In vitro evaluation of global coagulation potential in the copresence of emicizumab and warfarin in whole blood using rotational thromboelastometry

In vitro evaluation of global coagulation potential in the copresence of emicizumab and warfarin in whole blood using rotational thromboelastometry | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 17 February 2025 V1 Latest version Share on In vitro evaluation of global coagulation potential in the copresence of emicizumab and warfarin in whole blood using rotational thromboelastometry Authors : Takahiro Kajimoto , Yuto Nakajima 0000-0001-5422-2782 [email protected] , Nobuyuki Tsujii , and Keiji Nogami Authors Info & Affiliations https://doi.org/10.22541/au.173977041.11124252/v1 159 views 112 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background : Warfarin inhibits the generation of vitamin K-dependent proteins. Emicizumab can prevent bleeding episodes in persons with hemophilia A (PwHA). The hemostatic potential of emicizumab combined with warfarin in PwHA remains to be clarified. Aim : To assess the coagulation of emicizumab combined with warfarin in a simulated whole blood model of PwHA. Methods : We collect 19 whole blood samples from 10 patients taking warfarin and the prothrombin time-international normalized ratio (PT-INR) was used to defined groups as near-normal (INR 1.2-1.48; n=4), subtherapeutic (INR 1.56-1.9; n=7) and therapeutic (INR > 2.0; n=8). Factor (F)VIII activity (FVIII:C) was neutralized using an anti-FVIII inhibitor antibody (termed FVIII-depleted) prior to the addition of therapeutic concentrations emicizumab (50 µg/mL). Coagulation potential was then measured using Ca 2+ -triggered rotational thromboelastometry and compared with that determined in emicizumab-treated PwHA. The FII:C, FVII:C, FIX:C, FX:C, protein C (PC):C, and PS:C levels in warfarin-treated samples were also measured. Results : The average PT-INR in near-normal, subtherapeutic, and therapeutic groups were 1.3±0.1, 1.7±0.1, and 2.4±0.4, respectively, and levels of FII:C, FVII:C, FIX:C, FX:C, PC:C, and PS:C ranged from 50-70%, 35-50%, and 15-35%, respectively. The hemostatic potential in FVIII-depleted samples mixed with emicizumab in the near-normal group was comparable to that in emicizumab-treated PwHA. The coagulation potential in FVIII-depleted samples after the addition of emicizumab in subtherapeutic and therapeutic groups were lower than that in PwHA receiving emicizumab. Conclusion : Concentrations of vitamin K-dependent proteins influence hemostatic function in PwHA receiving emicizumab, and PT-INR monitoring could be informative in these individuals. In vitro evaluation of global coagulation potential in the copresence of emicizumab and warfarin in whole blood using rotational thromboelastometry Takahiro Kajimoto, 1 Yuto Nakajima, 1 Nobuyuki Tsujii, 1 Keiji Nogami 1 Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan Running title; Coagulation potential in emicizumab and warfarin Type of article; Research article Support; This research was partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to KN ( 21K07804, 24K10935 ) and YN (22K15928). Word counts: Abstract 246, Text 2,597 Reference counts: 31 Figure/Table: 4 figures and 2 tables Key words : hemophilia A, factor VIII, warfarin, emicizumab, rotational thromboelastometry Address correspondence Yuto Nakajima, M.D., Ph.D., Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Tel.: +81-744-29-8881; Fax.: +81-744-24-9222; E-mail: [email protected] PT-INR prothrombin time- international normalized ratio APTT activated partial thromboplastin time VK vitamin K ROTEM rotational thromboelastometry FII factor II FVII factor VII FVIII factor VIII FIX factor IX FX factor X PC protein C PS protein S :C activity Abstract Background : Warfarin inhibits the generation of vitamin K-dependent proteins. Emicizumab can prevent bleeding episodes in persons with hemophilia A (PwHA). The hemostatic potential of emicizumab combined with warfarin in PwHA remains to be clarified. Aim : To assess the coagulation of emicizumab combined with warfarin in a simulated whole