Search Results (56)

Gene therapy is reshaping the therapeutic paradigm in hemophilia by enabling sustained endogenous clotting factor production after a single administration. This approach moves disease management beyond lifelong replacement therapy. While clinical trials have demonstrated marked reductions in bleeding rates and treatment burden, real-world implementation has revealed emerging complexities. These include interindividual variability in transgene expression reflected by a progressive reduction in circulating FVIII or FIX activity over time, uncertainty regarding the long-term durability of expression, immune-mediated constraints, and episodes of transaminase elevation. This review addresses a critical transition point in the field: the shift from proof-of-concept efficacy toward integration of gene therapy into long-term hemophilia care. We examine determinants of therapeutic stability, host–vector immune interactions, and mechanisms underlying loss or fluctuation of expression, with emphasis on monitoring strategies and post-therapy management pathways. Immunogenic processes affecting vector transduction, hepatocellular responses, and transgene persistence are discussed alongside current approaches to immune modulation. This review uniquely focuses on post-gene therapy clinical integration rather than vector design or trial outcomes. Beyond direct factor correction, evolving therapeutic concepts targeting coagulation rebalancing and immune regulation are considered within a systems-based framework. Psychosocial adaptation and patient-reported outcomes are also explored, underscoring that therapeutic success extends beyond hemostatic control. In aggregate, these perspectives position gene therapy not as a singular curative event but as a component of an evolving, biologically integrated management strategy. Long-term follow-up translational research (LTFU) and coordinated global efforts will be essential to optimize durability, safety, and equitable access. Full article

Background/Objectives: Patients with severe hemophilia A on prophylaxis with emicizumab exhibit a mild/moderate bleeding phenotype that requires the use of either recombinant FVIII (rFVIII) or bypassing agents (BPAs) in patients with inhibitors, in the case of breakthrough bleeding or surgery. Since factor IX (FIX) limits the formation of the FIXa–emicizumab–FX complex, exogenously added FIX might enhance complex formation and thrombin generation. This study aimed to compare the procoagulant effects of various FIX concentrates with recombinant activated FVII (rFVIIa), activated prothrombin complex concentrate (aPCC), and rFVIII in SHA patients with and without inhibitors under emicizumab prophylaxis. Methods: Hemostatic changes were monitored using two optimized global coagulation assays: rotational thromboelastometry and calibrated automated thrombin generation. Tubes containing corn trypsin inhibitor (CTI) were used during blood collection to prevent activation. Low concentrations of tissue factor (TF) were used to trigger coagulation in both assays. Results: Ex vivo addition of recombinant FIX concentrates significantly increased the procoagulant activity of emicizumab, achieving levels comparable to therapeutic doses of rFVIIa or rFVIII, and the proportion of active FIXa within the concentrates is a major contributor to their procoagulant function. We assessed the influence of FVIII inhibitors on the hemostatic efficacy of rFIX concentrates and BPAs, finding that rFIX-induced thrombin generation increased in the presence of inhibitors, and no significant differences were observed with BPAs. Conclusions: These findings suggest that FIX concentrates could be an effective alternative to BPAs for emicizumab-treated patients, particularly those with inhibitors. Further studies are needed to confirm their in vivo efficacy and to evaluate thrombotic risk. Full article

Background/Objectives: Autoimmune factor VIII deficiency (AiFVIIID) is a rare disorder that causes severe bleeding. Emicizumab has recently been found to be effective in treating AiFVIIID; however, monitoring with standard coagulation tests presents challenges. Methods: Clot waveform analysis (CWA), which involves CWA-activated partial thromboplastin time (APTT), the CWA-small amount of tissue factor activation assay (sTF/FIXa), and clotting time using a small amount of thrombin (sTT), was used to both diagnose AiFVIIID and monitor emicizumab. Results: CWA-sTT reflects the residual FVIII activity in patients with AiFVIIID. Several tests were employed, including APTT, FVIII activity, CWA, mixing tests with normal plasma, FVIII inhibitor assays, and anti-FVIII antibody activity for the diagnosis of AiFVIID in three cases. However, the sensitivity of APTT reagents to AiFVIID differed between thrombocheck-APTT and APTT-SP. Emicizumab treatment was effective for major bleeding, and anti-FVIII antibody activity could be measured using CWA-sTT. Conclusions: The sensitivity of APTT reagents to AiFVIID varies. CWA-sTT may provide utility in the diagnosis of AiFVIIID. Emicizumab is useful for the treatment of AiFVIID, and anti-FVIII antibody activity can be measured even in patients treated with emicizumab. Full article

