Factor VIII Research Articles

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Genetic Variants Identified by Whole Exome Sequencing in a Large Italian Family with High Plasma Levels of Factor VIII and Von Willebrand Factor.

High plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain poorly known. In a large Italian family with high FVIII/VWF levels and thrombotic episodes, whole exome sequencing (WES) was performed on 12 family members to identify variants/genes involved in FVIII/VWF increase. Twenty variants spread over a 8300 Kb region on chromosome 5 were identified in 12 genes, including the low frequency rs13158382, located upstream of the MIR143/145 genes, which might affect miR-143/145 transcription or processing. The expression of miR-143/145 and VWF mRNA were evaluated in the peripheral blood mononuclear cells of six family members. Members with the variant (n = 3) showed lower levels of both miRNAs and higher levels of VWF mRNA compared to members without the variant (n = 3). An analysis of genetic and expression data from a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p-value = 0.023) in miR-143 in heterozygous (n = 35) compared to homozygous wild-type individuals (n = 386). This family-based study identified a new genetic variant potentially involved in VWF increase by affecting miR-143/145 expression.

Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System.

Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice's uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.

Acquired Isolated Factor VII Deficiency in Plasma Cell Dyscrasias: A Brief Presentation of Two Plasma-Cell-Leukemia-Related Cases and Review of Literature.

Acquired isolated factor VII (FVII) deficiency is a rare but important discovery in patients with plasma cell disorders with significant therapeutic and prognostic implications. The present analysis and review of cases reported in the literature is intended to highlight disease-related characteristics associated with this rare clotting defect, clinical manifestations and outcome, and potential underlying mechanisms, and to provide guidance on how to manage these patients in terms of prophylactic and therapeutic measures. The discovery of acquired FVII deficiency in a patient with multiple myeloma (MM) or monoclonal gammopathy of uncertain significance (MGUS) should prompt an evaluation for AL amyloidosis, particularly for amyloid hepatosplenic involvement, whenever not previously documented. Acquired FVII deficiency in patients with MM and AL amyloidosis is frequently associated with severe bleeding diathesis, also related to a number of concomitant predisposing factors, adversely affecting the outcome. The prompt institution of a rapidly acting therapy is crucial to prevent severe bleeding complications and positively impact outcome. Recombinant activated factor VII (rVIIa) may represent a useful supportive care measure, both in treating active bleeding and in the peri-procedural setting. However, further clinical experience is needed to optimize the therapeutic management of this rare disorder.

Mice possess a more limited natural antihuman factor VIII antibody repertoire than humans that is produced disproportionately by marginal zone B cells

BackgroundOne-third of patients with severe hemophilia A develop neutralizing antibodies to the factor VIII (FVIII) protein in response to intravenous replacement therapy. Patients may also generate natural, nonneutralizing antibodies to FVIII before FVIII exposure. These patients are at increased risk of developing neutralizing antibodies to FVIII. However, natural anti-FVIII antibodies are also present in healthy human donors. ObjectivesTo further characterize the natural antihuman (h) FVIII antibody repertoire in mice and humans. MethodsAn in-house ELISA was developed using a purified polyclonal immunoglobulin (Ig) standard to quantify anti-hFVIII Ig in cell culture supernatant or plasma from mice (wild-type and FVIII-/-) and adult human donors. ResultsAll naïve wild-type and FVIII-/- mice, as well as healthy human donors, possess natural anti-hFVIII antibodies. Mice only have natural anti-hFVIII IgM, which is present in germ-free mice, suggesting that they are germline encoded. Although murine marginal zone B cells (MZBs) contribute 44% to all circulating natural IgM, they contribute disproportionately to the anti-hFVIII IgM repertoire (82%). This naturally occurring murine MZB-derived IgM is not B-domain specific and is reduced by intravenously administered hFVIII, suggesting that it may form immune complexes immediately upon hFVIII administration. Natural anti-hFVIII antibodies of IgG, IgM, and IgA isotypes can be detected in adult human donors. There were increased levels of B-domain-favoring anti-hFVIII IgG in 14% of healthy donors, which were markedly different from the rest of the “low-titer” population. ConclusionsThere is a preponderance of natural anti-hFVIII antibodies in both mice and healthy adult human donors.

