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.

Emicizumab-mediated hemostatic function assessed by thrombin generation assay in an in vitro model of factor VIII-depleted thrombophilia plasma.

Patients with hemophilia A (PwHA) may have concurrent deficiency of representative anticoagulant proteins, protein (P)C, PS, and antithrombin (AT), which reduces bleeding frequency. However, emicizumab-driven hemostasis in PwHA with such thrombophilic potential remains unclarified. This study investigated the influence of natural anticoagulants on emicizumab-driven coagulation in HA model plasma. Various concentrations of PS and AT were added to PS-deficient plasma and AT-deficient plasma in the presence of anti-FVIII antibody (FVIIIAb; 10BU/mL). PC-deficient plasma was mixed with normal plasma at various concentrations in the presence of FVIIIAb. Emicizumab (50 µg/mL) was added to these thrombophilic HA model plasmas, prior to tissue factor/ellagic acid-triggered thrombin generation assays. Co-presence of emicizumab increased peak thrombin values (PeakTh) dependent on PS, AT, and PC concentrations. Maximum coagulation potentials in the PS-reduced HA model plasmas remained normal in the presence of emicizumab. PeakTh were close to normal in the presence of 50%AT irrespective of emicizumab, but were higher than normal in the presence of 25%AT. Addition of recombinant FVIIa (corresponding to an administered dose of 90 µg/kg) enhanced coagulation potential to normal levels. Our findings provide novel information on hemostatic regulation in emicizumab-treated PwHA with a possible thrombophilic disposition.

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.

[Elucidation of an altered anticoagulant function due to Factor V abnormality and development of a simple screening assay for thrombophilia].

Coagulation factor V (FV) is both procoagulant and anticoagulant functions. Congenital FV abnormality, which are caused by mutations in the FV gene, are characterized by a tendency to bleed. However, FV-R506Q (FVLeiden) is the most common FV abnormality that eliminates an activated protein C (APC) cleavage site, resulting in the occurrence of deep venous thrombosis (DVT). In Japan, the thrombotic predisposition caused by FVLeiden and FV molecular abnormalities was believed to be nonexistent. We did, however, report the first case in Japan of a young patient with FV abnormality-related thrombosis. The recurrent DVT in this case was caused by a novel mutation of FV-W1920R (FVNara), located in the C1 domain and far from the APC cleavage sites. We considered the possibility that there were cases of FV-related thrombotic predisposition that had gone undetected in Japan. We thoroughly examined FV-related anticoagulant function to understand the pathogenesis of thrombosis caused by FV abnormality. Furthermore, using recombinant thrombomodulin, we successfully developed a novel assay with clot waveform analysis for the rapid detection of FV deficiency with APC resistance. Other FV abnormality-related thrombosis has been reported in Japan in recent years, and we hope to further clarify the FV-related thrombotic predisposition in the future.

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.

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.

Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII.

Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production.

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.

Factor VIII mutated with Lys1813Ala within the factor IXa-binding region enhances intrinsic coagulation potential.

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

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.

The balance of comprehensive coagulation and fibrinolytic potential is disrupted in patients with moderate to severe COVID-19.

Coagulation and fibrinolytic mechanisms are enhanced in patients with coronavirus (COVID-19), but disturbances in the balance of both functions in COVID-19 patients remain unclear. We assessed global coagulation and fibrinolysis in plasma from 167 COVID-19 patients (mild/moderate/severe: 62/88/17, respectively) on admission using clot-fibrinolysis waveform analysis (CFWA). Maximum coagulation velocity (|min1|) and maximum fibrinolysis velocity (|FL-min1|) were expressed as ratios relative to normal plasma. Ten patients (6.0%) developed thrombosis, 5 (3.0%) had bleeding tendency, and 13 (7.8%) died during admission. FDP levels increased with severity of COVID-19 symptoms (mild/moderate/severe; median 2.7/4.9/9.9 µg/mL, respectively). The |min1| ratios were elevated in all categories (1.27/1.61/1.58) in keeping with enhanced coagulation potential, with significant differences between mild cases and moderate to severe cases. The |FL-min1| ratios were also elevated in all groups (1.19/1.39/1.40), reflecting enhanced fibrinolytic potential. These data identified coagulation dominance in moderate to severe cases, but balanced coagulation and fibrinolysis in mild cases. There were significant differences in FDP and TAT, but no significant differences in |min1| or |FL-min1| ratios, between patients with and without thrombosis. CFWA monitoring of coagulation and fibrinolysis dynamics could provide valuable data for understanding hemostatic changes and disease status in COVID-19 patients.

