FVIII Levels Research Articles

Prolonged α-thrombin-related activation and delayed active protein C-associated degradation confer mild phenotype in a patient with severe hemophilia A with F8 p.H118R.

In hemophilia A, bleeding mostly correlates with factor VIII activity (FVIII:C), although some patients show discrepancy in bleeding severity and FVIII:C. We report a novel procoagulant mechanism associated with F8 p.H118R (c.353A > G) in a young Japanese man with few bleeding episodes despite low levels of FVIII:C (< 1IU/dL). Plasma FVIII:C was < 1IU/dL measured by one-stage clotting assay (OSA) and chromogenic substrate assay (CSA), whereas FVIII antigen (FVIII:Ag) was 9.7%. The global coagulation assay showed higher max speed in clot waveform analysis (CWA), shorter clotting time in rotation thromboelastometry (ROTEM) (1605 vs. > 5000s), shorter lag time (4.87 vs. 12.47min) and larger ETP (207.9 vs. 53.3nM*min) in thrombin generation assay, compared with FVIII-deficient control. Expressed recombinant H118R mutant in culture media showed low FVIII:C (1-5IU/dL) by OSA, with non-hemophilia level of FVIII:Ag. Western blot analysis using recombinant H118R showed longer persistence of heavy-chain of H118R after incubation with α-thrombin, compared with wild-type. Incubation of H118R with activated protein C (APC) also showed longer persistence of A1-A2 domain. In conclusion, H118R showed prolonged activation by α-thrombin and delayed APC-related FVIII degradation. These properties may confer the procoagulant activity and few bleeding episodes despite low FVIII:C.

Screening of the protein C pathway abnormality-related thrombophilia by using thrombomodulin-mediated tissue factor-triggered clot waveform analysis.

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. 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. 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. rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

Clinical application of extended half-life factor VIII in children with severe haemophilia A.

Only few studies have presented results from real-world clinical use of Extended Half-Life (EHL) products in children with haemophilia (CWH). To retrospectively examine real-life experience with EHL factor VIII products use in CWH A, comparing with clinical experience from standard half-life products (SHL). A retrospective review of medical records of CWH A who have been prescribed EHL factor concentrates was conducted. All before/after comparisons were performed with the Wilcoxon matched-pairs signed-ranks test. Twenty-three children with severe haemophilia A were enrolled in the study (3-6 years old: n=4, 7-12 years old: n=7 and 13-18 years old: n=12). Median length of time that patients were treated with EHL products was 78weeks. Median dosing interval was significantly lengthened from 2.3 to 3.5days after switching from SHL to EHL concentrates. Mean trough FVIII levels were significantly increased from 2.3% to 4.1% after treatment with EHL products. Also, CWH A had a reduction of mean annual bleeding rate (ABR) and mean annual joint bleeding rate (AJBR) from 1 and .8 to .3 and .2, respectively, following treatment with EHL concentrates (ABR: p=.02, AJBR: p=.05). However, after switching to factor EHL, actual FVIII consumption, including bleeds, was significantly increased from 94IU/kg/week to 118IU/kg/week in CWH A. There was no inhibitor development. This study demonstrates the successful transition of 23 CWH A from SHL to EHL factor concentrates.

Management and outcomes of mild hemophiliacs and hemophilia carriers during pregnancy and peripartum period: a hemophilia treatment center experience in the United States

Background Pregnancy, peripartum management, and outcomes of mild hemophiliacs and hemophilia carriers in the United States are not well established. Aim To describe the management and outcomes of mild hemophiliacs and hemophilia carriers during assisted conception, pregnancy, peripartum and post-partum period at our hemophilia treatment center (HTC). Methods Retrospective review of electronic medical records of pregnant women with mild hemophilia A or B (Factor VIII [FVIII] or Factor IX [FIX] level <0.4 IU/mL) and hemophilia A and B carriers followed at our HTC from January 2008 to October 2020. Demographics, the reason for diagnosis, FVIII and FIX levels at baseline and third trimester, bleeding phenotype and genotype were obtained. Method of conception, factor replacement, iron supplementation, mode of delivery, type of anesthesia, peripartum complications, and offspring outcomes was recorded. Results There was a total of 18 pregnancies in 12 women (2 with mild hemophilia A, 2 mild hemophilia B, 6 hemophilia A carriers, and 2 hemophilia B carriers). Eleven pregnancies (61%) were conceived naturally and 7 (39%) via in-vitro fertilization (IVF). Eight (44.4%) and 10 (55.6%) pregnancies were vaginal and C-section deliveries, respectively. Neuraxial anesthesia was administered in 17 (94.4%) deliveries without complications. Four pregnancies (22.2%) had bleeding complications, 2 of which were post-partum hemorrhages not requiring transfusion. Conclusion In our case series of pregnant hemophilia carriers and mild hemophiliacs, successful outcomes were achieved with a carefully detailed multidisciplinary-driven approach.

Global Seroprevalence of Pre-existing Immunity Against AAV5 and Other AAV Serotypes in People with Hemophilia A.

