Factor V Research Articles (Page 1)

Congenital factor V (FV) deficiency, affecting approximately 1 in 1 million individuals worldwide, remains among the rare bleeding disorders (RBDs) without a licensed factor-specific replacement therapy. While other RBDs have successfully transitioned from plasma-based treatment to approved factor concentrates-exemplified by factor X deficiency's progression to US Food & Drug Administration (FDA)-approved Coagadex and two FDA-approved concentrates for factor XIII deficiency-FV deficiency treatment has remained unchanged for decades, relying solely on plasma and platelet transfusions. Two promising therapeutic candidates have emerged: a human plasma-derived FV concentrate demonstrating in vitro correction of severe deficiency, and an engineered activated FV (superFVa) showing potent hemostatic activity in preclinical models. This commentary outlines a pragmatic pathway to clinical trials, leveraging proven development strategies from other RBDs, existing registry infrastructure, and regulatory incentives for rare diseases. We propose phased trials combining pharmacodynamic endpoints with clinical outcomes, enabling feasible enrollment while generating decision-grade evidence. The time has come to extend modern therapeutic development to FV deficiency.

BackgroundFactor XI (FXI) deficiency is a rare bleeding disorder, common among Ashkenazi Jews. Affected individuals may present severe bleeding after trauma or surgery, which requires careful management. To date FXI deficiency has not been genetically or clinically described in the Jordanian population.Material and methodsSeventeen patients (median age 29) from 16 Jordanian Arab families, referred to the hemostasis and thrombosis laboratory at the University of Jordan, due to bleeding history or prolonged activated partial thromboplastin time. FXI deficiency was confirmed by factor assay (cutoff: ≤ 40% activity). Bleeding score was recorded, and Next Generation Sequencing of the F11 gene was utilized to detect causative mutations.ResultsOf the 17 patients, 14 had severe FXI deficiency. The median bleeding score was 6. Five patients carried the known p.Glu135Ter (type II), while ten patients had different point mutations. Three patients with severe FXI deficiency were found to carry previously undescribed variants, including one novel splice site mutation (c.1136-1G > C). No pathogenic variants were identified in two patients.DiscussionThis is the first study of FXI deficiency in Jordanian Arabs, which revealed fourteen patients with severe FXI deficiency. Ten mutations were identified, including one novel splice-site mutation. The relevance of type II mutation in our cohort suggested a founder effect similar to Jewish ancestry. Bleeding severity was incongruent with FXI activity. Despite the small cohort and lack of functional assays, this study expands the mutational spectrum and provides new insights into FXI deficiency in Arab populations.

BackgroundHemophilia A is a rare, severe X-linked recessive inherited hemorrhagic disorder caused by F8 gene dysfunction, which is characterized by spontaneous or post-traumatic bleeding tendencies. The pathogenic variants identified in the F8 gene contribute to prenatal diagnosis and genetic counseling services for patients and their families.MethodsWe used inverse shifting-PCR (IS-PCR), direct DNA sequencing, bioinformatics predictions, cDNA sequencing, and minigene splicing assays to explore candidate variants in a Chinese family with hemophilia A. The identified variant was classified in accordance with ACMG/AMP guidelines.ResultsA novel c.6115+5_6115+6delinsAG variant at 5′ splice sites (5’ss) in exon 19 was identified in a 14-year-old Chinese boy with hemophilia A by DNA sequencing, which is inherited from his asymptomatic carrier mother. Multiple bioinformatics prediction tools, including SD-Score, information content (Ri), varSEAK, and RDDC RNA splicer, predicted that this variant might affect the normal pre-mRNA splicing. Both cDNA sequencing and minigene splicing assays proved that the variant led to exon 19 skipping in the F8 gene, which was ultimately classified as pathogenic according to the ACMG/AMP guidelines.ConclusionThe c.6115+5_6115+6delinsAG variant in the F8 gene is considered to be responsible for hemophilia A in this family. This dinucleotide variant located at 5’ss of the gene is initially reported. Our study has expanded the mutation spectrum of F8 and provided a basis for prenatal and clinical diagnosis.

F8 gene variants influence the response to desmopressin in hemophilia A carriers.