blood model of PwHA. Methods : We collect 19 w hole blood samples from 10 patients taking warfarin and the prothrombin time- international normalized ratio (PT-INR) was used to defined groups as near-normal (INR 1.2-1.48; n=4), subtherapeutic (INR 1.56-1.9; n=7) and therapeutic (INR >2.0; n=8). Factor (F)VIII activity (FVIII:C) was neutralized using an anti-FVIII inhibitor antibody (termed FVIII-depleted) prior to the addition of therapeutic concentrations emicizumab (50 µg/mL). C oagulation potential was then measured using Ca 2+ -triggered rotational thromboelastometry and compared with that determined in emicizumab-treated PwHA. The FII:C, FVII:C, FIX:C, FX:C, protein C (PC):C, and PS:C levels in warfarin-treated samples were also measured. Results : The average PT-INR in near-normal, subtherapeutic, and therapeutic groups were 1.3±0.1, 1.7±0.1, and 2.4±0.4, respectively, and levels of FII:C, FVII:C, FIX:C, FX:C, PC:C, and PS:C ranged from 50-70%, 35-50%, and 15-35%, respectively. The hemostatic potential in FVIII-depleted samples mixed with emicizumab in the near-normal group was comparable to that in emicizumab-treated PwHA. The coagulation potential in FVIII-depleted samples after the addition of emicizumab in subtherapeutic and therapeutic groups were lower than that in PwHA receiving emicizumab. Conclusion : Concentrations of vitamin K-dependent proteins influence hemostatic function in PwHA receiving emicizumab , and PT-INR monitoring could be informative in these individuals. Introduction Cardiovascular diseases (CVDs) are major causes of death and health costs in developed countries. Patients with valvular heart disease and atrial fibrillation have a higher risk of developing venous thromboembolism (VTE) and usually receive oral anticoagulant therapy. 1 The standard approach to treatment and prevention of VTE in pediatric patients includes the use of unfractionated heparin, low molecular weight heparin, and vitamin K (VK) antagonists. 2,3 The VK antagonists such as warfarin reduce the synthesis of functional VK-dependent coagulation factor proteins, including factors (F)II, VII, IX, X, protein (P)C, and PS, by inhibiting the VK epoxide reductase activity, and are commonly used clinically to manage these patients. 4 Warfarin has a very narrow therapeutic range, however, and the efficacy can be affected by many diet–drug and drug–drug interactions. 5 Hemophilia A (HA) is characterized by low or dysfunctional plasma levels of coagulation FVIII and patients with HA (PwHA) may be considered as being naturally-anticoagulated due to the lack of FVIII. 6 A previous investigation suggested, however, that anticoagulation therapy should be considered for a significant number of non-severe PwHA. 7 This study has compared coagulation potentials among PwHA, patients receiving warfarin therapy, and healthy controls by thrombin generation assay (TGA) and the findings demonstrated that thrombin generation in severe PwHA was comparable to that in VK antagonists-treated patients with a therapeutic prothrombin time-international normalized ratio (PT-INR) of >2.0. TGA in PwHA with FVIII levels 1-19 IU/dL were considerably lower than in healthy controls, and were comparable to that in patients with subtherapeutic PT-INR (1.5-1.9). Consequently, FVIII level at therapeutic anticoagulation. 8,9 Emicizumab (Hemlibra ® ; Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) functions as a mimetic of FVIIIa cofactor function in the intrinsic tenase complex by binding to FIX/activated FIX (FIXa) and FX/FXa. 10 Emicizumab has good hemostatic efficacy in both children and adults with congenital PwHA, and emicizumab prophylaxis can prevent breakthrough bleedings in severe PwHA . 11 The hemostatic efficacy of emicizumab recorded in these clinical studies suggested that the trough emicizumab concentration in plasma (~50 µg/mL) appeared to correspond to approximately 10-15 IU/dL FVIII:C previously identified using an acquired HA primate model. 12 In addition, our earlier study suggested that in vitro coagulation potential at clinically therapeutic emicizumab concentrations were comparable to those in mild PwHA (median FVIII level 13 IU/dL). 