Background: Legg–Calvé–Perthes disease (LCPD) is a rare avascular osteonecrosis of the proximal femoral epiphysis and typically occurs during the childhood growth phase. LCPD is a complex illness of unknown origin, which is considered the main difficulty in the study of this disease. Various theories on LCPD etiology have been proposed; however, no consensus has been reached about its origin. Our research objective was to evaluate the polymorphisms FVL rs6025, FVIII rs5987061, FIX Malmö rs6048, PAI-1 rs1799889, eNOS rs17899983/rs2070744, IL-23R rs1569922/rs154655686/7539625, and TNF-α rs180062, and their relationship with LCPD. Methods: A blood sample was taken from each study participant. Complete blood count, coagulation times and factors, antithrombotic proteins, and homocysteine (Hcy) were determined using a coagulometric method. DNA was obtained and genotyped using real-time PCR with TaqMan probes. Genotypic and allelic distributions were analyzed using comparative analysis, the Hardy–Weinberg equilibrium, and OR. Results: This study included 46 children: 23 with LCPD (cases) and 23 without (controls). Statistically significant differences were found in Prothrombin Time, Factor V, and Factor IX activity, as well as Hcy concentration; these values suggest the presence of hypercoagulable states in patients, which can cause thrombotic events. On the other hand, significant differences were also found in the neutrophil–lymphocyte ratio and systemic immune-inflammation index, showing major inflammation states in the patient group. Moreover, statistically significant differences were found in the IL-23R rs1569922 polymorphism; it was found that carriers of the T/T and C/T genotypes have an increased risk of developing LCPD. Conclusions: Our results show greater hemostatic activity and inflammation in the group of patients included in this study, supporting various theories previously proposed. Therefore, we believe that LCPD is a multifactorial condition in which hemostatic, inflammatory, and genetic factors play a central and triggering role in the disease. Full article

Atherosclerosis as a multifactorial disease remains the first cause of death worldwide. Current oral lipid-lowering drugs (especially statins) reduce low-density lipoprotein cholesterol (LDLC) levels in the blood, but their clinical efficacy seems to be partially attributed to pleiotropic effects on different pathophysiologic factors of atherosclerosis extending beyond lipid-lowering properties such as anti-inflammatory, antithrombotic and antioxidative features. Novel drugs that interfere with proprotein convertase subtilisin/kexin type 9 (PCSK9) axis of LDL-C receptors (LDLRs) degradation, from the group of monoclonal antibodies (e.g., alirocumab, evolocumab) or small interfering RNA (siRNA), e.g., inclisiran, are effective in reducing LDLC as well. However, data depicting their antithrombotic and antiplatelet activity are scarce, whereas prothrombotic properties of PCSK9 are widely described. Thus, we performed a study to assess the effects of inclisiran on subclinical prothrombotic [fibrinogen, coagulation factor VIII (FVIII), plasminogen activator inhibitor-1 (PAI-1)] and platelet activation markers (platelet factor-4 (PF-4), soluble p-selectin (sCD62P)). Ten patients at high cardiovascular risk with concomitant heterozygous familial hypercholesterolemia (HeFH)—study group 1, and fourteen patients at very high cardiovascular risk without concomitant HeFH—study group 2, were recruited for the study. Lipid profile, subclinical prothrombotic and platelet activation markers were assessed at the beginning and after 3 months of therapy with inclisiran. During therapy, statistically significant reductions in both study groups were seen in total cholesterol levels (study group 1: from 287.6 ± 94.2 to 215.2 ± 89.1 (mg/dL), p = 0.022; study group 2: from 211.7 ± 52.7 to 147.6 ± 55.4 (mg/dL), p < 0.001) and LDL-c (study group 1: from 180.8 ± 73.3 to 114.7 ± 71.5 (mg/dL), p = 0.031; study group 2: from 129.6 ± 46.8 to 63.4 ± 43.6 (mg/dL), p < 0.001). Lipid profile changes were associated with significant decrease in the concentration of FVIII in both groups (study group 1: from 33.3 ± 22 to 22 ± 14.5 (ng/mL), p = 0.006; study group 2: from 37 ±16.9 to 29.3 ±16.4 (ng/mL), p = 0.002) and fibrinogen, but only in study group 2 (from 51.4 (33.2–72.7) to 42.6 (31.3–57.2) (µg/mL), p = 0.035). Among platelet activation markers, a significant decrease in PF-4 in study group 2 was noted (from 286 (272–295.5) to 272 (268–281.5) (ng/mL), p = 0.047). However, there were no statistically significant changes in PAI-1 and sCD62P throughout the study. In our study, inclisiran appeared to be an effective lipid-lowering drug in patients at high cardiovascular risk. Moreover, it was shown that beyond lipid-lowering properties, the drug may also partially affect thrombogenesis and platelet activation. Full article