Thrombophilia is influenced by genetic variants, such as Factor V Leiden (FVL) and the prothrombin G20210A mutation. In clinical settings, assessing numerous genetic factors can lead to diagnostic errors and unnecessary treatments. This meta-analysis examines gene variants associated with thrombosis in the Iranian population, where their role in thrombotic disorders remains underexplored. A systematic literature search was performed across PubMed, Scopus, and Web of Science, targeting case-control studies published up to July 2025. Studies were included if they evaluated thrombophilia-related polymorphisms in Iranian patients with various thrombotic conditions, such as recurrent pregnancy loss (RPL), venous thromboembolism (VTE), or deep vein thrombosis (DVT). Advanced statistical analyses, including random-effects models, fixed-effects models, and Bayesian meta-analysis, were used to compute odds ratios (ORs) and 95% confidence intervals (CIs). From 36 studies encompassing over 14 000 participants, significant associations emerged. For RPL, FVL G1691A heterozygote (OR: 1.998, 95% CI: 1.02-3.88), methylenetetrahydrofolate reductase (MTHFR) C677T heterozygote (OR: 1.77, 95% CI: 1.31-2.39), MTHFR A1298C heterozygote (OR: 3.10, 95% CI: 1.33-7.20) and homozygote (OR: 1.69, 95% CI: 1.05-2.70), prothrombin G20210A heterozygote (OR: 2.435, 95% CI: 1.09-5.39) and homozygote (OR: 0.487, 95% CI: 0.40-0.58), plasminogen activator inhibitor-1 (PAI-1) polymorphisms, factor V (FV) A4070G, FV 5279A/G, factor XIII (FXIII) Val34Leu, and integrin subunit beta-3 (ITGB3)1565T/C were linked to elevated RPL risk. Additionally, FVL G1691A heterozygote (OR: 5.25, 95% CI: 2.39-11.54) was associated with higher VTE risk, while MTHFR C677T heterozygote (OR: 1.404, 95% CI: 1.030-1.914) increased DVT risk. These ethnicity-specific findings highlight critical genetic risk factors for thrombotic disorders in Iranians, potentially guiding precise diagnostics and personalized interventions.

Coagulation factor (F) VII deficiency is the most frequent among the rare, inherited bleeding disorders and is predominantly caused by missense mutations in the F7 gene. The disease phenotype ranges from asymptomatic cases to extremely severe hemorrhagic forms, requiring prophylactic injections with plasma-derived or recombinant FVII concentrates. In response, we have developed an autologous cell-based approach that corrects the disease-causing mutation in patient-derived induced pluripotent stem cells (iPSCs) and generates therapeutic, three-dimensional hepatic organoids (HOs). We report the CRISPRmediated correction of homozygous c.718G>C (p.G240R), a missense mutation associated with a severe, life-threatening bleeding phenotype. The HOs contain all liver cell types and exhibit key liver functions, including coagulation factor production. After correction, our data indicate that the patient-derived HOs secrete consistent amounts of functional FVII protein, resulting in improved thrombin generation times. These results represent a significant milestone toward the establishment of an autologous cell-based therapy for patients with FVII- and other coagulation factor deficiencies.

Factor XI (FXI) deficiency is an autosomal bleeding disorder characterized by low FXI levels, resulting in bleeding after trauma or surgery. Genetic variants affecting FXI structure and function often result in bleeding diatheses.This study aimed to estimate the variant detection rate (VDR), and assess its correlation with FXI activity (FXI:C) in a large cohort of FXI-deficient patients.Genetic defects in the F11 gene were analyzed in 316 index patients (IPs) using Sanger or next-generation sequencing. Multiplex ligation-dependent probe amplification or copy number variation analysis was used to detect duplications and deletions.Genetic defects were identified in 249 IPs (VDR of 79%). A strong negative correlation (Pearson coefficient: -0.891) was found between FXI:C levels and VDRs: higher FXI:C levels corresponded to a lower likelihood of detecting genetic alterations, with a significant decline in VDR beyond 60 IU/dL. A total of 286 genetic variants were identified in F11 gene: 56% missense, 24% nonsense, 11% small deletions/insertions, and 6% splice-site variants. Large deletions were rare (3%). A total of 48 novel variants were detected. Ashkenazi Jewish founder variants were the most frequent (14.3%). Variants p.Gln134Ter, p.Ile215_Asp216del, and p.Glu315Lys (27% of cases) were recurrent. In four cases, large deletions extended beyond the F11 gene and included the neighboring KLKB1 gene, encoding prekallikrein.This study demonstrated a significant negative correlation between FXI:C levels and VDRs, underscoring the importance of genetic testing. Findings included combined deficiencies in FXI and prekallikrein due to large deletions affecting both F11 and KLKB genes.

