Plasma Factor VII Research Articles

ET1 acts as a potential plasma biomarker and therapeutic target in deep venous thrombosis rat model.

Deep venous thrombosis (DVT) is the third leading cause of death in cardiovascular disease, following heart attacks and strokes. Early diagnosis and intervention are crucial for effective DVT therapy. We aim to investigate whether endothelin-1 (ET-1) could serve as an early diagnostic marker or a potential therapeutic target in a DVT rat model. CCK8 assay, invasion assay, and flow cytometry were used to detect the proliferation, migration and apoptosis of HUVECs, respectively. Elisa assay was used to detect ET-1 and coagulation factor VII in cell supernatant and rat?s plasma. Western blot was used to detect antioxidant signaling protein. Inferior vena cava stenosis was used to construct the DVT rat model. Lentivirus mediated overexpression of ET-1 in HUVECs impaired the cell proliferation and migration, increased cell apoptosis, inhibited the antioxidant signaling pathway proteins expression (e.g., NQO1, GCLC, Nrf-2), and upregulated coagulation factor VII. Furthermore, overexpression of ET-1 further impaired antioxidant signaling pathway protein in response to H2O2 treatment. However, lentivirus mediated ET-1 knockdown and BQ123 (an ET-1 inhibitor), showed the opposite results with ET-1 overexpression. We then established a DVT rat model by inferior vena cava stenosis. The stenosis induced early expression of ET-1 and coagulation factor VII in plasma at day 1 and restore their level at day 10. BQ123 could downregulate the coagulation factor VII to ameliorate the stenosis effects. Our findings suggest that ET-1 might serve as an early diagnostic marker for DVT rat model and a potential therapeutic target for treating DVT.

Exploring the Causal Relationship Between Coagulation Function and Gestational Diabetes Mellitus Through Mendelian Randomization

To explore the causal association between coagulation function, including von Willebrand factor (vWF), a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13), activated partial thromboplastin time (aPTT), coagulation factor Ⅷ (FⅧ), coagulation factor Ⅺ (FⅪ), coagulation factor Ⅶ (FⅦ), coagulation factor Ⅹ (FⅩ), endogenous thrombin potential (ETP), plasminogen activator inhibitor-1 (PAI-1), protein C, and plasmin, and gestational diabetes mellitus (GDM) using two-sample two-way Mendelian randomization (MR), and to provide genetic evidence for the association between coagulation function and the pathogenesis of GDM. The IEU OpenGWAS database was accessed using the R package TwoSampleMR (v 0.5.6) to obtain the statistical data of the genome-wide association study (GWAS) summary of GDM. MR analysis of the causal association between 11 coagulation function and GDM was performed by the inverse-variance weighted method (IVW), the MR-Egger method, and the weighted median method (WM). In this study, the GWAS summary statistics of GDM (covering 5 687 cases and 117 892 controls) were used for MR analysis. It was found that there was a causal relationship between the predicted plasma FⅧ level and the risk for GDM (IVW: [odds ratio, OR]=0.28, 95% confidence interval [CI]: 0.10-0.75, P<0.001; WM: OR=0.30, 95% CI: 0.09-0.98, P<0.001). There was no causal relationship between other coagulation function and the risk for GDM (P>0.05). There is a significant causal relationship between the plasma FⅧ level and the risk for GDM. This finding highlights the complex interaction between coagulation function and glucose metabolism during pregnancy, but further research on this finding is warranted.

Evolution Directed Gene Engineering (EDGE) Identifies Single Amino Acid Substitutions in FVIII with Six-Fold Increased in Vitro and In Vivo Activity

