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.

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.

Activated partial thromboplastin time-based clot waveform analysis enables measurement of very low levels of factor IX activity in patients with severe hemophilia B.

The precise measurement of very low levels of factor IX activity (FIX:C < 1 IU/dL) is essential for understanding clinical severity and risk of inhibitor development in patients with severe hemophilia B (Pw-SHB). However, such measurement sensitivity has not yet been achieved. We aimed to establish a measurement method using clot waveform analysis (CWA). Residual FIX:C by adding anti-FIX monoclonal antibody, FIX:C by adding recombinant (r)FIX to the commercial Pw-SHB plasmas, and FIX:C in our Pw-SHB were determined by CS-2000i™/CS-2400™, followed by analysis of CWA parameters. The presence of anti-FIX antibody in the commercial Pw-SHB plasmas significantly decreased coagulation potential compared to its absence. The addition of rFIX to these innate plasma samples produced significant changes in three parameters upon adding FIX:C at 0.1-1 IU/dL, supporting the presence of trace FIX:C in Pw-SHB. Therefore, appropriate FIX-depleted plasma containing minimum residual FIX:C was chosen from reference curves of FIX:C (0.01-1 IU/dL). Among patients with untreated Pw-SHB, two had FIX:C 0.6-0.7 IU/dL and two had lower than detectable levels using FIX-depleted plasma. One of the latter had detectable trough levels post-rFIX administration. In conclusion, CWA enabled measurement of very low levels of FIX:C using appropriate FIX-deficient plasma.

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.

Clot waveform analysis for perioperative hemostatic monitoring of arthroscopic synovectomy in a pediatric patient with hemophilia A and inhibitor receiving emicizumab prophylaxis.

Emicizumab reduces bleeding in patients with hemophilia A and inhibitors (PwHA-I). Coagulation potential during the perioperative period in emicizumab-treated PwHA-I undergoing surgery remains to be evaluated. We describe a 14-year-old boy with HA-I receiving emicizumab prophylaxis who experienced arthroscopic synovectomy. He was treated with a bolus infusion of recombinant factor VIIa (rFVIIa; 80 µg/kg) immediately before surgery, and treatment continued at the same dose every 3 h on day 1, every 4 h on day 2, and every 6 h on day 3. Treatment with rFVIIa was discontinued on day 4. No perioperative bleeding or thrombotic events were observed. Coagulation potential throughout the perioperative period was retrospectively assessed with an easy-to-use clot waveform analysis (CWA). Measurements from CWA returned to within or near the normal range, suggesting successful hemostatic management. Coagulation potentials assessed by CWA showed a significant correspondence with those from a thrombin generation assay (TGA) that is already in use. CWA and TGA could both provide useful data for assessing coagulation potential in the perioperative hemostatic management of emicizumab-treated PwHA-I.

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.

How to recover lost vaccine acceptance? A multi-center survey on HPV vaccine acceptance in Japan.

BACKGROUND: The plummeting acceptance rate of the HPV vaccine in Japan is one of the most disappointing vaccine-related events in recent times. Since 2013, the national HPV vaccine coverage rate fell from more than 70% to less than 1%. This survey investigated parental HPV vaccine acceptance and the factors that influence it. METHODS: A multi-center survey was conducted in eight hospitals in Nara prefecture, Japan, from July 2019 to March 2020. Parents were asked to answer a series of questions in a survey that included information on the HPV vaccine. RESULTS: Among the 1884 parents who answered the questionnaire, 21.8% indicated that they had accepted the HPV vaccine even before reading the information provided in the questionnaire. The overall acceptance rate after everyone had read the information increased to 50.2% (p < 0.001). Among those who still did not accept the vaccine after reading the information (N = 925), 26.7% indicated that they might change their mind if more vaccine safety reports were to appear in the mass media; other potentially influencing factors were direct communication from health care providers (35.1%), a recommendation by government (19.5%), and peer behavior (16.8%). CONCLUSION: The study showed that providing appropriate medical information significantly improves HPV vaccine acceptance. To reverse the loss of HPV vaccine acceptance in Japan, a multi-discipline approach that includes the mass media, health care providers, the government and the general population will be needed.

Hemostatic assessment of combined anticoagulant therapy using warfarin and prothrombin complex concentrates in a case of severe protein C deficiency.

Patients with severe congenital protein (P)C deficiency require long-term anticoagulant management. Recombinant PC concentrates for prophylactic use are not available in Japan; prothrombin complex concentrates (PCC), containing factors (F)II, VII, IX, X, and PC (PPSB-HT®), have been used 'off-label' in a few patients. We investigated the combined use of prophylactic PCC and Warfarin (VKA; PT-INR 2.0-2.5) in a severely PC-deficient patient in whom VKA alone did not prevent recurrent purpura. Plasma VKA-dependent factor levels and global PC function (Thrombopath®) were assessed. Plasma activity levels of FII/FVII/FIX/FX post-infusion of PCC (6.3 unit/kg) increased 35/27/27/35 (initial level) to 59/60/38/83 IU/dl, respectively. FVII:C and FIX:C rapidly returned to baseline levels 12-24 h post-infusion, but FII:C and FX:C returned more slowly. PC antigen (< 5%) increased to ~ 15%, followed by return to baseline levels 24 h post-infusion. Global PC function was very low (%PiCi 24%), but improved post-PCC infusion. This potential was slightly detectable even at an undetectable PC level. At day 3, high levels of D-dimer and FDP were observed without thrombotic event, but these improved post-infusion. Although PCC restored VKA-dependent coagulation factors, PC contained in PCC significantly improved global anticoagulation, and was clinically beneficial in this severely deficient patient.