Adeno-associated virus (AAV)-mediated gene therapy may provide durable protection from bleeding events and reduce treatment burden for people with hemophilia A (HA). However, pre-existing immunity against AAV may limit transduction efficiency and hence treatment success. Global data on the prevalence of AAV serotypes are limited. In this global, prospective, noninterventional study, we determined the prevalence of pre-existing immunity against AAV2, AAV5, AAV6, AAV8, and AAVrh10 among people ≥12 years of age with HA and residual FVIII levels ≤2 IU/dL. Antibodies against each serotype were detected using validated, electrochemiluminescent-based enzyme-linked immunosorbent assays. To evaluate changes in antibody titers over time, 20% of participants were retested at 3 and 6 months. In total, 546 participants with HA were enrolled at 19 sites in 9 countries. Mean (standard deviation) age at enrollment was 36.0 (14.87) years, including 12.5% younger than 18 years, and 20.0% 50 years of age and older. On day 1, global seroprevalence was 58.5% for AAV2, 34.8% for AAV5, 48.7% for AAV6, 45.6% for AAV8, and 46.0% for AAVrh10. Considerable geographic variability was observed in the prevalence of pre-existing antibodies against each serotype, but AAV5 consistently had the lowest seroprevalence across the countries studied. AAV5 seropositivity rates were 51.8% in South Africa (n = 56), 46.2% in Russia (n = 91), 40% in Italy (n = 20), 37.2% in France (n = 86), 26.8% in the United States (n = 71), 26.9% in Brazil (n = 26), 28.1% in Germany (n = 89), 29.8% in Japan (n = 84), and 5.9% in the United Kingdom (n = 17). For all serotypes, seropositivity tended to increase with age. Serostatus and antibody titer were generally stable over the 6-month sampling period. As clinical trials of AAV-mediated gene therapies progress, data on the natural prevalence of antibodies against various AAV serotypes may become increasingly important.

Improved clinical outcome after PK-Guided Personalised Prophylaxis with my-PKFIT® in patients with hemophilia A without inhibitors

Objective: Prophylaxis is the gold standard in patients with severe hemophilia. In recent years, personalisation of prophylaxis treatment according to pharmacokinetic properties has been used in treatment. In this study, personalisation treatment experience based on the pharmacokinetic dosing tool my-PKfit results in pediatric and adult patients from three centers is shared. Material and Methods: myPKfit (www1.mypkfit.com) was used to evaluate pharmacokinetic parameters in hemophilia A patients receiving recombinant Factor VIII (Takeda Advate ®) prophylaxis. 75 samples in 34 patients (3 samples in 7 patients, 2 samples in 27 patients) were analysed for pharmacokinetic evaluation. Age, weight and baseline FVIII level of the patients were recorded. Pharmacokinetic curves were obtained after entering sampling times, factor dose and sample results. The annual bleeding rate (ABR) of the patients were evaluated before and after the changes made after the pharmacokinetic evaluation. Results: The median age of 34 patients with severe hemophilia A without inhibitors was 12.3±8.7 (1.5-37) years, and the mean weight was 40.0±22.0 (10-83) kg. All patients had a baseline FVIII level of less than or equal to 2 IU/dl. All patients were receiving primary or secondary/tertiary prophylaxis. The mean half-life of the factors of the patients was 9.6±1.4 (7.0-13.4) hours, and the mean time reached below 1 IU/dl was 48.9±11.2 (16.0-77.0) hours. Prophylactic factor therapy was changed in 17 patients after myPKfit, dose increased in 9 patients, the frequency increased in 6 patients, and both dose and frequency increased in 2 patients. With a mean follow-up period of 23.7 +16 (2-49) months, in 17 patients whose prophylaxis regimen was changed after the PK evaluation by myPkyfit, ABR was found to be significantly lower in the post-change period, compared to the last one year before the change of regimen (2.94 + 2.19 and 0.58 + 1.00 respectively) P: 0.028. Discussion: A pharmacokinetic study by the Bayesian method is an increasingly used method for personalised prophylaxis regimen. We believe that myPKfit is beneficial in providing effective and appropriate prophylaxis.

Immunostimulation and Coagulopathy in COVID-19 Compared to Patients With H1N1 Pneumonia or Bacterial Sepsis

Multiple reports from all over the world link COVID-19 with endothelial/coagulation disorders as well as a dysregulated immune response. This study tested the hypothesis that immunostimulation will be greater in COVID-19 patients than in patients with H1N1 infection or bacterial sepsis. Also, whether an increase in immune stimulation will be accompanied by a more severely affected endothelium/coagulation system was examined.Twenty-three septic patients, admitted in the Intensive Care Unit (ICU), were enrolled (9 with SARS-CoV-2, 5 with H1N1 pneumonia, 9 with bacterial sepsis). Myeloperoxidase (MPO) activity along with certain endothelial/coagulation factors were assessed on admission (time point 1) and at either improvement or deterioration (time point 2).MPO levels were significantly higher in COVID-19 patients compared to both other groups. Furthermore, in patients with COVID-19, vWF levels did not differ significantly, fVIII levels were lower while ADAMTS-13 activity was higher compared to patients with H1N1 pneumonia and bacterial sepsis (a trend in the latter).Increased immunostimulation was noted in COVID-19 patients compared to other septic patients; however, this was not accompanied by greater disturbance of the clotting system and/or more severe endothelial injury.