Background: Current AAV gene therapy for hemophilia A (HA) is limited by low sustained factor VIII (FVIII) levels. In contrast, AAV gene therapy for hemophilia B benefits from the widespread adoption of the single amino acid (AA) substituted high-specific-activity FIX variant R338L. We have previously shown that wild-type (WT) FIX has not evolved for maximal specific activity, but rather has evolutionary conserved AAs that limit procoagulant activity (Samelson-Jones et al. Blood Advances 2021). FVIII may have evolved with similar considerations. Based on this work, we developed a novel screening technique, termed Evolution Directed Gene Engineering (EDGE), to identify new hyperactive single AA substituted FVIII variant transgenes to address the current limitations of HA gene therapy. Aims: To find and characterize single AA substituted FVIII variants that can enhance gene transfer. Methods: EDGE evaluates AA positions fulfilling 2 criteria: 1) highly evolutionary conserved and 2) not associated with the disease phenotype. Here, EDGE compared 33 mammalian FVIII orthologues and then eliminated positions associated with known HA-causing missense mutations. FVIII variants were transiently expressed in Huh7 cells and cultured media was assayed for FVIII activity and antigen. Recombinant FVIII protein of lead candidates were purified to homogeneity and assayed for FVIII activity with one- and two-stage aPTT assays, thrombin generation assay (TGA), and enzyme kinetics studies. The in vivo efficacy of the variants was tested by tail clip assays of HA mice after retro-orbital delivery of the protein or liver-directed AAV gene therapy. Results: Out of 52 AA positions identified by EDGE, we found 12 AA positions in FVIII where single AA substitutions increased specific activity ≥2-fold (Figure 1A). The largest increase in specific activity was 6-fold, which occurred for multiple AA substitutions at position 659. Purified recombinant protein of FVIII-K659M and -K659V both demonstrated 6-fold increased activity in one- and two-stage aPTT assays, but comparable thrombin activation and A2-stability to FVIII-WT. TGA show the FVIII-K659M/V variants have greater than 6-fold improvements in endogenous thrombin potential and peak thrombin generation. Compared to FVIII-WT, FVIII-K659M/V proteins have an increased k cat and decreased appK D which suggests that the mechanism of increased specific activity is due to enhanced interactions with FIXa. This is similar to the mechanism of hyperactivity of FIX-R338L. FVIII-K659M/V also demonstrated enhanced procoagulant activity in vivo. In tail clip injuries of HA mice (n≥4) treated with purified protein, the FVIII-K659M/V proteins restored normal hemostasis at 5-fold lower concentrations than FVIII-WT. When mice were treated with equal doses of 2.5 µg/kg of protein, FVIII-K659M/V restored normal hemostasis while FVIII-WT had no significant change in blood loss (Figure 1B). As expected, 12.5 µg/kg of FVIII-WT protein was required to restore blood loss to WT mouse levels. FVIII-K659 substituted variants also enhanced AAV based gene therapy. HA mice (n=6) treated with AAV8 vectors expressing FVIII-K659C had 6-fold higher plasma FVIII activity than mice (n=6) treated at the same dose with AAV8 vectors expressing FVIII-WT. HA mice that received 4 x 10 12 vg/kg of AAV8 FVIII-K659C had less bleeding than HA mice that received the same dose of AAV8 FVIII-WT, but similar blood loss as WT mice after tail clip injury. Conclusions: The success of EDGE in identifying multiple new high specific activity FVIII variants supports the hypothesis that FVIII-WT is not optimized for specific activity and thus is amenable to bioengineering approaches. The ≥5-fold enhanced in vitro and in vivo hemostatic activity of the FVIII-K659 substituted variants and initial murine gene therapy studies support their translational potential to address current limitations of HA gene therapy. These results also implicate K659 in modulating the FVIIIa/FIXa interaction. EDGE may also be applicable to other gene therapy targets as well.

Acquired Postpartum Hemophilia A: About 3 Cases

Introduction: Acquired hemophilia A (AHA) is a rare but severe autoimmune disorder. It is caused by the acquisition of autoantibodies to factor VIII (FVIII). These post-partum autoantibodies account for 7-21% of all cases of AHA and most often develop after the first pregnancy. We report 3 new cases of postpartum-acquired hemophilia A. Observations: Three young women with no prior pathological history presented with a spontaneous and heterogeneous hemorrhagic syndrome, two cases of deep hematomas, and haematuria with ecchymosis in the 3rd patient. The diagnosis was made in all our patients based on plasma FVIII activity &lt;50% and the presence of an FVIII inhibitor. Immunological tests and neoplastic research were negative in all our patients. Hemostatic treatment combined with corticosteroid therapy and immunosuppressive drugs were used for the 3 patients. Conclusion: AHA is a rare disease, and its diagnosis is often underestimated in our population. It is essential to make clinicians aware of this pathology and to be able to evoke it in young patients of childbearing age. A relapse is possible in future pregnancies, so long-term follow-up is recommended.

Plasma Clearance of Coagulation Factor VIII and Extension of Its Half-Life for the Therapy of Hemophilia A: A Critical Review of the Current State of Research and Practice.

