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When patients with hemophilia A develop factor VIII (FVIII) inhibitors, FVIII replacement therapy becomes ineffective. Although immune-tolerance induction (ITI) therapy has been used to eradicate inhibitors, treatment is unsuccessful in approximately 30% of cases. However, the mechanism behind treatment failure remains unclarified. We retrospectively examined the longitudinal profiles of immunoglobulin G (IgG) subclasses and/or the inhibitory activities of FVIII in plasma samples from 14 Japanese patients with congenital hemophilia A during hemostatic, FVIII replacement, and/or ITI therapies. In five patients, an increase in IgG4 was observed simultaneously with a decrease in IgG1 when the patient had a history of relatively high FVIII inhibitor titers, reflecting an apparent change in humoral immunity. In addition, we examined the reactivity and specificity of the patients' anti-FVIII IgG1 and IgG4 to FVIII domains by immunoblotting. Under our experimental conditions, plasma from three patients with historically higher inhibitor titers appeared to have high titers of antibodies against the A2-a2 domain, which did not necessarily correlate with ITI failure. These observations may improve scientific understanding of the immune response to infused FVIII in patients with hemophilia A., (© 2022. Japanese Society of Hematology.)

Global coagulation potential was assessed in 59 patients with acquired hemophilia A (PwAHA) by clot waveform analysis (CWA) and/or thrombin and plasmin generation assay. Relationships between factor VIII activity (FVIII:C) and the parameters from CWA and T/P-GA in patients with congenital HA were compared by grading coagulation potential related to FVIII:C: T1 (FVIII:C < 1 IU/dL), T2 (1 ≤ , ≤ 5 IU/dL), T3 (5 < , 12 ≤ IU/dL), and T4 (12 < , ≤ 50 IU/dL). The median FVIII:C and inhibitor titers in PwAHA on admission were 3.3 IU/dL and 63.0 BU/mL, respectively, but global coagulation parameters corresponded to T1 or less. Median FVIII:C levels during follow-up in PwAHA were 1.7-9.6-6.7-40.0-21.7 IU/dL on days 0-14-28-56-93, respectively. CWA-based data corresponded to less than T2 until day 28, but more closely reflected FVIII:C after day 56. Peak thrombin was severely low (near T1) until day 28 and improved modestly after day 56 but remained less than T2. Peak plasmin was lower than T1 until day 56, and returned to T4 on day 93. In conclusion, global coagulation function in PwAHA was impaired to a greater extent than could be anticipated from assays of FVIII:C, until approximately 1 month after immunosuppression and treatment with FVIII-bypassing agents., (© 2021. Japanese Society of Hematology.)

Introduction: Acquired hemophilia A (AHA) is caused by autoantibodies against factor (F)VIII, and is characterized by severe, spontaneous bleeding, which can be life-threatening. Emicizumab, an anti-FIXa/FX bispecific antibody, significantly reduces bleeding events in congenital hemophilia A (HA) with and without inhibitors. The known pathophysiological mechanisms and current preclinical data in HA suggest that emicizumab could provide effective treatment for AHA, but the coagulation activities of emicizumab in these patients remain unknown., Aim: To evaluate the coagulant effects of emicizumab in plasma from AHA patients., Methods and Results: Tissue factor-triggered thrombin generation assays using normal plasma preincubated with anti-FVIII monoclonal antibodies recognizing different epitopes demonstrated that 20 µg/mL emicizumab recovered the depressed peak levels of thrombin generation to 46% to 72%. Further studies were devised, therefore, to simulate the clinical course in AHA patients, including during the acute phase for severe bleeding requiring FVIII-bypassing therapy, and during the subacute/chronic phase with less bleeding. Various concentrations of emicizumab were used to represent the potential changes in plasma levels based on the half-life of the antibody (~30 days). The ex vivo addition of emicizumab to plasma samples from AHA patients (n = 16) increased peak thrombin in all cases, irrespective of the inhibitor epitope specificity. Thrombin generation at 20 and 100 µg/mL emicizumab was restored to (median) 43.9% and 92.2%, respectively. Differences were evident in some cases, however, and recovery rates appeared likely to be greater in patients with type 2 inhibitor than those with type 1., Conclusion: Emicizumab improved ex vivo coagulation potential in plasma from AHA patients., (© 2020 International Society on Thrombosis and Haemostasis.)

