Correction: Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

[This corrects the article DOI: 10.1371/journal.pgen.1008690.].

Recombinant VWF fragments improve bioavailability of subcutaneous factor VIII in hemophilia A mice.

Conventional treatment of hemophilia A (HA) requires repetitive IV injection of coagulation factor VIII (FVIII). Subcutaneous administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PLs), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII-stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (1) no significant impact on rhFVIII recovery or half-life after a single IV administration; (2) enhanced bioavailability (up to 18.5%) of rhFVIII after subcutaneous administration; and (3) slow absorption (peak concentration, 6 hours) and prolonged half-life (up to 2.5-fold) of rhFVIII after subcutaneous administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 subcutaneously compared with rhFVIII IV. A single subcutaneous dose of rhFVIII/VWF-12 provided protection in the HA tail-bleeding model for up to 24 hours. In summary, recombinant VWF fragments support FVIII delivery through the subcutaneous space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after subcutaneous administration highlight the potential application of VWF fragments for subcutaneous FVIII prophylaxis in HA.

Prothrombotic immune thrombocytopenia after COVID-19 vaccination.

We report 5 cases of prothrombotic immune thrombocytopenia after exposure to the ChAdOx1 vaccine (AZD1222, Vaxzevria) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients presented 5 to 11 days after first vaccination. The spectrum of clinical manifestations included cerebral venous sinus thrombosis, splanchnic vein thrombosis, arterial cerebral thromboembolism, and thrombotic microangiopathy. All patients had thrombocytopenia and markedly elevated D-dimer. Autoantibodies against platelet factor 4 (PF4) were detected in all patients, although they had never been exposed to heparin. Immunoglobulin from patient sera bound to healthy donor platelets in an AZD1222-dependent manner, suppressed by heparin. Aggregation of healthy donor platelets by patient sera was demonstrated in the presence of buffer or AZD1222 and was also suppressed by heparin. Anticoagulation alone or in combination with eculizumab or intravenous immunoglobulin (IVIG) resolved the pathology in 3 patients. Two patients had thromboembolic events despite anticoagulation at a time when platelets were increasing after IVIG. In summary, an unexpected autoimmune prothrombotic disorder is described after vaccination with AZD1222. It is characterized by thrombocytopenia and anti-PF4 antibodies binding to platelets in AZD1222-dependent manner. Initial clinical experience suggests a risk of unusual and severe thromboembolic events.

Microembolic signal monitoring in patients with HeartMate 3 and HeartWare left ventricular assist devices: Association with antithrombotic treatment and cerebrovascular events.

BACKGROUND: In patients with left ventricular assist devices (LVADs), ischemic and hemorrhagic stroke are dreaded complications. Predictive markers for these events are lacking. This study aimed to investigate the prevalence and predictive value of microembolic signals (MES) for stroke, detected by Transcranial Doppler sonography (TCD) in patients with HeartMate 3 (HM 3) or HeartWare (HW). METHODS: A thirty-minute bilateral TCD monitoring of the middle cerebral artery (MCA) was performed in 62 outpatients with LVAD (HM 3 N = 31, HW N = 31) and 31 healthy controls. Prevalence and quantity of MES were investigated regarding clinical and laboratory parameters. Cerebrovascular events (CVE) were recorded on follow-up at 90 and 180 days. RESULTS: MES were detected in six patients with HM 3, three patients with HW, and three controls. Within the LVAD groups, patients on monotherapy with vitamin-K-antagonist (VKA) without antiplatelet therapy were at risk for a higher count of MES (negative binomial regression: VKA: 1; VKA + ASA: Exp(B) = 0.005, 95%CI 0.001-0.044; VKA + clopidogrel: Exp(B) = 0.012, 95%CI 0.002-0.056). There was no association between the presence of MES and CVE or death on follow-up (p > 0.05). CONCLUSION: For the first time, the prevalence of MES was prospectively investigated in a notable outpatient cohort of patients with HM 3 and HW. Despite optimized properties of the latest LVAD, MES remain detectable depending on antithrombotic therapy. No association between MES and CVE could be detected.

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.

Expression of human thrombomodulin on porcine endothelial cells can reduce platelet aggregation but did not reduce activation of complement or endothelium - an experimental study.