13 The coagulant function of emicizumab may also be dependent, however, on specific FIX and FX levels present the FIX-emicizumab-FX ternary complex. 14 In this context, therefore, the anticoagulant effect of warfarin in PwHA receiving emicizumab prophylaxis could contribute to pivotal mechanisms affecting clinical management. However, coagulation potential in PwHA receiving emicizumab and warfarin remains to be fully explored. Therefore, we have analyzed the effect of emicizumab combined with warfarin therapy in an in vitro simulated PwHA model using rotational thromboelastometry (ROTEM). Materials and Methods The study protocol has been approved by the Ethics Committee of Nara Medical University (No. 3480). We obtained informed consent from all patients in order to participate this study and collected blood samples from them according to the ethical guidelines of our university. Reagents – We purchased emicizumab (Chugai), prothrombin time (PT) and activated partial thromboplastin time (aPTT) reagents (Revohem PT ® and Thrombocheck APTT-SLA ® ; respectively, Sysmex Corp., Kobe, Japan) from the indicated vendor. Patients - A total of 10 children with cardiac disease receiving warfarin (19 samples) between March 1 st , 2023 to November 30 th , 2024 were included in this study. Residual whole blood was obtained from samples taken for the routine monitoring of PT-INR. The inclusion criteria were PT-INR >1.2 and patient’s weight over 8 kg. The conventional coagulation parameters, FII:C, FVII:C, FIX:C, and FX:C were measured by one-stage clotting assays using a STart4 ® hemostasis Analyzer (Diagnostica Stago, Asnieres, France). PC:C and PS:C were assessed using the CS2400i ® (Sysmex Corp. Kobe, Japan) analyzer using the HEMOCLOT TM kit (Sysmex). Plasma samples - Whole blood specimens were collected in plastic tubes containing 3.2% buffered sodium citrate at a 9:1 ratio of blood to anticoagulant ( Fuso Pharmaceutical Industries, Osaka, Japan ). Platelet poor plasma was obtained by centrifugation of the whole blood for 15 min at 2,000× g . All plasma samples were stored in a -80°C freezer and thawed rapidly at 37°C prior to analysis. In vitro simulated model of HA treated with concomitant emicizumab and warfarin - The in vitro simulated model of PwHA blood with concomitant emicizumab and warfarin treatment was prepared as follows. T he 19 warfarin-treated patients were categorized into three PT-INR groups: PT-INR 1.2-1.48 (near-normal group; n=4), PT-INR 1.56-1.9 (subtherapeutic group: n=7) and PT-INR >2.0 (therapeutic group: n=8) and each whole blood sample was preincubated with anti-FVIII polyclonal inhibitor antibody (10 BU/mL) to completely neutralize FVIII:C (termed ‘FVIII-depleted’) as previously described. 15 We confirmed the FVIII:C level <1 IU/dL of these samples by one-stage clotting assay. These samples were then further incubated with emicizumab at the clinically therapeutic concentration of 50 μg/m L for 30 min at room temperature (termed ‘FVIII-depleted with Emi). Rotational thromboelastometry (ROTEM) - Previous studies have shown that the NATEM mode of ROTEM can reflect near-physiological hemostatic status and global coagulation function in patients treated with emicizumab. 16,17 This method was adopted, therefore, using a Whole Blood Hemostasis Analyzer ® (Pentapharm, Munich, Germany).The citrated whole blood samples (280 μL) were incubated for 30 min at room temperature prior to the addition 20 μL CaCl 2 (final concentration; 12.5 mM) to initiate coagulation. Clot formation was evaluated using the parameters; clotting time (CT; the period until reaching a 2 mm amplitude), clot formation time (CFT; the period until reaching a 20 mm amplitude), and CT+CFT. These specific ROTEM parameters have been shown to reflect the coagulation function in PwHA receiving emicizumab. 16 The interquartile range (IQR) of CT( sec ) and CT+CFT(sec) in 20 healthy individuals were defined as 802-1,041 sec and 1,103-1,413 sec, respectively, as previously determined. 