Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors.

BACKGROUND: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. OBJECTIVES: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. METHODS: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. RESULTS: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. CONCLUSION: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Fitusiran reduces bleeding in factor X-deficient mice.

ABSTRACT: Factor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 µL to 590 ± 335 µL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Reduced plasma factor X is associated with a lack of response to recombinant activated factor VII in patients with hemophilia A and inhibitor, but does not impair emicizumab-driven hemostasis in vitro.

BACKGROUND: The hemostatic effect of recombinant (r) factor (F)VIIa after repetitive intermittent administration may be attenuated in patients with hemophilia A (PwHA) with inhibitors (PwHAwI) creating a clinically unresponsive status, although mechanism(s) remain to be clarified. In patients receiving prophylaxis treatment with emicizumab, concomitant rFVIIa is sometimes utilized in multiple doses for surgical procedures or breakthrough bleeding. AIM AND METHODS: We identified 'unresponsiveness' to rFVIIa, based on global coagulation function monitored using rotational thromboelastometry (ROTEM) in 11 PwHAwI and 5 patients with acquired HA, and investigated possible mechanisms focusing on the association between plasma FX levels and rFVIIa-mediated interactions. RESULTS: Our data demonstrated that FX antigen levels were lower in the rFVIIa-unresponsive group than in the rFVIIa-responsive group (0.46 ± 0.14 IU/mL vs. 0.87 ± 0.15 IU/mL, p < 0.01). This relationship was further examined by thrombin generation assays using a FX-deficient PwHAwI plasma model. The addition of FX with rFVIIa was associated with increased peak thrombin (PeakTh) generation. At low levels of FX (<0.5 IU/mL), rFVIIa failed to increase PeakTh to the normal range, consistent with clinical rFVIIa-unresponsiveness. In the presence of emicizumab (50 µg/mL), PeakTh was increased maximally to 80 % of normal, even at low levels of FX (0.28 IU/mL). CONCLUSIONS: Unresponsiveness to rFVIIa was associated with reduced levels of FX in PwHAwI. Emicizumab exhibited in vitro coagulation potential in the presence of FX at concentrations that appeared to limit the clinical response to rFVIIa therapy.

MAX controls meiotic entry in sexually undifferentiated germ cells.

Meiosis is a specialized type of cell division that occurs physiologically only in germ cells. We previously demonstrated that MYC-associated factor X (MAX) blocks the ectopic onset of meiosis in embryonic and germline stem cells in culture systems. Here, we investigated the Max gene's role in mouse primordial germ cells. Although Max is generally ubiquitously expressed, we revealed that sexually undifferentiated male and female germ cells had abundant MAX protein because of their higher Max gene expression than somatic cells. Moreover, our data revealed that this high MAX protein level in female germ cells declined significantly around physiological meiotic onset. Max disruption in sexually undifferentiated germ cells led to ectopic and precocious expression of meiosis-related genes, including Meiosin, the gatekeeper of meiotic onset, in both male and female germ cells. However, Max-null male and female germ cells did not complete the entire meiotic process, but stalled during its early stages and were eventually eliminated by apoptosis. Additionally, our meta-analyses identified a regulatory region that supports the high Max expression in sexually undifferentiated male and female germ cells. These results indicate the strong connection between the Max gene and physiological onset of meiosis in vivo through dynamic alteration of its expression.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Age-related variation in coagulation factors in non-valvular atrial fibrillation patients receiving direct oral anticoagulants.

Age is a significant risk factor for ischemic stroke. However, the influence of aging on coagulation parameters in non-valvular atrial fibrillation (NVAF) patients treated with direct oral anticoagulants (DOACs) remains unclear. A total of 775 samples were collected from 224 NVAF patients receiving apixaban, edoxaban or rivaroxaban. The samples were categorized into three age groups: (i) ≤ 64 years, (ii) 65-74 years, and (iii) ≥ 75 years (apixaban: N = 48, 108, 119; edoxaban: N = 63, 68, 126; rivaroxaban: N = 115, 90, 38, respectively). Coagulation parameters including fibrinogen (Fbg), factor II, factor V, factor VII, factor X, and D-dimer, were compared between the three age groups for each drug. The slopes in the correlation between drug concentrations and modified diluted prothrombin time (mdPT) were also assessed. Fbg and factor V increased with age, while factor II and factor X decreased. Factor VII and D-dimer showed no significant differences across age categories. The slope in response to drug concentrations was similar between the age groups. In NVAF patients treated with apixaban, edoxaban and rivaroxaban, some coagulation parameters exhibited age-related variation. However, the response of mdPT to drug concentration was consistent across age categories.