Molecular Genetic Analysis of Russian Patients with Coagulation Factor FVII Deficiency.

Coagulation factor VII (proconvertin) is one of the proteins starting the blood coagulation cascade. Plasma FVII concentration is regulated by different factors. A low level of FVII could also be a result of FVII deficiency (MIM# 227500), the rare autosomal recessive inherited disease caused by pathogenic variants in the F7 gene. The aim of this study was to describe a mutation spectrum of the F7 gene and genotype-phenotype relationship in patients with FVII deficiency in Russia for the first time. We studied the primary structure of the F7 gene of 54 unrelated patients with FVII deficiency by direct Sanger sequencing. Pathogenic variants in the F7 gene were detected in 37 (68.5%) of them. We identified 24 different mutations located mostly in the serine protease domain. Five pathogenic variants had never been reported before. A major mutation in the Russian population was c.1391delC (p. Pro464Hisfs*32), linked with rs36209567 and rs6046 functional polymorphisms, that is widely distributed in East Europe. As in other countries, the F7 genotypes poorly correlated with the severity of clinical manifestations but were quite well associated with FVII levels. Minor alleles of functional polymorphisms rs510335, rs5742910, rs561241, rs36209567, and rs6046 could also participate in the F7 genotype and influence FVII levels.

Rare factor deficiencies in children: A review of 23 cases from a single center

Objective: The prevalence of rare factor deficiency (RFD) is one in 500.000-2.000.000 in the general population. Different symptoms may occur from mild or moderate bleeding to severe and life-threatening bleeding. This study aimed to evaluate children with RFD in a single Turkish center.
 Materials and Methods: The records of children with RFD (Factor I, V, VII, X, XIII deficiency) were evaluated retrospectively.
 Results: Twenty-three cases (70% female) were reviewed. The mean age of patients was 9.52 years at review, and mean follow-up was 66.3 months. The most common factor (F) deficiencies were FVII (35%) and FX (35%). Parental consanguinity was present in 65%. The most common symptoms were mucocutaneous bleeding and epistaxis. Regarding treatment, fresh frozen plasma (FFP) was given to two patients, FXIII concentrate was given to one patient, and prothrombin complex concentrate (PCC) was given to two patients. Prophylaxis was started in patients with recurrent bleeding. Of the 16 receiving prophylaxis, three received FFP, seven received recombinant coagulation factor VIIa, and six received PCC.
 Conclusion: Treatment was given to a fifth of patients while nearly three-quarters received prophylaxis. As parental consanguinity was present in most of these patients, obtaining a detailed family history may aid in diagnosis.

Immunogenicity, safety, and efficacy of rurioctocog alfa pegol in previously untreated patients with severe hemophilia A: interim results from a phase 3, prospective, multicenter, open-label study

ABSTRACT Aim To determine the immunogenicity, safety, and efficacy of rurioctocog alfa pegol in previously untreated patients (PUPs) with severe hemophilia A (HA). Methods This prospective, phase 3 study (NCT02615691) was conducted in PUPs, or patients with ≤2 exposure days (EDs) prior to screening, aged <6 years with severe HA. The primary endpoint was incidence of factor VIII (FVIII) inhibitor development. This protocol-specified interim analysis was conducted after 50 patients had completed ≥50 EDs without developing FVIII inhibitors or had developed a confirmed inhibitor at any time. Results Of the enrolled patients, 59/80 (73.8%) received ≥1 dose of rurioctocog alfa pegol; 54 received prophylaxis, and 35 on-demand treatment. Incidence of inhibitor development was 0.19 (10/52). Total annualized bleeding rate (95% CIs) was 3.2 (2.0–5.0) for patients receiving prophylaxis and 3.2 (1.6–6.3) for on-demand treatment. Hemostatic efficacy of most bleedings was rated as ‘excellent’ or ‘good’ after 24 hours (122/131 [93.1%]) and at resolution (161/170 [94.7%]). Five patients received ≥1 dose of rurioctocog alfa pegol for immune tolerance induction (ITI) and 1 patient was defined as having ITI success. Thirteen patients experienced 14 treatment-related adverse events, including 10 cases of FVIII inhibitor development. Conclusion This is the first prospective study of rurioctocog alfa pegol for the treatment of PUPs with severe HA. Trial Registration This trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT02615691).

