Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity.

INTRODUCTION: Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA-I) are in progress. Most polyclonal anti-hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors. AIM: To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. METHODS: Three hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII-deficient samples. RESULTS: OSA demonstrated that the hybrid proteins avoided neutralization by anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti-A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). CONCLUSION: Hybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA-I by avoiding neutralization.

Peri-operative hemostatic management of tooth extraction in patients with hemophilia A, with and without inhibitors, receiving emicizumab prophylaxis.

INTRODUCTION: Emicizumab treatment may allow patients with hemophilia A without (PwHA) and with inhibitors (PwHA-I) to undergo some minor surgeries, such as tooth extraction, without peri-operative factor infusions. However, criteria for determining the necessity of factor infusions before minor surgeries are unknown. AIM: We report the peri-operative hemostatic management and outcomes of emicizumab-treated PwHA and PwHA-I cases who underwent tooth extractions using our institutional protocol. METHODS: We retrospectively evaluated PwHA and PwHA-I who underwent tooth extraction with emicizumab prophylaxis at our institution. Local bleeding risk was assessed based on the method, number, and site of tooth extraction. Hemostasis was monitored peri-operatively by rotational thromboelastometry (ROTEM). Hemostatic agents and a mouth splint were used. RESULTS: Twenty-nine extractions (17 interventions) were performed in eight PwHA and two PwHA-I. Based on ROTEM, pre-operative factor infusions were used in ten PwHA and four PwHA-I interventions. Among nine low local bleeding risk interventions, three (33.3%) each received no infusions, one dose of factor infusion pre-operatively, and pre- and post-operative factor infusions. All eight high local bleeding risk interventions involved planned factor infusions. Absorbable hemostats were used in all extractions. A mouth splint was used in 21/25 (84.0%) PwHA and in 4/4 (100%) PwHA-I extractions. No post-extraction bleeding or thrombotic events occurred. CONCLUSIONS: Use of a systemic hemostatic treatment plan according to the local bleeding risk, peri-operative coagulation status assessment using ROTEM, filling the extraction socket with hemostats, and use of a mouth splint can achieve effective and safe hemostatic management in emicizumab-treated PwHA and PwHA-I.

Anti-idiotype monoclonal antibodies against emicizumab enable accurate procoagulant and anticoagulant assays, irrespective of the test base, in the presence of emicizumab.

INTRODUCTION: Emicizumab markedly shortens the activated partial thromboplastin time (aPTT), resulting in inaccurate measurements of procoagulant and anticoagulant factor activities. We have recently reported that mixtures of two different anti-idiotype monoclonal antibodies against emicizumab (anti-emicizumab-mAbs) allow measurement of factor (F)VIII activity (FVIII:C) and FVIII inhibitor in emicizumab-containing plasmas. It is unknown whether anti-emicizumab mAbs can work for other aPTT-based procoagulant and anticoagulant assays. AIM: To investigate whether anti-emicizumab mAbs were measured by all of the aPTT-based assays tested. METHODS: Two anti-emicizumab-mAbs (300 µg/mL each) were preincubated with emicizumab (200 µg/mL)-spiked FVIII-deficient plasma; we then measured FVIII:C, FIX:C, FXI:C, FXII:C, protein (P)C:C, PS:C, global PC-FV (aPTT-based), and prothrombin time (PT), diluted Russel's viper venom time (dRVVT), chromogenic-based FVIII:C, FIX:C and PC:C (non-aPTT-based). Emicizumab (100 µg/mL)-spiked haemophilia (H)A plasmas from patients (n = 23) were also measured. RESULTS: Emicizumab shortened the clotting time in all aPTT-based assays, resulting in high levels of FVIII:C, FIX:C, FXI:C and FXII:C; low levels of PC:C and PS:C; and false-positive results for activated PC resistance. The addition of anti-emicizumab-mAbs to emicizumab-added plasma restored all factors to the initial levels without emicizumab. Chromogenic FVIII:C measurement by human FIXa/FX was affected by emicizumab, but anti-emicizumab mAbs cancelled this effect. PT-based assays and dRVVT, chromogenic FIX:C and PC:C assays showed no effect with emicizumab. Twenty-three plasma samples from HA patients also showed similar patterns. CONCLUSION: Anti-emicizumab mAbs in vitro could cancel the effect of emicizumab, irrespective of the test base, resulting in accurate measurements of procoagulant and anticoagulant factor activity.