Congenital factor VII deficiency is a rare autosomal recessive bleeding disorder characterized by markedly heterogeneous clinical phenotypes and reduced plasma factor VII activity. We present the 5-year-old Chinese boy who exhibited only mild intermittent epistaxis despite a factor VII:C level of 1.5% and compound heterozygous missense mutations in the F7 gene, c.722C>A (p.T241N) and c.1165T>G (p.C389G). Notably, no gastrointestinal, intracranial, or joint hemorrhages occurred. Bleeding symptoms were effectively controlled using fresh frozen plasma followed by recombinant activated factor VII. This case highlights the discordance between biochemical severity and clinical phenotype in factor VII deficiency and underscores the importance of early coagulation testing and molecular diagnosis in pediatric patients with unexplained mucosal bleeding.

Rare bleeding disorders (RBDs), defined as hereditary coagulation factor deficits other than haemophilia, are characterized by a heterogenous clinical phenotype ranging from life-threatening bleeding to thrombosis. There are uncertainties concerning treatment intensity and levels needed to achieve haemostasis, and epidemiological data from Germany, Austria, and Switzerland (GTH region) are scarce.We performed a narrative literature review, focusing on bleeding phenotype and thrombotic risk. Epidemiologic data, including adults and children, and general treatment approaches have been collected via an online survey among GTH haemophilia centres (all categories) and the general information service of the German national registry (Deutsches Hämophilieregister, DHR).We provided an overview on RBDs, revealing that especially in FV, FVII, and FXI deficiencies, the correlation between factor levels and bleeding phenotype is poor. A thrombotic risk needs to be considered in FVII deficiency and afibrinogenaemia or dysfibrinogenaemia. The survey was completed by 34 centres from Germany, Austria, and Switzerland, and compared with 137 centres reporting data to the DHR. FVII deficiency was confirmed to be the most frequent, and FII deficiency was the rarest RBD in this region. For treatment, single factor concentrates were preferred over multifactor concentrates or plasma, and tranexamic acid was often part of the treatment. Approximately 30, 40, and <10% of patients with severe FV, FVII, and FXI deficiency (defined as factor level <10%), respectively, were receiving prophylactic treatment, suggesting an overall milder bleeding phenotype.More detailed registry data could give insights into the treatment landscape of RBDs, considering the challenge of clinical trials in rare diseases.

Large deletions in the F8 gene predict immune tolerance induction failure in people with severe hemophilia A

Factor XI (FXI) deficiency, or hemophilia C, is a rare bleeding disorder resulting from reduced levels or dysfunctional FXI protein due to mutations in the F11 gene. This study investigated the correlation between FXI activity levels, F11 genotype, and bleeding phenotypes. Clinical and genetic characteristics of 93 individuals from southern Italy diagnosed with congenital FXI deficiency, including 39 index cases and their relatives, were evaluated. FXI:C plasma levels were measured. Sanger sequencing of F11 was performed, and the pathogenicity of variants identified was assessed using in silico tools. FXI activity levels ranged widely (1–69%), with most cases being heterozygous and showing moderate deficiency. Only 12 individuals had severe FXI deficiency, typically associated with homozygosity or compound heterozygosity. Bleeding symptoms varied from mild to severe and occurred in 31% of subjects, though only a minority of those with severe deficiency experienced spontaneous or surgery-related bleeding. Sanger sequencing revealed 24 distinct F11 gene variants, predominantly missense mutations, with three novel variants (p.Val89*, p.Leu306Pro, and p.Trp515Gly). Common mutations included p.Glu135* and p.Glu315Lys. Variants were distributed across the gene, with no domain-specific clustering. No clear genotype–phenotype correlation was observed. FXI levels alone did not reliably predict bleeding risk, highlighting the influence of additional factors such as age, gender, and clinical history. This study reinforces the allelic and clinical heterogeneity of FXI deficiency and the limited utility of FXI:C levels alone for predicting bleeding severity. Further research is needed to clarify the complex genotype–phenotype relationships in FXI deficiency.