16 The IQR of CT and CT+CFT values in emicizumab-treated PwHA were defined as 1,166-1,570 sec and 1,658-2,152 sec, respectively, as also previously described in 63 individuals. 13 Similarly, our previous study demonstrated that the IQR of CT and CT+CFT in severe PwHA were 3,787-6,242 sec and 4,568-7,562 sec, respectively. 16 On this basis, therefore, we defined their CT >3,800 sec or CT+CFT >4,600 sec as severe PwHA and all blood samples were monitored for 4,600 sec . Data analysis - The CT and CT+CFT in near normal, subtherapeutic and therapeutic INR groups were analyzed by one-way ANOVA with Tukey’s comparison test using KaleidaGraph software 5.0 (Synergy, Reading, PA). FII:C, FVII:C, FIX:C, FX:C, PC:C, and PS:C in warfarin samples are shown as the median and standard deviation. Patients’ profile Ten children receiving warfarin therapy were selected. Ages at the time of blood sampling ranged from 1 to 16 years. Conditions requiring warfarin therapy included four cases after Fontan surgery (Case 2, 3, 4, and 6), one case after mitral valve replacement (Case 9), one case after aortic valve replacement (Case 8), two cases of giant coronary artery aneurysm due to Kawasaki disease (Case 1, 7 and 10), and one case of multiple arterial aneurysms (Case 5). In six cases, warfarin was used in combination with aspirin at doses of 3-5 mg/kg/day (Case 1, 3, 4, 5, 6, 7 and 10). Table 1 shows the characteristics of the enrolled patients at baseline. Relationship between PT-INR and clotting factors in warfarin-treated patients Initial laboratory investigations included measurements of PT-INR, FII:C, FVII:C, FIX:C, FX:C, PC:C, and PS:C in all samples ( Table 2) . The average PT-INR in near-normal, subtherapeutic, and therapeutic PT-INR groups were 1.3±0.1, 1.7±0.1, and 2.4±0.4, respectively. In the near-normal group, the levels of FII:C, FVII:C, FIX:C, FX:C, PC:C, and PS:C were 58±15, 59±12, 60±15, 51±8 , 71±14, and 61±11 %, respectively. In the subtherapeutic group, these coagulation activities were 49±7, 36±13, 41±10, 34±7 , 40±17, and 45±7 %, respectively. In the therapeutic group, the corresponding assays of coagulation activity were 24±13, 25±8, 30±11, 21±6 , 14±8, and 34±11 %, respectively. These results confirmed that the reduced levels of VK-dependent clotting factors were proportionally related to the increase in PT-INR. Global coagulation function in the warfarin-treated HA model (FVIII-depleted) blood samples spiked with emicizumab. Coagulation potential was assessed by ROTEM measurement in the warfarin-treated patients and the corresponding HA model blood samples spiked with emicizumab. (A) Near-normal PT-INR group Representative data from the near-normal group are shown in Fig. 1A. The CT and CT+CFT in raw blood samples from the near-normal group tended to be longer relative to healthy controls, but shorter relative to that from severe PwHA receiving emicizumab prophylaxis (Fig. 2). In FVIII-depleted samples, the CT was consistently longer (>4,600 sec; data not shown) , indicating that FVIII:C was neutralized, and that the coagulation potential in these samples were consistent with severe PwHA phenotype . Moreover, in the experiments using FVIII-depleted samples spiked with emicizumab (50 μg/m L ), the CT and CT+CFT in this near-normal group were comparable to those in severe PwHA treated with emicizumab (Fig. 3) . These results suggested that warfarin treatment at near-normal levels of PT-INR appeared unlikely to affect coagulation potential in severe PwHA receiving emicizumab. (B) Subtherapeutic PT-INR group Representative data in the subtherapeutic groups are shown in Fig. 1B. The findings demonstrated that the CT and CT+CFT using raw samples from this group were prolonged relative to healthy controls, and were consistent with or longer than those in severe PwHA receiving emicizumab (Fig. 2) . With the FVIII-depleted samples, all CT were over 4,600 sec (data not shown). As for the FVIII-depleted samples supplemented with emicizumab, the CT and CT+CFT were longer than observed in the near-normal group, and were longer relative to those determined in severe PwHA receiving emicizumab (Fig. 3) . The CT as >3,800 