Hemophilia Gene Therapy: The End of the Beginning?

Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.

Midlife Hemostasis Measures, 20-Year Cognitive Decline, and Incident Dementia.

Blood concentrations of hemostatic factors affect thrombosis and bleeding diathesis and may contribute to cognitive impairment through modifiable vascular pathologies. Whether hemostasis, assessed in middle age, is associated with late-life cognitive impairment remains largely unknown in a community-dwelling population. Using data from 14,128 participants with cognitive function measurements in 1990-1992 from the Atherosclerosis Risk in Communities study, we assessed the associations of hemostasis measures with 20-year changes in cognitive performance and incident dementia. Activated partial thromboplastin time (aPTT) and level of fibrinogen, von Willebrand factor (VWF), factor VIII, factor VII, factor XI, d-dimer, and soluble thrombomodulin were measured in 1987-1989 or 1993-1995. Hemostasis measures were categorized into quintiles, with the lowest quintile indicating low coagulability. Cognitive performance was characterized using a combined z-score from 3 tests (that is, delayed word recall test [DWRT], digit symbol substitution [DSST], and word fluency test [WFT]), assessed in 1990-1992, 1996-1998, and 2011-2013. Dementia was determined either from in-person evaluations or using dementia surveillance through 2017. Mixed-effects models and Cox proportional hazards models were used to assess cognitive trajectories and risk of dementia, respectively. Among 12,765 participants with hemostasis measures in 1987-1989, who were aged 47-70 years at the first cognitive assessment, we observed significant trends of shorter aPTT (p for trend <0.001; difference in 20-year cognitive decline for fifth vs first quintile [Q5 vs Q1]: -0.104 [95% CI -0.160 to -0.048]) and higher levels of factor VII (p < 0.002; Q5 vs Q1: -0.085 [-0.142, -0.028]) and factor VIII (p = 0.033; Q4 vs Q1: -0.055 [-0.111, -0.000]) with greater 20-year cognitive declines. The associations with the decline in DSST were stronger than those with the decline in WFT or DWRT. Consistently, shorter aPTT and higher factor VIII levels were associated with higher dementia risk with HRs for Q5 vs Q1 of 1.23 (95% CI 1.07 to 1.42) and 1.17 (1.01-1.36), respectively, and p for trend of 0.008 and 0.024, respectively. Overall, our study found consistent trend associations of aPTT and factor VIII measured in midlife with cognitive decline and incident dementia over 20 years, likely driven by vascular pathologies.

Differences in coagulation-relevant parameters: Comparing cryoprecipitate and a human fibrinogen concentrate.