Insights From the First 820 Patients From the Brazilian Multicenter Registry of Hereditary Angioedema: The Key Role of Genetic Testing and Targeted Therapies.

Hemophilia A and B are X-linked bleeding disorders caused by mutations in the F8 and F9 genes, resulting in deficiencies of coagulation factors VIII (FVIII) and IX (FIX), respectively. A major complication of replacement therapy is the development of neutralizing antibodies (inhibitors), which occur in approximately 30% of patients with severe hemophilia A and about 3% of those with hemophilia B. The role of missense and nonsense mutations in inhibitor formation has been increasingly recognized. In hemophilia A, missense mutations within immunogenic domains may alter FVIII structure, eliciting immune responses. Nonsense mutations especially those located in the light chain are associated with higher inhibitor risk due to the production of truncated, non-functional proteins. In hemophilia B, missense mutations rarely result in inhibitor development, whereas nonsense mutations and large deletions carry a significantly higher risk. Molecular genotyping contributes to predicting inhibitor formation and supports individualized treatment planning.

To explore the clinical characteristics and genetic variants underlying Hereditary coagulation factor V (FV) deficiency in two Chinese pedigrees. Seventeen individuals from three generations of the two pedigrees affected with FV deficiency whom had visited Taizhou Hospital of Zhejiang Province respectively in March and June 2024 were recruited as study subjects. One hundred healthy individuals undergoing physical examinations have served as the controls. Relevant coagulation parameters were measured. Thrombin generation was assessed using the calibrated automated thrombogram (CAT) assay. All exons and flanking regions of the F5 gene were amplified by PCR and directly sequenced. Candidate variants were analyzed for evolutionary conservation and potential pathogenicity, and their effects on protein structure were predicted. This study was approved by the Medical Ethics Committee of Taizhou Hospital of Zhejiang Province (Ethics No.: 20230722). The FV activity (FV: C) and antigen levels (FV: Ag) of both probands showed concurrent decrease. By thrombin generation assay, both the lag time ratio and time to peak ratio were significantly increased. Genetic analysis revealed that proband A carried compound heterozygous missense variants c.911G>A (p.Gly304Glu) and c.1238T>C (p.Met413Thr), whilst Proband B carried compound heterozygous missense variants c.1258G>T (p.Gly420Cys) and c.1538G>A (p.Arg513Lys) of the F5 gene. Conservation analysis revealed that the amino acid residues p.Gly304, p.Gly420, and p.Arg513 are highly conserved across various species. Online bioinformatics tools predicted that both the p.Gly304Glu and p.Gly420Cys variants are pathogenic. Protein modeling demonstrated that all four variants can result in alterations of protein structure or disruption of hydrogen bonding. FV deficiency in these two pedigrees can be attributed to the compound heterozygous variants p.Gly304Glu/p.Met413Thr and p.Gly420Cys/p.Arg513Lys of the F5 gene.