Coagulant potentials of emicizumab in the plasmas from infant and toddler patients with hemophilia A.

BACKGROUND: Emicizumab significantly reduces bleedings in patients with hemophilia A (PwHA). A clinical study (HAVEN 7; NCT04431726) for PwHA aged less than or equal to 12 months is ongoing, but emicizumab-driven coagulation potential in PwHA in early childhood remains to be clarified. AIM: To investigate the in vitro or in vivo coagulation potential of emicizumab in plasmas obtained from infant and toddler PwHA. METHODS: Twenty-seven plasma samples from 14 infant/toddler PwHA (aged 0-42 months, median 19 months) who received emicizumab (n = 9), factor (F)VIII products (n = 8), or no treatment (n = 10) were obtained. FVIII activity in FVIII-treated plasmas was cancelled by the addition of anti-FVIII monoclonal antibody (mAb). Emicizumab-treated plasmas (in vivo) and emicizumab-spiked plasmas (in vitro) were analyzed. Emicizumab-untreated plasma or emicizumab-treated plasma supplemented with two anti-emicizumab mAbs were used as references. Adjusted maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (Peak-Th) by thrombin generation assay was assessed. RESULTS: Ad|min1| values in 24 samples were improved by the presence of emicizumab. Values did not improve in the three remaining samples (aged 1, 23, and 31 months). Although the presence of emicizumab showed an age-dependent increase in Peak-Th in 20 samples, this increase was not observed in seven samples (aged 0, 1, 1, 2, 8, 19, and 36 months). Emicizumab-dependent increases in both Ad|min1| and Peak-Th were shown in 18 samples, and increases in either parameter were shown in eight samples. One sample (from patient aged 1 month) showed no increase in both, however. CONCLUSION: Emicizumab could improve coagulant potential in plasmas from infant/toddler patients with hemophilia A.

Screening of the protein C pathway abnormality-related thrombophilia by using thrombomodulin-mediated tissue factor-triggered clot waveform analysis.

OBJECTIVES: Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V-abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA-based screening assay to distinguish PC pathway abnormality-related thrombophilia. METHODS: Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated. RESULTS: The peak ratio in healthy plasmas (n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high-FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC-deficient plasma or PS-deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose-dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV-deficient plasma spiked with 40 IU/dl rFV-R506Q (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios. CONCLUSIONS: rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

Heterogeneous coagulant potential of emicizumab in neonatal factor VIII-deficient plasma.

BACKGROUND: Emicizumab prophylaxis reduces bleeding in hemophilia A (HA) patients. However, there are few data on emicizumab treatment in neonates with HA (neonate-HA), and the procoagulant effects of emicizumab in these patients are unknown. AIM: To investigate the coagulation activity of emicizumab in vitro in a plasma model of neonate-HA. METHODS: Plasmas from 84 neonates with non-HA were enrolled. However, due to the limited plasma volumes in some cases, 50 plasmas were assigned to two different assay groups. To prepare the neonate-HA model, plasma was first preincubated with an antifactor (F) VIII A2 monoclonal antibody (mAb). After further incubation with emicizumab, global coagulation activity was measured: adjusted maximum coagulation velocity (Ad|min1|) in clot waveform analysis (CWA) and peak thrombin in thrombin generation assay (TGA). RESULTS: Because the addition of anti-FVIII mAb to 22 of 43 samples showed little decrease in Ad|min1|, the remaining 21 samples were analyzed by CWA. The addition of emicizumab increased Ad|min1| in 18 of the 19 cases (effective group) but not in the remaining 3 cases (noneffective group). Similarly, TGA found that emicizumab (effective group) improved peak thrombin in seven of the nine samples tested, but two cases did not respond (noneffective group). Although the effective group had lower levels of FX, there was no significant difference between the effective and noneffective groups in terms of FIX, protein S, protein C, antithrombin, and fibrinogen. CONCLUSIONS: The in vitro coagulant potentials of emicizumab in the neonate-HA model were more heterogeneous than those recorded in the adult-HA model.

