                        prothrombin consumption) in emicizumab- and Kovaltry-
    Frozen Hemophilia A plasma (FVIII<1%, 1 mL per vial)                plasma, thrombin generation in the absence and in the presence
was purchased from George King Bio-Medical (Overland Park,              of APC was assessed by a two-stage clotting assay as follows.
KS, USA). Three different batches of hemophilic plasma were             Six-hundred-forty μL prewarmed hemophilic plasma, freshly
used in our study.                                                      defibrinated as reported,16 was added into a test tube placed in a
    Emicizumab was obtained from Roche (Basel, Switzerland),            water bath at 37°C, followed by 20 μL emicizumab or Kovaltry
Kovaltry (recombinant full-length FVIII) from Bayer (Milan,             (50 μg/mL and 1IU/mL, respectively), 20 μL phospholipids (5
Italy), and Beriate (plasma-derived FVIII) from CSL Behring             μg/mL), 40 μL APC (0.4 μg/mL) or buffer, and 80 μL FXIa (10
(Marburg, Germany).                                                     nM). The reaction was started by the addition of 160 μL CaCl2
    The following reagents were purchased from the indicated            (16 mM). Then, 60 μL aliquots were transferred at intervals in a
sources: aPTT reagent (SynthasiL), human thromboplastin (Re-            prewarmed tube prefilled with 30 μL PBS containing bovine fib-
combiplastin), and S-2732 (FXa-specific substrate) from Instru-         rinogen (2 mg/mL) and sodium citrate (0.036 M), and the clot-
mentation Laboratory (Milan, Italy); reptilase from Bothrops            ting time was assessed manually by the tilting method.
atrox venom, fluorogenic thrombin substrate Z-Gly-Gly-Arg-7-            Thrombin activity in each subsample was calculated by refer-


                                          Bleeding, Thrombosis and Vascular Biology 2023; 2:89
50                                                         F. Mancazzo et al.




ence to a calibration curve constructed with increasing concen-        run in parallel, was assessed by paired Student t-test using the
trations of purified human thrombin. After thrombin activity had       MedCalc software (Mariakerke, Belgium). Two-tailed P<0.05
fallen to near zero, prothrombin (100 µg/mL) was added to the          was considered statistically significant.
reaction mixture, and the new thrombin formed was monitored
using the same two-stage assay.
                                                                       Results
FXa generation assay
                                                                       Effect of emicizumab on thrombin generation
    FXa generation was measured by a chromogenic assay as              in tissue factor-triggered hemophilic plasma
follows. Fifteen μL defibrinated hemophilic plasma, 5 μL emi-
cizumab (50 μg/mL) or Kovaltry (1 IU/mL), 5 μL phospholipids               Experiments were carried out using a low concentration of
(5 μg/mL), 5 μL buffer or APC (0.4 µg/mL), 15 μL FXIa (10              TF (1 pM) so that the intrinsic tenase complex, generated mainly
pM), 15 uL hirudin (200 U/mL) or buffer and 50 µL S-2732               through the Josso loop (FIX activation by TF/FVIIa), con-
(FXa-specific substrate, 1.6 mM) were added to round-bottom            tributed significantly to thrombin formation, as testified by the
96-well plates, after which the reaction was started by the addi-      very little thrombin formation in the absence of FVIII addition
tion of 10 μL CaCl2 (6 mM). Substrate cleavage was monitored           (not shown). Typical TG curves in the presence of increasing
by the change in absorption at 405 nm in a microplate reader           concentrations of APC are illustrated in Figure 1A. APC-induced
(Multiskan FC; Thermo Fisher Scientific, Waltham, MA, USA).            changes in TG parameters, calculated as APC ratio, showed that




                                                                                                 ly
                                                                       there was a concentration-dependent inhibition of TG in both
                                                                       emicizumab- and Kovaltry-plasma, as indicated by the progres-




                                                                                         on
Statistical analysis
                                                                       sive prolongation of lag time and reduction in ETP and thrombin
   Results are expressed as mean ± SD. The difference between          peak (Figure 1B). Although the changes in emicizumab-plasma
emicizumab- and Kovaltry-plasma samples, which were always             were slightly less pronounced than in Kovaltry-plasma, the dif-




                                                                                    e
                                                                                us
                                                                      al
                                                         ci
                                                       er
                                                      m
                                             om
                                       -c
                               on
                          N




Figure 1. Effect of increasing concentrations of activated protein C (APC) on tissue factor-triggered thrombin generation in hemophilic
plasma supplemented with emicizumab (50 µg/mL) or Kovaltry (1 IU/mL). A) Typical thrombin generation curves in emicizumab-
plasma (left) and Kovaltry-plasma (right); B) APC-induced changes in thrombin generation parameters calculated as APC ratio (value
in the presence of APC divided by value without APC). For lag time, the greater the ratio the stronger the anticoagulant effect of APC;
for Peak and endogenous thrombin potential (ETP), the lower the ratio, the stronger the APC effect. Data are the mean ± standard de-
viation of 6 independent experiments.