To explore the phenotypic and genotypic characteristics of a Chinese pedigree affected with Hereditary coagulation factor XI (FXI) deficiency. A female patient with FXI deficiency and her family members (five individuals from three generations) who presented at Quanzhou First Hospital Affiliated to Fujian Medical University on September 19, 2024 due to diarrhea and fever were selected as study subjects. A retrospective study was conducted to collect the patients' clinical data. Peripheral venous blood samples were collected from the patient and her family members. Genomic DNA was extracted, followed by sequencing of all exons and flanking sequences of the F11 gene. Candidate variants were validated by Sanger sequencing of the family members, and their pathogenicity was classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Quanzhou First Hospital [Approval No.: Quanyi Lun (2024) K281]. The patient exhibited significantly prolonged activated partial thromboplastin time (APTT) of 80.9 seconds, while FXI activity (FXI:C) and FXI antigen (FXI:Ag) levels were extremely low (2% and 3%, respectively). Genetic analysis revealed that the proband harbored homozygous c.896C>G (p.Thr299Ser) missense variant in exon 9 of the F11 gene, for which her son was heterozygous. The variant was located in a highly conserved domain. Although Mutation Taster predicted it as a polymorphism, SIFT, PolyPhen-2, and LRT analyses suggested it to be likely pathogenic. Protein modeling indicated that the p.Thr299Ser variant may alter the hydrogen bonds between amino acids, thereby affecting the structure and function of the FXI protein. According to the ACMG guidelines, c.896C>G was rated as a likely pathogenic variant (PM1+PM2_Supporting+PP1_Strong+PP3+PP4). The c.896C>G (p.Thr299Ser) missense variant of the F11 gene probably underlay the FXI deficiency in this pedigree. Above finding has enriched the mutational spectrum of the F11 gene and provided a basis for genetic counseling and prenatal diagnosis for this family.

Venoms of the Palearctic vipers in the Macrovipera genus cause severe procoagulant clinical effects, yet the precise molecular targets remain incompletely defined. To fill this toxicological knowledge gap, we tested five Macrovipera venoms—M. lebetina cernovi, M. l. obtusa, M. l. turanica (Turkmenistan and Uzbekistan localities), and M. schweizeri—using plasma clotting assays, Factors VII, X, XI, and XII and prothrombin zymogen activation assays, and SDS-PAGE to visualise Factor V (FV) cleavage. All venoms induced extremely rapid clot formation (10.5–12.5 s) compared with the negative control (spontaneous clotting) of 334.6 ± 3.6 s) and the positive control (kaolin trigger) of 55.8 ± 1.9 s. Activation of FVII or FXI was negligible, whereas consistent FX activation and species-variable FXII activation, both moderate, were observed. Prothrombin remained inert in the absence of cofactors, but the presence of FV or FVa elicited potent thrombin generation. SDS-PAGE confirmed proteolytic conversion of the 330 kDa FV zymogen into the ~105 kDa heavy and ~80 kDa light chains of FVa by the venoms of all species. This data demonstrates that Macrovipera venoms rely on a dual enzyme strategy: (i) activation of FV to FVa by serine proteases and (ii) FVa-dependent prothrombin activation by metalloproteases. These results reveal that prothrombin activation is the dominant procoagulant pathway and overshadows the historically emphasised FX activation. This mechanism mirrors, yet is evolutionarily independent from, the FXa:FVa prothrombinase formation seen in Australian elapid venoms, highlighting convergent evolution of cofactor-hijacking strategies among snakes. The discovery of potent FVa-mediated prothrombin activation in Macrovipera challenges existing paradigms of viperid venom action, prompts re-evaluation of related genera (e.g., Daboia), and underpins the design of targeted antivenom and therapeutic interventions.

We report on a 10-year-old female diagnosed with severe congenital FV deficiency who developed anti-FV inhibitory antibody 3 weeks following dental procedure hemorrhage treated with FFP. The patient presented a large spontaneous gluteal hematoma. Despite multiple FFP transfusions, FV levels remained critically low, prompting suspicion of FV inhibitor development, confirmed by ELISA. Bleeding control was achieved with recombinant factor VIIa. An immune tolerance induction protocol was initiated, and administration of FFP was continued. Factor V inhibitors became undetectable after 1 year and the patient had a favorable clinical evolution without further bleeding episodes. This case reports the successful eradication of FV inhibitor using FFP exposure and immunosuppressive therapy.