Clinical xenotransplantation will only be feasible when present limitations can be controlled sufficiently. Activation of endothelium and complement as well as coagulopathy and thrombotic microangiopathy (TMA) is important barriers. Transgenic expression of hTBM on porcine endothelial cells is a reasonable approach to reduce activation of haemostasis. Endothelial cells from wild-type pigs as well from pigs expressing hTBM alone or in combination with hCD46 and knockout of the alpha-1,3,-galactosyltransferase (GTKO) were perfused with platelet-rich plasma in a microfluidic flow chamber. Platelet aggregation and activation, coagulation, complement and endothelial cell activation were assessed. Perfusion of wild-type porcine aortic endothelial cells (PAEC) resulted in distinct platelet aggregation. Expression of hTBM in either mono-transgenic or triple-transgenic (GTKO/hCD46/hTBM) PAEC showed significantly reduced or absent platelet aggregation. Flow cytometric analysis of platelets showed an increased CD62P expression in wild-type PAEC and significantly reduced expression in mono- or triple-transgenic PAEC. Activation of coagulation measured by TAT occured in WT PAEC and was clearly reduced in hTBM and GTKO/hCD46/hTBM PAEC. Activation of complement and endothelial cells was only reduced in GTKO/hCD46/hTBM but not in PAEC expressing hTBM alone. Expression of hTBM was able to prevent activation of coagulation and platelet aggregation in mono- and triple-transgenic PAEC, while activation of complement and endothelial cells was not reduced in mono-transgenic PAEC.

Investigation of the influence of xenoreactive antibodies on activation of complement and coagulation in an ex vivo perfusion animal study using porcine kidneys.

During pig-to-primate xenotransplantation or perfusion of porcine organs with human blood, a xenogeneic coagulopathy with consecutive development of thrombotic microangiopathy (TMA) can be observed. The aim of this study was to elucidate the influence of the reduction of xenoreactive natural antibodies on the coagulopathy using an ex vivo perfusion system. Thirteen perfusion experiments using landrace wild-type porcine kidneys were performed in three different experimental groups: autologous, xenogeneic, and immunoadsorption. During and after perfusion, blood and tissue samples were collected to assess markers of coagulation, complement, inflammation, and endothelial activation. Immunoadsorption prior to perfusion did not prolong perfusion time (174 min ±28) compared to xenogeneic (182 min ±22) experiments, whereas autologous perfusion was possible for maximum of 240 min in all experiments. Activation of coagulation was similar comparing perfusions after immunoadsorption (D-Dimer 24 186 µg/l ±5813; TAT 566 µg/l ±34) to xenogeneic (D-Dimer 22 175 µg/l ±7826, TAT 600 µg/l ±0) experiments. But antibody-mediated complement activation was reduced in the immunoadsorption group. TNF-alpha and markers of endothelial cell activation were lower in the immunoadsorption group compared to the xenogeneic experiments. In this ex vivo perfusion model, we observed that marked removal of xenogeneic antibodies can reduce complement activation via the classical pathway as well as endothelial cell activation and inflammation. Immunoadsorption cannot prevent the activation of the terminal complement cascade and coagulation.

Anti-factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study.

Neutralizing autoantibodies against factor VIII (FVIII), also called FVIII inhibitors, are the cause of acquired hemophilia A (AHA). They are quantified in the Bethesda assay or Nijmegen-modified Bethesda assay by their ability to neutralize FVIII in normal human plasma. However, FVIII inhibitors do not represent the whole spectrum of anti-FVIII autoantibodies. Here, we studied isotypes, immunoglobulin G subclasses, and apparent affinities of anti-FVIII autoantibodies to assess their prognostic value for the outcome in AHA. We analyzed baseline samples from patients enrolled in the prospective GTH-AH 01/2010 study. Our data suggest that anti-FVIII immunoglobulin A (IgA) autoantibodies are predictors of poor outcome in AHA. Anti-FVIII IgA-positive patients achieved partial remission similar to anti-FVIII IgA-negative patients but had a higher risk of subsequent recurrence. Consequently, IgA-positive patients achieved complete remission less frequently (adjusted hazard ratio [aHR], 0.35; 95% confidence interval [CI], 0.18-0.68; P < .01) and had a higher risk of death (aHR, 2.62; 95% CI, 1.11-6.22; P < .05). Anti-FVIII IgA was the strongest negative predictor of recurrence-free survival after achieving partial remission and remained significant after adjustment for baseline demographic and clinical characteristics. In conclusion, anti-FVIII IgA represents a potential novel biomarker that could be useful to predict prognosis and tailor immunosuppressive treatment of AHA.