sec and/or the CT+CFT as >4,600 sec in 6 out of 7 of the FVIII-depleted samples spiked with emicizumab were recorded (Fig. 3) , indicating that coagulation potential in this sub therapeutic group was comparable to severe PwHA, even in the presence of emicizumab. These results suggested that warfarin treatment at a subtherapeutic level of PT-INR could moderate coagulation potential in severe PwHA receiving emicizumab. (C) Therapeutic PT-INR group Representative data from the therapeutic group are shown in Fig. 1C. Longer CT and CT+CFT were recorded with raw samples from this group relative to those in observed with healthy controls, near-normal patients and the severe PwHA receiving emicizumab (Fig. 2) . In addition, with the FVIII-depleted samples, all CT were again longer than 4,600 sec (data not shown). As for the FVIII-depleted samples spiked with emicizumab, both the CT and CT+CFT were longer than those in the severe PwHA samples spiked with emicizumab, and were markedly longer than those in near-normal group (Fig. 3) . All FVIII-depleted samples spiked with emicizumab in therapeutic group showed their CT similar to those determined in the subtherapeutic group, and confirmed that the hemostatic effects in FVIII-depleted samples mixed with emicizumab in therapeutic group was comparable to that in severe PwHA. Discussion VK antagonists, including warfarin, are commonly administered for minimizing the risk of thromboembolic events in pediatric patients diagnosed with coronary artery aneurysm caused by Kawasaki disease or in children that have undergone the Fontan procedure or heart valve replacement surgery. 18-20 The management of these disorders in PwHA is especially challenging, however, and demands an adequate balance between thrombotic and hemostatic interactions. Emicizumab bridges the enzyme FIXa and the substrate FX, resulting FX activation. 11 This novel bispecific antibody treatment has become the primary therapy for prophylaxis in PwHA with inhibitors. 21 Previous reports have recommended that anticoagulation therapy should be considered in PwHA with FVIII levels > 20%, 22 or at trough FVIII:C level of 30%. 23 In this context, the FVIII-equivalent coagulation activity of emicizumab is estimated to be approximately 15%, 12,13 and may not support adequate hemostasis during anticoagulation therapy. An earlier study demonstrated that the emicizumab-bridged ternary complex with FIX/FIXa and FX/FXa are formed in plasma at low concentrations of FX and FIX, 14 and we speculated, therefore, that warfarin therapy may adversely affect coagulation activity in PwHA treated with emicizumab. Specific data on emicizumab-treated PwHA during warfarin therapy is lacking, however. The current study protocol was designed to evaluate hemostatic efficacy in PwHA receiving emicizumab and warfarin using an in vitro model. To the best of our knowledge, this is the first report for assessing coagulation potential in emicizumab-treated PwHA in conjunction with warfarin therapy by in vitro study. The kinetics of FIX-emicizumab-FX ternary complex generation in these INR groups were estimated, therefore, as previously described (Fig. 4 ). 14 These calculations demonstrated, as expected, that the global coagulation potential in FVIII-depleted samples spiked with emicizumab in subtherapeutic and therapeutic PT-INR groups was depressed compared to that in the control emicizumab-treated PwHA. In contrast, generation of FIX-emicizumab-FX ternary complexes in the near-normal group appeared to be approximately one third of that in PwHA receiving emicizumab without warfarin ( see Fig. 3 ). Earlier studies have suggested that low doses of emicizumab may be helpful for reducing breakthrough bleeding in PwHA, 24,25 and the present data are in keeping with the concept that low concentrations of FIX/FIXa-emicizumab-FX/FXa complexes, to some extent may be sufficient for the prevention of bleedings in PwHA. In contrast, the ROTEM parameters in FVIII-depleted plasmas spiked with emicizumab in combination with warfarin in the subtherapeutic and therapeutic groups were markedly prolonged compared to those in the near-normal group ( see Fig. 3 ), suggesting that defective generation of the FIX-emicizumab-FX ternary complex may increase breakthrough bleeding in this category of PwHA. The target PT-INR in pediatric patients who undergo the Fontan procedure or are diagnosed with coronary artery aneurysm caused by Kawasaki disease is recommended to be 2.0-3.0. In addition, the target PT-INR in pediatric patients with mechanical heart valve replacement is defined as 2.5-3.5. 