Relationship Between Elevated FVIII Level and 3' UTR Variations of F8 Gene in Turkish Patients with Venous Thrombosis

Relationship Between Elevated FVIII Level and 3' UTR Variations of F8 Gene in Turkish Patients with Venous Thrombosis

Laboratory methods for monitoring argatroban in heparin-induced thrombocytopenia.

The Summary of Product Characteristics for the direct thrombin inhibitor argatroban states monitoring should be by activated partial thromboplastin time (APTT), with a target range of 1.5-3.0 times the patients' baseline APTT. APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Previous studies have shown sensitivity differences of APTT reagents to argatroban. Some recent publications have favoured the use of anti-IIa methods to determine the plasma drug concentration of argatroban. This study aims to compare the anti-IIa assays: Hemoclot thrombin inhibitor assay (HTI) and Ecarin chromogenic assay (ECA) alongside the APTT. Residual plasma of 25 samples from 8 patients (3 with COVID-19 and HIT: n=18, 5 with HIT: n=7) was tested at two sites: site A: Sysmex CS5100 by HTI and APTT (Actin FS and SynthASil), and also on Stago STA Compact Max: ECA and APTT (CK Prest); and site B: Stago STA R Max 2 by ECA and APTT (Cephascreen). Mean APTT ratio was 1.96 (Actin FS), 1.84 (SynthASil), 1.59 (CK Prest) and 2.48 (Cephascreen). Mean argatroban concentration by HTI was 0.60µg/mL and by ECA was 0.65µg/mL (site A) and 0.70µg/mL (site B). There was a poor correlation to HTI in APTT ratios (range r2 =.0235-0.4181) with stronger correlations between ECA methods to HTI (r2 =.8998 site A, r2 =.8734 site B). This study confirms previous publications on the unsuitability of APTT and expands this by being multisited and included APTT reagents that use mechanical clot detection. Both anti-IIa methods are more suitable.

Emicizumab Prophylaxis in Persons with Mild or Moderate Hemophilia A: Results from the Interim Analysis of the HAVEN 6 Study

Emicizumab Prophylaxis in Persons with Mild or Moderate Hemophilia A: Results from the Interim Analysis of the HAVEN 6 Study

Hemostatic Markers, Adamts-13 Profile and Anti-Sars-Cov-2 Antibody Levels in Patients with Immune Thrombotic Thrombocytopenic Purpura Receiving BNT162b2 Vaccination