Variable fibrinogen content within cryoprecipitate makes accurate dosing challenging in patients with coagulopathic bleeding, in addition to pathogen transmission risks associated with its administration. Purified and standardized human fibrinogen concentrates (HFCs) represent reliable alternatives. Full cryoprecipitate characterization is required to inform selection of an appropriate fibrinogen source for supplementation therapy. Extended biochemical comparison of pooled cryoprecipitate and HFC (Fibryga, Octapharma) was performed using commercially available assays to determine levels of variability in cryoprecipitate and HFC. In addition to standard procoagulant factors, measurements included activities of platelet-derived microparticles (PMPs) and plasminogen, and levels of fibrin degradation products. Cryoprecipitate contains lower fibrinogen levels than HFC (4.83 vs.19.73 g/L; p<0.001), translating to approximately half the amount of fibrinogen per standard cryoprecipitate dose (two pools, pre-pooled from five donations each) vs. HFC (2.14 vs. 3.95 g; p<0.001). Factor XIII (FXIII) levels were also lower in cryoprecipitate vs. HFC (192.17 vs. 328.33 IU/dL; p = 0.002). Levels of procoagulants in cryoprecipitate, such as von Willebrand Factor (VWF) and factor VIII (FVIII), were highly variable, as was PMP activity. A standard cryoprecipitate dose contains significantly higher levels of measured plasminogen and D-dimer fragments than a standard HFC dose. The tested HFC is a more reliable fibrinogen and FXIII source for accurate dosing compared with cryoprecipitate. Cryoprecipitate appears considerably less predictable for bleeding management due to wide variation in pro- and anticoagulation factors, the presence of PMPs, and the potential to elevate VWF and FVIII to prothrombotic levels.

Desmopressin as a Treatment in Patients With Von Willebrand Disease: A Systematic Review.

Von Willebrand disease (VWD) and hemophilia A are the most common inherited bleeding disorders. Quantitative or qualitative von Willebrand factor (VWF) anomalies cause this disorder in men and women. VWF, a plasma glycoprotein, relies on platelets for primary hemostasis. It also carries and stabilizes factor VIII in the blood. VWD has several categories. Types 1 and 3 have partial or total VWF quantitative deficiencies. However, type 2 and its subtypes have VWF quality issues. The major treatment is desmopressin (DDAVP), which replaces endogenous VWF and factor VIII (FVIII). Plasma-derived VWF/FVIII products may also be substituted exogenously. Treatment with plasma-derived or recombinant VWF concentrates without FVIII is also possible.The purpose of this retrospective, single-center research was to evaluate DDAVP's efficacy in treating VWD based on many criteria established in the current literature. We looked at the results on Google Scholar, the Cochrane Library, and PubMed/Medline. There were a total of 10 papers found, evaluated, and accepted for inclusion in this study. A comprehensive analysis of DDVAP's role in VWD was compiled from the aforementioned papers. Various aspects of DDVAP were captured by including an analysis of complementary treatments used in surgical and clinical settings. We also describe the treatment's intended impact on the different variations of the disease. Given these results, further investigation is required to determine the most effective method for managing VWD so that it may be included in standard clinical practice.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, andpeptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

Acquired haemophilia A: A 15-year population-based review of incidence rate, patient demographics and treatment outcomes.

Acquired haemophilia A (AHA) is a rare bleeding disorder characterized by autoantibodies against coagulation factor VIII (FVIII). Estimates of AHA incidence are largely based on registry data, which may be prone to referral bias. Population-based studies can enhance our understanding of the epidemiology, presentation and outcomes of AHA. We conducted a retrospective, population-based cohort study of all AHA diagnosed and treated in Manitoba, Canada over a 15-year period. Using records from the sole provincial reference laboratory, we identified all patients with FVIII inhibitors who did not have congenital haemophilia. Using a piloted case report form, patient data was ascertained from hospital and bleeding disorder clinic records. From 2006 to 2021, we identified 34 patients with AHA, corresponding to a population-based incidence rate of AHA of 1.78 cases per million per year. The median age at presentation was 76 years and most cases were idiopathic (79%). Almost all patients (97%) presented with bleeding, of which 58% were considered major bleeds and required haemostatic agents in 67%. Longstanding unexplained bleeding symptoms were commonly reported, suggesting delayed diagnosis. Immunosuppressive therapy (IST) was administered in 88% of patients. Remission was achieved in 79% of patients; median time to remission was 2.1 months. There were two deaths due to bleeding. No deaths due to IST were reported. The population-based incidence of AHA in Manitoba is 1.78 cases/million/year. Bleeding is common and can be life-threatening. AHA outcomes are encouraging with the use of haemostatic agents and IST. Serious treatment-associated morbidity and mortality is uncommon.