26 Our results provide evidence that the equivalent target PT-INRs in PwHA treated with emicizumab may require reassessment. Direct oral anticoagulants (DOACs), including direct inhibitors of FIIa (dabigatran) and FXa (rivaroxaban, apixaban and edoxaban), are relatively new agents approved for various thromboembolic disorders, and this type of therapy has become a more attractive as an alternative to VK antagonists or low molecular weight heparin for reducing the risk of thromboembolic complications. 27 Consequently, the use of DOACs is now generally recommended, especially in patients who have a high risk of bleeding. 27 Modern techniques of global coagulation function have demonstrated that in PwHA receiving emicizumab and DOACs, the TG parameters in PwHA receiving emicizumab and FXa inhibitor were impaired relative to those in PwHA with emicizumab alone. 28 Similar study also suggested the importance of FVIII supplementation in FVIII-deficient plasma containing emicizumab to counteract the anticoagulant effect caused by DOAC. 29 Furthermore, we have previously reported that reduced concentrations of FX antigen could weaken the coagulation potential in PwHA receiving emicizumab. 30 The use of low doses of DOACs might also be considered, therefore, as an alternative to warfarin in emicizumab-treated PwHA. Some limitations of the present study are recognized. Firstly, 10 out of 19 of the patient blood samples included those treated with a combination of warfarin and aspirin (3-5 mg/kg) and we did not investigate the influence of aspirin on global coagulation potential. However, previous studies have reported that platelet inhibition could lead to lower maximum clot firmness (MCF) in ROTEM, but did not influence on the CT and CFT in ROTEM. 31 Therefore, we speculate that the presence of aspirin appeared not to affect our analyses. Secondly, we did not investigate the difference between biventricular and univentricular circulation on global coagulation potential. Also, the number of plasma samples was limited. Nevertheless, our in vitro data clearly demonstrated that warfarin therapy could reduce the hemostatic efficacy in FVIII-depleted plasma containing emicizumab and further studies are required to consolidate the use of anticoagulant therapy in emicizumab-treated PwHA. Author Contributions TK; supported clinically, performed the assays, made the figures, and wrote the manuscript; YN; supported clinically, performed the assays, analyzed the results, wrote the manuscript, and approved the final version to be published; NT; supported clinically; KN; designed the study, analyzed the data, wrote the paper, and revised the manuscript. Disclosure of Conflict of Interests TK and NT have no disclosure of conflict of interest. YN has received grant from the Takeda Pharmaceutical Company. KN has received grants, personal fees, non-financial support from Chugai Pharmaceutical Co., Ltd., personal fees from F. Hoffmann-La Roche Ltd., grants, personal fees and non-financial support from Sysmex Co., grants and personal fees from Takeda Pharmaceutical Co., grants and personal fees from Sanofi S.A, personal fees from CSL Behring Co., grants and personal fees from KM Biologics Co., grants and personal fees from Novo Nordisk A/S, grants and personal fees from Bayer AG, grants and personal fees from Bioverativ Inc, and grants and personal fees from Shire Plc, and is an inventor of the patents relating to emicizumab. Data Availability Statement ; The dataset supporting the findings of this study can be obtained upon request from the corresponding author. The data are not publicly accessible due to privacy or ethical restrictions. References 1. Moon. I, Lee. S.R, Choi. E.K, et al., “Non-Vitamin K Antagonist Oral Anticoagulants in Patients with Atrial Fibrillation and Valvular Heart Disease,” J Clin Med 8, no.10 (2019): 1624. 