Prognostic factors for remission of and survival in acquired hemophilia A (AHA): results from the GTH-AH 01/2010 study.

Acquired hemophilia A (AHA) is caused by autoantibodies against factor VIII (FVIII). Immunosuppressive treatment (IST) results in remission of disease in 60% to 80% of patients over a period of days to months. IST is associated with frequent adverse events, including infections as a leading cause of death. Predictors of time to remission could help guide IST intensity but have not been established. We analyzed prognostic factors in 102 prospectively enrolled patients treated with a uniform IST protocol. Partial remission (PR; defined as no active bleeding, FVIII restored >50 IU/dL, hemostatic treatment stopped >24 hours) was achieved by 83% of patients after a median of 31 days (range 7-362). Patients with baseline FVIII <1 IU/dL achieved PR less often and later (77%, 43 days) than patients with ≥1 IU/dL (89%, 24 days). After adjustment for other baseline characteristics, low FVIII remained associated with a lower rate of PR (hazard ratio 0.52, 95% confidence interval 0.33-0.81, P < .01). In contrast, PR achieved on steroids alone within ≤21 days was more common in patients with FVIII ≥1 IU/dL and inhibitor concentration <20 BU/mL (odds ratio 11.2, P < .0001). Low FVIII was also associated with a lower rate of complete remission and decreased survival. In conclusion, presenting FVIII and inhibitor concentration are potentially useful to tailor IST in AHA.

Inhibition of complement component C5 prevents clotting in an ex vivo model of xenogeneic activation of coagulation.

BACKGROUND: Xenogeneic thrombotic microangiopathy (TMA) and acute vascular rejection (AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies (XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. METHODS: A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma (PRP) upon perfusion over wild-type (WT) or α-1,3- galactosyltransferase knockout (GTKO) and human CD46 (hCD46) transgenic porcine aortic endothelial cells (PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma (PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 µg/ml) was used to inhibit terminal complement activation in all experiments. RESULTS: WT-PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm(2) : 23 ± 10%; 10 dyn/cm(2) : 17 ± 10% of flow chamber viewing field). GTKO/hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm(2) : 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT-PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation (WT-PAEC: 1.6 ± 2% at 3 dyn/cm(2) and 0.24 ± 0.33% at 10 dyn/cm(2) , GTKO/hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm(2) ) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT-PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. CONCLUSION: Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function.

Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.

BACKGROUND: Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. METHODS: Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. RESULTS: Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. CONCLUSION: We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.

Laboratory diagnosis of acquired hemophilia A: limitations, consequences, and challenges.

Acquired hemophilia A (AHA) should be suspected in patients with a new onset of bleeding and an isolated prolongation of activated partial thromboplastin time. About 10% of patients do not bleed at the time of diagnosis, but are at risk of future bleeding, particularly during interventions or surgery. Diagnosis of AHA is confirmed by demonstrating markedly reduced factor VIII activity (FVIII:C) and neutralizing anti-FVIII antibodies, so-called inhibitors. Several limitations and pitfalls exist with the assays used to diagnose AHA. Interference can result from anticoagulants or lupus anticoagulant. The Bethesda assay used to measure inhibitor potency assumes a log-linear relationship between inhibitor concentration and effect on residual FVIII:C activity to allow exact quantification. However, this relationship is not present for the type 2 inhibitors typically seen in AHA. Therefore, this assay only provides a rough estimate of inhibitor potency. These limitations can explain, in part, why laboratory data, such as inhibitor potency, failed to predict bleeding or response to treatment in AHA. This article reviews the diagnostic approach to AHA, discusses assay-specific limitations and addresses some of the challenges for future research.

Recombinant human antithrombin prevents xenogenic activation of hemostasis in a model of pig-to-human kidney transplantation.