▪Introduction: Patients (pts) with immune thrombotic thrombocytopenic purpura (iTTP) are at high risk of severe COVID-19, therefore protection from SARS-CoV-2 by vaccination is particularly relevant in this setting, although concerns may exist on possible adverse reactions or disease relapse after vaccination. In this study, in a group of iTTP pts who received in-hospital COVID-19 vaccination in a special program for ‘fragile patients’, we prospectively evaluated over time the antibody response, the clinical and laboratory disease parameters and hemostatic biomarker levels.Methods: Twelve iTTP pts in clinical remission and regularly followed-up in our Center were enrolled in April 2021, all of them received 2 doses of BNT162b2 vaccine (Pfizer-BioNTech) over 21 days, and were followed-up for clinical and laboratory testing for 60 days. Blood samples were collected at enrollment (day 0, D0) before the 1 st vaccine dose; on day 21 (D21) before the 2 nd dose; and on day 60 (D60) after the 1 st dose. Blood cell counts, anti-Spike receptor-binding-domain protein (anti-S/RBD) IgG, ADAMTS-13 activity, and anti-ADAMTS-13 IgG (chromogenic assay and ELISA), were measured at each time point. Additionally, an extensive study of hemostatic markers (i.e. FVIII, von Willebrand Factor (vWF) antigen and activity, fibrinogen, D-dimer, tPA, PAI, and F1+2) was performed. Follow up is currently continuing.Results: Median age of our cohort was 65 years with M/F ratio of 4/8. Median time since last acute iTTP episode was 40 months, median follow up of the cohort was 71 months (95% CI 30-126). All pts were in clinical remission, except one patient (P1) who had an iTTP relapse after contracting SARS-CoV-2 infection, in Dec 2020, and was on low-dose steroids on D0. One patient (P2) had an ADAMTS-13 relapse in Jan 2021, and received pre-emptive rituximab. No other pts were on immunosuppressive therapy. Concerning the status of ADAMTS-13 activity on D0, 6 pts showed normal levels (>50%), while 5 had a moderate (50-20%) and 1 a complete (<10%) ADAMTS-13 deficiency. This latter patient (P3) had normal ADAMTS-13 activity before the pandemic. All patients were negative for anti-ADAMTS-13 inhibitor. Further, on D0, the anti-S/RBD IgG testing was positive in 3/12 pts (median 704,1 AU/mL), due to symptomatic infection in 1 case (P1), and asymptomatic in 2 (P3 and 1 pt with ADAMTS-13 activity of 54%, P4).The study of hemostatic markers on D0 showed an increase in median levels of FVIII and vWF antigen and activity. These parameters were altered in 7/12, 11/12 and 8/12 pts, respectively. Fibrinogen and D-dimer were increased in 3/12 and 2/12, respectively. Notably, P1, P3 and P4 presented the highest levels of FVIII and vWF antigen, associated with high levels of vWF activity in P1 and P3 (mean 233%); moreover, P3 showed higher levels of D-dimer (708 ng/mL) and tPA (13 ng/ml).After the 2 doses of BNT162b2, no significant clinical side effects were reported, and no changes in platelet counts. ADAMTS-13 activity and inhibitors did not significantly change on D21 and D60. A complete ADAMTS-13 activity deficiency persisted in P3 on D21 and D60, associated with anti-ADAMTS-13 IgG titer >15 U/ml, despite clinical remission. Overall, a significant increase in anti-S/RBD IgG level was observed on D21 (p = 0.0005) and D60 (p = 0.0005). Remarkably, only P2 did not show an increase in anti-S/RBD IgG titer after both doses of BNT162b2. Median levels of FVIII and vWF antigen did not significantly change during follow up, while increased vWF activity was seen on D60 (p = 0.05). Fibrinogen levels were stable, and an increase in D-dimer (>1000 ng/mL both on D21 and D60) was seen in P3. There were no changes in the other hemostatic parameters, and no thromboses were observed.Conclusions: In our cohort of iTTP pts, COVID-19 was associated with 1 clinical and 1 ADAMTS-13 relapse. Our data show that SARS-CoV-2 vaccination was effective in inducing an antibody response in all but one patient who received rituximab within 3 months before vaccination, confirming recent findings. Overall, vaccination had no relevant impact on the hemostatic profile of our pts, and did not appear to be a driver of iTTP relapses. However, anti-SARS-CoV-2 antibodies monitoring in iTTP pts may be useful after vaccination, as currently it is unknown how long the antibody titer may persist. Although small, this study is in favor of efficacy and safety of mRNA vaccines in pts with iTTP. DisclosuresFalanga: Bayer: Honoraria; Sanofi: Honoraria; Leo Pharma: Honoraria; Pfizer: Honoraria.

Optimization of Thrombin Generation in Hemophilia a

Background: Hemophilia A (HA) is a bleeding disorder characterized by decreased or absent FVIII. Clinical analysis of coagulation potential in this patient population is classically based on APTT based FVIII assays. Although both the one-stage FVIII assay and the chromogenic FVIII assay can measure FVIII concentrations reliably these types of assays only give insight on the initiation of coagulation. Global coagulation assays, like thrombin generation (TG), can be used to measure the full coagulation spectrum of initiation, amplification and propagation. However the frequently used commercially available TG kits lack sensitivity for measurements of hemophilia plasma within the lower FVIII ranges which are essential in explaining differences in bleeding phenotype.Aim: We aim to optimize the sensitivity of the TG-assay for measurements in hemophilia A patients, especially in the lower FVIII ranges.Methods: In order to minimize patient specific sensitivity a hemophilia A pool plasma (HAPP) was created. Analysis of the influence of pre-analytical variables, like contact activation inhibitors, on the assay was performed. Initiation of coagulation by different reagents was compared for sensitivity towards factor FVIII titrations in patient plasma. Other assay variables like phospholipids and temperature were adjusted to increase sensitivity even further.Results: Commonly used tissue factor (TF) initiated TG at varying concentrations was unable to significantly differentiate in FVIII levels below 20%. In contrast, TG activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. Additionally, a representative baseline TG-curve in severe HA plasma could only be produced using this dual TF/FXIa-activation. There was a value in the addition of contact activation inhibitors in the assay. Higher phospholipid concentrations seem to benefit this assay setup compared to a TF only setup.Conclusion: TF/FXIa dual activation thrombin generation increased assay sensitivity in severe hemophilia plasma, allows for dose-dependent measurements in low FVIII ranges and provides a solid baseline curve that can be used for further clinical evaluation of coagulation potential and possibly therapeutic monitoring in hemophilia A. [Display omitted] DisclosuresHackeng: ACS Biomarker BV: Current Employment, Current equity holder in publicly-traded company; Coagulation Profile BV: Current Employment, Current equity holder in publicly-traded company.

Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?

Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients.Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels.Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower.On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups.In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03).Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE.Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. [Display omitted] DisclosuresKruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.