A microchip flow-chamber assay screens congenital primary hemostasis disorders.

BACKGROUND: Von Willebrand disease (VWD) and platelet function disorders (PFDs) are congenital bleeding disorders caused by primary hemostasis defects. Platelet function tests are time-consuming and require considerable amounts of blood sample, and there have been no easy-to-use assays for assessing platelet function quickly and sensitively. We report the usefulness of a microchip flow-chamber system (T-TAS® ) for detecting and/or predicting clinical severity in patients with VWD type 1 and type 2N and platelet storage pool disease. Here, we developed an application of a screening assay for primary hemostasis disorders. METHODS: Microchips coated with collagen (PL-chip) and collagen/thromboplastin (AR-chip) were utilized to evaluate platelet thrombus formation (PTF) at high shear and fibrin-rich PTF at low shear, respectively, in whole blood samples from 22 patients with VWD (16 type 2A, four type 2B, two type 3) and four patients with PFDs (two BSS, two Glanzmann thrombasthenia). The time-to-increase by 10 kPa (T10 ) was calculated from flow pressure curves. Also, whole blood-induced platelet aggregation was assessed using Multiplate® analysis. RESULTS: PL-chip T10 values ≥10 min successfully distinguished patients with all types of VWD and PFDs from healthy controls, irrespective of age, bleeding scores, and von Willebrand factor levels. However, AR-chip assay incompletely distinguished between type 2A patients and healthy ones. Multiplate analysis permitted screening of PFDs and type 3 VWD, but values in type 2A partially overlapped with those in controls. PL-chip assay did not reflect the clinical severity in these patients. CONCLUSIONS: T-TAS with PL-chip could be a quick screening tool for congenital primary hemostasis disorder, VWD, and PFDs.

Prediction of the haemostatic effects of bypassing therapy using comprehensive coagulation assays in emicizumab prophylaxis-treated haemophilia A patients with inhibitors.

In emicizumab prophylaxis, the concomitant therapy using bypassing agents (BPAs) is required for breakthrough bleeding and invasive procedures with attention to thrombotic complications. To predict coagulant effects of BPAs in emicizumab-treated patients with haemophilia A (PwHA) with inhibitor (PwHAwI), blood samples from emicizumab-treated PwHAwI (n = 8) and PwHA without inhibitor (n = 2) in phase 1/2 and HAVEN 1 study, spiked with activated prothrombin complex concentrates (aPCC) or recombinant factor VIIa (rFVIIa) ex vivo, and blood samples from emicizumab-treated PwHAwI-receiving BPAs were analysed by Ca2+ -triggered rotational thromboelastometry (ROTEM) and ellagic acid/tissue factor-triggered clot waveform analysis (CWA). Spiked aPCC, corresponded to 10-100 U/kg, markedly shortened ROTEM parameters beyond the normal range, while spiked rFVIIa, corresponded to 90-270 µg/kg, shortened them within near-normal range. Each of the spiked BPA-improved adjusted maximum coagulation velocity of CWA to within or near the normal range. In blood samples at post-infusion of aPCC (44-73 U/kg) or rFVIIa (79-93 µg/kg), the parameters of both assays improved to approximately the normal range. Taken together, ex vivo results of spiking tests in ROTEM and CWA, except aPCC spiking test in ROTEM, were relatively consistent with in vivo ones, and could usefully predict the coagulant effects of concomitant bypassing therapy for emicizumab-treated PwHAwI.

Whole blood ristocetin-induced platelet impedance aggregometry does not reflect clinical severity in patients with type 1 von Willebrand disease.