Background and Aims: Congenital bleeding disorders, including hemophilia A (HA), hemophilia B (HB), and rare bleeding disorders (RBDs), often display variable clinical phenotypes not fully explained by a single pathogenic variant. Additional genetic variants modulating the coagulation cascade may influence bleeding severity. This study aimed to investigate such variants using a multigenic sequencing approach. Methods: Eighteen patients with a previously confirmed diagnosis of congenital bleeding disorder were selected for analysis. The cohort included individuals with HA, HB, FV deficiency, FXI deficiency, and plasminogen deficiency, all followed at the Hemophilia Center of Padua University Hospital. Genetic analysis was performed using a custom Next-Generation Sequencing (NGS) panel (xGen™ Hyb Panel, Integrated DNA Technologies) targeting 33 coagulation-related genes: A2M, CPB2, F2, F3, F5, F7, F8, F9, F10, F11, F12, F13A1, F13B, FGA, FGB, FGG, KLKB1, KNG1, PLAT, PLG, PROC, PROCR, PROS1, PROZ, SERPINA5, SERPINA10, SERPINC1, SERPIND1, SERPINE1, SERPINF2, TFPI, THBD, VWF. Sequencing was performed on the Illumina NextSeq550 platform. Variants with a minor allele frequency &lt;0.1% were classified according to ACMG criteria. Results: The study population consisted of 10 patients with HA (1 mild, 1 moderate, 8 severe), 2 with HB (1 mild, 1 severe), 1 with FV deficiency, 1 with FXI deficiency, and 4 with plasminogen deficiency. On average, 150 variants per patient were observed. NGS identified class 5 pathogenic variants in 8 patients with HA (F8, including intron 22 inversion) and in 2 with HB (F9), and class 4 likely pathogenic variants in 3 patients (F8 [n=2] and, F11 [n=1]). Although only class 3 variants were identified in patients with plasminogen deficiency (n=4) and FV deficiency (n=1), these were located in the gene associated with the clinical phenotype (PLG and F5, respectively). Moreover, two plasminogen-deficient patients harboured two distinct variants in PLG. Beyond the causative mutations, 11 out of 18 patients (61%) harboured at least one variant of class 3 or 4 with potential modulatory effect. These were found in F2, F5, F10, F13A1, KNG1, VWF, SERPINA5, SERPINC1, PROS1, and PLG and included well-characterized variants such as FV Leiden, the prothrombin G20210A mutation (prothrombotic), and the Friuli FX variant (bleeding phenotype). Moreover, two predisposing variants were identified in F13A1 (n=2, pro-hemorrhagic) and F2 (n=1, prothrombotic). The distribution of class 3, 4, and 5 variants and predisposing variants is illustrated in Figure 1. Conclusions: NGS identified the putative causative mutation in all patients and revealed additional coagulation-related variants in the majority of the cohort. These results support a multigenic model influencing the bleeding phenotype in congenital bleeding disorders. Functional and segregation studies are warranted to clarify the role and clinical impact of the predisposing variants identified.

Background and Aims: Hemophilia A (HA) is a rare X-linked recessive bleeding disorder caused by variants in the F8 gene, which encodes coagulation factor VIII (FVIII). It affects approximately one in 6,000 males. The severity of HA is determined by the level of plasma FVIII activity and is classified as severe (&lt;1%), moderate (1%–5%), or mild (5%–40%). In patients with HA, increasing FVIII activity to above 2–5% can significantly improve quality of life. The aim of this study was to restore FVIII levels to prevent spontaneous bleeding by correcting FVIII variants using CRISPR-Cas9 base editing in liver sinusoidal endothelial cells (LSECs), which are recognized as a key FVIII producers’ cells. Methods: Peripheral blood mononuclear cells (PBMC) from 3 HA patient and 2 healthy subjects were isolated and reprogrammed into induced pluripotent stem cells (iPSC) after transduction in a feeder-free culture with a non-integrative system with Sendai virus. The clones obtained were stabilized using an enzymatic method with EDTA and characterised for their pluripotency markers expression. Gene correction of the HA-iPSCs will be carried out using CRISPR-Cas9 DNA base editing technologies with specially target of FVIII variations. The following gene variants NM_000132.4:c.6593G&gt;T, NM_000132.4:c.4285C&gt;T and NM_000132.4:c.5373+ 1G&gt;A were considered. Subsequently, HA-iPSCs will be differentiated into liver sinusoidal endothelial cells (LSECs) and hepatocyte-like cells (HLCs) using an optimized differentiation protocol involving BMP4 and VEGF and others growth factors. Specific cells markers were analysed and the expression of FVIII, LSEC and HLCS markers, were evaluated using immunofluorescence (IF) and real-time PCR (RT-qPCR). Results: Obtained iPSCs was positive for alkaline phosphatase staining and immunofluorescence showed the expression of stem cells markers (Oct4, SeV, Sox2, Klf4, KOS and cMyc). The LSECs and HLCs differentiated obtained cells were stained positive for the expression of CD31, STAB2 and LYVE1 and cytokeratin 18 respectively. Following RT-qPCR and flow cytometry analysis expression of the above markers confirmed the obtained cells phenotype. Furthermore, the expression of FVIII were assessed by IF and flow cytometry in the HA-patients obtained cells before and after gene editing and the correction was confirmed. Conclusions: In this study, we generated specific iPSCs from HA-patients carrying different F8 gene variants, derived from PBMCs. These iPSCs were then differentiated in vitro into LSECs and HLCs using optimized protocols. CRISPR-Cas9 mediated gene correction enabled us to demonstrate that specific disease-causing variants can be effectively repaired, resulting in the restoration of FVIII expression. The LSECs and HLCs obtained through these protocols represent a useful in vitro model for evaluating FVIII restoration in HA-patients with different F8 gene variants. Given the limitations of current treatments, new strategies for a definitive cure are urgently needed. Gene and cell therapies offer promising avenues and could provide a powerful, personalized therapeutic approach to optimize treatment for each individual HA-patient.