Factor VIII (FVIII) is an important component of blood coagulation as its congenital deficiency results in life-threatening bleeding. Current prophylactic therapy of the disease (hemophilia A) is based on 3-4 intravenous infusions of therapeutic FVIII per week. This poses a burden on patients, demanding reduction of infusion frequency by using FVIII with extended plasma half-life (EHL). Development of these products requires understanding FVIII plasma clearance mechanisms. This paper overviews (i) an up-to-date state of the research in this field and (ii) current EHL FVIII products, including recently approved efanesoctocog alfa, for which the plasma half-life exceeds a biochemical barrier posed by von Willebrand factor, complexed with FVIII in plasma, which results in ~1 per week infusion frequency. We focus on the EHL FVIII products' structure and function, in particular related to the known discrepancy in results of one-stage clotting (OC) and chromogenic substrate (CS) assays used to assign the products' potency, dosing, and for clinical monitoring in plasma. We suggest a possible root cause of these assays' discrepancy that is also pertinent to EHL factor IX variants used to treat hemophilia B. Finally, we discuss approaches in designing future EHL FVIII variants, including those to be used for hemophilia A gene therapy.

Relationship between factor VIII levels and bleeding for rFVIII-SingleChain in severe hemophilia A: A repeated time-to-event analysis.

Publications on the exposure-effect relationships of factor concentrates for hemophilia treatment are limited, whereas such analyses give insight on treatment efficacy. Our objective was to examine the relationship between the dose, factor VIII (FVIII) levels and bleeding for rFVIII-SingleChain (lonoctocog alfa, Afstyla). Data from persons with severe hemophilia A on rFVIII-SingleChain prophylaxis from three clinical trials were combined. The published rFVIII-SingleChain population pharmacokinetic (PK) model was evaluated and expanded. The probability of bleeding was described with a parametric repeated time-to-event (RTTE) model. Data included 2080 bleeds, 2545 chromogenic stage assay, and 3052 one-stage assay FVIII levels from 241 persons (median age 19 years) followed for median 1090 days. The majority of the bleeds occurred in joints (65%) and the main bleeding reason was trauma (44%). The probability of bleeding decreased during follow-up and a FVIII level of 8.9IU/dL (95% confidence interval: 6.9-10.9) decreased the bleeding hazard by 50% compared to a situation without FVIII in plasma. Variability in bleeding hazard between persons with similar FVIII levels was large, and the pre-study annual bleeding rate explained part of this variability. When a FVIII trough level of 1 or 3IU/dL is targeted during prophylaxis, simulations predicted two (90% prediction interval [PI]: 0-17) or one (90% PI: 0-11) bleeds per year, respectively. In conclusion, the developed PK-RTTE model adequately described the relationship between dose, FVIII levels and bleeds for rFVIII-SingleChain. The obtained estimates were in agreement with those published for the FVIII concentrates BAY 81-8973 (octocog alfa) and BAY 94-9027 (damoctocog alfa pegol), indicating similar efficacy to reduce bleeding.

For people who have coagulopathic non-surgical bleeding and who undergo cardiac surgery, how does prothrombin complex concentrate (PCC) compare with fresh frozen plasma (FFP) or recombinant factor VIIa (rFVIIa)?

For people who have coagulopathic non-surgical bleeding and who undergo cardiac surgery, how does prothrombin complex concentrate (PCC) compare with fresh frozen plasma (FFP) or recombinant factor VIIa (rFVIIa)?

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Laboratory Testing for von Willebrand Factor: Factor VIII Binding for the Diagnosis or Exclusion of Type 2N von Willebrand Disease: An Update.

von Willebrand factor (VWF) is a large adhesive plasma protein that expresses several functional activities. One of these activities is to bind coagulation factor VIII (FVIII) and to protect it from degradation. Deficiency of, and/or defects in, VWF can give rise to a bleeding disorder called von Willebrand disease (VWD). The defect in VWF that affects its ability to bind to and protect FVIII is captured within type 2N VWD. In these patients, FVIII is produced normally; however, plasma FVIII quickly degrades as it is not bound to andprotected by VWF. These patients phenotypically resemble those with hemophilia A, where instead, FVIII is produced in lower amount. Both hemophilia A and 2N VWD patients therefore present with reduced levels of plasma FVIII relative to VWF level. However, therapy differs, since patients with hemophilia A are given FVIII replacement products, or FVIII mimicking products; instead, patients with 2N VWD require VWF replacement therapy, since FVIII replacement will only be effective for a short term, given this replacement product will quickly degrade in the absence of functional VWF. Thus, 2N VWD needs to be differentiated from hemophilia A. This can be achieved by genetic testing or by use of a VWF:FVIII binding assay. The current chapter provides a protocol for the performance of a commercial VWF:FVIII binding assay.