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. Full article

A 63-year-old woman with rheumatoid arthritis and Hashimoto’s thyroiditis was admitted to the emergency room, because of left leg pain associated with spontaneous subcutaneous hematomas, for 15 days. Their symptoms also occurred after the discontinuation of aspirin, which the patient had taken for a previous case of ocular papillitis. Laboratory tests showed anemia, a normal platelet count, but a prolonged activated partial thromboplastin time (aPTT) ratio; a computerized tomography scan of the left lower limb detected a recent hematoma in the left lateral rectus muscle, and subcutaneous soft tissue edema also involving the knee, without vascular involvement. Coagulation tests were performed showing normal levels of Lupus Anticoagulant, very low-factor FVIII activity (2.2%), normal FIX, FXI, and FXII activity, and the detection of FVIII inhibitors by a Bethesda assay (7.6 U). A diagnosis of acquired hemophilia A (AHA) was made, and hemostatic and immunosuppressive treatment was immediately started (activated prothrombin complex concentrates and methylprednisolone). Malignancies and infections were excluded. An autoantibodies panel confirmed the positivity to rheumatoid factor and anti-cyclic citrullinated peptide antibodies. In treatment, the patient did not present any new bruises, with aPTT normalizing, FVIII increasing, and inhibitors reducing until disappearance. A close follow-up continued every 1–2 week after discharge, with hemostatic treatment discontinuation and methylprednisolone decalage. Underlying autoimmune conditions induced this rare, autoimmune and life-threating disorder. Full article

Missense mutations are the most prevalent alterations in genetic disorders such as hemophilia A (HA), which results from coagulation factor VIII (FVIII) deficiencies. These mutations disrupt protein biosynthesis, folding, secretion, and function. Current treatments for HA are extremely expensive and inconvenient for patients. Small molecule drugs offer a promising alternative or adjunctive strategy due to their lower cost and ease of administration, enhancing accessibility and patient compliance. By screening drug/chemical libraries with cells stably expressing FVIII–Gaussia luciferase fusion proteins, we identified compounds that enhance FVIII secretion and activity. Among these, suberoylanilide hydroxamic acid (SAHA) improved the secretion and activity of wild-type FVIII and common HA-associated missense mutants, especially mild and moderate ones. SAHA increased FVIII interaction with the endoplasmic reticulum chaperone BiP/GRP78 but not with calreticulin. Lowering cellular BiP levels decreased SAHA-induced FVIII secretion and enhancing BiP expression increased FVIII secretion. SAHA also enhanced secretion and BiP interactions with individual domains of FVIII. In vivo, treating mice with SAHA or a BiP activator boosted endogenous FVIII activity. These findings suggest that SAHA serves as a proteostasis regulator, providing a novel therapeutic approach to improve the secretion and functionality of FVIII missense mutants prone to misfolding. Full article

Factor VIII (FVIII) is involved in several molecular pathways and biological processes; indeed, it has a role in the coagulative cascade, cardiovascular disease, hypertension, brain and renal function, cancer incidence and spread, macrophage polarization, and angiogenesis. Hemophilic patients usually present an increase in fracture risk, bone resorption, and an excess of osteoporosis as compared to healthy individuals. Several studies have tried to clarify their etiology but unfortunately it is still unclear. This review focuses on the role of FVIII in bone biology by summarizing all the knowledge present in the literature. We carried out a systematic review of the available literature following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Several studies demonstrated that FVIII is involved in different molecular pathways interfering with bone physiology; it exerts interesting effects on OPG/RANK/RANKL pathways and thrombin/PAR1 pathways. These data confirm a relationship between FVIII and bone metabolism; however, there are still many aspects to be clarified. This review highlights the role of the coagulation factor FVIII in bone metabolism, suggesting new hypotheses for future studies both in vitro and in vivo to better understand the important pleiotropic role of FVIII and hopefully to develop new therapeutic agents for skeletal diseases. Full article