Factor VIII Level in the Potential Female Carrier Mothers of Patients with Hemophilia A and Its Impact on Joint Health

Background Hemophilia A is an X-linked bleeding disorder. The characteristic bleeding phenotype in patients with severe hemophilia is recurrent spontaneous hemarthrosis with development of hemophilic arthropathy. Heterozygous female carriers with factor VIII (FVIII) activity level lower than 40% may be at risk for bleeding that is usually comparable to that seen in males with mild hemophilia. Aim of the Work To assess factor VIII level in mothers of known patients of hemophilia A correlating it with the frequency of clinically and radiologically documented arthropathy. Patients and Methods A cross sectional study included 40 mothers of patient with hemophilia A during the period from September 2020 to September 2021. Studied participants were subjected to detailed analysis of bleeding history, six major joints range of motion (ROM) examination, functional independence score in hemophilia (FISH), FVIII level one stage assessment and magnetic resonant imaging (MRI) of the reported affected joints. Results Mean age of mothers is 42.25 ± 7.74 years. Eleven mothers (27.5%) had no bleeding symptoms, 16 (40%) had one bleeding symptoms and 13 (32.5%) had two or more bleeding symptoms. As regard joint pain, 13 (32.5%) had severe pain, 3 (7.5%) had ankle pain and 2 (5%) had elbow pain with a mean pain score of 2.55±3.32, and 7 (17.5%) had bilateral pain. Mean FVIII activity was 43.33 IU (± 22.79), ranging from FVIII (0 &amp;lt; 30 IU) in 13 (32.5%), FVIII (30 &amp;lt;60) in 18 (45.0%) and FVIII ≥ 60 IU in 9 (22.5%) among the studied participants. Knees were the most affected joints with decreased mean ROM (±SD) in right and left knees of (123.25 (±12.06) and 120.00 (±15.81)) respectively. The median MRI scores were studied was 5 (2.6%) with median osteochondral changes of 4 (2.5%) and median soft tissue changes of 1 (0-1), their median FISH score of 8 (8-9). By comparing groups at the level of factor VIII, there was no significant difference according to anthropometric bleeding parameters, pain, and MRI score. None of participants needed hospital admission, factor VIII replacement or packed red cells replacement. Conclusion Potential female hemophilia A carriers are at risk of spontaneous bleeding which may entail bleeding in the joints, causing early joint damage. They showed reduced overall joint ROM and structural osteochondral changes in MRI joint examination which mandates further studies about early detection of potential female hemophilia carriers for the possible need of early medical intervention.

Damoctocog Alfa Pegol for Hemophilia A Prophylaxis: An Italian Multicenter Survey.

Hemophilia A is characterized by a deficiency of clotting factor VIII (FVIII) requiring lifelong prophylactic treatment, typically with recombinant FVIII. In recent years, drugs with extended half-lives have become available, including damoctocog alfa pegol (Bayer S.p.A.). The clinical efficacy and safety of damoctocog alfa pegol were demonstrated in the PROTECT VIII phase II/III development program. To assess the physicians' experience and to collect data on prophylactic treatment with damoctocog alfa pegol, a monitoring survey was carried out among 15 Italian hemophilia centers. A total of 149 patients on treatment with damoctocog alfa pegol for at least 6 months were considered. Zero bleeds were reported in 75% of patients treated with damoctocog alfa pegol in the last 6 months; zero hemarthroses were reported in 82% of the same patients. Overall, 86% of patients with damoctocog alfa pegol reduced their monthly infusions. The estimated average reduction in IU/kg during prophylaxis with damoctocog alfa pegol, both monthly and annually, was around 17.5%. All involved clinicians were satisfied with damoctocog alfa pegol. Survey results suggest that damoctocog alfa pegol reduced the number of weekly infusions, annual bleeding, and joint bleeding rate in the majority of patients, improving joint health and patients' quality of life.