Acquired Hemophilia a Relapse Is Related to Th2 Response, and Increased Expression of B-Cell Activating Factor (BAFF)

Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by the development of autoantibodies against the factor (F)VIII of coagulation with higher incidence in elderly individuals. The treatment includes immunosuppressive alternatives, and the majority of patients reach complete remission of symptoms. Although the role of immune T and B cells in the physiopathology of several autoimmune diseases is widely established, few studies have explored the T and B cells immune response pathway in AHA. This study aimed to determine the role of T cells through their cytokines expression and B cells through their expression of proteins that activate the B cell receptor (BCR), such as B-cell activating factor (BAFF) and A-proliferation inducing ligand (APRIL), in a longitudinal evaluation of patients with AHA.We included in this study 11 patients, 4 (36%) male, with the diagnosis of AHA, based on the occurrence of low FVIII levels (median FVIII level at the diagnosis was 0.10 IU/dL; range 0-18.8) and anti-FVIII inhibitor titer positive (≥0.6 Bethesda units (BU)/mL) (median inhibitor titer at the diagnosis was 60BU/mL; range 7.6-1000.2). The median age at the diagnosis was 59 years (range 9-75). Idiopathic AHA was evident in 72.7% of cases, while autoimmune diseases (18.2%), and pregnancy (9.1%) were the other underlying etiologies. All patients received immunosuppressive therapy (IST). Eight (72.7%) patients used corticosteroid (CS) plus cyclophosphamide (CP), one (9.1%) received only CP, one (9.1%) used CS plus rituximab and one (9.1%) received CS, CP and three cycles of rituximab. The response to IST was defined as complete remission (CR) with anti-FVIII < 0.6BU/mL and FVIII >50IU/dL, without further IST. Relapse was defined when anti-FVIII titer became >0.6BU/mL after previous remission. In this cohort, we observed 64% of sustained CR and 4(36%) AHA patients had at least one relapse, including two AHA patients considered idiopathic and two with other autoimmune diseases associated.Peripheral blood mononuclear cells (PBMC) were collected from all AHA patients at the diagnosis previously IST (baseline), when patient achieved CR, and at the occurrence of relapse. PBMCs were maintaining frozen in liquid nitrogen. Cells were taw and cultivated with RMPI-1640 medium in 7.5x10 5 cells/well in a 48-well plate. After 24h, cells were stimulated with a 1IU/well of a full-length recombinant FVIII concentrate (rFVIII) or phorbol myristate acetate (PMA) (1µM) with ionomycin (1µM). After 24h incubation, cells were analyzed by flow cytometry at BD FACSCalibur™.To determine T CD4 + cells' cytokine production profile, we stained T cells with anti-CD4, anti-interleukins (IL)-17a, IL-4, and IL-21, anti-TGF-b, and anti-TNF-a. We observed a significant increase of IL-17a production in the rFVIII-stimulated cells isolated at baseline from all patients compared to cells isolated from healthy individuals (P=0.01). However, IL-17a expression at baseline was not different between cells from patients who sustained CR to those who relapsed. Interestingly, when we compared cells isolated at baseline from patients that relapsed after different immunosuppressive attempts, we observed a significant difference for IL-4 (P=0.009), IL-21 (P=0.02), and TGF-b (P=0.01) expression in comparison to cells from healthy individuals. However, this difference was not observed in cells from sustained CR vs. cells from healthy individuals (figure 1). Thus, suggesting that all patients present Th17 response, while only patients that relapsed also showed Th2 response at the diagnosis.We also evaluated B cells' expression of BAFF and APRIL. The cells were stained with anti-CD19, BAFF, and APRIL. Only cells isolated at baseline from AHA relapsed patients after rFVIII stimulation or without any stimuli presented increased expression of BAFF (P=0.01 with rFVIII-stimulated and P=0.007 for unstimulated cells) when compared to PBMCs isolated from healthy individuals (figure 1). BAFF is responsible for controlling B-cell maturation and is associated with autoantibody production of different autoimmune conditions.Our finds reveal an increase in the Th17 response in patients with AHA at the diagnosis. However, the presence of Th2 response and increase of BAFF expression was observed only at the diagnosis of patients with recurrence of autoimmune response to FVIII, which suggests a potential biomarker for AHA evaluation. [Display omitted] DisclosuresOzelo: BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Other: Grant review.

Evaluation of Global Coagulation Tests for Monitoring Bleeding Phenotypes and Response to Treatments in FVII Deficiency

Evaluation of Global Coagulation Tests for Monitoring Bleeding Phenotypes and Response to Treatments in FVII Deficiency

Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the ‘cytokine storm’ resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program.Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer.Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group.Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups.Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. DisclosuresGligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

Flow-Cytometry Evaluation of Cellular Surface FVIII in Blood Cells and Its Correlation with FVIII Activity and Coagulation

