Abstract: 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 (FV Leiden ) or rFV‐W1920R (FV Nara ) showed 〉 90% peak ratios. Conclusions rTM‐mediated TF‐triggered CWA might be useful for screening PC pathway abnormality‐related thrombophilia.

Abstract: 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.

Abstract: Emicizumab prophylaxis dramatically reduces bleeding events in severe patients with hemophilia A (PwHA) with and without FVIII inhibitors. Recently, some real-world experiences on PwHA with emicizumab prophylaxis have been reported. The effectiveness of emicizumab could be attributed to an earlier report that the coagulant potential in emicizumab at clinical dosage appeared to correspond to 10-15 IU/dL of factor VIII activity (FVIII:C) (Muto, JTH 2014), indicating that emicizumab enables to transform severe PwHA to mild PwHA. However, the comparison of coagulation potential and bleeding characteristics in PwHA receiving emicizumab (Emi-PwHA) and mild PwHA (PwMHA) remains to be investigated. Here, we examined the clinical and laboratory characteristics in Emi-PwHA and PwMHA. Clinical data of bleeding episodes and coagulation potentials were collected from 64 Emi-PwHA and 15 PwMHA (median FVIII:C 13 IU/dL [IQR 8.5-17.0]). Comprehensive coagulation function was evaluated using Ca 2+-triggered rotational thromboelastometry (ROTEM) and/or ellagic acid/tissue factor-triggered clot waveform analysis (CWA). Furthermore, we compared the features of breakthrough bleeds in two groups. We first compared with the clinical characteristics and coagulation function between the Emi-PwHA with and without the experience of breakthrough bleeds. The age, treatment period, annual bleeding rates (ABR), the percentage of inhibitor-positive, and prophylaxis regimen were not significantly different between both groups. There was no significant difference in ROTEM and CWA-based coagulation potential between Emi-PwHA with and without breakthrough bleeds (median; CT+CFT 1,703s [IQR 1,547-1,849] and 1,837s [IQR 1,671-2,216] , Ad|min1| 4.92 [IQR 4.63-5.27] and 4.78 [IQR 4.4-5.11] , respectively). We examined clinical data and ROTEM-based coagulation function between the Emi-PwHA and PwMHA. The age in PwMHA (median 25 years [IQR 20-49]) was older than that in Emi-PwHA (median 13 years [IQR 6-25] ; p=0.004). The ABR and coagulation function in Emi-PwHA (median; ABR 0 [IQR 0-0.4], CT+CFT 1,795s [IQR 1,659-2,135] ) were not significantly different from that in PwMHA (median; ABR 0.3 [IQR 0-0.49], CT+CFT 2,077s [IQR 1,627-2,449] ), suggesting that the global coagulation potential in Emi-PwHA was equivalent to that in PwMHA. We next investigated bleeding features in Emi-PwHA (19 cases) and PwMHA (12 cases). Bleeding patterns of them were similar and divided into 3 groups; spontaneous, early post-traumatic (bleeding associated with trauma within 1-2 days) and late post-traumatic (bleeding involved with trauma within 1-2 weeks). Emi-PwHA experienced spontaneous (5/19), early post-traumatic (14/19) and late post-traumatic (3/19) bleedings, and PwMHA experienced spontaneous (4/12), early post-traumatic (7/12) and late post-traumatic (5/12) bleedings. Some patients of them had both spontaneous and early post-traumatic bleeding or both early and late post-traumatic bleeding. The majority of their bleedings was trauma-induced. Severe bleeding symptoms such as muscle bleeds, hemarthrosis or fracture in Emi-PwHA were required FVIII (total 60-200 IU/kg) and hospitalization for 3-5 days. In contrast, those severe breakthrough bleedings in PwMHA were required higher doses of FVIII (total 140-705 IU/kg) and hospitalization for 1-2 weeks. Although the coagulation potential in Emi-PwHA was similar to that in PwMHA, the treatment period and hospitalization for breakthrough bleeds in PwMHA appeared to be longer than those in Emi-PwHA. We speculated that this discrepancy might be due to the difference of physical activities or preventive managements for bleeding because Emi-PwHA was originally severe PwHA. In conclusion, the coagulation potential and bleeding characteristics in Emi-PwHA appeared to be similar to PwMHA, suggesting that emicizumab-mediated coagulation potential reflected the mild type in clinical severity. Disclosures Nakajima: Chugai Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical company: Research Funding. Mizumachi: Chugai Pharmaceutical Co., Ltd.: Research Funding. Shimonishi: CSL Bering: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Furukawa: Chugai Pharmaceutical Co., Ltd.: Research Funding. Ogiwara: Chugai Pharmaceutical Co., Ltd.: Research Funding. Takeyama: Chugai Pharmaceutical Co., Ltd.: Research Funding. Shima: Fujimoto Seiyaku: Consultancy, Speakers Bureau; Sanofi S.A.: Speakers Bureau; BioMarin Pharmaceutical Inc.: Membership on an entity's Board of Directors or advisory committees; Bayer AG: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk A/S: Honoraria, Speakers Bureau; Takeda: Research Funding; CSL Behring: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor of patents related to anti-FIXa/FX bispecific antibodies, Research Funding, Speakers Bureau. Nogami: Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk A/S: Honoraria, Research Funding, Speakers Bureau; Bayer AG: Honoraria, Research Funding, Speakers Bureau; Sanofi S.A.: Honoraria, Research Funding, Speakers Bureau; KM Biologics Co., Ltd.: Honoraria, Research Funding, Speakers Bureau.

Abstract: 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.

Abstract: 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.

Abstract: Emicizumab is an antifactor (F)IXa/FX bispecific antibody, mimicking FVIIIa cofactor function. Emi prophylaxis effectively reduces bleeding events in patients with haemophilia A. The physical properties of emicizumab‐induced fibrin clots remain to be investigated, however. Aim We have investigated the stability and structure of emicizumab‐induced fibrin clots. Methods Coagulation was initiated by activated partial thromboplastin time (aPTT) trigger and prothrombin time (PT)/aPTT‐mixed trigger in FVIII‐deficient plasma with various concentrations of emicizumab or recombinant FVIII. The turbidity and stability of fibrin clots were assessed by clot waveform and clot‐fibrinolysis waveform analyses, respectively. The resulting fibrin was analysed by scanning electron microscopy (SEM). Results Using an aPTT trigger, the turbidity was decreased and the fibrinolysis times were prolonged in the presence of emicizumab dose‐dependently. Scanning electron microscopy imaging demonstrated that emicizumab improved the structure of fibrin network with thinner fibres than in its absence. Although emicizumab shortened the aPTT dramatically, the nature of emicizumab‐induced fibrin clots did not reflect the hypercoagulable state. Similarly, using a PT/aPTT‐mixed trigger that could evaluate potential emicizumab activity, emicizumab improved the stability and structure of fibrin clot in a series of experiments. In this circumstance, fibrin clot properties with emicizumab at 50 and 100 µg/mL appeared to be comparable to those with FVIII at ~12 and ~24‐32 IU/dL, respectively. Conclusion Emicizumab effectively improved fibrin clot stability and structure in FVIII‐deficient plasma, and the physical properties of emicizumab‐induced fibrin clots were similar to those with FVIII.