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:89
                                  Influence of emicizumab on protein C-mediated clotting regulation                                 51



ferences between the two samples were not significant for any         centrations of TM (15 and 30 nM), we confirmed a striking dif-
TG parameters. Qualitatively similar results were obtained when       ference in TM-induced lag time prolongation between emi-
thrombin generation was performed in the presence of different        cizumab and Kovaltry but did not find significant differences in
concentrations of TM (Supplementary Figure 1) or when Ko-             Peak and ETP reduction at any TM concentration (Supplemen-
valtry was replaced by Beriate (1 IU/mL), a plasma-derived            tary Figure 2).
FVIII concentrate (not shown).                                            Qualitatively similar results were obtained upon the addition
                                                                      of APC or TM to plasma samples activated with diluted aPTT
Effect of emicizumab on thrombin generation                           reagent (not shown).
in contact-activated hemophilic plasma                                    Based on the above findings, all following experiments
                                                                      were performed in contact-activated hemophilic plasma added
     To make FXa generation exclusively dependent on FVIII            with APC.
(intrinsic tenase), we activated plasma with FXIa (10 pM) or di-
luted aPTT reagent (1/50 final dilution). Figure 2 illustrates the    Effect of emicizumab on FXa generation
results with FXIa-triggered plasma. In the absence of APC or
TM addition, emicizumab-plasma displayed a significantly                  FXa generation was evaluated in defibrinated plasma using
shorter lag time compared to Kovaltry-plasma (2.8±0.81 vs.            a chromogenic substrate (S-2732). The time course of substrate
5.1±0.84 min, P<0.001) most likely because emicizumab, con-           cleavage (optical density [OD] increase) in plasma challenged
trary to FVIII, does not need to be activated to function as co-      with FXIa showed remarkable differences between emicizumab-




                                                                                        ly
factor of FIXa. Upon addition of APC, the lag time prolongation       and Kovaltry-plasma (Figure 3). In the absence of APC, FXa




                                                                                on
was markedly and significantly less pronounced in emicizumab-         formation in emicizumab-plasma started earlier and increased
plasma than in Kovaltry-plasma at all tested APC concentra-           more rapidly than in Kovaltry-plasma; moreover, as expected,
tions. As to the reduction of ETP and thrombin peak, a                FXa generation in the presence of APC was marginally changed
                                                                      in emicizumab-plasma but markedly reduced in Kovaltry-




                                                                           e
significant difference between emicizumab and Kovaltry sam-
ples was only seen at the highest APC concentration (Figure 2).       plasma (Figures 3A and B). To evaluate the possible contribution
When the TG assay was performed in the presence of two con-          us
                                                                      of thrombin to FXa generation (via FVIII activation) and sub-
                                                                al
                                                  ci
                                                er
                                               m
                                     om
                               -c
                        on
                  N




Figure 2. Effect of increasing concentrations of activated protein C (APC) on FXIa-triggered thrombin generation in hemophilic plasma
supplemented with emicizumab (50 µg/mL) or Kovaltry (1 IU/mL). A) Typical thrombin generation curves in emicizumab-plasma (left)
and Kovaltry plasma (right); B) APC-induced changes in thrombin generation parameters calculated as APC ratio (see legend to Figure
1 for further details). Data are the mean ± standard deviation of 6 independent experiments. *p<0.05 by paired Student t-test.