The immunogenicity, safety, and efficacy of N8-GP in previously untreated patients with severe hemophilia A: pathfinder6 end-of-trial results

BackgroundThe pathfinder6 (NCT02137850) international phase 3 trial examined immunogenicity, safety, and efficacy of the extended half-life factor VIII (FVIII) replacement product N8-GP (turoctocog alfa pegol; Esperoct) in previously untreated patients (PUPs) with hemophilia A. ObjectivesWe present end-of trial results for extended PUP N8-GP treatment for up to a median (range) 2.5 (0.0; 7.4) years. Patients/MethodsLonger-term N8-GP treatment in PUPs with hemophilia A was examined. The prophylaxis regimen was ∼60 IU/kg N8-GP i.v. twice weekly, or every 3 or 7 days. The primary endpoint was the incidence of FVIII inhibitors. ResultsOverall, 81 patients received N8-GP and were included in this analysis. The inhibitor incidence was 30.0% (15.7% high-titer [>5 BU]) for the extension phase. Patients had a median (range) 2.9 (0.1; 7.2) years of prophylaxis following the pre-prophylaxis period. During prophylaxis, the median annualized bleeding rate (ABR) (interquartile range) was 1.4 (0.6; 3.5), 13% of patients experienced no bleeding episodes, and 55.1% of patients experienced no spontaneous bleeds. The proportion of patients without any spontaneous bleeding episodes increased after the first year of prophylaxis. The hemostatic success rate in the treatment of bleeding episodes was 87.6%. No additional safety concerns were observed in patients with previously reported observation of temporarily decreased incremental recovery (IR). ConclusionLong-term end-of-trial PUP N8-GP prophylaxis data indicate that PUPs respond well to long-term N8-GP treatment. The inhibitor incidence was consistent with previous results. Median ABR during prophylaxis was 1.4. There were no lasting clinical impacts or safety concerns for patients with an observation of temporarily decreased IR.

Coagulation factor VIIa enhances programmed death-ligand 1 expression and its stability in breast cancer cells to promote breast cancer immune evasion

BackgroundImmunotherapy for breast cancer has not gained significant success. Coagulation factor VIIa (FVIIa)-tissue factor (TF) mediated activation of protease-activated receptor 2 (PAR2) is shown to promote metastasis and secretion of the immune-modulatory cytokines but the role of FVIIa in cancer immunology is still not well understood. ObjectivesHere, we aim to investigate whether FVIIa protects breast cancer cells from CD8 T-cell–mediated killing. MethodsPeripheral blood mononuclear cell-derived CD8 T cells were cocultured with vehicle or FVIIa pretreated MDAMB468 cells. The proliferation and activity of CD8 T cells were measured by flow cytometry and ELISA. An allograft model, using wild-type or TF/PAR2-deleted 4T1 cells, was employed to determine the effect of FVIIa on breast cancer immune evasion in vivo. ResultsHere, we demonstrate that TF-FVIIa induces programmed death-ligand 1 (PD-L1) in breast cancer cells by activating PAR2. PAR2 activation triggers large tumor suppressor kinase 1 (LATS1) inactivation leading to loss of yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) phosphorylation and subsequent nuclear localization of YAP/TAZ. YAP/TAZ inhibition reduces PD-L1 expression and increases CD8 T-cell activity. We further demonstrate that, apart from transcriptional induction of PD-L1, PAR2 activation also increases PD-L1 stability by enhancing its glycosylation through N-glycosyltransferases STT3A and STT3B. ConclusionIn a mouse model of breast cancer, tumor cell-specific PAR2 depletion leads to PD-L1 downregulation and increases anti–PD-1 immunotherapy efficacy. In conclusion, we showed that FVIIa-mediated signaling cascade in cancer cells serves as a tumor intrinsic mechanism of immunosuppression to promote cancer immune evasion.

