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.

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.

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.

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.

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.

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.

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.

The safety of the combination therapy of recombinant factor VIIa and plasma-derived factor VIIa and factor X for refractory hemorrhage in acquired hemophilia A.

Acquired hemophilia A (AHA) is a rare, life-threatening hemorrhagic disease caused by autoantibodies against factor VIII (FVIII), and bypassing agents (BPA) are used to control bleeding. However, some cases need a change of BPA or BPAs given sequentially or in combination for refractory bleeding. A 71-year-old man was admitted with subcutaneous hemorrhage. Laboratory investigations showed prolongation of activated partial thromboplastin time (APTT) and low-coagulation FVIII activity and FVIII inhibitor; we, therefore, diagnosed AHA. He was treated with recombinant factor VIIa (rFVIIa) BPA and prednisolone. However, his symptoms did not improve sufficiently, thus we switched BPA to activated prothrombin complex concentrate. Unfortunately, this was not effective and he suffered hemorrhagic shock. Therefore, we selected rFVIIa, with plasma-derived FVIIa and factor X (pd-FVIIa/FX) as combination therapy, and hemostasis was achieved without thrombosis. This case suggests that the combination of rFVIIa and pd-FVIIa/FX short-term can be well tolerated for refractory hemorrhage in AHA.

Role of individual factor X concentrate pharmacokinetic studies in perioperative management of AL amyloidosis-associated acquired factor X deficiency.

BACKGROUND: AL amyloidosis is associated with acquired factor X (FX) deficiency. Experience related to its management is limited to case reports and series using prothrombin complex concentrate, fresh frozen plasma, plasma exchange, recombinant activated factor seven, and desmopressin with limited and variable efficacy. FX concentrate has not been widely used in its management. STUDY DESIGN AND METHODS: We report our experience with the perioperative use of FX concentrate (Coagadex) in two patients with AL amyloidosis-associated acquired FX deficiency requiring surgery, using their individual pharmacokinetic studies to manage perioperative hemostasis. Pharmacokinetic studies involved obtaining post-infusion FX activity at 10 min, 2, and 4 h following the administration of FX concentrate to calculate the FX half-life. RESULTS: Both patients' plasma FX activity was successfully increased to provide perioperative hemostatic support. Monitoring FX activity post-surgery was also utilized to maintain FX activity levels to prevent post-operative bleeding. CONCLUSION: Pharmacokinetic studies have a useful role in tailoring preoperative FX repletion in patients with AL amyloidosis associated with acquired FX deficiency.

Assay discrepancies using human coagulation factor VIII chromogenic kits: Results from a plasma-derived factor VIII collaborative study (BSP112).

Chromogenic assay discrepancies were reported at General European Official Medicines Control Laboratories Network (GEON) meetings by laboratories testing FVIII-products. The objectives of the present investigation were to carry out a controlled collaborative study to examine these reports and to delineate the reasons for these discrepancies by assessing affected and unaffected FVIII products. The laboratories followed a strict study protocol, which included assessing their own individual observed factor X (FX) activation times, i.e. the time to reach 50% of maximal FX activation (T1/2), for each chromogenic kit. This measurement was used, in parallel with the kit manufacturers' prescribed FX activation times, to assess the performance of the chromogenic potency assays on FVIII test products. This study confirmed a significant discrepancy between Coatest® and Coamatic® kits and between Siemens and Coamatic® kits when the kit manufacturers' prescribed T1/2 incubation times were followed. Coamatic® kits tended to produce higher potencies than the Coatest® or Siemens kits. Furthermore, FX activation assays revealed marked differences between individual laboratories for all three chromogenic kits in the observed T1/2 incubation times, which also did not correspond to the prescribed T1/2 incubation times. The resulting differences in potency between kits, in some cases, were significantly reduced when using the actual observed T1/2 incubation times instead of the prescribed T1/2 incubation times. The study showed that FVIII potency discrepancies can occur between chromogenic kits. To compensate for this, laboratories should ideally perform FX activation curves for each new chromogenic kit in order to determine the correct observed T1/2 incubation times, which can then be used to determine FVIII potencies in therapeutic concentrates.