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:89
52                                                         F. Mancazzo et al.




strate cleavage, the assay was also performed in the presence of       fell to near zero, we added purified prothrombin (100 µg/mL)
hirudin (200 U/ml). Under this condition, the OD increase in           and evaluated the second wave of thrombin generation by the
emicizumab-plasma was only slightly reduced compared to the            same clotting assay. As shown in Figure 4, the first thrombin
sample without hirudin, and the little difference between the ab-      curve of emicizumab-plasma showed a visibly shorter lag time
sence and presence of APC became even smaller (Figure 3C),             than Kovaltry-plasma but a similar thrombin peak. On the con-
suggesting a minimal contribution of thrombin to substrate             trary, the second curve displayed more marked differences, the
cleavage. On the contrary, in Kovaltry-plasma, substrate cleav-        rate and the amount of thrombin formed in emicizumab-plasma
age was markedly reduced in the absence of APC and practically         being much greater than those recorded in Kovaltry-plasma. No
absent in its presence (Figure 3D), a finding consistent with the      new thrombin formation was detected if the FXa inhibitor ri-
key role played by thrombin in FVIII activation and, conse-            varoxaban (10 µg/mL) was added along with purified prothrom-
quently, in FX activation by the intrinsic tenase complex.             bin (not shown).

Effect of emicizumab on the second burst
of thrombin generation                                                 Discussion
    The experiments with contact-activated plasma indicate that,           Our study shows that hemophilic plasma supplemented with
in the presence of APC, the accumulation of FXa in emi-                emicizumab displays, under certain conditions, a weaker re-
cizumab-plasma was much greater than in Kovaltry-plasma,               sponse to the anticoagulant effect of APC or TM addition com-




                                                                                                 ly
which is expected given the insensitivity of emicizumab to             pared to APC-sensitive FVIII preparations (Kovaltry and




                                                                                         on
degradation by APC. Because in our in vitro assay FX activation        Beriate). The response to the PC system was investigated by a
by FIXa persisted after the disappearance of thrombin, we              thrombin generation assay, which was performed using as co-
wished to determine how the difference in FXa accumulation             agulation trigger either TF or an activator of the contact system
                                                                       (FXIa or diluted aPTT reagent). The concentrations of emi-




                                                                                    e
between emicizumab- and Kovaltry-plasma would impact
thrombin generation after reconstitution of prothrombin levels,        cizumab and FVIII concentrates added to hemophilic plasma
thereby mimicking the in vivo conditions in which prothrombin
exhaustion is very unlikely to occur. For that purpose, we used
                                                                                us
                                                                       (50 µg/mL and 1 IU/mL, respectively) had a comparable effect
                                                                       on thrombin generation parameters, except for a shorter lag time
FXIa-triggered (defibrinated) plasma containing APC (0.4               of contact-activated plasma supplemented with emicizumab
                                                                      al
µg/mL) and determined the generation of thrombin by a clotting         (Figure 2). As to the response to the PC system, calculated as
                                                                       APC ratio or TM ratio, we had different results depending on
                                                         ci

assay on subsamples taken at intervals. Once thrombin activity
                                                       er
                                                      m
                                             om
                                       -c
                               on
                          N




Figure 3. Effect of activated protein C (0.4 µg/mL) on FXa generation in FXIa-triggered hemophilic plasma supplemented with emi-
cizumab (50 µg/mL, panels A and C) or Kovaltry (1 IU/mL, panels B and D). FXa generation was assessed in defibrinated plasma by
a chromogenic assay. The curves represent optical density at 405 nm (OD405 nm) increase over time. In panels C and D, experiments
were performed in the presence of the thrombin inhibitor hirudin (200 U/mL). Curves from an experiment representative of 4 independent
experiments with similar results are depicted.


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:89
                                   Influence of emicizumab on protein C-mediated clotting regulation                                   53



the coagulation trigger. In TF-triggered coagulation, the changes      our TG assay is that prothrombin is consumed rather quickly,
in thrombin generation parameters recorded in emicizumab-              whereas FXa formation continues beyond thrombin disappear-
plasma were fairly comparable to those observed in Kovaltry-           ance (compare Figure 2 with Figure 3), which is at variance with
plasma (Figure 1). On the contrary, when coagulation was               the in vivo situation where prothrombin exhaustion is very un-
activated via the contact system, we found that the lag time pro-      likely to occur. It is possible, therefore, that our in vitro model
longation induced by APC or TM addition to emicizumab-                 (closed system) did not allow us to fully appreciate the real im-
plasma was markedly and significantly smaller than that                pact of FXa formation on thrombin generation. In an attempt to
observed in Kovaltry-plasma at all tested concentrations of APC        overcome this limitation, we performed the TG assay by adding
and TM. This notwithstanding, no significant differences were          purified human prothrombin soon after endogenous thrombin
observed between emicizumab and Kovaltry samples as to the             had fallen to near zero, giving rise to two TG curves. In the pres-
reduction of ETP and thrombin peak by APC or TM, except for            ence of APC, while the first TG curve recapitulated the results
the highest APC concentration. This finding might suggest that         obtained with the fluorogenic assay, the second one showed a
the inactivation of FVa by APC plays a major role in controlling       marked difference between emicizumab- and Kovaltry-plasma,
the amount of thrombin formation.                                      in that the amount of thrombin generated in the former was sig-
     To further explore the influence of emicizumab on the con-        nificantly greater than that recorded in the latter, suggesting that
trol of coagulation by the PC system, we determined the gener-         the different accumulation of FXa has the potential to influence
ation of FXa in contact-activated plasma and found that, upon          not only the timing but also the amount of thrombin formation.
addition of APC, FXa formation was virtually unaffected in emi-