BACKGROUND: Xenogenic activation of hemostasis (XAH) represents a major hurdle for the transplantation of discordant animal organs into humans as it results in thrombotic microangiopathy (TMA). We have previously shown that recombinant human-activated protein C (rhAPC) mitigates XAH and TMA in an ex vivo model of pig-to-human kidney transplantation. However, the use of rhAPC may not be feasible in a perioperative setting due to possible bleeding complications. METHODS: Here, we explored the effects of another natural inhibitor of coagulation, human recombinant antithrombin (rhAT), in comparison with rhAPC. Unmodified porcine kidneys (n = 25) were perfused ex vivo with porcine blood, human blood, or human blood supplemented with rhAPC or rhAT. Surrogate parameters of organ survival, markers of XAH (D- Dimer, thrombin-antithrombin complex [TAT], fibrinogen, antithrombin activity, plasminogen), endothelial cell and platelet activation (E-selectin, P-selectin), platelet function tests and histological signs of TMA were evaluated. RESULTS: Perfusion was feasible for > 240 min in all experiments with autologous porcine blood, but limited to 126 ± 78 min with human blood due to increased vascular resistance. Addition of rhAT protected from TMA and allowed for perfusion times > 240 min. In addition, there were less signs of XAH with reduced release of P-selectin and overexpression of E-selectin, whereas the progressive loss of platelet function, observed during discordant perfusion, was prevented. The effect of rhAT was dose-dependent with maximum protection obtained at 3 IU/ml. CONCLUSION: In conclusion, in this ex vivo model of discordant xenotransplantation, rhAT reduced XAH and prevented TMA in doses that appear feasible for use in clinical or preclinical transplantation settings.

Emicizumab versus immunosuppressive therapy for the management of acquired hemophilia A.

BACKGROUND: Acquired hemophilia A (AHA) is an autoimmune bleeding disorder caused by neutralizing antibodies against coagulation factor VIII (FVIII). Immunosuppressive therapy (IST) is standard of care to eradicate autoantibody production and protect from further bleeding but carries a risk of severe infection and mortality in frail patients with AHA. Recently, emicizumab has been studied for its potential to reduce the need for early and aggressive IST. OBJECTIVE: To compare outcomes of two studies that used either IST (GTH-AH 01/2010; n=101) or prophylaxis with emicizumab (GHT-AHA-EMI; n=47) early after diagnosis of AHA. METHODS: Baseline characteristics were balanced by propensity score matching. Primary endpoint was the rate of clinically relevant new bleeds during the first 12 weeks; secondary endpoints were adverse events and overall survival. RESULTS: The negative binominal model-based bleeding rate was 68% lower with emicizumab as compared with IST (incident rate ratio 0.325, 95% confidence interval (CI) 0.182-0.581). No difference was apparent in the overall frequency of infections (emicizumab 21%, IST 29%) during the first 12 weeks, but infections were less often fatal in emicizumab treated patients (0%) compared with IST treated patients (11%). Thromboembolic events occurred less often with emicizumab (2%) than IST (7%). Overall survival after 24 weeks was better with emicizumab (90% versus 76%, HR 0.44; 95% CI 0.24-0.81). CONCLUSION: Using emicizumab instead of IST in the early phase after initial diagnosis of AHA reduced bleeding and fatal infections, and improved overall survival.

von Willebrand factor modulates immune complexes and the recall response against factor VIII in a murine hemophilia A model.

Achieving tolerance toward factor VIII (FVIII) remains an important goal of hemophilia treatment. Up to 40% of patients with severe hemophilia A (HA) develop neutralizing antibodies against FVIII, and the only proven treatment to achieve tolerance is infusion of FVIII over prolonged periods in the context of immune tolerance induction. Here, we addressed the role of von Willebrand factor (VWF) as a modulator of anti-FVIII antibody effector functions and the FVIII-specific recall response in an HA mouse model. Analytical ultracentrifugation was used to demonstrate formation of FVIII-containing immune complexes (FVIII-ICs). VWF did not fully prevent FVIII-IC formation but was rather incorporated into larger macromolecular complexes. VWF prevented binding of FVIII-ICs to complement C1q, most efficiently when it was preincubated with FVIII before the addition of antibodies. It also prevented binding to immobilized Fc-γ receptor and to bone marrow-derived dendritic cells. An in vitro model of the anti-FVIII recall response demonstrated that addition of VWF to FVIII abolished the proliferation of FVIII-specific antibody-secreting cells. After adoptive transfer of sensitized splenocytes into immunocompetent HA mice, the FVIII recall response was diminished by VWF. In summary, these data indicate that VWF modulates the formation and effector functions of FVIII-ICs and attenuates the secondary immune response to FVIII in HA mice.

Targets of autoantibodies in acquired hemophilia A are not restricted to factor VIII: data from the GTH-AH 01/2010 study.