Associations between coagulation factors and livedoid vasculopathy and the efficacy and safety of rivaroxaban in patients with livedoid vasculopathy: a retrospective study.

BACKGROUND: The mechanism of livedoid vasculopathy (LV) remains unknown. OBJECTIVES: To investigate the association between coagulation factors and LV and to assess the efficacy and safety of rivaroxaban in the treatment of patients with LV. METHODS: From May 2019 to July 2022, 89 patients with LV and 35 healthy controls were included in a cross-sectional cohort to measure the levels of coagulation factors. In addition, 55 patients with LV treated with rivaroxaban were included in a treatment cohort to assess the complete remission rate of ulcers (n = 44) and retiform purpura (n = 11) within 12 weeks. RESULTS: In the cross-sectional cohort, the activities of coagulation factor X in patients with LV were significantly higher than those in healthy controls: median 110.5% [interquartile range (IQR) 97.5-127.0%] vs. 101.3% (IQR 91.6-115.6); P = 0.05. In addition, coagulation factor X activities in the progressive stage were higher than at the stable stage: median 111.6% (IQR 102.3-132.5) vs. 105.4% (IQR 92.9-118.8); P = 0.04. Moreover, coagulation factor X activities were higher at the progressive stage than at the stable stage in a subgroup of 20 patients with LV (P = 0.04). In the treatment cohort taking rivaroxaban, 91% (40/44) of patients with ulcers achieved complete remission within 12 weeks, and 73% (8/11) of patients with retiform purpura achieved complete remission within 12 weeks. Mild side-effects occurred in 25% of patients (14/55), including menorrhagia (n = 10), gingival bleeding (n = 3) and haemorrhage (n = 1). CONCLUSIONS: Coagulation factor X was associated with the incidence and severity of LV in this study. In addition, rivaroxaban was an effective and safe treatment for ulcers and retiform purpura in people with LV.

Plasma-derived human factor X concentrate for the treatment of patients with hereditary factor X deficiency.

INTRODUCTION: Hereditary factor X (FX) deficiency (HFXD) is an autosomal recessive rare bleeding disorder that leads to defects in the FX protein. Depending on the degree of deficiency, patients may be at risk of life-threatening bleeding episodes. Historical treatments for FX deficiency include prothrombin complex concentrates, which can increase the risk of thrombosis, and fresh frozen plasma, which can cause volume overload and transfusion reactions. Plasma-derived FX (pdFX), a single-factor, high-purity, high-potency human FX treatment, was approved in 2015 in the United States and in 2016 in Europe for on-demand treatment and prophylaxis of bleeding episodes and perioperative management of patients with HFXD. METHODS: Five studies that examined the use of pdFX in patients with mild (plasma FX activity [FX:C] ≥5 IU/dL), moderate (FX:C ≥1 and <5 IU/dL), or severe (FX:C < 1 IU/dL) HFXD were reviewed: TEN01, TEN02 and TEN03 were prospective, open-label, multicentre, nonrandomised studies, and TEN05 and TEN06 were multicentre retrospective studies. RESULTS: When used as an on-demand treatment, pdFX was judged by investigators to be successful in treating 41/42 (97.6%), 2/3 (66.6%) and 79/79 (100%) bleeds in TEN01, TEN02 and TEN05, respectively. When used prophylactically, pdFX was judged 'excellent' for the prevention of bleeds in nine (100%) and eight (100%) patients in TEN02 and TEN05, respectively. Perioperative treatment and pharmacokinetics were also assessed. pdFX was safe and well tolerated. CONCLUSIONS: Together, these studies support the use of pdFX for on-demand treatment of bleeding, routine prophylaxis, and perioperative management of bleeding in patients with HFXD.

Five new F10 variants in hereditary factor x deficiency detected by high-throughput sequencing.