FVIII deficiency or inactivity leads to coagulation malfunction known as Hemophilia A (HA). FVIII overactivity on the contrary is commonly linked to a hypercoagulability state. In steady state, FVIII remains quiescent by forming a non-covalent complex with vWF, and upon coagulation cascade activation, thrombin liberates active FVIII (FVIIIa) from vWF. FVIIIa binds to the plasma membrane of the platelets and other blood cells via its C2 domain phospholipids binding motif to exert its coagulative role.Recently, we developed a reliable Flow-Cytometry (FC) platform for FVIII protein detection (Elnaggar M. et al, 2020). The assay is suitable to measure both intracellular and extracellular FVIII. When applied to human blood it showed that by overexpressing FVIII through a lentiviral-vector-based transduction, a high proportion of circulating FVIII is bound on leukocytes membranes. Leukocyte abundance is reported to be highly associated with thrombotic events but with no clear mechanistic explanation. Therefore, we sought to evaluate the percentage of FVIIIa bound on different leukocyte subsets (T cells, B cells, monocytes and granulocytes) in different clinical scenarios, including HA and other abnormal coagulation states and to correlate it to the plasmatic levels of FVIII.The analysis was conducted on 31 pediatric/young-adult subjects (median age 9y), including patients diagnosed with HA (n=8), patients with coagulopathies (von Willebrand Disease, menorrhagia and unexplained bleedings (n=11) and 1 patient with known high levels of FVIII), and patients without coagulopathies/healthy controls (n=11) referred to Hematology/Oncology clinic at Sidra Medicine-Qatar.Fresh blood samples were analyzed with a Sysmex XN1000 Hematologic Counter for complete blood count and with a BD-Symphony FC for the following surface markers: CD3/CD4/CD8/CD14/CD16/CD19/CD33/Live/Dead and for FVIII protein A2 domain (Ab clone GMA8024), after light-chain Zenon-labeling, with proper IgG controls, as described in the publication above. In parallel, plasma FVIII measurements were performed for clinical diagnostic purposes, and results were correlated.Expectedly, HA patients showed the lowest FVIII percentage in plasma (p<0.0001), while leukocytes' surface FVIII was generally low (on average <1%), trended to a lower rate in patients with coagulopathies and HA compared to healthy-controls and was significantly higher in the patient with hypercoagulability (p=0.0007). Surprisingly, among leukocytes' subpopulations, screened on patients with bleeding diathesis and HA subjects vs healthy-controls, only CD8-surface bound FVIIIa was significantly lower compared to healthy-controls (p=0.015 healthy-controls vs HA, and p=0,0093 healthy-controls vs bleeding diathesis). These results raise the possibility of a CD8-cell mediated controlled binding mechanism regulating FVIII availability in case of biological need of the protein, as bleeding.We did not find any significant correlation between leukocyte-surface bound FVIII and plasma FVIII level, and this further indicates that the differences in FVIIIa leukocyte surface binding are not directly proportional to FVIII availability in the circulation, rather actively regulated by a biological still unknown mechanism.While the study is still ongoing and recruiting other subjects with hypercoagulation state, we showed in this preliminary analysis that our in-house FC platform for FVIII protein detection screens in depth FVIII protein adherence to blood cells, may shed light on coagulation novel mechanisms, and potentially serve as a diagnostic and prognostic tool. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effectiveness of Monthly Low-Dose Emicizumab Prophylaxis without 4-Week Loading Doses Among Patients with Hemophilia a with and without Inhibitor: A Case Series Report