Extensive genetic screening of Iranian Factor FVII-deficient individuals unraveled several novel mutations and postulated founder effects in some cases

BackgroundAs the most frequent congenital rare bleeding disorder that transmits in an autosomal recessive manner, factor VII (FVII) deficiency is a serious bleeding complication in populations with high rate of in-marriages. While diagnosis mainly relies on clinical and laboratory phenotypes, plasma FVII antigen and activity levels do not often correlate with symptoms’ severity. ObjectivesGenetic profiling of the affected individuals potentially improves our biological understanding of this complicated rare disorder. MethodsConventional polymerase chain reaction-Sanger sequencing and whole-exome sequencing were applied for genetic profiling of F7 gene in 66 symptomatic FVII-deficient individuals from 62 independent pedigrees. Thirty-nine asymptomatic relatives of the patients were also studied. ResultsThirty different F7 pathogenic variations were identified in the studied cases of which 11 have not been reported before. The novel mutations include 5 missenses (c.715G>A, c.794T>C, c.1090C>G, c.1222C>A, c.1265T>C), 3 splicing (c.316+1G>T, c.682-2A>G, c.572-16C>G), 2 nonsenses (c.790delC, c.1248G>A), and 1 frameshift (c.1346delA). A founder effect is proposed for c.790delC that was detected in 8 independent pedigrees who were all from similar geographical regions and ethnic backgrounds. Homozygous c.790delC reduces plasma FVII activity to <1% and causes spontaneous intracranial hemorrhage in early infancy. ConclusionFrom the 66 studied symptomatic FVII-deficient individuals, 58 were homozygous carriers of the identified variations. Identification of homozygotes clarifies the potential role of nucleotide variations in reducing FVII activity and their contributions to a certain phenotype. Some of those variations, such as c.1A>G, c.509G>A, c.634C>T, and c.1285G>A have only been previously reported as heterozygous.

The Nonsynonymous-Single-Nucleotide Polymorphism (ns-SNP) 353 R>q in the Coagulation Factor VII (FVII) Gene, F7, is Associated with Decreased Plasma FVII Levels and a Decreased Risk of Coronary Heart Disease (CHD) in Mexican Americans of South Texas

The Nonsynonymous-Single-Nucleotide Polymorphism (ns-SNP) 353 R>q in the Coagulation Factor VII (FVII) Gene, F7, is Associated with Decreased Plasma FVII Levels and a Decreased Risk of Coronary Heart Disease (CHD) in Mexican Americans of South Texas

Personalization of Clotting Factor VIII Replacement Therapy Based on Population Pharmacokinetics in Adult Patients with Severe Hemophilia a Who Frequently Switch Factor VIII Concentrates

Interindividual pharmacokinetic differences in clotting factor VIII (FVIII) metabolism impede the dosing regimen optimization in patients with hemophilia A. The population pharmacokinetic (PK) analysis enables the estimation of FVIII pharmacokinetics in individual patients which is used to personalize dosing regimens. Therefore, we conducted a prospective study on adult patients with severe hemophilia A who received different FVIII concentrates for tertiary prophylaxis and whose dosage of FVIII was adjusted based on individual PK results. We aimed to compare the number of bleeding episodes and FVIII consumption before and after dosing adjustment. The study, conducted for 18 months for each participant, was divided into 3 phases: (1) pre-PK: assessments before FVIII dosing adjustment, (2) dosing adjustment based on the PK analysis of FVIII, and (3) assessments after FVIII dosing adjustment. All patients gave informed consent before enrolment, and all procedures were carried out according to the Declaration of Helsinki. During the study patients frequently changed FVIII concentrates (each patient used 3-5 different products) which reflects the standard of care in adult patients with hemophilia in Poland. During phase 1 of the study, over 5-13 months [median: 11.0 months, mean(standard deviation - SD): 10.8(1.9) months], patients recorded FVIII consumption and bleedings using electronic diaries (myPKFiT® licensed for octocog alfa). In phase 2 of the study, a PK analysis (clearance, half-time, time to reach the 1 IU/dl concentration) was performed: following the 72-hour washout period, blood samples were collected, and patients received the infusion of octocog alfa with the mean dose (SD) of 51.1(1.7) IU/kg. Two more blood samples were collected 3-4 h and 24-36 h after the infusion. Plasma FVIII activity was measured with a one-stage clotting assay, and the FVIII inhibitor with the Nijmegen modification of the standard Bethesda method. The myPKFiT® was used to obtain individualized FVIII dosing regimens based on PK analysis results. The dosing adjustment was offered to patients who experienced breakthrough bleeds during phase 1 or in whom the pre-PK treatment did not secure FVIII trough level of >1 IU/dl. In case of favorable PK results and no breakthrough bleeds during phase 1, a less intensive regimen was implemented. In phase 3 of the study (which lasted for 5-13 months, median: 7.7 months, mean(SD): 7.6(1.6) months), patients continued treatment with locally available FVIII concentrates and used electronic diaries to record FVIII consumption and bleedings. All bleeding rates were annualized (annualized bleeding rates - ABR). The final analysis set included 22 male patients, aged 23-68 years. The clearance of octocog alpha ranged from 0.014 to 0.051 dL/h/kg [mean(SD), 0.030±0.010 dL/h/kg; coefficient of variation (CV)=33%], half-life - from 9.1 to 27.9 h (14.6(4.3) h, CV=29%), and time to reach 1 IU/dl - from 51 to 179 h [91(29) h, CV=32%]. Three individuals were categorized as slow metabolizers (clearance of 0.014, 0.018, and 0.019 dL/h/kg), and 2 as fast metabolizers (clearance of 0.048 and 0.051 dL/kg/h). FVIII inhibitor was undetectable (<0.5 BU/ml) in all patients. After the PK analysis, 21 of 22 patients met the criteria of FVIII dosing adjustment. Four patients did not agree to change the regimen. Seventeen patients modified it according to the PK results (10 of them had FVIII trough levels of >1 IU/dl and 7 of them of <1 IU/dl in phase 1 of the study). Changing the dosing regimen in the 17 patients resulted in ABR reduction compared to the pre-PK phase: a decrease of 3.1-fold in total ABR (p=0.045 for dependent samples t-test); 3.2-fold in spontaneous ABR (p=0.046); and 3.1-fold in injury-related ABR (p=0.072). The consumption of FVIII due to bleeds had decreased by 77% (p=0.034). In conclusion, personalizing the FVIII replacement therapy based on population PK models helps to lower bleeding risk and drug consumption in patients with severe hemophilia A. Presented results suggest that prophylaxis regimen based on PK analysis carried out with octocog alfa and myPKFiT® application is clinically effective also with the use of other FVIII concentrates.