INTRODUCTION: Factor X deficiency is a rare inherited bleeding disorder. To date, 181 variants are reported in the recently updated F10-gene variant database. AIM: This study aimed to describe new F10 variants. METHOD: The F10 gene was analysed in 16 consecutive families with FX deficiency by a targeted high-throughput sequencing approach, including F10, F9, F8 genes, and 78 genes dedicated to haematological malignancies. RESULTS: We identified 19 variants (17 missense, one nonsense and one frameshift) and two copy number variations. Two patients presenting a combined FVII-FX deficiency showed a loss of one F10 gene copy (del13q34) associated with a missense variant on the remaining allele, leading to a FX:C significantly lower than the FVII:C level and explaining their unusual bleeding history. We reported five novel variants. Three missense variants (p.Glu22Val affecting the signal peptide cleavage site, p.Cys342Tyr removing the disulphide bond between the FX heavy and light chains, and p.Val385Met located in FX peptidase S1 domain) were detected at compound heterozygosis status in three patients with severe bleeding symptoms and FX:C level below 10 IU/dL. Two truncating variants p.Tyr279* and p.Thr434Aspfs*13 leading to an altered FX protein were found at heterozygous state in two patients with mild bleeding history. CONCLUSION: This study showed the feasibility and the interest of high-throughput sequencing approach for rare bleeding disorders, enabling the report of F10 gene screening in a 3-weeks delay, suitable for clinical use. The description of five new variants may contribute to a better understanding of the phenotype-genotype correlation in FX deficiency.

Postmortem diagnosis of severe factor X deficiency in a fetus with intracranial hemorrhage resulting in intrauterine death.

In patients with severe congenital factor X deficiency, spontaneous intracranial hemorrhage (ICH) is particularly frequent in early childhood. We describe a case of fetal death at 26 weeks due to massive ICH. Gene panel analysis of postmortem samples revealed homozygosity for a pathologic F10 gene variant (c.1210T>C, p.Cys404Arg), which impedes correct folding of the catalytic serine protease domain and, therefore, causes a significant reduction in FX levels. The parents, not consanguineous but of the same ethnic community, were found to be heterozygous for this variant and did not have any personal or family history of abnormal bleeding. To the best of our knowledge, this is the first reported case of severe FX deficiency resulting in ICH diagnosed through postmortem genetic analysis. It illustrates the importance of exploring the etiology of fetal or neonatal ICH, which may impact future pregnancies, and the treatment of a potential coagulopathy in the child.

Preclinical development and characterization of a human plasma-derived high-purity factor X concentrate for therapeutic use.

INTRODUCTION: Hereditary factor X deficiency is a rare bleeding disorder, with limited treatment options. This paper describes the approach to pre-clinical development and characterization of a high-purity plasma-derived factor X concentrate, to achieve orphan drug marketing authorization for the treatment of hereditary factor X deficiency. METHODS: A chromatographic process was developed, to purify factor X from human plasma for fractionation. The product was characterized using in vitro, in vivo and ex vivo tests for potency, purity, thrombogenicity, immunogenicity, toxicity and stability. RESULTS: The production process complied with good pharmaceutical manufacturing practice. It achieved 6000-fold purification and 100-fold concentration of the factor X protein compared to human plasma. The factor X protein was 94%-96% pure. Other residual plasma proteins were well below levels in plasma, minimizing potential interference in hemostasis after therapeutic administration. Effective virus-reduction during manufacture, and the absence of thrombogenicity, toxicity and immunogenic potential were confirmed, addressing the main safety concerns historically associated with plasma-derived therapeutics. The freeze-dried product remained stable between +2°C and +30°C for at least three years. After reconstitution with water for injections, the factor X activity was maintained for at least 48 h at +18°C to +22°C. CONCLUSION: Targeted pre-clinical development of the first highly-purified concentrate to treat hereditary factor X deficiency is described. Following international guidelines for nonclinical safety testing, particular strategies were adopted for unmodified products derived from human blood plasma. This approach may also be relevant to the development of other ultra-orphan medicinal products.

A novel factor modulating X chromosome dosage compensation in Anopheles.

Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies.1 In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex.2,3 The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway.4 It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs5 by a yet-unknown molecular mechanism distinct from that in D. melanogaster.6 Here we show that a male-specifically spliced gene we call 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes,7 introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control.

Clinical Values of Coagulation Factors X, XI, and XII in Cerebral Venous Sinus Thrombosis During Perinatal State.