Introduction: Emicizumab is a humanized, bispecific, monoclonal antibody proven as an effective prophylaxis for patients with hemophilia A with and without inhibitor. After the weekly loading of 3 mg/kg for 4 doses followed by maintenance dose of 1.5 to 6 mg/kg at 1 to 4-week intervals, the equivalent factor VIII was maintained at approximately 15% and showed an effective prophylaxis in reducing annual bleeding rate. Due to the health care resource constraint, the dose of emicizumab was reduced to maintain the prophylaxis factor VIII at 1-3%.Methods: The effectiveness of monthly low dose emicizumab prophylaxis without 4 loading doses was evaluated among patients with hemophilia A with and without inhibitor for at least 6 months. The bone mineral density (BMD), ultrasonography of bilateral ankle, elbow and knee as well as Hemophilia Joint Health Score (HJHS) were initially evaluated and planned to be repeated after one year treatment. The vitamin D level was determined initially, and vitamin D supplement was prescribed for 3 patients with low vitamin D levels at 13.3, 23.2 and 24.6 ng/mL. Moreover, the quality of life assessment using Hemo-QoL and CHO-KLAT was performed in 3-month intervals.Results: Five patients without inhibitor (3 severe, 2 moderate) and 1 patient with severe hemophilia A with inhibitor enrolled in the study. They all behaved in low bleeding risk circumstances such as avoidance of contact sport. Their ages ranged from 4 to 40 years of age. The youngest boy had difficulty at peripheral venous access for regular prophylaxis while the remaining 4 patients without inhibitor experienced frequent breakthrough bleeding episodes while receiving a low dose prophylaxis at 500 to 1,000 units of factor VIII concentrate twice weekly. All refused to receive more frequent prophylaxis. For patient with inhibitor, the high inhibitor titer of 65 Bethesda units (BU) declined during the 2 years of immune tolerance induction (ITI) at the intermediate dose of 100 units/kg of extended half-life factor VIII concentrate 3 times weekly. His lowest inhibitor was at 10 BU and was documented as failed ITI. He experienced frequent bleeding at the muscles and joints for which bypassing agents could not be adequately provided.For the baseline study, 2 patients had low BMD Z score ≤-2.0 while the remaining 4 patients had normalized BMD Z score >-2.0. The ultrasonography of bilateral ankle, elbow and knee at anterior, medial, lateral and posterior/inferior spaces revealed joint distension reflecting varying-degree synovial hypertrophy and/or bleeding component of 0-14.0, 0-9.8, 0-9.4, 0-12.8; 0-14.4, 0-17.8, 0-10.7, 0-18.6 and 0-23.8, 0-15.3, 0-17.1, 0-12.9 millimeter, respectively. Varying-degree hyperemia from the ultrasonography reflecting active inflammation of synovium was found in 4 ankles, 6 elbows and 4 knees of the total 36 joints in 4 of 6 patients. The HJHS scores ranged from 6 in the youngest patient to 56 in the patient with inhibitor. All patients except the 4-year-old boy had 1 to 3 target joints.All the studied patients received 1 each of the whole vial of emicizumab at 30, 75, 105 mg monthly except 3 patients receiving 60 mg monthly which was equal to 1.1 to 1.6 mg/kg of emicizumab. The monthly trough levels of emicizumab determined by a modified one stage factor VIII assay using emicizumab calibrator were maintained at 3.8 to 9.8 µg/ml which were equivalent to the levels of FVIII at 1 to 3% (0.3% FVIII per µg/ml of emicizumab). The bleeding rate was markedly decreased from 4-8 episodes monthly to 0-1 episode monthly. The monthly zero bleeding rate was found among 4 patients. Moreover, the swollen target joint gradually dissolved. Their quality of life markedly improved evaluated by the Hemo-QoL and CHO-KLAT. They could attend regular school and work happily. The HJHS and ultrasonography have not been repeated yet.Conclusion: The monthly low dose emicizumab prophylaxis of the whole vial at 1.1 to 1.6 mg/kg without loading dose could achieve emicizumab trough levels at 3.8 to 9.8 µg/ml which were equivalent to the levels of FVIII at 1 to 3%. It showed effective prophylaxis among patients with hemophilia with and without inhibitor who performed low bleeding risk activity. The 3 aspects of body functions and structures, activities and participation were gradually improved. DisclosuresNo relevant conflicts of interest to declare.

In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion

We have previously reported that in utero transplantation (IUTx) of sheep fetuses (n=14) with human placental cells (PLC) transduced with a lentiviral vector encoding mcoET3, an expression/secretion-optimized, bioengineered fVIII transgene (PLC-mcoET3) increased plasma FVIII activity levels by 57%, 42%, and 35% at 1, 2, and 3 years post-IUTx, respectively, without the development of FVIII/ET3 inhibitors. We also demonstrated that immune tolerance to the cell/gene product was maintained after postnatal administration of PLC-mcoET3 (cells producing 20 IU/kg/24h were administered i.v. for 3 consecutive weeks). However, when IUTx-treated animals received weekly i.v. infusions of purified ET3 protein (20IU/kg) for 5 weeks, all recipients developed a robust ET3-specific IgG response that appeared at week 3 of infusion at titers ranging from 1:70 to 1:857 and inhibitory antibodies that ranged from 3-36 BU. Here, we investigated differences in the immune responses of animals that received IUTx with PLC-mcoET3 and were boosted postnatally with PLC-mcoET3 (IUTx-PLC-mcoET3) vs. ET3 protein (IUTx-ET3) to define the pathways by which the immune system differentially responds to protein vs. cell-secreted ET3. A sheep-specific multiplex gene expression analysis with 165 genes involved in immune cell signaling pathways (NanoString) was used to evaluate mRNA isolated from peripheral blood mononuclear cells collected at Weeks (W) 0, 1, and 5 of postnatal infusions. Significant fold-change expression in these mRNA targets was determined using NanoString nSolver 4.0 software. Animals in the IUTx-PLC-mcoET3 group (known to be devoid of inhibitors to ET3 post-boosting) showed that immunoregulation and immune tolerance gene clusters were among the top three clusters that increased expression from W0 to W5 (adj. p-value<0.01). Differential expression of genes in pathways involved in Th1, Th2, and Th17 responses was also found, at differing levels, in the IUTx-PLC group, suggesting a balance between immunity and tolerance was maintained. Surprisingly, the IUTx-ET3 group, which developed inhibitory antibodies after ET3 boosting, also showed significantly increased expression of immune tolerance genes, and downregulation of Th1 and Th17 cell signaling, when evaluated by direct global significance score. Nevertheless, 66% of these animals had a significant upregulation of Th2 cell signaling by W1 vs W0. To determine if the increase in expression of immune tolerance genes was due to the IUTx treatment, we also evaluated a group of aged-matched, non-transplanted sheep that received ET3 protein under the same dose and schedule. Results from Gene Set Analysis (GSA) demonstrated significant upregulation of genes involved in interferon signaling, class I MHC antigen processing, and Th17 signaling in these animals, suggesting the potential involvement of Th17 cells in the immune response in this group. In conclusion, IUTx with PLC-mcoET3 induces the upregulation of genes associated with immune tolerance, providing an explanation for the long-lasting elevation in plasma FVIII levels in these animals in the absence of inhibitors. Nevertheless, despite the continued expression of tolerogenic genes, administration of purified ET3 protein to these IUTx recipients induced upregulation of Th2 signaling, a pathway that was not observed in animals that only received ET3 protein, demonstrating that the mechanism by which tolerance is broken in IUTx recipients differs from that by which an immune response to ET3 occurs in animals with no prior exposure. Of note is that animals that develop inhibitors by the Th17 pathway had considerably higher inhibitor titers than the IUTx recipients that responded to ET3 infusion by the Th2 pathway. These studies underscore the need for a more complete understanding of the mechanisms by which immune tolerance to FVIII develops during ontogeny. DisclosuresDoering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