Coagulation Factor VIII Enhances the Cleavage of Von Willebrand Factor By ADAMTS13 In Vivo

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Markedly Reduced FVIII Recovery Associated with Anti-FVIII Peg Antibodies after BNT162b2 Sars-Cov-2 Vaccination

Background The covalent link of polyethylene glycol (PEG) chains to recombinant factor VIII (FVIII) molecule is one of the most established approaches to produce novel therapeutic FVIII products with extended half-life (EHL). To date, three PEGyated-FVIII molecules, rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa pegol are available for treatment of hemophilia A patients. From the pivotal clinical studies data, they showed comparable safety profile and pharmacokinetic with a mean half-live 1.3-1.7 times longer than the standard non-PEGylated FVIII. Although the PEG polymer is considered non immunogenic, antibodies against PEG, called pre-existing anti-PEG antibodies (APAs), have been reported in persons never treated with PEGylated drugs owing to PEG exposure during the activities of daily living. These antibodies can impact the clinical efficacy of PEGylated drug therapies by accelerating the blood clearance and changing pharmacokinetic. Recently, small amounts of PEG are also included in the lipid nanoparticles of the mRNA SARS-Cov-2 vaccines, BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). Aim To describe a poor plasma FVIII recovery after the first infusion of a PEGylated FVIII product in two male patients with severe hemophilia A without inhibitor at about one month from the second dose of the BNT162b2 SARS-Cov-2 vaccination. Method About two months after the first dose of the SARS-Cov-2 vaccine and one month after the second, before switching from a standard half-life to a PEGylated FVIII product, patients underwent a PK study by infusion of 50 IU/kg of turoctocog alfa pegol (patient 1) or damoctocog alfa pegol (patient 2). FVIII activity was measured by Chromogenix Coamatic FVIII (Werfen) in plasma samples collected before and after the PEGylated FVIII infusion. Anti-PEGylated FVIII antibodies were searched by means of a home-made ELISA in plasma samples collected before administration of SARS-Cov-2 vaccine and at PK study. The assay was performed by using as capturing substrates each of the licensed PEGylated FVIII product (rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa). Moreover, a non-PEGylated standard FVIII (Advate®) was employed as control substrate. Results FVIII coagulant activity measured after the infusion of 50 IU/kg of the PEGylated FVIII product showed a very low plasma recovery, 5% for patient 1 and 4% for patient 2. Anti-PEGylated FVIII antibodies reacting with each PEGylated FVIII products were found in plasma samples collected at PK baseline and after FVIII infusion but not in a sample obtained before the SARS-Cov-2 vaccination. No reactivity was found in vitro against the non-PEGylated standard FVIII product (figure). Conclusions Our finding of antibodies reacting with the PEGylated part of the FVIII molecule induced or boosted by BNT162b2 SARS-Cov-2 vaccination supports the view that the administration of the mRNA vaccine could impair plasma FVIII recovery after infusion of a PEGylated product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rituximab and Bortezomib for Patients with Newly Diagnosed Acquired Haemophilia: Single Arm, Single Center, Prospective Phase 2 Study