                                                                                          ly
                                                                       Thus, at variance with Yada et al.,14 we were able to show that,
cizumab-plasma but markedly reduced in Kovaltry-plasma.                under specific experimental conditions, emicizumab attenuates




                                                                                  on
These results are well expected because, in contact-activated          the anticoagulant effect of PC thereby increasing thrombin gen-
plasma, FXa can only be generated through the intrinsic tenase,        eration and purportedly the thrombotic risk.
whose activity is strictly dependent on the availability of FVIIIa.




                                                                            e
Therefore, given that the FVIII-mimetic emicizumab is consti-
tutively active and insensitive to degradation by APC, FXa gen-
eration will be the same regardless of the presence of APC.
     An intriguing observation about the experiments with con-
                                                                      us
                                                                       Conclusions
                                                                           Safety concerns for an increased risk of thrombotic com-
tact-activated plasma containing APC is that, despite the much
                                                                 al
                                                                       plications in patients with hemophilia A under treatment with
greater generation of FXa in emicizumab-plasma, the only TG            emicizumab have been raised by the pivotal HAVEN 1 trial,1
                                                   ci

parameter that consistently showed a weaker response to APC,           which reported six episodes of thrombosis in patients with in-
compared to Kovaltry-plasma, was the lag time. One feature of          hibitors concomitantly treated with bypassing agents. There-
                                                 er


                                                                       fore, even though no evidence has been provided since then of
                                                                       emicizumab-related thrombosis, the resistance of emicizumab
                                                m




                                                                       to APC is perceived as potentially prothrombotic and has led
                                      om




                                                                       investigators to develop a next-generation FVIII-mimetic an-
                                                                       tibody equipped with a self-regulation mechanism that inacti-
                                                                       vates the antibody when enough thrombin is generated.17 Our
                                                                       in vitro study provides experimental evidence suggesting that
                                -c




                                                                       emicizumab may indeed tilt the coagulation balance towards
                        on




                                                                       a procoagulant state by attenuating the anticoagulant effect of
                                                                       the PC system when coagulation is activated via the intrinsic
                                                                       pathway. This finding is clinically relevant because the activa-
                   N




                                                                       tion of the contact system by cell-derived molecules, such as
                                                                       DNA and polyphosphate, has been shown to play a major role
                                                                       both in venous and arterial thrombosis.18,19 Further studies in
                                                                       samples from hemophilic patients under treatment with emi-
                                                                       cizumab are warranted to confirm our results with spiked
                                                                       plasma samples.



Figure 4. Effect of activated protein C (APC 0.4 µg/mL) on             References
thrombin generation in FXIa-triggered hemophilic plasma after
replenishment of prothrombin. Thrombin generation was eval-             1. Oldenburg J, Mahlangu JN, Kim B, et al. Emicizumab pro-
uated by a two-stage clotting assay as detailed in Methods. In             phylaxis in hemophilia A with inhibitors. N Engl J Med
the absence of APC, the only appreciable difference was a                  2017;377:809-18.
shorter lag time in the first thrombin generation curve of emi-         2. Mahlangu JN, Oldenburg J, Paz-Priel I, et al. Emicizumab
cizumab-plasma. Therefore, for the sake of clarity, only the data
in the presence of APC are shown. Curves from an experiment                prophylaxis in patients who have hemophilia A without In-
representative of 4 independent experiments with similar results           hibitors. N Engl J Med 2018;379:811-22.
are depicted. Qualitatively similar results were obtained when          3. Kitazawa T, Igawa T, Sampei Z, et al. A bispecific antibody
prothrombin was added to emicizumab plasma soon after en-                  to factors IXa and X restores factor VIII hemostatic activity
dogenous thrombin disappearance (7 min in this experiment).
                                                                           in a hemophilia A model. Nat Med 2012;18:1570-4.