A case of acquired hemophilia A (AHA) that developed in a patient with autoimmune pancreatitis (AIP) is presented. A 64-year-old woman was diagnosed with AIP in 2007. The symptoms resolved with prednisolone (PSL). Although the dose of PSL was tapered to 7.5 mg/day for maintenance, serum IgG4 levels remained high. She suddenly presented with subcutaneous bleeding in 2015. Her activated partial thromboplastin time was prolonged (80.0 s). A mixing test showed an inhibitor pattern, factor VIII (FVIII) activity was less than 1%, and FVIII inhibitor was 290 BU/mL. She was diagnosed with AHA. Her serum IgG4 was elevated to 133 mg/dL. She was treated first with PSL alone, but she developed bladder tamponade. Cyclophosphamide and activated prothrombin complex concentrate were combined with PSL. She then achieved hemostasis, and FVIII inhibitor disappeared. FVIII inhibitor had been detected since PSL was tapered and AHA recurred two months later. An enzyme-linked immunosorbent assay showed that the inhibitor was mainly IgG4 and IgG1. This case suggests that elevation of IgG4 may be associated with the development of both AHA and AIP.

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 Ca 2+ -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., (© 2018 John Wiley & Sons Ltd.)

We describe an 8-year-old boy with acquired hemophilia A (AHA) associated with streptococcal infection and amoxicillin. Laboratory data revealed low factor VIII activity (FVIII:C, 1.5 IU/dl), and FVIII inhibitor (15.9 BU/ml). Comprehensive coagulation function assays, including rotation thromboelastometry (ROTEM ® ), revealed a markedly prolonged clotting time. Thrombin and plasmin generation (TG/PG) appeared to be moderately impaired. The inhibitor epitope of his anti-FVIII autoantibody recognized light and heavy chains. He was treated with Novoseven ® and prednisolone, resulting in rapid improvement. ROTEM showed the return of coagulation time to normal level on day 20, and TG gradually improved. PG was moderately reduced in the clinical early phase, but improved at day 20. The patient's IgG subtype was IgG 4 at onset. IgG 1 was transiently positive on day 20, but negative on day 46. FVIII inhibitor gradually decreased and was completely absent after day 46, along with the elevated FVIII:C. IgG4 was again elevated on day 83, followed by a rapid decrease, indicative of the presence of non-neutralizing antibody, which remains currently undetected. We for the first time report changes in comprehensive coagulation function and IgG subtype of anti-FVIII antibody in a rare pediatric case of AHA.

PT, APTT, and coagulation factor activity are measured with clotting time-based methods. When coagula- tion and fibrinolysis are enhanced, TAT and PIC are detected, but evaluations of their hypofunctional state are difficult. We devised a new method that can be used to comprehensively and continuously evaluate coagula- tion and fibrinolysis in real time. We elucidated the clinical phenotype of congenital and acquired coagulation disorders using the following methods; (1) Clot waveform analysis (CWA) to evaluate fibrin formation; (2) Thrombin generation test (TGT) to monitor one-step before fibrin formation. (3) Thrombin/Plasmin generation assay (T/P-G) to evaluate fibrinolysis simultaneously with TGT. The results revealed that (1) CWA enabled the measurement of a very low FVIII activity level (FVIII:C 0.2-1.0 IU/dL) and detected a markedly severe type with FVIII:C <0.2 IU/dL. (2) For TGT, acquired hemophilia A showed a much lower value than that of congenital severe hemophilia A, being consistent with its severe bleeding. (3) CWA parameters for acquired factor V inhibitors in patients with bleeding symptoms were more impaired than with non-bleeding. Taken together these comprehensive assays can reflect the clinical phenotype and make it possible to analyze unidentified coagulation/fibrinolysis abnormality.