2. Lassandro. G, Palmieri. V.V, Palladino. V, Amoruso. A, Faienza. 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S, et al., Effectiveness of monthly low dose emicizumab prophylaxis without 4-week loading doses among patients with haemophilia A with and without inhibitors: A case series report. Haemophilia 29, no.1 (2023): 382-385. 26. Streif. W, Andrew. M, Marzinotto. V, et al., “Analysis of warfarin therapy in pediatric patients: A prospective cohort study of 319 patients,” Blood 94, no.9 (1999): 3007-3014. 27. Chen. A, Stecker. E, A. Warden. B, “Direct Oral Anticoagulant Use: A Practical Guide to Common Clinical Challenges,” J Am Heart Assoc 9, no.13 (2020): e017559. 28. Dix C, McFadyen. J.D, and Tran. H.A, “Exploring the effects of Factor Xa inhibitors on thrombin generation in people with haemophilia,” Thromb Res 237, (2024): 148-153. 29. Lamoine. S, Jury. V, Fourneyron. V, et al., “Thrombin generation to evaluate the complex hemostatic balance of hemophilia A plasma containing direct oral anticoagulant and supplemented by factor VIII,” Res Pract Thromb Haemost 8, no.7 (2024): 102576. 30. Yada. K, Fujitate. N, Ogiwara. K, Soeda. T, Kitazawa. T, and Nogami. K, “Reduced plasma factor X is associated with a lack of response to recombinant activated factor VII in patients with hemophilia A and inhibitor, but does not impair emicizumab-driven hemostasis in vitro,” Thromb Res 237, (2024): 37-45. 31. Abdelfattah. K, and Cripps. M.W, “Thromboelastography and Rotational Thromboelastometry use in trauma,” Int J Surg 33, no.Pt B (2016): 196-201. Figure Legends Figure 1. Rotational thromboelastometry (ROTEM) using whole blood from warfarin-treated patients in various PT-INR groups, and in the equivalent FVIII-depleted blood (HA model) spiked with emicizumab The global coagulation potential of raw whole blood samples, FVIII-depleted whole blood samples (simulated HA model), and FVIII-depleted whole blood samples spiked with emicizumab (50 μg/m L ) from patients with warfarin in near-normal PT-INR group ( panel A ), subtherapeutic PT-INR group ( panel B), and therapeutic PT-INR group ( panel C) were evaluated by Ca 2+ -triggered rotational thromboelastometry (ROTEM) . T he representative ROTEM data in raw whole blood samples, FVIII-depleted whole blood samples, and FVIII-depleted whole blood samples spiked with emicizumab are shown. Abbreviation; HA; Hemophilia A, PT-INR; prothrombin time- international normalized ratio . Figure 2. ROTEM parameters of raw whole blood samples in warfarin-treated patients in various PT-INR groups The CT and CT+CFT of raw whole blood samples in warfarin-treated patients are illustrated. CT and CFT results between 3,800 sec and 4,600 sec, and >4600 sec respectively were recorded as 3,800 sec and 4,600 sec based on the data from severe PwHA ( see Methods ) . The CT and CT+CFT in all groups (near-normal, subtherapeutic, and therapeutic PT-INR groups) were analyzed by one-way ANOVA with Tukey’s comparison test. Significant differences were defined as p < 0.05 (*p <0.05). The dashed line represents the IQR of CT and CT+CFT in normal controls. The dotted line represents the IQR of CT and CT+CFT in severe PwHA receiving emicizumab. The straight line represents the 25 th percentile of CT and CT+CFT in severe PwHA. Abbreviation; CT; clotting time, CFT; clot formation time, PT-INR; prothrombin time- international normalized ratio, IQR; interquartile range, PwHA; patients with hemophilia A, ns; not significant. Figure 3. ROTEM parameters of FVIII-depleted whole blood samples spiked with emicizumab in various PT-INR groups of warfarin-treated patients The CT and CT+CFT of FVIII-depleted whole blood samples spiked with emicizumab in warfarin-treated patients were classified as above. The CT and CT+CFT in all groups (near-normal, subtherapeutic, and therapeutic PT-INR group) were analyzed by one-way ANOVA with Tukey’s