Cerebral venous sinus thrombosis (CVST) has become a rare but potentially life-threatening condition in perinatal women. Early and rapid identification of CVST in pregnant women is a challenge for frontline clinical workers. In this study, 40 perinatal patients with CVST in our hospital were included in the five-year period, and 120 normal perinatal pregnant women in the obstetrics and gynecology department of our hospital were randomly enrolled in the five-year period as the control group, including 60 cases in pregnancy and puerperium. 5 mL of fasting venous blood was collected from puerperal CVST patients in the acute phase of onset (within 72 h of onset) and the recovery phase (fourth week of treatment). In the control group, 5 mL of fasting venous blood was collected. Coagulation factors X, XI, and XII, plasma D-Dimer were analyzed and compared. Coagulation factors X, XI, and XII in plasma of CVST patients were significantly increased compared with controls. Plasma coagulation factors X, XI, and XII and their combined detection (Union Model = 0.056 * FX: C + 0.046 * FXI: C + 0.081 * FXII: C) have diagnostic values for perinatal CVST. Plasma coagulation factors X, XI, and XII were significantly positively correlated with plasma D-dimer levels in perinatal CVST patients. Plasma coagulation factors X, XI, and XII have diagnostic values for perinatal CVST.

Establishment of SV40 Large T-Antigen-Immortalized Yak Rumen Fibroblast Cell Line and the Fibroblast Responses to Lipopolysaccharide.

The yak lives in harsh alpine environments and the rumen plays a crucial role in the digestive system. Rumen-associated cells have unique adaptations and functions. The yak rumen fibroblast cell line (SV40T-YFB) was immortalized by introducing simian virus 40 large T antigen (SV40T) by lentivirus-mediated transfection. Further, we have reported the effects of lipopolysaccharide (LPS) of different concentrations on cell proliferation, extracellular matrix (ECM), and proinflammatory mediators in SV40T-YFB. The results showed that the immortalized yak rumen fibroblast cell lines were identified as fibroblasts that presented oval nuclei, a fusiform shape, and positive vimentin and SV40T staining after stable passage. Chromosome karyotype analysis showed diploid characteristics of yak (n = 60). LPS at different concentrations inhibited cell viability in a dose-dependent manner. SV40T-YFB treated with LPS increased mRNA expression levels of matrix metalloproteinases (MMP-2 and MMP-9), inflammatory cytokines (TNF-α, IL-1ß, IL-6), and urokinase-type plasminogen activator system components (uPA, uPAR). LPS inhibits the expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), plasminogen activator inhibitor-2 (PAI-2), fibronectin (FN), anti-inflammatory factor IL-10, and collagen I (COL I) in SV40T-YFB. Overall, these results suggest that LPS inhibits cell proliferation and induces ECM degradation and inflammatory response in SV40T-YFB.

Long-Term Exposure to Microcystin-LR Induces Gastric Toxicity by Activating the Mitogen-Activated Protein Kinase Signaling Pathway.

Previous studies have primarily concentrated on the hepatotoxicity of MC-LR, whereas its gastric toxicity effects and mechanisms of long-term exposure under low dosage remain unknown. Herein, the gastric tissue from C57BL/6 mice fed with drinking water contaminated by low-dose MC-LR (including 1, 60, and 120 µg/L) was investigated. The results obtained showed that exposure to different concentrations of MC-LR resulted in significant shedding and necrosis of gastric epithelial cells in mice, and a down-regulation of tight junction markers, including ZO-1, Claudin1, and Occludin in the stomach, which might lead to increased permeability of the gastric mucosa. Moreover, the protein expression levels of p-RAF/RAF, p-ERK1/2/ERK1/2, Pink1, Parkin, and LC3-II/LC-3-I were increased in the gastric tissue of mice exposed to 120 µg/L of MC-LR, while the protein expression level of P62 was significantly decreased. Furthermore, we found that pro-inflammatory factors, including IL-6 and TNF-ɑ, were dramatically increased, while the anti-inflammatory factor IL-10 was significantly decreased in the gastric tissue of MC-LR-exposed mice. The activation of the MAPK signaling pathway and mitophagy might contribute to the development of gastric damage by promoting inflammation. We first reported that long-term exposure to MC-LR induced gastric toxicity by activating the MAPK signaling pathway, providing a new insight into the gastric toxic mechanisms caused by MC-LR.