Comparisons of global coagulation potential and bleeding episodes in emicizumab-treated hemophilia A patients and mild hemophilia A patients.

Emicizumab reduces bleeding events in patients with severe hemophilia A (HA). The coagulation potential of emicizumab at a clinical dose appears to correspond to about 15 IU/dL of factor VIII activity (FVIII:C), the equivalent of converting from a severe to mild phenotype. However, the clinical and laboratory characteristics of HA patients receiving emicizumab (Emi-PwHA) compared with patients with mild HA (PwMHA) remain to be determined. We reviewed clinical data from Emi-PwHA (n = 63) and PwMHA (n = 15) and evaluated comprehensive coagulation function using Ca2+-triggered rotational thromboelastometry (ROTEM) and ellagic acid/tissue factor-triggered clot waveform analysis (modified CWA). The median FVIII:C in PwMHA was 13.0 (IQR 8.5-17.0) IU/dL. Bleeding patterns in both groups were similar and classified into three categories: (1) spontaneous bleeding, post-traumatic, (2) bleeding within 1-2 days, and (3) delayed bleeding after 1-2 weeks. The coagulation potential in Emi-PwHA with and without breakthrough bleeds was comparable. Furthermore, coagulation function in Emi-PwHA was equivalent to PwMHA, although time between treatment and hospitalization for breakthrough bleeds in PwMHA appeared to be longer than those in Emi-PwHA. The coagulation potential and bleeding patterns appeared to be similar in Emi-PwHA and PwMHA, indicating that emicizumab-driven coagulation potential reflected mild HA.

Ex Vivo Prediction of Comprehensive Coagulation Potential Using Simulated Blood Concentrations of Emicizumab in Patients with Acquired Hemophilia A.

INTRODUCTION: Emicizumab prophylaxis improves coagulation function in congenital hemophilia A, regardless of inhibitor presence. We recently reported that emicizumab enhanced the coagulant potentials, ex vivo, in plasmas from patients with acquired hemophilia A (PwAHAs) at diagnosis. However, coagulant effects of emicizumab in PwAHAs during the clinical course remain unclear. AIM: To assess ex vivo coagulant effects of emicizumab in PwAHAs during the clinical course. METHODS/RESULTS: Blood samples were obtained from 14 PwAHAs on (median) days 0 and 6 during a severe-bleeding phase, and days 27 and 59 during a reduced-bleeding phase with elevated endogenous factor VIII (FVIII) and decreased inhibitor titers. If administered a single dose of 3 or 6 mg/kg, or two doses at 6 mg/kg followed by 3 mg/kg, estimated plasma emicizumab concentrations (10/5/2.5, 20/10/5, and 30/15/7.5 µg/mL on days 0-7/30/60, respectively) could be used to represent potential changes, based on the half-life (T 1/2: ∼30 days). Emicizumab concentrations that covered maximum plasma concentrations of each dosage were used for spiking on day 0. Ex vivo addition of estimated emicizumab to PwAHA's plasma containing endogenous FVIII and/or inhibitor, without and with recombinant (r)FVIIa administration during immunosuppressive therapy, increased the calculated Ad|min1| values, assessed by clot waveform analysis, and their coagulant potentials were below normal levels. Rotational thromboelastometry revealed that ex vivo emicizumab addition resulted in the further improvement of coagulant potentials in whole bloods when combined with rFVIIa administration. CONCLUSION: Based on ex vivo and in vitro data, emicizumab has the potential to be effective in clinical situations for PwAHAs.