Hemophilia A (HA) is associated with FVIII coagulation insufficiency or inactivity leading to excessive bleeding. Elevated FVIII, on the contrary, is associated with thrombophilia, thrombosis, myocardial infarctions, and stroke. Active FVIII (aFVIII) uses its C2 domain to bind to blood cells’ membranes, consequently carrying out its coagulative function. We developed a reliable flow cytometry (FC) method for FVIII detection that can be utilized for assessing surface-bound FVIII on leukocytes in different coagulation/clinical states; we analyzed 49 pediatric subjects, encompassing patients with HA, other coagulopathies, venous thrombosis, and normal coagulation. Interestingly, the total leukocyte surface FVIII showed a declining trend across thrombosis, normal, and hypo-coagulation states. As expected, the leukocytes of HA patients displayed significantly lower levels of cellular-surface FVIII in comparison to patients with thrombosis. However, no significant correlation was observed between circulating levels of FVIII in plasma and the levels of FVIII bound to leukocytes, indicating that the differences in FVIII surface binding are not directly proportional to the availability of FVIII in the circulation and suggesting a specific binding mechanism governing the interaction between FVIII and leukocytes. Intriguingly, when analyzing the distinct blood subpopulations, we observed that surface FVIII levels were significantly elevated in classical monocytes of thrombosis patients compared to HA patients, healthy controls, and patients with other coagulopathies. Our study highlights the reliability of our FC platform in assessing FVIII abundance on leukocytes’ membranes across coagulation states. Monocytes, particularly in cases of thrombosis, exhibit active binding of FVIII on their surface, suggesting a potential role in the pathophysiology of thrombosis that requires further investigation. Full article

Background/Objectives: Hemophilia A is associated with frequent bleeding episodes, joint damage, and reduced bone mineral density (BMD). The role of coagulation factors and inflammatory cytokines on bone metabolism, particularly on osteoblast function, is of increasing interest. However, significant inter-species differences in bone remodeling raise concerns about the translatability of findings from murine models to human systems. This study aims to investigate the effects of human coagulation factors and cytokines on bone formation, focusing on inter-species differences in the cell viability and mineralization of murine and human osteoblasts. Methods: Murine MC3T3-E1 and human SaOs-2 osteoblasts were cultured in osteoblast differentiation medium supplemented with various coagulation factors (FVIII, vWF, vWF-FVIII, FIX, FX, thrombin, and FVIII-thrombin) or cytokines (IL-6, TNF-α). Cell viability was assessed at both two-week and three-week time points using the CCK-8 assay, and mineralization was evaluated via Alizarin red S staining. Results: Coagulation factors significantly enhanced cell viability in human osteoblasts but had no effects on the murine counterpart. FX inhibited mineralization in human cells, while murine cells showed no significant changes. TNF-α stimulated mineralization in murine osteoblasts but inhibited it in human cells, highlighting species-specific responses to inflammatory cytokines. Similar trends in response patterns were observed at two and three weeks, with greater consistency at the later time point. Conclusions: These findings reveal critical inter-species differences in osteoblast responses to coagulation factors and cytokines, raising questions about the validity of using murine models to study human bone metabolism. Future research must account for these differences to ensure that preclinical models accurately reflect human pathophysiology, particularly in the context of hemophilia A. Full article