BACKGROUND: The haemorrhagic phenotype in patients with von Willebrand disease (VWD) is heterogeneous, and assays of von Willebrand factor ristocetin cofactor activity (VWF:RCo) do not always reflect clinical severity, especially in those individuals classed as type 1 VWD. Recent studies have shown that whole blood ristocetin-induced platelet agglutination (WB-RIPA) using an easy-to-use analyzer, Multiplate® platelet impedance technique, could be informative as a diagnostic test in VWD, although inconsistencies were evident in patients with the type 1 disorder, possibly associated with clinical symptoms. AIM: To investigate the relationship between WB-RIPA, bleeding scores (BS) and VWF-related measurements in type 1 VWD. METHODS: WB-RIPA assay using the Multiplate® was performed using whole blood from 55 patients with type 1 VWD. BS was determined using a standardized questionnaire. RESULTS: WB-RIPA values were significantly lower in type 1 VWD than in healthy controls (P < 0.0001). Weak correlations were apparent between WB-RIPA and VWF:RCo or VWF antigen (VWF:Ag; r = 0.22 or 0.28, respectively). There were significant differences in VWF:RCo (P = 0.036) and VWF:Ag (P = 0.0013) between patients with BS ≥4 (defined as abnormal bleeding tendency) and BS <4 (defined as no abnormal bleeding tendency), respectively. However, no significant difference was observed in WB-RIPA between the BS ≥4 group and BS <4 group. Overall, VWD patients with a WB-RIPA level >70 U did not seem to have an abnormal bleeding tendency, but low levels of WB-RIPA did not correlate with BS. CONCLUSION: WB-RIPA did not reflect clinical severity in type 1 VWD patients.

Factor (F)VIII/VIIa enhances global haemostatic function in the co-presence of bypassing agents and FVIII among patients with haemophilia A with inhibitor.

Bypassing therapy is essential for the haemostatic management of patients with haemophilia A with inhibitor (PWHA-inh), but the therapeutic effects are inconsistent. We previously reported that activated prothrombin complex concentrates (aPCC) activated factor (F)VIIIin vitro, and was mediated mainly by the activated FVII (FVIIa) contained in aPCC. We have extended those studies to assess global coagulation in whole blood from 18 PWHA-inh in the co-presence of aPCC and FVIII using Ca2+ -triggered rotational thromboelastometry. The clot times (CTs) in the presence of both aPCC (0·05 iu/ml) and recombinant (r)FVIII (1 iu/ml) ex vivo were shortened compared to the aPCC alone (P < 0·01). These enhancing effects of rFVIII were observed, irrespective of recognizing inhibitor epitopes; however, the clot formation time and 'α'-angle were not significantly different. In samples from 7 PWHA-inh post-infusion of aPCC (70-80 iu/kg), only the CTs were shortened in the presence of rFVIIIex vivo compared to its absence (P < 0·05), indicating that the enhanced activity centred on the initiation phase of coagulation. Furthermore, experiments in the co-presence of rFVIIa and rFVIII demonstrated that FVIII accelerated only the CTs. We concluded that FVIII/FVIIa-related coagulation mechanism enhanced global haemostatic function by the co-presence of bypassing agents and FVIII in PWHA-inh.

Liquid chromatography-mass spectrometry with triazole-bonded stationary phase for N-methyl-D-aspartate receptor-related amino acids: development and application in microdialysis studies.

We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as D-Ser, Gly, and L-Glu, which were involved in neurotransmission via the N-methyl-D-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p < 0.0001) between relative standard deviation (%) of the area ratio and the analyte-IS retention time differences. Using the proposed method, we were able to accurately analyze trace amounts of amino acids in MD samples using ISs that were structurally similar to the analytes. Furthermore, we observed that the peripheral administration of S-methyl-L-cysteine, which was an inhibitor of the amino acid transporter Asc-1, caused some amino acid level changes in the rat brain. The proposed LC-MS/MS method can be applied in vivo to study the effects of novel therapeutic agents with monitoring the levels of amino acid neuromodulators, such as Glu, Gly, GABA, and D-Ser, in the brain. Graphical abstract LC-MS/MS analysis of amino acid enantiomers in microdialysis samples from rat striatum using triazole-bonded stationary phase.

Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

BACKGROUND: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa. AIM: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798. METHODS AND RESULTS: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC50; 19.2 and 42.9 µM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction. CONCLUSION: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.

Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

High levels of factor VIII activity in patients with acquired hemophilia A in remission are associated with unusually low coagulation potentials.

BACKGROUND: Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated. AIM: To evaluate comprehensive coagulation potentials in PwAHA. METHODS: We investigated coagulation function in eleven PwAHA with high FVIII:C (> 150 IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII. RESULTS: The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206 IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171 IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C. CONCLUSION: PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

Decrease in in vivo coagulant potential of emicizumab in a patient with hemophilia A and inhibitor complicated with infectious mononucleosis.