Inhibitor Index in the Clot Waveform Analysis-Based Mixing Test Differentiates among Hemophilia A without and with Inhibitors, and Lupus Anticoagulant.

BACKGROUND: The mixing test is used to identify the pathway to follow-up testing and is also useful for the investigation of lupus anticoagulant (LA) positivity. "To completely correct" indicates clotting factor deficiency, while "to not correct" indicates the presence of a clotting factor inhibitor including LA. "Index of circulation anticoagulant" and/or "percent correction" is used to interpret the results of mixing studies, but it does not accurately differentiate factor inhibitors from LA. AIM: To precisely differentiate hemophilia A (HA), HA with inhibitor (HA-inh), and LA using the clot waveform analysis (CWA)-based mixing test. METHODS: Plasma samples from HA, LA, and HA-inh including acquired HA were incubated with normal plasma in 9:1, 1:1, and 1:9 mix ratios. From activated partial thromboplastin time CWA at 0-minute (immediately) and 12-minute incubation, the ratios of CWA parameters at 12 minutes/0 minute (inhibitor index) were assessed. RESULTS: The inhibitor index values of CWA parameters obtained using the mixing test in a 1:1 ratio demonstrated a significant difference between HA-inh and LA but could not differentiate LA from HA-inh completely. Plasmas used for the mixing tests in 9:1 and 1:9 ratios were able to fully distinguish between HA-inh (>0.5 BU/mL) and LA. These indices significantly correlated with inhibitor titer below 40 BU/mL (r > 0.90), possibly estimating FVIII inhibitor titer from the inhibitor index. Plasmas in HA and LA could be distinguished by mixing in a 1:1 ratio at 0 minute (immediately). CONCLUSION: The inhibitor index from CWA-based mixing tests with a 12-minute incubation could differentiate among HA, HA-inh, and LA quickly.

A Novel Assessment of Factor VIII Activity by Template Matching Utilizing Weighted Average Parameters from Comprehensive Clot Waveform Analysis.

BACKGROUND: Activated partial thromboplastin time (aPTT)-based clot waveform analysis is used to evaluate the comprehensive dynamics of fibrin clot formation. In addition, the technique can be usefully utilized for the rapid assessment of factor (F)VIII procoagulant activity in various clinical settings in patients with hemophilia A (HA). We defined a novel algorithm based on the weighted average parameters from aPTT-based waveforms to devise a template-matching procedure for assessing FVIII activity (FVIII:C). METHODS: The first derivatives of original clot waveforms triggered by the aPTT reagent (Coagpia APTT-N) were used to determine weighted averages of areas surrounded by the waveform at different percentages of maximum height in various clotting factor-deficient plasmas. Prepared templates based on 50 weighted average-related parameters were compared with 78 aPTT-prolonged plasmas. RESULTS: Original nonsmoothed waveforms of the various clotting factor-deficient plasmas with prolonged aPTTs demonstrated a variety of shapes. The weighted averages were calculated after adjustments for different baselines, and the patterns seemed to be governed by the specific clotting factor deficiency. The weighted average-related parameters including baseline wedge (r 2 = 0.998) and aspect ratio (r 2 = 0.998) were highly correlated with FVIII:C levels. Template-matching analyses based on weighted average-related waveform parameters obtained from 158 samples demonstrated that the sensitivity was 97.2% and specificity was 83.3% in aPTT-prolonged plasmas (n = 78). CONCLUSION: This novel algorithm based on weighted averages of aPTT-based waveforms together with template-matching may support clinical usefulness for judging of HA and may aid clinical management in the patients in the absence of specific clotting factor assays.

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.