comparison test. Significant differences were defined as p < 0.05 (*p <0.05). The dashed line represents the IQR of CT and CT+CFT in normal controls. The dot line represents the IQR of CT and CT+CFT in severe PwHA receiving emicizumab. The straight line represents the 25 th percentile of CT and CT+CFT in severe PwHA. Abbreviation - CT; clotting time, CFT; clot formation time, PT-INR; prothrombin time- international normalized ratio, IQR; interquartile range, PwHA; patients with hemophilia A, ns; not significant. Figure 4. Simulated prediction of the concentrations of FIX-emicizumab-FX ternary complexes in PwHA treated with emicizumab and warfarin Estimated based on the different PT-INR groups are illustrated; blue , normal FIX:C (100 %) and FX:C (100 %); red , the average FIX:C and FX:C in near-normal group (PT-INR 1.2-1.5); green , the average FIX:C and FX:C in subtherapeutic group (PT-INR 1.5-2.0); purple , the average FIX:C and FX:C in therapeutic group (PT-INR 2.0<). The dashed line represents the FIX-emicizumab-FX ternary complex in emicizumab (50 µg/mL). Abbreviation : FIX; factor IX, FX; factor X, PT-INR; prothrombin time- international normalized ratio . Table 1. Characteristic profiles of enrolled pediatric patients on warfarin therapy A 1 (case 1) 9 1.2 CAL Biventricule Warfarin + aspirin 2 (case 2) 5 1.24 Fontan Univentricule Warfarin 3 (case 3) 5 1.41 Fontan Univentricule Warfarin + aspirin 4 (case 4) 6 1.48 Fontan Univentricule Warfarin + aspirin B 5 (case 5) 7 1.56 MAA Biventricule Warfarin + aspirin 6 (case 6) 3 1.69 Fontan Univentricule Warfarin + aspirin 7 (case 7) 10 1.76 CAL Biventricule Warfarin + aspirin 8 (case 8) 16 1.74 AVR Biventricule Warfarin 9 (case 1) 10 1.76 CAL Biventricule Warfarin + aspirin 10 (case 9) 9 1.77 MVR Biventricule Warfarin 11 (case 3) 5 1.9 Fontan Univentricule Warfarin + aspirin C 12 (case 8) 16 2.05 AVR Biventricule Warfarin 13 (case 2) 5 2.07 Fontan Univentricule Warfarin 14 (case 9) 9 2.21 MVR Biventricule Warfarin 15 (case 9) 8 2.33 MVR Biventricule Warfarin 16 (case 6) 2 2.52 Fontan Univentricule Warfarin + aspirin 17 (case 10) 1 2.57 CAL Biventricule Warfarin + aspirin 18 (case 8) 16 2.69 AVR Biventricule Warfarin 19 (case 8) 16 3.12 AVR Biventricule Warfarin Group: A: Near-normal PT-INR, B; subtherapeutic PT-INR, C; therapeutic PT-INR ( see in Methods ) Abbreviation: CAL: coronary artery lesions, MAA: multiple arterial aneurysms, AVR: aortic valve replacement, MVR: mitral valve replacement, PT-INR; prothrombin time- international normalized ratio. Table 2. Vitamin K-dependent clotting factors in enrolled patients with warfarin therapy (%) (%) (%) (%) (%) (%) Near-normal group (n=4) 1.3 ± 0.1 58 ± 15 59 ± 12 60 ± 15 51 ± 8 71 ± 14 61 ± 11 Subtherapeutic group (n=7) 1.7 ± 0.1 49 ± 7 36 ± 13 41 ± 10 34 ± 7 40 ± 17 45 ± 7 Therapeutic group (n=8) 2.4 ± 0.3 24 ± 13 25 ± 8 30 ± 11 21 ± 6 14 ± 8 34 ± 11 Abbreviation: PT-INR; prothrombin time- international normalized ratio, FII; factor II, FVII; factor VII, FIX; factor IX, FX; factor X, PC; protein C, PS; protein S, :C; Activity Information & Authors Information Version history V1 Version 1 17 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords clotting factor related research hematology hemophilia hemostatis & thrombosis Authors Affiliations Takahiro Kajimoto Nara Kenritsu Ika Daigaku View all articles by this author Yuto Nakajima 0000-0001-5422-2782 [email protected] Nara Kenritsu Ika Daigaku View all articles by this author Nobuyuki Tsujii Nara Kenritsu Ika Daigaku View all articles by this author Keiji Nogami Nara Kenritsu Ika Daigaku View all articles by this author Metrics & Citations Metrics Article Usage 159 views 112 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Takahiro Kajimoto, Yuto Nakajima, Nobuyuki Tsujii, et al. In vitro evaluation of global coagulation potential in the copresence of emicizumab and warfarin in whole blood using rotational thromboelastometry. Authorea . 17 February 2025. DOI: https://doi.org/10.22541/au.173977041.11124252/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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