Administration of FVIII-Expressing Human Placental Cells to Juvenile Sheep Yields Multi-Organ Engraftment, Therapeutic Plasma FVIII Levels and Alter Immune Signaling Pathways to Evade FVIII Inhibitor Induction

We have previously reported that normal juvenile sheep that received weekly intravenous (IV) infusions of human (n=3) or an expression/secretion-optimized, bioengineered human/porcine hybrid (ET3) FVIII protein (n=3) for 5 weeks (20 IU/kg) developed anti-FVIII inhibitory antibodies (10-116 BU, and IgG titers of 1:20-1:245) by week 3 of infusion. By contrast, the IV infusion, or IP administration, of human placental mesenchymal cells (PLC) transduced with a lentiviral vector encoding a myeloid codon-optimized ET3 transgene (PLC-mcoET3) to produce high levels of ET3 protein (4.9-6IU/10^6 cells/24h) enabled the delivery of FVIII without eliciting antibodies, despite using PLC-mcoET3 doses that provided ~20-60 IU/kg ET3 each 24h to mirror the amount of FVIII protein infused. In addition, we showed that the route of PLC-mcoET3 administration (IP vs IV) did not impact the resultant plasma FVIII levels, with animals in these two groups exhibiting mean increases in FVIII activity (quantified by aPTT) of 30.9% and 34.2%, respectively, at week 15 post-treatment. Here, we investigated whether the sites and levels of PLC-mcoET3 engraftment were dependent upon the route of administration and performed s sheep-specific multiplexed transcriptomic analysis (NanoString) to define the immune signaling pathways that thwarted FVIII/ET3 protein immune response when ET3 was delivered through PLC. Tissue samples were collected from various organs at euthanasia and RT-qPCR performed using primers specific to the mcoET3 transgene, to the human housekeeping transcript GAPDH, and to sheep GAPDH, to quantify PLC-mcoET3 tissue engraftment, and normalize the results. RT-qPCR demonstrated PLC-mcoET3 engrafted, in both IP and IV groups, in all the organs evaluated (liver, lung, lymph nodes, thymus, and spleen). Animals that received PLC-mcoET3 via the IP route displayed higher overall levels of engraftment than their IV counterparts. The spleen was the preferential organ of engraftment for both IP and IV groups (IP:2.41±1.97%; IV: 0.64±0.54%). The IP group exhibited significantly higher engraftment in the left lobe of the liver (IP: 1.36±0.35%; IV: 0.041±0.022%), which was confirmed by immunohisto-chemistry (IHC) with an antibody to the human nuclear antigen Ku80 and ImageJ analysis (IP:5.24±3.36%; IV: 0±0). Of note is that the IP route resulted in higher levels of engraftment in the thymus, while IV infusion yielded higher levels of PLC-mcoET3 in lymph nodes. Analysis of H&E-stained tissues demonstrated they were devoid of any abnormal histologic changes and exhibited no evidence of hyperplasia or neoplasia, supporting the safety of the cell platform, irrespective of the route of administration. To date, NanoString analysis of PBMC collected at day 0, week 1, and week 5 post-infusion demonstrated that animals who received FVIII protein had upregulation of UBA5 and BATF, genes involved in antigen processing and Th17 signaling pathways, respectively. Although both IV and IP recipients of PLC-mcoET3 also had an increase in BATF, the IV group exhibited upregulation of BTLA, a gene involved in immune-tolerance, and downregulation of NOTCH and DDL1, involved in T cell differentiation, as well as MAPK12 and PLCG1, genes involved in proinflammatory cytokine regulation and T signaling within the Th17 signature. In IP recipients, BTLA, NOTCH, and DLL1 were all downregulated. Since ET3-reactive Th 1 cells were not present in any of the treated animals, it is possible that the Th17 cells are responsible for the inhibitory antibodies seen in the juvenile sheep treated with FVIII/ET3 protein, while in animals receiving PLC-mcoET3, downregulation of genes involved in T cell differentiation and proinflammatory cytokine signaling keeps the immune system in check to avoid an immune response. DisclosuresDoering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.