Factor-mimetic and rebalancing therapies in hemophilia A and B: the end of factor concentrates?

Hemophilia A (HA) and B are inherited bleeding disorders caused by a deficiency of factor VIII or factor IX, respectively. The current standard of care is the administration of recombinant or purified factor. However, this treatment strategy still results in a high economic and personal burden to patients, which is further exacerbated by the development of inhibitors—alloantibodies to factor. The treatment landscape is changing, with nonfactor therapeutics playing an increasing role in what we consider to be the standard of care. Emicizumab, a bispecific antibody that mimics the function of factor VIIIa, is the first such nonfactor therapy to gain US Food and Drug Administration approval and is rapidly changing the paradigm for HA treatment. Other therapies on the horizon seek to target anticoagulant proteins in the coagulation cascade, thus “rebalancing” a hemorrhagic tendency by introducing a thrombotic tendency. This intricate hemostatic balancing act promises great things for patients in need of more treatment options, but are these other therapies going to replace factor therapy? In light of the many challenges facing these therapies, should they be viewed as a replacement of our current standard of care? This review discusses the background, rationale, and potential of nonfactor therapies as well as the anticipated pitfalls and limitations. This is done in the context of a review of our current understanding of the many aspects of the coagulation system.

The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

Clinical, humanistic, and economic burden of severe haemophilia B in adults receiving factor IX prophylaxis: findings from the CHESS II real-world burden of illness study in Europe

Background Real-world studies of the burden of severe haemophilia B in the context of recent therapeutic advances such as extended half-life (EHL) factor IX (FIX) products are limited. We analysed data from the recent CHESS II study to better understand the clinical, humanistic, and economic burden of severe haemophilia B in Europe. Data from male adults with severe haemophilia B receiving prophylaxis were analysed from the retrospective cross-sectional CHESS II study conducted in Germany, France, Italy, Spain and the United Kingdom. Inhibitors were exclusionary. Patients and physicians completed questionnaires on bleeding, joint status, quality of life, and haemophilia-related direct and indirect costs (2019–2020). All outcomes were summarised using descriptive statistics. Results A total of 75 CHESS II patients were eligible and included; 40 patients (53%) provided self-reported outcomes. Mean age was 36.2 years. Approximately half the patients were receiving EHL versus standard half-life (SHL) prophylaxis (44% vs 56%). Most patients reported mild or moderate chronic pain (76%) and had ≥ 2 bleeding events per year (70%), with a mean annualised bleed rate of 2.4. Mean annual total haemophilia-related direct medical cost per patient was €235,723, driven by FIX costs (€232,328 overall, n = 40; €186,528 for SHL, €290,620 for EHL). Mean annual indirect costs (€8,973) were driven by early retirement or work stoppage due to haemophilia. Mean quality of life (EQ-5D) score was 0.67. Conclusions These data document a substantial, persistent real-world burden of severe haemophilia B in Europe. Unmet needs persist for these patients, their caregivers, and society.

The coagulation factor IX (F9) loss of function prevents the cell cycle arrest induced by CDK4/6 inhibitors treatment

During this last decade the development of pro-senescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and have showed efficacy in reducing tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their anti-tumour activity. Here, using a functional genome wide CRISPR/Cas9 genetic screen we found several genes that synergistically participate in the proliferation arrest induced by the CDK4/6 inhibitor, Palbociclib. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout reduces senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitors response that will be useful to design new therapeutic strategies in personalized medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

Sensitivity analysis of a reduced model of thrombosis under flow: Roles of Factor IX, Factor XI, and γ‘-Fibrin

A highly reduced extrinsic pathway coagulation model (8 ODEs) under flow considered a thin 15-micron platelet layer where transport limitations were largely negligible (except for fibrinogen) and where cofactors (FVIIa, FV, FVIII) were not rate-limiting. By including thrombin feedback activation of FXI and the antithrombin-I activities of fibrin, the model accurately simulated measured fibrin formation and thrombin fluxes. Using this reduced model, we conducted 10,000 Monte Carlo (MC) simulations for ±50% variation of 5 plasma zymogens and 2 fibrin binding sites for thrombin. A sensitivity analysis of zymogen concentrations indicated that FIX activity most influenced thrombin generation, a result expected from hemophilia A and B. Averaging all MC simulations confirmed both the mean and standard deviation of measured fibrin generation on 1 tissue factor (TF) molecule per μm2. Across all simulations, free thrombin in the layer ranged from 20 to 300 nM (mean: 50 nM). The top 2% of simulations that produced maximal fibrin were dominated by conditions with low antithrombin-I activity (decreased weak and strong sites) and high FIX concentration. In contrast, the bottom 2% of simulations that produced minimal fibrin were dominated by low FIX and FX. The percent reduction of fibrin by an ideal FXIa inhibitor (FXI = 0) ranged from 71% fibrin reduction in the top 2% of MC simulations to only 34% fibrin reduction in the bottom 2% of MC simulations. Thus, the antithrombotic potency of FXIa inhibitors may vary depending on normal ranges of zymogen concentrations. This reduced model allowed efficient multivariable sensitivity analysis.