Background and Objectives: Sustained virologic responses (SVRs) lead to a decrease in portal hypertension, the regression of fibrosis, and the improvement in the hepatic synthesis of procoagulant and anticoagulant factors. We aimed to assess the influence of SVR on coagulation parameters in cirrhotic patients with HCV treated with DAAs. Methods: We performed a prospective study in the Institute of Gastroenterology and Hepatology Iasi, Romania, between January 2022 and February 2024. We included patients diagnosed with compensated and decompensated HCV-related liver cirrhosis, treated with direct antivirals (PrOD ± RBV or SOF/LED ± RBV) for 12/24 weeks. Blood samples for biochemical, immunological, and coagulation tests were collected at the baseline, end of treatment (EOT), and once sustained virological response had been achieved over a period of 12/24 weeks (SVR12/24). Results: We analyzed a group of 52 patients with HCV-related liver cirrhosis, predominantly female (68.0%), and the degree of severity of cirrhosis placed the patients mainly in Child–Pugh classes B (40%) and C (36%). All patients achieved SVRs. The MELD score decreased at EOT (13.48 ± 4.273; p = 0.001) and SVR (9.88 ± 2.774; p = 0.000), compared to the baseline (14.92 ± 4.707). The FibroScan values decreased at SVR (17.596 ± 3.7276; p = 0.000) compared to the baseline (26.068 ± 7.0954). For all common coagulation parameters (platelets, INR, PT, fibrinogen, aPTT), there was a trend towards improvement during treatment, including changes which were statistically significant for the majority of patients. Factor II was low at the baseline (75.40 ± 7.506) but increased at EOT (87.40 ± 9.587) and, later, at SVR (99.12 ± 11.695; p = 0.000). The FVIII values increased at the baseline (175.52 ± 16.414) and decreased at EOT (151.48 ± 13.703) and SVR (143.40 ± 13.937). The FvW values decreased during treatment (146.84 ± 9.428, at baseline; 141.32 ± 9.690, p = 0.000, at EOT; and 126.68 ± 17.960, at SVR). In regard to the anticoagulant factors (PC, PS, ATIII), a significant improvement was brought on by SVR. Advanced stages of liver disease showed the most diminished FII activity, while at the baseline and in Child–Pugh C patients we recorded the highest values of FVIII and FvW. Conclusions: Our study proved that the “reset” of coagulopathy might be due to the improvement in liver function due to viral eradication secondary to AAD therapy. Full article

Hemophilia A is an X-linked disorder characterized by quantitative deficiency of coagulation factor VIII (FVIII) caused by pathogenic variants in the factor 8 (F8) gene. Our study’s primary objective was to identify genetic variants within the exonic region of F8 in 50 Colombian male participants with severe hemophilia A (HA). Whole-exome sequencing and bioinformatics analyses were performed, and bivariate analysis was used to evaluate the relationship between identified variants, disease severity, and inhibitor risk formation. Out of the 50 participants, 21 were found to have 17 different pathogenic F8 variants (var). It was found that 70% (var = 12) of them were premature truncation variants (nonsense, frameshift), 17.6% (var = 3) were missense mutations, and 11.7% (var = 2) were splice-site variants. Interestingly, 35% (var = 6) of the identified variants have not been previously reported in the literature. All patients with a history of positive inhibitors (n = 4) were found to have high-impact genetic variants (nonsense and frameshift). When investigating the relationship between variant location (heavy versus light chain) and specific inhibitor risk, 75% (n = 3) of the inhibitor participants were found to have variants located in the F8 light chain (p = 0.075), suggesting that conserved domains are associated with higher inhibitor risk. In summary, we identified genetic variants within the F8 that can possibly influence inhibitor development in Colombian patients with severe HA. Our results provide a basis for future studies and the development of further personalized treatment strategies in this population. Full article

The presence of skin bacteria capable of forming biofilm, exhibiting antibiotic resistance, and displaying virulence represents a significant challenge in the field of transfusion medicine. This underscores the necessity of enhancing the microbiological safety of blood and blood components against pathogens with virulent characteristics. The aim of this work was to demonstrate bacterial inactivation in plasma by using a photoinactivation method against virulent bacteria and to evaluate coagulation factors before and after treatment. Logarithmic loads of biofilm-producing, antibiotic-resistant, and virulent bacteria isolated from skin (Enterobacter cloacae, Klebsiella ozaenae, and Staphylococcus epidermidis) were used in artificial contamination assays of fresh frozen plasma bags and subjected to photoreduction. FVIII and FI activity were evaluated before and after photoinactivation. The photoinactivation of plasma was demonstrated to be an effective method for the elimination of these bacteria. However, the efficiency of this method was found to be dependent on the bacterial load and the type of test microorganism. Conversely, decay of coagulation factors was observed with net residual activities of 61 and 69% for FVIII and FI, respectively. The photoinactivation system could have a bias in its effectiveness that is dependent on the test pathogen. These findings highlight the importance of employing technologies that increase the safety of the recipient of blood and/or blood components, especially against virulent bacteria, and show the relevance of the role of photoinactivation systems as an option in transfusion practice. Full article

Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies. Full article