Introduction Acquired haemophilia A (AHA) is a rare autoimmune disorder caused by neutralizing autoantibodies against factor VIII (FVIII). The most common IST treatment used are Steroids-based regimens. However, patients with AHA are often elderly with comorbidities. Steroids-based regimens have significant side effects in patients with AHA. In particular, the risk of dying from infection appears to be higher than the risk of fatal bleeds today. This dilemma highlights the lack of safe and rapid response IST therapy in AHA. Bortezomib targets plasma cells rather than B cells and has reported efficacy in refractory AHA. It is an attractive agent because it depletes plasma cells that can continue to produce autoantibody even after CD20-positive B cells have been depleted by rituximab. The combination of bortezomib and rituximab regimen showed rapid response without severe adverse event in refractory and relapsed warm autoimmune hemolytic anemia. This phase 2, prospective, single-center study was designed to evaluate the efficacy and safety of rituximab and bortezomib (RB) treatment regimen in newly diagnosed AHA patients. Methods All patients 18 years of age or older who met the following criteria were considered as eligible candidates for this study: 1) plasma FVIII activity (FVIII:C) <50 l/dl, detectable inhibitor to FVIII. 2) newly diagnosed without any IST treatment. After obtaining the written informed consent from the patients, the information of demographic and medical history were collected and laboratory tests were performed.The patients were received the following regimens: Rituximab 375mg/m2 intravenous infusion on day 0 and bortezomib 1.3 mg/m2 subcutaneous on day 1,4,8,11. Best supportive care, including administration of hemostatic treatment and blood product transfusions if needed were used. Safety was evaluated according to NCI-CTCAE v4.0. Results From March 2018 to May 2022, 30 patients were eligible for the study. The baseline characteristics of the patients are summarized in Table 1. Overall, 19 patients were male (63.3%). The median age was 53.5 years (range 22 - 90 years). The underlying disorders that could be associated with AHA included rheumatoid arthritis, systemic lupus erythematosus and pemphigus. One patient was diagnosed at her postpartum. One patient was diagnosed soon after surgery. Traditional Chinese medicine was suspected as the inducement in one patient. No patient in our cohort was reported to have coexisting malignancy. 24 patients (80.0%) were regarded as idiopathic AHA. The median time from disease onset to the diagnosis was 36.5 days (range 10-249 days). The median FVIII activity at baseline was 1.5 IU/ml (range 0 to 10.4 IU/ml), and the median inhibitor concentration was 25.6 BU/ ml (range 2.7 to 600 BU/ ml). The bleeding event that precipitated diagnosis was reported as spontaneous in 27 patients (90.0%), by surgery in 3 patients (10.0%). All patients presented with bleeding diathesis as listed in Table 1. Sixteen patients (61.5%) received haemostatic treatment for initial bleeding, all of them received prothrombin complex concentrate (PCC). Nine patients experienced further bleeding after the treatment of BR. All patients completed protocol treatment. Overall, 23 patients (76.7%) achieved CR, 6 patients (20.0%) achieved PR and 1 patients died of cardiac events 3 month after last dose of bortezomib. The ORR was 96.7%. Median time to CR was 74 days (range 12-179 days). After a median follow-up of 39 months (range 3-50 months), one patient had relapse of the disease, 2 months after achieved CR. Major toxicities and adverse events are summarized in Table 2. One patient (3.8%) had grade 3 neutropenia; One patient (3.8%) had grade 4 thrombocytopenia and grade 3 thrombocytopenia each. Eight patients experienced infectious fever, including 2 grade 3 and 2 (7.7%) grade 4. Five patients (19.2%) developed grade 1-2 peripheral neuropathy.No treatment-related deaths occurred. Conclusion Here we report the results of a phase 2 prospective study of newly diagnosed AHA. This study confirmed that rituximab plus bortezomib was an efficient and safe regimen with ORR of 96.7%, and the median time to CR was 74 days.This trial was registered at www.clinicaltrials.gov as #NCT03700229. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phenotypic variation in severe hemophilia A is related to endogenous thrombin potential and plasma levels of factor VII.