The root cause of autoantibody formation against factor VIII (FVIII) in acquired hemophilia A (AHA) remains unclear. We aimed to assess whether AHA is exclusively associated with autoantibodies toward FVIII or whether patients also produce increased levels of autoantibodies against other targets. A case-control study was performed enrolling patients with AHA and age-matched controls. Human epithelial cell (HEp-2) immunofluorescence was applied to screen for antinuclear (ANA) and anticytoplasmic autoantibodies. Screening for autoantibodies against extractable nuclear antigens was performed by enzyme immunoassay detecting SS-A/Ro, SS-B/La, U1RNP, Scl-70, Jo-1, centromere B, Sm, double-stranded DNA, and α-fodrin (AF). Patients with AHA were more often positive for ANA than control patients (64% vs 30%; odds ratio [OR] 4.02, 1.98-8.18) and had higher ANA titers detected than controls. Cytoplasmic autoantibodies and anti-AF immunoglobulin A autoantibodies were also more frequent in patients with AHA compared with controls. Autoantibodies against any target other than FVIII were found in 78% of patients with AHA compared with 46% of controls (OR 4.16, 1.98-8.39). Results were similar preforming sensitivity analyses (excluding either subjects with autoimmune disorders, cancer, pregnancy, or immunosuppressive medication at baseline) and in multivariable binary logistic regression. To exclude that autoantibody staining was merely a result of cross-reactivity of anti-FVIII autoantibodies, we tested a mix of 7 well-characterized monoclonal anti-FVIII antibodies. These antibodies did not stain HEp-2 cells used for ANA detection. In conclusion, a diverse pattern of autoantibodies is associated with AHA, suggesting that a more general breakdown of immune tolerance might be involved in its pathology.

Emicizumab prophylaxis in patients with acquired haemophilia A (GTH-AHA-EMI): an open-label, single-arm, multicentre, phase 2 study.

BACKGROUND: Acquired haemophilia A is caused by neutralising autoantibodies against coagulation factor VIII, leading to severe bleeding. Standard treatment involves immunosuppressive therapy, which is associated with adverse events and mortality in the frail population of patients with acquired haemophilia A. This study investigated whether emicizumab, a factor VIIIa mimetic antibody, protects patients with acquired haemophilia A from bleeding and allows deferral of immunosuppression during the first 12 weeks after diagnosis. METHODS: We report final results of an open-label, single-arm, phase 2 clinical trial. Adult patients with acquired haemophilia A from 16 haemophilia treatment centres in Germany and Austria were eligible if they had not previously received immunosuppression. Patients received emicizumab subcutaneously (6 and 3 mg/kg on days 1 and 2, 1·5 mg/kg weekly until week 12), but no immunosuppression. Follow-up was until week 24. The primary endpoint was the number of clinically relevant bleeds per patient-week until week 12. Emicizumab was considered effective if the mean bleeding rate was significantly below 0·15 bleeds per patient-week, the rate observed in a previous study of patients with acquired haemophilia A treated with bypassing agents and immunosuppression but no emicizumab. The study is registered with clinicaltrials.gov, NCT04188639 and is complete. FINDINGS: Of 49 patients screened from March 25, 2021, to June 10, 2022, 47 were enrolled (23 women, 24 men). Median age was 76 years (IQR 66-80), 46 (98%) of 47 patients were White, median factor VIII activity was 1·4 IU/dL (0·3-5·6), and median inhibitor concentration was 11·4 Bethesda units per mL (3·9-42·7). Mean breakthrough bleeding rate was 0·04 bleeds per patient-week (upper 97·5% CI 0·06). 33 (70%) of 47 patients had no bleeding events, seven patients (15%) had one bleed, six patients (13%) had two bleeds, and one patient (2%) had three bleeds. Adverse events of grade 3 or worse included COVID-19 (n=2), acute kidney injury (n=2), and stroke (n=1). Four of 47 patients died, including two deaths related to bleeding, one from COVID-19, and one from cardiac arrest (none were judged as related to emicizumab). INTERPRETATION: This study suggests that emicizumab prophylaxis prevents bleeding in patients with acquired haemophilia A and that immunosuppressive therapy can be deferred while patients are receiving this treatment. The low number of thromboembolic events, severe infections, and fatalities observed in this study are promising. FUNDING: This study was supported by funding from Hoffman-La Roche.

Long-Term Outcomes after Vaccine-Induced Thrombotic Thrombocytopenia.