Some hemophilia A patients who have developed inhibitors are poorly responsive to activated prothrombin complex concentrates (aPCC) after daily dosage, but the mechanism(s) underlying this remain unknown. We examined two representative cases. In case 1, we found that changing to recombinant factor VIIa (rFVIIa) therapy was more effective, and the response to aPCC was restored within ~2 weeks. Tissue factor (TF)-triggered thrombin generation demonstrated a prolonged lag-time and decreased peak thrombin, and this impairment was focused on TF pathway inhibitor (TFPI). Plasma-free TFPI was elevated post-infusion of aPCC, while this was unaffected by rFVIIa. TFPI returned to normal range within 2-3 weeks. Plasmas obtained from patients with poor or good response to aPCC (aPCC-poor or aPCC-good), and good response to rFVIIa (FVIIa-good) demonstrated that free TFPI levels are increased in both aPCC groups, but not in FVIIa-good. TFPI levels pre- and post-infusion in aPCC-poor were significantly higher than those in aPCC-good. Addition of anti-TFPI antibody to the reaction samples demonstrated a greater increase of peak thrombin in aPCC-poor compared to aPCC-good, showing the higher TFPI activity in aPCC-poor. Free TFPI contained in aPCC corresponded to the increasing levels in plasma. In conclusion, TFPI in aPCC attenuated thrombin generation, and the reduced effectiveness of therapy in these circumstances appeared to be related to TFPI activity.

Normal haemostasis is maintained by a controlled balance between coagulation and fibrinolysis, involving thrombin and plasmin the respective key enzymes. Simultaneous evaluation of both enzymes facilitates, therefore, an overall understanding of normal and pathological haemostasis. Combined thrombin and plasmin generation (T/P-G) assays have been recently described, and we have adapted the technique to investigate the interplay between coagulation and fibrinolysis in patients with various haemostatic disorders. Our modified T/P-G was initiated by the addition of a mixture of optimised lower concentrations of tissue factor and tissue-type plasminogen activator. Thrombin generation (TG) and plasmin generation (PG) were monitored simultaneously using individual fluorescent substrates in separate microtitre wells. The relationship between coagulation and fibrinolysis was demonstrated by analysing the effects of thrombin inhibitors, activated protein C and thrombomodulin. The most evident impairments in TG were observed with plasma samples deficient of coagulation factors participating in the prothrombinase complex. Defects in PG were observed with deficiencies of factor (F)V, FX, fibrinogen, and plasminogen. TG appeared to be a prerequisite for the initiation of PG, and overall PG was governed by fibrinogen concentration. TG in patients with haemophilia A correlated with levels of FVIII activity, but there was no significant relationship between PG and FVIII:C, confirming that the abnormal haemostasis in haemophilia A results in a severe imbalance between coagulation and fibrinolysis. The findings demonstrate that global haemostasis depends on a sensitive balance between coagulation and fibrinolysis, and that the modified T/P-G assay could provide an enhanced understanding of haemorrhage and thrombosis in clinical practice.

Acquired haemophilia A (AHA) is caused by the development of factor (F)VIII autoantibodies, demonstrating type 1 or type 2 inhibitory behaviour, and results in more serious haemorrhagic symptoms than in congenital severe HA. The reason(s) for this remains unknown, however. The global coagulation assays, thrombin generation tests and clot waveform analysis, demonstrated that coagulation parameters in patients with AHA-type 2 inhibitor were more significantly depressed than those in patients with moderate HA with similar FVIII activities. Thrombin and intrinsic FXa generation tests were significantly depressed in AHA-type 1 and AHA-type 2 compared to severe HA, and more defective in AHA-type 1 than in AHA-type 2. To investigate these inhibitory mechanism(s), anti-FVIII autoantibodies were purified from AHA plasmas. AHA-type 1 autoantibodies, containing an anti-C2 ESH4-epitope, blocked FVIII(a)-phospholipid binding, whilst AHA-type 2, containing an anti-C2 ESH8-epitope, inhibited thrombin-catalysed FVIII activation. The coagulation function in a reconstituted AHA-model containing exogenous ESH4 or ESH8 was more abnormal than in severe HA. The addition of anti-FIX antibody to FVIII-deficient plasma resulted in lower coagulation function than its absence. These results support the concept that global coagulation might be more suppressed in AHA than in severe HA due to the inhibition of FIXa-dependent FX activation by steric hindrance in the presence of FVIII-anti-C2 autoantibodies. Additionally, AHA-type 1 inhibitors prevented FVIIIa-phospholipid binding, essential for the tenase complex, whilst AHA-type 2 antibodies decreased FXa generation by inhibiting thrombin-catalysed FVIII activation. These two distinct mechanisms might, in part, contribute to and exacerbate the serious haemorrhagic symptoms in AHA.