                                         Bleeding, Thrombosis and Vascular Biology 2023; 2:89
54                                                                 Autore et al.




 4. Lenting PJ, Denis CV, Christophe OD. Emicizumab, a bis-                          measurement of activated protein C resistance. Blood Co-
    pecific antibody recognizing coagulation factors IX and X:                       agul Fibrinolysis 1996;7:761-5.
    how does it actually compare to factor VIII? Blood 2017;                   13.   Kyrle PA, Minar E, Hirschl M, et al. High plasma levels of
    130:2463-68.                                                                     factor VIII and the risk of recurrent venous thromboem-
 5. Esmon CT. The protein C pathway. Chest 2003;124:26s-32s.                         bolism. N Engl J Med 2000;343:457-62.
 6. Bertina RM, Koeleman BP, Koster T, et al. Mutation in                      14.   Yada K, Nogami K, Shinozawa K, et al. Emicizumab-me-
    blood coagulation factor V associated with resistance to ac-                     diated haemostatic function in patients with haemophilia A
    tivated protein C. Nature 1994;369:64-7.                                         is down-regulated by activated protein C through inactiva-
 7. Nicolaes GA, Dahlbäck B. Factor V and thrombotic disease:                        tion of activated factor V. Br J Haematol 2018;183:257-66.
    description of a janus-faced protein. Arterioscler Thromb                  15.   Hemker HC, Al Dieri R, De Smedt E, Béguin S. Thrombin
    Vasc Biol 2002;22:530-8.                                                         generation, a function test of the haemostatic-thrombotic
 8. Dahlbäck B, Villoutreix BO. Regulation of Blood Coagula-                         system. Thromb Haemost 2006;96:553-61.
    tion by the Protein C Anticoagulant Pathway. Arterioscler                  16.   Ammollo CT, Semeraro F, Incampo F, et al. Dabigatran en-
    Thromb Vasc Biol 2005;25:1311-20.                                                hances clot susceptibility to fibrinolysis by mechanisms de-
 9. Shen L, Dahlbäck B. Factor V and protein S as synergistic                        pendent on and independent of thrombin-activatable
    cofactors to activated protein C in degradation of factor                        fibrinolysis inhibitor. J Thromb Haemost 2010;8:790-8.
    VIIIa. J Biol Chem 1994;269:18735-8.                                       17.   Muczynski V, Smith A, Sefiane T, et al. A FVIII-Mimetic
10. Wilhelm AR, Parsons NA, Samelson-Jones BJ, et al. Acti-                          Bispecific Antibody with an Embedded Self-Regulation




                                                                                                            ly
    vated protein C has a regulatory role in factor VIII function.                   Mechanism Reduces the Risk of Prothrombotic Events for




                                                                                                   on
    Blood 2021;137:2532-43.                                                          the Treatment of Haemophilia A. Blood 2022;140:677-8.
11. de Visser MC, Rosendaal FR, Bertina RM. A reduced sen-                     18.   Grover SP, Mackman N. Intrinsic Pathway of Coagulation
    sitivity for activated protein C in the absence of factor V Lei-                 and Thrombosis. Arterioscler Thromb Vasc Biol 2019;39:




                                                                                             e
    den increases the risk of venous thrombosis. Blood 1999;                         331-8.
    93:1271-6.                                                                 19.   Fredenburgh JC, Weitz JI. News at XI: moving beyond fac-
12. Laffan MA, Manning R. The influence of factor VIII on                             us
                                                                                     tor Xa inhibitors. J Thromb Haemost 2023;21:1692-702.
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Online supplementary material:
Figure S1. Effect of increasing concentrations of thrombomodulin (TM) on tissue factor-triggered thrombin generation in hemophilic plasma supplemented
    with emicizumab (50 µg/mL) or Kovaltry (1 IU/mL).
Figure S2. Effect of two different concentrations of thrombomodulin (TM) on FXIa-triggered thrombin generation in hemophilic plasma supplemented
    with emicizumab (50 µg/mL) or Kovaltry (1 IU/mL).


                                           Bleeding, Thrombosis and Vascular Biology 2023; 2:89