Evaluation of bacterial growth, effects on albumin, and coagulation factors in canine fresh frozen plasma administered as continuous rate infusion exposed to room temperature for 12hours.

To determine the risk of bacterial growth and to analyze the stability of albumin and coagulation factors in canine fresh frozen plasma (FFP) units exposed to room temperature (24°C) administered as a continuous rate infusion (CRI) for 12hours. Ex vivo study. University teaching hospital and pet blood bank. None. None. An FFP CRI was simulated to replicate the standard routine procedure used in dogs. Plasma samples were collected before starting the CRI (H0), after 4hours (H4), and after 12hours (H12). Bacterial culture of FFP was performed and albumin concentration and specific activity levels for factors V, VII, VIII, and IX were measured and compared. All plasma culture results were negative. There were no statistically significant differences at any time point in the factor VIII activity (median 105.5% [range, 75.6%-142.0%] at H0; median 107.8% [range, 75.0%-172.7%] at H4; and median 112.1% [range, 81.7%-171.0%] at H12); factor IX activity (median 119.3% [range, 89.1%-175.9%] at H0; median 123.1% [range, 72.5%-172.7%] at H4; and median 118.3% [range, 86.6%-177.5%] at H12); or albumin concentration (median 21.0g/L [range, 17.0-23.0g/L] at H0 and median 20.0g/L [range, 17.0-24.0g/L] at H12). A slight but significant increase in factor V activity was observed when comparing H0 (median 107.0% [range, 71.0%-159.0%]) to H4 (median 117.7% [range, 71.0%-176.7%]) (P=0.002) or H12 (median 116.2% [range, 71.0%-191.6%]) (P=0.001). A slight but significant increase in factor VII activity was observed when comparing H0 (median 115.4% [range, 70.6%-183.7%]) to H4 (median 118.2% [range, 82.7%-194.6%]) (P=0.005); H0 to H12 (median 128.7% [range, 86.4%-200.0%]) (P<0.001); and H4 to H12 (P=0.002). FFP CRI at room temperature for 12hours could be considered safe with regard to risk for bacterial growth and also effective by providing albumin and clotting factors.

Management of surgery in persons with hemophilia A receiving emicizumab prophylaxis: data from a national hemophilia treatment center

BackgroundPersons with hemophilia A may require surgical procedures. Real-world data on invasive procedures in persons with hemophilia A receiving emicizumab prophylaxis are limited. ObjectivesTo evaluate the safety of invasive procedures in persons with hemophilia A receiving emicizumab prophylaxis and their outcomes in a longitudinally followed cohort. MethodsData from medical records of persons with hemophilia A with and without factor VIII (FVIII) inhibitors longitudinally followed at our tertiary center, who received emicizumab prophylaxis and underwent all types of invasive procedures, were retrieved. Outcomes of interest were bleeding and thrombotic complications. ResultsOverall, 35 patients underwent 56 invasive procedures, 18 (32.1%) were major. The median age was 36.3 years (IQR, 8.8-55.9 years); 12 patients (34.3%) were younger than 18 years at the time of procedure; 17 (48.6%) were patients with FVIII inhibitors. Among major procedures, orthopedic surgeries prevailed. All patients who underwent major procedures received factor replacement with either recombinant activated factor VII (patients with inhibitors) or FVIII (patients without inhibitors). Factor concentrates were administered prior to 32 (84.2%) of the minor procedures. Repeated doses were given according to international expert opinion recommendations and patients’ condition.There were 7 bleeding events in 6 patients, 5 were major bleeds, including 1 patient who underwent a minor procedure without factor replacement. None of the patients experienced a thrombotic complication. ConclusionInvasive procedures can be performed safely in patients receiving emicizumab prophylaxis with close surveillance after surgery. Factor concentrates may be advised in selected patients undergoing minor procedures.