We have utilized high-dose factor VIII (FVIII) concentrates in 4 hemophilia A patients with inhibitors prior to surgery for the insertion of a central venous access device. In total, 3 patients out of 4 had high responding inhibitors. Dosing algorithms for this type of therapy have not been previously validated and established. We devised an effective formula to calculate the initial dose required to neutralize the inhibitors, although some of the patients demonstrated a lower recovery of FVIII than expected. An anamnestic inhibitor response was evident in 3 cases, but overall our strategy provided a reliable hemostatic effect for at least 4 days after surgery. In addition, our protocol appeared to be more cost-effective than FVIII bypass therapy. The financial saving in 1 case for the initial 3 days was estimated to be approximately US$49 122. Our results demonstrated that high-dose FVIII therapy provided clinically effective and economically viable results even in high responders.

The patient was a 36-year-old man who had been aware of a mild bleeding tendency since childhood, but did not show any history of severe bleeding. After lumbar epidural block was performed for pain caused by lumbar disc herniation, the patient developed paraplegia due to an acute epidural hematoma, and rectovesical disorder. He was admitted to our hospital, and wide fenestration and hematoma evacuation were performed. Because of persistent bleeding, reoperation was performed to achieve hemostasis. Although factor VIII antigen was 138%, its activity was decreased to 18% of normal. A diagnosis of cross-reacting material positive mild hemophilia A was made. Postoperative injection of a factor VIII preparation resulted in complete hemostasis. The activated partial thromboplastin time was within normal range. A thrombin generation test showed reduced endogenous thrombin potential, peak thrombin levels, and prolonged time-to-peak levels. The thrombin generation test, which allows comprehensive assessment of the coagulation profile, was useful for diagnosis and treatment of this case.

Discrepancies between low levels of FVIII:C and clinical symptoms in severe hemophilia A are well-known. We have recently demonstrated that levels of FVIII:C < 0.2 IU/dl were consistent with clinical phenotype by clot waveform analysis, suggesting that precise measurement of very low levels of FVIII:C was clinically important. Thrombin generation tests (TGTs) triggered by tissue factor (TF) have been recently utilized to monitor coagulation function in hemophilia A. We examined whether TGT was useful for evaluating hemophilia A patients with very low levels of FVIII:C. TGTs in 40 hemophilia A plasmas with FVIII:C < 0.2-17 IU/dl (measured by clot waveform analysis using MDA-II) were performed using TF and/or ellagic acid (ELG). The lagtime in ELG-TGT at very low levels of FVIII:C was shortened dose-dependently, whilst this parameter in TF-TGT was not significantly affected. Other parameters (endogenous thrombin potential, peak thrombin, time to peak) correlated with FVIII:C levels to some extent in both assays (r = 0.4-0.7). Using a TF/ELG mixture in TGT, however, the correlation coefficients increased to ~0.85. TGT parameters correlated well with levels of FVIII:C > 0.2 IU/dl, although the lagtime was not especially informative. We conclude that modified TGT, using a TF/ELG mixture as the trigger, is useful for monitoring coagulation function at very low levels of FVIII:C in hemophilia A.