Hemophilia A is a bleeding disorder caused by deficiency or low activity of circulating factor VIII characterized by prolonged blood coagulation time and often spontaneous bleeding. Patients with the severe form of the disease may present considerable heterogeneity in the occurrence of bleeding episodes and some of them have a mild hemophilia A phenotype. This study aimed to evaluate the association of biomarkers and coagulation parameters to the differential hemorrhagic profile of severe hemophilia A patients. Polymorphisms in the genes of proteins C and S, factors V and VII and prothrombin were evaluated in a group of severe hemophilia A patients with a broad spectrum of bleeding profile. Plasma levels of coagulation factors and thrombin generation were also analyzed. This study included 59 Brazilian hemophilia A patients who were allocated into low bleeding profile (LBP; n = 33) and high bleeding profile (HBP; n = 26) groups based on their joint and muscle bleeding episodes requiring treatment in the 5 years before inclusion in the study. Results evidenced that endogenous thrombin potential (ETP) and plasma factor VII levels were significantly higher in the LBP group. Results indicate a prominent importance of FVII plasma activity and endogenous thrombin potential on the differential bleeding phenotype of hemophilia A patients.

CLINICAL AND HEMORRHAGIC PROFILES OF HEMOPHILIA A FEMALE CARRIERS OF INTRON 22 INVERSION

According to the Ministry of Health, Brazil had 10,821 patients diagnosed with hemophilia A (HA) in 2019 and with Minas Gerais had the third largest population of HA patients. Fundação Hemominas is a reference treatment center in this state for the clinical and laboratory care of these patients. HA is an X-linked coagulopathy caused by a deficiency of coagulation factor VIII (FVIII). Almost all patients are male, and females are considered carriers if they have an X chromosome with a mutation in the FVIII gene. In addition, it is estimated that there are 1.6 female carriers for every male HA patient. HA Carriers are classified into five categories based on residual plasma FVIII level (FVIII:C) and phenotype: (i) FVIII:C > 40% with bleeding phenotypes are classified as symptomatic; (ii) FVIII:C > 40% without bleeding phenotypes are classified as asymptomatic; (iii) FVIII:C > 5% to 40% are classified as mild HA; (iv) FVIII:C 1% to 5% are classified as moderate HA; and (v) FVIII:C < 1% are classified as severe HA. The aim of this study was to analyze the clinical and hemorrhagic profiles of female relatives of patients with severe HA with intron-22 inversion treated at Fundação Hemominas and older than 12 years. To date, sixteen participants have been enrolled in the study after signing an informed consent form. Participants answered two questionnaires: 1) evaluation of bleeding - ISTH (BAT-ISTH); and 2) evaluation of arthralgia. Peripheral blood samples were collected in a citrate tube and processed for DNA extraction. Genotyping to identify intron 22 inversion was performed by inverse PCR. Of the 16 participants, seven (44%) were identified as carriers of the mutation. Among the carriers, the median (MED) age was 35 years (IQR 34 - 35.5) and the bleeding score MED was 6 (IQR 4.8 - 9). In addition, carriers reported pain in the joints of the shoulders (3 – 42.9%), hips (2 – 28.6%), knees (2 – 28.6%), and ankles (2 – 28.6%). For participants who were not carriers, the age MED was 36 years (IQR 30 - 56) and the bleeding score MED was 4 (IQR 1 - 7). In addition, participants who were not carriers had pain in all joints studied, i.e., shoulders (3 – 33.33%), elbows (1 – 11.11%), hips (2 – 22.22%), knees (4 – 44.44%), and ankles (2 – 22.22%). Molecular diagnosis was important to identify family members with intron-22 inversion and to provide appropriate genetic counseling for family planning. A significant statistical difference (p = 0.019) was found between carriers and non-carriers in bleeding score. This is an ongoing study and the number of participants needs to be increased in order to draw conclusions. We thank all participants, Fapemig, Fundação Hemominas, and the CGSH of the Ministry of Health.