Background: Hemostasis is governed by a dynamic balance between procoagulant and anticoagulant forces. While inherited bleeding and thrombotic disorders are typically studied as discrete entities, rare cases of co-inheritance may produce unique clinical phenotypes due to compensatory interactions. Among these, hemophilia A and antithrombin (AT) deficiency represent prototypical yet contrasting conditions: patients with severe hemophilia A often experience spontaneous joint and muscle bleeding, requiring lifelong prophylaxis or on-demand therapy with FVIII concentrates, whereas AT deficiency is associated with a markedly increased risk of venous thromboembolism (VTE), particularly in high-risk situations such as surgery, trauma, or prolonged immobilization. Understanding how opposing defects interact within the same individual may inform individualized management and therapeutic innovation. Case Report: We describe the case of a 19-year-old male with severe hemophilia A (FVIII &lt;1%, no inhibitors), carrying a mutation in exon 3 of the F8 gene (NM_000132.4:c.296A&gt;G, resulting in p.His99Arg), co-inherited with congenital AT deficiency (~50% activity). At birth, neonatal screening was prompted by a maternal family history of hemophilia A and a paternal history of thrombophilia. The patient’s father had been diagnosed with AT deficiency following a life-threatening episode of VTE involving extensive ilio-caval thrombosis and pulmonary embolism. Despite the severe FVIII deficiency, the patient has remained asymptomatic, with an annual bleeding rate (ABR) of zero and no history of spontaneous hemorrhage. He has only received perioperative FVIII prophylaxis and has remained free of thrombotic complications. Thrombin generation assay (TGA) showed delayed initiation and propagation, reduced peak and velocity index, but preserved endogenous thrombin potential (ETP), suggesting a compensatory procoagulant effect from AT deficiency (Figure 1). Although the patient demonstrated impaired thrombin amplification – consistent with FVIII deficiency – the preserved ETP suggests that decreased anticoagulant activity due to AT deficiency provides sufficient compensatory procoagulant effect to maintain clinical hemostasis. This dynamic interplay reflects a finely tuned compensatory mechanism that may alter the clinical expression of inherited bleeding disorders. Conclusions: This case exemplifies a naturally rebalanced hemostatic state resulting from the dual inheritance of opposing coagulation traits, and aligns with previously reported cases. The preserved ETP despite FVIII deficiency supports the concept of compensatory interplay and provides a rationale for rebalancing therapies such as Fitusiran, a small interfering RNA (siRNA) agent that suppresses hepatic AT production. By mimicking this compensatory mechanism, Fitusiran enhances thrombin generation and may improve coagulation in hemophilia patients, including those with inhibitors. Recognition of such rare but informative genetic constellations not only improves diagnostic accuracy, but also has significant implications for therapeutic decision-making. Comprehensive coagulation profiling, including genetic and functional assays, is essential for characterizing atypical presentations and guiding personalized treatment strategies.