Emicizumab prophylaxis significantly reduces bleeding episodes in patients with hemophilia A (PwHA). There is little information on coagulant potentials in emicizumab-treated PwHA with infection, however. We encountered an emicizumab-treated PwHA with inhibitor, complicated with Epstein-Barr virus-associated infectious mononucleosis (IM) in phase 1/2 study (ACE001JP/ACE002JP). Although it was a typical clinical course of IM, activated partial thromboplastin time was mildly prolonged but rotational thromboelastometry revealed severely impaired coagulant potential. The blood concentration of emicizumab decreased moderately in the low concentration range, resulting in an increased risk of bleeding and possibly leading to severe ileocecal bleeds requiring coil embolization. The blood concentrations of factors IX/X little decreased and antiemicizumab antibodies did not develop, however. After the influence by IM resolved, his coagulant potentials gradually recovered with the recovery of emicizumab concentration, and parameters by global coagulation assays improved. An IM case for emicizumab-treated PwHA may need to monitor using global coagulation assays.

Two pediatric cases of severe hemophilia A in which emicizumab prophylaxis failed to prevent traumatic extra-articular hemorrhage.

Emicizumab reduces bleeding episodes in patients with severe hemophilia A (PwHA). Little information is available on hemostatic management of severe traumatic hemorrhages in emicizumab-treated pediatric PwHA. We assessed therapeutic efficacy and global coagulation potentials in two pediatric cases of emicizumab-treated pediatric PwHA with intracranial or retroperitoneal/iliopsoas hemorrhage. A modified clot waveform analysis (CWA) triggered by mixtures of tissue factor and ellagic acid was used to assess coagulant potentials, and maximum coagulant velocity (Ad|min1|) was calculated. One patient with intracranial hemorrhage was treated with continuous infusions of recombinant factor VIII (rFVIII) at a dose of 4-4.6 IU/kg/hr for 9 days, followed by bolus infusion at 66 IU/kg/day for 2 days and 33 IU/kg/day for an additional 2 days. The Ad|min1| was increased from 5.5 (at baseline) to 7.0-8.1 under concomitant treatment and maintained within or near normal range (IQR; 6.9-7.7). The other patient with retroperitoneal/iliopsoas hemorrhage received bolus infusions of rFVIII at 50 IU/kg/day for 20 days and every-other-day infusion of rFVIII for 8 days. The Ad|min1| was increased from 5.2 (at baseline) to 5.8-6.8 under concomitant treatment and maintained within the normal range. We successfully managed a treatment plan for severe traumatic bleeding in emicizumab-treated pediatric PwHA using modified CWA.

Longitudinal dynamic changes in factor VIII inhibitor titers in patients with hemophilia A and inhibitors receiving emicizumab prophylaxis.

Emicizumab prophylaxis dramatically reduces bleeding events in patients with hemophilia A (PwHA) with or without factor VIII (FVIII) inhibitors. However, long-term dynamic changes in FVIII inhibitor titers during emicizumab prophylaxis remain to be investigated. We conducted a retrospective follow-up study of FVIII inhibitor titers after initiation of emicizumab prophylaxis in 25 PwHA carrying current or historical FVIII inhibitors. Nineteen PwHA had FVIII inhibitors at initiation of emicizumab prophylaxis (age: median 22 [range 4-60] years and inhibitor titer: 30 [1.0-1450] BU/mL). In 17 of the 19 patients, the inhibitor titers markedly decreased to a median of 1.2 (< 0.6-58) BU/mL at a median follow-up of 71 (38-111) months. In two patients, titers were slightly elevated after initiation of emicizumab but decreased in the long term. The remaining six patients had negative inhibitor status (< 0.6 BU/mL) when switched to emicizumab from FVIII prophylaxis. Five patients maintained negative titers. One patient had inhibitor recurrence, with a peak titer of 1.6 BU/mL that decreased to 0.9 BU/mL. In most cases, FVIII inhibitor titers can be expected to decrease spontaneously during emicizumab prophylaxis, but regular follow-up is necessary to manage breakthrough bleeds.