Treatment tactics for a patient with acquired hemophilia: continuous infusion of recombinant activated coagulation factor VII and the inhibitor eradication

Introduction. Acquired hemophilia is a rare autoimmune disease caused by an inhibitor to clotting factor VIII (FVIII). It complicates the course of many diseases, in particular autoimmune diseases, and in women is often associated with pregnancy.Aim — to present a case of successful treatment of a patient with acquired hemophilia using long-term continuous infusion of rFVIIa and inhibitor eradication as a result of immunosuppressive therapy.Main findings. A clinical observation of severe hemorrhagic syndrome in a patient with acquired hemophilia associated with pregnancy is presented. Uterine bleeding in the patient after spontaneous delivery, refractory to standard hemorrhagic syndrome treatment, required multiple surgical interventions. Laboratory tests showed prolongation of APTT, CT in the INTEM rotational thromboelastometry test, a decrease in plasma FVIII activity and presence of inhibitor to FVIII. Vacuum-assisted closure was applied to treat infected laparotomy wound. Therapy for acquired hemophilia consisted of hemostatic therapy and the inhibitor eradication. Hemostatic therapy included a continuous infusion of rFVIIa at a rate of 30 μg/kg/h with a gradual decrease up to 9.6 μg/kg/h, as well as its fractional administration before every surgery at a dosage of 80 μg/kg. The effectiveness of the therapy was assessed by the dynamics of CT in the INTEM test and the shortening of the APTT. The inhibitor eradication was achieved by prednisolone therapy, combined immunosuppression with rituximab and azathioprine, followed by its replacement with cyclophosphamide. As a result, the hemorrhage was stopped, reference plasma activity of FVIII and eradication of the inhibitor were reached.

A prospective, multicenter, clinical Study to evaluate the Safety, Pharmacokinetics, and Efficacy of Bleed Outcomes, with HemoRel-A® in severe Hemophilia A Patients.

To evaluate efficacy for an on-demand treatment of acute bleeding events, pharmacokinetics, safety, and tolerability of HemoRel-A® in severe hemophilia A. A total of 44 male subjects with severe hemophilia A with an annualized bleed rate of 12 while on-demand treatment with factor VIII (FVIII) were enrolled in the study and received HemoRel-A® for bleed treatment. The efficacy of HemoRel-A® was evaluated based on a four-point scale (excellent, good, moderate, or none). Six-point pharmacokinetic (PK) assessment was performed following a single dose of 50 IU/kg in 12 subjects after a 7-day wash-out period. Safety evaluations were performed at each visit and inhibitor testing was performed in all patients at screening and end of study. Forty-four male subjects received at least a single dose of the study medication and were included in the intent-to-treat (ITT) analysis and safety outcome. In 23 (7.52%) out of the 306 bleeding events, HemoRel-A® efficacy was rated as excellent, in 272 (88.89 %) bleeds it was rated as good, and in 11 (3.68%) bleeding events it was rated as moderate. No failure of efficacy was noted in any of the bleeding events. Thus overall out of 306 bleeding events, 295 (96.41%) showed excellent or good efficacy. Pharmacokinetic assessment based on plasma FVIII activity measured by the chromogenic assay in 12 patients showed comparative results similar to FVIII preparations. A total of 12 adverse events (AEs) were reported in this study. There was no inhibitor development in this previously treated patients (PTP) cohort. HemoRel-A® was established to be efficacious and safe in the treatment of acute bleeding events in subjects with severe hemophilia A. CTRI/2018/05/013790. Registration date: 9th May 2018.

Influence of N-glycosylation in the A and C domains on the immunogenicity of factor VIII

AbstractThe most significant complication in hemophilia A treatment is the formation of inhibitors against factor VIII (FVIII) protein. Glycans and glycan-binding proteins are central to a properly functioning immune system. This study focuses on whether glycosylation of FVIII plays an important role in induction and regulation of anti-FVIII immune responses. We investigated the potential roles of 4 N-glycosylation sites, including N41 and N239 in the A1 domain, N1810 in the A3 domain, and N2118 in the C1 domain of FVIII, in moderating its immunogenicity. Glycomics analysis of plasma-derived FVIII revealed that sites N41, N239, and N1810 contain mostly sialylated complex glycoforms, while high mannose glycans dominate at site N2118. A missense variant that substitutes asparagine (N) to glutamine (Q) was introduced to eliminate glycosylation on each of these sites. Following gene transfer of plasmids encoding B domain deleted FVIII (BDD-FVIII) and each of these 4 FVIII variants, it was found that specific activity of FVIII in plasma remained similar among all treatment groups. Slightly increased or comparable immune responses in N41Q, N239Q, and N1810Q FVIII variant plasmid-treated mice and significantly decreased immune responses in N2118Q FVIII plasmid-treated mice were observed when compared with BDD-FVIII plasmid-treated mice. The reduction of inhibitor response by N2118Q FVIII variant was also demonstrated in AAV-mediated gene transfer experiments. Furthermore, a specific glycopeptide epitope surrounding the N2118 glycosylation site was identified and characterized to activate T cells in an FVIII-specific proliferation assay. These results indicate that N-glycosylation of FVIII can have significant impact on its immunogenicity.