Vaccine-induced thrombotic thrombocytopenia (VITT), or thrombosis with thrombocytopenia syndrome (TTS), is a rare but serious complication of adenovirus-based vaccines against severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Observation of long-term outcomes is important to guide treatment of affected patients. This single-center consecutive cohort study included all patients diagnosed based on (1) vaccination 4 to 21 days before symptom onset, (2) signs or symptoms of venous or arterial thrombosis, (3) thrombocytopenia < 150/nL, (4) positive anti-platelet factor 4 (PF4) antibody, and (5) elevated D-Dimer > 4 times the upper limit of normal. Nine patients were enrolled. Acute management consisted of parenteral anticoagulants, corticosteroids, intravenous immunoglobulin (IVIG), and/or eculizumab. Eculizumab was successfully used in two patients with recurrent thromboembolic events after IVIG. Direct oral anticoagulants were given after hospital discharge. Median follow-up duration was 300 days (range 153 to 380). All patients survived the acute phase of the disease and were discharged from hospital. One patient died from long-term neurological sequelae of cerebral venous sinus thrombosis 335 days after diagnosis. Eight out of nine patients were alive at last follow-up, and seven had fully recovered. Anti-PF4 antibodies remained detectable for at least 12 weeks after diagnosis, and D-Dimer remained elevated in some patients despite oral anticoagulation. No recurrent thromboembolic events, other signs of VITT relapse, or bleeding complications occurred after discharge. In conclusion, VITT appears to be a highly prothrombotic condition. IVIG is not always successful, and eculizumab may be considered a rescue agent. Long-term management with direct oral anticoagulants appears to be safe and effective.

DNA analysis from stool samples: a fast and reliable method avoiding invasive sampling methods in mouse models of bleeding disorders.

Mouse models with targeted disruptions of coagulation factor genes are used to study disorders of haemostasis such as haemophilia. Standard protocols to obtain biopsies for genotyping in breeding programmes are based on invasive sampling methods such as tail clipping. These procedures imply a high risk of fatal bleeding, especially in haemophilic mouse models. Here we used a non-invasive sampling method obtaining stool samples for DNA isolation in a breeding programme, aiming to introduce targeted disruptions of Fc receptor genes (Fc gamma receptor IIB and III) into the haemophilia A mouse model (factor VIII deficiency). Faecal pellets were reliably obtained from individual mice and high-quality DNA was extracted with a mean yield of 7.1 microg/pellet. Polymerase chain reaction amplification of wild-type and knockout alleles for Fc receptor and factor VIII genes was similar, comparing stool and peripheral blood as the source of genomic DNA. Definite genotype identification was achieved in a first attempt in 336 of 352 analyses (95%). Repeated analysis of homozygous knockout animals confirmed the first result in all cases. No animal was lost due to bleeding from the procedure. In conclusion, DNA isolation from stool is a preferable method for genotyping in laboratory animals, especially in models of bleeding disorders. Avoiding loss of animals due to bleeding implies a substantial improvement in animal welfare by reducing the number of animals used and may also advance the effectiveness of breeding programmes in these disease models.

CD32 inhibition and high dose of rhFVIII suppress murine FVIII-specific recall response by distinct mechanisms in vitro.

Development of neutralising antibodies (inhibitors) against factor VIII (FVIII) is a frequent and severe complication of replacement therapy in haemophilia A. Previous data from haemophilia A mouse model demonstrates that both CD32 inhibition and high doses of rhFVIII prevent the differentiation of FVIII-specific memory B cells (MBCs) into antibody secreting cells (ASCs). Here, cellular targets responsible for the suppression of ASC formation by means of CD32 inhibition and high dose of rhFVIII were analysed. We investigated apoptosis on FVIII-specific MBCs using a pan caspases inhibitor, and screened for defects in rhFVIII presentation by analysing T cell release of Th1- and Th2-cytokines in vitro. Although high dose of rhFVIII suppressed ASC formation, cytokine response was not affected. Upon re-stimulation of splenocytes with high dose of rhFVIII, prevention of apoptosis fully restored the FVIII-specific recall response. In contrast, genetic deletion or inhibition of CD32 significantly altered Th1- and Th2-response. CD32 blockade and inhibition of apoptosis resulted in a partial rescue of FVIII-specific ASCs. Normal cytokine secretion could not be restored. In conclusion, suppression of FVIII-specific recall response by CD32 and high doses of rhFVIII is mediated by distinct mechanisms. High dose of rhFVIII induces apoptosis in FVIII-specific MBCs but does not influence FVIII-specific T cell response. CD32 blockade, however, may suppress the FVIII-specific recall response by two ways: i) increasing apoptosis of FVIII-specific MBCs and ii) disturbing FVIII-specific T cell response by modulating presentation of rhFVIII to CD4+ T cells in vitro.